Compositions for enhancing nucleic acid delivery

Abstract

The present invention relates to a composition for enhancing the delivery of an active agent or therapeutic agent, such as a therapeutic nucleic acid, comprising: (A) an active agent or therapeutic agent, preferably comprising a nucleic acid; (B) an amphiphilic block copolymer; (C) a cationic lipid; and (D) a non-cationic lipid. The invention also provides methods for making, delivering, and using such compositions, as well as the use of the compositions for the treatment and / or prevention of related diseases or conditions.

Description

Compositions for enhancing nucleic acid delivery Field of the Invention

[0001] The present invention relates to compositions for enhancing the delivery of active or therapeutic agents, such as therapeutic nucleic acids. Background Art

[0002] After being successfully introduced into the cells of an organism, biological macromolecules such as proteins, DNA, siRNA, and in vitro transcribed messenger RNA (mRNA) can exert their efficacy by producing or acting on specific protein functions, thereby achieving the purpose of disease prevention or treatment. These biological macromolecules are becoming an important method for preventing and treating a variety of diseases. Among them, vaccines developed based on mRNA technology have shown great potential in combating the novel coronavirus (SARS-CoV-2) pandemic that broke out in 2019 with extremely high protection rates. Currently, two new coronavirus (COVID-19) mRNA vaccines have been approved for marketing by the US FDA. In addition to the new coronavirus mRNA vaccine, mRNA technology also has great application potential in tumor immunotherapy, gene therapy, infectious disease prevention, gene editing, genetic disease treatment and other fields.

[0003] mRNA technology has many advantages: it is manufactured in a cell-free manner, which enables rapid, economical and efficient production, and has a unique advantage in quickly responding to large-scale outbreaks of sudden infectious diseases; mRNA is almost never integrated into the genome, is highly safe, and avoids the possibility of insertion mutations; in addition, a single mRNA vaccine can encode multiple antigens, enhance the immune response against adaptive pathogens, and can target multiple microorganisms or viral variants with a single formulation.

[0004] mRNA therapy involves administering specific mRNA to a subject in need of the therapy, thereby producing the protein encoded by the mRNA within the patient's body. Using a safe delivery system to efficiently and precisely deliver mRNA to target tissues and cells, maximizing its expression efficiency, is key to the successful clinical translation of these drugs or vaccines. The successful expression of mRNA in the body is a systemic, cascading process, with key challenges: 1) mRNA is easily degraded by widespread RNases, and its structural stability is a prerequisite for its biological effects; 2) mRNA is easily cleared from the body, resulting in a short half-life and difficulty reaching specific target sites; and 3) due to its large molecular weight, strong hydrophilicity, and electronegativity, mRNA itself struggles to effectively cross various physiological barriers in the body, such as mucosal barriers, cell membranes, and lysosomal barriers. Therefore, the effective targeted delivery of mRNA drugs / vaccines has always been challenging in medicine.

[0005] Currently, commonly used gene therapy delivery systems are categorized into two types: viral and non-viral vectors. Early attempts at using viral vectors as a gene delivery method focused on their relatively high in vivo transfection efficiency. However, major challenges with viral vectors, such as host immune responses to the viral vectors themselves, difficulty with repeated administration, potential activation of oncogenes that cause malignancies, and inflammatory complications, have significantly hindered their development. In contrast, non-viral vectors offer numerous advantages over viral vectors, including improved safety, reduced immunogenicity, reduced pathogenicity, reduced insertional mutagenesis, and ease of large-scale preparation. Nanoparticles, developed from nanobiomaterials, are a typical type of non-viral vector. Common non-viral vectors include lipid nanoparticles (LNPs) and cationic polymer-based formulations such as chitosan, polyethyleneimine, and dendrimers. The LNP system is, to date, the most common, most advanced, and the only mRNA drug delivery system to have successfully achieved clinical translation.

[0006] Although LNP has developed into the "gold standard" delivery technology in the mRNA field due to its many advantages, the system still has many defects: for example, the LNP system usually needs to be administered through injection routes such as intramuscular injection, subcutaneous injection, or intravenous injection, and can only produce efficient gene transfection effects in tissues with large gaps such as the liver and spleen, as well as in the intramuscular injection site (Pardi, N. et al. Expression kinetics of nucleoside-modified mRNA delivered in lipid nanoparticles to mice by various routes. J. Control. Release. 217, 345–351 (2015)). Studies have shown that LNP preparations only mediate very low mRNA gene expression in mice after respiratory administration (Zhang, N.-N. et al. A thermostable mRNA vaccine against COVID-19. Cell. 182, 1271-1283 (2020)). Secondly, LNP preparations have the problem of severe allergic reactions and pose risks in high-dose administration regimens (Ndeupen, S. et al. The mRNA-LNP platform's lipid nanoparticle component used in preclinical vaccine studies is highly inflammatory. iScience. 24, 103479 (2021); Landesman-Milo, D. & Peer, D. Toxicity profiling of several common RNAi-based nanomedicines: a comparative study. Drug Deliv. Transl. Res. 4, 96–103 (2014);). In the field of mRNA delivery, most successful application progress based on LNP systems is still limited to delivery to liver tissue (Samaridou, E. et al. Lipid nanoparticles for nucleic acid delivery: Current perspectives. Adv Drug Deliv Rev. 154-155, 37–63 (2020)), which greatly limits the widespread application of mRNA / LNP preparations in other target organ disease fields, such as the lungs, nose, gastrointestinal tract, reproductive tract and other mucosal sites.

[0007] Taking the lungs as an example, mRNA drugs have great potential for treating many lung diseases that currently have no cure, such as cystic fibrosis (CF), chronic obstructive pulmonary disease (COPD), α1-trypsin deficiency, asthma, pulmonary hypertension, primary ciliary dyskinesia, and idiopathic pulmonary fibrosis. The unique physiological structure of the lungs provides many advantages for its drug delivery route, making it an ideal expression site for mRNA drugs (Suberi, A., et al. Polymer nanoparticles deliver mRNA to the lung for mucosal vaccination. Sci Transl Med. 15, eabq0603 (2023)). Drugs delivered through the respiratory tract have a simple administration method, good patient compliance, are suitable for multiple repeated dosing regimens, and can also make the drug preparations evenly distributed in the bronchial and alveolar epithelial tissues (Patel, AK et al. Inhaled Nanoformulated mRNA Polyplexes for Protein Production in Lung Epithelium. Adv. Mater. e1805116, (2019)). Compared with the injection administration method, it can minimize potential systemic side effects and reduce problems such as cross-infection caused by needle contamination. The lung airway has a large absorption surface area and a rich capillary network, which is conducive to the absorption and efficient transfection of gene drugs. In addition, the lungs have a strong angiogenesis capacity, which can mediate the secretory protein to enter the circulatory system to exert its effect. Therefore, the lungs are an ideal target for protein replacement therapy based on mRNA drugs.

[0008] Secondly, mRNA vaccines have great application prospects in inducing antigen-specific mucosal immunity in the respiratory mucosa (e.g., nasal-associated lymphoid tissue NALT and bronchus-associated lymphoid tissue BALT). More than 90% of pathogens invade the human body through the mucosal parts (e.g., respiratory tract, gastrointestinal tract, reproductive tract, etc.), including the new coronavirus (SARS-CoV-2), severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS), influenza virus, respiratory syncytial virus, and Mycobacterium tuberculosis. A variety of virulent pathogens mainly infect the host through the respiratory mucosa (Lavelle, EC & Ward, RW Mucosal vaccines-fortifying the frontiers. Nat. Rev. Immunol. 22, 236-250 (2022)), which means that in the induction stage and the effect stage, the interaction between the pathogen and the host immune system first occurs in the respiratory mucosa. Taking the COVID-19 vaccine as an example, the vast majority of marketed vaccines are administered by intramuscular injection. This route can only induce a systemic immune response dominated by serum IgG antibodies. The resulting IgG neutralizing antibodies are difficult to reach and remain on the surface of the respiratory mucosa, making it difficult to eliminate the virus remaining in this area. Moreover, vaccines administered by intramuscular injection rarely induce a mucosal immune response, and thus fail to produce secretory IgA (sIgA) antibodies to protect the host from initial infection in the mucosal area. The virus may still replicate in the respiratory mucosa and cause infection in others. Studies have shown that the early specific humoral response in patients infected with SARS-CoV-2 is dominated by sIgA antibodies produced by the mucosal immune response (Sterlin, D. et al. IgA dominates the early neutralizing antibody response to SARS-CoV-2. Sci Transl Med. 13, eabd22234 (2021)). Moreover, the average neutralizing efficacy of sIgA against the new coronavirus is more than seven times that of IgG, and it plays a dominant role in the virus neutralization process (Wang, Z. et al. Enhanced SARS-CoV-2 neutralization by dimeric IgA. Sci Transl Med. 13, eabf1555 (2021)).Therefore, an efficient mucosal immune response is of great value to the efficacy of vaccines related to respiratory invasive pathogens and may be the key to eradicating the infection and spread of related pathogens (Jeyanathan, M. et al. Immunological considerations for COVID-19 vaccine strategies. Nat. Rev. Immunol. 20, 615–632 (2020)).

[0009] Although administering mRNA drugs / vaccines through the respiratory route has many advantages, how to achieve efficient delivery of mRNA in the respiratory tract is a difficult problem that has not yet been overcome worldwide. Due to the complex microenvironment of the respiratory mucosal tissue and the presence of a large number of enzymes, antigen molecules are easily degraded. The respiratory system has evolved over a long period of time to produce strong physiological barriers (such as bronchial structural barriers, mucus barriers, immune barriers and cell membrane barriers, etc.) and physiological mechanisms that can efficiently clear exogenous foreign bodies (such as ciliary clearance). It is difficult for mRNA molecules to reach the effector target, which places extremely high demands on mRNA delivery technology. As mentioned earlier, even "gold standard" delivery systems such as LNPs still find it difficult to mediate satisfactory mRNA gene transfection in the respiratory tract. Therefore, there is an urgent need to develop new delivery systems that can efficiently deliver mRNA molecules to mucosal sites such as the respiratory tract.

[0010] SUMMARY OF THE INVENTION

[0011] The present invention surprisingly found that adding amphiphilic block copolymers such as poloxamine or ) and or poloxamer or ) and other components, the resulting novel formulation can significantly improve the efficiency of gene transfection of nucleic acid molecules, such as mRNA, in animals mediated by such formulations, particularly when delivered through the mucosal membranes of the organism (e.g., the respiratory tract). When the formulation of the present invention is administered via the respiratory tract, the gene transfection effect in the animal's lungs / respiratory mucosal tissue is significantly superior to that of formulations such as LNP formulations, poloxamine, and poloxamer, thereby resolving the low delivery efficiency issue in the prior art.

[0012] Accordingly, in a first aspect, the present invention provides a polymer-lipid composition comprising:

[0013] (A) an active or therapeutic agent, preferably comprising a nucleic acid;

[0014] (B) amphiphilic block copolymers;

[0015] (C) cationic lipids; and

[0016] (D) non-cationic lipids,

[0017] Wherein the composition is formulated for delivery through a mucosal site of an organism, such as respiratory tract delivery, oral mucosal delivery, gastrointestinal tract delivery, ocular mucosal delivery, ear mucosal delivery, urethral delivery, or reproductive tract delivery, preferably the composition is formulated for delivery through the respiratory tract.

[0018] In some embodiments, the nucleic acid comprises at least one selected from the group consisting of messenger RNA (mRNA), self-amplifying RNA (saRNA), circular RNA (circRNA), small interfering RNA (siRNA), short hairpin RNA (shRNA) and microRNA (miRNA), primary-miRNA, antisense oligonucleotide (ASO), transfer RNA (tRNA), plasmid DNA (pDNA), single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), deoxyribozyme (DNAzyme), ribozyme (RNAzyme), nucleic acid aptamer (aptamer), clustered regularly interspaced short palindromic repeats (CRISPR)-related nucleic acid, single guide RNA (sgRNA), CRISPR-RNA (crRNA), trans-activating crRNA (tracrRNA), guide RNA, single-stranded RNA (ssRNA) and double-stranded RNA (dsRNA).

[0019] In some embodiments, the nucleic acid is a therapeutic nucleic acid. More preferably, the nucleic acid comprises mRNA.

[0020] In some embodiments of the compositions of the present invention, the amphiphilic block copolymer comprises 0.1%-98.0% by weight of the composition, for example, 0.5%-95.0% by weight, 1%-90.0% by weight, 10%-80.0% by weight, 20%-80.0% by weight, 30%-80.0% by weight, 40%-80.0% by weight, 40%-70.0% by weight, or 50%-60.0% by weight.

[0021] In some embodiments of the composition of the present invention, the cationic lipid comprises at least one selected from the group consisting of permanent cationic lipids, ionizable cationic lipids, cholesterol-derived cationic lipids, and dendrimers or dendrons. Preferably, the cationic lipid comprises an ionizable cationic lipid.

[0022] In some embodiments, the cationic lipid comprises 23 mol%-83 mol% of the total lipids present in the composition, e.g., 30 mol%-80 mol%, 30 mol%-70 mol%, or 40 mol%-60 mol%, or the cationic lipid comprises about 25 mol%, about 30 mol%, about 35 mol%, about 40 mol%, about 45 mol%, about 50 mol%, about 55 mol%, about 60 mol%, about 65 mol%, about 70 mol%, about 75 mol%, or about 80 mol% of the total lipids present in the composition.

[0023] In some embodiments of the compositions of the present invention, the non-cationic lipid comprises at least one selected from the group consisting of anionic lipids, zwitterionic lipids, and neutral lipids, preferably, the non-cationic lipid comprises a neutral lipid. In some embodiments, the neutral lipid comprises 19 mol% to 75 mol% of the total lipids present in the composition.

[0024] In some embodiments, the neutral lipid comprises:

[0025] Cholesterol or cholesterol-derived neutral lipids;

[0026] phospholipids; or

[0027] A mixture of cholesterol or cholesterol-derived neutral lipids and phospholipids.

[0028] In some embodiments, the cholesterol comprises 14 mol% to 70 mol% of the total lipids in the composition.

[0029] In some embodiments, the phospholipids comprise about 5 mol% to about 30 mol% or 30 mol% to about 75 mol% of the total lipids in the composition.

[0030] In some embodiments of the composition of the present invention, it further comprises a lipid conjugate, wherein the lipid conjugate comprises at least one selected from the group consisting of: a PEG-lipid conjugate, an ATTA-lipid conjugate, a polysarcosine-lipid conjugate, a polypeptide / protein-lipid conjugate, a cation-polymer-lipid conjugate (CPL), and derivatives thereof. Preferably, the lipid conjugate comprises a PEG-lipid conjugate.

[0031] In some embodiments, the lipid conjugate comprises 0.1 mol%-10.0 mol% of the total lipids in the composition.

[0032] In some embodiments of the compositions of the present invention, the compositions comprise:

[0033] (1) an amphiphilic block copolymer, a cationic lipid, a phospholipid, cholesterol, and a lipid conjugate, such as a PEG-lipid conjugate, wherein the cationic lipid comprises 30.0 mol%-80.0 mol% of the total lipid present in the composition, the phospholipid comprises 5.0 mol%-50.0 mol% of the total lipid, the cholesterol comprises 14.0 mol%-64.0 mol% of the total lipid, the lipid conjugate comprises 0.1 mol%-8.0 mol% of the total lipid, and the amphiphilic block copolymer comprises 0.1%-95.0% by weight (e.g., 1%-90.0% by weight, 10%-80.0% by weight, 20%-80.0% by weight, 30%-80.0% by weight, or 40%-70.0% by weight) of the composition;

[0034] (2) an amphiphilic block copolymer, a cationic lipid, a phospholipid, cholesterol, and a lipid conjugate, such as a PEG-lipid conjugate, wherein the cationic lipid comprises 23.0 mol%-75.0 mol% of the total lipid present in the composition, the phospholipid comprises 10.0 mol%-62.0 mol% of the total lipid, the cholesterol comprises 14.0 mol%-46.0 mol% of the total lipid, the lipid conjugate comprises 0.1 mol%-8.0 mol% of the total lipid, and the amphiphilic block copolymer comprises 0.1%-95.0% by weight (e.g., 1%-90.0% by weight, 10%-80.0% by weight, 20%-80.0% by weight, 30%-80.0% by weight, or 40%-70.0% by weight) of the composition;

[0035] (3) amphiphilic block copolymers, cholesterol-derived cationic lipids, phospholipids, and lipid conjugates, such as PEG-lipid conjugates, wherein the cholesterol-derived cationic lipids comprise 29.0 mol%-80.0 mol% of the total lipids present in the composition, the phospholipids comprise 19.0 mol%-70.0 mol% of the total lipids, the lipid conjugates comprise 0.1 mol%-8.0 mol% of the total lipids, and the amphiphilic block copolymers comprise 0.1%-95.0% by weight (e.g., 1%-90.0% by weight, 10%-80.0% by weight, 20%-80.0% by weight, 30%-80.0% by weight, or 40%-70.0% by weight) of the composition;

[0036] (4) an amphiphilic block copolymer, a cationic lipid, cholesterol, and a lipid conjugate, such as a PEG-lipid conjugate, wherein the cationic lipid comprises 25.0 mol%-80.0 mol% of the total lipid present in the composition, the cholesterol comprises 15.0 mol%-50.0 mol% of the total lipid, the lipid conjugate comprises 0.1 mol%-8.0 mol% of the total lipid, and the amphiphilic block copolymer comprises 0.1%-95.0% by weight (e.g., 1%-90.0% by weight, 10%-80.0% by weight, 20%-80.0% by weight, 30%-80.0% by weight, or 40%-70.0% by weight) of the composition;

[0037] (5) amphiphilic block copolymers, cationic lipids, phospholipids, and lipid conjugates such as PEG-lipid conjugates, wherein the cationic lipids comprise 30.0 mol%-80.0 mol% of the total lipids present in the composition, the phospholipids comprise 10.0 mol%-50.0 mol% of the total lipids, the lipid conjugates comprise 0.1 mol%-8.0 mol% of the total lipids, and the amphiphilic block copolymers comprise 0.1%-95.0% by weight (e.g., 1%-90.0% by weight, 10%-80.0% by weight, 20%-80.0% by weight, 30%-80.0% by weight, or 40%-70.0% by weight) of the composition;

[0038] (6) an amphiphilic block copolymer, a cationic lipid, a phospholipid, and cholesterol, wherein the cationic lipid accounts for 30.0 mol% to 80.0 mol% of the total lipids present in the composition, the phospholipids account for 5.0 mol% to 50.0 mol% of the total lipids, and the cholesterol accounts for 15.0 mol% to 50.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 0.1% to 95.0% by weight of the composition (e.g., 1% to 90.0% by weight, 10% to 80.0% by weight, 20% to 80.0% by weight, 30% to 80.0% by weight, or 40% to 70.0% by weight); or

[0039] (7) an amphiphilic block copolymer, a cholesterol-derived cationic lipid, and a phospholipid, wherein the cholesterol-derived cationic lipid accounts for 30.0 mol%-70.0 mol% of the total lipids present in the composition, the phospholipids account for 30.0 mol%-70.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 0.1%-95.0% by weight of the composition (e.g., 1%-90.0% by weight, 10%-80.0% by weight, 20%-80.0% by weight, 30%-80.0% by weight, or 40%-70.0% by weight).

[0040] In some embodiments of the composition of the present invention, the amphiphilic block copolymer is a tetrafunctional amphiphilic block copolymer, wherein the tetrafunctional amphiphilic block copolymer comprises a block copolymer of four branches each comprising at least one hydrophilic block and at least one hydrophobic block, or the amphiphilic block copolymer is a linear amphiphilic block copolymer, wherein the linear amphiphilic block copolymer comprises a block copolymer of at least one hydrophilic block and at least one hydrophobic block.

[0041] In some embodiments, the hydrophilic block is selected from polyoxyalkylenes, polyvinyl alcohol, polyvinyl pyrrolidone, poly(2-methyl-2-oxazoline) and sugars, and / or the hydrophobic block is selected from polyoxyalkylenes, fatty chains, alkylene polyesters, polyethylene glycol with benzyl polyether ends and cholesterol, preferably, the hydrophilic block comprises polyethylene oxide units and the hydrophobic block comprises polypropylene oxide units.

[0042] In some embodiments, the amphiphilic block copolymer comprises at least one selected from the group consisting of poloxamine or ), poloxamer or ), polyoxyethylene glycol dehydrated alcohol alkyl esters (polysorbates), polyvinyl pyrrolidone (PVP), polyethylene glycol ether (BRIJ), polyoxyethylene fatty acid esters, polyoxyethylene fatty alcohol ethers, sorbitan and their derivatives.

[0043] In some embodiments, the amphiphilic block copolymer comprises poloxamine or ), for example, the poloxamine is selected from poloxamine 304, poloxamine 701, poloxamine 704, poloxamine 901, poloxamine 904, poloxamine 908, poloxamine 1107, poloxamine 1301, poloxamine 1304, poloxamine 1307, poloxamine 90R4, poloxamine 150R1, or a combination thereof.

[0044] In some embodiments, the amphiphilic block copolymer comprises poloxamer or ), for example, the poloxamer is selected from poloxamer 84, poloxamer 101, poloxamer 105, poloxamer 108, poloxamer 122, poloxamer 123, poloxamer 124, poloxamer 181, poloxamer 182, poloxamer 183, poloxamer 184, poloxamer 185, poloxamer 188, poloxamer 212, poloxamer 215, poloxamer 217, poloxamer Poloxamer 231, Poloxamer 234, Poloxamer 235, Poloxamer 237, Poloxamer 238, Poloxamer 282, Poloxamer 284, Poloxamer 288, Poloxamer 304, Poloxamer 331, Poloxamer 333, Poloxamer 334, Poloxamer 335, Poloxamer 338, Poloxamer 401, Poloxamer 402, Poloxamer 403, Poloxamer 407, or a combination thereof.

[0045] In some embodiments of the compositions of the present invention, the cationic lipid comprises at least one selected from the group consisting of DOTMA, DOSPA, DOTAP, ePC, DODAP, DODMA, DDAB, DSDMA, DODAC, DOAP, DMRIE, DOGS, DMOBA, HGT5000, HGT5001, HGT5002, HGT4001, HGT4002, HGT4003, HGT4005, DLin-MC3-DMA, DLin-KC2-DMA, Acuitas ALC-0315, Acuitas A9, Acuitas Lipid 2,2, Moderna Lipid H (SM-102), Moderna Lipid 5. A2-Iso5-2DC18, BAME-O16B, 9A1P9, C12-200, cKK-E12, OF-Deg-Lin, 306Oi10, TT3, FTT5, Lipid319, 5A2-SC8, Genevant CL1, DLinDMA, DLenDMA, ClinDMA, CpLinDMA, imidazole cholesteryl ester (ICE), RE-1, RE-2, RE-3, GL-67, 5A2-SC8, Acuitas A9, Arcturus Lipid2,2(8,8)4C CH3, OF-02, A18-Iso5-2DC18, BAME-O16B, A6, 98N12-5, L319, L343, 304O13, 306O138, 306O12B, 306-O12B, LP01, G0-C14, 7C1, Cephalin, Dlin-EG-DMA, DLinAP, DLin-MPZ, DLin-C-DAP, DLin-2-DMAP, Dlin-S-DMA, DLinDAP, DLin-MA, DLin-DAC, DLin-K-DMA, DLin-K-MP Z, DLin-K-DMA, DLin-K6-C4-DMA, DLin-K-C4-DMA, DLin-K-C3-DMA, CpLinDMA, DOcarbDAP, DLincarbDAP, C12-(2-3-2), Genevant Lipid CL1, XTC, ALNY-100, NC98-5 and their derivatives.

[0046] In some embodiments of the compositions of the present invention, the cholesterol-derived cationic lipid comprises at least one selected from the group consisting of DC-Choi (N,N-dimethyl-N-ethylcarboxamide cholesterol), 1,4-bis(3-N-oleylamino-propyl)piperazine, N4-argininocholesterol carbonylamide (GL67), a cholesterol derivative coupled to a basic amino acid sequence, and imidazole cholesterol ester (ICE).

[0047] In some embodiments of the composition of the present invention, the phospholipid comprises at least one selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine, lysophosphatidylcholine, lysophosphatidylethanolamine, phosphatidylserine, dioleoylphosphatidylserine (DOPS), phosphatidylinositol, sphingomyelin, egg yolk sphingomyelin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebroside, dihexadecyl phosphate, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylethanolamine (DOPE), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleoyl-phosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), palmitoyloleoyl-phosphatidylglycerol (POPG), dioleoylphosphatidylethanolamine 4-(N-maleimidomethyl) )-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoyl-phosphatidylethanolamine (DMPE), distearoylphosphatidylethanolamine (DSPE), monomethyl-phosphatidylethanolamine, dimethyl-phosphatidylethanolamine, dioleoyl-phosphatidylethanolamine (DEPE), stearoyloleoyl-phosphatidylethanolamine (SOPE), lysophosphatidylcholine, egg yolk phosphatidylcholine ( 1-Hydroxy-1-nitro-2-nitro-1,2-diamino-1-nitro-2-nitro-1-phosphocholine (EPC), dilinoleylphosphatidylcholine, 1,2-dipalmitoyl-sn-glycero-3-O-4'-(N,N,N-trimethyl)-homoserine (DGTS), monogalactosyldiacylglycerol (MGDG), diacetyldiacylglycerol (DGDG), sulfaquinolinediacylglycerol (SQDG), 1-palmitoyl-2-cis-9,10-methylenehexyl-decanoyl-sn-glycero-3-phosphocholine (Cyclo PC), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE and their derivatives.

[0048] In some embodiments of the composition of the present invention, the cholesterol-derived neutral lipid comprises at least one selected from the group consisting of cholestanol, cholestanone, cholestenone, coprostanol, cholesteryl-2'-hydroxyethyl ether, cholesteryl-4'-hydroxybutyl ether, BHEM-cholesterol, β-sitosterol, 20α-hydroxycholesterol, cholesterol covalently linked to a polypeptide / protein, and derivatives thereof, preferably the cholesterol-derived neutral lipid comprises β-sitosterol.

[0049] In some embodiments of the composition of the present invention, the PEG-lipid conjugate comprises at least one selected from the group consisting of 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2K), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000](DMPE-PEG2K), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000](DSPE-PEG2K), a conjugate of DSPE-PEG2K and mannose (DSPE-PEG2K-Mannose), 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-5000 (DMG-PEG5K), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] glycol)-5000](DMPE-PEG5K), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-5000](DSPE-PEG5K), and the conjugate of DSPE-PEG5K and mannose (DSPE-PEG5K-Mannose).

[0050] In some embodiments of the polymer-lipid-containing compositions of the present invention, the composition comprises an amphiphilic block copolymer and the following components:

[0051] (1) DLin-MC3-DMA, ALC-0315, SM-102, C12-200, cKK-E12, HGT5000 or HGT5001; DSPC, DPPC, DOPS, SOPE, DOPG, DSPE, ESM or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K, DMG-PEG5K or DSPE-PEG2K-Mannose, wherein the DLin-MC3-DMA, ALC-0315, SM-102, C12-200, cKK-E12, HGT5000 or HGT5001 The amphiphilic block copolymer accounts for 40.0 mol% to 70.0 mol% of the total lipids present in the composition, DSPC, DPPC, DOPS, SOPE, DOPG, DSPE, ESM or DOPE accounts for 8.0 mol% to 39.0 mol% of the total lipids, cholesterol or β-sitosterol accounts for 20.0 mol% to 40.0 mol% of the total lipids, DMG-PEG2K, DMG-PEG5K or DSPE-PEG2K-Mannose accounts for 0.1 mol% to 5.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 15.0% to 90.0% by weight of the composition;

[0052] (2) DLin-MC3-DMA, ALC-0315, SM-102, C12-200, cKK-E12, HGT5000 or HGT5001; DSPC, DPPC, DOPS, SOPE, DOPG, DSPE, ESM or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K, DMG-PEG5K or DSPE-PEG2K-Mannose, wherein the DLin-MC3-DMA, ALC-0315, SM-102, C12-200, cKK-E12, HGT5000 or HGT5001 1 accounts for 30 mol%-60 mol% of the total lipids present in the composition, DSPC, DPPC, DOPS, SOPE, DOPG, DSPE, ESM or DOPE accounts for 10.0 mol%-49.0 mol% of the total lipids, cholesterol or β-sitosterol accounts for 20.0 mol%-40.0 mol% of the total lipids, DMG-PEG2K, DMG-PEG5K or DSPE-PEG2K-Mannose accounts for 0.1 mol%-5.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 20.0%-90.0% by weight of the composition;

[0053] (3) DLin-MC3-DMA, ALC-0315, SM-102, C12-200, cKK-E12, HGT5000 or HGT5001; DSPC, DPPC, DOPS, SOPE, DOPG, DSPE, ESM or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K, DMG-PEG5K or DSPE-PEG2K-Mannose, wherein the DLin-MC3-DMA, ALC-0315, SM-102, C12-200, cKK-E12, HGT5000 or HGT5001 accounts for 24.0 mol%-40.0 mol% of the total lipids present in the composition, DSPC, DPPC, DOPS, SOPE, DOPG, DSPE, ESM or DOPE account for 30.0 mol%-64.0 mol% of the total lipids, cholesterol or β-sitosterol account for 15.0 mol%-40.0 mol% of the total lipids, DMG-PEG2K, DMG-PEG5K or DSPE-PEG2K-Mannose account for 0.1 mol%-5.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 20.0%-90.0% by weight of the composition;

[0054] (4) DOTAP, DODAP, DOTMA or DOSPA; DSPC, DPPC, DOPS, SOPE, DOPG, DSPE, ESM or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K, DMG-PEG5K or DSPE-PEG2K-Mannose, wherein the DOTAP, DODAP, DOTMA or DOSPA accounts for 23.0 mol%-60 mol% of the total lipids present in the composition, DSPC, DPPC, DOPS, SOPE, DOPG, DSPE, ESM or DOPE accounts for 14.0 mol%-60.0 mol% of the total lipids, cholesterol or β-sitosterol accounts for 15.0 mol%-50.0 mol% of the total lipids, DMG-PEG2K, DMG-PEG5K or DSPE-PEG2K-Mannose accounts for 0.1 mol%-5.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 30.0%-90.0% by weight of the composition;

[0055] (5) GL67, ICE, or HGT4002; DSPC, DPPC, DOPS, SOPE, DOPG, DSPE, ESM, or DOPE; and DMG-PEG2K, DMG-PEG5K, or DSPE-PEG2K-Mannose, wherein the GL67, ICE, or HGT4002 accounts for 40.0 mol% to 80.0 mol% of the total lipids present in the composition, DSPC, DPPC, DOPS, SOPE, DOPG, DSPE, ESM, or DOPE accounts for 10.0 mol% to 50.0 mol% of the total lipids, DMG-PEG2K, DMG-PEG5K, or DSPE-PEG2K-Mannose accounts for 0.1 mol% to 5.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 30.0% to 90.0% by weight of the composition;

[0056] (6) cKK-E12, DLin-MC3-DMA, ALC-0315, SM-102, C12-200, DOTAP, DODAP, DOTMA, DOSPA, HGT5000 or HGT5001; cholesterol or β-sitosterol; and DMG-PEG2K, DMG-PEG5K or DSPE-PEG2K-Mannose, wherein the cKK-E12, DLin-MC3-DMA, ALC-0315, SM-102, C12-200, DOTAP , DODAP, DOTMA, DOSPA, HGT5000 or HGT5001 account for 45.0 mol%-75.0 mol% of the total lipids present in the composition, cholesterol or β-sitosterol account for 20.0 mol%-45.0 mol% of the total lipids, DMG-PEG2K, DMG-PEG5K or DSPE-PEG2K-Mannose account for 0.1 mol%-5.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 30.0%-90.0% weight percentage of the composition;

[0057] (7) DLin-MC3-DMA, ALC-0315, SM-102, C12-200, cKK-E12, HGT5000 or HGT5001; cholesterol or β-sitosterol; and DMG-PEG2K, DMG-PEG5K or DSPE-PEG2K-Mannose, wherein the DLin-MC3-DMA, ALC-0315, SM-102, C12-200, cKK-E12, HGT5000 or HGT5001 00 or HGT5001 accounts for 30.0mol%-65.0mol% of the total lipids present in the composition, cholesterol or β-sitosterol accounts for 20.0mol%-40.0mol% of the total lipids, DMG-PEG2K, DMG-PEG5K or DSPE-PEG2K-Mannose accounts for 0.1mol%-5.0mol% of the total lipids, and the amphiphilic block copolymer accounts for 40.0%-90.0% weight percentage of the composition;

[0058] (8) DLin-MC3-DMA, ALC-0315, SM-102, C12-200, cKK-E12, HGT5000 or HGT5001; DSPC, DPPC, DOPS, SOPE, DOPG, DSPE, ESM or DOPE; and DMG-PEG2K, DMG-PEG5K or DSPE-PEG2K-Mannose, wherein the DLin-MC3-DMA, ALC-0315, SM-102, C12-200, cKK-E12, HGT50 00 or HGT5001 accounts for 35.0 mol%-70.0 mol% of the total lipids present in the composition, DSPC, DPPC, DOPS, SOPE, DOPG, DSPE, ESM or DOPE accounts for 10.0 mol%-40.0 mol% of the total lipids, DMG-PEG2K, DMG-PEG5K or DSPE-PEG2K-Mannose accounts for 0.1 mol%-5.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 40.0%-90.0% weight percentage of the composition;

[0059] (9) GL67, ICE, or HGT4002; and DSPC, DPPC, DOPS, SOPE, DOPG, DSPE, ESM, or DOPE, wherein the GL67, ICE, or HGT4002 comprises 40.0 mol% to 80.0 mol% of the total lipids present in the composition, DSPC, DPPC, DOPS, SOPE, DOPG, DSPE, ESM, or DOPE comprises 10.0 mol% to 50.0 mol% of the total lipids, and the amphiphilic block copolymer comprises 40.0% to 90.0% by weight of the composition; or

[0060] (10) DLin-MC3-DMA, ALC-0315, SM-102, cKK-E12, HGT5000 or HGT5001; DSPC, DPPC, DOPS, SOPE, DOPG, DSPE, ESM or DOPE; and cholesterol or β-sitosterol, wherein the DLin-MC3-DMA, ALC-0315, SM-102, cKK-E12, HGT5000 or HGT5001 accounts for 30.0 mol%-60.0 mol% of the total lipids present in the composition, DSPC, DPPC, DOPS, SOPE, DOPG, DSPE, ESM or DOPE accounts for 20.0 mol%-45.0 mol% of the total lipids, cholesterol or β-sitosterol accounts for 20.0 mol%-45.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 30.0%-90.0% by weight of the composition.

