WO2024094027A1 - Séquence de blocage, kit correspondant et procédé pour son utilisation - Google Patents

Séquence de blocage, kit correspondant et procédé pour son utilisation Download PDF

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WO2024094027A1
WO2024094027A1 PCT/CN2023/128795 CN2023128795W WO2024094027A1 WO 2024094027 A1 WO2024094027 A1 WO 2024094027A1 CN 2023128795 W CN2023128795 W CN 2023128795W WO 2024094027 A1 WO2024094027 A1 WO 2024094027A1
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PCT/CN2023/128795
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唐劼
肖晶
李航
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深圳鸿生生物科技有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/24—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing atoms other than carbon, hydrogen, oxygen, halogen, nitrogen or sulfur, e.g. cyclomethicone or phospholipids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/34—Macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyesters, polyamino acids, polysiloxanes, polyphosphazines, copolymers of polyalkylene glycol or poloxamers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system

Definitions

  • the present invention relates to compositions for enhancing the delivery of active or therapeutic agents, such as therapeutic nucleic acids.
  • biological macromolecules such as proteins, DNA, siRNA, and in vitro transcribed messenger RNA (mRNA) can produce or act on specific protein functions to exert their efficacy, thereby achieving the purpose of disease prevention or treatment.
  • mRNA messenger RNA
  • These biological macromolecules are becoming an important method for preventing and treating a variety of diseases.
  • 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.
  • SARS-CoV-2 novel coronavirus
  • two COVID-19 mRNA vaccines have been approved by the US FDA for marketing worldwide.
  • mRNA technology also has great application potential in tumor immunotherapy, gene therapy, infectious disease prevention, gene editing, and genetic disease treatment.
  • 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, has high safety, 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.
  • mRNA therapy involves administering specific mRNA to a subject in need of the therapy to produce proteins encoded by the mRNA in the patient. How to use a safe delivery system to efficiently and accurately deliver mRNA to target tissues and target cells and maximize its expression efficiency is the key to achieving successful clinical transformation of such drugs or vaccines.
  • mRNA is easily degraded by widely existing RNases, and its structural stability is a prerequisite for exerting biological effects; 2) mRNA is easily cleared after entering the body, has a short half-life in the body, and is difficult to reach specific target sites; 3) Due to its large relative molecular mass, strong hydrophilicity and electronegativity, mRNA itself is difficult 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.
  • viral vectors Due to the relatively high in vivo transfection efficiency, early attempts to use viral vectors as a delivery method for gene delivery were made.
  • main problems of viral vectors such as the host's immune response to the viral vector itself, difficulty in repeated administration, possible activation of oncogenes that cause malignant tumors, and complications of inflammatory responses, have significantly hindered their development.
  • non-viral vectors have many advantages over viral vectors, such as high safety, low immunogenicity, reduced pathogenicity, reduced ability to insert mutations, and convenient large-scale preparation.
  • Nanoparticles developed based on nanobiomaterials are a typical type of non-viral vector.
  • Common non-viral vectors include lipid nanoparticles (LNP) and preparations based on cationic polymers, such as chitosan, polyethyleneimine, and dendrimers.
  • LNP lipid nanoparticles
  • preparations based on cationic polymers such as chitosan, polyethyleneimine, and dendrimers.
  • cationic polymers such as chitosan, polyethyleneimine, and dendrimers.
  • the LNP system is the most common, most advanced, and only mRNA drug delivery system that has successfully achieved clinical transformation.
  • LNP has developed into the "gold standard" delivery technology in the field of mRNA 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 and 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 after LNP preparations are administered via the respiratory route, they only mediate very low mRNA gene expression in mice (Zhang, N.-N.
  • mRNA drugs have great potential in the treatment of many lung diseases that have no clinical cure, such as cystic fibrosis (CF), chronic obstructive pulmonary disease (COPD), ⁇ 1-trypsin deficiency, asthma, pulmonary hypertension, primary ciliary dyskinesia, and dyskinesia) and idiopathic pulmonary fibrosis.
  • CF cystic fibrosis
  • COPD chronic obstructive pulmonary disease
  • ⁇ 1-trypsin deficiency asthma
  • pulmonary hypertension pulmonary hypertension
  • primary ciliary dyskinesia and dyskinesia
  • idiopathic pulmonary fibrosis idiopathic pulmonary fibrosis.
  • the special physiological structure of the lungs brings many advantages to its route of administration, and makes 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,
  • Drugs delivered through the respiratory tract have simple administration methods, good patient compliance, are suitable for repeated administration regimens, and can also make the drug preparations evenly distributed in bronchial and alveolar epithelial tissues (Patel, AK et al. Inhaled Nanoformulated mRNA Polyplexes for Protein Production in Lung Epithelium. Adv. Mater. e1805116, (2019)).
  • Potential systemic side effects can be minimized, and problems such as cross infection caused by needle contamination can also be reduced.
  • the lung airway has a large absorption surface area and a rich capillary network, which is conducive to gene drug absorption and efficient transfection.
  • the lungs also have strong angiogenesis capabilities, which can mediate secretory proteins to enter the circulatory system to exert their effects. Therefore, the lungs are an ideal target for mRNA-based protein replacement therapy.
  • mRNA vaccines have great application prospects in inducing antigen-specific mucosal immunity in respiratory mucosal sites (e.g., nasal-associated lymphoid tissue NALT and bronchial-associated lymphoid tissue BALT). More than 90% of pathogens invade the human body through mucosal sites (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.
  • SARS-CoV-2 new coronavirus
  • SARS-CoV severe acute respiratory syndrome coronavirus
  • MERS Middle East respiratory syndrome coronavirus
  • influenza virus respiratory syncytial virus
  • Mycobacterium tuberculosis Mycobacterium tuberculosis.
  • vaccines administered by intramuscular injection can hardly induce mucosal immune responses, and thus cannot 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 to others.
  • Studies have shown that the early specific humoral response in patients infected with SARS-CoV-2 is dominated by sIgA antibodies produced by mucosal immune responses (Sterlin, D. et al. IgA dominates the early neutralizing antibody response to SARS-CoV-2. Sci Transl Med. 13, eabd22234 (2021)).
  • the average neutralization potency 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, 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 infection and transmission of related pathogens (Jeyanathan, M. et al. Immunological considerations for COVID-19 vaccine strategies. Nat. Rev. Immunol. 20, 615–632(2020)).
  • the present invention surprisingly found that adding amphiphilic block copolymers such as poloxamine or ) and or poloxamer or ) and other components, the obtained novel preparation can greatly improve the gene transfection efficiency of nucleic acid molecules such as mRNA mediated by such preparations in animals, especially when delivered through the mucosal part of the organism (such as respiratory delivery).
  • the preparation of the present invention is administered via the respiratory route, the gene transfection effect produced in the animal lung/respiratory mucosal tissue is significantly better than that of LNP preparations, poloxamine and poloxamer, thereby solving the problem of low delivery efficiency in the art.
  • the present invention provides a composition comprising a polymer-lipid, the composition comprising:
  • an active agent or therapeutic agent preferably the active agent or therapeutic agent comprises a nucleic acid
  • composition is formulated for delivery through a mucosal site of an organism, such as the respiratory tract, oral mucosa, gastrointestinal tract, ocular mucosa, ear mucosa, urethra, or reproductive tract, preferably the composition is formulated for delivery through the respiratory tract.
  • 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 micro RNA (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).
  • the nucleic acid is a therapeutic nucleic acid. More preferably, the nucleic acid comprises mRNA.
  • the amphiphilic block copolymer accounts for 0.1%-98.0% weight percent of the composition, for example, 0.5%-95.0% weight percent, 1%-90.0% weight percent, 10%-80.0% weight percent, 20%-80.0% weight percent, 30%-80.0% weight percent, 40%-80.0% weight percent, 40%-70.0% weight percent or 50%-60.0% weight percent.
  • 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.
  • the cationic lipid comprises an ionizable cationic lipid.
  • the cationic lipid accounts for 23 mol%-83 mol% of the total lipids present in the composition, such as 30 mol%-80 mol%, 30 mol%-70 mol% or 40 mol%-60 mol%, or the cationic lipid accounts for 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.
  • 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.
  • the neutral lipid accounts for 19 mol%-75 mol% of the total lipids present in the composition.
  • the neutral lipid comprises:
  • a mixture of cholesterol or cholesterol-derived neutral lipids and phospholipids is provided.
  • the cholesterol comprises 14 mol%-70 mol% of the total lipids in the composition.
  • the phospholipids comprise about 5 mol% to about 30 mol% or 30 mol% to about 75 mol% of the total lipids in the composition.
  • composition of the present invention further comprises a lipid conjugate, wherein the lipid conjugate comprises at least one selected from the group consisting of: PEG-lipid conjugate, ATTA-lipid conjugate, polysarcosine-lipid conjugate, polypeptide/protein-lipid conjugate, cation-polymer-lipid conjugate (CPL) and derivatives thereof.
  • the lipid conjugate comprises a PEG-lipid conjugate.
  • the lipid conjugate comprises 0.1 mol%-10.0 mol% of the total lipids in the composition.
  • compositions of the present invention comprise:
  • 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 lipid present in the composition, the phospholipid accounts for 5.0 mol%-50.0 mol% of the total lipid, the cholesterol accounts for 14.0 mol%-64.0 mol% of the total lipid, the lipid conjugate accounts for 0.1 mol%-8.0 mol% of the total lipid, and the amphiphilic block copolymer accounts for 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;
  • a lipid conjugate such as a PEG-lipid conjugate
  • amphiphilic block copolymers cationic lipids, phospholipids, cholesterol, and lipid conjugates such as PEG-lipid conjugates, wherein the cationic lipids account for 23.0 mol%-75.0 mol% of the total lipids present in the composition, the phospholipids account for 10.0 mol%-62.0 mol% of the total lipids, the cholesterol accounts for 14.0 mol%-46.0 mol% of the total lipids, the lipid conjugates account for 0.1 mol%-8.0 mol% of the total lipids, and the amphiphilic block copolymers account 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);
  • 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 accounts for 29.0 mol%-80.0 mol% of the total lipids present in the composition, the phospholipids account for 19.0 mol%-70.0 mol% of the total lipids, the lipid conjugates account 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 (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). Compare);
  • 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%-80.0 mol% of the total lipids present in the composition, the cholesterol accounts for 15.0 mol%-50.0 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 (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;
  • a lipid conjugate such as a PEG-lipid conjugate
  • amphiphilic block copolymers cationic lipids, phospholipids and lipid conjugates such as PEG-lipid conjugates, wherein the cationic lipids account for 30.0 mol%-80.0 mol% of the total lipids present in the composition, the phospholipids account for 10.0 mol%-50.0 mol% of the total lipids, the lipid conjugates account for 0.1 mol%-8.0 mol% of the total lipids, and the amphiphilic block copolymers account 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);
  • an amphiphilic block copolymer a cationic lipid, a phospholipid, and cholesterol
  • the cationic lipid accounts for 30.0 mol%-80.0 mol% of the total lipids present in the composition
  • the phospholipids account for 5.0 mol%-50.0 mol% of the total lipids
  • the cholesterol accounts for 15.0 mol%-50.0 mol% of the total lipids
  • the amphiphilic block copolymer accounts for 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; or
  • 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% weight percent (e.g., 1%-90.0% weight percent, 10%-80.0% weight percent, 20%-80.0% weight percent, 30%-80.0% weight percent or 40%-70.0% weight percent) of the composition.
  • 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
  • the amphiphilic block copolymer accounts for 0.1%-95.0% weight percent (e.g., 1%-90.0% weight percent, 10%-
  • 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.
  • the hydrophilic block is selected from polyoxyalkylenes, polyvinyl alcohol, polyvinyl pyrrolidone, poly(2-methyl-2-oxazoline) and sugars
  • the hydrophobic block is selected from polyoxyalkylenes, lipids, Fatty chains, alkylene polyesters, polyethylene glycol with benzyl polyether ends, and cholesterol
  • the hydrophilic block comprises polyethylene oxide units
  • the hydrophobic block comprises polypropylene oxide units.
  • 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 ethers (BRIJ), polyoxyethylene fatty acid esters, polyoxyethylene fatty alcohol ethers, sorbitan and their derivatives.
  • 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.
  • 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 402, Poloxa
  • 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,
  • 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), a cholesterol derivative coupled to a basic amino acid sequence, and imidazole cholesterol ester (ICE).
  • DC-Choi N,N-dimethyl-N-ethylformamide cholesterol
  • 1,4-bis(3-N-oleylamino-propyl)piperazine N4-argininocholesterol carbonylamide
  • GL67 N4-argininocholesterol carbonylamide
  • ICE imidazole cholesterol ester
  • 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), dipalmitoylphosphat
  • 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, ⁇ -sitosterol, 20 ⁇ -hydroxycholesterol, cholesterol covalently linked to a polypeptide/protein, and derivatives thereof, preferably the cholesterol-derived neutral lipid comprises ⁇ -sitosterol.
  • the PEG-lipid conjugate comprises at least one selected from the following: 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
  • compositions comprise an amphiphilic block copolymer and the following components:
  • composition comprises 40.0 mol% to 70.0 mol% of the total lipids, DSPC, DPPC, DOPS, SOPE, DOPG, DSPE, ESM or DOPE comprises 8.0 mol% to 39.0 mol% of the total lipids, cholesterol or ⁇ -sitosterol comprises 20.0 mol% to 40.0 mol% of the total lipids, DMG-PEG
  • GL67, ICE, or HGT4002 DSPC, DPPC, DOPS, SOPE, DOPG, DSPE, ESM, or DOPE
  • 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
  • the amphiphilic block copolymer accounts for 30.0%-90.0% by weight of the composition
  • composition comprises the following components:
  • an amphiphilic block copolymer DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or ⁇ -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 ⁇ -sitosterol account for 38.5 mol% of the total lipids
  • DMG-PEG2K or DMG-PEG5K account for 1.5 mol% of the total lipids
  • the amphiphilic block copolymer accounts for 89.9% by weight of the composition
  • an amphiphilic block copolymer (2) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or ⁇ -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 ⁇ -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;
  • an amphiphilic block copolymer DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or ⁇ -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 ⁇ -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;
  • an amphiphilic block copolymer DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or ⁇ -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 ⁇ -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;
  • an amphiphilic block copolymer DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or ⁇ -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 ⁇ -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;
  • mol% DSPC, DPPC or DOPE account for 14.1mol% of the total lipids
  • cholesterol or ⁇ -sitosterol account for 35.0mol% of the total lipids
  • DMG-PEG2K or DMG-PEG5K account for 0.9mol% of the total lipids
  • amphiphilic block copolymer accounts for 43.3% by weight of the composition
  • an amphiphilic block copolymer DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or ⁇ -sitosterol; and DMG-PEG2K
  • 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 ⁇ -sitosterol account for 35.0 mol% of the total lipids
  • DMG-PEG2K or DMG-PEG5K account for 0.7 mol% of the total lipids
  • the amphiphilic block copolymer accounts for 48.4% by weight of the composition
  • an amphiphilic block copolymer DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or ⁇ -sitosterol; and DMG-PEG2K
  • 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 ⁇ -sitosterol account for 35.0 mol% of the total lipids
  • DMG-PEG2K or DMG-PEG5K account for 0.5 mol% of the total lipids
  • the amphiphilic block copolymer accounts for 43.6% by weight of the composition
  • an amphiphilic block copolymer DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or ⁇ -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 ⁇ -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;
  • an amphiphilic block copolymer (10) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or ⁇ -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 ⁇ -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;
  • Sterol accounts for 30.0 mol% of the total lipids
  • DMG-PEG2K or DSPE-PEG2K-Mannose accounts for 1.0 mol% of the total lipids
  • the amphiphilic block copolymer accounts for 41.9% by weight of the composition
  • an amphiphilic block copolymer DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or ⁇ -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 ⁇ -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;
  • an amphiphilic block copolymer DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or ⁇ -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 ⁇ -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;
  • an amphiphilic block copolymer DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or ⁇ -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 ⁇ -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;
  • an amphiphilic block copolymer DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or ⁇ -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 ⁇ -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;
  • an amphiphilic block copolymer DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or ⁇ -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 ⁇ -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 The amphiphilic block copolymer accounts for 74.5% by weight of the composition;
  • an amphiphilic block copolymer DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or ⁇ -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 ⁇ -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;
  • an amphiphilic block copolymer DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or ⁇ -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 ⁇ -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;
  • an amphiphilic block copolymer DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or ⁇ -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 ⁇ -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;
  • an amphiphilic block copolymer DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or ⁇ -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 ⁇ -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;
  • an amphiphilic block copolymer DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or ⁇ -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 ⁇ -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;
  • an amphiphilic block copolymer DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or ⁇ -sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 40.6 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 30.1 mol% of the total lipids, cholesterol or ⁇ -sitosterol account for 28.4 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 36.9% by weight of the composition;
  • Amphiphilic block copolymers DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or ⁇ -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 ⁇ -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;
  • an amphiphilic block copolymer DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or ⁇ -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 ⁇ -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;
  • an amphiphilic block copolymer DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or ⁇ -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 ⁇ -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;
  • an amphiphilic block copolymer DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or ⁇ -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 ⁇ -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;
  • an amphiphilic block copolymer DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or ⁇ -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 ⁇ -sitosterol account for 20.0 mol% of the total lipids
  • DMG-PEG2K or DMG-PEG5K account for 0.9 mol% of the total lipids
  • the amphiphilic block copolymer accounts for 21.0% by weight of the composition
  • 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;
  • an amphiphilic block copolymer (4) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; cholesterol or ⁇ -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 ⁇ -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;
  • an amphiphilic block copolymer cKK-E12, DLin-MC3-DMA, ALC-0315, SM-102 or C12-200; DOTAP, DODAP, DOTMA or DOSPA; cholesterol or ⁇ -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 ⁇ -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
  • An amphiphilic block copolymer DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; and cholesterol or ⁇ -sitosterol, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 40.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 32.0 mol% of the total lipids, cholesterol or ⁇ -sitosterol account for 28.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 50.0% weight percent of the composition.