[0061] In some embodiments of the compositions of the present invention, the compositions comprise the following components:

[0062] (1) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 50.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 10.0 mol% of the total lipids, cholesterol or β-sitosterol account for 38.5 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 1.5 mol% of the total lipids, and the amphiphilic block copolymer accounts for 89.9% by weight of the composition;

[0063] (2) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 50.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 10.6 mol% of the total lipids, cholesterol or β-sitosterol account for 38.5 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 0.9 mol% of the total lipids, and the amphiphilic block copolymer accounts for 72.9% by weight of the composition;

[0064] (3) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DSPE-PEG2K-Mannose, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 50.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 12.5 mol% of the total lipids, cholesterol or β-sitosterol account for 36.0 mol% of the total lipids, DMG-PEG2K or DSPE-PEG2K-Mannose account for 1.5 mol% of the total lipids, and the amphiphilic block copolymer accounts for 43.0% by weight of the composition;

[0065] (4) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DSPE-PEG2K-Mannose, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 50.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 14.5 mol% of the total lipids, cholesterol or β-sitosterol account for 34.0 mol% of the total lipids, DMG-PEG2K or DSPE-PEG2K-Mannose account for 1.5 mol% of the total lipids, and the amphiphilic block copolymer accounts for 81.8% by weight of the composition;

[0066] (5) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DSPE-PEG2K-Mannose, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 50.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 14.0 mol% of the total lipids, cholesterol or β-sitosterol account for 35.0 mol% of the total lipids, DMG-PEG2K or DSPE-PEG2K-Mannose account for 1.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 69.2% by weight of the composition;

[0067] (6) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 50.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 14.1 mol% of the total lipids, cholesterol or β-sitosterol account for 35.0 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 0.9 mol% of the total lipids, and the amphiphilic block copolymer accounts for 43.3% by weight of the composition;

[0068] (7) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 50.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 14.3 mol% of the total lipids, cholesterol or β-sitosterol account for 35.0 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 0.7 mol% of the total lipids, and the amphiphilic block copolymer accounts for 48.4% by weight of the composition;

[0069] (8) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 50.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 14.5 mol% of the total lipids, cholesterol or β-sitosterol account for 35.0 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 0.5 mol% of the total lipids, and the amphiphilic block copolymer accounts for 43.6% by weight of the composition;

[0070] (9) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 48.5 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 17.0 mol% of the total lipids, cholesterol or β-sitosterol account for 32.5 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 1.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 42.3% by weight of the composition;

[0071] (10) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 46.5 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 20.0 mol% of the total lipids, cholesterol or β-sitosterol account for 32.5 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 1.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 52.9% by weight of the composition;

[0072] (11) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DSPE-PEG2K-Mannose, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 49.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 20.0 mol% of the total lipids, cholesterol or β-sitosterol account for 30.0 mol% of the total lipids, DMG-PEG2K or DSPE-PEG2K-Mannose account for 1.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 41.9% by weight of the composition;

[0073] (12) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DSPE-PEG2K-Mannose, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 49.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 20.1 mol% of the total lipids, cholesterol or β-sitosterol account for 30.0 mol% of the total lipids, DMG-PEG2K or DSPE-PEG2K-Mannose account for 0.9 mol% of the total lipids, and the amphiphilic block copolymer accounts for 40.3% by weight of the composition;

[0074] (13) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 51.6 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 20.0 mol% of the total lipids, cholesterol or β-sitosterol account for 27.5 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 0.9 mol% of the total lipids, and the amphiphilic block copolymer accounts for 42.9% by weight of the composition;

[0075] (14) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 46.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 23.0 mol% of the total lipids, cholesterol or β-sitosterol account for 30.0 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 1.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 50.2% by weight of the composition;

[0076] (15) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DSPE-PEG2K-Mannose, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 46.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 23.0 mol% of the total lipids, cholesterol or β-sitosterol account for 30.0 mol% of the total lipids, DMG-PEG2K or DSPE-PEG2K-Mannose account for 1.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 66.1% by weight of the composition;

[0077] (16) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 46.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 23.0 mol% of the total lipids, cholesterol or β-sitosterol account for 30.0 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 1.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 74.5% by weight of the composition;

[0078] (17) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 46.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 23.0 mol% of the total lipids, cholesterol or β-sitosterol account for 30.0 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 1.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 79.6% by weight of the composition;

[0079] (18) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 46.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 23.0 mol% of the total lipids, cholesterol or β-sitosterol account for 30.0 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 1.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 83.0% by weight of the composition;

[0080] (19) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DSPE-PEG2K-Mannose, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 46.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 23.0 mol% of the total lipids, cholesterol or β-sitosterol account for 30.0 mol% of the total lipids, DMG-PEG2K or DSPE-PEG2K-Mannose account for 1.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 88.2% by weight of the composition;

[0081] (20) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 43.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 23.3 mol% of the total lipids, cholesterol or β-sitosterol account for 33.0 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 0.7 mol% of the total lipids, and the amphiphilic block copolymer accounts for 39.1% by weight of the composition;

[0082] (21) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 44.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 25.0 mol% of the total lipids, cholesterol or β-sitosterol account for 30.0 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 1.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 63.6% by weight of the composition;

[0083] (22) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 39.5 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 25.3 mol% of the total lipids, cholesterol or β-sitosterol account for 34.5 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 0.7 mol% of the total lipids, and the amphiphilic block copolymer accounts for 37.2% by weight of the composition;

[0084] (23) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 41.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 28.0 mol% of the total lipids, cholesterol or β-sitosterol account for 30.0 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 1.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 66.0% by weight of the composition;

[0085] (24) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 39.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 28.0 mol% of the total lipids, cholesterol or β-sitosterol account for 32.0 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 1.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 36.3% by weight of the composition;

[0086] (25) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 39.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 30.0 mol% of the total lipids, cholesterol or β-sitosterol account for 30.0 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 1.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 62.9% by weight of the composition;

[0087] (26) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 accounts for 40.6 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE accounts for 30.1 mol% of the total lipids, cholesterol or β-sitosterol accounts for 28.4 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K accounts for 0.9 mol% of the total lipids, and the amphiphilic block copolymer accounts for 36.9% by weight of the composition;

[0088] (27) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 34.7 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 40.1 mol% of the total lipids, cholesterol or β-sitosterol account for 24.3 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 0.9 mol% of the total lipids, and the amphiphilic block copolymer accounts for 32.5% by weight of the composition;

[0089] (28) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 29.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 50.1 mol% of the total lipids, cholesterol or β-sitosterol account for 20.0 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 0.9 mol% of the total lipids, and the amphiphilic block copolymer accounts for 28.0% by weight of the composition;

[0090] (29) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 35.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 17.6 mol% of the total lipids, cholesterol or β-sitosterol account for 46.5 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 0.9 mol% of the total lipids, and the amphiphilic block copolymer accounts for 35.9% by weight of the composition;

[0091] (30) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 55.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 16.9 mol% of the total lipids, cholesterol or β-sitosterol account for 27.2 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 0.9 mol% of the total lipids, and the amphiphilic block copolymer accounts for 44.6% by weight of the composition;

[0092] (31) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 60.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 14.9 mol% of the total lipids, cholesterol or β-sitosterol account for 24.2 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 0.9 mol% of the total lipids, and the amphiphilic block copolymer accounts for 46.5% by weight of the composition;

[0093] (32) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 65.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 14.1 mol% of the total lipids, cholesterol or β-sitosterol account for 20.0 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 0.9 mol% of the total lipids, and the amphiphilic block copolymer accounts for 21.0% by weight of the composition;

[0094] (33) an amphiphilic block copolymer; GL67, ICE, or HGT4002; DSPC, DPPC, or DOPE; and DMG-PEG2K or DMG-PEG5K, wherein the GL67, ICE, or HGT4002 accounts for 70.0 mol% of the total lipids present in the composition, DSPC, DPPC, or DOPE accounts for 28.5 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K accounts for 1.5 mol% of the total lipids, and the amphiphilic block copolymer accounts for 48.1% by weight of the composition;

[0095] (34) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; cholesterol or β-sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 61.3 mol% of the total lipids present in the composition, cholesterol or β-sitosterol account for 37.6 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 1.1 mol% of the total lipids, and the amphiphilic block copolymer accounts for 41.9% by weight of the composition;

[0096] (35) an amphiphilic block copolymer; cKK-E12, DLin-MC3-DMA, ALC-0315, SM-102, or C12-200; DOTAP, DODAP, DOTMA, or DOSPA; cholesterol or β-sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the cKK-E12, DLin-MC3-DMA, ALC-0315, SM-102, or C12-200 accounts for 30.0 mol% of the total lipids present in the composition, DOTAP, DODAP, DOTMA, or DOSPA accounts for 39.0 mol% of the total lipids, cholesterol or β-sitosterol accounts for 30.0 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K accounts for 1.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 57.7% by weight of the composition; or

[0097] (36) An amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; and cholesterol or β-sitosterol, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 accounts for 40.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE accounts for 32.0 mol% of the total lipids, cholesterol or β-sitosterol accounts for 28.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 50.0% by weight of the composition.

[0098] In some embodiments of the compositions of the invention, the molar ratio of nitrogen (amine) groups in the cationic lipid to phosphate groups in the nucleic acids (N / P ratio) in the composition is from about 1.0 to about 30.0, from about 4.0 to about 15.0, from about 6.0 to about 8.0, or from about 6.7 to about 7.6.

[0099] In some embodiments of the compositions of the invention, the ratio of lipid to nucleic acid in the composition (mass / mass ratio) is about 2 (2:1) to about 100 (100:1), about 5 (5:1) to about 60 (60:1), about 15 (15:1) to about 45 (45:1), or about 20 (20:1) to about 30 (30:1).

[0100] In some embodiments of the composition of the present invention, the active agent or therapeutic agent further comprises a protein or polypeptide, and in some embodiments, the protein is a protein related to translation or transcription. In some embodiments, the protein is related to the CRISPR process. In some embodiments, the protein is a protein related to CRISPR. In some embodiments, the therapeutic agent is a protein or polypeptide. In some embodiments, the composition comprises both a protein and a nucleic acid. In some embodiments, the therapeutic agent is a small molecule. In some embodiments of the composition of the present invention, the composition further comprises a targeting portion to target the composition to a target organ, tissue or cell in a subject, preferably the targeting portion comprises at least one selected from the following: a glycosyl, a lipid, a nucleic acid aptamer, a small molecule therapeutic agent, a vitamin, a polypeptide and a protein such as an antibody.

[0101] In some embodiments of the composition of the present invention, the composition further comprises an adjuvant, preferably the adjuvant comprises at least one selected from the group consisting of: CpG oligodeoxynucleotides, polyinosinic:polycytidylic acid, saponin extract (QS-21 extract), aluminum adjuvant, squalene, α-tocopherol, Tween, Span, lipopolysaccharide LPS, Pam3CSK4 triacyl lipopeptide, cyclic adenosine diphosphate (c-di-AMP), 2′3′-cyclic guanosine monophosphate adenosine monophosphate (cGAMP), monophosphoryl-lipid A, MPL lipid, flagellin or immunomodulatory proteins such as IL-2, IL-12, GM-CSF, TSLP and nucleic acids encoding these immunomodulatory proteins.

[0102] In some embodiments of the composition of the present invention, the composition further comprises a transfection enhancer, preferably the transfection enhancer comprises at least one selected from the group consisting of: pulmonary surfactant protein, cell-penetrating peptide, amphiphilic polypeptide, mucolytic enzyme, 1,2-propylene glycol, cellulose (such as carboxymethyl cellulose or hydroxypropyl cellulose), hyaluronate, alginate, pectin, polyethylene glycol, poloxamer, poloxamine, glucose, fructose, sucrose, trehalose, dextran, polyvinyl pyrrolidone, chitosan, polyvinyl alcohol, polyvinyl acetate, lectin, polylactic acid, polyhydroxybutyric acid, tromethamine, benzalkonium chloride, modified arginine, cetylpyridinium chloride, L-lysine monohydrate, and polylactic-co-glycolic acid or salt solution.

[0103] In some embodiments of the compositions of the present invention, the compositions are in the form of nanoparticles having an average size of about 1000 nm or less. In some embodiments, the nanoparticles have an average size of about 500 nm or less, 400 nm or less, 300 nm or less, 200 nm or less, 150 nm or less, 125 nm or less, 100 nm or less, 75 nm or less, or about 50 nm or less.

[0104] In some embodiments, about 30% to about 100%, about 70% to about 100%, about 90% to about 100%, about 50% to about 90%, about 70% to about 90%, or about 80% to about 90% of the nanoparticles have the active or therapeutic agent encapsulated therein.

[0105] In some embodiments of the composition of the present invention, the composition is formulated as a solution, dry powder, atomization or spray. In some embodiments, the composition is formulated for pulmonary and / or nasal administration by aerosolization, dry powder, inhalation, nebulization or instillation.

[0106] In a second aspect, the present invention provides a method for preparing a composition according to the first aspect of the present invention, the method comprising:

[0107] (A) mixing a solution comprising the active agent or therapeutic agent and a solution comprising the lipid in the presence of an amphiphilic block copolymer to form the composition; or

[0108] (B) mixing a solution containing the active agent or therapeutic agent and a solution containing the lipid to form lipid nanoparticles encapsulating the active agent or therapeutic agent, and then mixing the amphiphilic block copolymer with the lipid nanoparticle solution to form the composition.

[0109] In some embodiments, the method comprises:

[0110] 1) adding the amphiphilic block copolymer to a solution comprising the active agent or therapeutic agent and / or a solution comprising the lipid, and

[0111] 2) mixing the solution comprising the active agent or therapeutic agent and the solution comprising the lipid,

[0112] Thereby forming the composition.

[0113] In other embodiments, the method comprises:

[0114] 1) in a solution comprising the lipids, allowing the lipids to pre-form into lipid nanoparticles without an active agent or therapeutic agent; and

[0115] 2) mixing a solution comprising the active agent or therapeutic agent and the amphiphilic block copolymer with the lipid nanoparticle solution,

[0116] Thereby forming the composition.

[0117] In other embodiments, the method comprises:

[0118] 1) preforming the lipid and amphiphilic block copolymer into polymer-lipid nanoparticles without active agent or therapeutic agent; and

[0119] 2) mixing a solution containing the active agent or therapeutic agent with the polymer-lipid nanoparticle solution,

[0120] Thereby forming the composition.

[0121] In some embodiments, the method further comprises the step of removing free lipid components and / or amphiphilic block copolymers, preferably by dialysis and / or tangential flow filtration.

[0122] In some embodiments, the method further comprises the step of adding the amphiphilic block copolymer again after removing the free lipid component and / or the amphiphilic block copolymer.

[0123] In a third aspect, the present invention provides a pharmaceutical composition comprising the composition according to the first aspect of the present invention and a pharmaceutically acceptable carrier and / or excipient.

[0124] In a fourth aspect, the present invention provides a method for delivering an active agent or therapeutic agent to a target cell, the method comprising: contacting the cell with the composition according to the first aspect of the present invention under conditions sufficient to cause the active agent or therapeutic agent to be taken up into the cell, preferably the cell is a mammalian cell.

[0125] In a fifth aspect, the present invention provides a method for preventing and / or treating a disease or condition in a mammal, the method comprising administering to a subject in need thereof a pharmaceutically effective amount of a composition according to the first aspect of the present invention or a pharmaceutical composition according to the third aspect of the present invention, wherein the composition or pharmaceutical composition comprises an active agent or therapeutic agent for the disease or condition. In some embodiments, the disease or condition is selected from an immune system disease, a metabolic disease, a genetic disease, a cancer, a blood disease, a bacterial infection, or a viral infection.

[0126] In a sixth aspect, the present invention provides a composition according to the first aspect of the present invention or a pharmaceutical composition according to the third aspect of the present invention for use in the preparation of a medicament for preventing and / or treating a disease in a subject, wherein the composition or pharmaceutical composition comprises an active agent or therapeutic agent for the disease or condition. In some embodiments, the disease or condition is selected from an immune system disease, a metabolic disease, a genetic disease, a cancer, a blood disease, a bacterial infection, or a viral infection.

[0127] Other objects, features and advantages of the present disclosure will become apparent from the following detailed description. However, it should be understood that although the detailed description and specific examples indicate certain embodiments of the present disclosure, they are given by way of illustration only, as those skilled in the art will appreciate various changes and modifications within the spirit and scope of the present disclosure from this detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0128] Figure 1: Structural characterization of polymer-lipid composites (PoLixNano) and studies in in vitro models.

[0129] A. Transmission electron microscopy (TEM) images of LNP nanoparticles prepared with different lipid ratios and PoLixNano nanoparticles prepared with different formulations. Scale bar = 100 nm. B. Uptake and transfection efficiency of PoLixNano formulations in cultured 16HBE cells, DC2.4 cells, and BMDCs (n = 6). C. Toxicity assessment of different formulations in the aforementioned in vitro cell models. Cell viability was analyzed after incubation of 16HBE cells, DC2.4 cells, and BMDCs with PoLixNano formulations, Lipofecatmine 2000 (Lipo2000), and brPEI for 4 hours (n = 6). D. The PoLixNano preparation penetrated the Calu-3 cell monolayer in the Transwell chamber and successfully transfected DC2.4 cells cultured in the basal layer, verifying the efficiency of the PoLixNano preparation in crossing the mucus barrier and the cell monolayer (n=3); E. BMDC cell differentiation and maturation experiment: BMDC cells were incubated with naked mRNA, LNP preparation, and PoLixNano preparation for 24 hours, and the expression of BMDC cell-specific markers CD40+, CD80+, CD86+, and MHCII+ was detected by flow cytometry (n=3).

[0130] Figure 2: Study on the transfection efficiency of mRNA encoding firefly luciferase (Fluc-mRNA) in mice 6 hours after intratracheal spray (it) administration of PoLixNano formulation and LNP control formulation. A. Firefly luciferase (Fluc) transfection signal assay in live mice using Poloxamine 904 formulation (T904), Poloxamine 704 formulation (T704), PoLixNano formulation formulated with T904 (PoLixNano(T904)), PoLixNano formulation formulated with T704 (PoLixNano(T704)), and LNP control formulation (LNP), with naked Fluc-mRNA (naked-mRNA) and PBS-administered groups serving as control groups, respectively; B. Quantitative determination of Fluc bioluminescent signals mediated by the formulations described in A in isolated mouse lungs (n=3); C. Exemplary transfection conditions (left) and Fluc bioluminescent signal quantification results (right) of PoLixNano formulations prepared using trace amounts of T904 (0.025 mg / mL) and T704 (0.025 mg / mL) and LNP control formulations in live mice and isolated lungs (n=3).

[0131] Figure 3: Transfection efficiency study of PoLixNano formulations prepared with different types and concentrations of amphiphilic block copolymers in mice 6 hours after intratracheal spray (it) administration. A. Determination of Fluc expression levels in live mice and isolated lungs of PoLixNano formulations containing different concentrations of T904 and a control LNP formulation (n=3); B. Determination of Fluc expression levels in live mice and isolated lungs of PoLixNano formulations containing different concentrations of T704 and a control LNP formulation (n=3); C. Determination of Fluc expression levels in live mice and isolated lungs of PoLixNano formulations containing different concentrations of T90R4, T304, T901, and Tween 80 and a control LNP formulation (n=3); D. Determination of Fluc expression levels in live mice and isolated lungs of PoLixNano formulations containing different concentrations of poloxamer 407, poloxamer 338, poloxamer 124, poloxamer 237, poloxamer 188, and poloxamer P105 and a control LNP formulation (n=3).

[0132] Figure 4: In vivo transfection efficiency of PoLixNano formulations formulated with different nitrogen-to-phosphorus ratios (N / P) and different lipid ratios in mice 6 hours after intratracheal aerosol (IT) administration. A. Fluid expression levels in live mice and isolated lungs of PoLixNano formulations with different nitrogen-to-phosphorus ratios and control LNP formulations (n=3); B: Fluid expression levels in live mice and isolated lungs of PoLixNano formulations formulated with different molar ratios of lipid components (Dlin-MC3-DMA:DSPC:Chol:DMG-PEG2000) and control LNP formulations (n=3).

[0133] Figure 5: Study on the transfection efficiency of PoLixNano formulations containing different types of lipids and lipid ratios in mice 6 hours after intratracheal spray (it) administration. A. Determination of the Fluc expression level of PoLixNano formulations containing different types of cationic lipids (Dlin-MC3-DMA, ALC-0315, SM-102, C12-200 and HGT5000) and control LNP formulations in living mice and isolated lungs (n=3); B. Determination of the Fluc expression level of PoLixNano formulations containing different types of phospholipids (DSPC, DPPC, DOPE and ESM) and control LNP formulations in living mice and isolated lungs (n=3); C. Determination of the Fluc expression level of PoLixNano formulations containing different types of phospholipids (DSPC, DPPC, DOPE and ESM) and control LNP formulations in living mice and isolated lungs (n=3); A. Determination of the Fluc expression levels in the lungs of living mice and ex vivo by PoLixNano formulations containing a mixture of PEG lipids (cholesterol, β-sitosterol, and β-sitosterol: cholesterol = 1:1) and a control LNP formulation (n = 3); D. Determination of the Fluc expression levels in the lungs of living mice and ex vivo by PoLixNano formulations containing different types of PEG lipids (DMG-PEG2000, DSPE-PEG-Mannose, and DMG-PEG5000) and different molar ratios of DMG-PEG5000 and a control LNP formulation (n = 3).

[0134] Figure 6: Transfection of PoLixNano formulations prepared with different formulations in mice 6 hours after intranasal administration. A. Quantitative determination of Fluc bioluminescent signals mediated by PoLixNano formulations containing different concentrations of T704 (3 mg / mL-40 mg / mL), T904 (3 mg / mL), poloxamer 237 (10 mg / mL), poloxamer 338 (3 mg / mL) and different concentrations of poloxamer 124 (3 mg / mL and 20 mg / mL) and LNP control formulation in isolated mouse lungs (n=3); B. Quantitative determination of different molar ratios of Dlin-MC3-D MA: DSPC: Chol: DMG-PEG2000 formulated PoLixNano preparations and LNP control preparations mediated Fluc bioluminescence signals in isolated mouse lungs (n = 3); C. Quantitative determination of the Fluc bioluminescence signals mediated by PoLixNano preparations containing different types of cationic lipids, different types of neutral lipids, different types of PEG lipids and different molar ratios of DMG-PEG5000 and LNP control preparations in isolated mouse lungs (n = 3).

[0135] Figure 7: Study on the efficient transfection of Fluc-mRNA in mice by LNP and PoLixNano formulations (LNP+KG41, LNP+T904, LNP+10% sucrose, PoLixNano+KG41, PoLixNano+T904, and PoLixNano+10% sucrose) obtained by physical mixing with different enhancers (KG41 polypeptide (KG41), T904, and 10% sucrose solution (10% sucrose)) via different administration routes (it or in). A. Quantitative determination of the Fluc bioluminescent signal in living mice and isolated organs (lung, liver, and spleen) 6 hours after administration of the above formulations via intratracheal spray (it) (n=3); B. Quantitative determination of the Fluc bioluminescent signal in living mice and isolated organs (lung, liver, and spleen) 6 hours after administration of the above formulations via nasal route (in) (n=3).

[0136] Figure 8: In vivo transfection efficiency study of PoLixNano formulations containing unconventional lipid components and compositions in mice. A. Flu expression levels in live mice and isolated lungs were measured by intratracheal spray (it) of PoLixNano formulations containing both CKK-E12 and DOTAP cationic lipids (CKK-E12: DOTAP: Chol: DMG-PEG2000 = 30:39:30:1) and LNP control formulations (n ​​= 3). B. Flu expression levels in live mice and isolated lungs were measured by intratracheal spray (it) of PoLixNano formulations containing GL67 cationic lipids: DOPE: DMG-PEG2000 = 70:28.5:1.5 and LNP control formulations (n ​​= 3). A. The expression level of Fluc in the isolated lungs of mice was determined by intratracheal spray (it) and nasal route (in) of PoLixNano formulations without DMG-PEG2000 lipid components and LNP control formulations in living mice and in isolated lungs and livers (n=3); D. The expression level of Fluc in the isolated lungs and livers of PoLixNano formulations without phospholipid components and LNP control formulations was determined by intratracheal spray (it) and nasal route (in) (n=3).

[0137] Figure 9: PoLixNano formulation mediated efficient Fluc-mRNA transfection in mice after administration via nebulization, as well as the study of the physicochemical properties of nanoparticles. A. Schematic diagram of the mouse nebulization apparatus and research process; B. Transmission electron microscopy (TEM) images of LNP nanoparticles (top, scale bar = 200 nm) and PoLixNano nanoparticles (bottom, scale bar = 100 nm) before and after nebulization, showing comparison of nanoparticle morphology; C. Quantitative determination of the Fluc bioluminescent signal in isolated lungs of PoLixNano formulations containing different types of amphiphilic block copolymers (poloxamine 704, poloxamer 188, poloxamer 237, poloxamer 338, poloxamer 124, poloxamer P105, and poloxamer 407) and LNP control formulations 6 hours after nebulization (n = 3); D. Quantitative determination of the Fluc bioluminescent signal in isolated mouse lungs 6 hours after nebulization of PoLixNano formulations containing different types of cationic lipids and different types of neutral lipids and LNP control formulations (n ​​= 3).

[0138] Figure 10: PoLixNano formulations containing different types of amphiphilic block copolymers, different molar ratios of lipid compositions and different dosages efficiently mediated Fluc-mRNA transfection in mice after administration via nebulization. A. Quantitative determination of the Fluc bioluminescence signal in isolated lungs 6 h after nebulization administration of PoLixNano formulations containing different molar ratios of DMG-PEG2000 formulated with poloxamer 188 (left) or poloxamine 704 (right) and LNP control formulations (n=3); B. Quantitative determination of the Fluc bioluminescence signal in isolated lungs 6 h after nebulization administration of PoLixNano formulations containing different molar ratios of lipid compositions formulated with poloxamer 188 (left) or poloxamine 704 (right) and LNP control formulations (n=3); C. Investigation of the Fluc bioluminescence signal in live mice and isolated lungs 24 h after nebulization administration of PoLixNano formulations at different Fluc-mRNA doses (25 μg / mouse, 50 μg / mouse, and 150 μg / mouse) (n=3).

[0139] Figure 11: Study on the distribution, uptake and transfection efficiency of PoLixNano preparations in mice. A. After 6 hours of administration of DIR-labeled PoLixNano preparations via intratracheal spray (it), nasal route (in), and nebulization route (nebulization), in vivo imaging technology (IVIS) was used to detect the distribution of PoLixNano preparations in living mice (left) and in vitro organs (right). The figure shows representative results; B. RT-qPCR was used to determine the content of IVT-mRNA (encoding the receptor binding domain RBD of the new coronavirus spike protein) loaded by PoLixNano preparations in different organs of mice 6 hours after administration via intratracheal spray (it) (n=3); C. Flow cytometry was used to detect DID-labeled PoLixNano preparations and DID-labeled LNPs administered via intratracheal spray (it) route. Uptake of the preparation in lung DC cells (CD11c+), endothelial cells (CD31+), epithelial cells (CD326+), type I alveolar cells (Podoplanin+) and macrophages (F4 / 80+) (n=3); D. Kinetic curve of protein expression of PoLixNano preparation in living mice (Mice) and different organs (Lung, Liver and Spleen) over time after intratracheal spray (it) inoculation (n=5); E. Kinetic curve of protein expression of PoLixNano preparation loaded with circular RNA (circRNA) encoding Fluc in living mice (Mice) after intratracheal spray (it) administration (n=3).

[0140] Figure 12: Transfection of PoLixNano formulations prepared with different formulations in mice after administration via different routes (it, in and nebulization). A. Example results of transfection of PoLixNano formulations containing poloxamer 188 (3 mg / mL), T304 (3 mg / mL) and poloxamer 338 (3 mg / mL) and LNP control formulations in living mice and isolated organs 6 hours after intratracheal nebulization (it); B. Quantitative determination of Fluc bioluminescent signal mediated by the formulations described in A in isolated mouse lungs (n=3); C. Example of transfection of PoLixNano formulations containing T904 (3 mg / mL), T704 (6 mg / mL), 407 (3 mg / mL), Tween80 (3 mg / mL) and LNP control formulations in living mice and isolated organs 6 hours after intratracheal nebulization (it); L) Representative results of transfection induced by the PoLixNano formulation and LNP control formulation in live mice and isolated organs 6 hours after intratracheal aerosol administration (it); D. Quantitative determination of the Fluc bioluminescent signal mediated by the formulations in C in different isolated organs (n=3); E. Representative results of transfection induced by the PoLixNano formulation and LNP control formulation in live mice and isolated organs 6 hours after nasal administration (in); F. Quantitative determination of the Fluc bioluminescent signal mediated by the formulations in E in different isolated organs (n=3). G. Representative results of transfection induced by the PoLixNano formulation and LNP control formulation in live mice and isolated organs 6 hours after nebulization administration; H. Quantitative determination of the Fluc bioluminescent signal mediated by the formulations in G in isolated mouse lungs (n=3).

[0141] Figure 13: Safety evaluation of PoLixNano formulations in mice. A. Representative H&E staining results of mouse lung tissue sections 6 hours after administration of PoLixNano formulations via different routes (intratracheal spray, intranasal administration, and nebulization). Control samples of PBS solution and LNP formulations inoculated via the intratracheal route served as negative and positive controls, respectively. Scale bar = 50 μm. B. Representative H&E staining results of mouse lung tissue sections 6 hours after administration of PoLixNano formulations prepared with different concentrations of poloxamer 237 and poloxamer 338 via the intratracheal route. Control samples of PBS solution and LNP formulations inoculated via the intratracheal route served as negative and positive controls, respectively. Scale bar = 50 μm; C. Expression of inflammatory factors such as IL-4, IL-6, IL-17, and TNF-α in the abrasive fluid of mouse lung tissue 48 hours after inoculation with PBS solution, PoLixNano preparation, and LNP preparation via the it route (n = 3); D. After inoculation with PoLixNano preparation via the it route, serum was collected at different time points to detect the levels of various biochemical indicators in mice (n = 3); E. Serum was collected on days 7, 14, and 21, and the immunogenicity of the polymer component (T904) and lipid component (empty-LNP) of PoLixNano nanoparticles in mice was detected by ELISA (n = 3).

[0142] Figure 14: Flux mRNA transfection mediated by PoLixNano formulations and LNP control formulations in C57BL / 6 mice and Sprague-Dawley (SD) rats after administration via different routes (it, in, and nebulization). A. Exemplary results of Flux mRNA transfection mediated by the formulations in C57BL / 6 mice and in isolated lungs, livers, and spleens 6 hours after administration via intratracheal spray (it), nasal (in), and nebulization (nebulization) (top) and quantitative results of Flux bioluminescence signal in isolated lungs (bottom) (n=3); B. Flux bioluminescence signal measurement in SD rats and in isolated lungs, livers, and spleens 6 hours after administration via it (20 μg Flux mRNA / rat), in (same dose as it), and nebulization (45 μg Flux mRNA / rat).

[0143] Figure 15: Antigen-specific humoral immune response induced in mice after intratracheal spray (it) inoculation of PoLixNano preparation (RBD-mRNA / PoLixNano) loaded with mRNA encoding the SARS-CoV-2 RBD protein (RBD-mRNA). A. ELISA was used to detect the RBD antigen-specific IgG antibody titers in the serum samples of mice 14 days, 28 days and 280 days after the first immunization of PBS buffer (PBS), LNP preparation (LNP(it)), PoLixNano preparation and LNP preparation (LNP(im)) inoculated by the it route (n=5). At the same time, the RBD antigen-specific IgG antibody titers in the bronchoalveolar lavage fluid (BALF) and nasal lavage fluid (NLF) samples of mice on day 28 after the first immunization were detected. B. ELISA was used to detect the RBD antigen-specific secretory sIgA antibody titers in the BALF and NLF samples of mice collected 28 days and 280 days after the first immunization of the above-mentioned PBS, LNP(it), LNP(im) and PoLixNano groups (n=5). C. Pseudovirus neutralization experiments were performed to verify the efficacy of the serum and BALF samples of mice immunized with the above-mentioned preparations 28 days after the first immunization against the original strain of the new coronavirus (ancestral strain) and the omicron mutant strain (n=8); D. The content of IgG- and IgA-antibody-secreting cells (ASC) in the spleens and mediastinal lymph nodes (MLN) samples of mice 28 days after priming with the above-mentioned preparations was detected by ELIspot method (n=3); E. The proportion of Tfh and GCB-positive cells in MLN samples of mice 10 days after priming with the above-mentioned samples was detected by flow cytometry (n=3).

[0144] Figure 16: Intratracheal (it) administration of the RBD-mRNA / PoLixNano formulation induced a highly efficient and sustained adaptive cellular immune response in mice. A. Splenocytes and lung lymphocytes were collected 28 days after priming with PBS, LNP (it), LNP (im), and the PoLixNano formulation. After 24 hours of stimulation with the RBD overlapping peptide library, the production of cytokines IFN-γ, IL-4, and IL-17 was assessed by ELISpot (n=5). B. IFN-γ secretion was assessed by flow cytometry in lung lymphocytes from immunized mice 6 hours after stimulation with the RBD overlapping peptide library (n=5). C. Lung lymphocytes were collected 28 and 280 days after priming, and the proportions of tissue-resident memory T cells (Trm), effector memory T cells (Tem), and central memory T cells (Tcm) in the samples were assessed by flow cytometry (n=5). D. PoLixNano formulation induced trained innate immune responses in mice. BALF samples were collected from mice 11 days after primary immunization with LNP(it), LNP(im) and PoLixNano formulations, and the ratio of MHC II+ alveolar macrophages (AM) or MHC II+ interstitial macrophages (IM) was detected by flow cytometry (n=5).

[0145] Figure 17: Challenge study of lethal ancestral strains and omicron mutants of SARS-CoV-2 in mice immunized with PoLixNano formulations via intratracheal aerosol (IT) administration. A. Survival rate, weight change curves, and SARS-CoV-2 RNA loads in the lungs or nasal turbinates of mice immunized with PBS, LNP (im), LNP (it), and PoLixNano formulations after challenge with the ancestral strain of SARS-CoV-2 (n=8). Samples from mice that were not challenged and did not receive any intervention served as controls. B. Survival rate, weight change curves, and SARS-CoV-2 RNA loads in the lungs and nasal turbinates of mice immunized with PBS, LNP (im), LNP (it), and PoLixNano formulations after challenge with the omicron mutant of SARS-CoV-2 (n=8). Samples from mice that were not challenged with the virus and did not receive any intervention served as controls (Control). C. Representative H&E-stained sections (top) and representative immunohistochemical sections (bottom) of lung tissue from mice immunized with different preparations for 2019-nCoV 3 days after challenge with the ancestral strain of the novel coronavirus. Scale bar = 200 μm (n = 8). D. Representative H&E-stained sections (top) and representative immunohistochemical sections (bottom) of lung tissue from mice immunized with different preparations for 2019-nCoV 3 days after challenge with the omicron mutant strain. Scale bar = 200 μm (n = 8).

[0146] Figure 18: Study on the transfection of Fluc-mRNA in mice and the generation of efficient adaptive immune responses to RBD-mRNA mediated by PoLixNano formulation after intranasal administration (in). A. Kinetic curve of protein expression in live mice and isolated lungs after in vitro administration of PoLixNano formulation over time (n=3); B. ELISA method was used to detect the RBD antigen-specific IgG antibody titer in serum samples collected from mice immunized with PBS buffer (PBS), LNP control formulation (LNP) and PoLixNano formulation intranasally at 7 days, 14 days, 21 days, 28 days, 35 days, 42 days and 49 days after the first immunization, and at the same time, the RBD antigen-specific IgG antibody titer in the BALF of mice was detected 28 days after the first immunization. C. Levels of RBD antigen-specific secretory sIgA antibodies in lung lymphocytes of mice immunized with the above-mentioned preparations 28 days after priming were detected by ELISpot assay (n=5); D. The proportions of tissue-resident memory T cells (Trm) and effector memory T cells (Tem) in lung lymphocytes of mice collected 28 days after priming were determined by flow cytometry (n=5).

[0147] Figure 19: Study on the transfection of Fluc-mRNA in mice and the induction of adaptive immune response mediated by PoLixNano formulations via intramuscular injection (im). A. Representative images of Fluc bioluminescence (left) and quantitative analysis of related signals (right) in living mice and different isolated organs (liver, spleen) after intramuscular injection (im) of PoLixNano formulations loaded with Fluc-mRNA and LNP control formulations (n=3); B. Representative images of Fluc bioluminescence (left) and quantitative analysis of related Fluc signals in living mice and different isolated organs (liver, spleen) after intramuscular injection (im) of PoLixNano formulations not containing DMG-PEG2000 lipid components and LNP control formulations (n=3). Figure 3. Analysis results (right) (n=3); C. After two intramuscular (im) vaccinations (days 0 and 21) of the RBD-mRNA / PoLixNano formulation and the LNP control formulation, the ELISA method was used to detect the RBD-specific IgG antibody titers in the mouse serum samples collected 14 days, 21 days, 28 days and 35 days after the first immunization (n=3); D. The ELIspot method was used to detect the number of cytokine IL-4 and IL-17 spots produced by mouse splenic lymphocytes after stimulation with the RBD overlapping peptide library 28 days after the initial immunization with the formulation described in C (n=3).

[0148] All statistical data in the figures are presented as mean ± standard deviation (mean ± SD). Statistical analysis was performed using GraphPad Prism 8 software and two-tailed t-test. ns indicates no significant difference, *p < 0.05, **p < 0.01, ***p < 0.001.

[0149] Detailed Description of the Invention

[0150] definition

[0151] When used in conjunction with the term "comprising" in the claims and / or the specification, the use of the word "a" or "an" can mean "one", but it is also consistent with the meaning of "one or more", "at least one", and "one or more than one".

[0152] Throughout this document, the term "about" is used to indicate that a value includes the inherent variation of error for the device or method being employed to determine the value, or the variation that exists between study subjects.

[0153] As used herein, the term "substantially" refers to the qualitative condition of exhibiting all or nearly all of the extent or degree of a characteristic or property of interest. One of ordinary skill in the life sciences will understand that biological and chemical phenomena rarely, if ever, complete and / or proceed to completion or achieve or avoid an absolute result. Therefore, the term "substantially" is used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena.

[0154] The terms "comprise," "have," and "include" are open-ended linking verbs. Any form or tense of one or more of these verbs, such as "comprise," "have," and "include," is also open-ended. For example, any method that "comprises," "has," or "includes" one or more steps is not limited to having only those one or more steps and also encompasses other, unlisted steps.

[0155] As used in this specification and / or claims, the term "effective" means sufficient to achieve a desired, expected, or intended result. When used in the context of treating a patient or subject with a compound, "effective amount," "therapeutically effective amount," or "pharmaceutically effective amount" means an amount of the compound that, when administered to a subject or patient for treating a disease, is sufficient to achieve such treatment for the disease.

[0156] As used herein, the terms "improve," "increase," or "decrease," or grammatical equivalents, refer to values ​​relative to a baseline measurement, such as the measurement of the same individual before the start of a treatment described herein, or the measurement of a control sample or subject (or multiple control samples or subjects) in the absence of a treatment described herein. A "control sample" is a sample that has been subjected to the same conditions as the test sample, except for the test article. A "control subject" is a subject having the same form of disease as the subject being treated and who is about the same age as the subject being treated.

[0157] As used herein, the term "in vitro" refers to events that occur in an artificial environment, such as in a test tube or reaction vessel, in cell culture, etc., rather than in a multicellular organism.

[0158] As used herein, the term "in vivo" refers to events that occur within multicellular organisms such as humans and non-human animals. In the context of cell-based systems, the term can be used to refer to events that occur within living cells (as opposed to, for example, in vitro systems).

[0159] The term "gene product" as used herein refers to the product of a gene such as an RNA transcript, protein, or polypeptide.

[0160] The term "lipid" refers to a group of organic compounds that include, but are not limited to, esters of fatty acids and are characterized by being insoluble in water but soluble in many organic solvents. They are generally divided into at least three categories: (1) "simple lipids," which include fats and oils as well as waxes; (2) "compound lipids," which include phospholipids and glycolipids; and (3) "derivative lipids" such as steroids.