  • the molar ratio of nitrogen (amine) groups in the cationic lipids in the composition to phosphate groups in the nucleic acids is about 1.0 to about 30.0, about 4.0 to about 15.0, about 6.0 to about 8.0, or about 6.7 to about 7.6.
  • the ratio of lipid to nucleic acid (mass/mass ratio) in the composition 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).
  • 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 protein and nucleic acid. In some embodiments, the therapeutic agent is a small molecule.
  • 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 following: glycosyl, lipid, nucleic acid aptamer, small molecule therapeutic agent, vitamin, polypeptide and protein such as antibody.
  • the composition further comprises an adjuvant, preferably the adjuvant comprises at least one selected from the following: CpG oligodeoxynucleotides, polyinosinic: polycytidylic acid, saponin extract (QS-21 extract), aluminum adjuvant, squalene, ⁇ -tocopherol, Tween, Span, lipopolysaccharide LPS, Pam 3 CSK 4 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 immunomodulator proteins.
  • the adjuvant comprises at least one selected from the following: CpG oligodeoxynucleotides, polyinosinic: poly
  • the composition further comprises a transfection enhancer, preferably the transfection enhancer comprises at least one selected from the following: 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, cetyl pyridinium chloride, L-lysine monohydrate, and polylactic acid-glycolic acid copolymer or salt solution.
  • pulmonary surfactant protein preferably the transfection enhancer comprises at least
  • the compositions are in the form of nanoparticles having an average size of about 1000 nm or less.
  • 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.
  • 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.
  • the composition is formulated in the form of 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, atomization or instillation.
  • the present invention provides a method for preparing a composition according to the first aspect of the present invention, the method comprising:
  • the method comprises:
  • the method comprises:
  • the method comprises:
  • the method further comprises the step of removing free lipid components and/or amphiphilic block copolymers, preferably by dialysis and/or tangential flow filtration. component and/or amphiphilic block copolymer.
  • 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.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising the composition according to the first aspect of the present invention and a pharmaceutically acceptable carrier and/or excipient.
  • the present invention provides a method for delivering an active agent or a 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 the therapeutic agent to be taken up into the cell, preferably the cell is a mammalian cell.
  • the present invention provides a method for preventing and/or treating a disease or condition in a mammal, the method comprising administering 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 to a subject in need thereof, wherein the composition or pharmaceutical composition comprises an active agent or therapeutic agent for the disease or condition.
  • 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.
  • 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.
  • 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.
  • Figure 1 Structural characterization of polymer-lipid composites (PoLixNano) and studies in in vitro models.
  • TEM Transmission electron microscopy
  • Figure 2 Study on the transfection efficiency of mRNA encoding firefly luciferase (Fluc-mRNA) in mice mediated by PoLixNano formulation and LNP control formulation via intratracheal spray (i.t.) administration for 6 hours.
  • Fluc-mRNA firefly luciferase
  • FIG. 3 Transfection efficiency study of PoLixNano formulations prepared with different types and concentrations of amphiphilic block copolymers in mice 6 h after intratracheal spray (i.t.) administration.
  • C Transfection efficiency study of PoLixNano formulations prepared with different types and concentrations of amphiphilic block copolymers in mice 6 h after intratracheal spray (i.t.) administration.
  • Figure 4 Study on the transfection efficiency of PoLixNano preparations prepared with different nitrogen-phosphorus ratios (N/P) and different lipid ratios in mice after 6 hours of intratracheal spray (it) administration.
  • Figure 5 Study on the transfection efficiency of PoLixNano formulations containing different types of lipids and lipid ratios in mice 6 hours after administration via intratracheal spray (i.t.).
  • A. Determination of Fluc expression levels in live mice and isolated lungs of PoLixNano formulations containing different types of cationic lipids (Dlin-MC3-DMA, ALC-0315, SM-102, C12-200 and HGT5000) and control LNP formulations (n 3)
  • FIG. 6 Transfection status of PoLixNano preparations prepared with different formulations in mice 6 hours after intranasal (i.n.) administration.
  • A. Quantitative determination of the Fluc bioluminescent signals mediated by PoLixNano preparations 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 preparations in the isolated lungs of mice (n 3); B.
  • Figure 7 Study on the efficient transfection of Fluc-mRNA in mice mediated by the preparations (LNP+KG41, LNP+T904, LNP+10% sucrose, PoLixNano+KG41, PoLixNano+T904 and PoLixNano+10% sucrose) obtained by physical mixing of LNP preparations and PoLixNano preparations with different enhancers (KG41 polypeptide (KG41), T904 and sucrose solution with a final concentration of 10%) through different administration methods (i.t. or i.n.).
  • the preparations obtained by physical mixing of LNP preparations and PoLixNano preparations with different enhancers (KG41 polypeptide (KG41), T904 and sucrose solution with a final concentration of 10%
  • FIG. 8 Transfection efficacy of PoLixNano formulations containing unconventional lipid components and compositions in mice
  • CKK-E12: DOTAP: Chol: DMG-PEG2000 30:39:30:1
  • A. The Fluc expression level in the isolated lung of mice was determined by intratracheal spray (it) and nasal route (in) of PoLixNano preparations without DMG-PEG2000 lipid components and LNP control preparations in living mice and isolated lungs and livers (n 3);
  • FIG. 9 After the PoLixNano preparation was administered via nebulization, it mediated efficient Fluc-mRNA transfection in mice and studied the physicochemical properties of the nanoparticles.
  • A Schematic diagram of the mouse nebulization device and research process;
  • TEM Transmission electron microscopy
  • Figure 10 A study on the efficient mediating Fluc-mRNA transfection in mice of PoLixNano preparations containing different types of amphiphilic block copolymers, different molar ratios of lipid compositions and different dosages after administration via nebulization.
  • A. Quantitative determination of the Fluc bioluminescent signal in the isolated lungs of PoLixNano preparations containing different molar ratios of DMG-PEG2000 prepared with poloxamer 188 (left) or poloxamine 704 (right) and LNP control preparations 6 hours after administration via nebulization (n 3);
  • Fluc bioluminescent signal in the isolated lungs of PoLixNano preparations containing different molar ratios of lipid compositions prepared with poloxamer 188 (left) or poloxamine 704 (right) and LNP control preparations 6 hours after administration via nebulization (n 3);
  • FIG. 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 (i.t.), nasal route (i.n.), and 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.
  • IVIS in vivo imaging technology
  • FIG. 12 Transfection of PoLixNano preparations prepared with different formulations in mice after administration via different routes (i.t., i.n. and nebulization).
  • Exemplary results of the transfection mediated by the PoLixNano formulation and the LNP control formulation in living mice and isolated organs 6 hours after administration via the nasal route i.n.
  • G Representative results of the transfection of the PoLixNano formulation and the LNP control formulation in living mice and isolated organs 6 hours after administration via nebulization inhalation route (nebulization);
  • FIG. 13 Safety study of PoLixNano preparation in mice.
  • A. Six hours after administration of PoLixNano preparation via different routes (intratracheal spray, intranasal spray, and nebulization), the lungs of mice Representative H&E staining results of tissue sections, using control samples of PBS solution and LNP preparation inoculated by it route as negative control and positive control, respectively, scale bar 50 ⁇ m;
  • Figure 14 Fluc-mRNA transfection mediated by PoLixNano formulation and LNP control formulation in C57black 6 (C57BL/6) mice and Sprague-Dawley (SD) rats after administration via different routes (i.t., i.n. and nebulization).
  • A. Exemplary results of transfection induced by the above-mentioned preparations in living C57BL/6 mice and in ex vivo lungs, livers and spleens 6 h after administration by intratracheal spray (i.t.), nasal (i.n.) and nebulization (nebulization) routes (top) and quantitative results of Fluc bioluminescent signals in ex vivo lungs (bottom) (n 3);
  • B Exemplary results of transfection induced by the above-mentioned preparations in living C57BL/6 mice and in ex vivo lungs, livers and spleens 6 h after administration by intratracheal spray (i.t.), nasal (i.n.) and
  • 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).
  • PBS PBS buffer
  • LNP preparation LNP (it)
  • PoLixNano preparation and LNP preparation LNP (im)
  • FIG. 16 RBD-mRNA/PoLixNano formulation induced efficient and long-lasting adaptive cellular immune response in mice after intratracheal spray (i.t.) inoculation.
  • Figure 17 Challenge study of lethal SARS-CoV-2 ancestral strain and omicron mutant strain against mice immunized with PoLixNano preparations inoculated via intratracheal spray (i.t.).
  • A. Survival rate, weight change curve of mice immunized with PBS, LNP (i.m.), LNP (i.t.) and PoLixNano preparations after challenge with SARS-CoV-2 ancestral strain, and SARS-CoV-2 RNA load in lung tissue (Lungs) or nasal turbinates (Nasal turbinates) of the above mice 3 days after challenge (n 8). Samples of mice that were not challenged and did not undergo any intervention were used as controls (Control); B.
  • FIG. 18 PoLixNano preparation mediated Fluc-mRNA transfection in mice and mediated RBD-mRNA to produce efficient adaptive immune response after nasal administration (in).
  • the kinetic curve of protein expression in live mice and isolated lungs after administration of the preparation in vivo (n 3);
  • the 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 preparation (LNP) and PoLixNano preparation 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 BALF of mice 28 days after the first immunization was detected.
  • PBS PBS buffer
  • LNP LNP control preparation
  • PoLixNano preparation 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 BALF
  • Figure 19 Study on the transfection of Fluc-mRNA in mice and the induction of adaptive immune response mediated by PoLixNano preparations after intramuscular injection (i.m.).
  • A. Representative images of Fluc bioluminescence in live mice and different ex vivo organs (liver, spleen) and quantitative analysis of related signals (right) of PoLixNano preparations loaded with Fluc-mRNA and LNP control preparations after intramuscular injection (i.m.) (n 3);
  • the term “substantially” refers to the qualitative condition of exhibiting a target feature or characteristic in full or close to full extent or degree.
  • biological and chemical phenomena rarely, if ever, complete and/or proceed to complete or reach 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.
  • the term “effective” means sufficient to achieve a desired, expected, or intended result.
  • “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.
  • control sample is a sample subjected to the same conditions as a test sample except for the test article.
  • control subject is a subject having the same form of disease as a treated subject and whose age is about the same as that of the treated subject.
  • 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.
  • in vivo refers to events that occur within multicellular organisms such as humans and non-human animals.
  • the term can be used to refer to events that occur within living cells (as opposed to, for example, in vitro systems).
  • gene product refers to the product of a gene such as an RNA transcript, protein, or polypeptide.
  • 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.
  • Lipid granule 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.
  • an active agent or therapeutic agent such as a nucleic acid (e.g., mRNA)
  • 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 them are disclosed in, e.g., U.S. Patent Publication Nos. 20040142025 and 20070042031, the disclosures of which are incorporated herein by reference for all purposes.
  • PoLixNano refers to a stable polymer-lipid composition or polymer-lipid particles or polymer-lipid compositions of nucleic acid loads or polymer-lipid particles of nucleic acid loads.
  • PoLixNano represents a composition made of amphiphilic block copolymers and lipids (e.g., cationic lipids, non-cationic lipids, and lipid conjugates to prevent particle aggregation), wherein the nucleic acid (e.g., mRNA, gRNA, siRNA, aiRNA, miRNA, ssDNA, dsDNA, ssRNA, short hairpin RNA (shRNA), dsRNA, or plasmid, including plasmids transcribed by it interfering RNA) is completely or partially encapsulated in lipids.
  • nucleic acid e.g., mRNA, gRNA, siRNA, aiRNA, miRNA, ssDNA, dsDNA, ssRNA,
  • PoLixNano is a term for referring to nucleic acid-polymer-lipid compositions comprising nucleic acids (e.g., mRNA) encapsulated in polymer-lipid compositions.
  • PoLixNano typically contains amphiphilic block copolymers, cationic lipids, non-cationic lipids, and lipid conjugates (e.g., PEG-lipid conjugates).
  • 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 solutions.
  • PoLixNano is very effective for mucosal applications because they can efficiently penetrate the mucus (mucus penetration) present in large quantities in mucosal tissue sites, 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 silencing of target gene expression in these mucosal-related sites mediated by the therapeutic agent.
  • the loaded therapeutic agent e.g., mRNA
  • target target cells e.g., respiratory epithelial cells, dendritic cells, etc.
  • PoLixNano can also mediate the accumulation of therapeutic agents (e.g., mRNA) in distant sites (e.g., sites on the body separated from the site of administration), and it can mediate the expression of transfected genes or silencing of target gene expression at these distant sites.
  • therapeutic agents e.g., mRNA
  • the polymer-lipid composition particles of the invention 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.
  • the term "delivery” encompasses local delivery and systemic delivery.
  • the delivery of mRNA encompasses situations where the mRNA is delivered to a target tissue and the encoded protein or peptide is expressed and retained in 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 is distributed throughout the body and absorbed by other tissues (also referred to as “systemic distribution” or “systemic delivery”).
  • the patient's circulatory system e.g., serum
  • Distal site refers to a separate site in the body that is not limited to adjacent capillary beds but includes sites that are widely distributed throughout the organism.
  • the term "encapsulation” or grammatical equivalents refers to the process of confining individual mRNA molecules within polymer-lipid nanoparticles.
  • encapsulated can refer to providing a fully encapsulated, partially encapsulated, or both active or therapeutic agents, such as lipid particles of nucleic acids (e.g., mRNA).
  • nucleic acids e.g., mRNA
  • nucleic acids are fully encapsulated in lipid particles (e.g., to form PoLixNano or other nucleic acid-nano particles).
  • cationic lipid refers to any of a number of lipid species carrying a net positive charge at a selected pH value, such as a physiological pH value (e.g., pH is about 7.0). It has been unexpectedly found that cationic lipids comprising alkyl chains with multiple unsaturated sites, for example, at least 2 or 3 unsaturated sites, are 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 Nos.
  • the cationic lipid comprises a protonatable tertiary amine (e.g., pH titrable) head group, a C 18 alkyl chain, an ether bond between the head group and the alkyl chain, and 0 to 3 double bonds.
  • lipids include, for example, DSDMA, DLinDMA, DLenDMA, and DODMA.
  • amphiphilic lipid refers in part to any suitable material, wherein 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 comes from the presence of polar or charged groups such as sugars, 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 hydrocarbons and such groups substituted by one or more aromatic, alicyclic or heterocyclic groups.
  • Examples of amphiphilic compounds include, but are not limited to, phospholipids, amino lipids and sphingolipids.
  • phospholipids include, but are not limited to, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine, Phosphatidylethanolamine, dipalmitoylphosphatidylcholine, dioleoylphosphatidylcholine, distearoylphosphatidylcholine and dilinoleoylphosphatidylcholine.
  • amphipathic lipids Other compounds lacking phosphorus, such as sphingolipids, sphingolipid families, diacylglycerols and beta-acyloxy acids are also included in the group known as amphipathic lipids.
  • amphipathic lipids can be mixed with other lipids, including triglycerides and sterols.
  • neutral lipid refers to any of a number of lipid species that exist as uncharged or neutral zwitterionic forms at a selected pH.
  • lipids include, for example, diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, cephalin, cholesterol, cerebrosides, and diacylglycerol.
  • non-cationic lipid refers to any amphipathic lipid as well as any other neutral lipid or anionic lipid.
  • 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.
  • phosphatidylglycerol cardiolipin
  • diacylphosphatidylserine diacylphosphatidic acid
  • N-dodecanoylphosphatidylethanolamine N-succinylphosphatidylethanolamine
  • N-glutarylphosphatidylethanolamine N-glutarylphosphatidylethanol
  • lipid conjugate refers to a bound lipid that inhibits aggregation of lipid particles.
  • the lipid conjugate includes, but is not limited to, polyamide oligomers (e.g., ATTA-lipid conjugates), poly (ethylene glycol) -lipid conjugates (PEG-lipid conjugates), 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.
  • PEG can be directly conjugated to a lipid or can be connected to a lipid via a linker moiety.
  • Any linker moiety suitable for connecting PEG and a 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.
  • N/P ratio refers to the molar ratio of the positively charged molecular units in the cationic lipids in the polymer-lipid composition relative to the negatively charged molecular units in the mRNA encapsulated in the polymer-lipid composition. Therefore, the N/P ratio is usually calculated as the ratio of the molar number of amine groups in the cationic lipids in the polymer-lipid composition relative to the molar number of phosphate groups in the mRNA encapsulated in the polymer-lipid composition.
  • 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 by one or more aromatic, alicyclic or heterocyclic groups. Suitable examples include, but are not limited to, diacylglycerols, dialkylglycerols, N-N-dialkylamino, 1,2-diacyloxy-3-aminopropane, and 1,2-dialkyl-3-aminopropane.
  • 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 cell nucleus, or the like.
  • aqueous solution refers to a composition comprising, in whole or in part, water.
  • organic lipid solution refers to a composition comprising in whole or in part an organic solvent with lipids.
  • “Isomers” of a first compound are individual compounds wherein each molecule contains the same constituent atoms as the first compound, but wherein the three-dimensional configuration of those atoms is different.
  • 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.
  • the patient or subject is a primate.
  • Non-limiting examples of human subjects are adults, adolescents, infants, and fetuses.
  • 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.
  • “Pharmaceutically acceptable salt” refers to a salt of a compound of the present disclosure that is pharmaceutically acceptable as defined above and that has 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 , cinnamic acid,
  • Pharmaceutically acceptable salts also include those in which the acidic protons present are capable of The base addition salt that can be formed when reacting with an inorganic base or an organic base.
  • 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, etc. It should be recognized that the specific anion or cation that forms a part of any salt of the present disclosure is not critical, as long as the salt is pharmacologically acceptable as a whole.
  • Other examples of pharmaceutically acceptable salts and their preparation methods and methods of use are presented in Handbook of Pharmaceutical Salts: Properties, and Use (PH Stahl & C.G. Wermuth, Verlag Helvetica Chimica Acta, 2002).
  • pharmaceutically acceptable carrier refers to a pharmaceutically acceptable material, composition or vehicle involved in carrying or transporting a medicament, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material.
  • 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, isotonic agents, thickeners or emulsifiers, preservatives, binders, lubricants or coloring agents, sweeteners or flavoring agents, stabilizers, antioxidants, antimicrobial or antifungal agents, osmotic pressure regulators, pH regulators, buffers, chelating agents, cold protection agents and/or plasticizers for a particular dosage form.
  • 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 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 does not yet experience or display any or all of the symptoms or signs of the disease.
  • 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 signs), (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 signs), 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.
  • a “repeat unit” is the simplest structural entity of some material, e.g., a framework and/or a polymer, whether organic, inorganic, or metallo-organic.
  • the repeat units are linked together one after another along the chain, like the beads of a necklace.
  • the repeating unit is -CH2CH2- .
  • the subscript "n” indicates the degree of polymerization, that is, the number of repeating units linked together. When the value of "n" is undefined or in the absence of "n", it simply indicates the repetition of the formula within the brackets and the polymeric nature of the material.
  • repeating unit also applies where the connectivity between repeating units extends three-dimensionally, such as in metal organic frameworks, modified polymers, thermosetting polymers, etc.
  • repeating units may also be described as branching units, inner layers, or generations.
  • end-capping groups may also be described as surface groups.
  • the present invention encompasses the unexpected discovery that the addition of amphiphilic block copolymers such as poloxamine or styrene 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 performance of the LNP formulations.
  • 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/
  • the obtained novel composite preparation (referred to herein as a composition comprising polymer-lipid or PoLixNano) can greatly improve the gene transfection efficiency of nucleic acid molecules (such as mRNA) mediated by such preparations in animals (especially in cells related to respiratory tissues) after administration via the mucosal site of an organism, especially the respiratory tract (tracheal spray IT or nasal drops IN or atomized inhalation), so that the nucleic acid molecules can efficiently produce functional proteins in animal respiratory tract-related tissues and non-lung cells/tissues, thereby achieving the purpose of disease prevention or treatment.
  • nucleic acid molecules such as mRNA
  • Polymer-lipid preparations are particularly suitable for treating or preventing diseases related to respiratory tissue via non-invasive routes of administration (e.g., tracheal spray it, nasal drops in, and atomized inhalation, etc.).
  • non-invasive routes of administration e.g., tracheal spray it, nasal drops in, and atomized inhalation, etc.
  • 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 (e.g., new coronavirus vaccines, influenza virus vaccines, tuberculosis vaccines, etc.), lung gene therapy (e.g., cystic fibrosis, ⁇ 1-trypsin deficiency, etc.), and refractory lung diseases (e.g., lung cancer, pulmonary fibrosis, asthma, chronic obstructive pulmonary disease, etc.), solving the problem that there is currently no ideal delivery system in these fields.
  • mucosal vaccines e.g., new coronavirus vaccines, influenza virus vaccines, tuberculosis vaccines, etc.
  • lung gene therapy e.g., cystic fibrosis, ⁇ 1-trypsin deficiency, etc.
  • refractory lung diseases e.g., lung cancer, pulmonary fibrosis, asthma, chronic obstructive pulmonary disease, etc.
  • polymer-lipid nanoformulations of specific formulations delivered via the respiratory route can be translocated after pulmonary delivery, that is, partially or completely moved from the site of administration (lungs) to the systemic blood supply by active or passive means, and then deposited 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 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.
  • mRNA active ingredients
  • the main transfection organs of LNP preparations or poloxamine or poloxamer preparations administered under the same conditions are limited to the lungs.
  • the present invention provides a composition comprising a polymer-lipid, the composition comprising:
  • an active agent or therapeutic agent preferably the active agent or therapeutic agent comprises a nucleic acid
  • composition is formulated for delivery through a mucosal site of an organism, such as the respiratory tract, oral mucosa, gastrointestinal tract, ocular mucosa, ear mucosa, urethra, or reproductive tract, preferably the composition is formulated for delivery through the respiratory tract.
  • 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.
  • RNA ribonucleic acid
  • mRNA messenger RNA
  • 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 micro RNA (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).
  • the composition comprises a first nucleic acid and a second nucleic acid.
  • the first nucleic acid is an mRNA.
  • the second nucleic acid is a single guide RNA.
  • the first nucleic acid is a messenger RNA (mRNA) and a single guide RNA (sgRNA).
  • RNA is a single-stranded RNA.
  • single-stranded RNA means a single continuous ribonucleotide chain, which is distinguished from RNA of a double-stranded molecule formed by hybridization of two or more separated chains.
  • single-stranded RNA does not exclude that the single-stranded molecule itself forms a double-stranded structure, such as a secondary (e.g., ring and stem-loop) structure or a tertiary structure.
  • RNA encompasses RNAs that encode amino acid sequences as well as RNAs that do not encode amino acid sequences.
  • 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.
  • mRNA Messenger RNA
  • mRNA is a copolymer constructed from phosphate nucleoside building blocks, mainly adenosine, cytidine, uridine and guanosine, which acts as an intermediate carrier to introduce genetic information from DNA in the cell nucleus into the cytoplasm, where it is translated into protein. Therefore, mRNA is suitable as a substitute for gene expression.
  • mRNA should be understood to mean any polyribonucleotide molecule, if it enters a cell, is suitable for protein or its fragmentary expression, or can be translated into protein or its fragment.
  • Term " protein" covers 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.
  • mRNA contains a ribonucleotide sequence, which encodes a protein or a fragment thereof of a function needed or beneficial in or near a cell.
  • mRNA may contain a sequence of a complete protein or its functional variant.
  • a ribonucleotide sequence may encode a protein or its functional fragment acting as a factor, an inducer, a regulator, a stimulant or an enzyme, wherein such a protein is a protein necessary for its function to compensate for an obstacle (particularly a metabolic disorder) or to initiate 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.
  • a functional variant means the 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.
  • mRNA synthesis includes the addition of a "cap” on the 5' end and a “tail” on 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” serves to protect the mRNA from exonuclease degradation.
  • the mRNA includes a 5' cap structure.
  • the mRNA includes a 5' and/or 3' untranslated region.
  • the 5' untranslated region includes one or more elements that affect the stability or translation of the mRNA.
  • the 3' untranslated region includes one or more polyadenylation signals, protein binding sites that affect the localization stability of the mRNA in the cell, or one or more miRNA binding sites.
  • the term mRNA used herein encompasses modified mRNA, i.e., the mRNA can be a modified mRNA.
  • the present invention can be used to formulate and encapsulate unmodified mRNA or mRNA containing one or more modifications that generally enhance stability.
  • the modification is selected from modified nucleotides, modified sugar-phosphate backbones, and 5' and/or 3' untranslated regions.
  • the modification of mRNA may include modification of nucleotides of RNA.
  • the modified mRNA according to the present invention may include, for example, backbone modifications, sugar modifications, phosphate modifications, or base modifications.
  • mRNA may be synthesized from naturally occurring nucleotides and/or nucleotide analogs (modified nucleotides), the naturally occurring nucleotides and/or nucleotide analogs 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.
  • RNA preferably mRNA
  • 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.
  • 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 connected. Nucleotides with different functional groups can also be used to provide binding sites for connecting different groups.
  • 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 ( ⁇ ), 5-methyluridine 5'-triphosphate (m5U), N-1-methyl-pseudouridine (N1m ⁇ ), 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'
  • the RNA, preferably 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'NH 2 ), 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'-triphosphat
  • the RNA, preferably 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'NH 2 ), 2'-azido-2'-deoxyadenosine 5'-triphosphate (A2'N 3 ) and 2'-fluoro-2'-deoxyadenosine 5'-triphosphate (A2'F).
  • m6A N6-methyladenosine 5'-triphosphate
  • m1A N1-methyladenosine 5'-triphosphate
  • A2'm 2'-O-methyladenosine 5'-triphosphate
  • 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'NH 2 ), 2'-azido-2'-deoxyguanosine 5'-triphosphate (G2'N 3 ), 2'-fluoro-2'-deoxyguanosine 5'-triphosphate (G2'F).
  • m1G N1-methylguanosine 5-triphosphate
  • G2'm 2-amino-2-deoxyguanosine 5'-triphosphate
  • G2'N 3 2-azido-2'-deoxyguanosine 5'-triphosphate
  • G2'F 2'-fluoro-2'-deoxyguanosine 5'-triphosphate
  • the invention provides oligonucleotides comprising nucleosides connected.
  • nucleosides can be linked together using any nucleoside bond.
  • the two main categories of nucleoside linking groups are limited by the presence or absence of a phosphorus atom. Compared with natural phosphodiester bonds, modified bonds can be used to change (usually increase) the nuclease resistance of oligonucleotides.
  • nucleoside 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 nucleoside bonds are well known to those skilled in the art.
  • 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,
  • the mRNA is an mRNA containing a combination of modified and unmodified nucleotides.
  • 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.
  • mRNA contains labeled nucleic acid (preferably, nucleotides and/or ribonucleotides), such as, for example, isotope- and/or fluorescently labeled nucleotides.
  • labeled mRNA molecules play an important role in, for example, studying the intracellular conformation of RNA and DNA molecules and the distribution in animals and or cells.
  • mRNA can be synthesized according to any of a variety of known methods.
  • mRNA according to the present invention can be synthesized via in vitro transcription (IVT).
  • 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 chemical synthesis of the corresponding DNA sequence and subsequent transcription thereof in vitro or in vivo.
  • 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.
  • 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.
  • the RNA can be combined with a microRNA or shRNA downstream of the 3' polyA tail.
  • 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).
  • the present invention can be used to prepare and encapsulate mRNAs of various lengths.
  • the present invention can be used to prepare and encapsulate in vitro synthesized mRNAs having a 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.
  • the present invention can be used to prepare and encapsulate in vitro synthesized mRNAs having a length of 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.
  • the present invention can achieve the effect of disease treatment, disease prevention and disease diagnosis through the interaction of ribonucleic acid with cell molecules and organelles.
  • This interaction can activate the innate immune system alone, such as certain CpG oligonucleotides and sequences designed to interact specifically with toll-like receptors and other extracellular or intracellular receptors.
  • ribonucleic acid preferably mRNA
  • the intake or introduction of ribonucleic acid (preferably mRNA) in the cell can be intended to cause nucleotide sequences, such as the expression of genes included in ribonucleic acid (preferably mRNA); it can be intended to be used for the expression of endogenous genes caused by the presence of introduced exogenous nucleic acid in the cell, silencing or knocking down; or it can be intended for the modification of endogenous nucleic acid sequences, such as the repair, excision, insertion or exchange of the whole stretches of the selected base or endogenous nucleic acid sequence; or it can be intended for the presence and interaction of introduced exogenous ribonucleic acid (preferably mRNA) in the cell and actually interfere with any cellular process.
  • nucleotide sequences such as the expression of genes included in ribonucleic acid (preferably mRNA)
  • endogenous genes caused by the presence of introduced exogenous nucleic acid in the cell, sile
  • the overexpression of the exogenous ribonucleic acid (preferred mRNA) introduced can be intended to compensate or complement endogenous gene expression, particularly in the case of endogenous gene defect or silence, causing gene expression product to be absent, insufficient or defective or malfunctioning, multiple metabolic and hereditary diseases, such as cystic fibrosis, hemophilia or muscular dystrophy, etc. are often such situations.
  • the overexpression of the exogenous ribonucleic acid (preferred mRNA) introduced can also be intended to make expression product interact with any endogenous cell process or interfere with any endogenous cell process, such as the regulation, signal transduction and other cell processes of gene expression.
  • the overexpression of the exogenous ribonucleic acid (preferred mRNA) introduced can also be intended to cause immune response in the background of the organism in which the cell of transfection or transduction resides or resides.
  • Example is the genetic modification of antigen presenting cells (such as dendritic cells) so that it presents antigens for vaccination purposes.
  • Other examples are the overexpression of cytokines in tumors, so as to cause tumor-specific immune response.
  • overexpression of introduced exogenous ribonucleic acid (preferably mRNA) can also be aimed at generating transiently genetically modified cells in vivo or ex vivo for cell therapy, such as modified T cells or precursors or stem cells or other cells for regenerative medicine.
  • the present invention can also realize the downregulation, silencing or knocking down of endogenous gene expression for therapeutic purposes by RNA interference (RNAi), using ribozymes, antisense oligonucleotides, tRNA, long double-stranded RNA.
  • RNA interference RNA interference
  • the downregulation, silencing or knocking down of endogenous gene expression of endogenous or pre-existing gene expression can be used to treat acquired, hereditary or spontaneous diseases, including viral infection and cancer.
  • nucleic acid can be introduced into cells as a preventive measure to practice, to prevent, for example, viral infection or tumor formation.
  • gene repair, base or sequence changes can be realized at the genome level and mRNA level (including exon skipping).
  • Base or sequence changes can be realized, for example, by RNA-guided site-specific DNA cutting, by utilizing trans-splicing, trans-splicing ribozymes, chimeric repair bodies (chimeraplasts), the shear and paste mechanism of RNA trans-splicing mediated by spliceosomes, or by utilizing class II or re-targeted introns, or by utilizing viral-mediated insertion mutagenesis or utilizing the targeted genome insertion using prokaryotic, eukaryotic or viral integrase systems.
  • RNA preferably mRNA therapeutic methods.
  • the disorder caused by a single gene mutation such as cystic fibrosis, hemophilia and multiple other diseases can be dominant or recessive.
  • polygenic disorders are caused by two or more genes, and the manifestation of the corresponding disease is often variable and related to environmental factors.
  • the example of polygenic disorder is hypertension, elevated cholesterol levels, cancer, neurodegenerative diseases, mental illness, etc.
  • the therapeutic RNA, preferably mRNA, representing one or more of these genes can be beneficial to those patients.
  • genetic disorders are not necessarily transmitted from parental genes, and may also be caused by new mutations.
  • the therapeutic RNA, preferably mRNA, representing the correct gene sequence can be beneficial to patients.
  • the coding sequence of the RNA, preferably mRNA, of the present invention can be transcribed and translated into a partial or full-length protein comprising a level of cellular activity equal to or greater than that of the native protein.