[0161] "Lipid granule", "lipid nanoparticle" or "LNP" as used herein refers to a lipid formulation that can be used to deliver an active agent or therapeutic agent, such as a nucleic acid (e.g., mRNA), to a target site. In the lipid granules typically formed by cationic lipids, non-cationic lipids and lipid conjugates that prevent particle aggregation, the active agent or therapeutic agent can be encapsulated in a lipid, thereby protecting the agent from enzymatic degradation. Nucleic acid-lipid granules and methods for preparing the same are disclosed in, for example, U.S. Patent Publication Nos. 20040142025 and 20070042031, the disclosures of which are incorporated herein by reference for all purposes.

[0162] When used herein, the term "PoLixNano" refers to a stable polymer-lipid composition or polymer-lipid particles or a polymer-lipid composition of a load nucleic acid or a polymer-lipid particle of a load nucleic acid. PoLixNano represents a composition made of amphiphilic block copolymers and lipids (e.g., cationic lipids, non-cationic lipids, and lipid conjugates for preventing particle aggregation), wherein the nucleic acid (e.g., mRNA, gRNA, siRNA, aiRNA, miRNA, ssDNA, dsDNA, ssRNA, short hairpin RNA (shRNA), dsRNA, or plasmid, including a plasmid for transcribing RNA interference thereof) is completely or partially encapsulated in lipid. When used herein, the term "PoLixNano" is a term for referring to a nucleic acid-polymer-lipid composition comprising a nucleic acid (e.g., mRNA) encapsulated in a polymer-lipid composition. PoLixNano typically contains amphiphilic block copolymers, cationic lipids, non-cationic lipids, and lipid conjugates (e.g., PEG-lipid conjugates). In addition, nucleic acids, when present in the polymer-lipid particles and / or lipid particles of the present invention, are resistant to degradation by nucleases in aqueous solution. PoLixNano is very effective for mucosal applications because it can efficiently penetrate the mucus (mucus penetration) present in large quantities in the mucosal tissue site, thereby safely delivering the loaded therapeutic agent (e.g., mRNA) to the target target cells (e.g., respiratory epithelial cells, dendritic cells, etc.), ultimately mediating the expression of transfected genes or the silencing of target gene expression in these mucosal-related sites. At the same time, by using a specific formulation, PoLixNano can also mediate the accumulation of therapeutic agents (e.g., mRNA) in remote sites (e.g., sites separated from the administration site on the body), and it can mediate the expression of transfected genes or the silencing of target gene expression at these remote sites.

[0163] The polymer-lipid composition particles of the invention (e.g., PoLixNano) typically have a median 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, or about 80 nm to about 100 nm, and are substantially non-toxic.

[0164] As used herein, the term "delivery" encompasses both local and systemic delivery. For example, delivery of mRNA encompasses situations where the mRNA is delivered to a target tissue and the encoded protein or peptide is expressed and retained within the target tissue (also referred to as "local distribution" or "local delivery"), and situations where the mRNA is delivered to a target tissue and the encoded protein or peptide is expressed and secreted into the patient's circulatory system (e.g., serum) and distributed throughout the body and taken up by other tissues (also referred to as "systemic distribution" or "systemic delivery").

[0165] As used herein, "distal site" refers to a separate location on the body that is not limited to adjacent capillary beds but includes sites that are widely distributed throughout the organism.

[0166] As used herein, the term "encapsulation" or grammatical equivalents refers to the process of confining individual mRNA molecules within polymer-lipid nanoparticles.

[0167] When used herein, " encapsulated " can refer to providing fully encapsulated, partially encapsulated, or both active agents or therapeutic agents, such as the lipid particles of nucleic acids (e.g., mRNA). In preferred embodiments, nucleic acid is fully encapsulated in lipid particles (e.g., to form PoLixNano or other nucleic acid-nano particles).

[0168] The term "cationic lipid" refers to any of the many lipid species carrying a net positive charge at selected pH, such as physiological pH (e.g., pH is about 7.0). It has been unexpectedly found that the cationic lipid comprising an alkyl chain with multiple unsaturated sites, e.g., at least 2 or 3 unsaturated sites, is particularly effective for forming lipid particles with increased membrane fluidity. Many cationic lipids and related analogs that are also effective for the present invention have been described in U.S. Patent Publication Nos. 20060083780 and 20060240554; U.S. Patent No. 5,208,036; 5,264,618; 5,279,833; 5,283,185; 5,753,613; and 5,785,992; and PCT Publication No. WO 96 / 10390, the disclosure of which is incorporated herein by reference for all purposes. The limiting examples of cationic lipids are described in detail in this article. In some cases, the cationic lipid comprises a protonatable tertiary amine (e.g., pH titratable) head group, C 18 An alkyl chain, an ether bond between the head group and the alkyl chain, and 0 to 3 double bonds. Such lipids include, for example, DSDMA, DLinDMA, DLenDMA, and DODMA.

[0169] The term "amphiphilic lipid" refers, in part, to any suitable material in which the hydrophobic portion of the lipid material is oriented into the hydrophobic phase, while the hydrophilic portion is oriented into the aqueous phase. The hydrophilic nature results from the presence of polar or charged groups such as carbohydrates, phosphates, carboxyls, sulfates, aminos, sulfhydryls, nitros, hydroxyls, and other similar groups. Hydrophobicity can be imparted by the inclusion of non-polar groups, including, but not limited to, long-chain saturated and unsaturated aliphatic hydrocarbon groups and such groups substituted with one or more aromatic, alicyclic, or heterocyclic groups. Examples of amphiphilic compounds include, but are not limited to, phospholipids, amino lipids, and sphingolipids.

[0170] The representative examples of phospholipids include, but are not limited to, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine, dipalmitoylphosphatidylcholine, dioleoylphosphatidylcholine, distearoylphosphatidylcholine and dilinoleoylphosphatidylcholine. Other compounds lacking phosphorus, such as sphingolipids, sphingolipid family, diacylglycerol and β-acyloxy acid are also included in the group that is called as amphipathic lipid. In addition, above-mentioned amphipathic lipid can be mixed with other lipids, and described other lipids include triglycerides and sterols.

[0171] The term "neutral lipid" refers to any of a number of lipid species that exist as uncharged or neutral zwitterionic forms at a selected pH. At physiological pH, such lipids include, for example, diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, cephalin, cholesterol, cerebrosides, and diacylglycerol.

[0172] The term "non-cationic lipid" refers to any amphipathic lipid as well as any other neutral lipid or anionic lipid.

[0173] The term "anionic lipid" refers to any lipid that is negatively charged at physiological pH. These lipids include, but are not limited to, phosphatidylglycerol, cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid, N-dodecanoylphosphatidylethanolamine, N-succinylphosphatidylethanolamine, N-glutarylphosphatidylethanolamine, lysylphosphatidylglycerol, palmitoyloleoylphosphatidylglycerol (POPG), and other anionic modifying groups attached to neutral lipids.

[0174] The term "lipid conjugate" refers to a conjugated lipid that inhibits aggregation of lipid particles. The lipid conjugate includes, but is not limited to, a polyamide oligomer (e.g., ATTA-lipid conjugate), a poly (ethylene glycol) -lipid conjugate (PEG-lipid conjugate), such as PEG coupled to a dialkoxypropyl group, PEG coupled to diacylglycerol, PEG coupled to cholesterol, PEG coupled to phosphatidylethanolamine, PEG coupled to ceramide (see, e.g., U.S. Patent No. 5,885,613, and International Patent PCT / US2016 / 000129, the disclosure of which is incorporated herein by reference for all purposes), polysarcosine-lipid conjugates, cationic PEG lipids, and mixtures thereof. PEG can be directly conjugated to the lipid or can be connected to the lipid through a linker moiety. Any linker moiety suitable for connecting PEG and lipid can be used, such as a linker moiety without an ester and a linker moiety containing an ester. In a preferred embodiment, a linker moiety without an ester is used.

[0175] As used herein, the term "N / P ratio" refers to the molar ratio of positively charged molecular units in the cationic lipid in the polymer-lipid composition relative to the negatively charged molecular units in the mRNA encapsulated within the polymer-lipid composition. Thus, the N / P ratio is typically calculated as the ratio of the molar number of amine groups in the cationic lipid in the polymer-lipid composition relative to the molar number of phosphate groups in the mRNA encapsulated within the polymer-lipid composition.

[0176] The term "hydrophobic lipid" refers to a compound having a non-polar group, including, but not limited to, long-chain saturated and unsaturated aliphatic groups and such groups optionally substituted with one or more aromatic, alicyclic or heterocyclic groups. Suitable examples include, but are not limited to, diacylglycerols, dialkylglycerols, NN-dialkylamino, 1,2-diacyloxy-3-aminopropane, and 1,2-dialkyl-3-aminopropane.

[0177] The term "fusogenic" refers to the ability of polymer lipid particles, such as PoLixNano, to fuse with a cell membrane. The membrane may be a plasma membrane or a membrane surrounding a cell organelle, such as an endosome, a lysosome, a nucleus, or the like.

[0178] As used herein, the term "aqueous solution" refers to a composition comprising, in whole or in part, water.

[0179] As used herein, the term "organic lipid solution" refers to a composition comprising, in whole or in part, an organic solvent with lipids.

[0180] An "isomer" of a first compound is an individual compound wherein each molecule contains the same constituent atoms as the first compound, but wherein the three-dimensional configuration of those atoms is different.

[0181] As used herein, the term "patient" or "subject" refers to a living mammalian organism, such as a human, monkey, cow, sheep, goat, dog, cat, mouse, rat, guinea pig, or a transgenic species thereof. In certain embodiments, the patient or subject is a primate. Non-limiting examples of human subjects are adults, adolescents, infants, and fetuses.

[0182] As used generally herein, "pharmaceutically acceptable" refers to compounds, materials, compositions and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues, organs and / or body fluids of human beings and animals without excessive toxicity, irritation, allergic response or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[0183] "Pharmaceutically acceptable salts" refers to salts of the compounds of the present disclosure that are pharmaceutically acceptable as defined above and that possess the desired pharmacological activity. Such salts include acid addition salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like; or organic acids such as 1,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, 2-naphthalenesulfonic acid, 3-phenylpropionic acid, 4,4'-methylenebis(3-hydroxy-2-ene-1-carboxylic acid), 4-methylbicyclo[2.2.2]oct-2-ene-1-carboxylic acid, acetic acid, aliphatic mono- and dicarboxylic acids, aliphatic sulfuric acid, aromatic sulfuric acid, benzenesulfonic acid, benzoic acid, camphorsulfonic acid, carbonic acid, Pharmaceutically acceptable salts include cinnamic acid, citric acid, cyclopentanepropionic acid, ethanesulfonic acid, fumaric acid, glucoheptonic acid, gluconic acid, glutamic acid, glycolic acid, heptanoic acid, hexanoic acid, hydroxynaphthoic acid, lactic acid, lauryl sulfate, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, muconic acid, o-(4-hydroxybenzoyl)benzoic acid, oxalic acid, p-chlorobenzenesulfonic acid, phenyl-substituted alkanoic acid, propionic acid, p-toluenesulfonic acid, pyruvic acid, salicylic acid, stearic acid, succinic acid, tartaric acid, tert-butylacetic acid, trimethylacetic acid, and the like. Pharmaceutically acceptable salts also include base addition salts that can be formed when the acidic protons present are capable of reacting with inorganic or organic bases. Acceptable inorganic bases include sodium hydroxide, sodium carbonate, potassium hydroxide, aluminum hydroxide, and calcium hydroxide. Acceptable organic bases include ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine, and the like. It will be appreciated that the particular anion or cation forming part of any salt of the present disclosure is not critical so long as the salt as a whole is pharmacologically acceptable. Other examples of pharmaceutically acceptable salts and methods for their preparation and use are presented in Handbook of Pharmaceutical Salts: Properties, and Use (P.H. Stahl & C.G. Wermuth, eds., Verlag Helvetica Chimica Acta, 2002).

[0184] As used herein, the term "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition, or vehicle that participates in carrying or transporting a pharmaceutical agent, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material. As used herein, the term "excipient" includes, but is not limited to, any and all solvents, dispersion media or other liquid vehicles, dispersing or suspending agents, diluents, granulating and / or dispersing agents, surfactants, isotonicity agents, thickening or emulsifying agents, preservatives, binders, lubricants or coloring agents, sweeteners or flavoring agents, stabilizers, antioxidants, antimicrobial or antifungal agents, osmotic pressure regulators, pH regulators, buffers, chelating agents, cryoprotectants, and / or plasticizers required for the particular dosage form desired. Various excipients used to formulate pharmaceutical preparation compositions and techniques for preparing such compositions are known in the art (see Remington: The Science and Practice of Pharmacy, 21st ed. AR. Gennaro (Lippincott, Williams & Wilkins, Baltimore, MD, 2006; herein incorporated by reference in its entirety).

[0185] "Prevention" includes: (1) inhibiting the onset of a disease in a subject or patient who may be at risk for and / or susceptible to the disease but who does not yet experience or display any or all of the symptoms or signs of the disease; and / or (2) slowing the onset of symptoms or signs of a disease in a subject or patient who may be at risk for and / or susceptible to the disease but who does not yet experience or display any or all of the symptoms or signs of the disease.

[0186] "Treating" includes (1) inhibiting the disease in a subject or patient who is experiencing or exhibiting symptoms or signs of the disease (e.g., arresting further development of the symptoms and / or symptoms), (2) ameliorating the disease in a subject or patient who is experiencing or exhibiting symptoms or signs of the disease (e.g., reversing the symptoms and / or symptoms), and / or (3) achieving any measurable reduction in the disease in a subject or patient who is experiencing or exhibiting symptoms or signs of the disease.

[0187] A "repeat unit" is the simplest structural entity of some material, for example, a framework and / or a polymer, whether organic, inorganic, or metallo-organic. In the case of a polymer chain, the repeat units are linked together sequentially along the chain, like beads on a necklace. For example, in polyethylene -[-CH2CH2-] n‐, the repeating unit is ‐CH2CH2‐. The subscript "n" indicates the degree of polymerization—that is, the number of linked repeating units. When the value of "n" is undefined or absent, it simply indicates the repetition of the formula within the brackets and the polymeric nature of the material. The concept of a repeating unit also applies when the connectivity between repeating units extends three-dimensionally, such as in metal-organic frameworks, modified polymers, and thermosets. In the context of dendrimers, repeating units can also be described as branching units, inner layers, or generations. Similarly, end-capping groups can also be described as surface groups.

[0188] The above definitions supersede any conflicting definitions in any references incorporated herein by reference. However, the fact that certain terms are defined should not be construed as indicating that any undefined term is undefined. Rather, all terms used are considered to describe the present disclosure in a manner that allows one of ordinary skill in the art to understand the scope and practice the present disclosure.

[0189] The present invention encompasses the unexpected discovery that the addition of amphiphilic block copolymers such as poloxamine or dapoxetine to LNP formulations (e.g., four-component LNP formulations consisting of ionizable / cationic lipids, neutral lipids, cholesterol, and PEG-lipids; three-component LNP formulations consisting of ionizable / cationic lipids, neutral lipids, and cholesterol; three-component LNP formulations comprising cholesterol-derivatized cationic lipids, neutral lipids, and PEG lipids; two-component LNP formulations comprising cholesterol-derivatized cationic lipids and neutral lipids) can enhance the effectiveness of the LNP formulation. ) and / or poloxamer or ) components, the resulting novel composite formulation (referred to herein as a polymer-lipid composition or PoLixNano) can significantly enhance the gene transfection efficiency of nucleic acid molecules (such as mRNA) mediated by such formulations in animals (especially in cells associated with the respiratory tract) after administration via the mucosal site of an organism, particularly the respiratory tract (tracheal spray IT, nasal drops IN, or atomized inhalation), thereby enabling the nucleic acid molecules to efficiently produce functional proteins in the animal's respiratory tract-related tissues and non-lung cells / tissues, thereby achieving the purpose of preventing or treating diseases.

[0190] Polymer-lipid preparations are particularly suitable for treating or preventing diseases related to respiratory tissue via non-invasive routes of administration (such as tracheal spray it, nasal drops in, and atomized inhalation, etc.). After the polymer-lipid composition loaded with mRNA is administered via the respiratory route, the gene transfection effect produced in the lung / respiratory mucosal tissue of animals is significantly better than that of classic LNP preparations, poloxamine and poloxamer preparations. mRNA delivered via the respiratory route is expected to make breakthroughs in the fields of mucosal vaccines (such as new coronavirus vaccines, influenza virus vaccines, tuberculosis vaccines, etc.), lung gene therapy (such as cystic fibrosis, α1-trypsin deficiency, etc.), and refractory lung diseases (such as lung cancer, pulmonary fibrosis, asthma, chronic obstructive pulmonary disease, etc.), solving the problem that there is currently no ideal delivery system in these fields.

[0191] Secondly, polymer-lipid nanoformulations of specific formulations delivered via the respiratory route are able to translocate after pulmonary delivery, that is, to move partially or completely from the site of administration (lungs) to the systemic blood supply by active or passive means, and then deposit in different non-lung cells or tissues, for example, the liver and / or spleen. This transport of polymer-lipid compositions containing mRNA encoding therapeutic proteins (such as cystic fibrosis transmembrane conductance regulator CFTR, anti-α1-trypsin, etc.) or specific antigens (such as tumor-specific antigens, new coronavirus spike proteins, etc.) constitutes non-invasive systemic delivery of active ingredients (i.e., mRNA) outside the lungs, resulting in the production of functional proteins in non-lung cells or tissues accessible to the whole body. Under the same conditions, the main transfection organs of LNP preparations or poloxamine or poloxamer preparations administered are limited to the lungs.

[0192] Accordingly, in one aspect, the present invention provides a polymer-lipid composition comprising:

[0193] (A) an active or therapeutic agent, preferably comprising a nucleic acid;

[0194] (B) amphiphilic block copolymers;

[0195] (C) cationic lipids; and

[0196] (D) non-cationic lipids,

[0197] Wherein the composition is formulated for delivery through a mucosal site of an organism, such as respiratory tract delivery, oral mucosal delivery, gastrointestinal tract delivery, ocular mucosal delivery, ear mucosal delivery, urethral delivery, or reproductive tract delivery, preferably the composition is formulated for delivery through the respiratory tract.

[0198] Nucleic Acids

[0199] The composition according to the present invention may preferably comprise ribonucleic acid (RNA), such as single-stranded RNA, more preferably messenger RNA (mRNA), such as in vitro transcribed mRNA.

[0200] In some embodiments, the nucleic acid comprises at least one selected from the group consisting of messenger RNA (mRNA), self-amplifying RNA (saRNA), circular RNA (circRNA), small interfering RNA (siRNA), short hairpin RNA (shRNA) and microRNA (miRNA), primary-miRNA, antisense oligonucleotide (ASO), transfer RNA (tRNA), plasmid DNA (pDNA), single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), deoxyribozyme (DNAzyme), ribozyme (RNAzyme), nucleic acid aptamer (aptamer), clustered regularly interspaced short palindromic repeats (CRISPR)-related nucleic acid, single guide RNA (sgRNA), CRISPR-RNA (crRNA), trans-activating crRNA (tracrRNA), guide RNA, single-stranded RNA (ssRNA) and double-stranded RNA (dsRNA). In some embodiments, the nucleic acid is a therapeutic nucleic acid. More preferably, the nucleic acid comprises mRNA.

[0201] In some embodiments, the composition comprises a first nucleic acid and a second nucleic acid. In some embodiments, the first nucleic acid is an mRNA. In some embodiments, the second nucleic acid is a single guide RNA. In some embodiments, the first nucleic acid is a messenger RNA (mRNA) and a single guide RNA (sgRNA).

[0202] In principle, any type of RNA can be used in the context of the present invention. In a preferred embodiment, the RNA is single-stranded RNA. The term "single-stranded RNA" means a single continuous ribonucleotide chain, which is distinguished from RNA that is a double-stranded molecule formed by hybridization of two or more separate chains. The term "single-stranded RNA" does not exclude that the single-stranded molecule itself forms a double-stranded structure, such as a secondary (e.g., loop and stem-loop) structure or a tertiary structure.

[0203] The term "RNA" encompasses RNA that encodes an amino acid sequence as well as RNA that does not encode an amino acid sequence. RNA can be prepared by synthetic chemistry and enzymatic methods known to those of ordinary skill in the art, or by using recombinant technology, or can be isolated from natural sources, or by a combination thereof.

[0204] Messenger RNA (mRNA) is a copolymer composed of nucleoside phosphate building blocks, primarily adenosine, cytidine, uridine, and guanosine. It acts as an intermediate to carry genetic information from DNA in the cell nucleus into the cytoplasm, where it is translated into proteins. Therefore, mRNA is a suitable surrogate for gene expression.

[0205] In the context of the present invention, mRNA should be understood to mean any polyribonucleotide molecule, if it enters the cell, is then suitable for protein or its fragmentary expression, or can be translated into protein or its fragment.Term " protein " contains any kind of amino acid sequence in this article, i.e. two or more amino acid whose chains connected by peptide bonds separately, and also comprises peptide and fusion protein.

[0206] MRNA contains a ribonucleotide sequence that encodes a protein or a fragment thereof of a function needed or useful in or near a cell. MRNA can contain the sequence of a complete protein or its functional variant. Further, a ribonucleotide sequence can encode a protein or its functional fragment that acts as a factor, an inducer, a regulator, a stimulant or an enzyme, wherein such protein is a protein necessary for its function to make up for an obstacle (particularly a metabolic disorder) or to start a process in the body (such as the formation of new blood vessels, tissues, etc.) or to induce the immune system to produce an adaptive immune response. Herein, functional variant means following fragment: it can assume the function of a protein in a cell, the function of the protein is needed in a cell, or the form of the absence or defect of the protein is pathogenic.

[0207] Typically, mRNA synthesis includes adding a "cap" to the 5' end and a "tail" to the 3' end. The presence of the cap is important for providing resistance to nucleases present in most eukaryotic cells. The presence of the "tail" is used to protect the mRNA from exonuclease degradation. Therefore, in some embodiments, mRNA includes a 5' cap structure. In some embodiments, the mRNA includes a 5' and / or 3' untranslated region. In some embodiments, the 5' untranslated region includes one or more elements that affect the stability or translation of the mRNA. In some embodiments, the 3' untranslated region includes one or more polyadenylation signals, protein binding sites that affect the stability of the mRNA location in the cell, or one or more miRNA binding sites. As described above, unless otherwise noted in the specific context, the term mRNA used herein encompasses modified mRNA, i.e., the mRNA can be a modified mRNA.

[0208] The present invention can be used to formulate and encapsulate unmodified mRNA or mRNA containing one or more modifications that generally enhance stability. In some embodiments, the modifications are selected from modified nucleotides, modified sugar-phosphate backbones, and 5' and / or 3' untranslated regions.

[0209] In some embodiments, the modification of mRNA may include modification of the nucleotides of RNA. Modified mRNA according to the present invention may include, for example, backbone modifications, sugar modifications, phosphate modifications, or base modifications. In some embodiments, mRNA may be synthesized from naturally occurring nucleotides and / or nucleotide analogs (modified nucleotides), including but not limited to purines (adenine (A), guanine (G)) or pyrimidines (thymine (T), cytosine (C), uracil (U)) and modified nucleotide analogs or derivatives of purines and pyrimidines, the preparation of such analogs being known to those skilled in the art, for example, from U.S. Patent No. 4,373,071, U.S. Patent No. 4,373,071. No. 4,401,796, U.S. Patent No. 4,415,732, U.S. Patent No. 4,458,066, U.S. Patent No. 4,500,707, U.S. Patent No. 4,668,777, U.S. Patent No. 4,973,679, U.S. Patent No. 5,047,524, U.S. Patent No. 5,132,418, U.S. Patent No. 5,153,319, U.S. Patent Nos. 5,262,530 and 5,700,642, the entire disclosures of which are incorporated herein by reference.

[0210] For RNA, preferably mRNA, according to the present invention, all uridine nucleotides and cytidine nucleotides can be modified in the same form, or a mixture of modified nucleotides can be used for each. The modified nucleotides can have natural or non-naturally occurring modifications. Mixtures of various modified nucleotides can be used.

[0211] In one embodiment of the invention, one type of nucleotide uses at least two different modifications, wherein the modified nucleotide of one type has a functional group through which other groups can be attached. Nucleotides with different functional groups can also be used to provide binding sites for attaching different groups.

[0212] In a preferred embodiment, the RNA, preferably mRNA, according to the invention is characterized in that the modified uridine is selected from the group consisting of 2-thiouridine, 5-methyluridine, pseudouridine (ψ), 5-methyluridine 5'-triphosphate (m5U), N-1-methyl-pseudouridine (N1mΨ), N-1-methyl-pseudouridine-triphosphate, 2-thiouridine 5'-triphosphate (S2U), 5-iodouridine 5'-triphosphate (I5U), 4-thiouridine 5'-triphosphate (S4U), 5-bromouridine 5'-triphosphate (Br5U), 2'-methyl-2'-deoxyuridine 5'-triphosphate (U2'm), 2'-amino-2'-deoxyuridine 5'-triphosphate (U2'NH2), 2'-azido-2'-deoxyuridine 5'-triphosphate (U2'N3) and 2'-fluoro-2'-deoxyuridine 5'-triphosphate (U2'F).

[0213] In another preferred embodiment, the RNA, preferably the mRNA, according to the invention is characterized in that the modified cytidine is selected from the group consisting of 5-methylcytidine, 5-hydroxymethylcytidine, 5-methoxycytidine, 3-methylcytidine, 2-thio-cytidine, 2'-methyl-2'-deoxycytidine 5'-triphosphate (C2'm), 2'-amino-2'-deoxycytidine 5'-triphosphate (C2'NH2), 2'-fluoro-2'-deoxycytidine 5'-triphosphate (C2'F), 5-iodocytidine 5'-triphosphate (I5C), 5-bromocytidine 5'-triphosphate (Br5C), 5-methylcytidine 5'-triphosphate (m5C), 2-thiocytidine 5'-triphosphate (S2C) and 2'-azido-2'-deoxycytidine 5'-triphosphate (C2'N3).

[0214] In another preferred embodiment, the RNA, preferably the mRNA, according to the invention is characterized in that the modified adenosine is selected from the group consisting of N6-methyladenosine 5'-triphosphate (m6A), N1-methyladenosine 5'-triphosphate (m1A), 2'-O-methyladenosine 5'-triphosphate (A2'm), 2'-amino-2'-deoxyadenosine 5'-triphosphate (A2'NH2), 2'-azido-2'-deoxyadenosine 5'-triphosphate (A2'N3) and 2'-fluoro-2'-deoxyadenosine 5'-triphosphate (A2'F).

[0215] In another preferred embodiment, the RNA, preferably mRNA, according to the invention is characterized in that the modified guanosine is selected from N1-methylguanosine 5-triphosphate (m1G), 2'-O-methylguanosine 5'-triphosphate (G2'm), 2-amino-2-deoxyguanosine 5'-triphosphate (G2'NH2), 2'-azido-2'-deoxyguanosine 5'-triphosphate (G2'N3), and 2'-fluoro-2'-deoxyguanosine 5'-triphosphate (G2'F).

[0216] Representative U.S. patents that teach the preparation of some of the above-mentioned modified nucleobases, as well as other modified nucleobases, include, but are not limited to, U.S. Patents 3,687,808; 4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121; 5,596,091; 5,614,617; 5,645,985; 5,681,941; 5,750,692; 5,763,588; 5,830,653 and 6,005,096, each of which is incorporated herein by reference in its entirety.

[0217] In some embodiments, the invention provides oligonucleotides comprising connected nucleosides. In such embodiments, nucleosides can be linked together using any internucleoside bond. The two main categories of internucleoside linking groups are defined by the presence or absence of a phosphorus atom. Compared to natural phosphodiester bonds, modified bonds can be used to change (usually increase) the nuclease resistance of oligonucleotides. In some embodiments, internucleoside bonds with chiral atoms can be prepared as racemic mixtures or separate enantiomers. Representative chiral bonds include, but are not limited to, alkyl phosphonates and thiophosphates. The preparation methods of phosphorus-containing and non-phosphorus-containing internucleoside bonds are well known to those skilled in the art.

[0218] Representative U.S. patents that teach the preparation of such oligonucleotide conjugates include, but are not limited to, U.S. Patents 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717; 5,580,731; 5,580,731; 5,591,584; 5,109,124; 5,118, 802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958, 013; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241; 5,391,723; 5,416, 5,599,923; 5,599,928 and 5,688,941, each of which is incorporated herein by reference.

[0219] In a preferred embodiment, the mRNA is an mRNA containing a combination of modified and unmodified nucleotides. Preferably, it is an mRNA containing a combination of modified and unmodified nucleotides as described in WO2011 / 012316. The mRNA described therein shows improved stability and reduced immunogenicity.

[0220] In another embodiment, the mRNA contains labeled nucleic acids (preferably nucleotides and / or ribonucleotides), such as, for example, isotope- and / or fluorescently labeled nucleotides. Labeled mRNA molecules play an important role, for example, in studying the intracellular conformation of RNA and DNA molecules and their distribution in animals and / or cells.

[0221] mRNA can be synthesized according to any of a variety of known methods.For example, mRNA according to the present invention can be synthesized via in vitro transcription (IVT).

[0222] Furthermore, modified RNA, preferably mRNA molecules can be chemically synthesized, for example, by conventional chemical synthesis on an automated nucleotide sequence synthesizer using a solid support and standard techniques, or by chemically synthesizing the corresponding DNA sequence and subsequently transcribing it in vitro or in vivo.

[0223] While in some embodiments mRNA provided by an in vitro transcription reaction is desirable, other sources of mRNA are contemplated within the scope of the present invention, including mRNA produced from bacteria, fungi, plants, and / or animals.

[0224] In another preferred embodiment, the mRNA can be combined with a target binding site, a targeting sequence and / or with a microRNA binding site to allow the activity of the desired mRNA only in the relevant cells. In a further preferred embodiment, the RNA can be combined with a microRNA or shRNA downstream of the 3' polyA tail.

[0225] In some embodiments, in vitro synthesized mRNA can be purified prior to formulation and encapsulation to remove undesirable impurities (including various enzymes and other reagents used in the mRNA synthesis process).

[0226] The present invention can be used to formulate and encapsulate mRNA of various lengths. In some embodiments, the present invention can be used to formulate and encapsulate in vitro synthesized mRNA of length equal to or greater than about 0.1 kb, 0.5 kb, 1 kb, 1.5 kb, 2 kb, 2.5 kb, 3 kb, 3.5 kb, 4 kb, 4.5 kb, 5 kb, 6 kb, 7 kb, 8 kb, 9 kb, 10 kb, 11 kb, 12 kb, 13 kb, 14 kb, 15 kb or 20 kb. In some embodiments, the present invention can be used to formulate and encapsulate in vitro synthesized mRNA ranging in length from about 0.1-2 kb, about 1-20 kb, about 1-15 kb, about 1-10 kb, about 5-20 kb, about 5-15 kb, about 5-12 kb, about 5-10 kb, about 8-20 kb, or about 8-15 kb.

[0227] In general, the present invention can achieve the effect of disease treatment, disease prevention and disease diagnosis by the interaction of ribonucleic acid with cellular molecules and organelles. This interaction can activate the innate immune system alone, for example, for certain CpG oligonucleotides and sequences designed to interact specifically with toll-like receptors and other extracellular or intracellular receptors, namely belong to this class of situations. In addition, the intake or introduction of ribonucleic acid (preferably mRNA) in the cell can be intended to cause nucleotide sequences, such as the expression of the gene included in the ribonucleic acid (preferably mRNA); it can be intended to lower the expression of endogenous genes caused by the presence of the exogenous nucleic acid introduced in the cell, silence or knock down; or it can be intended to be used for the modification of endogenous nucleic acid sequences, such as the repair, excision, insertion or exchange of the entire sequence (whole stretches) of the selected base or endogenous nucleic acid sequence; or it can be intended to be used for the presence and interaction of the exogenous ribonucleic acid (preferably mRNA) introduced in the cell and actually interfere with any cellular process. The cross expression of the exogenous ribonucleic acid (preferred mRNA) introduced can be intended to compensate or complementary endogenous gene expression, particularly under the situation of endogenous gene defect or silence, cause gene expression product to not exist, not enough or defective or malfunction, multiple metabolic and hereditary diseases, such as cystic fibrosis, hemophilia or muscular dystrophy etc. are usually such situations. The cross expression of the exogenous ribonucleic acid (preferred mRNA) introduced can also be intended to make expression product and any endogenous cell process interact or disturb any endogenous cell process, such as the regulation and control of gene expression, signal transduction and other cellular processes. The cross expression of the exogenous ribonucleic acid (preferred mRNA) introduced can also be intended to cause immune response under the background of the organism that the cell of transfection or transduction resides or makes it reside. Example is the genetic modification of antigen presenting cells (such as dendritic cells), so that it presents antigens for vaccination purpose. Other examples are the cross expression of cytokines in the tumor, to cause tumor-specific immune response. Furthermore, overexpression of introduced exogenous ribonucleic acids (preferably mRNA) can also be aimed at generating transiently genetically modified cells for cell therapy in vivo or ex vivo, such as modified T cells or precursors or stem cells or other cells for regenerative medicine.

[0228] In addition, the present invention can also realize the downward regulation of endogenous gene expression, silence or strike low for therapeutic purposes by RNA interference (RNAi), use ribozymes, antisense oligonucleotides, tRNA, long double-stranded RNA.The downward regulation of endogenous or pre-existing gene expression, silence or strike low can be used for treating acquired, hereditary or spontaneous diseases, including viral infection and cancer. It can also be imagined that nucleic acid can be introduced into the cell and put into practice as a preventive measure, to prevent, for example, viral infection or tumor formation. Similarly, gene repair, base or sequence changes can be realized at genome level and mRNA level (including exon skipping).Base or sequence changes can be for example guided by RNA site-specific DNA cutting, by utilizing trans-splicing, trans-splicing ribozymes, chimeric prosthetic body (chimeraplasts), the shear and paste mechanism of the RNA trans-splicing mediated by spliceosome or by utilizing II class or the intron of re-targeting or by utilizing by virus-mediated insertion mutagenesis or utilizing the targeted genome insertion using protokaryon, eukaryotic or viral integrase system to realize.

[0229] In addition, a variety of genetic disorders caused by mutations in a single gene are known and are candidates for RNA, preferably mRNA treatment methods. Regarding the possibility that a certain trait will occur in offspring, the disorder caused by a single gene mutation, such as cystic fibrosis, hemophilia and a variety of other diseases can be dominant or recessive. In contrast, polygenic disorders are caused by two or more genes, and the manifestation of the corresponding disease is often variable and related to environmental factors. Examples of polygenic disorders are high blood pressure, elevated cholesterol levels, cancer, neurodegenerative diseases, mental illnesses, etc. In these cases as well, therapeutic RNA, preferably mRNA, representing one or more of these genes can be beneficial to those patients. In addition, genetic disorders are not necessarily transmitted from parental genes, but may also be caused by new mutations. In these cases as well, therapeutic RNA, preferably mRNA, representing the correct gene sequence can be beneficial to patients.

[0230] An online catalogue with 22,993 entries for human genes and genetic disorders with descriptions of their corresponding genes and their phenotypes is available at the ONIM (Online Mendelian Inheritance in Man) website (http: / / onim.org); each sequence is available from the Uniprot database (http: / / www.uniprot.org).

[0231] In some embodiments, the coding sequence of the RNA, preferably mRNA, of the present invention can be transcribed and translated into a partial or full-length protein that comprises a level of cellular activity equal to or greater than that of the native protein.

[0232] In some embodiments, a genetic disease may be involved, for example, one that affects the lungs, such as SPB (surfactant protein B) deficiency, ABCA3 deficiency, cystic fibrosis (CF), primary ciliary dyskinesia, asthma, chronic obstructive pulmonary disease (COPD), and alpha 1-antitrypsin deficiency, or one that affects plasma proteins, such as congenital hemochromatosis (hepcidin deficiency), thrombotic thrombocytopenic purpura (TPP, ADAMTS 13 deficiency) and causes coagulation defects (e.g., hemophilia a and b) and complement deficiencies (e.g., protein C deficiency), immunodeficiencies such as, for example, SCID (caused by mutations in various genes such as RAG1, RAG2, JAK3, IL7R, CD45, CD3δ, CD3ε) or deficiency, due to a deficiency of adenosine deaminase, for example (ADA-SCID), septic granulomatosis (e.g., caused by mutations in the gp-91-phox gene, the p47-phox gene, the p67-phox gene, or the p33-phox gene), and storage diseases such as Gaucher's disease, Fabry's disease, Krabbe's disease, MPS I, MPS II (Hunter syndrome), MPS VI, glycogen storage disease type II, or muccopolysacchaidoses.

[0233] Other diseases for which the RNA, preferably mRNA, of the present invention may be therapeutic include, for example, SMN1-related spinal muscular atrophy (SMA); amyotrophic lateral sclerosis (ALS); GALT-related galactosemia; SLC3A1-related disorders, including cystinuria; COL4A5-related disorders, including Alport syndrome; galactocerebrosidase deficiency; X-linked leukoreflexia and adrenoneuropathy; Friedreich's ataxia; Perelman-Merzheimer disease; TSC1 and TSC2-related tuberous sclerosis; Sanfilip B syndrome (MPS) IIIB); CTNS-related cystinosis; FMR1-related disorders, including fragile X syndrome, fragile X-linked tremor / ataxia syndrome, and fragile X premature ovarian failure syndrome; Prader-Willi syndrome; hereditary hemorrhagic telangiectasia (AT); Nieto-Philosophyll disease type C1; neuronal ceroid lipofuscinosis-related disorders, including juvenile neuronal ceroid lipofuscinosis (JNCL), juvenile Batten disease, Santavuori-Haltia disease, Jansky-Bielschowsky disease, and PTT-1 and TPP1 deficiency; EIF2B1-, EIF2B2-, EIF2B3-, EIF2B4-, and EIF2B5-related childhood ataxia with hypomyelination / vanishing white matter in the central nervous system; CACNA1A and CAC NB4-related paroxysmal ataxia type 2; MECP2-related disorders, including classic Rett syndrome, MECP2-related severe neonatal encephalopathy, and PPM-X syndrome; CDKL5-related atypical Rett syndrome; Kennedy disease (SBMA); Notch-3-related cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL); SCN1A and SCN1B-related seizure disorders; polymerase G-related disorders, including Alpers-Huttenlocher syndrome, POLG-related sensory ataxia neuropathy, dysphonia, and ophthalmoparesis, and autosomal dominant and recessive progressive external ophthalmoplegia with mitochondrial DNA deletions; X-linked adrenal hypoplasia; X-linked agammaglobulinemia; Fabry disease; and Wilson disease.

[0234] In all of these diseases, a protein, such as an enzyme, is defective and can be treated by treatment with RNA, preferably mRNA, encoding any of the above proteins of the present invention, which makes the protein encoded by the defective gene or a functional fragment thereof available. Transcript replacement therapy / enzyme replacement therapy does not affect the underlying genetic defect, but increases the concentration of the enzyme that the patient lacks. As an example, in Pompe disease, transcript replacement therapy / enzyme replacement therapy replaces the deficient lysosomal enzyme acid α-glucosidase (GAA).

[0235] Thus, non-limiting examples of proteins that can be encoded by the mRNA according to the invention are erythropoietin (EPO), growth hormone (somatotropin, hGH), cystic fibrosis transmembrane conductance regulator (CFTR), growth factors such as GM-SCF, G-CSF, MPS, protein C, hepcidin, ABCA3 and surfactant protein B. Other examples of diseases that can be treated with the RNA according to the invention are hemophilia A / B, Fabry disease, CGD, ADAMTS13, Hurler's disease, X-linked A-gammaglobulinemia, adenosine deaminase-related immunodeficiency and respiratory distress syndrome in newborns, which is associated with SP-B. Particularly preferably, the RNA according to the invention, preferably the mRNA, contains a coding sequence for cystic fibrosis transmembrane conductance regulator (CFTR), alpha 1-antitrypsin, dynein axonemal intermediate chain 1 (DNAI1), surfactant protein B (SP-B) or erythropoietin. Further examples of proteins that can be encoded by the inventive RNA, preferably mRNA, according to the invention are growth factors, such as the human growth hormone hGH, BMP-2 or angiogenic factors.

[0236] Alternatively, the RNA, preferably mRNA, may contain a ribonucleotide sequence encoding a full-length antibody or nanobody (e.g., both heavy and light chains) that can be used in therapeutic settings, such as to confer immunity to a subject. Corresponding antibodies and their therapeutic application(s) are known in the art.

[0237] In another embodiment, the RNA, preferably mRNA, can encode a functional monoclonal or polyclonal antibody that can be used to target and / or inactivate a biological target (e.g., a stimulatory cytokine such as tumor necrosis factor). Similarly, the RNA, preferably mRNA sequence can encode a functional anti-nephrotic factor antibody, for example, for the treatment of type II membranoproliferative glomerulonephritis or acute hemolytic uremic syndrome, or alternatively can encode an anti-vascular endothelial growth factor (VEGF) antibody for the treatment of VEGF-mediated diseases such as cancer.

[0238] In another embodiment, RNA, preferably mRNA, may contain a ribonucleotide sequence encoding a polypeptide or protein that can be used for genome editing technology. A variety of genome editing systems utilizing different polypeptides or proteins are known in the art, i.e., for example, CRISPR-Cas systems, large-range nucleases (homing nucleases, meganucleases), zinc finger nucleases (ZFNs), and nucleases (TALENs) based on transcription activator-like effectors. Trends in Biotechnology, 2013, 31 (7), 397-405 summarizes methods for genome engineering.

[0239] Therefore, in a preferred embodiment, RNA, preferably mRNA, may contain a ribonucleotide sequence encoding a polypeptide or protein of the Cas (CRISPR-associated protein) protein family, preferably Cas9 (CRISPR-associated protein 9). Proteins of the Cas protein family, preferably Cas9, can be used in CRISPR / Cas9-based methods and / or CRISPR / Cas9 genome editing technologies. Nat. Biotechnol., 2014, 32(4): 347-355, which reviews CRISPR-Cas systems for genome editing, regulation, and targeting.

[0240] In another preferred embodiment, the RNA, preferably the mRNA, may contain a ribonucleotide sequence encoding a transcription activator-like effector nuclease (TALEN). In another preferred embodiment, the RNA, preferably the mRNA, may contain a ribonucleotide sequence encoding a zinc finger nuclease (ZFN). In another preferred embodiment, the RNA, preferably the mRNA, may contain a ribonucleotide sequence encoding a meganuclease.

[0241] As above-mentioned optional mode, RNA contains the ribonucleotide sequence that is not expressed into protein or polypeptide.Therefore, term RNA should not be merely interpreted as and mean any such polynucleotide molecule: if it is introduced into cell, then can be translated into polypeptide / protein or its fragment.To be precise, it is also considered that RNA contains the ribonucleotide sequence that is only transcribed into (functional) RNA, wherein said RNA is final product (and therefore, does not need to be translated).Under this background, it is envisioned that RNA contains ribonucleotide sequence, and it preferably provides the genetic information of siRNA sequence or another desired ribonucleotide sequence.

[0242] In certain embodiments, the present invention provides a method for preparing a therapeutic composition comprising a polymer-lipid composition as described herein for delivering a specific antigen and / or a nucleic acid encoding a specific antigen, wherein the antigen can be an antigen from bacteria, virus, fungus, or cancer cells.

[0243] The term "antigen" refers to a peptide or nucleotide-based biological material (natural, recombinant, or synthetic) that stimulates a protective immune response in an animal. Antigens suitable for the present invention can be amino acid sequences such as peptides or proteins, or nucleic acid sequences such as genomic DNA, cDNA, mRNA, saRNA, circRNA, tRNA, rRNA, small interfering RNA (iRNA) hybridization sequences, or modified or unmodified synthetic or semisynthetic oligonucleotide sequences.

[0244] Antigens suitable for the present invention may be obtained from an organism selected from the group consisting of bacteria, viruses, parasites, rickettsiae, protozoa and cancer cells.

[0245] The polymer-lipid compositions of the present invention can be used to treat or prevent a number of diseases and disorders, such as:

[0246] Diseases and disorders involving the following viruses: Retroviridae (e.g., human immunodeficiency virus, including HIV-1); Flaviviridae (e.g., dengue virus, encephalitis virus, yellow fever virus); Coronaviridae (e.g., coronavirus); Rhabdoviridae (e.g., vesicular stomatitis virus, rabies virus); Filoviridae (e.g., Ebola virus); Paramyxoviridae (e.g., parainfluenza virus, mumps virus, measles virus, respiratory syncytial virus); Orthomyxoviridae (e.g., influenza virus); Reoviridae (e.g., reovirus, orbivirus, and rotavirus); Binaviridae; Hepadnaviridae (hepatitis B virus); Parvoviridae (parvovirus); Herpesviridae (herpes simplex virus (HSV) 1 and 2, varicella-zoster virus, cytomegalovirus (CMV), herpes simplex virus); Poxyiridae (monkeypox virus, smallpox virus, vaccinia virus, Poxvirus); and Iridoviridae (e.g., African swine fever virus); and unclassified viruses (e.g., causative agents of spongiform encephalopathies, agents of hepatitis delta (thought to be defective satellites of hepatitis B virus), HCV virus (causing non-A, non-B hepatitis); Norwalk and related viruses and astroviruses). HIV, hepatitis A, hepatitis B, hepatitis C, coronavirus, rabies virus, poliovirus, influenza virus, meningitis virus, measles virus, mumps virus, rubella, pertussis, encephalitis virus, papillomavirus, yellow fever virus, respiratory syncytial virus, parvovirus, chikungunya virus, hemorrhagic fever virus and herpes virus (particularly varicella), cytomegalovirus and Epstein-Barr virus are particularly preferred. In the above embodiments, the antigens selected for use in the polymer-lipid composition are derived from those antigens present in naturally occurring viruses (or expressed / induced during infection) (or designed with reference to those antigens).

[0247] Diseases and disorders involving the following Gram-negative and Gram-positive bacteria: Helicobacter pylori, Legionella pneumophilia, Mycobacterium (e.g., M. tuberculosis), Staphylococcus aureus, Neisseria gonorrhoeae, Neisseria meningitidis, Listeria monocytogenes, Streptococcus pyogenes (Group A Streptococcus), Streptococcus agalactiae (Group B Streptococcus), Streptococcus viridans, Streptococcus pneumoniae, Klebsiella spp.) (including Klebsiella pneumoniae (K. pneumoniae)), Rickettsia spp., and Actinomyces spp. (including A. israelii). In the above embodiments, the antigens selected for use in the polymer-lipid composition are derived from those antigens present in naturally occurring bacteria (or expressed / induced during infection) (or artificially designed with reference to those antigens).

[0248] Diseases and disorders involving the following fungi: Cryptococcus neoformans, Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis, Chlamydia trachomatis, and Candida albicans, in which embodiments the antigens selected for use in the vaccine are derived from (or designed with reference to) those antigens present in naturally occurring fungi (or expressed / induced during infection).

[0249] Diseases and disorders involving the following protozoa: Plasmodium spp. (including Plasmodium falciparum, Plasmodium malariae, Plasmodium ovale, and Plasmodium vivax), Toxoplasma spp. (including T. gondii and T. cruzii), and Leishmania spp.

[0250] Diseases and disorders involving cancer cells of the blood and lymphatic systems (including Hodgkin's disease, leukemias, lymphomas, multiple myeloma, and Diseases), melanoma (including melanoma of the eye), adenoma, sarcoma, cancer of solid tissue, melanoma, lung cancer, thyroid cancer, salivary gland cancer, leg cancer, tongue cancer, lip cancer, bile duct cancer, pelvic cancer, mediastinal cancer, urethral cancer, Kaposi's sarcoma (e.g., when associated with AIDS); skin cancer (including malignant melanoma), digestive tract cancer (including head and neck cancer, esophageal cancer, stomach cancer, pancreatic cancer, liver cancer, colon and rectal cancer, anal cancer), reproductive and urinary tract cancer (including kidney cancer, bladder cancer, testicular cancer, prostate cancer), female cancer (including breast cancer, cervical cancer, ovarian cancer, gynecological cancer and choriocarcinoma) and brain cancer, bone carcinoid tumor, nasopharyngeal cancer, retroperitoneal tumor, thyroid cancer, soft tissue tumor and unknown primary site cancer. In the above embodiment, the antigen selected for the vaccine is a homologous neoantigen or tumor-associated antigen present in malignant cells and / or tissues.

[0251] Diseases and disorders involving multicellular parasites such as helminths (eg, Schistosoma spp.).

[0252] In a preferred embodiment, the antigen useful in the present invention is mRNA.

[0253] The antigen is naturally associated with the polymer-lipid composition of the present invention in an immunogenic effective amount. An "immunogenic effective amount" means that when a polymer-lipid composition of the present invention and an antigen (e.g., mRNA) are administered to an individual, the antigen contains a protective component at a concentration sufficient to protect the animal from the target disease. As an example of an immunogenic effective amount of an antigen, an amount of 0.01-100 μg can be mentioned.

[0254] In certain embodiments, the present invention provides a method for preparing a composition as described herein comprising delivering an immunomodulator and / or mRNA encoding an immunomodulator. The immunomodulator includes, but is not limited to, interleukin 2 (IL-2), interleukin 12 (IL-12), granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin 23 (IL-23), CC domain chemokine ligand 28 (CCL28), interleukin 36γ (IL-36γ), constitutively active variants of stimulator of interferon genes (STING) protein, etc.

[0255] It will be understood that particles for use in the context of the present invention may comprise a single type of RNA, but may alternatively comprise a combination of two or more types of RNA, for example in the form of particles comprising two or more types of RNA in a single particle, or in the form of a mixture of particles comprising different types of RNA.

[0256] protein

[0257] In some embodiments, the polymer-lipid composition may further comprise one or more proteins. Some proteins may include enzymes such as nucleases. The compositions described herein may include one or more CRISPR-related proteins (e.g., CRISPR enzymes), including Cas proteins. These enzymes are known; for example, the amino acid sequence of the Streptococcus pyogenes (S. pyogenes) Cas9 protein can be found in the SwissProt database under accession number Q99ZW2.

[0258] The protein in the compositions described herein can be Cas9 (e.g., from Streptococcus pyogenes or Streptococcus pneumoniae (S. pneumonia)). The CRISPR enzyme can guide the cleavage of one or both chains at the target sequence position, such as within the target sequence and / or within the complement of the target sequence. The CRISPR enzyme can be mutated relative to the corresponding wild-type enzyme so that the mutated CRISPR enzyme lacks the ability to cut one or both chains of the target polynucleotide containing the target sequence. For example, an aspartic acid-to-alanine substitution (D10A) in the RuvC I catalytic domain of Cas9 from Streptococcus pyogenes converts Cas9 from a nuclease that cuts two chains to a nickase (cutting a single strand). In some embodiments, the Cas9 nickase can be used in combination with a guide sequence (e.g., two guide sequences) that target the sense and antisense strands of a DNA target, respectively. This combination allows both chains to be nicked and used to induce NHEJ or HDR.

[0259] In some embodiments, the present disclosure provides compounds containing one or more therapeutic proteins. Therapeutic proteins that can be included in the composition include a wide range of molecules such as cytokines, chemokines, interleukins, interferons, growth factors, coagulation factors, anticoagulants, blood factors, bone morphogenic proteins, immunoglobulins, and enzymes. Some non-limiting examples of specific therapeutic proteins include erythropoietin (EPO), granulocyte colony-stimulating factor (G-CSF), α-galactosidase A, α-L-iduronidase, thyrotropin α, N-acetylgalactosamine-4-sulfatase (rhASB), dornase alfa, tissue plasminogen activator (TPA) Activase, glucocerebrosidase, interferon (IF) β-1a, interferon β-1b, interferon γ, interferon α, TNF-α, IL-1 to IL-36, human growth hormone (rHGH), human insulin (BHI), human chorionic gonadotropin α, darbepoetin α, follicle-stimulating hormone (FSH), and factor VIII.

[0260] Amphiphilic block copolymers

[0261] A variety of amphiphilic block copolymers can be used to practice the present invention. In some embodiments, the amphiphilic block copolymers are also referred to as "surfactants" or "nonionic surfactants" or "nonionic amphiphilic block copolymers" or "block copolymers" or "amphiphilic polymers" or "polymers."

[0262] As used herein, the term "block copolymer" refers to a polymer comprising groups or blocks of at least two polymerized monomer units. "Block" refers to a motif obtained by polymerizing monomers, which may be repeated within the polymer. A block copolymer must contain blocks of at least two different types of polymerized monomers.

[0263] As used herein, the term "amphiphilic block copolymer" refers to a block copolymer comprising at least one hydrophilic block and at least one hydrophobic block. In some embodiments, the blocks are nonionic, ie, do not comprise ion-forming moieties.

[0264] In some embodiments, the amphiphilic block copolymer is a tetrafunctional amphiphilic block copolymer, wherein the tetrafunctional amphiphilic block copolymer comprises a block copolymer of four branches each comprising at least one hydrophilic block and at least one hydrophobic block, or the amphiphilic block copolymer is a linear amphiphilic block copolymer, wherein the linear amphiphilic block copolymer comprises a block copolymer of at least one hydrophilic block and at least one hydrophobic block.

[0265] In some embodiments, the hydrophilic block is selected from polyoxyalkylenes, polyvinyl alcohol, polyvinyl pyrrolidone, poly(2-methyl-2-oxazoline) and sugars, and / or the hydrophobic block is selected from polyoxyalkylenes, fatty chains, alkylene polyesters, polyethylene glycol with benzyl polyether ends and cholesterol, preferably, the hydrophilic block comprises polyethylene oxide units and the hydrophobic block comprises polypropylene oxide units.

[0266] In some preferred embodiments, the amphiphilic block copolymer comprises at least one selected from the group consisting of poloxamine or ), poloxamer or ), polyoxyethylene glycol dehydrated alcohol alkyl esters (polysorbates), polyvinyl pyrrolidone (PVP), polyethylene glycol ether (BRIJ), polyoxyethylene fatty acid esters, polyoxyethylene fatty alcohol ethers, sorbitan and their derivatives.

[0267] In some embodiments, the amphiphilic block copolymer comprises 0.1%-98.0% by weight of the composition, e.g., 0.5%-95.0% by weight, 5.0%-85.0% by weight, 10.0%-80.0% by weight, 20.0%-75.0% by weight, 30.0%-70.0% by weight, or 40.0%-60.0% by weight.

[0268] Poloxamine (or )

[0269] The poloxamines suitable for the present invention are also known as tetrafunctional nonionic amphiphilic block copolymers.

[0270] The tetrafunctional nonionic amphiphilic block copolymer of the present invention preferably comprises a hydrophilic block and a hydrophobic block in a hydrophilic block / hydrophobic block ratio of 0.5 to 1.5, preferably 0.8 to 1.3, more preferably 0.8 to 1.2.

[0271] The tetrafunctional nonionic amphiphilic tetrafunctional block copolymer useful in the present invention can be a (AB)nC branched block copolymer, wherein A represents a hydrophilic block, B represents a hydrophobic block, C represents a connecting portion, and n is 4 and represents the number of (AB) groups connected to C.

[0272] Preferably, the hydrophilic block A is a polyethylene oxide block (PEO), and the hydrophobic block B is a polypropylene oxide block (PPO).

[0273] The linking moiety C may be an alkylenediamine moiety, preferably an ethylenediamine moiety.

[0274] The tetrafunctional nonionic amphiphilic block copolymers useful in the present invention may have the following structure:

[0275] where R A 、R B 、R C 、R D Independently express

[0276] in

[0277] i has a value of about 2 to about 225, particularly about 10 to about 100, more particularly about 10 to about 60, and j has a value of 2 to about 185, particularly about 10 to about 50, particularly about 10 to about 20, more particularly equal to or greater than 13,

[0278] R* is an alkylene group having 2 to 6 carbons, a cycloalkylene group having 5 to 8 carbons or a phenylene group, and is preferably an ethylene group,

[0279] For R 1 and R 2 , or (a) both are hydrogen, or (b) one is hydrogen and the other is methyl,

[0280] For R 3 and R 4 , or (a) both are hydrogen, or (b) one is hydrogen and the other is methyl, and

[0281] If R 3 and R 4 If both are hydrogen, then R 5 and R 6 One is hydrogen and the other is methyl, or if R 3 and R 4 If one of them is methyl, then R 5 and R 6 All are hydrogen.

[0282] More preferably, the nonionic amphiphilic tetrafunctional block copolymer useful in the present invention may be of the following structure:

[0283] in

[0284] The value of i is from about 2 to about 225, particularly from about 10 to about 100, more particularly from about 10 to about 60, and

[0285] The value of j is from about 2 to about 185, particularly from about 10 to about 50, especially from about 10 to about 20, and more particularly equal to or greater than 13,

[0286] and where for each R 1 、R2 Yes, one should be hydrogen and the other should be methyl.

[0287] Preferably, i may be from about 5 to about 125, particularly from about 10 to about 100, more particularly from about 10 to about 60, and j may be from about 5 to about 50, particularly from about 10 to about 25, particularly from about 10 to about 20, more particularly equal to or greater than 13.

[0288] In some embodiments, the nonionic amphiphilic tetrafunctional block copolymers described herein have the structure of the following formula:

[0289] in

[0290] The value of i is from about 2 to about 225, particularly from about 10 to about 100, more particularly from about 10 to about 60, and

[0291] The value of j is from about 2 to about 185, particularly from about 10 to about 50, especially from about 10 to about 20, and more particularly equal to or greater than 13,

[0292] and where for each R 1 、R 2 Yes, one should be hydrogen and the other should be methyl.

[0293] Preferably, i may be from about 5 to about 125, particularly from about 10 to about 100, more particularly from about 10 to about 60, and j may be from about 5 to about 50, particularly from about 10 to about 25, particularly from about 10 to about 20, more particularly equal to or greater than 13.

[0294] The molecular weight of the nonionic amphiphilic tetrafunctional block copolymer of the present invention may be 1,000 to 35,000, particularly 4,500 to 30,000, and more particularly 5,000 to 25,000.

[0295] As a preferred embodiment of the present invention, the nonionic amphiphilic tetrafunctional block copolymer may be more specifically mentioned as having a molecular weight of 1650 g / mol and a PEO / PPO ratio of 15:16 (e.g., Poloxamine 304); or having a molecular weight of 5500 g / mol and a PEO / PPO ratio of 50:56 (e.g., Poloxamine

[0296] 704); or a molecular weight of 6700 g / mol and a PEO / PPO ratio of 61:68 (e.g., poloxamine 904)

[0297] The nonionic amphiphilic tetrafunctional block copolymers of the present invention may comprise, preferably consist of, an ethylene oxide unit content of about 40%, particularly about 45%, in particular about 45% to about 80%, in particular about 45-70%, more particularly about 45-60%, more preferably about 50%.

[0298] Many of the tetrafunctional nonionic amphiphilic block copolymers of the present invention, particularly the nonionic amphiphilic tetrafunctional block copolymers, are generally known by the trade names "poloxamines" or Commercially available.

[0299] Further details of poloxamines suitable for use in the present invention can be found in Surfactant Systems, Eds. Attwood and Florence, Chapman and Hall, London 1983, p 356-361; The Condensed Encyclopaedia of Surfactants, Ed. Ash and Ash, Edward Arnold, London, 1989; Non-ionic Surfactants, pp. 300-371, Ed. Nace, Dekker, New York, 1996; Santon, Am. Perfumer Cosmet. 72(4): 54-58 (1958); (Dekker, NY, 1967); or US 6,353,055.

[0300] In some embodiments, a suitable poloxamine is poloxamine 304. In some embodiments, a suitable poloxamine is poloxamine 701. In some embodiments, a suitable poloxamine is poloxamine 704. In some embodiments, a suitable poloxamine is poloxamine 901. In some embodiments, a suitable poloxamine is poloxamine 904. In some embodiments, a suitable poloxamine is poloxamine 908. In some embodiments, a suitable poloxamine is poloxamine 1107. In some embodiments, a suitable poloxamine is poloxamine 1301. In some embodiments, a suitable poloxamine is poloxamine 1304. In some embodiments, a suitable poloxamine is poloxamine 1307. In some embodiments, a suitable poloxamine is poloxamine 90R4. In some embodiments, a suitable poloxamine is poloxamine 150R1. In some embodiments, suitable poloxamines are combinations thereof.

[0301] In some embodiments, the amphiphilic block copolymers of the present invention (e.g., poloxamine) can improve their targeting characteristics by chemical structure modification, such as glycosylation modification, protein targeting ligand modification, antibody modification, polypeptide modification, folic acid modification, growth factor modification, cytokine modification, vitamin modification, and integrin modification. Taking glycosylation modification as an example, the glycosylated amphiphilic block copolymer comprises at least one terminal block conjugated to a glycosyl portion, preferably a terminal hydrophilic block, more preferably at least 25%, particularly at least 50%, particularly at least 75%, and more particularly at least 100% of the terminal blocks of the glycosylated amphiphilic block copolymer are conjugated to the glycosyl portion. The glycosyl portion can be conjugated to the block copolymer of the present invention by a covalent bond formed between a functional group of the glycosyl portion and a functional group of the block copolymer. The covalent bond can be formed by a reaction between two functional groups that are themselves modified to be reactive, and the glycosyl portion can be directly conjugated to the block copolymer. Alternatively, the glycosyl portion can be conjugated to the block copolymer through a spacer.

[0302] Poloxamer (Poloxamer or )

[0303] In some embodiments, suitable amphiphilic polymers are poloxamer. For example, suitable poloxamer has the following structure (PEO-PPO-PEO, i.e., polyethylene oxide-polypropylene oxide-polyethylene oxide structure):

[0304] or a salt or isomer thereof, wherein a is an integer between 10 and 150, and b is an integer between 20 and 60. For example, a is about 12 and b is about 20, or a is about 80 and b is about 27, or a is about 64 and b is about 37, or a is about 141 and b is about 44, or a is about 101 and b is about 56.

[0305] In some embodiments, poloxamers suitable for the present invention have from about 10 to about 150 ethylene oxide units. In some embodiments, poloxamers have from about 10 to about 100 ethylene oxide units.

[0306] In some embodiments, a "reverse-poloxamer" (PPO-PEO-PPO, i.e., a poloxamer having a polypropylene oxide-polyethylene oxide-polypropylene oxide structure) having a similar structure to poloxamer is also suitable for use in the present invention. For example, a suitable "reverse-poloxamer" has the following structure (PPO-PEO-PPO):

[0307] or a salt or isomer thereof, wherein a is an integer between 10 and 150, and b is an integer between 20 and 60.

[0308] In some embodiments, "reverse-poloxamers" suitable for the present invention have from about 10 to about 150 ethylene oxide units. In some embodiments, "reverse-poloxamers" have from about 10 to about 100 ethylene oxide units.

[0309] In some embodiments, a suitable poloxamer is poloxamer 84. In some embodiments, a suitable poloxamer is poloxamer 101. In some embodiments, a suitable poloxamer is poloxamer 105. In some embodiments, a suitable poloxamer is poloxamer 108. In some embodiments, a suitable poloxamer is poloxamer 122. In some embodiments, a suitable poloxamer is poloxamer 123. In some embodiments, a suitable poloxamer is poloxamer 124. In some embodiments, a suitable poloxamer is poloxamer 181. In some embodiments, a suitable poloxamer is poloxamer 182. In some embodiments, a suitable poloxamer is poloxamer 183. In some embodiments, a suitable poloxamer is poloxamer 184. In some embodiments, a suitable poloxamer is poloxamer 185. In some embodiments, a suitable poloxamer is poloxamer 188. In some embodiments, a suitable poloxamer is poloxamer 212. In some embodiments, a suitable poloxamer is poloxamer 215. In some embodiments, a suitable poloxamer is poloxamer 217. In some embodiments, a suitable poloxamer is poloxamer 231. In some embodiments, a suitable poloxamer is poloxamer 234. In some embodiments, a suitable poloxamer is poloxamer 235. In some embodiments, a suitable poloxamer is poloxamer 237. In some embodiments, a suitable poloxamer is poloxamer 238. In some embodiments, a suitable poloxamer is poloxamer 282. In some embodiments, a suitable poloxamer is poloxamer 284. In some embodiments, a suitable poloxamer is poloxamer 288. In some embodiments, a suitable poloxamer is poloxamer 304. In some embodiments, a suitable poloxamer is poloxamer 331. In some embodiments, a suitable poloxamer is poloxamer 333. In some embodiments, a suitable poloxamer is poloxamer 334. In some embodiments, a suitable poloxamer is poloxamer 335. In some embodiments, a suitable poloxamer is poloxamer 338. In some embodiments, a suitable poloxamer is poloxamer 401. In some embodiments, a suitable poloxamer is poloxamer 402. In some embodiments, a suitable poloxamer is poloxamer 403. In some embodiments, a suitable poloxamer is poloxamer 407. In some embodiments, suitable poloxamers are combinations thereof.

[0310] In some embodiments, suitable poloxamers have an average molecular weight of about 4,000 g / mol to about 20,000 g / mol. In some embodiments, suitable poloxamers have an average molecular weight of about 1,000 g / mol to about 50,000 g / mol. In some embodiments, suitable poloxamers have an average molecular weight of about 1,000 g / mol. In some embodiments, suitable poloxamers have an average molecular weight of about 2,000 g / mol. In some embodiments, suitable poloxamers have an average molecular weight of about 3,000 g / mol. In some embodiments, suitable poloxamers have an average molecular weight of about 4,000 g / mol. In some embodiments, suitable poloxamers have an average molecular weight of about 5,000 g / mol. In some embodiments, suitable poloxamers have an average molecular weight of about 6,000 g / mol. In some embodiments, suitable poloxamers have an average molecular weight of about 7,000 g / mol. In some embodiments, suitable poloxamers have an average molecular weight of about 8,000 g / mol. In some embodiments, suitable poloxamers have an average molecular weight of about 9,000 g / mol. In some embodiments, suitable poloxamers have an average molecular weight of about 10,000 g / mol. In some embodiments, suitable poloxamers have an average molecular weight of about 20,000 g / mol. In some embodiments, suitable poloxamers have an average molecular weight of about 30,000 g / mol. In some embodiments, suitable poloxamers have an average molecular weight of about 40,000 g / mol. In some embodiments, suitable poloxamers have an average molecular weight of about 50,000 g / mol.

[0311] In some embodiments, the amphiphilic block copolymers of the present invention (e.g., poloxamer) can improve their targeting characteristics by chemical structure modification, such as glycosylation modification, protein targeting ligand modification, antibody modification, polypeptide modification, folic acid modification, growth factor modification, cytokine modification, vitamin modification, and integrin modification. Taking glycosylation modification as an example, the glycosylated amphiphilic block copolymer comprises at least one terminal block conjugated to a glycosyl moiety, preferably a terminal hydrophilic block, more preferably at least 25%, particularly at least 50%, particularly at least 75%, and more particularly at least 100% of the terminal blocks of the glycosylated amphiphilic block copolymer are conjugated to the glycosyl moiety. The glycosyl moiety can be conjugated to the block copolymer of the present invention by a covalent bond formed between a functional group of the glycosyl moiety and a functional group of the block copolymer. The covalent bond can be formed by a reaction between two functional groups that are themselves modified to be reactive, and the glycosyl moiety can be directly conjugated to the block copolymer. Alternatively, the glycosyl moiety can be conjugated to the block copolymer through a spacer.

[0312] Other amphiphilic block copolymers

[0313] In some embodiments, the amphiphilic block copolymer is polyvinylpyrrolidone (PVP), such as PVP having a molecular weight of 3 kDa, 10 kDa, or 29 kDa.

[0314] In some embodiments, the amphiphilic block copolymer is polyethylene glycol ether (BRIJ), polysorbate, sorbitan, and their derivatives. In some embodiments, the amphiphilic polymer is a polysorbate, such as PS 20.

[0315] In some embodiments, the amphiphilic block copolymer is a polyethylene glycol ether. In some embodiments, a suitable polyethylene glycol ether is a compound of formula (S-1):

[0316] or a salt or isomer thereof, wherein t is an integer between 1 and 100; R 1BRIJ Independently C 10-40 Alkyl, C 10-40 Alkenyl or C 10-40 Alkynyl; and optionally, R 5PEG The one or more methylene groups are independently C 3-10 Carbocyclylene, 4- to 10-membered heterocyclylene, C 6‐10 Arylene, 4- to 10-membered heteroarylene, -N(R N )‐, ‐O‐, ‐S‐, ‐C(O)‐, ‐C(O)N(R N )-、-NR NC(O)‐,‐NR C(O)N(R)‐,‐C(O)O‐‐OC(O)‐,‐OC(O)O‐‐OC(O)N(R N )-、-NR N C(O)O‐‐C(O)S‐‐SC(O)‐,‐C(=NR N )-, -C(=NR)N(R)-, -NR N C(=NR N )-NR N C(=NR N )N(R N )-, -C(S)-, -C(S)N(R N )-、-NR N C(S)-,-NR N C(S)N(R N )‐, ‐S(O)‐, ‐OS(O)‐, ‐S(O)O‐‐OS(O)O‐‐OS(O)2‐‐S(O)2O‐‐OS(O)2O‐‐N(R N )S(O)-、-S(O)N(R N )-N(R N )S(O)N(R N )--OS(O)N(R N )-N(R N )S(O)O‐‐S(O)2‐‐N(R N )S(O)2‐S(O)2N(R N )-、-N(R N )S(O)2N(R N )--OS(O)2N(R N )-or-N(R N )S(O)2O-substituted; and R N Each instance of is independently hydrogen, C 1-6 Alkyl or nitrogen protecting group.

[0317] In some embodiments, R 1BRIJ For example, the polyethylene glycol ether is a compound of formula (S-1a):

[0318] or a salt or isomer thereof, wherein S is an integer between 1 and 100.

[0319] In some embodiments, R 1BRIJ For example, a suitable polyethylene glycol ether is a compound of formula (S-1b):

[0320] or a salt or isomer thereof, wherein S is an integer between 1 and 100.

[0321] In some embodiments, the amphiphilic block copolymers of the present invention (e.g., PVP, BRIJ, polysorbate, sorbitol, etc.) can improve their targeted delivery characteristics by chemical structure modification, such as glycosylation modification, protein targeting ligand modification, antibody modification, polypeptide modification, folic acid modification, growth factor modification, cytokine modification, vitamin modification, and integrin modification. Taking glycosylation modification as an example, the glycosylated amphiphilic block copolymer comprises at least one terminal block conjugated to a glycosyl portion, preferably a terminal hydrophilic block, more preferably at least 25%, particularly at least 50%, particularly at least 75%, and more particularly at least 100% of the terminal blocks of the glycosylated amphiphilic block copolymer are conjugated to the glycosyl portion. The glycosyl portion can be conjugated to the block copolymer of the present invention by a covalent bond formed between a functional group of the glycosyl portion and a functional group of the block copolymer. The covalent bond can be formed by a reaction between two functional groups that are themselves modified to be reactive, and the glycosyl portion can be directly conjugated to the block copolymer. Alternatively, the glycosyl moiety can be conjugated to the block copolymer via a spacer.

[0322] Cationic lipids

[0323] As used herein, the term "cationic lipid" refers to any one of many lipids and lipidoids with a net positive charge at a selected pH (such as physiological pH). Cationic lipids of the present invention include at least one selected from the group consisting of permanent cationic lipids, ionizable cationic lipids, cholesterol-derived cationic lipids and dendrimers or dendrons. Such lipids include, but are not limited to, DOTMA, DOSPA, DOTAP, ePC, DODAP, DODMA, DDAB, DSDMA, DODAC, DOAP, DMRIE, DOGS, DMOBA, HGT5000, HGT5001, HGT5002, HGT4001, HGT4002, HGT4003, HGT4005, DLin-MC3-DMA, DLin-KC2-DMA, Acuitas ALC-0315, Acuitas A9, Acuitas Lipid 2,2, Moderna Lipid H (SM-102), Moderna Lipid 5, A2-Iso5-2DC18, BAME-O16B, 9A1P9, C12-200, cKK-E12, OF-Deg-Lin, 306Oi10, TT3, FTT5, Lipid319, 5A2-SC8, Genevant CL1, DLinDMA, DLenDMA, ClinDMA, CpLinDMA, imidazole cholesterol ester (ICE), DC-Choi (N,N-dimethyl-N-ethylformamide cholesterol), 1,4-bis(3-N-oleylamino-propyl)piperazine, N4-argininocholesterol carbonylamide (GL67), cholesterol derivatives coupled to basic amino acid sequences, RE-1, RE-2, RE-3 and GL-67, 5A2-SC8, Acuitas A9, Arcturus Lipid 2,2(8,8)4CCH3, OF-02, A18-Iso5-2DC18, BAME-O16B, A6, 98N12-5, L319, L343, 304O13, 306O138, 306O12B, 30 6-O12B, LP01, G0-C14, 7C1, Cephalin, Dlin-EG-DMA, DLinAP, DLin-MPZ, DLin-C-DAP, DLin-2-DMAP , Dlin-S-DMA, DLinDAP, DLin-MA, DLin-DAC, DLin-K-DMA, DLin-K-MPZ, DLin-K-DMA, DLin-K6-C4-DMA, DLin-K-C4-DMA, DLin-K-C3-DMA, CpLinDMA, DOcarbDAP, DLincarbDAP, C12-(2-3-2), Genevant Lipid CL1, XTC, ALNY-100, NC98-5, and mixtures thereof. Many of these lipids and related analogs have been described in U.S. Patent Publication Nos. 20220160633, 20220168234, 20220062175, 20220370624, 20210137840, 20210378980, 20210316008, 20210369866, 20180153822, 20170151333, 2 0100036115, 20120202871, 20130064894, 20130129785, 20130150625, 20130178541, 20130225836, 20060083780, and 20060240554; U.S. Patent Nos. 11045418, 10245229, 10130649, 108211 86, 9801944, 8058069, 9364435, 9567296, 10980895, 10233148, 10961188, 9365610, 9404127, 10940207, 5208036, 5264618, 5279833, 5283185, 5753613, 7893302, 7404969, 828 3333, 8466122, 5785992 and PCT Publication Nos. WO2022032154, WO2022040641, WO2022235935, WO20220378702, WO20220389422, WO2021216577, WO2021226463, WO2021141944, WO2021016430,WO2021222801, WO2020097520, WO2020051220, WO2019246203, WO2019141814, WO2018183901, WO2017201091, WO2017048789, WO2017201 076, WO2017075531, WO2015061467, WO2015199952, WO2013149140, WO2013086373, WO2013086354, WO2013116126, WO20130225836, WO96 / 10390, WO2012170889, WO2012170930, WO2012040184, WO2012061259, WO2012054365, WO2012044638, WO2011153120, WO2011149733, WO2011090965, WO2011043913, WO2011022460, WO2010080724, WO201021865, WO2008103276, WO2010054401, the disclosures of which are incorporated herein by reference in their entirety for all purposes. In addition, many commercial cationic lipid formulations are available and can be used in the present invention. These include, for example, (Commercially available cationic liposomes containing DOTMA and DOPE from GIBCO / BRL, Grand Island, New York, USA); (commercially available cationic liposomes containing DOSPA and DOPE from GIBCO / BRL); and (Commercially available cationic liposomes containing DOGS were obtained from Promega Corp., Madison, Wisconsin, USA).