  • 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 of the immune system, such as hemophilia a and b, and complement deficiencies (such as protein C deficiency), immunodeficiencies such as SCID (caused by mutations in various genes such as RAG1, RAG2, JAK3, IL7R, CD45, CD3 ⁇ , CD3 ⁇ ) or deficiency, due to a lack of adenosine deaminase, such as (ADA-SCID), septic granulomatosis (caused by mutations in the gp-91-pho
  • SPB
  • RNA preferably mRNA
  • Other diseases for which the RNA, preferably mRNA, of the present invention may exert a therapeutic effect 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 leukoreflex atrophy and adrenoleukodystrophy; Friedreich's ataxia; Perelman-Merzbacher 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 tel
  • a protein such as an enzyme
  • RNA preferably mRNA
  • Transcript replacement therapy/enzyme replacement therapy does not affect the underlying genetic defect, but increases the concentration of the enzyme that the patient lacks.
  • GAA acid alpha-glucosidase
  • EPO erythropoietin
  • somatotropin 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.
  • RNA according to the invention contains a coding sequence for cystic fibrosis transmembrane conductance regulator (CFTR), ⁇ 1-antitrypsin, Dynein axonemal intermediate chain 1 (DnaI1), surfactant protein B (SP-B) or erythropoietin.
  • 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.
  • the RNA may contain a ribonucleotide sequence encoding a full-length antibody or nanobody (e.g., both heavy and light chains), which may be used in therapeutic settings, such as to confer immunity to a subject.
  • a full-length antibody or nanobody e.g., both heavy and light chains
  • a full-length antibody or nanobody e.g., both heavy and light chains
  • the RNA 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).
  • a biological target e.g., a stimulatory cytokine such as tumor necrosis factor
  • 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.
  • VEGF vascular endothelial growth factor
  • RNA may contain a ribonucleotide sequence encoding a polypeptide or protein that can be used for genome editing technology.
  • a variety of genome editing systems using 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.
  • CRISPR-Cas systems large-range nucleases (homing nucleases, meganucleases), zinc finger nucleases (ZFNs), and nucleases (TALENs) based on transcription activator-like effectors.
  • ZFNs zinc finger nucleases
  • TALENs nucleases
  • RNA may contain a ribonucleotide sequence encoding a polypeptide or protein of the Cas (CRISPR-associated protein) protein family, preferably Cas9 (CRISPR-associated protein 9).
  • Cas Cas9
  • Proteins of the Cas protein family, preferably Cas9 may be used in CRISPR/Cas9-based methods and/or CRISPR/Cas9 genome editing techniques. Nat. Biotechnol., 2014, 32(4): 347-355 reviews CRISPR-Cas systems for genome editing, regulation and targeting.
  • the RNA may contain a ribonucleotide sequence encoding a transcription activator-like effector nuclease (TALEN).
  • the RNA preferably the mRNA, may contain a ribonucleotide sequence encoding a zinc finger nuclease (ZFN).
  • the RNA preferably the mRNA, may contain a ribonucleotide sequence encoding a meganuclease.
  • RNA contains the ribonucleotide sequence that is not expressed into protein or polypeptide.
  • RNA should not be merely interpreted as meaning any such polynucleotide molecule: if it is introduced into a cell, it can be translated into polypeptide/protein or its fragment.
  • RNA contains the ribonucleotide sequence that is only transcribed into (functional) RNA, wherein the RNA is the final product (and therefore, does not need to be translated).
  • RNA contains the ribonucleotide sequence, which preferably provides the genetic information of the siRNA sequence or another desired ribonucleotide sequence.
  • the present invention provides a method for preparing a therapeutic composition comprising a polymer-lipid as described herein for delivering a specific antigen and/or a nucleic acid encoding a specific antigen, wherein the antigen may be an antigen from bacteria, virus, fungus or cancer cell.
  • 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.
  • 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.
  • polymer-lipid compositions of the invention can be used to treat or prevent a number of diseases and disorders, such as:
  • 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
  • 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
  • the antigens selected for 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).
  • H. pylori Helicobacter pylori, Legionella pneumophilia, Mycobacterium (e.g., M. tuberculosis, Staphylococcus aureus, Neisseria gonorrhoeae, Neisseria meningitidis, Listeria monocytogenes, Streptococcus pyogenes (Group A Streptococcus), Streptococcus agalactiae (Streptococcus agalactiae) (Group B Streptococcus), Streptococcus viridans, Streptococcus pneumoniae, Klebsiella spp. (including K.
  • Mycobacterium e.g., M. tuberculosis, Staphylococcus aureus, Neisseria gonorrhoeae, Neisseria meningitidis, Listeria monocytogenes, Streptococcus pyogenes
  • the antigens selected for 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).
  • Cryptococcus neoformans Cryptococcus neoformans
  • Histoplasma capsulatum Coccidioides immitis
  • Blastomyces dermatitidis Chlamydia trachomatis
  • Candida albicans in which the antigen selected for the vaccine is derived from an existing Those antigens that exist in naturally occurring fungi (or are expressed/induced during infection) (or designed with reference to those antigens).
  • Plasmodium spp. including Plasmodium falciparum, Plasmodium malariae, Plasmodium ovale, and Plasmodium vivax
  • Toxoplasma spp. including T. gondii and T. cruzii
  • Cancers of the blood and lymphatic system including Hodgkin's disease, leukemia, lymphoma, 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 system cancer (including kidney cancer, bladder cancer, testicular cancer, prostate cancer), female cancer (including breast cancer, cervical cancer, ovarian cancer, gynecological cancer and chorio
  • helminths e.g. Schistosoma spp.
  • the antigen useful in the present invention is mRNA.
  • the antigen is naturally combined with the polymer-lipid composition of the present invention in an immunogenic effective amount.
  • 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 protective components at a concentration sufficient to protect the animal from the target disease.
  • an immunogenic effective amount of an antigen an amount of 0.01-100 ⁇ g can be mentioned.
  • the present invention provides a method for preparing a composition as described herein comprising delivering an immunomodulator and/or an mRNA encoding an immunomodulator.
  • the immunomodulator includes, but is not limited to, for example, interleukin 2 (IL-2), interleukin 12 (IL-12), granulocyte-macrophage colony stimulating factor (GM-CSF), interleukin 23 (IL-23), C-C domain chemokine ligand 28 (CCL28), interleukin 36 ⁇ (IL-36 ⁇ ), constitutively active variants of interferon gene (STING) protein stimulators, etc.
  • IL-2 interleukin 2
  • IL-12 interleukin 12
  • GM-CSF granulocyte-macrophage colony stimulating factor
  • IL-23 interleukin 23
  • CCL28 C-C domain chemokine ligand 28
  • IL-36 ⁇ constitutively active variants of interferon gene (STING) protein stimulators, etc.
  • 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, e.g. two or more types of RNA in a single particle.
  • the present invention may be in the form of particles containing one or more types of RNA, or in the form of a mixture of particles containing different types of RNA.
  • the polymer-lipid composition may further comprise one or more proteins.
  • 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 Streptococcus pyogenes (S. pyogenes) Cas9 protein can be found in the SwissProt database under accession number Q99ZW2.
  • the protein in the compositions described herein can be Cas9 (e.g., from Streptococcus pyogenes or Streptococcus pneumoniae (S. pneumonia)).
  • the CRISPR enzyme can direct the cleavage of one or both strands at the location of the target sequence, 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 cleave one or both strands of a target polynucleotide containing the target sequence.
  • an aspartate-to-alanine substitution (D10A) in the RuvC I catalytic domain of Cas9 from Streptococcus pyogenes converts Cas9 from a nuclease that cleaves both strands to a nickase (cleaving a single strand).
  • 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 strands to be nicked and used to induce NHEJ or HDR.
  • 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.
  • EPO erythropoietin
  • G-CSF granulocyte colony stimulating factor
  • ⁇ -galactosidase A ⁇ -L-iduronidase
  • rhASB N-acetylgalactosamine-4-sulfatase
  • TPA tissue-type plasminogen activator
  • IF interferon
  • IF interferon
  • rHGH human growth hormone
  • BHI human insulin
  • FSH follicle-stimulating hormone
  • amphiphilic block copolymers can be used to practice the present invention.
  • 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”.
  • block copolymer refers to a polymer comprising at least two groups or blocks of polymerized monomer units.
  • Block refers to a motif obtained by polymerization of monomers, which may be repeated within the polymer.
  • a block copolymer must contain blocks of at least two different types of polymerized monomers.
  • amphiphilic block copolymer refers to a block copolymer comprising at least one hydrophilic block and at least one hydrophobic block.
  • the blocks are nonionic, ie, do not comprise ion-forming moieties.
  • the amphiphilic block copolymer is a tetrafunctional amphiphilic block copolymer, wherein the tetrafunctional amphiphilic block copolymer comprises four branched block copolymers 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.
  • 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.
  • the hydrophilic block comprises polyethylene oxide units and the hydrophobic block comprises polypropylene oxide units.
  • 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 ethers (BRIJ), polyoxyethylene fatty acid esters, polyoxyethylene fatty alcohol ethers, sorbitan and their derivatives.
  • 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.
  • the poloxamines suitable for the present invention are also known as tetrafunctional nonionic amphiphilic block copolymers.
  • 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.
  • the tetrafunctional nonionic amphiphilic tetrafunctional block copolymer useful in the present invention can be a (A-B)n-C 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 (A-B) groups connected to C.
  • the hydrophilic block A is a polyethylene oxide block (PEO)
  • the hydrophobic block B is a polypropylene oxide block (PPO).
  • the linking moiety C may be an alkylenediamine moiety, preferably an ethylenediamine moiety.
  • the tetrafunctional nonionic amphiphilic block copolymer useful in the present invention may be of the following structure:
  • 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,
  • 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
  • R1 and R2 either (a) both are hydrogen, or (b) one is hydrogen and the other is methyl,
  • R3 and R4 either (a) both are hydrogen, or (b) one is hydrogen and the other is methyl, and
  • R3 and R4 are both hydrogen, then one of R5 and R6 is hydrogen and the other is methyl, or if one of R3 and R4 is methyl, then R5 and R6 are both hydrogen.
  • nonionic amphiphilic tetrafunctional block copolymer useful in the present invention may be of the following structure:
  • 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
  • the value of j is about 2 to about 185, particularly about 10 to about 50, particularly about 10 to about 20, and more particularly equal to or greater than 13,
  • R1 , R2 pair wherein for each R1 , R2 pair, one shall be hydrogen and the other shall be methyl.
  • i may be about 5 to about 125, particularly about 10 to about 100, more particularly about 10 to about 60, and j may be about 5 to about 50, particularly about 10 to about 25, particularly about 10 to about 20, more particularly equal to or greater than 13.
  • nonionic amphiphilic tetrafunctional block copolymers described herein have the structure of the following formula:
  • 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
  • the value of j is about 2 to about 185, particularly about 10 to about 50, particularly about 10 to about 20, and more particularly is equal to or greater than 13,
  • R1 , R2 pair wherein for each R1 , R2 pair, one shall be hydrogen and the other shall be methyl.
  • i may be about 5 to about 125, particularly about 10 to about 100, more particularly about 10 to about 60, and j may be about 5 to about 50, particularly about 10 to about 25, particularly about 10 to about 20, more particularly equal to or greater than 13.
  • 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.
  • the nonionic amphiphilic tetrafunctional block copolymer can 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
  • the nonionic amphiphilic tetrafunctional block copolymer of the present invention may comprise, preferably consists of, an ethylene oxide unit content of about 40%, particularly about 45%, particularly about 45% to about 80%, particularly about 45-70%, more particularly about 45-60%, more preferably about 50%.
  • tetrafunctional nonionic amphiphilic block copolymers of the present invention are generally known by the trade names "poloxamines" or Commercially available.
  • poloxamines suitable for use in the present invention may 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, N.Y., 1967); or US6,353,055.
  • 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, the suitable poloxamine is poloxamine 150R1. In some embodiments, the suitable poloxamine is a combination thereof.
  • amphiphilic block copolymers of the present invention 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.
  • 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.
  • the glycosylated amphiphilic block copolymer comprises at least one terminal block conjugated to a glycosyl moiety, preferably a terminal hydrophilic block, and more preferably at least 25%, especially at least 50%, especially at least 75%, and more especially 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 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.
  • Poloxamer (Poloxamer or )
  • suitable amphiphilic polymers are poloxamer.
  • suitable poloxamer has the following structure (PEO-PPO-PEO, i.e., polyethylene oxide-polypropylene oxide-polyethylene oxide structure):
  • a is an integer between 10 and 150
  • b is an integer between 20 and 60.
  • 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.
  • 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.
  • "reverse-poloxamer” i.e., poloxamer having a polypropylene oxide-polyethylene oxide-polypropylene oxide structure
  • a suitable "reverse-poloxamer” has the following structure (PPO-PEO-PPO):
  • a is an integer between 10 and 150, and b is an integer between 20 and 60.
  • 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.
  • 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.
  • 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.
  • 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 one 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the amphiphilic block copolymers of the present invention 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.
  • 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.
  • 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, and more preferably at least 25%, especially at least 50%, of the glycosylated amphiphilic block copolymer.
  • the glycosyl moiety may be conjugated to the block copolymer of the invention via a covalent bond formed between one functional group of the glycosyl moiety and one functional group of the block copolymer.
  • the covalent bond may be formed by a reaction between two functional groups which are themselves modified to be reactive, and the glycosyl moiety may be conjugated directly to the block copolymer.
  • the glycosyl moiety may be conjugated to the block copolymer via a spacer.
  • the amphiphilic block copolymer is polyvinylpyrrolidone (PVP), such as PVP having a molecular weight of 3 kDa, 10 kDa, or 29 kDa.
  • PVP polyvinylpyrrolidone
  • 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.
  • amphiphilic block copolymer is a polyethylene glycol ether.
  • a suitable polyethylene glycol ether is a compound of formula (S-1):
  • R 1BRIJ is C alkyl.
  • the polyethylene glycol ether is a compound of formula (S-1a):
  • S is an integer between 1 and 100.
  • R 1BRIJ is C alkenyl.
  • a suitable polyethylene glycol ether is a compound of formula (S-1b):
  • S is an integer between 1 and 100.
  • amphiphilic block copolymers of the present invention 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.
  • 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.
  • the glycosylated amphiphilic block copolymer comprises at least one terminal block conjugated to a glycosyl portion, preferably a terminal hydrophilic block, and more preferably at least 25%, especially at least 50%, especially at least 75%, and more especially 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 modified to be reactive, and the glycosyl portion can be directly conjugated to the block copolymer.
  • the glycosyl moiety can be conjugated to the block copolymer via a spacer.
  • cationic lipid refers to any of a number of lipid and lipidoid species having a net positive charge at a selected pH, such as physiological pH.
  • the cationic lipid of the present invention comprises at least one selected from the group consisting of permanent cationic lipids, ionizable cationic lipids, cholesterol-derived cationic lipids, and dendritic polymers 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-016B, 9A1P9, C12-200, cKK-E12, OF-Deg-Lin, 306Oi10, TT3, FTT5, Lipid319, 5A2-SC8, Genevan CL1, DLinDMA, DLenDMA, ClinDMA, CpLinDMA, imidazole cholesterol ester (ICE), DC-Choi (N
  • cationic lipid preparations are available and can be used in the present invention. These include, for example, (commercially available cationic liposomes comprising DOTMA and DOPE from GIBCO/BRL, Grand Island, New York, USA); (commercially available cationic liposomes comprising DOSPA and DOPE from GIBCO/BRL); and (Commercially available cationic liposomes containing DOGS are from Promega Corp., Madison, Wisconsin, USA).
  • the cationic lipids included in the compositions and methods of the present invention can be defined as compounds having the following structure or salts thereof:
  • R1 and R2 are independently selected and are H or C1 - C3 alkyl
  • R3 and R4 are independently selected and are alkyl groups having from about 10 to about 20 carbon atoms, and at least one of R3 and R4 includes at least 2 sites of unsaturation.
  • R 3 and R 4 are the same, that is, R 3 and R 4 are linoleyl (C 18 ) and the like. In some other instances, R 3 and R 4 are different, that is, R 3 is tetradectrienyl (C 14 ) and R 4 is linoleyl (C 18 ).
  • the cationic lipid of Formula I is symmetrical, that is, R 3 and R 4 are the same. In another preferred embodiment, R 3 and R 4 both include at least 2 unsaturated sites.
  • R 3 and R 4 are independently selected from the group consisting of dodecadienyl, tetradecadienyl, hexadecadienyl, linoleyl, and icosadienyl. In a preferred embodiment, R 3 and R 4 are linoleyl. In some embodiments, R3 and R4 include at least 3 sites of unsaturation and are independently selected from, for example, dodecatrienyl, tetradecatrienyl, hexadecatrienyl, linolenyl, and icosatrienyl.
  • the cationic lipid of Formula I is 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA) or 1,2-dilinolenoyloxy-N,N-dimethylaminopropane (DLenDMA).
  • DLinDMA 1,2-dilinoleyloxy-N,N-dimethylaminopropane
  • DLenDMA 1,2-dilinolenoyloxy-N,N-dimethylaminopropane
  • a cationic lipid having the following structure (or a salt thereof) can be effectively used in the present invention.
  • R1 and R2 are the same or different and are independently optionally substituted C12 - C24 alkyl, optionally substituted C12 - C24 alkenyl, optionally substituted C12 - C24 alkynyl, or optionally substituted C12 - C24 acyl;
  • R3 and R4 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 R3 and R4 may combine to form an optionally substituted heterocyclic ring of 4-6 carbon atoms and 1 or 2 heteroatoms selected from nitrogen and oxygen;
  • R 5 is absent or is hydrogen or C 1 -C 6 alkyl to provide a quaternary amine
  • n, 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.
  • 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]-di
  • compositions and methods of the present invention include permanent cationic lipids comprising quaternary ammonium ions.
  • permanent cationic lipids have the following general formula:
  • R1 and R2 are each independently C8 - C24 alkyl, C8 - C24 alkenyl, or a substituted form of either group;
  • R 3 , R 3 ′ and R 3 ′′ are each independently alkyl (C ⁇ 6) or substituted alkyl (C ⁇ 6) ;
  • X ⁇ is a monovalent anion
  • the cationic lipids of the compositions and methods of the present invention are further defined as compounds having the following structure or salts thereof:
  • R1 and R2 are each independently C8 - C24 alkyl, C8 - C24 alkenyl, or a substituted form of either group;
  • R 3 , R 3 ′ and R 3 ′′ are each independently alkyl (C ⁇ 6) or substituted alkyl (C ⁇ 6) ;
  • R4 is alkyl (C ⁇ 6) or substituted alkyl (C ⁇ 6) ;
  • X ⁇ is a monovalent anion
  • the cationic lipids of the compositions and methods of the present invention are further defined as compounds having the following structure or salts thereof:
  • R1 and R2 are independently selected and are H or C1 - C3 alkyl
  • R3 and R4 are independently selected and are alkyl groups having from about 10 to about 20 carbon atoms, and at least one of R3 and R4 includes at least 2 sites of unsaturation.
  • R 3 and R 4 are all the same, that is, R 3 and R 4 are all linoleyl (C 18 ), etc. In some other examples, R 3 and R 4 are different, that is, R 3 is tetradecatrienyl (C 14 ) and R 4 is linoleyl (C 18 ).
  • the cationic lipid of the present invention is symmetrical, that is, R 3 and R 4 are all the same. In another preferred embodiment, R 3 and R 4 both include at least 2 unsaturated sites.
  • R 3 and R 4 are independently selected from the group consisting of dodecadienyl, tetradecadienyl, hexadecadienyl, linoleyl and eicosadienyl. In a preferred embodiment, R 3 and R 4 are all linoleyl. In some embodiments, R 3 and R 4 include at least 3 unsaturated sites and are independently selected from, for example, dodecatrienyl, tetradecatrienyl, hexadecatrienyl, linoleyl and eicosadienyl.
  • the cationic lipids of the compositions and methods of the present invention are further defined as compounds having the following structure or salts thereof:
  • R 4 and R 4 ' are each independently C 6 -C 24 alkyl, C 6 -C 24 alkenyl, or a substituted form of either group;
  • R 4 ′′ is alkyl (C ⁇ 24) , alkenyl (C ⁇ 24) , or a substituted form of either group;
  • R 4 ′′′ is C 1 -C 8 alkyl, C 2 -C 8 alkenyl, or a substituted form of either group;
  • X2 is a monovalent anion.
  • the permanent cationic lipid of the compositions and methods of the present invention is further defined as a compound having the following structure or a salt thereof:
  • Y 1 , Y 2 or Y 3 are each independently X 1 C(O)R 1 or X 2 N + R 3 R 4 R 5 ; provided that at least one of Y 1 , Y 2 and Y 3 is X 2 N + R 3 R 4 R 5 ;
  • R1 is C1 - C24 alkyl, C1 - C24 substituted alkyl, C1 - C24 alkenyl, C1 - C24 substituted alkenyl;
  • X1 is O or NR a , wherein R a is hydrogen, C 1 ⁇ C 4 alkyl or C 1 ⁇ C 4 substituted alkyl;
  • X2 is C1 - C6 alkanediyl or C1 - C6 substituted alkanediyl;
  • R 3 , R 4 and R 5 are each independently C 1 -C 24 alkyl, C 1 -C 24 substituted alkyl, C 1 -C 24 alkenyl, C 1 -C 24 substituted alkenyl;
  • A1 is an anion having a charge equal to the number of X2N + R3R4R5 groups in the compound.
  • Suitable cationic lipids for use in the compositions and methods of the present invention include cationic lipids as described in International Patent Publication WO 2010/144740 and U.S. Patent 8,058,069, which are incorporated herein by reference.
  • 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.
  • compositions and methods of the present invention include a cationic lipid of one of the following formulas:
  • R 1 and R 2 are each independently selected from the group consisting of hydrogen, optionally substituted different saturated or unsaturated C 1 -C 20 alkyl groups, and optionally substituted different saturated or unsaturated C 6 -C 20 acyl groups; wherein L 1 and L 2 are each independently selected from the group consisting of hydrogen, optionally substituted C 1 -C 30 alkyl groups, optionally substituted different unsaturated C 1 -C 30 alkenyl groups, and optionally substituted C 1 -C 30 alkynyl groups; wherein m and o are each independently selected from the group consisting of: zero and any positive integer (eg, where m is three); and where n is zero or any positive integer (eg, where n is one).
  • the polymer-lipid composition provided herein comprises a cationic lipid of the formula:
  • p is an integer between 1 and 9, inclusive;
  • R 2 is independently hydrogen or optionally substituted C 1-6 alkyl
  • R 6 and R 7 are independently a group of formula (i), (ii) or (iii);
  • R' is independently hydrogen or optionally substituted alkyl
  • X is O, S or NR x , wherein RX 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;
  • Y is O, S or NR Y , wherein RY 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;
  • R 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.
  • protecting group; and RL is optionally substituted C1-50 alkyl, optionally substituted C2-50 alkenyl, optionally substituted C2-50 alkynyl, optionally substituted heteroC1-50 alkyl, optionally substituted heteroC2-50 alkenyl, optionally substituted heteroC2-50 alkynyl, or a polymer.
  • compositions and methods of the invention include a cationic lipid having the formula:
  • 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.
  • compositions and methods of the present invention include a cationic lipid having the formula:
  • each R 1 and R 2 are independently H or C 1 -C 6 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 is independently an ester, a thioester, a disulfide bond or an anhydride group; each L 2 is independently C 2 -C 10 aliphatic; each X 1 is independently H or OH; and each R 3 is independently C 6 -C 20 aliphatic.
  • compositions and methods of the present invention include cationic lipids as described in International Patent Publication WO2017/075531, which is incorporated herein by reference.
  • compositions and methods of the present invention include cationic lipids having the formula:
  • 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.
  • compositions and methods of the invention include a compound having the following structure: BL 1 -S,
  • B is a basic functional group, wherein the protonated form has a pKa of no more than about 8.0;
  • L 1 is an optionally substituted linking group, which is a C 1 -C 20 alkylene group or a 2 to 20-membered heteroalkylene group;
  • B is an optionally substituted 5-membered or 6-membered nitrogen-containing heteroaryl.
  • B is a group selected from pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, pyridyl, pyrimidinyl, pyrazinyl and pyridazinyl, each of which is optionally substituted.
  • L1 comprises a moiety that is an ester, amide, carbamate, carbonate or urea group, and L1 does not comprise a substituent having the structure -N(R') 2 , or a positively charged form thereof, wherein each R' is independently hydrogen or an optionally substituted C1 - C20 alkyl group.
  • 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.
  • S is a sterol selected from the group consisting of cholesterol, an oxidized form of cholesterol, a reduced form of cholesterol, an alkyl lithochlate, stigmasterol, stigmasterol, campesterol, ergosterol and sitosterol.
  • compositions and methods of the invention include a compound having the structure
  • n is 0 or 1, preferably 0,
  • R 1 is a group –(CH 2 ) q -NH 2 or a group –(CH 2 ) r -NH-(CH 2 ) s -NH 2 ,
  • R 2 is a group –(CH 2 ) t -NH 2 or a group –(CH 2 ) u -NH-(CH 2 ) w -NH 2 ,
  • t, u and w are independently integers from 2 to 6,
  • R3 is a straight chain alkanediyl group having 1 to 4 carbon atoms
  • cationic lipids based on cholesterol can be used as a replacement of cationic lipids as described herein or in addition to cationic lipids as described herein.
  • Suitable cationic lipids based on sterols are cationic lipids based on sterols containing dialkylamino, imidazoles, basic amino acid sequences and guanidine.
  • compositions comprising one or more cationic lipids based on sterols comprising imidazoles, such as imidazole cholesterol ester or "ICE" lipid (3S, 10R, 13R, 17R)-10,13-dimethyl-17-((R)-6-methylheptane-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopentadien[a]phenanthrene-3-yl 3-(1H-imidazole-4-yl) propionate.
  • imidazoles such as imidazole cholesterol ester or "ICE" lipid
  • imidazoles such as imidazole cholesterol ester or "ICE" lipid
  • the cholesterol-derived cationic lipid comprises at least one selected from the following: 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. Patent No. 5,744,335), N4-arginine cholesterol carbonylamide (GL67), cholesterol derivatives coupled to basic amino acid sequences, imidazole cholesterol ester (ICE) and their derivatives.
  • 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,
  • 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.
  • compositions and methods of the present invention include a cationic lipid having the formula:
  • R 1 is selected from the group consisting of imidazole, guanidine, amino, imine, enamine, optionally substituted alkylamino (e.g., alkylamino such as dimethylamino) and pyridyl; wherein R 2 is selected from the group consisting of one of the following two general formulae:
  • R 3 and R 4 are each independently selected from the group consisting of: optionally substituted different saturated or unsaturated C 6 _C 20 alkyl groups and optionally substituted different saturated or unsaturated C 6 _C 20 acyl groups; 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).
  • 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.
  • the compositions and methods of the present invention include cationic lipids, which are any of the general formulas described in WO2019222424A1 or structures (1a)-(21a) and (1b)-(21b) and (22)-(237).
  • the compositions and methods of the present invention include cationic lipids having a structure according to the formula,
  • RX is independently -H, -L1-R1 or -L5A-L5B-B'; L1 , L2 and L3 are each independently a covalent bond, -C(O)-, -C(O)O-, -C(O)S- or -C(O)NRL-; each L4A and L5A are independently -C(O)-, -C(O)O- or -C(O)NRL-; each L4B and L5B are independently C1 -C20 alkylene; C2 - C20 alkenylene; or C2 -C20 alkynylene; each B and B' is NR4R5 or a 5-10 membered nitrogen-containing heteroaryl group; each R1, R2 and R3 are independently C6 - C30 alkyl, C6-C30 alkenyl or C6- C30 alkynyl; each R4 and R5 are independently C6- C30
  • each RL is independently hydrogen, C 1 -C 20 alkyl, C 2 -C 20 alkenyl or C 2 -C 20 alkynyl.
  • 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.
  • the cationic lipid compositions and methods of the present invention comprise one or more lipid-modified spermine derivatives having the following general formula:
  • X 1 is -(CH 2 ) n - or a carbonyl group, wherein n is 1, 2 or 3;
  • X 2 is selected from -(CH 2 )-, an ester group, an amide group, oxygen or sulfur;
  • R1 and R2 are independently selected from C6 - C18 alkyl, C6 - C18 alkyl containing olefinic bonds or lipophilic cholesterol molecules;
  • X1 and X2 are both -( CH2 )-, and R1 and R2 are independently selected from C10 - C18 alkyl groups.
  • X 1 is -(CH 2 ) 2 -
  • X 2 is oxygen
  • R 1 and R 2 are the same C 12 -C 18 alkyl group or a C 6 -C 18 alkyl group containing an olefinic bond.
  • X 1 is a carbonyl group
  • X 2 is -(CH 2 )-
  • R 1 and R 2 are the same C 12 -C 18 alkyl group or a C 6 -C 18 alkyl group containing an olefinic bond.
  • X 1 is -(CH 2 ) 2 -
  • X 2 is an ester group
  • R 1 and R 2 are the same C 12 -C 18 alkyl group or a C 6 -C 18 alkyl group containing an olefinic bond.
  • X 1 is -(CH 2 )-
  • X 2 is an amide group
  • R 1 and R 2 are the same C 12 -C 18 alkyl group or a C 6 -C 18 alkyl group containing an olefinic bond.
  • the lipid-modified spermine derivative of the invention is composed of spermine and oleyl alcohol coupled by different chemical bonds, wherein spermine is used as a head group with positive charge, and oleyl alcohol is linked to two tertiary amine groups in the middle.
  • Patent application publication number CN104876831 teaches some non-limiting examples of lipid-modified spermine derivatives that can be used in the present disclosure, and the patent application publication number CN104876831 is incorporated herein by reference.
  • compositions and methods of the 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).
  • DOTMA cationic lipid N-[l-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride
  • 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-carboxyamide)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”).
  • DOGS 5-carboxysperminylglycine dioctadecylamide
  • DOSPA 2,3-dioleyloxy-N-[2-(spermine
  • 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-di
  • the one or more cationic lipids comprise at least one of an imidazole, dialkylamino, or guanidinium moiety.
  • one or more cationic lipids suitable for use in the compositions and methods of the invention include 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane ("XTC"); (3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)-octadecane-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").
  • XTC 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-diox
  • Suitable cationic lipids for the compositions and methods of the present invention include compounds of the following formula and pharmaceutically acceptable salts thereof:
  • RCOO is selected from the list comprising: myristoyl, alpha-D-tocopheryl succinyl, linoleoyl, and oleoyl; and X is selected from the list comprising:
  • 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.
  • Suitable cationic lipids for use in the compositions and methods of the present invention include those described in J. McClellan, M.C. King, Cell 2010, 141, 210-217 and Whitehead et al., Nature Communications (2014) 5:4277, which are incorporated herein by reference.
  • 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.
  • 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.
  • 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.
  • one or more of the cationic lipids may exist as structural isomers.
  • the compound has the same chemical formula, but is different from the connectivity of the nitrogen atom of the core.
  • structural isomers may present a fully reacted primary amine, and then present a mixture of reacted secondary amines.
  • Chemical formula for representing cationic lipid of the present invention will usually only show one of several possible different tautomers.For example, many types of keto groups are known to exist in equilibrium with the corresponding enol groups. Similarly, many types of imino groups exist in equilibrium with the enamine groups. Whether for a given formula, which tautomer is described, and whether which tautomer is the most common, all tautomers of a given chemical formula are meant.
  • the atoms constituting the cationic lipids of the present invention are intended to include all isotopic forms of such atoms.
  • Isotopes used herein include those atoms with the same atomic number but with different mass numbers.
  • the isotopes of hydrogen include tritium and deuterium
  • the isotopes of carbon include 13 C and 14 C.
  • the cationic lipid comprises about 23 mol% to about 83 mol% of the total lipids present in the composition.
  • the cationic lipid accounts for 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.
  • the cationic lipid accounts for 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.
  • the cationic lipid accounts for about 25 mol% to about 60 mol%, such as 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.
  • the cationic lipid accounts for about 25 mol% to about 50 mol%, such as about 30 mol% to about 50 mol%, about 35 mol% to about 50 mol%, or about 50 mol% of the total lipids present in the composition. About 40 mol% to about 50 mol% or about 45 mol% to about 50 mol%.
  • 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.
  • 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 (such as in the case of nucleic acid loading) assays, etc.
  • ERP endosomal release parameter
  • in vivo gene transfection efficiency such as in the case of nucleic acid loading
  • 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.
  • the phrase "non-cationic lipid” refers to any neutral, zwitterionic or anionic lipid.
  • anionic lipid refers to any of a variety of lipid species that carry a net negative charge at a selected pH such as a physiological pH.
  • the neutral lipids account for 19 mol%-75 mol% of the total lipids present in the composition, such as 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%.
  • the neutral lipids account for 20 mol%-65 mol% of the total lipids present in the composition, 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%.
  • the neutral lipids account for 20 mol%-60 mol% of the total lipids present in the composition, 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%.
  • the neutral lipids account for 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.
  • the neutral lipids account for 20% of the total lipids present in the composition.
  • mol%-45mol% for example 25mol%-45mol%, 30mol%-45mol%, 35mol%-45mol% or 40mol%-45mol%, or 20mol%-40mol%, for example 25mol%-40mol%, 30mol%-40mol% or 35mol%-40mol%.
  • the neutral lipid comprises:
  • a mixture of cholesterol or cholesterol-derived neutral lipids and phospholipids is provided.