[0324] In certain embodiments, the compositions and methods of the present invention include cationic lipids that can be defined as compounds having the following structure or salts thereof:

[0325] in,

[0326] R 1 and R 2 are independently selected and are H or C1-C3 alkyl;

[0327] R 3 and R 4 are independently selected and are alkyl groups having from about 10 to about 20 carbon atoms, and R 3 and R 4 At least one of the amino acids comprises at least 2 sites of unsaturation.

[0328] In some instances, R 3 and R 4 are the same, that is, R 3 and R 4 All are linoleyl (C 18 ) etc. In certain other instances, R 3 and R 4 Different, that is, R 3 Is tetradectrienyl (C 14 ) and R 4 Linoleyl (C 18 In a preferred embodiment, the cationic lipid of Formula I is symmetrical, i.e., R 3 and R 4 In another preferred embodiment, R 3 and R 4 In some embodiments, R 3 and R 4 are independently selected from the group consisting of dodecadienyl, tetradecadienyl, hexadecadienyl, linoleyl, and icosadienyl. 3 and R 4 In some embodiments, R 3 and R 4 Includes at least 3 sites of unsaturation and is independently selected from, for example, dodecatrienyl, tetradecatrienyl, hexadecatrienyl, linolenyl, and icosatrienyl.

[0329] In a preferred embodiment, the cationic lipid of Formula I is 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA) or 1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA).

[0330] In addition, cationic lipids having the following structure (or salts thereof) can be effectively used in the present invention.

[0331] in,

[0332] R 1 and R 2 The same or different and independently optionally substituted C 12 -C24 Alkyl, optionally substituted C 12 -C 24 Alkenyl, optionally substituted C 12 -C 24 Alkynyl, or optionally substituted C 12 -C 24 acyl group;

[0333] R 3 and R 4 are the same or different and are independently optionally substituted C1-C6 alkyl, optionally substituted C1-C6 alkenyl, or optionally substituted C1-C6 alkynyl or R 3 and R 4 may combine to form an optionally substituted heterocyclic ring of 4 to 6 carbon atoms and 1 or 2 heteroatoms selected from nitrogen and oxygen;

[0334] R 5 is absent or is hydrogen or a C1-C6 alkyl group to provide a quaternary amine;

[0335] m, n, and p are the same or different and are independently 0 or 1, provided that m, n, and p are not simultaneously 0; q is 0, 1, 2, 3, or 4; and Y and Z are the same or different and are independently 0, S, or NH.

[0336] In some embodiments, the cationic lipid having the above structure is 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-K-C2-DMA), 2,2-dilinoleyl-4-(3-dimethylaminopropyl)-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-dilinoleyl-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-dilinoleyl-3-morphocyanopropane (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 (DLin-TMA.Cl), 1,2-dilinoleyl-3-trimethylaminopropane chloride (DLin-TAP.Cl), 1,2-dilinoleyloxy-3-(N-methylpiperazine)propane (DLin-MPZ), 3-(N,N-dilinoleylamino)-1,2-propanediol (DLinAP), 3-(N,N-dioleylamino)-1,2-propanediol (DOAP), 1,2-dilinoleyloxy-3-(2-N-dimethylamino)ethoxypropane (DLin-EG-DMA), or a mixture thereof. In a preferred embodiment, the cationic lipid having the above structure is DLin-K-C2-DMA.

[0337] In certain embodiments, the compositions and methods of the present invention include permanent cationic lipids comprising quaternary ammonium ions. In certain embodiments, such permanent cationic lipids have the following general formula:

[0338] in,

[0339] R1 and R2 are each independently C8-C 24 Alkyl, C8-C 24 alkenyl or a substituted form of either group;

[0340] R3, R3' and R3" are each independently an alkyl group (C≤6) or substituted alkyl (C≤6) ;

[0341] X ‐ It is a monovalent anion.

[0342] In certain embodiments, the compositions and methods of the present invention further define the cationic lipid as a compound having the following structure:

[0343] in,

[0344] R1 and R2 are each independently C8-C 24 Alkyl, C8-C 24 alkenyl or a substituted form of either group;

[0345] R3, R3' and R3" are each independently an alkyl group (C≤6) or substituted alkyl (C≤6) ;

[0346] R4 is an alkyl group (C≤6) or substituted alkyl (C≤6) ;

[0347] And X ‐ It is a monovalent anion.

[0348] In certain embodiments, the compositions and methods of the present invention further define the cationic lipid as a compound having the following structure:

[0349] in,

[0350] R 1 and R 2 are independently selected and are H or C1-C3 alkyl;

[0351] R 3 and R 4 are independently selected and are alkyl groups having from about 10 to about 20 carbon atoms, and R 3 and R 4 At least one of the amino acids comprises at least 2 sites of unsaturation.

[0352] In some instances, R 3 and R 4 are the same, that is, R 3 and R 4 All are linoleyl (C 18 ), etc. In certain other instances, R 3 and R 4 Different, that is, R 3 Is tetradecatrienyl (C14 ) and R 4 Linoleyl (C 18 In a preferred embodiment, the cationic lipids of the present invention are symmetrical, i.e., R 3 and R 4 In another preferred embodiment, R 3 and R 4 In some embodiments, R 3 and R 4 In a preferred embodiment, R 3 and R 4 In some embodiments, R 3 and R 4 Includes at least 3 sites of unsaturation and is independently selected from, for example, dodecatrienyl, tetradecatrienyl, hexadecatrienyl, linolenyl, and eicosatrienyl.

[0353] In certain embodiments, the compositions and methods of the present invention further define the cationic lipid as a compound having the following structure:

[0354] in,

[0355] R4 and R4' are each independently C6-C 24 Alkyl, C6-C 24 alkenyl or a substituted form of either group;

[0356] R4" is an alkyl group (C≤24) , alkenyl (C≤24) or a substituted form of any group;

[0357] R4'' is C1-C8 alkyl, C2-C8 alkenyl, or a substituted form of either group;

[0358] and X2 is a monovalent anion.

[0359] In certain embodiments, the compositions and methods of the present invention further define the permanent cationic lipid as a compound having the following structure or a salt thereof:

[0360] wherein Y1, Y2 or Y3 are each independently X1C(O)R1 or X2N + R3R4R5; as long as at least one of Y1, Y2 and Y3 is X2N + R3R4R5;

[0361] R1 is C1-C 24Alkyl, C1-C 24 Substituted alkyl, C1-C 24 Alkenyl, C1-C 24 substituted alkenyl;

[0362] X1 is O or NR a , where R a is hydrogen, C1-C4 alkyl or C1-C4 substituted alkyl;

[0363] X2 is a C1-C6 alkanediyl group or a C1-C6 substituted alkanediyl group;

[0364] R3, R4 and R5 are each independently C1-C 24 Alkyl, C1-C 24 Substituted alkyl, C1-C 24 Alkenyl, C1-C 24 substituted alkenyl;

[0365] A1 is an anion with a charge equal to that of X2N in the compound. + The number of R3R4R5 groups.

[0366] Suitable cationic lipids for use in the compositions and methods of the invention include those described in International Patent Publication No. WO 2010 / 144740 and US Patent No. 8,058,069, which are incorporated herein by reference.

[0367] Other suitable cationic lipids for use in the compositions and methods of the present invention include ionizable cationic lipids as described in International Patent Publication WO2013 / 149140, which is incorporated herein by reference. In some embodiments, the compositions and methods of the present invention include a cationic lipid of one of the following formulas:

[0368] or a pharmaceutically acceptable salt thereof,

[0369] wherein R1 and R2 are each independently selected from the group consisting of hydrogen, optionally substituted different saturated or unsaturated C1-C 20 alkyl and optionally substituted different saturated or unsaturated C6-C 20 Acyl; wherein L1 and L2 are each independently selected from the group consisting of hydrogen, optionally substituted C1-C 30 Alkyl, optionally substituted different unsaturated C1-C 30 Alkenyl and optionally substituted C1-C 30 Alkynyl; wherein m and o are each independently selected from the group consisting of zero and any positive integer (eg, wherein m is three); and wherein n is zero or any positive integer (eg, wherein n is one).

[0370] In some embodiments, the polymer-lipid compositions provided herein comprise a cationic lipid of the formula:

[0371] or a pharmaceutically acceptable salt thereof,

[0372] Wherein, p is an integer between 1 and 9, inclusive;

[0373] R 2 Each instance of is independently hydrogen or optionally substituted C 1‐6 alkyl;

[0374] R 6 and R 7 Each instance of is independently a group of formula (i), (ii) or (iii);

[0375] Formulas (i), (ii) and (iii) are:

[0376] wherein each instance of R' is independently hydrogen or optionally substituted alkyl;

[0377] X is O, S or NR X , where R X is hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, or a nitrogen protecting group;

[0378] Y is O, S or NR Y , where R Y is hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, or a nitrogen protecting group;

[0379] R P is hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, an oxygen protecting group attached to an oxygen atom, a sulfur protecting group attached to a sulfur atom, or a nitrogen protecting group attached to a nitrogen atom; and R L is optionally substituted C 1‐50 Alkyl, optionally substituted C 2‐50 Alkenyl, optionally substituted C 2‐50 Alkynyl, optionally substituted heteroC 1‐50 Alkyl, optionally substituted heteroC 2‐50 Alkenyl, optionally substituted heteroC 2‐50 Alkynyl, or polymer.

[0380] In some embodiments, the compositions and methods of the invention include a cationic lipid having the formula:

[0381] or a pharmaceutically acceptable salt thereof, wherein each X is independently O or S; each Y is independently O or S; each m is independently 0 to 20; each n is independently 1 to 6; each R A are independently hydrogen, optionally substituted C 1‐50 Alkyl, optionally substituted C 2‐50 Alkenyl, optionally substituted C 2‐50 Alkynyl, optionally substituted C 3‐10 Carbocyclyl, optionally substituted 3-14 membered heterocyclyl, optionally substituted C 6‐14 aryl, optionally substituted 5-14 membered heteroaryl or halogen; and each R B are independently hydrogen, optionally substituted C 1‐50 Alkyl, optionally substituted C 2‐50 Alkenyl, optionally substituted C 2‐50 Alkynyl, optionally substituted C 3‐10 Carbocyclyl, optionally substituted 3-14 membered heterocyclyl, optionally substituted C 6‐14 aryl, optionally substituted 5-14 membered heteroaryl or halogen.

[0382] Other suitable cationic lipids for use in the compositions and methods of the present invention include cationic lipids as described in PCT Application Publication No. WO2020097384A1, the disclosure of which is incorporated herein by reference. In some embodiments, the compositions and methods of the present invention include a cationic lipid having the formula:

[0383] or a pharmaceutically acceptable salt thereof, wherein each R 1 and R 2 is independently H or C1-C6 aliphatic; each m is independently an integer having a value of 1 to 4; each A is independently a covalent bond or an arylene group; each L 1 are independently an ester, thioester, disulfide bond or anhydride group; each L 2 Independently C2-C 10 Aliphatic; each X 1 is independently H or OH; and each R 3 Independently C6-C 20 aliphatic.

[0384] Other suitable cationic lipids for use in the compositions and methods of the present invention include cationic lipids as described in International Patent Publication WO2017 / 075531, which is incorporated herein by reference. In some embodiments, the compositions and methods of the present invention include a cationic lipid having the formula:

[0385] or a pharmaceutically acceptable salt thereof, wherein L 1 or L 2 One of them is ‐O(C=O)‐, ‐(C=O)O‐, ‐C(=O)‐, ‐O‐, ‐S(O) x , ‐S‐S‐, ‐C(=O)S‐, ‐SC(=O)‐, ‐NR a C(=O)-, -C(=O)NR a ‐、NR a C(=O)NR a ‐、‐OC(=O)NR a ‐or ‐NR a C(=O)O-; and L 1 or L 2 The other one is ‐O(C=O)‐, ‐(C=O)O‐, ‐C(=O)‐, ‐O‐, ‐S(O) x , ‐S‐S‐, ‐C(=O)S‐, SC(=O)‐, ‐NR a C(=O)-, -C(=O)NR a ‐、NR a C(=O)NR a ‐、‐OC(=O)NR a ‐or ‐NR a C(=O)O- or direct bond; G 1 and G 2 Each independently is an unsubstituted C1-C 12 Alkylene or C1-C 12 Alkenylene; G 3 It is C1-C 24 Alkylene, C1-C 24 Alkenylene, C3-C8 cycloalkylene, C3-C8 cycloalkenylene; R a Is H or C1-C 12 Alkyl; R 1 and R 2 Each is independently C6-C 24 Alkyl or C6-C 24 Alkenyl; R 3 It is H, OR 5 、CN、-C(=O)OR 4 ,-OC(=O)R 4 or NR5 C(=O)R 4 ; R 4 It is C1-C 12 Alkyl; R 5 is H or C1-C6 alkyl; and x is 0, 1 or 2.

[0386] Other suitable cationic lipids for use in the compositions and methods of the invention include cholesterol-based cationic lipids as described in International Patent Publications WO2018 / 089790 and WO2022 / 032154, which are incorporated herein by reference.

[0387] In certain embodiments, the compositions and methods of the present invention include a compound having the following structure: BL 1 -S,

[0388] or a pharmaceutically acceptable salt thereof,

[0389] in,

[0390] B is a basic functional group, wherein the protonated form has a pKa of no more than about 8.0;

[0391] L 1 is an optionally substituted linking group, which is C1-C 20 Alkylene or 2- to 20-membered heteroalkylene;

[0392] And S is a sterol.

[0393] Preferably, B is an optionally substituted 5-membered or 6-membered nitrogen-containing heteroaryl group. B is a group selected from pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, pyridyl, pyrimidinyl, pyrazinyl and pyridazinyl, each of which is optionally substituted.

[0394] Preferably, L 1 -X 1 -C(X 3 )-X 2 、-(C1-C 19 Alkylene)-X 1 -C(X 3 )-X 2 、-X 1 -C(X 3 )-X 2 (C1-C 19 Alkylene)-, -(C1-C 19 Alkylene)-X1-, -X1-(C1-C19 alkylene)-, wherein each X 1 and X 2 are independently a covalent bond, -O-, -S- or -NH-; X 3are independently ═O, ═S or ═NH; and wherein said C1-C 19 Alkylene groups are optionally substituted.

[0395] More preferably, L1 comprises a moiety that is an ester group, an amide group, a carbamate group, a carbonate group, or a urea group. 1 does not contain a substituent having the structure -N(R')2, or a positively charged version thereof, wherein each R' is independently hydrogen or an optionally substituted C1-C 20 alkyl.

[0396] Preferably, S is a zoosterol, or an oxidized or reduced form thereof; S is a phytosterol, or an oxidized or reduced form thereof; S is a synthetic sterol, or an oxidized or reduced form thereof.

[0397] More preferably, S is a sterol selected from the group consisting of cholesterol, an oxidized form of cholesterol, a reduced form of cholesterol, an alkyl lithocholate, stigmasterol, stigmasterol, campesterol, ergosterol and sitosterol.

[0398] In certain embodiments, the compositions and methods of the present invention include a compound having the structure,

[0399] or a pharmaceutically acceptable salt thereof,

[0400] wherein n is 0 or 1, preferably 0,

[0401] R 1 It is the group –(CH2) q -NH2 or group –(CH2) r -NH-(CH2) s -NH2,

[0402] wherein q, r and s are independently integers from 2 to 6,

[0403] R 2 It is the group –(CH2) t -NH2 or group –(CH2) u -NH-(CH2) w -NH2,

[0404] wherein t, u and w are independently integers from 2 to 6,

[0405] R3 is a straight-chain alkanediyl group having 1 to 4 carbon atoms;

[0406] In some embodiments, as a replacement of cationic lipid as described herein or in addition to cationic lipid as described herein, a cationic lipid based on cholesterol (sterol) can be used. Suitable cationic lipid based on sterol is a cationic lipid based on sterol containing dialkylamino, imidazoles, basic amino acid sequence and guanidine. For example, some embodiments relate to compositions comprising one or more cationic lipids based on sterol comprising imidazoles, such as imidazole cholesterol ester or " ICE " lipid (3S, 10R, 13R, 17R) -10,13- dimethyl -17- ((R) -6- methylheptane -2- bases) -2,3,4,7,8,9,10,11,12,13,14,15,16,17- tetradecahydro -1H- cyclopenta [a] phenanthren -3- bases 3- (1H- imidazoles -4- bases) propionate.

[0407] In some embodiments of the composition of the present invention, the cholesterol-derived cationic lipid comprises at least one selected from the group consisting of DC-Choi (N,N-dimethyl-N-ethylformamide cholesterol), 1,4-bis(3-N-oleylamino-propyl)piperazine (Gao, et al. Biochem. Biophys. Res. Comm. 179, 280 (1991); Wolf et al. BioTechniques 23, 139 (1997); U.S. Pat. No. 5,744,335), N4-argininocholesterol carbonylamide (GL67), cholesterol derivatives coupled to basic amino acid sequences, imidazole cholesterol ester (ICE), and derivatives thereof.

[0408] Other suitable cationic lipids for use in the compositions and methods of the present invention include cleavable cationic lipids as described in International Patent Publication WO2012 / 170889, which is incorporated herein by reference. In some embodiments, the compositions and methods of the present invention include a cationic lipid having the formula:

[0409] wherein R1 is selected from the group consisting of imidazole, guanidine, amino, imine, enamine, optionally substituted alkylamino (e.g., alkylamino such as dimethylamino), and pyridyl; wherein R2 is selected from the group consisting of one of the following two general formulae:

[0410] and wherein R3 and R4 are each independently selected from the group consisting of: optionally substituted different saturated or unsaturated C6-C 20 alkyl and optionally substituted different saturated or unsaturated C6-C 20acyl; and wherein n is zero or any positive integer (e.g., one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, or more).

[0411] Other suitable cationic lipids for use in the compositions and methods of the present invention include degradable cationic lipids, as described in PCT Application Publication No. WO2019222424A1, which is incorporated herein by reference. In certain embodiments, the compositions and methods of the present invention include a cationic lipid, the cationic lipid being any one of the general formula described in WO2019222424A1 or structures (1a)-(21a) and (1b)-(21b) and (22)-(237). In certain embodiments, the compositions and methods of the present invention include a cationic lipid having a structure according to the formula,

[0412] and pharmaceutically acceptable salts thereof. X are independently -H, -L1-R1 or -L5A-L5B-B'; L 1 , L 2 and L 3 are each independently a covalent bond, ‐C(O)‐, ‐C(O)O‐, ‐C(O)S‐, or ‐C(O)NRL‐; each L 4A and L 5A are independently -C(O)-, -C(O)O- or -C(O)NRL-; each L 4B and L 5B Independently C1-C 20 Alkylene; C2-C 20 Alkenylene; or C2-C 20 Alkynylene; each B and B' is NR 4 R 5 or a 5-10 membered nitrogen-containing heteroaryl group; each R 1 、R 2 and R 3 Independently C6-C 30 Alkyl, C6-C 30 Alkenyl or C6-C 30 Alkynyl; each R 4 and R 5 are independently hydrogen, C1-C 10 Alkyl; C2-C 10 Alkenyl; or C2-C 10 Alkynyl; and each RL is independently hydrogen, C1-C 20 Alkyl, C2-C 20 Alkenyl or C2-C 20 Alkynyl.

[0413] In certain embodiments, the cationic lipid in the compositions and methods of the present invention is a dendritic polymer or dendron as described in International Patent Publication No. WO2020051220A1, which is incorporated herein by reference.

[0414] In certain embodiments, the cationic lipid compositions and methods of the present invention comprise one or more lipid-modified spermine derivatives having the following general formula:

[0415] Where X1 is -(CH2) n - or carbonyl, wherein n is 1, 2 or 3;

[0416] X2 is selected from -(CH2)-, an ester group, an amide group, oxygen or sulfur;

[0417] R1 and R2 are independently selected from C6-C 18 Alkyl, C6-C containing olefinic bond 18 Alkyl or lipophilic cholesterol-like molecules;

[0418] Furthermore, X1 and X2 are both -(CH2)-, R1 and R2 are independently selected from C 10 -C 18 alkyl.

[0419] Furthermore, X1 is -(CH2)2-, X2 is oxygen, R1 and R2 are the same C 12 -C 18 Alkyl or C6-C containing olefinic bond 18 alkyl.

[0420] Preferably, X1 is a carbonyl group, X2 is -(CH2)-, R1 and R2 are the same C 12 -C 18 Alkyl or C6-C containing olefinic bond 18 alkyl.

[0421] Preferably, X1 is -(CH2)2-, X2 is an ester group, R1 and R2 are the same C 12 -C 18 Alkyl or C6-C containing olefinic bond 18 alkyl.

[0422] Preferably, X1 is -(CH2)-, X2 is an amide group, R1 and R2 are the same C 12 -C 18 Alkyl or C6-C containing olefinic bond 18 alkyl.

[0423] or a pharmaceutically acceptable salt thereof.

[0424] The lipid-modified spermine derivative of the present invention is composed of spermine and oleyl alcohol coupled via different chemical bonds, wherein spermine serves as a positively charged head group and oleyl alcohol is linked to two tertiary amine groups in the middle.

[0425] Some non-limiting examples of lipid-modified spermine derivatives that can be used in the present disclosure are taught in patent application publication number CN104876831, which is incorporated herein by reference.

[0426] In some embodiments, the compositions and methods of the present invention include the cationic lipid N-[l-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride ("DOTMA") (Feigner et al., Proc. Natl. Acad. Sci. 84, 7413 (1987); U.S. Pat. No. 4,897,355, which is incorporated herein by reference). Other cationic lipids suitable for use in the compositions and methods of the present invention include, for example, 5-carboxysperminylglycine dioctadecylamide ("DOGS"); 2,3-dioleyloxy-N-[2-(spermine-carboxamide)ethyl]-N,N-dimethyl-1-propylammonium ("DOSPA") (Behr et al., Proc. Natl. Acad. Sci. 86, 6982 (1989), U.S. Pat. No. 5,171,678; U.S. Pat. No. 5,334,761); 1,2-dioleoyl-3-dimethylammonium-propane ("DODAP"); 1,2-dioleoyl-3-trimethylammonium-propane ("DOTAP").

[0427] Additional exemplary cationic lipids suitable for use in the compositions and methods of the present invention include: 1,2-distearoyloxy-N,N-dimethyl-3-aminopropane ("DSDMA"); 1,2-dioleyloxy-N,N-dimethyl-3-aminopropane ("DODMA"); 1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane ("DLinDMA"); 1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane ("DLenDMA"); N-dioleyl-N,N-dimethylammonium chloride ("DODAC"); N,N-distearoyl- N,N-dimethylammonium bromide ("DDAB"); N-(1,2-dimyristyloxypropyl-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide ("DMRIE"); 3-dimethylamino-2-(cholest-5-en-3-β-oxybutane-4-oxy)-1-(cis,cis-9,12-octadecadienyloxy)propane ("CLinDMA"); 2-[5'-(cholest-5-en-3-β-oxy)-3'-oxopentyloxy)-3-dimethyl-1-(cis,cis-9',12'-octadecadienyloxy)propane ("CpLinDMA"); N,N-dimethyl-3,4-dioleyloxybenzylamine ("DMOBA"); 1,2-N,N'-dioleylcarbamoyl-3-dimethylaminopropane ("DOcarbDAP"); 2,3-dilinoleoyloxy-N,N-dimethylpropylamine ("DLinDAP"); 1,2-N,N'-dilinoleylcarbamoyl-3-dimethylaminopropane ("DLincarbDAP"); 1,2-dilinoleoylcarbamoyl-3-dimethylaminopropane ("DLinCDAP"); 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane ("DLin-K-DMA"); 2-((8-[(3P)-cholest-5-en-3-yloxy]octyl)oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadecanoyl- 9,12-dien-1-yloxy]propane-1-amine ("octyl-CLinDMA"); (2R)-2-((8-[(3β)-cholest-5-en-3-yloxy]octyl)oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadec-9,12-dien-1-yloxy]propane-1-amine ("octyl-CLinDMA(2R)"); (2S)-2- ((8-[(3P)-cholest-5-en-3-yloxy]octyl)oxy)-N,fsl-dimethyl 3-[(9Z,12Z)-octadec-9,12-dien-1-yloxy]propane-1-amine ("octyl-CLinDMA(2S)"); 2,2-dilinoleyl-4-dimethylaminoethyl-[l,3]-dioxolane ("DLin-K-XTC2-DMA");and 2-(2,2-di((9Z,12Z)-octadeca-9,12-dien-1-yl)-1,3-dioxolane-4-yl)-N,N-dimethylethylamine ("DLin-KC2-DMA") (see, WO 2010 / 042877, incorporated herein by reference; Semple et al., Nature Biotech. 28:172-176 (2010); Heyes, J., et al., J Controlled Release 107:276-287 (2005); Morrissey, DV. et al., Nat. Biotechnol. 23(8):1003-1007 (2005); International Patent Publication No. WO 2005 / 121348). In some embodiments, the one or more cationic lipids comprise at least one of an imidazole, dialkylamino, or guanidinium moiety. ;

[0428] In some embodiments, one or more cationic lipids suitable for use in the compositions and methods of the present invention include 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane ("XTC"); (3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine ("ALNY-100") and / or 4,7,13-tris(3-oxo-3-(undecylamino)propyl)-N1,N16-di-undecyl-4,7,10,13-tetraazahexadecane-1,16-diamide ("NC98-5").

[0429] In some embodiments, other suitable cationic lipids for the compositions and methods of the present invention include compounds of the following formula and pharmaceutically acceptable salts thereof:

[0430] in,

[0431] RCOO is selected from the list comprising myristoyl, α-D-tocopheryl succinyl, linoleoyl, and oleoyl; and X is selected from the list comprising:

[0432] Other suitable cationic lipids for use in the compositions and methods of the invention include those described in International Patent Publications WO2010 / 053572, WO2013 / 063468, WO2015 / 184256, WO2015 / 199952, WO2015 / 095340, WO2016 / 118725, WO2016 / 205691, WO2016 / 004202, WO2017 / 004143, WO2017 / 117528, WO2017 / 049245, WO2017 / 173054, which are incorporated herein by reference.

[0433] Other suitable cationic lipids for use in the compositions and methods of the invention include those described in J. McClellan, MCKing, Cell 2010, 141, 210-217 and Whitehead et al., Nature Communications (2014) 5:4277, which are incorporated herein by reference.

[0434] In some embodiments of the compositions of the present invention, the cationic lipid comprises at least one selected from the group consisting of permanent cationic lipids, ionizable cationic lipids, cholesterol-derived cationic lipids, and dendrimers or dendrons. In preferred embodiments, the cationic lipid comprises an ionizable cationic lipid.

[0435] In some embodiments of the compositions of the present invention, the cationic lipid may contain one or more asymmetrically substituted carbon or nitrogen atoms and may be separated in an optically active or racemic form. Thus, unless otherwise specified, all chiral, diastereoisomer, racemic, epimeric, and all geometric isomeric forms of a chemical formula are intended. Cationic lipids may exist as racemates and racemic mixtures, single enantiomers, diastereomeric mixtures, and each diastereomer. In some embodiments, a single diastereomer is obtained. The chiral center of the cationic lipid of the present invention may have an S or R configuration. In addition, it is contemplated that one or more of the cationic lipids may exist as structural isomers. In some embodiments, the compound has the same chemical formula, but is different from the connectivity of the nitrogen atom at the core. Without wishing to be bound by any theory, it is believed that such cationic lipids exist because the starting monomer first reacts with a primary amine and then statistically reacts with any secondary amine present. Therefore, structural isomers may present a fully reacted primary amine and then a mixture of reacted secondary amines.

[0436] Chemical formula for representing cationic lipids of the present invention will usually only show one of several possible different tautomers. For example, it is known that many types of keto groups exist in equilibrium with the corresponding enol groups. Similarly, many types of imino groups exist in equilibrium with the enamine groups. No matter which tautomer is described for a given formula, and no matter which tautomer is the most common, all tautomers of a given chemical formula are meant.

[0437] In addition, the atoms constituting the cationic lipids of the present invention are intended to include all isotopic forms of such atoms. Isotopes as used herein include those atoms having the same atomic number but different mass numbers. As a general example and not by way of limitation, isotopes of hydrogen include tritium and deuterium, and isotopes of carbon include 13 C and 14 C.

[0438] It should be appreciated that the specific anion or cation forming part of any salt form of the cationic ionizable lipids provided herein is not critical, so long as the salt as a whole is pharmacologically acceptable. Other examples of pharmaceutically acceptable salts and methods for their preparation and use are presented in Handbook of Pharmaceutical Salts: Properties, and Use (2002), which is incorporated herein by reference.

[0439] In some embodiments, the cationic lipid comprises about 23 mol% to about 83 mol% of the total lipids present in the composition.

[0440] In some embodiments, the cationic lipid comprises about 25 mol% to about 80 mol%, e.g., about 30 mol% to about 80 mol%, about 35 mol% to about 80 mol%, about 40 mol% to about 80 mol%, about 45 mol% to about 80 mol%, about 50 mol% to about 80 mol%, about 55 mol% to about 80 mol%, about 60 mol% to about 80 mol%, about 65 mol% to about 80 mol%, about 70 mol% to about 80 mol%, or about 75 mol% to about 80 mol% of the total lipids present in the composition.

[0441] In some embodiments, the cationic lipid comprises about 25 mol% to about 70 mol%, e.g., about 30 mol% to about 70 mol%, about 35 mol% to about 70 mol%, about 40 mol% to about 70 mol%, about 45 mol% to about 70 mol%, about 50 mol% to about 70 mol%, about 55 mol% to about 70 mol%, about 60 mol% to about 70 mol%, or about 65 mol% to about 70 mol% of the total lipids present in the composition.

[0442] In some embodiments, the cationic lipid comprises about 25 mol% to about 60 mol%, e.g., about 30 mol% to about 60 mol%, about 35 mol% to about 60 mol%, about 40 mol% to about 60 mol%, about 45 mol% to about 60 mol%, about 50 mol% to about 60 mol%, or about 55 mol% to about 60 mol% of the total lipids present in the composition.

[0443] In some embodiments, the cationic lipid comprises about 25 mol% to about 50 mol%, e.g., about 30 mol% to about 50 mol%, about 35 mol% to about 50 mol%, about 40 mol% to about 50 mol%, or about 45 mol% to about 50 mol% of the total lipids present in the composition.

[0444] In some embodiments, the cationic lipid comprises about 23 mol%, 25 mol%, about 30 mol%, about 35 mol%, about 40 mol%, about 45 mol%, about 50 mol%, about 55 mol%, about 60 mol%, about 65 mol%, about 70 mol%, about 75 mol%, about 80 mol%, or about 83 mol% of the total lipids present in the composition.

[0445] It will be clear to those skilled in the art that, depending on the intended application of the particles, the proportions of the components of the composition may vary and the delivery efficiency of a particular formulation may be measured using, for example, endosomal release parameter (ERP) assays and / or in vivo gene transfection efficiency (e.g., in the case of nucleic acid loading) assays.

[0446] Non-cationic / helper lipids

[0447] In some embodiments of the compositions comprising polymer-lipids of the present invention, the non-cationic lipid comprises at least one selected from the group consisting of anionic lipids, zwitterionic lipids, and neutral lipids, preferably, the non-cationic lipid comprises a neutral lipid. As used herein, the phrase "non-cationic lipid" refers to any neutral, zwitterionic, or anionic lipid. As used herein, the phrase "anionic lipid" refers to any of a variety of lipid species that carry a net negative charge at a selected pH, such as physiological pH.

[0448] In some embodiments, the neutral lipids comprise 19 mol%-75 mol%, e.g., 25 mol%-70 mol%, 30 mol%-70 mol%, 35 mol%-70 mol%, 40 mol%-70 mol%, 45 mol%-70 mol%, 50 mol%-65 mol%, or 55 mol%-60 mol%, of the total lipids present in the composition.

[0449] In some embodiments, the neutral lipids comprise 20 mol%-65 mol%, e.g., 25 mol%-65 mol%, 30 mol%-65 mol%, 35 mol%-65 mol%, 40 mol%-65 mol%, 45 mol%-65 mol%, 50 mol%-65 mol%, or 55 mol%-65 mol%, of the total lipids present in the composition.

[0450] In some embodiments, the neutral lipids comprise 20 mol%-60 mol%, e.g., 25 mol%-60 mol%, 30 mol%-60 mol%, 35 mol%-60 mol%, 40 mol%-60 mol%, 45 mol%-60 mol%, 50 mol%-60 mol%, or 55 mol%-60 mol%, of the total lipids present in the composition.

[0451] In some embodiments, the neutral lipids comprise 20 mol%-55 mol%, e.g., 25 mol%-55 mol%, 30 mol%-55 mol%, 35 mol%-55 mol%, 40 mol%-55 mol%, 45 mol%-55 mol%, 50 mol%-55 mol%, or 20 mol%-50 mol%, e.g., 25 mol%-50 mol%, 30 mol%-50 mol%, 35 mol%-50 mol%, 40 mol%-50 mol%, or 45 mol%-50 mol%, of the total lipids present in the composition.

[0452] In some embodiments, the neutral lipids comprise 20 mol%-45 mol%, e.g., 25 mol%-45 mol%, 30 mol%-45 mol%, 35 mol%-45 mol%, or 40 mol%-45 mol%, or 20 mol%-40 mol%, e.g., 25 mol%-40 mol%, 30 mol%-40 mol%, or 35 mol%-40 mol%, of the total lipids present in the composition.

[0453] In some embodiments, the neutral lipid comprises:

[0454] Cholesterol or cholesterol-derived neutral lipids;

[0455] phospholipids; or

[0456] A mixture of cholesterol or cholesterol-derived neutral lipids and phospholipids.

[0457] In some embodiments, the cholesterol comprises 14 mol%-70 mol%, e.g., 20 mol%-70 mol%, 25 mol%-70 mol%, 30 mol%-70 mol%, 35 mol%-70 mol%, 40 mol%-70 mol%, 15 mol%-60 mol%, 20 mol%-60 mol%, 25 mol%-60 mol%, 30 mol%-60 mol%, 35 mol%-60 mol%, 40 mol%-60 mol%, 15 mol%-50 mol%, 20 mol%-50 mol%, 25 mol%-50 mol%, 30 mol%-50 mol%, 35 mol%-50 mol%, 15 mol%-40 mol%, 20 mol%-40 mol%, 25 mol%-40 mol%, 30 mol%-40 mol%, or 35 mol%-40 mol%.

[0458] In some embodiments, the phospholipids comprise about 5 mol% to about 75 mol% of the total lipids in the composition, e.g., 5 mol% to 70 mol%, 8 mol% to 65 mol%, 10 mol% to 60 mol%, 10 mol% to 50 mol%, 10 mol% to 40 mol%, 10 mol% to 30 mol%, 10 mol% to 20 mol%, 15 mol% to 55 mol%, 15 mol% to 50 mol%, 15 mol% to 45 mol%, 1 %-40mol%, 15mol%-35mol%, 15mol%-30mol%, 15mol%-25mol%, 20mol%-45mol%, 20mol%-45mol%, 20mol%-40mol%, 20mol%-35mol%, 20mol%-30mol%, 20mol%-25mol%, 25mol%-40mol%, 25mol%-35mol% or 25mol%-30mol%.

[0459] In some embodiments of the composition of the present invention, the phospholipid comprises at least one selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine, lysophosphatidylcholine, lysophosphatidylethanolamine, phosphatidylserine, dioleoylphosphatidylserine (DOPS), phosphatidylinositol, sphingomyelin, egg yolk sphingomyelin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebroside, dicetyl phosphate, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylethanolamine (DOPE), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleoyl-phosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleoyl-phosphatidylcholine (DPPC), dioleoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dioleoylphosphatidylglycerol (DPPG), palmitoyloleoylphosphatidylcholine (DPPC), di ... phosphatidylethanolamine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), palmitoyloleoylphosphatidylglycerol (POPG), dioleoylphosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), distearoylphosphatidylethanolamine (DSPE), monomethylphosphatidylethanolamine, dimethylphosphatidylethanolamine, di-elaidoylphosphatidylethanolamine (DEPE), stearoyloleoylphosphatidylethanolamine (SOPE), lysophosphatidylcholine, egg yolk phosphatidylcholine (EPC), and dilinoleoylphosphatidylcholine. 1,2-Dipalmitoyl-sn-glycero-3-O-4'-(N,N,N-trimethyl)-homoserine (DGTS), monogalactosyldiacylglycerol (MGDG), diacetyldiacylglycerol (DGDG), sulfaquinolinediacylglycerol (SQDG), 1-palmitoyl-2-cis-9,10-methylenehexyl-decanoyl-sn-glycero-3-phosphocholine (Cyclo PC), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE and their derivatives.

[0460] In some embodiments, the compositions and methods of the invention include a non-cationic lipid having the formula:

[0461] Wherein R1 and R2 are each independently C8-C 24 Alkyl, C8-C 24 alkenyl or a substituted form of any group; R3, R3' and R3" are each independently alkyl (C≤6) or substituted alkyl (C≤6) ; and X ‐ It is a monovalent anion.