  • the cholesterol accounts for 14 mol%-70 mol% of the total lipids in the composition, for example, 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%.
  • the phospholipids account for about 5 mol% to about 75 mol% of the total lipids in the composition, such as 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%, 10 mol% to 50 mol%, 10 ...
  • %-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%.
  • 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), dipalmitoylphosphatidylphosphatidylcholine (DPSC), dioleoylphosphatidylethanolamine (DOPE), dioleo
  • DPPC dioleoylphosphatidylglycerol
  • DOPG dipalmitoylphosphatidylglycerol
  • POPC palmitoyloleoyl-phosphatidylcholine
  • POPE palmitoyloleoyl-phosphatidylethanolamine
  • POPG palmitoyloleoyl-phosphatidylglycerol
  • DGTS 1,2-Dipalmitoyl-sn-glycero-3-O-4'-(N,N,N-trimethyl)-homoserine
  • MGDG monogalactosyldiacylglycerol
  • DGDG diacetyldiacylglycerol
  • SQLDG sulfaquinolinediacylglycerol
  • 16-O-monomethyl PE 16-O-dimethyl PE, 18-1-trans PE and their derivatives.
  • compositions and methods of the invention include a non-cationic lipid having the formula:
  • R 1 and R 2 are each independently C 8 -C 24 alkyl, C 8 -C 24 alkenyl or a substituted form of either group;
  • R 3 , R 3 ′ and R 3 ′′ are each independently alkyl (C ⁇ 6) or substituted alkyl (C ⁇ 6) ;
  • X ⁇ is a monovalent anion.
  • R 1 is C 8 -C 24 alkenyl or substituted C 8 -C 24 alkenyl.
  • R 2 is C 8 -C 24 alkenyl or substituted C 8 -C 24 alkenyl.
  • R 1 is C 8 -C 24 alkyl or substituted C 8 -C 24 alkyl.
  • R 2 is C 8 -C 24 alkyl or substituted C 8 -C 24 alkyl.
  • both R 1 and R 2 are the same.
  • R 3 , R 3 ′, and R 3 ′′ are each the same. In some embodiments, R 3 , R 3 ′, and R 3 ′′ are each methyl. In some embodiments, X - is a halide anion such as bromide or chloride.
  • compositions and methods of the invention include a phosphoglyceride or a salt thereof of the formula:
  • R4 is a straight chain alkyl group having 10 to 24 carbon atoms or a straight chain alkyl group having 1 to 3 double bonds and Straight-chain alkenyl groups of 10 to 24 carbon atoms;
  • R5 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;
  • compositions and methods of the invention include an anionic lipid of the formula:
  • R1 and R2 are each independently alkyl (C8-C24) , alkenyl (C8-C24) or a substituted form of either group;
  • R3 is hydrogen, alkyl (C ⁇ 6) or substituted alkyl (C ⁇ 6) or -Y1 - R4 , wherein: Y1 is alkanediyl (C ⁇ 6) or substituted alkanediyl (C ⁇ 6) ; and R4 is acyloxy (C ⁇ 8-24) or substituted acyloxy (C ⁇ 8-24) .
  • non-cationic lipids suitable for use in the present invention include phosphorus-free lipids, such as stearylamide, dodecylamine, hexadecylamine, acetylapalmitate, glyceryl ricinoleate, hexadecyl stearate, isopropyl myristate, amphoteric acrylic acid polymers, triethanolamine-lauryl sulfate, alkyl-aryl sulfate polyethoxylated fatty acid amides, distearyl dimethyl bromide, ceramides, sphingomyelin and their derivatives, etc.
  • phosphorus-free lipids such as stearylamide, dodecylamine, hexadecylamine, acetylapalmitate, glyceryl ricinoleate, hexadecyl stearate, isopropyl myristate, amphoteric acrylic acid
  • 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, ⁇ -sitosterol, 20 ⁇ -hydroxycholesterol, polypeptide/protein covalently modified cholesterol, and derivatives thereof, wherein the synthesis of cholesteryl-2'-hydroxyethyl ether is described in U.S. Pat. No. 8,058,069, the disclosure of which is incorporated herein by reference for all purposes.
  • the cholesterol-derived lipid comprises ⁇ -sitosterol.
  • compositions and methods of the present invention include a steroid having a ring structure comprising three fused cyclohexyl rings and a fused cyclopentyl ring, as shown in the following formula:
  • the steroid derivative comprises a moiety having one or more non-alkyl substitutions.
  • the steroid or steroid derivative is a sterol, wherein the formula is further defined as:
  • the steroid or steroid derivative of the compositions and methods of the present invention is cholestane or a cholestane derivative.
  • the ring structure is further defined by the formula:
  • cholestane derivatives include non-alkyl substitutions of one or more of the above ring systems.
  • the cholestane or cholestane derivative is cholestene or a cholestene derivative or a sterol or a sterol derivative.
  • the cholestane or cholestane derivative is cholestene and a sterol or a derivative thereof.
  • the composition further comprises a lipid conjugate, wherein the lipid conjugate comprises at least one selected from the group consisting of a poly(ethylene glycol)-lipid conjugate (PEG-lipid conjugate or PEG-lipid), an ATTA-lipid conjugate, a polysarcosine-lipid conjugate, a polypeptide/protein-lipid conjugate, and a cation-polymer-lipid conjugate (CPL), preferably, the lipid conjugate comprises a PEG-lipid conjugate.
  • PEG-lipid conjugate or PEG-lipid poly(ethylene glycol)-lipid conjugate
  • ATTA-lipid conjugate ATTA-lipid conjugate
  • a polysarcosine-lipid conjugate a polypeptide/protein-lipid conjugate
  • CPL cation-polymer-lipid conjugate
  • the lipid conjugate is a PEG-lipid.
  • PEG-lipids include, but are not limited to, PEG coupled to a dialkoxypropyl group (PEG-DAA) as described in, for example, PCT Publication No. WO 05/026372, PEG coupled to diacylglycerol (PEG-DAG) as described in, for example, U.S. Patent Publication Nos. 20030077829 and 2005008689, PEG coupled to a phospholipid such as phosphatidylethanolamine (PEG-PE), 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.
  • PEG-lipids include, but are not limited to, PEG coupled to a dialkoxypropyl group (PEG-DAA) as described in, for example, PCT Publication No. WO 05/026372, PEG coupled to diacylglycerol (PEG
  • 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-diethylacetamide), and mixtures thereof.
  • the PEG-lipid described herein can be synthesized as described in International Patent PCT/US2016/000129.
  • the PEG-lipid that can be used in the present invention can be a PEGylated lipid described in International Patent WO2012099755, the disclosures of which are incorporated herein by reference in their entirety for all purposes.
  • 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.
  • 6,774,180 and 7,053,150 are also effective for preparing the PEG-lipid conjugates of the present invention.
  • mPEG (20 KDa) amine e.g., 6,774,180 and 7,053,150
  • mPEG (20 KDa) amine e.g., 6,774,180 and 7,053,150
  • mPEG (20 KDa) amine e.g., 6,774,180 and 7,053,150
  • mPEG (20 KDa) amine e.g., mPEG (20 KDa) amine
  • MePEG-CH 2 COOH monomethoxypolyethylene glycol-acetic acid
  • the PEG moiety of the PEG-lipid conjugate 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 a preferred embodiment, the PEG moiety has an average molecular weight of about 5,000 Daltons or about 2,000 Daltons or about 750 Daltons.
  • PEG can be optionally substituted with alkyl, alkoxy, acyl or aryl groups.
  • PEG can be directly conjugated to the lipid or can be connected to the lipid through a linker moiety.
  • Any linker moiety suitable for coupling PEG to the lipid can be used including, for example, non-ester-containing linker moieties and ester-containing linker moieties.
  • the linker moiety is a non-ester-containing linker moiety.
  • non-ester-containing linker moiety refers to a linker moiety that does not contain a carboxylate bond (-OC(O)-).
  • Suitable non-ester-containing linkers are preferably non-ester-containing linkers.
  • 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)-), succinamido (-NHC(O) CH2CH2C (O)NH-), and combinations thereof (such as linkers comprising both carbamate linker moieties and amine linker moieties).
  • a carbamate linker is used to couple the PEG and lipid.
  • 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.
  • Phosphatidylethanolamines with various acyl chain groups of different chain lengths and degrees of saturation can be conjugated with PEG to form lipid conjugates.
  • Such phosphatidylethanolamines are commercially available, or can be separated or synthesized using conventional techniques known to those skilled in the art.
  • Phosphatidyl-ethanolamines containing saturated or unsaturated fatty acids with a preferred carbon chain length in the range of C10 - C20 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), dioleoyl-phosphatidylethanolamine (DOPE) and distearoyl-phosphatidylethanolamine (DSPE).
  • DMPE dimyristoyl-phosphatidylethanolamine
  • DPPE dipalmitoyl-phosphatidylethanolamine
  • DOPE dioleoyl-phosphatidylethanolamine
  • DSPE distearoyl-phosphatidylethanolamine
  • the PEG-lipid conjugate of the compositions and methods of the present invention comprises a PEGylated phosphoglyceride of the formula:
  • p is an integer from 5 to 200, preferably from 10 to 170, and most preferably from 10 to 140;
  • R6 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;
  • R7 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;
  • the compositions and methods of the invention has the formula:
  • R 12 and R 13 are each independently alkyl (C ⁇ 24) , alkenyl (C ⁇ 24) , or a substituted form of any of these groups; Re is hydrogen, alkyl (C ⁇ 8) , or substituted alkyl (C ⁇ 8) ; and x is 1-250. In some embodiments, Re is alkyl (C ⁇ 8) such as methyl. R 12 and R 13 are each independently 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.
  • the lipid conjugate of the compositions and methods of the invention has the formula:
  • n1 is an integer between 1-100, and n2 and n3 are each independently selected from an integer between 1-29.
  • 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.
  • n1 is about 30 to about 50.
  • n2 is 5-23.
  • n2 is 11 to about 17.
  • n3 is 5-23.
  • n3 is 11 to about 17.
  • R is a member selected from the group consisting of hydrogen, alkyl, and acyl
  • R1 is a member selected from the group consisting of hydrogen and alkyl; or optionally, R and R1 and the nitrogen to which they are attached form an azide moiety
  • R2 is a member selected from the group consisting of hydrogen, optionally substituted alkyl, optionally substituted aryl, and an amino acid side chain
  • R3 is a member selected from the group consisting of hydrogen, halogen, hydroxyl, alkoxy, thiol, hydrazine, amino, and NR4R5 , wherein R4 and R5 are independently hydrogen or alkyl
  • n is 4-80
  • m is 2-6
  • p is 1-4
  • q is 0 or 1.
  • diacylglycerol refers to a compound having two fatty acyl chains, R1 and R2 , each independently having 2-30 carbons, which are bound to the 1- and 2-positions of glycerol via ester bonds.
  • the acyl groups may be saturated or have varying degrees of unsaturation. Suitable acyl groups include, but are not limited to, lauryl ( C12 ), myristyl ( C14 ), palmityl ( C16 ), stearyl ( C18 ), and eicosyl ( C20 ).
  • R1 and R2 are the same, i.e., both R1 and R2 are myristyl (i.e., dimyristyl), both R1 and R2 are stearyl (i.e., distearyl), etc.
  • Diacylglycerol has the following general formula:
  • dialkoxypropyl refers to a compound having two alkyl chains, R1 and R2 , each independently having 2-30 carbons.
  • the alkyl group may be saturated or have varying degrees of unsaturation.
  • Dialkoxypropyl has the following general formula:
  • the PEG-lipid is a PEG-DAA conjugate having the following general formula:
  • 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 ).
  • R 1 and R 2 are the same, i.e., both R 1 and R 2 are myristyl (i.e., dimyristyl), both R 1 and R 2 are stearyl (i.e., distearyl), etc.
  • PEG has an average molecular weight in 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 a preferred embodiment, PEG has an average molecular weight of about 2,000 daltons or about 750 daltons. PEG can be optionally substituted with an alkyl, alkoxy, acyl or aryl group. In certain embodiments, the terminal hydroxyl group is substituted with an ethoxy or methyl group.
  • 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.
  • the non-ester-containing linker moiety is a carbamate
  • the non-ester-containing linker moiety is an amine linker moiety (i.e., a PEG-A-DAA conjugate).
  • the non-ester-containing linker moiety is a succinyl linker moiety (i.e., a PEG-S-DAA conjugate).
  • the PEG-lipid conjugate is selected from:
  • the PEG-DAA conjugates are synthesized using standard techniques and reagents known to those skilled in the art. It should be recognized that the PEG-DAA conjugates will contain a variety of amide, amine, ether, thio, carbamate, and urea bonds. Those skilled in the art should recognize 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 Edition (Longman 1989).
  • the PEG-DAA conjugate is a dilauryloxypropyl (C 12 )-PEG conjugate, a dimyristyloxypropyl (C 14 )-PEG conjugate, a dipalmityloxypropyl (C 16 )-PEG conjugate, or a distearyloxypropyl (C 18 )-PEG conjugate.
  • C 12 dilauryloxypropyl
  • C 14 dimyristyloxypropyl
  • C 16 dipalmityloxypropyl
  • C 18 distearyloxypropyl
  • hydrophilic polymers may be Instead of PEG.
  • suitable polymers that can be used instead of PEG include, but are not limited to, polyvinyl pyrrolidone, polymethyl oxazoline, polyethyl oxazoline, polyhydroxypropyl methacrylamide, polymethacrylamide and polydimethyl acrylamide, polylactic acid, polyglycolic acid, poloxamer, poloxamine, derivatized cellulose such as hydroxymethyl cellulose or carboxyethyl cellulose, polysarcosine-lipid conjugates, and conjugates of polysarcosine with lipid-like substances.
  • polysarcosine-lipid conjugate or the conjugate of polysarcosine with lipid-like substances can be selected from the following group: polysarcosine-diacylglycerol conjugate, polysarcosine-dialkoxypropyl conjugate, polysarcosine-phospholipid conjugate, polysarcosine-ceramide conjugate, and mixtures thereof.
  • Suitable polysarcosine-lipid conjugates or conjugates of polysarcosine with lipid-like substances for use in the present invention, and methods for preparing and using polysarcosine-lipid conjugates or conjugates of polysarcosine with lipid-like substances are disclosed in, for example, U.S. Patent No.
  • suitable polysarcosine-lipid conjugates for use in the compositions and methods of the present invention include polysarcosine lipids as described in International Patent Publication WO2020070040, which is incorporated herein by reference.
  • the polymer-lipid composition (e.g., PoLixNano) of the present invention may also include a cation-polymer-lipid conjugate (CPL) (see, e.g., Chen et al., Bioconj. Chem., 11: 433-437 (2000)).
  • CPL cation-polymer-lipid conjugate
  • 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.
  • the polycationic moiety may have a connected ligand, such as a targeting ligand or a chelating moiety for complexing calcium.
  • a connected ligand such as a targeting ligand or a chelating moiety for complexing calcium.
  • the cationic moiety maintains a positive charge.
  • the connected 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, radioactive markers, fluorophores, biotin, drugs, haptens, DNA, RNA, polysaccharides, liposomes, virosomes, micelles, immunoglobulins, functional groups, other targeting moieties, or toxins.
  • the present invention also contemplates the use of PEG-modified phospholipids and derivatized lipids such as derivatized ceramides (PEG-CER), including N-octanoyl-sphingosine-l-[succinyl(methoxypolyethylene glycol)-2000] (C8PEG-2000 ceramide).
  • PEG-CER derivatized ceramides
  • Contemplated PEG-modified lipids include But not limited to a polyethylene glycol chain of up to 2 kDa, up to 3 kDa, up to 4 kDa, or up to 5 kDa in length, covalently attached to a lipid having an alkyl chain of C 6 -C 20 in length.
  • 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).
  • DMPE-PEG2K 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-PEG2000
  • the addition of such components can prevent complex aggregation, and can also provide a method for increasing the circulation life and increasing the delivery of compositions comprising polymer-lipids to target tissues (Klibanov et al.
  • Suitable PEG-modified lipids for the compositions and methods of the present invention include PEG lipids as described in International Patent Publication WO 2020061295 and U.S. Patent Publication US 20220016029, which are incorporated herein by reference.
  • the lipid conjugates of the present invention 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.
  • 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.
  • 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.
  • the covalent bond can be formed by the reaction between two functional groups that are modified to be reactive, and the glycosyl moiety can be directly conjugated to the PEG lipid.
  • the glycosyl moiety can be conjugated to the PEG lipid through a spacer.
  • the lipid conjugates of the 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, radioactive labels, fluorophores, biotin, drugs, haptens, DNA, RNA, polysaccharides, liposomes, virosomes, micelles, immunoglobulins, functional groups, toxins or other targeting moieties.