[0462] In some embodiments, R1 is C8-C 24 Alkenyl or substituted C8-C 24 In some embodiments, R2 is C8-C 24Alkenyl or substituted C8-C 24 In other embodiments, R1 is C8-C 24 Alkyl or substituted C8-C 24 In other embodiments, R2 is C8-C 24 Alkyl or substituted C8-C 24 In some embodiments, both R1 and R2 are the same.

[0463] In some embodiments, R3, R3′, and R3″ are each the same. In some embodiments, R3, R3′, and R3″ are each methyl. In some embodiments, X ‐ is a halide anion such as bromide or chloride.

[0464] In some embodiments, the compositions and methods of the present invention include a phosphoglyceride or a salt thereof of the formula:

[0465] Among them, R 4 is a straight-chain alkyl group having 10 to 24 carbon atoms or a straight-chain alkenyl group having 1 to 3 double bonds and 10 to 24 carbon atoms;

[0466] R 5 is a straight-chain alkyl group having 10 to 24 carbon atoms or a straight-chain alkenyl group having 1 to 3 double bonds and 10 to 24 carbon atoms;

[0467] In some embodiments, the compositions and methods of the invention include an anionic lipid of the formula:

[0468] Wherein R1 and R2 are each independently an alkyl (C8‐C24) , alkenyl (C8‐C24) or a substituted form of any group; R3 is hydrogen, alkyl (C≤6) or substituted alkyl (C≤6) or -Y1-R4, wherein: Y1 is an alkanediyl (C≤6) or substituted alkanediyl (C≤6) ; and R4 is an acyloxy group (C≤8‐24) or substituted acyloxy (C≤8‐24) .

[0469] Other examples of non-cationic lipids suitable for use in the present invention include lipids that do not contain phosphorus, such as stearamide, dodecylamine, hexadecylamine, acetyl palmitate, glyceryl ricinoleate, hexadecyl stereate, isopropyl myristate, amphoteric acrylic acid polymers, triethanolamine-lauryl sulfate, alkyl-aryl sulfate polyethoxylated fatty acid amides, dioctadecyl dimethyl bromide, ceramide, sphingomyelin and derivatives thereof, and the like.

[0470] In some embodiments of the composition of the present invention, the cholesterol-derived neutral lipid comprises at least one selected from the group consisting of cholestanol, cholestanone, cholestenone, coprostanol, cholesteryl-2'-hydroxyethyl ether, cholesteryl-4'-hydroxybutyl ether, BHEM-cholesterol, β-sitosterol, 20α-hydroxycholesterol, polypeptide / protein covalently modified cholesterol, and derivatives thereof, wherein the synthesis of cholesteryl-2'-hydroxyethyl ether is described in U.S. Patent No. 8,058,069, the disclosure of which is incorporated herein by reference in its entirety for all purposes. Preferably, the cholesterol-derived lipid comprises β-sitosterol.

[0471] In some embodiments, the compositions and methods of the present invention comprise a steroid having a ring structure comprising three fused cyclohexyl rings and a fused cyclopentyl ring, as shown in the following formula:

[0472] In some embodiments, the steroid derivative comprises the above ring structure with one or more non-alkyl substitutions. In some embodiments, the steroid or steroid derivative is a sterol, wherein the formula is further defined as:

[0473] In some embodiments, the steroid or steroid derivative of the compositions and methods of the present invention is cholestane or a cholestane derivative. In cholestane, the ring structure is further defined by the formula:

[0474] As described above, cholestane derivatives include non-alkyl substitutions of one or more of the above-mentioned ring systems. In some embodiments, the cholestane or cholestane derivative is cholestene or a cholestene derivative or a sterol or a sterol derivative. In other embodiments, the cholestane or cholestane derivative is cholestene and a sterol or a derivative thereof.

[0475] In some embodiments of the polymer-lipid composition of the present invention, the composition further comprises a lipid conjugate, wherein the lipid conjugate comprises at least one selected from the group consisting of: poly(ethylene glycol)-lipid conjugate (PEG-lipid conjugate or PEG-lipid), ATTA-lipid conjugate, polysarcosine-lipid conjugate, polypeptide / protein-lipid conjugate and cation-polymer-lipid conjugate (CPL), preferably, the lipid conjugate comprises a PEG-lipid conjugate.

[0476] In a preferred embodiment, the lipid conjugate is a PEG-lipid. Examples of PEG-lipids include, but are not limited to, PEG (PEG-DAA) coupled to a dialkoxypropyl group as described in, for example, PCT Publication No. WO 05 / 026372, PEG (PEG-DAG) coupled to diacylglycerol as described in, for example, U.S. Patent Publication Nos. 20030077829 and 2005008689, PEG (PEG-PE) coupled to phospholipids such as phosphatidylethanolamine, PEG coupled to ceramide as described in, for example, U.S. Patent No. 5,885,613, PEG conjugated to cholesterol or its derivatives, and mixtures thereof. The disclosures of these patent documents are incorporated herein by reference in their entirety for all purposes. Additional PEG-lipids include, but are not limited to, PEG-C-DOMG, DMG-PEG2000 ((R)-2,3-bis(myristoyloxy)propyl-1-(methoxy poly(ethylene glycol)2000)carbamate, DMG-PEG2K), ALC-0159 (2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide; 2-[(polyethylene glycol)-2000]-N,N-didecyl acetamide), and mixtures thereof. The PEG-lipids described herein can be synthesized as described in International Patent Application PCT / US2016 / 000129. In some embodiments, the PEG-lipids useful in the present invention may be the PEGylated lipids described in International Patent Application WO2012099755, the disclosures of which are incorporated herein by reference in their entirety for all purposes.

[0477] PEG is a linear, water-soluble polymer of ethylene PEG repeating units with two terminal hydroxyl groups. PEG is classified by its molecular weight; for example, PEG2000 (PEG2K) has an average molecular weight of about 2,000 daltons, and PEG5000 (PEG5K) has an average molecular weight of about 5,000 daltons. PEG is commercially available from Sigma-Aldrich Chemical Co. and other companies and includes, for example, the following: monomethoxy polyethylene glycol (MePEG-OH), monomethoxy polyethylene glycol-succinate (MePEG-S), monomethoxy polyethylene glycol-succinimidyl succinate (MePEG-S-NHS), monomethoxy polyethylene glycol-amine (MePEG-NH2), monomethoxy polyethylene glycol-trifluoroethanesulfonate (MePEG-TRES), and monomethoxy polyethylene glycol-imidazolyl-carboxyl (MePEG-IM). Other PEGs such as those described in U.S. Patent Nos. 6,774,180 and 7,053,150 (e.g., mPEG (20 kDa) amine) are also effective for preparing the PEG-lipid conjugates of the present invention. The disclosures of these patents are incorporated herein by reference in their entirety for all purposes. In addition, monomethoxypolyethylene glycol-acetic acid (MePEG-CH2COOH) is particularly effective for preparing PEG-lipid conjugates including, for example, PEG-DAA conjugates.

[0478] The PEG moiety of the PEG-lipid conjugates described herein can include an average molecular weight in the range of about 550 daltons to about 10,000 daltons. In certain instances, the PEG moiety has an average molecular weight of about 750 daltons to about 5,000 daltons (e.g., about 1,000 daltons to about 5,000 daltons, about 1,500 daltons to about 3,000 daltons, about 750 daltons to about 3,000 daltons, about 750 daltons to about 2,000 daltons, etc.). In preferred embodiments, the PEG moiety has an average molecular weight of about 5,000 daltons, or about 2,000 daltons, or about 750 daltons.

[0479] In some instances, PEG can be optionally substituted with an alkyl, alkoxy, acyl, or aryl group. PEG can be directly conjugated to the lipid or can be connected to the lipid via a linker moiety. Any linker moiety suitable for coupling PEG to the lipid can be used, including, for example, a linker moiety that does not contain an ester and a linker moiety that contains an ester. In a preferred embodiment, the linker moiety is a linker moiety that does not contain an ester. As used herein, the term "linker moiety that does not contain an ester" refers to a linker moiety that does not contain a carboxylate bond (-OC(O)-). Suitable non-ester containing linker moieties include, but are not limited to, amine (-C(O)NH-), amino (-NR-), carboxyl (-C(O)-), carbamate (-NHC(O)O-), urea (-NHC(O)NH-), disulfide (-SS-), ether (-O-), succinyl (-(O)CCH2CH2C(O)-), succinyl (-NHC(O)CH2CH2C(O)-), and combinations thereof (such as linkers comprising both a carbamate linker moiety and an amine linker moiety). In a preferred embodiment, a carbamate linker is used to couple the PEG and lipid.

[0480] In other embodiments, an ester-containing linker moiety is used to couple the PEG and lipid. Suitable ester-containing linker moieties include, for example, carbonate (—OC(O)O—), succinyl, phosphate (—O—(O)POH—O—), sulfonate, and combinations thereof.

[0481] Phosphatidylethanolamines with various acyl chain groups of varying chain lengths and degrees of saturation can be conjugated to PEG to form lipid conjugates. Such phosphatidylethanolamines are commercially available or can be isolated or synthesized using conventional techniques known to those skilled in the art. 10 -C 20 Phosphatidylethanolamines containing saturated or unsaturated fatty acids can be used. Phosphatidylethanolamines with mono- or di-unsaturated fatty acids and mixtures of saturated and unsaturated fatty acids can also be used. Suitable phosphatidylethanolamines include, but are not limited to, dimyristoyl-phosphatidylethanolamine (DMPE), dipalmitoyl-phosphatidylethanolamine (DPPE), dioleoylphosphatidylethanolamine (DOPE), and distearoyl-phosphatidylethanolamine (DSPE).

[0482] In some embodiments, the PEG-lipid conjugate of the compositions and methods of the present invention comprises a PEGylated phosphoglyceride of the formula:

[0483] in,

[0484] p is an integer from 5 to 200, preferably from 10 to 170, and most preferably from 10 to 140;

[0485] R 6 is a straight-chain alkyl group having 10 to 20 carbon atoms or a straight-chain alkenyl group having 1 to 3 double bonds and 10 to 20 carbon atoms;

[0486] R 7 is a straight-chain alkyl group having 10 to 20 carbon atoms or a straight-chain alkenyl group having 1 to 3 double bonds and 10 to 20 carbon atoms;

[0487] In some embodiments, the compositions and methods of the present invention, the PEG-lipid conjugate has the formula:

[0488] in,

[0489] R 12 and R 13 are each independently an alkyl (C≤24) , alkenyl (C≤24) or a substituted form of any of these groups; R e Is hydrogen, alkyl (C≤8) or substituted alkyl (C≤8) ; and x is 1-250. In some embodiments, R e is an alkyl group (C≤8) Such as methyl. 12 and R 13 are each independently an alkyl (C≤4‐20) In some embodiments, x is 5-250. In one embodiment, x is 5-125 or x is 100-250. In some embodiments, the PEG lipid is 1,2-dimyristoyl-sn-glycerol, methoxypolyethylene glycol.

[0490] In some embodiments, the lipid conjugate of the compositions and methods of the present invention has the formula:

[0491] in,

[0492] n1 is an integer between 1 and 100, and n2 and n3 are each independently selected from an integer between 1 and 29. In some embodiments, n1 is 5, 10, 15, 20, 25, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100, or any range derivable therein. In some embodiments, n1 is about 30 to about 50. In some embodiments, n2 is 5 to 23. In some embodiments, n2 is 11 to about 17. In some embodiments, n3 is 5 to 23. In some embodiments, n3 is 11 to about 17.

[0493] The term "ATTA" or "polyamide" refers to, but is not limited to, the compounds described in U.S. Patent Nos. 6,320,017 and 6,586,559, the disclosures of which are incorporated herein by reference in their entirety for all purposes. These compounds include compounds having the formula:

[0494] wherein R is a member selected from the group consisting of hydrogen, alkyl and acyl; R 1 is a member selected from the group consisting of hydrogen and alkyl; or optionally, R and R 1 and the nitrogen to which they are attached form an azide moiety; R 2 is a member selected from the group consisting of hydrogen, optionally substituted alkyl, optionally substituted aryl, and an amino acid side chain; R 3 is selected from hydrogen, halogen, hydroxy, alkoxy, thiol, hydrazine, amino and NR 4 R 5 The members of the group, where R 4 and R 5 is independently hydrogen or alkyl; n is 4-80; m is 2-6; p is 1-4; and q is 0 or 1. It will be apparent to those skilled in the art that other polyamides may be used in the compounds of the present invention.

[0495] The term "diacylglycerol" refers to a fatty acid having two acyl chains, R 1 and R 2 The compound, said R 1 and R 2 Both independently have 2-30 carbon atoms, which are bound to the 1- and 2-positions of glycerol via ester bonds. The acyl group may be saturated or have varying degrees of unsaturation. Suitable acyl groups include, but are not limited to, lauryl (C 12 ), myristyl (C 14 ), palmityl (C 16 ), stearyl (C 18 ), and eicosyl (C20 ). In a preferred embodiment, R 1 and R 2 Same, that is, R 1 and R 2 are myristyl (i.e., dimyristyl), R 1 and R 2 are all stearyl (i.e., distearyl), etc. Diacylglycerol has the following general formula:

[0496] The term "dialkoxypropyl" refers to a group having two alkyl chains, R 1 and R 2 The compound, said R 1 and R 2 Both independently have 2-30 carbon atoms. The alkyl group can be saturated or have varying degrees of unsaturation. The dialkoxypropyl group has the following general formula:

[0497] In a preferred embodiment, the PEG-lipid is a PEG-DAA conjugate having the following general formula:

[0498] where R 1 and R 2 are independently selected and are long chain alkyl groups having from about 10 to about 22 carbon atoms; PEG is polyethylene glycol; and L is a non-ester containing linker moiety or an ester containing linker moiety as described above. The long chain alkyl group may be saturated or unsaturated. Suitable alkyl groups include, but are not limited to, lauryl (C 12 ), myristyl (C 14 ), palmityl (C 16 ), stearyl (C 18 ), and eicosyl (C 20 ). In a preferred embodiment, R 1 and R 2 Same, that is, R 1 and R 2 are myristyl (i.e., dimyristyl), R 1 and R 2 are all stearyl (ie, distearyl), etc.

[0499] In the above formula, PEG has an average molecular weight within the range of about 550 daltons to about 10,000 daltons. In some instances, PEG has an average molecular weight of about 750 daltons to about 5,000 daltons (e.g., about 1,000 daltons to about 5,000 daltons, about 1,500 daltons to about 3,000 daltons, about 750 daltons to about 3,000 daltons, about 750 daltons to about 2,000 daltons, etc.). In preferred embodiments, PEG has an average molecular weight of about 2,000 daltons or about 750 daltons. PEG can optionally be substituted with an alkyl, alkoxy, acyl or aryl group. In certain embodiments, the terminal hydroxyl group is substituted with an ethoxy or methyl group.

[0500] In a preferred embodiment, "L" is a non-ester containing linker moiety. Suitable non-ester containing linkers include, but are not limited to, amine linker moieties, amino linker moieties, carboxyl linker moieties, carbamate linker moieties, urea linker moieties, ether linker moieties, disulfide linker moieties, succinyl linker moieties, and combinations thereof. In a preferred embodiment, the non-ester containing linker moiety is a carbamate linker moiety (i.e., a PEG-C-DAA conjugate). In another preferred embodiment, the non-ester containing linker moiety is an amine linker moiety (i.e., a PEG-A-DAA conjugate). In another preferred embodiment, the non-ester containing linker moiety is a succinyl linker moiety (i.e., a PEG-S-DAA conjugate).

[0501] In particular embodiments, the PEG-lipid conjugate is selected from:

[0502] PEG-DAA conjugates are synthesized using standard techniques and reagents known to those skilled in the art. It will be appreciated that PEG-DAA conjugates may contain a variety of amide, amine, ether, thio, carbamate, and urea bonds. It will be appreciated that methods and reagents for forming these bonds are well known and readily available. See, for example, March, ADVANCED ORGANIC CHEMISTRY (Wiley 1992); Larock, COMPREHENSIVE ORGANIC TRANSFORMATIONS (VCH 1989); and Furniss, VOGEL'S TEXTBOOK OF PRACTICAL ORGANIC CHEMISTRY, 5th ed. (Longman 1989). It will also be appreciated that any functional groups present may require protection and deprotection at various points in the synthesis of the PEG-DAA conjugate. It will be appreciated that such techniques are well known. See, for example, Green and Wuts, PROTECTIVE GROUPS IN ORGANIC SYNTHESIS (Wiley 1991).

[0503] In some embodiments, the PEG-DAA conjugate is dilauryloxypropyl (C 12 )-PEG conjugate, dimyristyloxypropyl (C 14 )-PEG conjugate, dipalmityloxypropyl (C 16 )-PEG conjugate, or distearyloxypropyl (C 18 Those skilled in the art will readily appreciate that other dialkoxypropyl groups can be used in the PEG-DAA conjugates of the present invention.

[0504] In addition to the foregoing, it should be readily apparent to those skilled in the art that other hydrophilic polymers can be used in place of PEG. Examples of suitable polymers that can be used in place of PEG include, but are not limited to, polyvinylpyrrolidone, polymethyloxazoline, polyethyloxazoline, polyhydroxypropylmethacrylamide, polymethacrylamide and polydimethylacrylamide, polylactic acid, polyglycolic acid, poloxamers, poloxamines, derivatized celluloses such as hydroxymethylcellulose or carboxyethylcellulose, polysarcosine-lipid conjugates, and conjugates of polysarcosine with lipid-like substances. The polysarcosine-lipid conjugates or conjugates of polysarcosine with lipid-like substances can be selected from the group consisting of polysarcosine-diacylglycerol conjugates, polysarcosine-dialkoxypropyl conjugates, polysarcosine-phospholipid conjugates, polysarcosine-ceramide conjugates, and mixtures thereof. Suitable polysarcosine-lipid conjugates or conjugates of polysarcosine with lipid-like substances for use in the present invention, and methods of making and using polysarcosine-lipid conjugates or conjugates of polysarcosine with lipid-like substances, are disclosed, for example, in U.S. Patent No. 17 / 281,697 and PCT Publication No. PCT / EP2019 / 076369, the disclosures of which are incorporated herein by reference in their entireties for all purposes. In some embodiments, suitable polysarcosine-lipid conjugates for use in the compositions and methods of the present invention include polysarcosine lipids as described in International Patent Publication No. WO2020070040, which is incorporated herein by reference.

[0505] In addition to the aforementioned components, the polymer-lipid compositions of the present invention (e.g., PoLixNano) may also include a cation-polymer-lipid conjugate (CPL) (see, e.g., Chen et al., Bioconj. Chem., 11: 433-437 (2000)). Suitable PoLixNano-CPLs for use in the present invention, and methods for preparing and using PoLixNano-CPLs are disclosed in, e.g., U.S. Pat. No. 6,852,334 and PCT Publication No. WO 00 / 62813, the disclosures of which are incorporated herein by reference in their entirety for all purposes.

[0506] In some instances, the polycationic moiety may have a ligand attached, such as a targeting ligand or a chelating moiety for complexing calcium. Preferably, the cationic moiety maintains a positive charge after the ligand is attached. In some instances, the attached ligand has a positive charge. Suitable ligands include, but are not limited to, compounds or devices with reactive functional groups and include lipids, amphipathic lipids, carrier compounds, bioaffinity compounds, biomaterials, biopolymers, biomedical devices, compounds that can be analyzed and detected, therapeutically active compounds, enzymes, peptides, proteins, antibodies, immunostimulants, radiolabels, fluorophores, biotin, drugs, haptens, DNA, RNA, polysaccharides, liposomes, virions, micelles, immunoglobulins, functional groups, other targeting moieties, or toxins.

[0507] In some embodiments, the present invention also contemplates the use of PEG-modified phospholipids and derivatized lipids, such as derivatized ceramides (PEG-CERs), including N-octanoyl-sphingosine-l-[succinyl(methoxypolyethylene glycol)-2000] (C8PEG-2000 ceramide). Contemplated PEG-modified lipids include, but are not limited to, polyethylene glycol chains of up to 2 kDa, up to 3 kDa, up to 4 kDa, or up to 5 kDa in length, covalently linked to a C6-C 20 In some embodiments, the PEG-modified or PEGylated lipid is PEGylated cholesterol. In some embodiments, the PEG-modified or PEGylated lipid is 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-PEG (DMPE-PEG), wherein the PEG portion contains 10 to 140 repeating units, more preferably 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-PEG2000 (DMPE-PEG2K). The addition of such components can prevent complex aggregation and can also provide a method for increasing circulation life and increasing the delivery of compositions comprising polymers-lipids to target tissues (Klibanov et al. (1990) FEBS Letters, 268 (1): 235-237), or it can be selected as a rapid replacement formulation in vivo (see U.S. Patent No. 5,885,613). Particularly useful exchangeable lipids are those with shorter acyl chains (e.g., C 14 or C 18 Suitable PEG-ceramides for use in the compositions and methods of the present invention include PEG lipids as described in International Patent Publication No. WO 2020061295 and U.S. Patent Publication No. US 20220016029, which are incorporated herein by reference.

[0508] In some embodiments, the lipid conjugates of the present invention (e.g., PEG lipids) can improve their targeting characteristics by chemical structural modification, such as glycosylation modification, protein targeting ligand modification, antibody modification, polypeptide modification, folic acid modification, growth factor modification, cytokine modification, vitamin modification, and integrin modification. Taking glycosylation modification as an example, the glycosylated PEG lipid comprises at least one terminal block conjugated to a glycosyl moiety, preferably a terminal hydrophilic block, and the glycosyl moiety can be conjugated to the PEG lipid of the present invention by a covalent bond formed between a functional group of the glycosyl moiety and a functional group of the PEG lipid. For example, commercial distearoylphosphatidylethanolamine-polyethylene glycol 2000 (polyethylene glycol 2000)-mannose (mannose) (DSPE-PEG2K-Mannose), the covalent bond can be formed by a reaction between two functional groups that are modified to be reactive, and the glycosyl moiety can be directly conjugated to the PEG lipid. Alternatively, the glycosyl moiety can be conjugated to the PEG lipid through a spacer.

[0509] In some embodiments, the lipid conjugates of the present invention may have a ligand attached. Suitable ligands include, but are not limited to, compounds or devices having reactive functional groups and include lipids, carrier compounds, bioaffinity compounds, biomaterials, biopolymers, biomedical devices, analytically detectable compounds, therapeutically active compounds, enzymes, immunostimulants, radiolabels, fluorophores, biotin, drugs, haptens, DNA, RNA, polysaccharides, liposomes, virosomes, micelles, immunoglobulins, functional groups, toxins, or other targeting moieties.

[0510] In some embodiments, the lipid conjugate comprises 0.1 mol%-10.0 mol%, e.g., 0.1 mol%-10.0 mol%, 1 mol%-10.0 mol%, 2 mol%-10.0 mol%, 3 mol%-10.0 mol%, 5 mol%-10.0 mol%, 0.1 mol%-5 mol%, 1 mol%-5 mol%, 2 mol%-5 mol%, or 3 mol%-5.0 mol% of the total lipids in the composition.

[0511] In some embodiments, the compositions comprising polymer-lipids as described herein contain less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2% or less than 0.1% of the lipid conjugates or PEG modified by mole or weight of total lipids. In some embodiments, the compositions comprising polymer-lipids as described herein contain 0.4% or less of PEG-modified lipids or PEG, 0.3% or less of PEG-modified lipids or PEG, 0.2% or less of PEG-modified lipids or PEG, or 0.1% or less of PEG-modified lipids or PEG modified by mole or weight of total lipids. In some embodiments, the compositions comprising polymer-lipids as described herein contain 0.01% or less of PEG-modified lipids or PEG modified by mole or weight of total lipids. In some embodiments, the compositions of polymer-lipids as described herein do not comprise lipid conjugates (e.g., PEG-lipids) or PEG.

[0512] It will be understood by those skilled in the art that the concentration of the lipid conjugate may vary depending on the rate at which the lipid conjugate and the polymer-lipid composition used form fusions.

[0513] By controlling the composition and concentration of the lipid conjugate, one can control the rate at which the lipid conjugate is exchanged from the polymer-lipid composition, and consequently the rate at which the nucleic acid / polymer-lipid composition forms a fusion. For example, when a PEG-phosphatidylethanolamine conjugate or a PEG-ceramide conjugate is used as the lipid conjugate, the rate at which the nucleic acid / polymer-lipid composition forms a fusion can be varied, for example, by changing the lipid conjugate concentration, by changing the PEG molecular weight, or by changing the chain length and degree of saturation of the acyl chain groups on the phosphatidylethanolamine or ceramide. In addition, other variables include, for example, pH, temperature, ionic strength, etc., which can be used to change and / or control the rate at which the nucleic acid / polymer-lipid composition forms a fusion. Other methods that can be used to control the rate at which the nucleic acid / polymer-lipid composition forms a fusion will be apparent to those skilled in the art upon reading the present disclosure.

[0514] In some embodiments of the polymer-lipid composition of the present invention, the PEG-lipid conjugate comprises at least one selected from the group consisting of DMG-PEG2K, DMPE-PEG2K, DSPE-PEG2K, DSPE-PEG2K-Mannose, DMG-PEG5K, DMPE-PEG5K, DSPE-PEG5K-Mannose, and DSPE-PEG5K.

[0515] Composition

[0516] In some embodiments of the compositions of the present invention, the compositions comprise:

[0517] (1) an amphiphilic block copolymer, a cationic lipid, a phospholipid, cholesterol, and a lipid conjugate, such as a PEG-lipid conjugate, wherein the cationic lipid accounts for 30.0 mol%-80.0 mol% of the total lipids present in the composition, the phospholipid accounts for 5.0 mol%-50.0 mol% of the total lipids, the cholesterol accounts for 14.0 mol%-64.0 mol% of the total lipids, the lipid conjugate accounts for 0.1 mol%-8.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 0.1%-95.0% by weight of the composition;

[0518] (2) an amphiphilic block copolymer, a cationic lipid, a phospholipid, cholesterol, and a lipid conjugate, such as a PEG-lipid conjugate, wherein the cationic lipid accounts for 23.0 mol% to 75.0 mol% of the total lipids present in the composition, the phospholipid accounts for 10.0 mol% to 62.0 mol% of the total lipids, the cholesterol accounts for 14.0 mol% to 46.0 mol% of the total lipids, the lipid conjugate accounts for 0.1 mol% to 8.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 0.1% to 95.0% by weight of the composition;

[0519] (3) an amphiphilic block copolymer, a cholesterol-derived cationic lipid, a phospholipid, and a lipid conjugate, such as a PEG-lipid conjugate, wherein the cholesterol-derived cationic lipid comprises 29.0 mol% to 80.0 mol% of the total lipids present in the composition, the phospholipids comprise 19.0 mol% to 70.0 mol% of the total lipids, the lipid conjugates comprise 0.1 mol% to 8.0 mol% of the total lipids, and the amphiphilic block copolymer comprises 0.1% to 95.0% by weight of the composition;

[0520] (4) an amphiphilic block copolymer, a cationic lipid, cholesterol, and a lipid conjugate, such as a PEG-lipid conjugate, wherein the cationic lipid accounts for 25.0 mol% to 80.0 mol% of the total lipids present in the composition, the cholesterol accounts for 15.0 mol% to 50.0 mol% of the total lipids, the lipid conjugate accounts for 0.1 mol% to 8.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 0.1% to 95.0% by weight of the composition;

[0521] (5) amphiphilic block copolymers, cationic lipids, phospholipids, and lipid conjugates such as PEG-lipid conjugates, wherein the cationic lipids account for 30.0 mol% to 80.0 mol% of the total lipids present in the composition, the phospholipids account for 10.0 mol% to 50.0 mol% of the total lipids, the lipid conjugates account for 0.1 mol% to 8.0 mol% of the total lipids, and the amphiphilic block copolymers account for 0.1% to 95.0% by weight of the composition;

[0522] (6) an amphiphilic block copolymer, a cationic lipid, a phospholipid, and cholesterol, wherein the cationic lipid accounts for 30.0 mol% to 80.0 mol% of the total lipids present in the composition, the phospholipids account for 5.0 mol% to 50.0 mol% of the total lipids, the cholesterol accounts for 15.0 mol% to 50.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 0.1% to 95.0% by weight of the composition; or

[0523] (7) an amphiphilic block copolymer, a cholesterol-derived cationic lipid, and a phospholipid, wherein the cholesterol-derived cationic lipid accounts for 30.0 mol%-70.0 mol% of the total lipids present in the composition, the phospholipids account for 30.0 mol%-70.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 0.1%-95.0% by weight of the composition.

[0524] In some embodiments of the compositions of the present invention, the compositions comprise an amphiphilic block copolymer and the following components:

[0525] (1) DLin-MC3-DMA, ALC-0315, SM-102, C12-200, cKK-E12, HGT5000 or HGT5001; DSPC, DPPC, DOPS, SOPE, DOPG, DSPE, ESM or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K, DMG-PEG5K or DSPE-PEG2K-Mannose, wherein the DLin-MC3-DMA, ALC-0315, SM-102, C12-200, cKK-E12, HGT5000 or HGT5001 The amphiphilic block copolymer accounts for 40.0 mol% to 70.0 mol% of the total lipids present in the composition, DSPC, DPPC, DOPS, SOPE, DOPG, DSPE, ESM or DOPE accounts for 8.0 mol% to 39.0 mol% of the total lipids, cholesterol or β-sitosterol accounts for 20.0 mol% to 40.0 mol% of the total lipids, DMG-PEG2K, DMG-PEG5K or DSPE-PEG2K-Mannose accounts for 0.1 mol% to 5.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 15.0% to 90.0% by weight of the composition;

[0526] (2) DLin-MC3-DMA, ALC-0315, SM-102, C12-200, cKK-E12, HGT5000 or HGT5001; DSPC, DPPC, DOPS, SOPE, DOPG, DSPE, ESM or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K, DMG-PEG5K or DSPE-PEG2K-Mannose, wherein the DLin-MC3-DMA, ALC-0315, SM-102, C12-200, cKK-E12, HGT5000 or HGT5001 1 accounts for 30 mol%-60 mol% of the total lipids present in the composition, DSPC, DPPC, DOPS, SOPE, DOPG, DSPE, ESM or DOPE accounts for 10.0 mol%-49.0 mol% of the total lipids, cholesterol or β-sitosterol accounts for 20.0 mol%-40.0 mol% of the total lipids, DMG-PEG2K, DMG-PEG5K or DSPE-PEG2K-Mannose accounts for 0.1 mol%-5.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 20.0%-90.0% by weight of the composition;

[0527] (3) DLin-MC3-DMA, ALC-0315, SM-102, C12-200, cKK-E12, HGT5000 or HGT5001; DSPC, DPPC, DOPS, SOPE, DOPG, DSPE, ESM or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K, DMG-PEG5K or DSPE-PEG2K-Mannose, wherein the DLin-MC3-DMA, ALC-0315, SM-102, C12-200, cKK-E12, HGT5000 or HGT5001 accounts for 24.0 mol%-40.0 mol% of the total lipids present in the composition, DSPC, DPPC, DOPS, SOPE, DOPG, DSPE, ESM or DOPE account for 30.0 mol%-64.0 mol% of the total lipids, cholesterol or β-sitosterol account for 15.0 mol%-40.0 mol% of the total lipids, DMG-PEG2K, DMG-PEG5K or DSPE-PEG2K-Mannose account for 0.1 mol%-5.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 20.0%-90.0% by weight of the composition;

[0528] (4) DOTAP, DODAP, DOTMA or DOSPA; DSPC, DPPC, DOPS, SOPE, DOPG, DSPE, ESM or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K, DMG-PEG5K or DSPE-PEG2K-Mannose, wherein the DOTAP, DODAP, DOTMA or DOSPA accounts for 23.0 mol%-60 mol% of the total lipids present in the composition, DSPC, DPPC, DOPS, SOPE, DOPG, DSPE, ESM or DOPE accounts for 14.0 mol%-60.0 mol% of the total lipids, cholesterol or β-sitosterol accounts for 15.0 mol%-50.0 mol% of the total lipids, DMG-PEG2K, DMG-PEG5K or DSPE-PEG2K-Mannose accounts for 0.1 mol%-5.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 30.0%-90.0% by weight of the composition;

[0529] (5) GL67, ICE, or HGT4002; DSPC, DPPC, DOPS, SOPE, DOPG, DSPE, ESM, or DOPE; and DMG-PEG2K, DMG-PEG5K, or DSPE-PEG2K-Mannose, wherein the GL67, ICE, or HGT4002 accounts for 40.0 mol% to 80.0 mol% of the total lipids present in the composition, DSPC, DPPC, DOPS, SOPE, DOPG, DSPE, ESM, or DOPE accounts for 10.0 mol% to 50.0 mol% of the total lipids, DMG-PEG2K, DMG-PEG5K, or DSPE-PEG2K-Mannose accounts for 0.1 mol% to 5.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 30.0% to 90.0% by weight of the composition;

[0530] (6) cKK-E12, DLin-MC3-DMA, ALC-0315, SM-102, C12-200, DOTAP, DODAP, DOTMA, DOSPA, HGT5000 or HGT5001; cholesterol or β-sitosterol; and DMG-PEG2K, DMG-PEG5K or DSPE-PEG2K-Mannose, wherein the cKK-E12, DLin-MC3-DMA, ALC-0315, SM-102, C12-200, DOTAP , DODAP, DOTMA, DOSPA, HGT5000 or HGT5001 account for 45.0 mol%-75.0 mol% of the total lipids present in the composition, cholesterol or β-sitosterol account for 20.0 mol%-45.0 mol% of the total lipids, DMG-PEG2K, DMG-PEG5K or DSPE-PEG2K-Mannose account for 0.1 mol%-5.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 30.0%-90.0% weight percentage of the composition;

[0531] (7) DLin-MC3-DMA, ALC-0315, SM-102, C12-200, cKK-E12, HGT5000 or HGT5001; cholesterol or β-sitosterol; and DMG-PEG2K, DMG-PEG5K or DSPE-PEG2K-Mannose, wherein the DLin-MC3-DMA, ALC-0315, SM-102, C12-200, cKK-E12, HGT5000 or HGT5001 00 or HGT5001 accounts for 30.0mol%-65.0mol% of the total lipids present in the composition, cholesterol or β-sitosterol accounts for 20.0mol%-40.0mol% of the total lipids, DMG-PEG2K, DMG-PEG5K or DSPE-PEG2K-Mannose accounts for 0.1mol%-5.0mol% of the total lipids, and the amphiphilic block copolymer accounts for 40.0%-90.0% weight percentage of the composition;

[0532] (8) DLin-MC3-DMA, ALC-0315, SM-102, C12-200, cKK-E12, HGT5000 or HGT5001; DSPC, DPPC, DOPS, SOPE, DOPG, DSPE, ESM or DOPE; and DMG-PEG2K, DMG-PEG5K or DSPE-PEG2K-Mannose, wherein the DLin- MC3-DMA, ALC-0315, SM-102, C12-200, cKK-E12, HGT5000 or HGT5001 account for 35.0 mol%-70.0 mol% of the total lipids present in the composition, DSPC, DPPC, DOPS, SOPE, DOPG, DSPE, ESM or DOPE account for 10.0 mol%-40.0 mol% of the total lipids, DMG-PEG2K, DMG-PEG5K or DSPE-PEG2K-Mannose account for 0.1 mol%-5.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 40.0%-90.0% by weight of the composition;

[0533] (9) GL67, ICE, or HGT4002; and DSPC, DPPC, DOPS, SOPE, DOPG, DSPE, ESM, or DOPE, wherein the GL67, ICE, or HGT4002 comprises 40.0 mol% to 80.0 mol% of the total lipids present in the composition, DSPC, DPPC, DOPS, SOPE, DOPG, DSPE, ESM, or DOPE comprises 10.0 mol% to 50.0 mol% of the total lipids, and the amphiphilic block copolymer comprises 40.0% to 90.0% by weight of the composition; or

[0534] (10) DLin-MC3-DMA, ALC-0315, SM-102, cKK-E12, HGT5000 or HGT5001; DSPC, DPPC, DOPS, SOPE, DOPG, DSPE, ESM or DOPE; and cholesterol or β-sitosterol, wherein the DLin-MC3-DMA, ALC-0315, SM-102, cKK-E12, HGT5000 or HGT5001 accounts for 30.0 mol%-60.0 mol% of the total lipids present in the composition, DSPC, DPPC, DOPS, SOPE, DOPG, DSPE, ESM or DOPE accounts for 20.0 mol%-45.0 mol% of the total lipids, cholesterol or β-sitosterol accounts for 20.0 mol%-45.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 30.0%-90.0% by weight of the composition.