  • the lipid conjugate accounts for 0.1 mol%-10.0 mol% of the total lipids in the composition, e.g., 0.1 mol%-10.0 mol%, 1 mol%-10.0 mol%, 2 mol%-10.0 mol%, 3mol%-10.0mol%, 5mol%-10.0mol%, 0.1mol%-5mol%, 1mol%-5mol%, 2mol%-5mol% or 3mol%-5.0mol%.
  • compositions comprising polymer-lipids 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 total lipids by mole or weight. In some embodiments, the compositions comprising polymer-lipids 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 by mole or weight of total lipids.
  • compositions comprising polymer-lipids described herein contain 0.01% or less of PEG-modified lipids or PEG by mole or weight of total lipids. In some embodiments, the compositions of polymer-lipids described herein do not include lipid conjugates (e.g., PEG-lipids) or PEG.
  • the concentration of the lipid conjugate may vary, depending on the rate at which the lipid conjugate and polymer-lipid composition used form fusions.
  • the rate at which the lipid conjugate is exchanged from the polymer-lipid composition can be controlled, and the rate at which the nucleic acid/polymer-lipid composition forms a fusion.
  • 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.
  • 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 clear to those skilled in the art when reading the disclosure 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.
  • compositions of the present invention comprise:
  • amphiphilic block copolymers cationic lipids, phospholipids, cholesterol and lipid conjugates such as PEG-lipid conjugates, wherein the cationic lipid accounts 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 percent of the composition;
  • 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%-75.0 mol% of the total lipid present in the composition, the phospholipid accounts for 10.0 mol%-62.0 mol% of the total lipid, the cholesterol accounts for 14.0 mol%-46.0 mol% of the total lipid, the lipid conjugate accounts for 0.1 mol%-8.0 mol% of the total lipid, and the amphiphilic block copolymer accounts for 0.1%-95.0% by weight of the composition;
  • amphiphilic block copolymers cholesterol-derived cationic lipids, phospholipids, and lipid conjugates such as PEG-lipid conjugates, wherein the cholesterol-derived cationic lipids account for 29.0 mol%-80.0 mol% of the total lipids present in the composition, the phospholipids account for 19.0 mol%-70.0 mol% of the total lipids, the lipid conjugates account for 0.1 mol%-8.0 mol% of the total lipids, and the amphiphilic block copolymers account for 0.1%-95.0% by weight of the composition;
  • 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%-80.0 mol% of the total lipids present in the composition, cholesterol accounts for 15.0 mol%-50.0 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;
  • amphiphilic block copolymers cationic lipids, phospholipids and lipid conjugates such as PEG-lipid conjugates, wherein the cationic lipids account for 30.0 mol%-80.0 mol% of the total lipids present in the composition, the phospholipids account for 10.0 mol%-50.0 mol% of the total lipids, the lipid conjugates account for 0.1 mol%-8.0 mol% of the total lipids, and the amphiphilic block copolymers account for 0.1%-95.0% by weight of the composition;
  • an amphiphilic block copolymer a cationic lipid, a phospholipid, and cholesterol
  • the cationic lipid accounts for 30.0 mol%-80.0 mol% of the total lipids present in the composition
  • the phospholipids account for 5.0 mol%-50.0 mol% of the total lipids
  • the cholesterol accounts for 15.0 mol%-50.0 mol% of the total lipids
  • the amphiphilic block copolymer accounts for 0.1%-95.0% by weight of the composition
  • 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.
  • the composition comprises an amphiphilic block copolymer and the following components:
  • composition comprises 40.0 mol% to 70.0 mol% of the total lipids, DSPC, DPPC, DOPS, SOPE, DOPG, DSPE, ESM or DOPE comprises 8.0 mol% to 39.0 mol% of the total lipids, cholesterol or ⁇ -sitosterol comprises 20.0 mol% to 40.0 mol% of the total lipids, DMG-PEG
  • DOTAP DODAP
  • DOTMA DOSPA
  • DSPC DPPC
  • DOPS SOPE
  • DOPG DOPG
  • DSPE ESM or DOPE
  • cholesterol or ⁇ -sitosterol DOTAP, DODAP, DOTMA or DOSPA
  • DSPC DPPC
  • DOPS SOPE
  • DOPG DOPG
  • DSPE ESM or DOPE
  • cholesterol or ⁇ -sitosterol accounts for 15.0mol%-50.0mol% of the total lipids
  • DMG-PEG2K, DMG-PEG5K or DSPE-PEG2K-Mannose accounts for 0.1mol%-5.0mol% of the total lipids
  • amphiphilic block copolymer accounts for 30.0%-90.
  • GL67, ICE, or HGT4002 DSPC, DPPC, DOPS, SOPE, DOPG, DSPE, ESM, or DOPE
  • 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
  • the amphiphilic block copolymer accounts for 30.0%-90.0% by weight of the composition
  • compositions comprise the following components:
  • an amphiphilic block copolymer DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or ⁇ -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 ⁇ -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;
  • an amphiphilic block copolymer (2) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or ⁇ -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 ⁇ -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;
  • an amphiphilic block copolymer DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or ⁇ -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 ⁇ -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;
  • an amphiphilic block copolymer DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or ⁇ -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 ⁇ -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;
  • an amphiphilic block copolymer DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or ⁇ -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 ⁇ -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;
  • an amphiphilic block copolymer DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or ⁇ -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 ⁇ -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;
  • an amphiphilic block copolymer DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or ⁇ -sitosterol; and DMG-PEG2K
  • 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 ⁇ -sitosterol account for 35.0 mol% of the total lipids
  • DMG-PEG2K or DMG-PEG5K account for 0.7 mol% of the total lipids
  • the amphiphilic block copolymer accounts for 48.4% by weight of the composition
  • Amphiphilic block copolymers DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or ⁇ -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 ⁇ -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;
  • an amphiphilic block copolymer DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or ⁇ -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 ⁇ -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;
  • an amphiphilic block copolymer (10) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or ⁇ -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 ⁇ -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;
  • an amphiphilic block copolymer DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or ⁇ -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 ⁇ -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;
  • an amphiphilic block copolymer DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or ⁇ -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 ⁇ -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;
  • an amphiphilic block copolymer DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or ⁇ -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 ⁇ -sitosterol account for 27.5 mol% of the total lipids
  • DMG-PEG2K or DMG-PEG5K account for 0.9 mol% of the total lipids
  • the amphiphilic block copolymer accounts for 42.9% by weight of the composition;
  • an amphiphilic block copolymer DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or ⁇ -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 ⁇ -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;
  • an amphiphilic block copolymer DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or ⁇ -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 ⁇ -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;
  • an amphiphilic block copolymer DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or ⁇ -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 ⁇ -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;
  • an amphiphilic block copolymer DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or ⁇ -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 ⁇ -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;
  • an amphiphilic block copolymer (18) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or ⁇ -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 ⁇ -sitosterol account for 30.0mol% of the total lipids
  • DMG-PEG2K or DMG-PEG5K account for 1.0mol% of the total lipids
  • amphiphilic block copolymer accounts for 83.0% by weight of the composition
  • an amphiphilic block copolymer DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or ⁇ -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 ⁇ -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;
  • an amphiphilic block copolymer DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or ⁇ -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 ⁇ -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;
  • 30.0 mol% of lipids, DMG-PEG2K or DMG-PEG5K accounts for 1.0 mol% of total lipids, and the amphiphilic block copolymer accounts for 66.0% by weight of the composition;
  • an amphiphilic block copolymer DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or ⁇ -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 ⁇ -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;
  • an amphiphilic block copolymer DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or ⁇ -sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 40.6 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 30.1 mol% of the total lipids, cholesterol or ⁇ -sitosterol account for 28.4 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 36.9% by weight of the composition;
  • an amphiphilic block copolymer DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or ⁇ -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 ⁇ -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;
  • an amphiphilic block copolymer DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or ⁇ -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 ⁇ -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 The amphiphilic block copolymer accounts for 28.0% by weight of the composition;
  • an amphiphilic block copolymer DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or ⁇ -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 ⁇ -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;
  • an amphiphilic block copolymer DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or ⁇ -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 ⁇ -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;
  • an amphiphilic block copolymer DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or ⁇ -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 ⁇ -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;
  • 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;
  • Amphiphilic block copolymers DLin-MC3-DMA, ALC-0315 or SM-102; cholesterol Or ⁇ -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 ⁇ -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;
  • an amphiphilic block copolymer cKK-E12, DLin-MC3-DMA, ALC-0315, SM-102 or C12-200; DOTAP, DODAP, DOTMA or DOSPA; cholesterol or ⁇ -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 ⁇ -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
  • An amphiphilic block copolymer DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; and cholesterol or ⁇ -sitosterol, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 40.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 32.0 mol% of the total lipids, cholesterol or ⁇ -sitosterol account for 28.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 50.0% weight percent of the composition.
  • the molar ratio of nitrogen (amine) groups in the cationic lipid to phosphate groups of 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.
  • the ratio of lipid to nucleic acid in the composition 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).
  • a mixture of non-identical polymer-lipid compositions formed from at least a first and a second separate entity is included.
  • Each polymer-lipid composition comprises an mRNA and one or more cationic lipids, wherein the first polymer-lipid composition comprises 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 after administration of the pharmaceutical composition to a subject
  • the expression of the mRNA encoding protein or peptide is at least about two-fold greater than that of an otherwise identical amount administered with the first lipid nanoparticle but without the second lipid nanoparticle.
  • the first polymer-lipid composition and the second polymer-lipid composition comprise one or more non-cationic lipids.
  • the first non-cationic lipid and the second non-cationic lipid are not the same; and wherein the expression of a protein or peptide encoded by an mRNA after administering the pharmaceutical composition to a subject exceeds the expression of an otherwise identical amount of mRNA-encoded protein or peptide administered with the first polymer-lipid composition but not with the second polymer-lipid composition by at least about two times.
  • 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 not identical; and wherein the expression of a protein or peptide encoded by the mRNA after administration of the pharmaceutical composition to a subject exceeds the expression of an otherwise identical amount of mRNA-encoded protein or peptide administered with the first polymer-lipid composition but without the second polymer-lipid composition by at least about two times.
  • 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 not the same; and wherein the expression of a protein or peptide encoded by the mRNA after administration of the pharmaceutical composition to a subject exceeds the expression of an otherwise identical amount of mRNA-encoded protein or peptide administered with the first polymer-lipid composition but without the second polymer-lipid composition by at least about two times.
  • first polymer-lipid composition and the second polymer-lipid composition comprise one or more amphiphilic block copolymers.
  • first amphiphilic block copolymer and the second amphiphilic block copolymer are not the same; and wherein the expression of a protein or peptide encoded by mRNA after administering the pharmaceutical composition to a subject exceeds the expression of an otherwise identical amount of mRNA encoded protein or peptide administered with the first polymer-lipid composition but not with the second polymer-lipid composition by at least about two times.
  • 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.
  • cationic lipids (or non-lipid cationic agents) that can be distributed on the outer surface of polymer-lipid composition nanoparticles can be additionally added to the polymer-lipid composition.
  • the additionally added cationic lipid can be a sterolamine, such as GL67, ICE, etc.
  • the additional non-lipid cationic agent may be tromethamine, benzalkonium chloride, modified arginine, cetylpyridinium chloride, L-lysine monohydrate, etc.
  • the contact 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.
  • 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 ⁇ -tocopherol polyethylene glycol succinate (TPGS).
  • 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 buffer solution, such as PBS and Tris buffer.
  • Suitable mixtures of polymer-lipid compositions for use in the compositions and methods of the present invention include synergistically enhanced nucleic acid delivery mixed formulations as described in International Patent Publications WO 2014144196, WO 2022032154 and U.S. Patent US 10,130,649, which are incorporated herein by reference.
  • 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.
  • 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 homologue thereof, or a modified form thereof.
  • the protein is Cas9.
  • the protein or polypeptide is present in a molar ratio of about 1:1 to about 1:20 to the nucleic acid. 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.
  • the therapeutic agent is a protein or polypeptide.
  • the composition comprises both a protein and a nucleic acid.
  • the composition comprises a Cas9 protein and a single guide nucleic acid.
  • the composition comprises a Cas9 protein, a single guide nucleic acid, and a donor DNA.
  • the therapeutic agent is a small molecule such as a small molecule selected from the group consisting of: anticancer anti-inflammatory drugs, anti-fungal drugs, psychiatric drugs such as analgesics, consciousness-altering agents such as anesthetics or hypnotics, nonsteroidal anti-inflammatory drugs (NSAIDS), anthelmintics, anti-acne agents, anti-angina agents, anti-arrhythmic agents, anti-asthma drugs, anti-bacterial agents, anti-benign prostatic hypertrophy agents, anticoagulants, antidepressants, anti-diabetic agents, antiemetics, anti-epileptic drugs, anti-gout drugs, anti-hypertensive agents, anti-inflammatory agents, anti-malarials, anti-migraine drugs, anti-muscarinics, anti-neoplastic agents, anti-obesity agents, anti-osteoporosis agents, anti-Parkinson's syndrome agents, anti- prolifer, anti- pro
  • 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 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.
  • a targeting moiety 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.
  • the targeting moiety is preferably selected from the following group: an epithelial cell ligand, in particular a respiratory epithelial cell ligand, a gastrointestinal epithelial cell ligand, a reproductive epithelial cell ligand or a microfolding cell ligand; an immune cell ligand, in particular a dendritic cell ligand, a T cell ligand, a B cell ligand or a macrophage ligand; an endothelial cell ligand, in particular a lung endothelial cell ligand or a liver endothelial cell ligand; a tumor cell ligand, in particular a melanoma, a lung or liver tissue-associated tumor cell ligand; and/or a skin cell ligand, in particular a dermal fibroblast ligand or a keratinocyte ligand.
  • an epithelial cell ligand in particular a respiratory epithelial cell ligand, a gastrointestinal epit
  • the composition further comprises an adjuvant, preferably the adjuvant comprises at least one selected from the following: CpG oligodeoxynucleotides, polyinosinic: polycytidylic acid, saponin extract (QS-21 extract), aluminum adjuvant, squalene, ⁇ -tocopherol, Tween, Span, lipopolysaccharide LPS, Pam 3 CSK 4 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 immunomodulator proteins.
  • the adjuvant comprises at least one selected from the following: CpG oligodeoxynucleotides, polyinosinic: poly
  • Suitable adjuvants may include, but are not limited to, inorganic salts (e.g., AlK(SO 4 ) 2 , AlNa(SO 4 ) 2 , AlNH(SO 4 ) 2 , silica, aluminum, aluminum hydroxide, Ca 3 (PO 4 ) 2 , kaolin or carbon), polynucleotides with or without immune stimulating complexes (ISCOMs) (e.g., CpG oligonucleotides, such as Chuang, TH et al., (2002) J. Leuk. Biol. 71(3):538-44; Ahmad Nejad, P. et al., (2003) J. Leuk. Biol. 71(3):538-44). (2002) Eur.
  • inorganic salts e.g., AlK(SO 4 ) 2 , AlNa(SO 4 ) 2 , AlNH(SO 4 ) 2 , silica, aluminum, aluminum hydroxide, Ca 3 (PO 4 ) 2
  • the adjuvant is another RNA.
  • a suitable adjuvant is aluminum phosphate.
  • a suitable adjuvant is aluminum hydroxide.
  • a suitable adjuvant is a combination of aluminum phosphate and aluminum hydroxide.
  • the composition further comprises a transfection enhancer, preferably the transfection enhancer comprises at least one selected from the group consisting of a pulmonary surfactant protein, a cell-penetrating peptide, an amphiphilic polypeptide, a mucolytic enzyme, 1,2-propylene glycol, a cellulose (such as carboxymethyl cellulose or hydroxypropyl cellulose), a hyaluronate, alginate, pectin, polyethylene glycol, a poloxamer, a poloxamine, glucose, fructose, sucrose, trehalose, dextran, polyvinyl pyrrolidone, chitosan, polyvinyl alcohol, polyvinyl acetate, agglutinin, polylactic acid, polyhydroxybutyric acid, tromethamine, benzalkonium chloride, modified arginine, cetylpyridinium chloride, L-lysine
  • the compositions are in the form of nanoparticles having an average size of about 1000 nm or less.
  • 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.
  • 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.
  • the composition comprising polymer-lipid of the present invention
  • the composition is formulated as a solution, dry powder, atomization or spray.
  • the composition is formulated to be administered to the lung and/or nose by aerosolization, dry powder, inhalation, atomization or instillation.
  • the present invention particularly provides mRNA-polymer-lipid compositions prepared using the methods of the present invention as described herein.
  • 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.
  • an amphiphilic polymer is present in the mRNA solution before mixing.
  • the amphiphilic polymer is present in the lipid solution before mixing.
  • an amphiphilic polymer is added during the mixing of the mRNA solution and the lipid solution.