[0535] In some embodiments of the polymer-lipid-containing compositions of the present invention, the compositions comprise the following components:

[0536] (1) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 50.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 10.0 mol% of the total lipids, cholesterol or β-sitosterol account for 38.5 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 1.5 mol% of the total lipids, and the amphiphilic block copolymer accounts for 89.9% by weight of the composition;

[0537] (2) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 50.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 10.6 mol% of the total lipids, cholesterol or β-sitosterol account for 38.5 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 0.9 mol% of the total lipids, and the amphiphilic block copolymer accounts for 72.9% by weight of the composition;

[0538] (3) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DSPE-PEG2K-Mannose, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 50.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 12.5 mol% of the total lipids, cholesterol or β-sitosterol account for 36.0 mol% of the total lipids, DMG-PEG2K or DSPE-PEG2K-Mannose account for 1.5 mol% of the total lipids, and the amphiphilic block copolymer accounts for 43.0% by weight of the composition;

[0539] (4) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DSPE-PEG2K-Mannose, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 50.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 14.5 mol% of the total lipids, cholesterol or β-sitosterol account for 34.0 mol% of the total lipids, DMG-PEG2K or DSPE-PEG2K-Mannose account for 1.5 mol% of the total lipids, and the amphiphilic block copolymer accounts for 81.8% by weight of the composition;

[0540] (5) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DSPE-PEG2K-Mannose, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 50.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 14.0 mol% of the total lipids, cholesterol or β-sitosterol account for 35.0 mol% of the total lipids, DMG-PEG2K or DSPE-PEG2K-Mannose account for 1.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 69.2% by weight of the composition;

[0541] (6) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 50.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 14.1 mol% of the total lipids, cholesterol or β-sitosterol account for 35.0 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 0.9 mol% of the total lipids, and the amphiphilic block copolymer accounts for 43.3% by weight of the composition;

[0542] (7) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 50.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 14.3 mol% of the total lipids, cholesterol or β-sitosterol account for 35.0 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 0.7 mol% of the total lipids, and the amphiphilic block copolymer accounts for 48.4% by weight of the composition;

[0543] (8) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 50.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 14.5 mol% of the total lipids, cholesterol or β-sitosterol account for 35.0 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 0.5 mol% of the total lipids, and the amphiphilic block copolymer accounts for 43.6% by weight of the composition;

[0544] (9) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 48.5 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 17.0 mol% of the total lipids, cholesterol or β-sitosterol account for 32.5 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 1.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 42.3% by weight of the composition;

[0545] (10) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 46.5 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 20.0 mol% of the total lipids, cholesterol or β-sitosterol account for 32.5 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 1.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 52.9% by weight of the composition;

[0546] (11) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DSPE-PEG2K-Mannose, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 49.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 20.0 mol% of the total lipids, cholesterol or β-sitosterol account for 30.0 mol% of the total lipids, DMG-PEG2K or DSPE-PEG2K-Mannose account for 1.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 41.9% by weight of the composition;

[0547] (12) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DSPE-PEG2K-Mannose, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 49.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 20.1 mol% of the total lipids, cholesterol or β-sitosterol account for 30.0 mol% of the total lipids, DMG-PEG2K or DSPE-PEG2K-Mannose account for 0.9 mol% of the total lipids, and the amphiphilic block copolymer accounts for 40.3% by weight of the composition;

[0548] (13) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 51.6 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 20.0 mol% of the total lipids, cholesterol or β-sitosterol account for 27.5 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 0.9 mol% of the total lipids, and the amphiphilic block copolymer accounts for 42.9% by weight of the composition;

[0549] (14) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 46.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 23.0 mol% of the total lipids, cholesterol or β-sitosterol account for 30.0 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 1.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 50.2% by weight of the composition;

[0550] (15) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DSPE-PEG2K-Mannose, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 46.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 23.0 mol% of the total lipids, cholesterol or β-sitosterol account for 30.0 mol% of the total lipids, DMG-PEG2K or DSPE-PEG2K-Mannose account for 1.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 66.1% by weight of the composition;

[0551] (16) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 46.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 23.0 mol% of the total lipids, cholesterol or β-sitosterol account for 30.0 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 1.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 74.5% by weight of the composition;

[0552] (17) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 46.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 23.0 mol% of the total lipids, cholesterol or β-sitosterol account for 30.0 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 1.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 79.6% by weight of the composition;

[0553] (18) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 46.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 23.0 mol% of the total lipids, cholesterol or β-sitosterol account for 30.0 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 1.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 83.0% by weight of the composition;

[0554] (19) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DSPE-PEG2K-Mannose, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 46.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 23.0 mol% of the total lipids, cholesterol or β-sitosterol account for 30.0 mol% of the total lipids, DMG-PEG2K or DSPE-PEG2K-Mannose account for 1.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 88.2% by weight of the composition;

[0555] (20) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 43.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 23.3 mol% of the total lipids, cholesterol or β-sitosterol account for 33.0 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 0.7 mol% of the total lipids, and the amphiphilic block copolymer accounts for 39.1% by weight of the composition;

[0556] (21) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 44.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 25.0 mol% of the total lipids, cholesterol or β-sitosterol account for 30.0 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 1.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 63.6% by weight of the composition;

[0557] (22) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 39.5 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 25.3 mol% of the total lipids, cholesterol or β-sitosterol account for 34.5 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 0.7 mol% of the total lipids, and the amphiphilic block copolymer accounts for 37.2% by weight of the composition;

[0558] (23) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 41.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 28.0 mol% of the total lipids, cholesterol or β-sitosterol account for 30.0 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 1.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 66.0% by weight of the composition;

[0559] (24) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 39.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 28.0 mol% of the total lipids, cholesterol or β-sitosterol account for 32.0 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 1.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 36.3% by weight of the composition;

[0560] (25) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 39.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 30.0 mol% of the total lipids, cholesterol or β-sitosterol account for 30.0 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 1.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 62.9% by weight of the composition;

[0561] (26) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 accounts for 40.6 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE accounts for 30.1 mol% of the total lipids, cholesterol or β-sitosterol accounts for 28.4 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K accounts for 0.9 mol% of the total lipids, and the amphiphilic block copolymer accounts for 36.9% by weight of the composition;

[0562] (27) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 accounts for 34.7 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE accounts for 40.1 mol% of the total lipids, cholesterol or β-sitosterol accounts for 24.3 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K accounts for 0.9 mol% of the total lipids, and the amphiphilic block copolymer accounts for 32.5% by weight of the composition;

[0563] (28) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 29.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 50.1 mol% of the total lipids, cholesterol or β-sitosterol account for 20.0 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 0.9 mol% of the total lipids, and the amphiphilic block copolymer accounts for 28.0% by weight of the composition;

[0564] (29) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 35.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 17.6 mol% of the total lipids, cholesterol or β-sitosterol account for 46.5 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 0.9 mol% of the total lipids, and the amphiphilic block copolymer accounts for 35.9% by weight of the composition;

[0565] (30) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 55.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 16.9 mol% of the total lipids, cholesterol or β-sitosterol account for 27.2 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 0.9 mol% of the total lipids, and the amphiphilic block copolymer accounts for 44.6% by weight of the composition;

[0566] (31) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 60.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 14.9 mol% of the total lipids, cholesterol or β-sitosterol account for 24.2 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 0.9 mol% of the total lipids, and the amphiphilic block copolymer accounts for 46.5% by weight of the composition;

[0567] (32) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 65.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 14.1 mol% of the total lipids, cholesterol or β-sitosterol account for 20.0 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 0.9 mol% of the total lipids, and the amphiphilic block copolymer accounts for 21.0% by weight of the composition;

[0568] (33) an amphiphilic block copolymer; GL67, ICE, or HGT4002; DSPC, DPPC, or DOPE; and DMG-PEG2K or DMG-PEG5K, wherein the GL67, ICE, or HGT4002 accounts for 70.0 mol% of the total lipids present in the composition, DSPC, DPPC, or DOPE accounts for 28.5 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K accounts for 1.5 mol% of the total lipids, and the amphiphilic block copolymer accounts for 48.1% by weight of the composition;

[0569] (34) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; cholesterol or β-sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 61.3 mol% of the total lipids present in the composition, cholesterol or β-sitosterol account for 37.6 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 1.1 mol% of the total lipids, and the amphiphilic block copolymer accounts for 41.9% by weight of the composition;

[0570] (35) an amphiphilic block copolymer; cKK-E12, DLin-MC3-DMA, ALC-0315, SM-102, or C12-200; DOTAP, DODAP, DOTMA, or DOSPA; cholesterol or β-sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the cKK-E12, DLin-MC3-DMA, ALC-0315, SM-102, or C12-200 accounts for 30.0 mol% of the total lipids present in the composition, DOTAP, DODAP, DOTMA, or DOSPA accounts for 39.0 mol% of the total lipids, cholesterol or β-sitosterol accounts for 30.0 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K accounts for 1.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 57.7% by weight of the composition; or

[0571] (36) An amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; and cholesterol or β-sitosterol, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 accounts for 40.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE accounts for 32.0 mol% of the total lipids, cholesterol or β-sitosterol accounts for 28.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 50.0% by weight of the composition.

[0572] In some embodiments of the composition of the invention, the molar ratio of nitrogen (amine) groups in the cationic lipid to phosphate groups in the nucleic acid (N / P ratio) in the composition is about 0.5 to about 40, about 1 to about 30, about 2 to about 25, about 3 to about 20, about 4 to about 15, about 5 to about 10, about 6 to about 8, about 2 to about 12, about 4 to about 8, about 5 to about 8, about 6 to about 7.8, about 6.7 to about 7.6, or about 6.8 to about 7.5.

[0573] In some embodiments of the compositions of the invention, the ratio of lipid to nucleic acid in the composition (mass / mass ratio) is about 1 (1:1) to about 100 (100:1), about 5 (5:1) to about 90 (90:1), about 1 (1:1) to about 50 (50:1), about 5 (5:1) to about 45 (45:1), about 10 (10:1) to about 40 (40:1), about 15 (15:1) to about 35 (35:1), about 20 (20:1) to about 30 (30:1), about 1 (1:1) to about 25 (25:1), about 5 (5:1) to about 30 (30:1), about 5 (5:1) to about 20 (20:1), about 5 (5:1) to about 15 (15:1) or about 5 (5:1) to about 10 (10:1).

[0574]

[0014] The present invention encompasses embodiments comprising a mixture of at least first and second separately formed non-identical polymer-lipid compositions.

[0575] Each polymer-lipid composition comprises an mRNA and one or more cationic lipids, wherein the first polymer-lipid composition includes a first cationic lipid and the second polymer-lipid composition comprises a second cationic lipid; wherein the first cationic lipid and the second cationic lipid are not the same; and wherein expression of a protein or peptide encoded by the mRNA following administration of the pharmaceutical composition to a subject exceeds expression of an otherwise identical amount of the mRNA-encoded protein or peptide by at least about two-fold compared to expression of the protein or peptide encoded by the mRNA administered with the first lipid nanoparticle but without the second lipid nanoparticle.

[0576] In some embodiments encompassed by the present invention, the first polymer-lipid composition and the second polymer-lipid composition comprise one or more non-cationic lipids, wherein the first non-cationic lipid and the second non-cationic lipid are different, and wherein expression of a protein or peptide encoded by the mRNA after administration of the pharmaceutical composition to a subject exceeds expression of an otherwise identical amount of the mRNA-encoded protein or peptide administered with the first polymer-lipid composition but without the second polymer-lipid composition by at least about two-fold.

[0577] In some embodiments encompassed by the present invention, the first polymer-lipid composition and the second polymer-lipid composition comprise one or more phospholipids, wherein the first phospholipid and the second phospholipid are different, and wherein expression of a protein or peptide encoded by the mRNA after administration of the pharmaceutical composition to a subject exceeds expression of an otherwise identical amount of the mRNA-encoded protein or peptide administered with the first polymer-lipid composition but without the second polymer-lipid composition by at least about two-fold.

[0578] In some embodiments encompassed by the present invention, the first polymer-lipid composition and the second polymer-lipid composition comprise one or more lipid conjugates, wherein the first lipid conjugate and the second lipid conjugate are different, and wherein expression of a protein or peptide encoded by the mRNA after administration of the pharmaceutical composition to a subject exceeds expression of an otherwise identical amount of the mRNA-encoded protein or peptide administered with the first polymer-lipid composition but without the second polymer-lipid composition by at least about two-fold.

[0579] In some embodiments encompassed by the present invention, wherein the first polymer-lipid composition and the second polymer-lipid composition comprise one or more amphiphilic block copolymers, wherein the first amphiphilic block copolymer and the second amphiphilic block copolymer are not identical, and wherein expression of a protein or peptide encoded by the mRNA after administration of the pharmaceutical composition to a subject exceeds expression of an otherwise identical amount of the mRNA-encoded protein or peptide administered with the first polymer-lipid composition but without the second polymer-lipid composition by at least about two-fold.

[0580] In some embodiments of the present invention, the ratio of the first polymer-lipid composition to the second polymer-lipid composition in the pharmaceutical composition is about 1:1, or about 2:1, or about 3:1, or about 4:1.

[0581] In some embodiments included in the present invention, cationic lipids (or non-lipid cationic agents) that can be distributed on the outer surface of the polymer-lipid composition nanoparticles can be additionally added to the polymer-lipid composition. In some embodiments, the additionally added cationic lipid can be a sterolamine, such as GL67, ICE, etc. In some embodiments, the additionally added non-lipid cationic agent can be tromethamine, benzalkonium chloride, modified arginine, cetylpyridinium chloride, L-lysine monohydrate, etc. In other embodiments, the contacting of the polymer-lipid composition with the cationic lipid (or non-lipid cationic agent) comprises dissolving the cationic lipid (or non-lipid cationic agent) in a non-ionic excipient. In some embodiments, the non-ionic excipient is selected from polyethylene glycol (15)-hydroxystearate ( HS 15), 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2K), polyoxyethylene sorbitan monooleate (Tween-80), sorbitan monooleate (Span-85), polyoxyethylene fatty acid esters (such as Myrij 52), polyoxyethylene fatty alcohol ethers (such as Brij 35) and α-tocopheryl polyethylene glycol succinate (TPGS). In some embodiments, contacting the polymer-lipid composition with the cationic lipid (or non-lipid cationic agent) comprises dissolving the cationic lipid (or non-lipid cationic agent) in a buffer solution, such as PBS and Tris buffer.

[0582] Suitable mixtures of polymer-lipid compositions for use in the compositions and methods of the present invention include synergistically enhanced nucleic acid delivery hybrid formulations as described in International Patent Publication Nos. WO 2014144196, WO 2022032154, and U.S. Patent No. 10,130,649, which are incorporated herein by reference.

[0583] In some embodiments of the compositions of the present invention, the active agent or therapeutic agent further comprises a protein or polypeptide. In some embodiments, the protein is a protein related to translation or transcription. In some embodiments, the protein is related to the CRISPR process. In some embodiments, the protein is a CRISPR-related protein. In some embodiments, the protein is Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Cas10, Cas12a, Cas13a, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, a homolog thereof, or a modified form thereof. In some embodiments, the protein is Cas9.

[0584] In some embodiments, the protein or polypeptide and the nucleic acid are present in a molar ratio of about 1:1 to about 1:20. In some embodiments, the molar ratio is about 1:1 to about 1:10. In some embodiments, the molar ratio is about 1:3 to about 1:8.

[0585] In some embodiments, the therapeutic agent is a protein or polypeptide. In some embodiments, the composition comprises both a protein and a nucleic acid. In some embodiments, the composition comprises a Cas9 protein and a single guide nucleic acid. In some embodiments, the composition comprises a Cas9 protein, a single guide nucleic acid, and a donor DNA.

[0586] In some embodiments, the therapeutic agent is a small molecule such as a small molecule selected from the group consisting of anticancer agents, antifungal agents, psychiatric agents such as analgesics, consciousness-altering agents such as anesthetics or hypnotics, nonsteroidal anti-inflammatory drugs (NSAIDS), anthelmintics, anti-acne agents, antianginal agents, antiarrhythmic agents, antiasthmatic agents, antibacterial agents, anti-benign prostatic hyperplasia agents, anticoagulants, antidepressants, antidiabetic agents, antiemetics, antiepileptics, antigout agents, antihypertensive agents, anti-inflammatory agents, antimalarials, antimigraine agents, antimuscarinics, antitumor agents, antiobesity agents, anti Osteoporotic agents, antiparkinsonian drugs, antiproliferative agents, antiprotozoal agents, antithyroid agents, antitussives, anti-incontinence agents, antiviral agents, anxiolytics, appetite suppressants, beta-blockers, cardiac inotropes, chemotherapy drugs, cognitive enhancers, contraceptives, corticosteroids, Cox-2 inhibitors, diuretics, erectile dysfunction medications, expectorants, gastrointestinal agents, histamine receptor antagonists, immunosuppressants, keratolytics, lipid-regulating agents, leukotriene inhibitors, macrolides, muscle relaxants, neuroleptics, nutritional supplements, narcotic analgesics, protease inhibitors, or sedatives.

[0587] In some embodiments of the composition of the present invention, the composition further comprises a targeting moiety to target the composition to a target organ, tissue or cell in a subject, preferably the targeting moiety comprises at least one selected from the group consisting of a glycosyl, a lipid, a nucleic acid aptamer, a small molecule therapeutic agent, a vitamin, a polypeptide and a protein such as an antibody. Preferably, the targeting moiety is selected from the group consisting of an epithelial cell ligand, particularly a respiratory epithelial cell ligand, a gastrointestinal epithelial cell ligand, a reproductive epithelial cell ligand or a microfold cell ligand; an immune cell ligand, particularly a dendritic cell ligand, a T cell ligand, a B cell ligand or a macrophage ligand; an endothelial cell ligand, particularly a lung endothelial cell ligand or a liver endothelial cell ligand; a tumor cell ligand, particularly a melanoma, lung or liver tissue-associated tumor cell ligand; and / or a skin cell ligand, particularly a dermal fibroblast ligand or a keratinocyte ligand.

[0588] In some embodiments of the composition of the present invention, the composition further comprises an adjuvant, preferably the adjuvant comprises at least one selected from the group consisting of: CpG oligodeoxynucleotides, polyinosinic:polycytidylic acid, saponin extract (QS-21 extract), aluminum adjuvant, squalene, α-tocopherol, Tween, Span, lipopolysaccharide LPS, Pam3CSK4 triacyl lipopeptide, cyclic adenosine diphosphate (c-di-AMP), 2′3′-cyclic guanosine monophosphate adenosine monophosphate (cGAMP), monophosphoryl-lipid A, MPL lipid, flagellin or immunomodulatory proteins such as IL-2, IL-12, GM-CSF, TSLP and nucleic acids encoding these immunomodulatory proteins.

[0589] Suitable adjuvants may include, but are not limited to, inorganic salts (e.g., AlK(SO4)2, AlNa(SO4)2, AlNH(SO4)2, silica, aluminum, aluminum hydroxide, Ca3(PO4)2, kaolin, or carbon), polynucleotides with or without immune stimulating complexes (ISCOMs) (e.g., CpG oligonucleotides such as those described in Chuang, TH et al. (2002) J. Leuk. Biol. 71(3):538-44; Ahmad Nejad, P. et al. (2002) Eur. J. Immunol. 32(7):1958-68); poly IC or poly AU acids, poly arginine with or without CpG (also known in the art as IC31; see Schellack, C. et al. (2003) Proceedings of the 34th Annual Meeting of the German Society of Immunology; Lingnau, K. et al. (2002) Vaccine 20(29-30):3498-508), JuvaVax™ (U.S. Pat. No. 6,693,086), certain natural substances (e.g., wax D from Mycobacterium tuberculosis, substances present in Cornyebacterium parvum, Bordetella pertussis, or members of the genus Brucella), flagellin (a ligand for Toll-like receptor 5; see McSorley, SJ et al. (2002) J. Immunol. 169(7):3914-9), saponins (such as QS21, QS17, and QS7) (U.S. Pat. Nos. 5,057,540; 5,650,398; 6,524,584; 6,645,495), monophosphoryl lipid A (specifically, 3 -de-O-acylated monophosphoryl lipid A (3D-MPL)), imiquimod (also known in the art as IQM and commercially available as; U.S. Patent Nos. 4,689,338; 5,238,944; Zuber, AK et al. (2004) 22(13-14):1791-8); the CCR5 inhibitor CMPD167 (see Veazey, RS et al. (2003) J. Exp. Med. 198:1551-1562); and squalene emulsion, i.e., MF59.

[0590] In some embodiments, the adjuvant is another RNA.

[0591] In the past, the adjuvant widely used in humans was aluminum. In some embodiments, a suitable adjuvant is aluminum phosphate. In some embodiments, a suitable adjuvant is aluminum hydroxide. In some embodiments, a suitable adjuvant is a combination of aluminum phosphate and aluminum hydroxide. Saponins and their purified components, Quil A, Freund's complete adjuvant, and other adjuvants are commonly used in research and veterinary applications; however, new chemically defined preparations, such as muramyl dipeptide, monophosphoryl lipid A, phospholipid conjugates, such as those described by Goodman-Snitkoff et al. J. Immunol. 147: 410-415 (1991), are incorporated herein by reference.

[0592] In some embodiments of the polymer-lipid composition of the present invention, the composition further comprises a transfection enhancer, preferably the transfection enhancer comprises at least one selected from the group consisting of: pulmonary surfactant protein, cell-penetrating peptide, amphiphilic polypeptide, mucolytic enzyme, 1,2-propylene glycol, cellulose (such as carboxymethyl cellulose or hydroxypropyl cellulose), hyaluronate, alginate, pectin, polyethylene glycol, poloxamer, poloxamine, glucose, fructose, sucrose, trehalose, dextran, polyvinyl pyrrolidone, chitosan, polyvinyl alcohol, polyvinyl acetate, lectin, polylactic acid, polyhydroxybutyric acid, tromethamine, benzalkonium chloride, modified arginine, cetylpyridinium chloride, L-lysine monohydrate, and polylactic-co-glycolic acid or salt solution.

[0593] In some embodiments of the polymer-lipid compositions of the present invention, the compositions are in the form of nanoparticles having an average size of about 1000 nm or less. In some embodiments, the nanoparticles have an average size of about 500 nm or less, 400 nm or less, 300 nm or less, 200 nm or less, 150 nm or less, 125 nm or less, 100 nm or less, 75 nm or less, or about 50 nm or less.

[0594] In some embodiments, about 30% to about 100%, about 70% to about 100%, about 90% to about 100%, about 50% to about 90%, about 70% to about 90%, or about 80% to about 90% of the nanoparticles have the active agent or therapeutic agent encapsulated therein.

[0595] In some embodiments of the polymer-lipid compositions of the present invention, the compositions are formulated as solutions, dry powders, atomized or sprayed forms. In some embodiments, the compositions are formulated for pulmonary and / or nasal administration by aerosolization, dry powder, inhalation, atomization or instillation.

[0596] Preparation method

[0597] The present invention particularly provides mRNA-polymer-lipid compositions prepared using the methods of the present invention as described herein. In some embodiments, the method for encapsulating mRNA as described herein includes a step of mixing a lipid solution with an mRNA solution in the presence of an amphiphilic polymer (e.g., poloxamine and / or poloxamer) to form polymer-lipid nanoparticles that encapsulate mRNA. In some embodiments, the amphiphilic polymer is present in the mRNA solution before mixing. In some embodiments, the amphiphilic polymer is present in the lipid solution before mixing. In some embodiments, the amphiphilic polymer is added during the mixing of the mRNA solution and the lipid solution. In some embodiments, the amphiphilic polymer is added after the mixing of the mRNA solution and the lipid solution. In some embodiments, the step of mixing the lipid solution with the mRNA solution in the presence of an amphiphilic polymer to form polymer-lipid nanoparticles that encapsulate mRNA, and after purification and dialysis, the amphiphilic polymer is further added to the solution of the polymer-lipid nanoparticles.

[0598] In some embodiments, the lipid solution in suitable polymer-lipid includes cationic lipids and non-cationic lipids (also referred to as helper lipids). In some embodiments, suitable lipid solution includes cationic lipids, non-cationic lipids and PEG-modified lipids or PEG. In some embodiments, suitable lipid solution includes cationic lipids, non-cationic lipids, cholesterol-based lipids and PEG-modified lipids or PEG. Various lipids can be dissolved in a suitable solvent with the required respective amount and / or ratio to prepare a lipid solution for methods described herein. A variety of methods can be used to prepare a suitable lipid solution. Exemplary methods are described in US 2016 / 0038432, US 2018 / 0153822 and US 2018 / 0125989, which are incorporated herein by reference.

[0599] In some embodiments, the lipid solution in the suitable polymer-lipid comprises the lipid modified by one or more cationic lipids, non-cationic lipids, cholesterol and / or PEG-. For example, the polymer-lipid can comprise at least one of the following cationic lipids: DLin-MC3-DMA, ALC-0315, SM-102, cKK-E12, A2-Iso5-2DC18, BAME-O16B, 9A1P9, OF-Deg-Lin, 306Oi10, TT3, FTT5, C12-200, DLin-KC2-DMA, DODAP, HGT4003, ICE, GL67, HGT5000 or HGT5001 etc. In some embodiments, the polymer-lipid may comprise at least one of the following non-cationic lipids: DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine), DOPE (1,2-dioleyl-sn-glycero-3-phosphoethanolamine), DPPE (1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine), DMPE (1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine), DOPG (2-dioleoyl-sn-glycero-3-phosphoethanolamine). In some embodiments, the polymer-lipid may comprise cholesterol and / or β-sitosterol, etc. In some embodiments, the polymer-lipid comprises PEG-modified / modified lipids, and the PEG-modified lipids may include (but not exclusively) covalently linked to C6-C 20In some embodiments, the polymer-lipid comprises lipids with alkyl chains of lengths up to 5 kDa, PEG chains of lengths up to 5 kDa. In some embodiments, the polymer-lipid comprises DMG-PEG2K, DMPE-PEG2K, DSPE-PEG2K, DSPE-PEG2K-Mannose, DMG-PEG5K, DMPE-PEG5K, DSPE-PEG5K, DSPE-PEG5K-Mannose, etc. In certain embodiments, the polymer-lipid comprises one of the following lipid formulations: DLin-MC3-DMA, DSPC, cholesterol, DMG-PEG2K; DLin-MC3-DMA, DPPC, cholesterol, DMG-PEG2K; DLin-MC3-DMA, DOPE, cholesterol, DMG-PEG2K; DLin-MC3-DMA, DSPC, cholesterol, DMG-PEG5K; DLin-MC3-DMA, DPPC, cholesterol, DMG-PEG5K; DLin-MC3-DMA, DOPE, cholesterol, DMG-PEG5K; ALC-0315, DSPC, cholesterol, DMG-PEG2K; ALC-0315, DPPC, cholesterol, DMG-PEG2K; ALC-0 315, DOPE, cholesterol, DMG-PEG2K; ALC-0315, DSPC, cholesterol, DMG-PEG5K; ALC-0315, DPPC, cholesterol, DMG-PEG5K; ALC-0315, DOPE, cholesterol, DMG-PEG5K; SM-102, DSPC, cholesterol, DMG-PEG2K; SM-102, DPPC, cholesterol, DMG-PEG2K; SM-102, DOPE, cholesterol, DMG-PEG2K; SM-102, DSPC, cholesterol, DMG-PEG5K; SM-102, DPPC, cholesterol, DMG-PEG5K; SM-102, DOPE, cholesterol, DMG-PEG5K; C12-200, DOPE, cholesterol, DMG-PEG2K; DODAP, DOPE, cholesterol, DMG-PEG2K; HGT5000, DOPE, cholesterol, DMG-PEG2K; HGT5001, DOPE, cholesterol, DMG-PEG2K; GL67, DPPC, DMG-PEG2K; cKK-E12, DOTAP, cholesterol, DMG-PEG2K; SM-102, DSPC, cholesterol; SM-102, DOPS, cholesterol; SM-102, DOPE, cholesterol; SM-102, cholesterol, DMG-PEG2K; SM-102, DSPC, DMG-PEG2K.

[0600] In some embodiments, the method comprises:

[0601] (A) mixing a solution comprising the active agent or therapeutic agent and a solution comprising the lipid in the presence of an amphiphilic block copolymer to form the composition; or

[0602] (B) mixing a solution containing the active agent or therapeutic agent and a solution containing the lipid to form lipid nanoparticles encapsulating the active agent or therapeutic agent, and then mixing the amphiphilic block copolymer with the lipid nanoparticle solution to form the composition.

[0603] In some embodiments, the method comprises:

[0604] 1) adding the amphiphilic block copolymer to a solution comprising the active agent or therapeutic agent and / or a solution comprising the lipid, and

[0605] 2) mixing the solution comprising the active agent or therapeutic agent and the solution comprising the lipid,

[0606] Thereby forming the composition.

[0607] In some embodiments, the method comprises:

[0608] 1) in a solution comprising the lipids, allowing the lipids to pre-form into lipid nanoparticles without an active agent or therapeutic agent; and

[0609] 2) mixing a solution comprising the active agent or therapeutic agent and the amphiphilic block copolymer with the lipid nanoparticle solution,

[0610] Thereby forming the composition.

[0611] In other embodiments, the method comprises:

[0612] 1) preforming the lipid and amphiphilic block copolymer into polymer-lipid nanoparticles without active agent or therapeutic agent; and

[0613] 2) mixing a solution containing the active agent or therapeutic agent with the polymer-lipid nanoparticle solution,

[0614] Thereby forming the composition.

[0615] In some embodiments, the method further comprises the step of removing free lipid components and / or amphiphilic block copolymers, preferably by dialysis and / or tangential flow filtration.

[0616] In some embodiments, the method further comprises the step of adding the amphiphilic block copolymer again after removing the free lipid component and / or the amphiphilic block copolymer.

[0617] In some embodiments, mRNA solution or lipid solution or both can be heated to a predetermined temperature higher than ambient temperature before mixing. In some embodiments, mRNA solution and lipid solution are heated to a predetermined temperature respectively before mixing. In some embodiments, mRNA solution and lipid solution are mixed at ambient temperature, and then heated to a predetermined temperature after mixing. In some embodiments, lipid solution is heated to a predetermined temperature, and mixed with mRNA solution at ambient temperature. In some embodiments, mRNA solution is heated to a predetermined temperature, and mixed with lipid solution at ambient temperature. In some embodiments, compared with other identical methods without a heating step, a heating step is included in the method process (before, during, or after formation) to provide a higher mRNA encapsulation efficiency. In some embodiments, the encapsulation of mRNA in polymer-lipid can be further enhanced by heating a formulation solution comprising mRNA-polymer-lipid and some free mRNA that is not encapsulated in the polymer-lipid formation solution to a predetermined temperature as described herein.

[0618] In some embodiments, the mRNA solution is heated to a predetermined temperature by adding an mRNA stock solution at ambient temperature to a buffer solution that is heated to reach the desired predetermined temperature.

[0619] As used herein, the term "ambient temperature" refers to the temperature in a room, or the temperature surrounding an object of interest without heating or cooling. In some embodiments, the ambient temperature maintained by one or more solutions is at or below about 35°C, 30°C, 25°C, 20°C, or 16°C. In some embodiments, the ambient temperature maintained by one or more solutions is in the range of about 15-35°C, about 15-30°C, about 15-25°C, about 15-20°C, about 20-35°C, about 25-35°C, about 30-35°C, about 20-30°C, about 25-30°C, or about 20-25°C. In some embodiments, the ambient temperature maintained by one or more solutions is 20-25°C.

[0620] Thus, the predetermined temperature above ambient temperature is typically greater than about 25° C. In some embodiments, the predetermined temperature suitable for the present invention is or is greater than about 30° C., 37° C., 40° C., 45° C., 50° C., 55° C., 60° C., 65° C., or 70° C. In some embodiments, the predetermined temperature suitable for the present invention is in the range of about 25-70° C., about 30-70° C., about 35-70° C., about 40-70° C., about 45-70° C., about 50-70° C., or about 60-70° C. In a specific embodiment, the predetermined temperature suitable for the present invention is about 65° C.

[0621] Methods that can be used to prepare the polymer-lipid compositions described herein are described, for example, in U.S. Patent Nos. 8,058,069; 5,753,613; 5,785,992; 5,705,385; 5,976,567; 5,981,501; 6,110,745; and 6,320,017; and PCT Publication Nos. WO 2022 / 032154 and WO 96 / 40964, the disclosures of which are each incorporated herein by reference in their entirety for all purposes.

[0622] In some embodiments, a pump is used to mix the mRNA solution and the lipid solution. Since the encapsulation procedure with this type of mixing can be carried out on various scales, different types of pumps can be used to adapt to the required scale. However, it is generally desirable to use a pulseless flow pump. As used herein, a pulseless flow pump refers to any pump that can establish a continuous flow rate with a stable flow rate. The type of suitable pump can include, but is not limited to, a gear pump and a centrifugal pump.

[0623] The mRNA solution and lipid solution can be mixed at various flow rates. Generally, the mRNA solution can be mixed at a flow rate greater than the flow rate of the lipid solution. For example, the mRNA storage solution can be mixed at a flow rate at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 times greater than the flow rate of the lipid solution.

[0624] The suitable flow rate for mixing can be determined based on scale. In some embodiments, the lipid solution is mixed at a flow rate within the range of about 1-4 ml / min, 2-6 ml / min, 3-8 ml / min, 5-10 ml / min, 10-20 ml / min, 15-30 ml / min, 25-75 ml / min, 20-50 ml / min, 25-75 ml / min, 30-90 ml / min, 40-100 ml / min, 50-110 ml / min, 75-200 ml / min, 200-350 ml / min, 350-500 ml / min, 500-650 ml / min, 650-850 ml / min, or 850-1000 ml / min. In some embodiments, the mRNA solution is mixed at a flow rate in the range of about 1000-2000 ml / min, 2000-3000 ml / min, 3000-4000 ml / min, or 4000-5000 ml / min.

[0625] In some embodiments, the present polymer-lipid composition can be prepared using nanoprecipitation, which can be performed by the following unit operation, wherein the polymer-lipid composition is mixed by kinetics, and then matured and serially diluted from its individual lipid component self-assembly. This unit operation generally includes a continuous online combination of three liquid streams and an online maturation step: an aqueous buffer is mixed with a lipid stock solution, matured via a controlled residence time, and the nanoparticles are diluted. The nanoprecipitation itself occurs in a mixer suitable for scale, which is designed to allow the continuous, high-energy combination of an aqueous solution and a lipid stock solution dissolved in ethanol. Throughout this operation, the aqueous solution and the lipid stock solution all flow continuously into the mixer at the same time. The ethanol content keeping the lipids dissolved is suddenly reduced, and the lipids are all precipitated one another. Thus the nanoparticles self-assemble in the mixing chamber.

[0626] In certain embodiments, the present invention provides a polymer-lipid composition produced by a continuous mixing method, for example, a process comprising providing an aqueous solution (which may include an amphiphilic block polymer) containing a nucleic acid, such as mRNA, in a first reservoir, providing an organic lipid solution (which may include an amphiphilic block polymer) in a second reservoir, and mixing the aqueous solution and the organic lipid solution so that the organic lipid solution mixes with the aqueous solution to substantially immediately produce a polymer-lipid composition encapsulating the nucleic acid (e.g., mRNA). This process and an apparatus for performing the process are described in detail in U.S. Patent Publication No. 20040142025, the disclosure of which is incorporated herein by reference in its entirety for all purposes.

[0627] In another embodiment, the present invention provides a polymer-lipid composition produced by a direct dilution method comprising forming a lipid solution and immediately and directly introducing the lipid solution into a collection vessel containing a controlled amount of dilution buffer. In a preferred aspect, the collection vessel comprises one or more components configured to stir the contents of the collection vessel to promote dilution. In one aspect, the amount of dilution buffer present in the collection vessel is substantially equal to the volume of the lipid solution introduced therein. As a non-limiting example, a liposome solution in 45% ethanol will advantageously produce smaller particles when introduced into a collection vessel containing an equal volume of dilution buffer.

[0628] In another embodiment, the present invention provides polymer-lipid compositions produced by a direct dilution method, wherein a third reservoir containing a dilution buffer is fluidly connected to the second mixing zone. These methods and apparatus for performing these direct dilution methods are described in detail in U.S. Patent Publication No. 20070042031, the disclosure of which is incorporated herein by reference in its entirety for all purposes.

[0629] In contrast to other preparation techniques (such as film rehydration / extrusion), ethanol drop precipitation has become the industry standard for preparing nucleic acid lipid nanoparticles. The precipitation reaction is favored because of its continuity, scalability and ease of use. This type of method typically uses a high-energy mixer (such as a T-joint, a restricted impact jet, a microfluidic mixer, a vortex mixer) to introduce lipids (in ethanol) into a suitable reverse phase solvent (i.e., water) in a controllable manner, thereby driving the liquid to be supersaturated and spontaneously precipitated into lipid particles. In some embodiments, the vortex mixer used is described in U.S. patent applications 62 / 799,636 and 62 / 886,592, which are incorporated herein by reference in their entirety. In some embodiments, the microfluidic mixer used can adopt the mixer described in PCT patent WO / 2014 / 172045, which is incorporated herein by reference in its entirety. In some embodiments, the mixing step is performed using a T-junction, a confined impinging jet, a microfluidic mixer, or a vortex mixer. In some embodiments, the loading step is performed using a T-junction, a confined impinging jet, a microfluidic mixer, or a vortex mixer.

[0630] In another embodiment, the present invention provides PoLixNano-CPLs (polymer-lipid compositions containing cation-polymer-lipid conjugates CPL) and / or PoLixNano-sterolamines (e.g., polymer-lipid compositions containing sterolamine GL67) prepared by "standard" techniques and "post-insertion" techniques, wherein the CPL or GL67 is inserted into, for example, a pre-formed polymer-lipid composition (PoLixNano), and the "standard" technique, wherein the CPL or GL67 is included in the lipid mixture, for example, during the PoLixNano formation step. Methods for preparing PoLixNano-CPL are described, for example, in U.S. Patent Nos. 5,705,385; 6,586,410; 5,981,501; 6,534,484; and 6,852,334; U.S. Patent Publication No. 20020072121; and PCT Publication No. WO 00 / 62813, the disclosures of which are incorporated herein by reference in their entireties for all purposes. Methods for preparing PoLixNano-GL67 are described in PCT Publication No. WO 2022 / 032154, the disclosures of which are incorporated herein by reference in their entireties for all purposes.

[0631] If desired, the polymer-lipid compositions of the present invention can be sized by any method that can be used to adjust the size of liposomes. Size adjustment can be performed to obtain a desired size range and a relatively narrow particle size distribution.

[0632] Several techniques exist for adjusting particles to a desired size, such as ultrasonic bath / probe sonication, homogenization, extrusion, etc. One sizing method, which is used for liposomes and is also applicable to the polymer-lipid compositions of the present invention, is described in U.S. Pat. No. 4,737,323, the disclosure of which is incorporated herein by r...

Claims

1. A polymer-lipid composition comprising: (A) an active or therapeutic agent, preferably comprising a nucleic acid; (B) amphiphilic block copolymers; (C) cationic lipids; and (D) non-cationic lipids, Wherein the composition is formulated for delivery through a mucosal site of an organism, such as respiratory tract delivery, oral mucosal delivery, gastrointestinal tract delivery, ocular mucosal delivery, ear mucosal delivery, urethral delivery, or reproductive tract delivery, preferably the composition is formulated for delivery through the respiratory tract.

2. The composition of claim 1, wherein the nucleic acid comprises at least one selected from the group consisting of messenger RNA (mRNA), self-amplifying RNA (saRNA), circular RNA (circRNA), small interfering RNA (siRNA), short hairpin RNA (shRNA) and microRNA (miRNA), primary-miRNA, antisense oligonucleotide (ASO), transfer RNA (tRNA), plasmid DNA (pDNA), single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), deoxyribozyme (DNAzyme), ribozyme (RNAzyme), nucleic acid aptamer (aptamer), clustered regularly interspaced short palindromic repeats (CRISPR)-related nucleic acid, single guide RNA (sgRNA), CRISPR-RNA (crRNA), trans-activating crRNA (tracrRNA), guide RNA, single-stranded RNA (ssRNA) and double-stranded RNA (dsRNA).

3. The composition of claim 2, wherein the nucleic acid is a therapeutic nucleic acid, preferably wherein the nucleic acid comprises mRNA.

4. The composition of any one of claims 1 to 3, wherein the amphiphilic block copolymer comprises at least 0.1% by weight of the composition, preferably 0.1% to 98.0% by weight, such as 1% to 90.0% by weight, 40.0% to 80.0% by weight, 50.0% to 70.0% by weight, or 50.0% to 60.0% by weight.

5. The composition of any one of claims 1 to 4, wherein the cationic lipid comprises at least one selected from the group consisting of a permanent cationic lipid, an ionizable cationic lipid, a cholesterol-derived cationic lipid, and a dendrimer or dendron, preferably, the cationic lipid comprises an ionizable cationic lipid.

6. The composition of any one of claims 1-5, wherein the cationic lipid accounts for 23.0 mol%-83.0 mol%, preferably 30.0 mol%-80.0 mol%, 30.0 mol%-70 mol% or 40.0 mol%-60 mol% of the total lipids present in the composition.

7. The composition of any one of claims 1 to 6, wherein the non-cationic lipid comprises at least one selected from the group consisting of anionic lipids, zwitterionic lipids and neutral lipids, preferably, the non-cationic lipid comprises a neutral lipid, preferably, the neutral lipid accounts for 19.0 mol% to 75.0 mol% of the total lipids present in the composition.

8. The composition of claim 7, wherein the neutral lipid comprises: Cholesterol or cholesterol-derived neutral lipids; phospholipids; or A mixture of cholesterol or cholesterol-derived neutral lipids and phospholipids.

9. The composition of claim 8, wherein the cholesterol accounts for 14.0 mol% to 70.0 mol% of the total lipids in the composition.

10. The composition of claim 8, wherein the phospholipids account for 5.0 mol% to 75.0 mol% of the total lipids in the composition.

11. The composition of any one of claims 1 to 10, further comprising a lipid conjugate, wherein the lipid conjugate comprises at least one selected from the group consisting of a PEG-lipid conjugate, an ATTA-lipid conjugate, a polysarcosine-lipid conjugate, a polypeptide / protein-lipid conjugate, a cation-polymer-lipid conjugate (CPL), and derivatives thereof, preferably, the lipid conjugate comprises a PEG-lipid conjugate, preferably, the lipid conjugate accounts for 0.1 mol%-10.0 mol% of the total lipids in the composition.

12. The composition of any one of claims 1-3, wherein the composition comprises: (1) amphiphilic block copolymers, cationic lipids, phospholipids, cholesterol, and lipid conjugates such as PEG-lipid conjugates, wherein the cationic lipids account for 30.0% of the total lipids present in the composition mol%-80.0mol%, phospholipids account for 5.0mol%-50.0mol% of the total lipids, cholesterol accounts for 14.0mol%-64.0mol% of the total lipids, lipid conjugates account for 0.1mol%-8.0mol% of the total lipids, and the amphiphilic block copolymer accounts for 0.1%-95.0% weight percentage of the composition; (2) an amphiphilic block copolymer, a cationic lipid, a phospholipid, cholesterol, and a lipid conjugate, such as a PEG-lipid conjugate, wherein the cationic lipid accounts for 23.0 mol% to 75.0 mol% of the total lipids present in the composition, the phospholipid accounts for 10.0 mol% to 62.0 mol% of the total lipids, the cholesterol accounts for 14.0 mol% to 46.0 mol% of the total lipids, the lipid conjugate accounts for 0.1 mol% to 8.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 0.1% to 95.0% by weight of the composition; (3) an amphiphilic block copolymer, a cholesterol-derived cationic lipid, a phospholipid, and a lipid conjugate, such as a PEG-lipid conjugate, wherein the cholesterol-derived cationic lipid comprises 29.0 mol% to 80.0 mol% of the total lipids present in the composition, the phospholipids comprise 19.0 mol% to 70.0 mol% of the total lipids, the lipid conjugates comprise 0.1 mol% to 8.0 mol% of the total lipids, and the amphiphilic block copolymer comprises 0.1% to 95.0% by weight of the composition; (4) an amphiphilic block copolymer, a cationic lipid, cholesterol, and a lipid conjugate, such as a PEG-lipid conjugate, wherein the cationic lipid accounts for 25.0 mol% to 80.0 mol% of the total lipids present in the composition, the cholesterol accounts for 15.0 mol% to 50.0 mol% of the total lipids, the lipid conjugate accounts for 0.1 mol% to 8.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 0.1% to 95.0% by weight of the composition; (5) amphiphilic block copolymers, cationic lipids, phospholipids, and lipid conjugates such as PEG-lipid conjugates, wherein the cationic lipids account for 30.0 mol% to 80.0 mol% of the total lipids present in the composition, the phospholipids account for 10.0 mol% to 50.0 mol% of the total lipids, the lipid conjugates account for 0.1 mol% to 8.0 mol% of the total lipids, and the amphiphilic block copolymers account for 0.1% to 95.0% by weight of the composition; (6) an amphiphilic block copolymer, a cationic lipid, a phospholipid, and cholesterol, wherein the cationic lipid accounts for 30.0 mol% to 80.0 mol% of the total lipids present in the composition, the phospholipids account for 5.0 mol% to 50.0 mol% of the total lipids, the cholesterol accounts for 15.0 mol% to 50.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 0.1% to 95.0% by weight of the composition; or (7) an amphiphilic block copolymer, a cholesterol-derived cationic lipid, and a phospholipid, wherein the cholesterol-derived cationic lipid accounts for 30.0 mol%-70.0 mol% of the total lipids present in the composition, the phospholipids account for 30.0 mol%-70.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 0.1%-95.0% by weight of the composition.

13. The composition of any one of claims 1 to 12, wherein the amphiphilic block copolymer is a tetrafunctional amphiphilic block copolymer, wherein the tetrafunctional amphiphilic block copolymer comprises a block copolymer of four branches each comprising at least one hydrophilic block and at least one hydrophobic block, or the amphiphilic block copolymer is a linear amphiphilic block copolymer, wherein the linear amphiphilic block copolymer comprises a block copolymer of at least one hydrophilic block and at least one hydrophobic block.

14. The composition of claim 13, wherein the hydrophilic block is selected from the group consisting of polyoxyalkylenes, polyvinyl alcohol, polyvinyl pyrrolidone, poly(2-methyl-2-oxazoline) and sugars, and / or the hydrophobic block is selected from the group consisting of polyoxyalkylenes, fatty chains, alkylene polyesters, polyethylene glycol with benzyl polyether ends and cholesterol, preferably, the hydrophilic block comprises polyethylene oxide units and the hydrophobic block comprises polypropylene oxide units.

15. The composition of any one of claims 1 to 14, wherein the amphiphilic block copolymer comprises at least one selected from the group consisting of poloxamine or ), poloxamer or ), polyoxyethylene glycol dehydrated alcohol alkyl esters (polysorbates), polyvinyl pyrrolidone (PVP), polyethylene glycol ether (BRIJ), polyoxyethylene fatty acid esters, polyoxyethylene fatty alcohol ethers, dehydrated sorbitan esters and their derivatives.

16. The composition of any one of claims 1-14, wherein the amphiphilic block copolymer comprises poloxamine or ), for example, the poloxamine is selected from poloxamine 304, poloxamine 701, poloxamine 704, poloxamine 901, poloxamine 904, poloxamine 908, poloxamine 1107, poloxamine 1301, poloxamine 1304, poloxamine 1307, poloxamine 90R4, poloxamine 150R1, or a combination thereof.

17. The composition of any one of claims 1-14, wherein the amphiphilic block copolymer comprises poloxamer or ), for example, the poloxamer is selected from poloxamer 84, poloxamer 101, poloxamer 105, poloxamer 108, poloxamer 122, poloxamer 123, poloxamer 124, poloxamer 181, poloxamer 182, poloxamer 183, poloxamer 184, poloxamer 185, poloxamer 188, poloxamer 212, poloxamer 215, poloxamer 217, poloxamer Poloxamer 231, Poloxamer 234, Poloxamer 235, Poloxamer 237, Poloxamer 238, Poloxamer 282, Poloxamer 284, Poloxamer 288, Poloxamer 304, Poloxamer 331, Poloxamer 333, Poloxamer 334, Poloxamer 335, Poloxamer 338, Poloxamer 401, Poloxamer 402, Poloxamer 403, Poloxamer 407, or a combination thereof.

18. The composition of any one of claims 1-17, wherein the cationic lipid comprises at least one selected from the group consisting of DOTMA, DOSPA, DOTAP, ePC, DODAP, DODMA, DDAB, DSDMA, DODAC, DOAP, DMRIE, DOGS, DMOBA, HGT5000, HGT5001, HGT5002, HGT4001, HGT4002, HGT4003, HGT4005, DLin-MC3-DMA, DLin-KC2-DMA, Acuitas ALC-0315, Acuitas A9, Acuitas Lipid 2,2, Moderna Lipid H (SM-102), Moderna Lipid 5. A2-Iso5-2DC18, BAME-O16B, 9A1P9, C12-200, cKK-E12, OF-Deg-Lin, 306Oi10, TT3, FTT5, Lipid319, 5A2-SC8, Genevant CL1, DLinDMA, DLenDMA, ClinDMA, CpLinDMA, imidazole cholesteryl ester (ICE), RE-1, RE-2, RE-3, GL-67, 5A2-SC8, Acuitas A9, Arcturus Lipid 2,2(8,8)4C CH3, OF-02, A18-Iso5-2DC18, BAME-O16B, A6, 98N12-5, L319, L343, 304O13, 306O138, 306O12B, 30 6-O12B, LP01, G0-C14, 7C1, Cephalin, Dlin-EG-DMA, DLinAP, DLin-MPZ, DLin-C-DAP, DLin-2-DMAP , Dlin-S-DMA, DLinDAP, DLin-MA, DLin-DAC, DLin-K-DMA, DLin-K-MPZ, DLin-K-DMA, DLin-K6-C4-DMA, DLin-K-C4-DMA, DLin-K-C3-DMA, CpLinDMA, DOcarbDAP, DLincarbDAP, C12-(2-3-2), Genevant Lipid CL1, XTC, ALNY-100, NC98-5 and their derivatives.

19. The composition of any one of claims 5-18, wherein the cholesterol-derived cationic lipid comprises at least one selected from the group consisting of DC-Choi (N,N-dimethyl-N-ethylformamide cholesterol), 1,4-bis(3-N-oleylamino-propyl)piperazine, N4-argininocholesterol carbonylamide (GL67), cholesterol derivatives coupled to basic amino acid sequences, imidazole cholesterol ester (ICE), and derivatives thereof.

20. The composition of any one of claims 8 to 19, wherein the phospholipid comprises at least one selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine, lysophosphatidylcholine, lysophosphatidylethanolamine, phosphatidylserine, dioleoylphosphatidylserine (DOPS), phosphatidylinositol, sphingomyelin, egg yolk sphingomyelin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebroside, dihexadecyl phosphate, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylethanolamine (DOPE), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleoyl-phosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), palmitoyloleoyl-phosphatidylglycerol (POPG), dioleoylphosphatidylethanolamine 4-(N-maleimide) methyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoyl-phosphatidylethanolamine (DPPE), dimyristoyl-phosphatidylethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), monomethyl-phosphatidylethanolamine, dimethyl-phosphatidylethanolamine, dioleoyl-phosphatidylethanolamine (DEPE), stearoyloleoyl-phosphatidylethanolamine (SOPE), lysophosphatidylcholine, egg yolk phosphatidyl Choline (EPC), dilinoleylphosphatidylcholine, 1,2-dipalmitoyl-sn-glycero-3-O-4'-(N,N,N-trimethyl)-homoserine (DGTS), monogalactosyldiacylglycerol (MGDG), diacetyldiacylglycerol (DGDG), sulfaquinolinediacylglycerol (SQDG), 1-palmitoyl-2-cis-9,10-methylenehexyl-decanoyl-sn-glycero-3-phosphocholine (Cyclo PC), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE and their derivatives.

21. The composition of any one of claims 8 to 20, wherein the cholesterol-derived neutral lipid comprises at least one selected from the group consisting of cholestanol, cholestanone, cholestenone, coprostanol, cholesteryl-2'-hydroxyethyl ether, cholesteryl-4'-hydroxybutyl ether, BHEM-cholesterol, β-sitosterol, 20α-hydroxycholesterol, cholesterol covalently linked to a polypeptide / protein, and derivatives thereof, preferably the cholesterol-derived neutral lipid comprises β-sitosterol.

22. The composition of any one of claims 11-21, wherein the PEG-lipid conjugate comprises at least one selected from the group consisting of DMG-PEG2K, DMPE-PEG2K, DSPE-PEG2K, DSPE-PEG2K-Mannose, DMG-PEG5K, DMPE-PEG5K, DSPE-PEG5K-Mannose, and DSPE-PEG5K.

23. The composition of any one of claims 1-3, wherein the composition comprises an amphiphilic block copolymer and the following components: (1) DLin-MC3-DMA, ALC-0315, SM-102, C12-200, cKK-E12, HGT5000 or HGT5001; DSPC, DPPC, DOPS, SOPE, DOPG, DSPE, ESM or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K, DMG-PEG5K or DSPE-PEG2K-Mannose, wherein the DLin-MC3-DMA, ALC-0315, SM-102, C12-200, cKK-E12, HGT5000 or HGT5001 account for 40.0 mol% to 70.0 mol% of the total lipids present in the composition, and DSPC, DPPC, DOPS, SOPE, DOPG, DSPE, ESM or DOPE account for 8.0 mol%-39.0 mol% of the total lipids, cholesterol or β-sitosterol account for 20.0 mol%-40.0 mol% of the total lipids, DMG-PEG2K, DMG-PEG5K or DSPE-PEG2K-Mannose account for 0.1 mol%-5.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 15.0%-90.0% by weight of the composition; (2) DLin-MC3-DMA, ALC-0315, SM-102, C12-200, cKK-E12, HGT5000 or HGT5001; DSPC, DPPC, DOPS, SOPE, DOPG, DSPE, ESM or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K, DMG-PEG5K or DSPE-PEG2K-Mannose, wherein the DLin-MC3-DMA, ALC-0315, SM-102, C12-200, cKK-E12, HGT5000 or HGT5001 1 accounts for 30 mol%-60 mol% of the total lipids present in the composition, DSPC, DPPC, DOPS, SOPE, DOPG, DSPE, ESM or DOPE accounts for 10.0 mol%-49.0 mol% of the total lipids, cholesterol or β-sitosterol accounts for 20.0 mol%-40.0 mol% of the total lipids, DMG-PEG2K, DMG-PEG5K or DSPE-PEG2K-Mannose accounts for 0.1 mol%-5.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 20.0%-90.0% by weight of the composition; (3) DLin-MC3-DMA, ALC-0315, SM-102, C12-200, cKK-E12, HGT5000 or HGT5001; DSPC, DPPC, DOPS, SOPE, DOPG, DSPE, ESM or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K, DMG-PEG5K or DSPE-PEG2K-Mannose, wherein the DLin-MC3-DMA, ALC-0315, SM-102, C12-200, cKK-E12, HGT5000 or HGT5001 accounts for 24.0 mol%-40.0 mol% of the total lipids present in the composition, DSPC, DPPC, DOPS, SOPE, DOPG, DSPE, ESM or DOPE account for 30.0 mol%-64.0 mol% of the total lipids, cholesterol or β-sitosterol account for 15.0 mol%-40.0 mol% of the total lipids, DMG-PEG2K, DMG-PEG5K or DSPE-PEG2K-Mannose account for 0.1 mol%-5.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 20.0%-90.0% by weight of the composition; (4) DOTAP, DODAP, DOTMA or DOSPA; DSPC, DPPC, DOPS, SOPE, DOPG, DSPE, ESM or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K, DMG-PEG5K or DSPE-PEG2K-Mannose, wherein the DOTAP, DODAP, DOTMA or DOSPA accounts for 23.0 mol%-60 mol% of the total lipids present in the composition, DSPC, DPPC, DOPS, SOPE, DOPG, DSPE, ESM or DOPE accounts for 14.0 mol%-60.0 mol% of the total lipids, cholesterol or β-sitosterol accounts for 15.0 mol%-50.0 mol% of the total lipids, DMG-PEG2K, DMG-PEG5K or DSPE-PEG2K-Mannose accounts for 0.1 mol%-5.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 30.0%-90.0% by weight of the composition; (5) GL67, ICE, or HGT4002; DSPC, DPPC, DOPS, SOPE, DOPG, DSPE, ESM or DOPE; and DMG-PEG2K, DMG-PEG5K or DSPE-PEG2K-Mannose, wherein the GL67, ICE, or HGT4002 accounts for 40.0 mol%-80.0 mol% of the total lipids present in the composition, DSPC, DPPC, DOPS, SOPE, DOPG, DSPE, ESM or DOPE accounts for 10.0 mol%-50.0 mol% of the total lipids, DMG-PEG2K, DMG-PEG5K or DSPE-PEG2K-Mannose accounts for 0.1 mol%-5.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 30.0%-90.0% weight percent of the composition; (6) cKK-E12, DLin-MC3-DMA, ALC-0315, SM-102, C12-200, DOTAP, DODAP, DOTMA, DOSPA, HGT5000 or HGT5001; cholesterol or β-sitosterol; and DMG-PEG2K, DMG-PEG5K or DSPE-PEG2K-Mannose, wherein the cKK-E12, DLin-MC3-DMA, ALC-0315, SM-102, C12-200, DOTAP , DODAP, DOTMA, DOSPA, HGT5000 or HGT5001 account for 45.0 mol%-75.0 mol% of the total lipids present in the composition, cholesterol or β-sitosterol account for 20.0 mol%-45.0 mol% of the total lipids, DMG-PEG2K, DMG-PEG5K or DSPE-PEG2K-Mannose account for 0.1 mol%-5.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 30.0%-90.0% weight percentage of the composition; (7) DLin-MC3-DMA, ALC-0315, SM-102, C12-200, cKK-E12, HGT5000 or HGT5001; cholesterol or β-sitosterol; and DMG-PEG2K, DMG-PEG5K or DSPE-PEG2K-Mannose, wherein the DLin-MC3-DMA, ALC-0315, SM-102, C12-200, cKK-E12, HGT5000 or HGT5001 00 or HGT5001 accounts for 30.0mol%-65.0mol% of the total lipids present in the composition, cholesterol or β-sitosterol accounts for 20.0mol%-40.0mol% of the total lipids, DMG-PEG2K, DMG-PEG5K or DSPE-PEG2K-Mannose accounts for 0.1mol%-5.0mol% of the total lipids, and the amphiphilic block copolymer accounts for 40.0%-90.0% weight percentage of the composition; (8) DLin-MC3-DMA, ALC-0315, SM-102, C12-200, cKK-E12, HGT5000 or HGT5001; DSPC, DPPC, DOPS, SOPE, DOPG, DSPE, ESM or DOPE; and DMG-PEG2K, DMG-PEG5K or DSPE-PEG2K-Mannose, wherein the DLin-MC3-DMA, ALC-0315, SM-102, C12-200, cKK-E12, HGT50 00 or HGT5001 accounts for 35.0 mol%-70.0 mol% of the total lipids present in the composition, DSPC, DPPC, DOPS, SOPE, DOPG, DSPE, ESM or DOPE accounts for 10.0 mol%-40.0 mol% of the total lipids, DMG-PEG2K, DMG-PEG5K or DSPE-PEG2K-Mannose accounts for 0.1 mol%-5.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 40.0%-90.0% weight percentage of the composition; (9) GL67, ICE, or HGT4002; and DSPC, DPPC, DOPS, SOPE, DOPG, DSPE, ESM, or DOPE, wherein the GL67, ICE, or HGT4002 accounts for 40.0 mol%-80.0 mol% of the total lipids, DSPC, DPPC, DOPS, SOPE, DOPG, DSPE, ESM or DOPE account for 10.0 mol%-50.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 40.0%-90.0% weight percentage of the composition; or (10) DLin-MC3-DMA, ALC-0315, SM-102, cKK-E12, HGT5000 or HGT5001; DSPC, DPPC, DOPS, SOPE, DOPG, DSPE, ESM or DOPE; and cholesterol or β-sitosterol, wherein the DLin-MC3-DMA, ALC-0315, SM-102, cKK-E12, HGT5000 or HGT5001 accounts for 30.0 mol%-60.0 mol% of the total lipids present in the composition, DSPC, DPPC, DOPS, SOPE, DOPG, DSPE, ESM or DOPE accounts for 20.0 mol%-45.0 mol% of the total lipids, cholesterol or β-sitosterol accounts for 20.0 mol%-45.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 30.0%-90.0% by weight of the composition.

24. The composition of any one of claims 1-3, wherein the composition comprises the following components: (1) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 50.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 10.0 mol% of the total lipids, cholesterol or β-sitosterol account for 38.5 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 1.5 mol% of the total lipids, and the amphiphilic block copolymer accounts for 89.9% by weight of the composition; (2) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 50.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 10.6 mol% of the total lipids, cholesterol or β-sitosterol account for 38.5 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 0.9 mol% of the total lipids, and the amphiphilic block copolymer accounts for 72.9% by weight of the composition; (3) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DSPE-PEG2K-Mannose, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 50.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 12.5 mol% of the total lipids, cholesterol or β-sitosterol account for 36.0 mol% of the total lipids, DMG-PEG2K or DSPE-PEG2K-Mannose account for 1.5 mol% of the total lipids, and the amphiphilic block copolymer accounts for 43.0% by weight of the composition; (4) Amphiphilic block copolymers: DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DSPE-PEG2K-Mannose, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 50.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 14.5 mol% of the total lipids, cholesterol or β-sitosterol account for 34.0 mol% of the total lipids, DMG-PEG2K or DSPE-PEG2K-Mannose account for 1.5 mol% of the total lipids, and the amphiphilic block copolymer accounts for 81.8% by weight of the composition; (5) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DSPE-PEG2K-Mannose, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 50.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 14.0 mol% of the total lipids, cholesterol or β-sitosterol account for 35.0 mol% of the total lipids, DMG-PEG2K or DSPE-PEG2K-Mannose account for 1.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 69.2% by weight of the composition; (6) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 50.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 14.1 mol% of the total lipids, cholesterol or β-sitosterol account for 35.0 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 0.9 mol% of the total lipids, and the amphiphilic block copolymer accounts for 43.3% by weight of the composition; (7) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 50.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 14.3 mol% of the total lipids, cholesterol or β-sitosterol account for 35.0 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 0.7 mol% of the total lipids, and the amphiphilic block copolymer accounts for 48.4% by weight of the composition; (8) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 50.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 14.5 mol% of the total lipids, cholesterol or β-sitosterol account for 35.0 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 0.5 mol% of the total lipids, and the amphiphilic block copolymer accounts for 43.6% by weight of the composition; (9) amphiphilic block copolymers; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein The DLin-MC3-DMA, ALC-0315 or SM-102 account for 48.5 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 17.0 mol% of the total lipids, cholesterol or β-sitosterol account for 32.5 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 1.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 42.3% by weight of the composition; (10) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 46.5 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 20.0 mol% of the total lipids, cholesterol or β-sitosterol account for 32.5 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 1.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 52.9% by weight of the composition; (11) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DSPE-PEG2K-Mannose, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 49.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 20.0 mol% of the total lipids, cholesterol or β-sitosterol account for 30.0 mol% of the total lipids, DMG-PEG2K or DSPE-PEG2K-Mannose account for 1.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 41.9% by weight of the composition; (12) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DSPE-PEG2K-Mannose, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 49.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 20.1 mol% of the total lipids, cholesterol or β-sitosterol account for 30.0 mol% of the total lipids, DMG-PEG2K or DSPE-PEG2K-Mannose account for 0.9 mol% of the total lipids, and the amphiphilic block copolymer accounts for 40.3% by weight of the composition; (13) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 51.6 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 20.0 mol% of the total lipids, cholesterol or β-sitosterol account for 27.5 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 0.9 mol% of the total lipids, and the amphiphilic block copolymer accounts for 42.9% by weight of the composition; (14) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 accounts for 46.0% of the total lipids present in the composition. mol%, DSPC, DPPC or DOPE account for 23.0mol% of the total lipids, cholesterol or β-sitosterol account for 30.0mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 1.0mol% of the total lipids, and the amphiphilic block copolymer accounts for 50.2% by weight of the composition; (15) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DSPE-PEG2K-Mannose, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 46.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 23.0 mol% of the total lipids, cholesterol or β-sitosterol account for 30.0 mol% of the total lipids, DMG-PEG2K or DSPE-PEG2K-Mannose account for 1.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 66.1% by weight of the composition; (16) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 46.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 23.0 mol% of the total lipids, cholesterol or β-sitosterol account for 30.0 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 1.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 74.5% by weight of the composition; (17) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 46.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 23.0 mol% of the total lipids, cholesterol or β-sitosterol account for 30.0 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 1.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 79.6% by weight of the composition; (18) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 46.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 23.0 mol% of the total lipids, cholesterol or β-sitosterol account for 30.0 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 1.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 83.0% by weight of the composition; (19) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DSPE-PEG2K-Mannose, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 46.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 23.0 mol% of the total lipids, and cholesterol or β-sitosterol account for 1. Sterols account for 30.0 mol% of the total lipids, DMG-PEG2K or DSPE-PEG2K-Mannose accounts for 1.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 88.2% by weight of the composition; (20) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 43.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 23.3 mol% of the total lipids, cholesterol or β-sitosterol account for 33.0 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 0.7 mol% of the total lipids, and the amphiphilic block copolymer accounts for 39.1% by weight of the composition; (21) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 44.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 25.0 mol% of the total lipids, cholesterol or β-sitosterol account for 30.0 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 1.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 63.6% by weight of the composition; (22) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 39.5 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 25.3 mol% of the total lipids, cholesterol or β-sitosterol account for 34.5 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 0.7 mol% of the total lipids, and the amphiphilic block copolymer accounts for 37.2% by weight of the composition; (23) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 41.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 28.0 mol% of the total lipids, cholesterol or β-sitosterol account for 30.0 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 1.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 66.0% by weight of the composition; (24) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 39.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 28.0 mol% of the total lipids, cholesterol or β-sitosterol account for 32.0 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 1.0 mol% of the total lipids, and the The amphiphilic block copolymer accounts for 36.3% by weight of the composition; (25) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 39.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 30.0 mol% of the total lipids, cholesterol or β-sitosterol account for 30.0 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 1.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 62.9% by weight of the composition; (26) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 accounts for 40.6 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE accounts for 30.1 mol% of the total lipids, cholesterol or β-sitosterol accounts for 28.4 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K accounts for 0.9 mol% of the total lipids, and the amphiphilic block copolymer accounts for 36.9% by weight of the composition; (27) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 accounts for 34.7 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE accounts for 40.1 mol% of the total lipids, cholesterol or β-sitosterol accounts for 24.3 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K accounts for 0.9 mol% of the total lipids, and the amphiphilic block copolymer accounts for 32.5% by weight of the composition; (28) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 29.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 50.1 mol% of the total lipids, cholesterol or β-sitosterol account for 20.0 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 0.9 mol% of the total lipids, and the amphiphilic block copolymer accounts for 28.0% by weight of the composition; (29) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 35.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 17.6 mol% of the total lipids, cholesterol or β-sitosterol account for 46.5 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 0.9 mol% of the total lipids, and the amphiphilic block copolymer accounts for 35.9% by weight of the composition; (30) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 55.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 16.9 mol% of the total lipids, cholesterol or β-sitosterol account for 27.2 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 0.9 mol% of the total lipids, and the amphiphilic block copolymer accounts for 44.6% by weight of the composition; (31) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 60.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 14.9 mol% of the total lipids, cholesterol or β-sitosterol account for 24.2 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 0.9 mol% of the total lipids, and the amphiphilic block copolymer accounts for 46.5% by weight of the composition; (32) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 65.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 14.1 mol% of the total lipids, cholesterol or β-sitosterol account for 20.0 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 0.9 mol% of the total lipids, and the amphiphilic block copolymer accounts for 21.0% by weight of the composition; (33) an amphiphilic block copolymer; GL67, ICE, or HGT4002; DSPC, DPPC, or DOPE; and DMG-PEG2K or DMG-PEG5K, wherein the GL67, ICE, or HGT4002 accounts for 70.0 mol% of the total lipids present in the composition, DSPC, DPPC, or DOPE accounts for 28.5 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K accounts for 1.5 mol% of the total lipids, and the amphiphilic block copolymer accounts for 48.1% by weight of the composition; (34) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; cholesterol or β-sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 61.3 mol% of the total lipids present in the composition, cholesterol or β-sitosterol account for 37.6 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 1.1 mol% of the total lipids, and the amphiphilic block copolymer accounts for 41.9% by weight of the composition; (35) an amphiphilic block copolymer; cKK-E12, DLin-MC3-DMA, ALC-0315, SM-102, or C12-200; DOTAP, DODAP, DOTMA, or DOSPA; cholesterol or β-sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the cKK-E12, DLin-MC3-DMA, ALC- 0315, SM-102 or C12-200 accounts for 30.0 mol% of the total lipids present in the composition, DOTAP, DODAP, DOTMA or DOSPA accounts for 39.0 mol% of the total lipids, cholesterol or β-sitosterol accounts for 30.0 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K accounts for 1.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 57.7% by weight of the composition; or (36) An amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; and cholesterol or β-sitosterol, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 accounts for 40.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE accounts for 32.0 mol% of the total lipids, cholesterol or β-sitosterol accounts for 28.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 50.0% by weight of the composition.

25. The composition of any of claims 1-24, wherein the molar ratio of nitrogen (amine) groups in the cationic lipid to phosphate groups in the nucleic acids in the composition (N / P ratio) is from about 1.0 to about 30.0, from about 3.0 to about 15.0, from about 4.0 to about 10.0, from about 6.0 to about 8.

0.

26. The composition of any one of claims 1-25, wherein the ratio of lipid to nucleic acid in the composition (mass / mass ratio) is about 2 (2:1) to about 100 (100:1), about 5 (5:1) to about 60 (60:1), about 15 (15:1) to about 45 (45:1), or about 20 (20:1) to about 30 (30:1).

27. The composition of any one of claims 1-26, wherein the active or therapeutic agent further comprises a protein or polypeptide, preferably the protein or polypeptide is present in a molar ratio of about 1:1 to about 1:20 to the nucleic acid.

28. The composition of any of the preceding claims, wherein the composition further comprises a targeting moiety to target the composition to a target organ, tissue or cell in a subject, preferably the targeting moiety comprises at least one selected from the group consisting of a glycosyl, a lipid, a nucleic acid aptamer, a small molecule therapeutic, a vitamin, a polypeptide and a protein such as an antibody.

29. The composition of any one of the preceding claims, wherein the composition further comprises an adjuvant, preferably the adjuvant comprises at least one selected from the group consisting of: CpG oligodeoxynucleotides, polyinosinic:polycytidylic acid, saponin extract (QS-21 extract), aluminum adjuvant, manganese adjuvant, zinc adjuvant, squalene, α-tocopherol, Tween, Span, lipopolysaccharide LPS, Pam3CSK4 triacyl lipopeptide, cyclic adenosine diphosphate (c-di-AMP), 2′3′-cyclic guanosine monophosphate adenosine monophosphate (cGAMP), monophosphoryl-lipid A, MPL lipid, flagellin or immunomodulatory proteins such as IL-2, IL-12, GM-CSF, TSLP and nucleic acids encoding these immunomodulatory proteins.

30. The composition of any of the preceding claims, wherein the composition further comprises a transfection enhancer, preferably the transfection enhancer comprises at least one selected from the group consisting of pulmonary surfactant protein, cell-penetrating peptide, amphiphilic polypeptide, mucolytic enzyme, 1,2-propylene glycol, cellulose (such as carboxymethyl cellulose or hydroxypropyl cellulose), hyaluronate, alginate, pectin, polyethylene glycol, poloxamer, poloxamine, glucose, fructose, sucrose, trehalose, dextran, sucrose, trehalose, mannose, polyvinyl pyrrolidone, chitosan, polyvinyl alcohol, polyvinyl acetate, lectin, polylactic acid, polyhydroxybutyric acid, tromethamine, benzalkonium chloride, modified arginine, cetylpyridinium chloride, L-lysine monohydrate, and poly(lactic-co-glycolic acid) copolymer or a salt solution.

31. The composition of any of the preceding claims, wherein the composition is in the form of nanoparticles having an average size of about 1000 nm or less.

32. The composition of claim 31, wherein the nanoparticles have an average size of about 500 nm or less, about 400 nm or less, about 300 nm or less, about 200 nm or less, about 150 nm or less, about 125 nm or less, about 100 nm or less, about 75 nm or less, or about 50 nm or less.

33. The composition of claim 31 or 32, wherein about 30% to about 100%, about 70% to about 100%, about 90% to about 100%, about 50% to about 90%, about 70% to about 90%, or about 80% to about 90% of the nanoparticles have an active or therapeutic agent encapsulated therein.

34. The composition of any one of claims 1-33, wherein the composition is formulated as a solution, dry powder, atomization, or spray.

35. The composition of any one of claims 1-34, formulated for administration to the respiratory tract, lungs, trachea, bronchus and / or nose by aerosolization, dry powder, inhalation, nebulization or instillation.

36. A method of preparing the composition of any one of claims 1 to 35, comprising: (A) mixing a solution comprising the active agent or therapeutic agent and a solution comprising the lipid in the presence of an amphiphilic block copolymer to form the composition; or (B) mixing a solution containing the active agent or therapeutic agent and a solution containing the lipid to form lipid nanoparticles encapsulating the active agent or therapeutic agent, and then mixing the amphiphilic block copolymer with the lipid nanoparticle solution to form the composition.

37. The method of claim 36, comprising: 1) adding the amphiphilic block copolymer to a solution comprising the active agent or therapeutic agent and / or a solution comprising the lipid, and 2) mixing the solution comprising the active agent or therapeutic agent and the solution comprising the lipid, Thereby forming the composition.

38. The method of claim 36, comprising: 1) in a solution comprising the lipid, allowing the lipid to pre-form into lipid nanoparticles without an active agent or therapeutic agent; and 2) mixing a solution comprising the active agent or therapeutic agent and the amphiphilic block copolymer with the lipid nanoparticle solution, thereby forming the composition. or The method comprises: 1) preforming the lipid and amphiphilic block copolymer into polymer-lipid nanoparticles without active agent or therapeutic agent; and 2) mixing a solution containing the active agent or therapeutic agent with the polymer-lipid nanoparticle solution, Thereby forming the composition.

39. The method of any one of claims 36-38, further comprising the step of removing free lipid components and / or amphiphilic block copolymers, preferably by dialysis and / or tangential flow filtration.

40. The method of claim 39, further comprising the step of adding the amphiphilic block copolymer again after removing the free lipid component and / or the amphiphilic block copolymer.

41. A pharmaceutical composition comprising: the composition of any one of claims 1 to 35 and a pharmaceutically acceptable carrier and / or excipient.

42. A method of delivering an active agent or therapeutic agent to a target cell, the method comprising: The cell, preferably a mammalian cell, is contacted with the composition of any one of claims 1 to 35 under conditions sufficient to cause uptake of the active or therapeutic agent into the cell.

43. A method for preventing and / or treating a disease or condition in a mammal, the method comprising administering to a subject in need thereof a pharmaceutically effective amount of the composition according to any one of claims 1 to 35 or the pharmaceutical composition according to claim 41, wherein the composition or pharmaceutical composition comprises the active agent or therapeutic agent for the disease or condition.

44. Use of the composition of any one of claims 1 to 35 or the pharmaceutical composition of claim 41 in the preparation of a medicament for preventing and / or treating a disease in a subject, wherein the composition or pharmaceutical composition comprises an active agent or therapeutic agent for the disease or condition.

45. The method of claim 43 or the use of claim 44, wherein the disease or condition is selected from an immune system disease, a metabolic disease, a genetic disease, cancer, a blood disease, a bacterial infection, or a viral infection.

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