  • an amphiphilic polymer is added after the mixing of the mRNA solution and the lipid solution.
  • the lipid solution in a suitable polymer-lipid comprises a cationic lipid and a non-cationic lipid (also referred to as a helper lipid).
  • a suitable lipid solution comprises a cationic lipid, a non-cationic lipid and a PEG-modified lipid or PEG.
  • a suitable lipid solution comprises a cationic lipid, a non-cationic lipid, a cholesterol-based lipid and a PEG-modified lipid 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 the 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.
  • the lipid solution in a suitable polymer-lipid comprises one or more cationic lipids, non-cationic lipids, cholesterol and/or PEG-modified lipids.
  • 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-016B, 9A1P9, OF-Deg-Lin, 306Oi10, TT3, FTT5, C12-200, DLin-KC2-DMA, DODAP, HGT4003, ICE, GL67, HGT5000 or HGT5001, etc.
  • the polymer-lipid may include 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).
  • DSPC 1,2-distearoyl-sn-glycero-3-phosphocholine
  • DPPC 1,2-dipalmitoyl-sn-glycero-3-phosphocholine
  • DOPE 1,2-dioleyl-sn-glycero-3-phosphoethanolamine
  • the polymer-lipid may include cholesterol and/or ⁇ -sitosterol, etc.
  • the polymer-lipid includes PEG-modified/modified lipids, and the PEG-modified lipids may include (but not exclusively) lipids covalently linked to alkyl chains having a length of C 6 -C 20 , and PEG chains up to 5 kDa.
  • the polymer-lipid comprises DMG-PEG2K, DMPE-PEG2K, DSPE-PEG2K, DSPE-PEG2K-Mannose, DMG-PEG5K, DMPE-PEG5K, DSPE-PEG5K, DSPE-PEG5K-Mannose, etc.
  • 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-PEG
  • the method comprises:
  • the method comprises:
  • the method comprises:
  • the method comprises:
  • the method further comprises the step of removing free lipid components and/or amphiphilic block copolymers, preferably by dialysis and/or tangential flow filtration.
  • 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.
  • 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 heating steps, a heating step is included in the method process (before, during or after formation) to provide a higher encapsulation rate of mRNA.
  • the encapsulation of mRNA in polymer-lipid can be further enhanced by heating a formulation solution comprising mRNA-polymer-lipid and some free mRNA not encapsulated in polymer-lipid forming solution to a predetermined temperature as described herein.
  • 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.
  • the term "ambient temperature” refers to the temperature in a room, or the temperature surrounding an object of interest without heating or cooling.
  • the ambient temperature maintained by one or more solutions is or is less than about 35°C, 30°C, 25°C, 20°C, or 16°C.
  • 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.
  • the ambient temperature maintained by one or more solutions is 20-25°C.
  • the predetermined temperature above ambient temperature is typically greater than about 25°C.
  • 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.
  • 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.
  • the predetermined temperature suitable for the present invention is about 65°C.
  • a pump is used to mix the mRNA solution and the lipid solution. Since the encapsulation procedure with such 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 at a stable flow rate. The type of suitable pump may include, but is not limited to, a gear pump and a centrifugal pump.
  • the mRNA solution and lipid solution can be mixed at various flow rates.
  • the mRNA solution can be mixed at a flow rate greater than the flow rate of the lipid solution.
  • the mRNA storage solution can be mixed at a flow rate greater than 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.
  • the suitable flow rate for mixing can be determined based on the scale.
  • 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.
  • 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.
  • 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.
  • the 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.
  • the aqueous solution and the lipid stock solution are all continuously flowed into the mixer at the same time.
  • the ethanol content of the lipid dissolution is kept to decrease suddenly, and the lipids are all precipitated from each other.
  • the nanoparticles are self-assembled in the mixing chamber.
  • the present invention provides a polymer-lipid composition produced by a continuous mixing method, for example, a process comprising the steps of 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).
  • aqueous solution which may include an amphiphilic block polymer
  • organic lipid solution which may include an amphiphilic block polymer
  • the present invention provides polymer-lipid compositions produced by direct dilution method, and the direct dilution method comprises forming lipid solution and immediately and directly introducing lipid solution into the collection container containing controlled amount of dilution buffer.
  • the collection container comprises one or more components configured to stir the collection container contents to promote dilution.
  • the amount of dilution buffer present in the collection container is substantially equal to the volume of lipid solution introduced therein.
  • the liposome solution in 45% ethanol will advantageously produce smaller particles when introduced into the collection container containing equal volume of dilution buffer.
  • the present invention provides a polymer-lipid composition produced by a direct dilution method, wherein a third reservoir containing a dilution buffer is fluidically connected to the second mixing zone.
  • ethanol-drop precipitation has become the industry standard for preparing nucleic acid lipid nanoparticles. Precipitation reactions are favored for their continuity, scalability, and ease of use.
  • Such methods typically use high-energy mixers (e.g., T-junctions, confined impinging jets, microfluidic mixers, vortex mixers) to introduce lipids (in ethanol) into a suitable reverse phase solvent (i.e., water) in a controlled manner, thereby driving liquid supersaturation and spontaneous precipitation into lipid particles.
  • the vortex mixer used is described in U.S.
  • the microfluidic mixer used may employ the mixer described in PCT patent WO/2014/172045, which is incorporated herein by reference in its entirety.
  • the mixing step is performed using a T-junction, a confined impinging jet, a microfluidic mixer, or a vortex mixer.
  • the loading step is performed using a T-junction, a confined impinging jet, a microfluidic mixer, or a vortex mixer.
  • 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 "post-insertion” techniques, i.e., CPL or GL67 is inserted into, e.g., a preformed polymer-lipid composition (PoLixNano), and the "standard” techniques, wherein CPL or GL67 is included in the lipid mixture during, e.g., the PoLixNano formation step.
  • PoLixNano-CPLs polymer-lipid compositions containing cation-polymer-lipid conjugates CPL
  • PoLixNano-sterolamines e.g., polymer-lipid compositions containing sterolamine GL67
  • Methods for preparing PoLixNano-CPLs Methods for preparing PoLixNano-GL67 are described in, for example, 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 entirety 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 entirety for all purposes.
  • the polymer-lipid compositions of the present invention can be sized by any method useful for adjusting the size of liposomes. Size adjustment can be performed to obtain a desired size range and a relatively narrow particle size distribution.
  • nucleic acids in a polymer-lipid composition are pre-concentrated, for example, as described in U.S. Patent Application No. 09/744,103, the disclosure of which is incorporated herein by reference in its entirety for all purposes.
  • the method provided by the present invention also includes adding a transfection enhancer to achieve enhanced nucleic acid transfection effect.
  • suitable transfection enhancers include pulmonary surfactant proteins, cell-penetrating peptides, amphipathic polypeptides, mucolytic enzymes, 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, cetyl pyridinium chloride, L-lysine monohydrate, and polylactic acid-glycoli
  • the method may further include adding a non-lipid polycation, which is used to achieve lipid transfection of cells using the composition of the present invention.
  • suitable non-lipid polycations include hexadimethrine bromide (trade name from Sigma-Aldrich Chemical Co.) or other hexadimethrine salts.
  • suitable polycations include, but are not limited to, for example, poly-L-ornithine, poly-L-arginine, poly-D-arginine, poly-L-lysine, poly-D-lysine, polyallylamine, chitosan, and polyethyleneimine and pharmaceutically acceptable salts thereof. These polycations are preferably added after the particles have been formed.
  • the inventive method as herein described comprises the step of removing lipid and/or amphipathic polymer (for example poloxamine and/or poloxamer) that do not form particles.
  • free lipid and/or amphipathic polymer can be removed by buffer exchange techniques such as dialysis.
  • polymer-lipid formation solution is exchanged in the solution constituting the product formulation solution.
  • the mixture of the polymer-lipid nanoparticle formed can be dialyzed in one or more formulation solutions to remove free lipid and/or amphipathic polymer present during the formation of the polymer-lipid nanoparticle.
  • the solution comprising polymer-lipid nanoparticles can be replaced from polymer-lipid forming solution to formulation solution by any of a variety of buffer exchange techniques known in the art.
  • the step of replacing the polymer-lipid forming solution into formulation solution is accompanied by the purification and/or concentration of polymer-lipid.
  • Various methods can be used to realize the replacement of solution and the purification of polymer-lipid or the concentration of polymer-lipid in solution. For example, in some embodiments, this solution replacement is realized by diafiltration.
  • the solution is replaced, and tangential flow filtration (TFF, also referred to as cross-flow filtration) is used to purify polymer-lipid.
  • TMF tangential flow filtration
  • TFF non-desired permeate passes through the filter, and the desired retentate (polymer-lipid nanoparticles and free mRNA) is passed along the filter and collected downstream.
  • desired retentate polymer-lipid nanoparticles and free mRNA
  • Exemplary TFF purification methods are described in US2016/0040154 and US2015/0376220, which are incorporated herein by reference.
  • the residual amount of the amphiphilic polymer remains in the formulation after removal.
  • residual amount means the remaining amount after substantially all substances (amphiphilic polymers such as poloxamine and/or poloxamer described herein) are removed in the composition.
  • the residual amount can be detected qualitatively or quantitatively using known techniques. The residual amount may not be detected using known techniques.
  • excess mRNA is also removed along with the amphiphilic polymer (eg, poloxamine and/or poloxamer) present during mRNA-polymer-lipid formation.
  • amphiphilic polymer eg, poloxamine and/or poloxamer
  • a suitable formulation solution comprises only residual citrate. In some embodiments, a suitable formulation solution comprises only residual non-aqueous solvents such as ethanol. In some embodiments, a suitable formulation solution contains less than about 10 mM citrate. In some embodiments, a suitable formulation solution contains less than about 25% non-aqueous solvents such as ethanol. In some embodiments, a suitable formulation solution does not require any further lyophilization prior to lyophilization.
  • the formulation solution does not require any further downstream processing (e.g., buffer exchange and/or further purification steps and/or additional excipients) prior to administration to a sterile fill in a vial, syringe, or other container. In some embodiments, the formulation solution does not require any further downstream processing prior to administration to a subject.
  • further downstream processing e.g., buffer exchange and/or further purification steps and/or additional excipients
  • a suitable formulation solution after freeze-thawing, can improve or enhance the mRNA encapsulation capacity of the polymer-lipid composition after heating.
  • a suitable formulation solution is 10% sucrose and can be freeze-stable.
  • the polymer-lipid composition in the suitable formulation solution after heating can be stably frozen (e.g., retaining enhanced encapsulation) in about 1%, about 3%, about 5%, about 7%, about 9%, about 10%, about 13%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45% or about 50% sucrose solution.
  • the suitable formulation solution may not require any downstream purification or processing and can be stably stored in frozen form.
  • suitable formulation solutions may include buffers or salts.
  • Exemplary buffers may include 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), calcium chloride, ammonium sulfate, magnesium sulfate, sodium bicarbonate, sodium citrate, sodium acetate, potassium phosphate, and sodium phosphate.
  • Exemplary salts may include sodium chloride, magnesium chloride, lithium acetate, lithium chloride, lithium formate, lithium nitrate, lithium sulfate, sodium malonate, sodium nitrate, sodium sulfate, and potassium chloride.
  • suitable formulation solutions may include a combination of cations and anions contained in the above-mentioned buffer salts.
  • a suitable formulation solution comprises one or more protective agents, and each of the one or more protective agents is independently a polyol (e.g., a diol or triol, such as propylene glycol (i.e., 1,2-propylene glycol)), 1,3-propylene glycol, glycerol, (+/-)-2-methyl-2,4-pentanediol, 1,6-hexanediol, 1,2-butanediol, 2,3-butanediol, ethylene glycol, or diethylene glycol), a non-detergent sulfobetaine (e.g., NDSB-201 (3-(1-pyridyl)-1-propanesulfonate), an osmotic agent (e.g., L-proline or trimethylamine N-oxide dihydrate), a polymer (e.g., polyethylene glycol 200 (PEG 200), PEG 400, PEG 600, PEG 1000,
  • suitable formulation solutions are aqueous solutions comprising pharmaceutically acceptable excipients (including but not limited to cryoprotectants).
  • suitable formulation solutions are aqueous solutions comprising pharmaceutically acceptable excipients, and the excipients include but are not limited to sugars, such as one or more of trehalose, sucrose, mannose, lactose and mannitol.
  • suitable formulation solutions include trehalose.
  • suitable formulation solutions include sucrose.
  • suitable formulation solutions include mannose.
  • suitable formulation solutions include lactose.
  • suitable formulation solutions include mannitol.
  • a suitable formulation solution is an aqueous solution containing 1% to 20% weight/volume sugars, such as trehalose, sucrose, mannose, lactose, and mannitol. In some embodiments, a suitable formulation solution is an aqueous solution containing 1% to 20% weight/volume trehalose. In some embodiments, a suitable formulation solution is an aqueous solution containing 1% to 20% weight/volume sucrose. In some embodiments, a suitable formulation solution is an aqueous solution containing 1% to 20% weight/volume mannose. In some embodiments, a suitable formulation solution is an aqueous solution containing 1% to 20% weight/volume lactose.
  • a suitable formulation solution is an aqueous solution containing 1% to 20% weight/volume mannitol.
  • a suitable formulation solution is an aqueous solution containing about 10% weight/volume sugars, such as trehalose, sucrose, mannose, lactose, and mannitol.
  • a suitable formulation solution is an aqueous solution containing about 10% weight/volume trehalose.
  • a suitable formulation solution is an aqueous solution containing about 10% weight/volume sucrose.
  • a suitable formulation solution is an aqueous solution comprising about 10% weight/volume mannose.
  • a suitable formulation solution is an aqueous solution comprising about 10% weight/volume lactose.
  • a suitable formulation solution is an aqueous solution comprising about 10% weight/volume mannitol.
  • suitable formulation solutions have a pH between pH 4.5 and pH 7.5. In some embodiments, suitable formulation solutions have a pH between pH 5.0 and pH 7.0. In some embodiments, suitable formulation solutions have a pH between pH 5.5 and pH 7.0. In some embodiments, suitable formulation solutions have a pH higher than pH 4.5. In some embodiments, suitable formulation solutions have a pH higher than pH 5.0. In some embodiments, suitable formulation solutions have a pH higher than pH 5.5. In some embodiments, suitable formulation solutions have a pH higher than pH 6.0. In some embodiments, suitable formulation solutions have a pH higher than pH 6.5.
  • the present invention also provides the polymer-lipid composition in the form of a kit.
  • the kit may include containers divided into various components (e.g., various lipids, polymer components of active agents or therapeutic agents such as nucleic acids and particles) for accommodating the polymer-lipid composition.
  • the kit also includes an endosomal membrane destabilizer (e.g., calcium ions).
  • the kit typically includes the polymer-lipid composition of the present invention preferably in a dehydrated form and instructions for their rehydration and administration.
  • polymer-lipid compositions of the present invention can be customized to preferentially target special target tissues, organs or tumors.
  • the preferential targeting of polymer-lipid compositions can be carried out by controlling the particle components themselves. For example, as described in Example 11 and Example 16, it has been found that (PoLixNano+T904) physical mixed formulations can be used for preferentially targeting liver and spleen, and polymer-lipid composition formulations can be used for preferentially targeting lungs.
  • a targeting moiety may be desirable to attach to the surface of the polymer-lipid composition to further enhance the targeting of the particle.
  • Methods for attaching a targeting moiety e.g., a glycosyl, polypeptide, antibody, protein, etc.
  • a lipid or amphiphilic polymer such as those used in the particles of the present invention
  • the polymer-lipid composition of the present invention is effective for introducing an active agent or therapeutic agent (e.g., mRNA) into a cell. Therefore, the present invention also provides a method for introducing an active agent or therapeutic agent such as a nucleic acid (e.g., mRNA) into a cell. The method is performed in vitro or in vivo by the following steps: first forming a particle as described above, then contacting the particle with a cell to be sufficient to cause an active agent or therapeutic agent to be introduced into a cell for a period of time.
  • Polymer-lipid compositions of the present invention can be adsorbed on almost any cell type mixed or contacted therewith. Once adsorbed, the particle can be endocytosed by a part of the cell, exchange lipids with the cell membrane, or merge with the cell. Transfer or combination of the active agent or therapeutic agent (e.g., nucleic acid) part of the particle can occur by any of these approaches. Specifically, when fusion occurs, the particle membrane is integrated into the cell membrane and the content of the particle is combined with the intracellular fluid.
  • the active agent or therapeutic agent e.g., nucleic acid
  • the polymer-lipid composition of the present invention can be administered alone or in a mixture with a pharmaceutical carrier (e.g., saline or phosphate buffered saline PBS), which is selected according to the route of administration and standard pharmaceutical practice.
  • a pharmaceutical carrier e.g., saline or phosphate buffered saline PBS
  • standard buffered saline e.g., 135-150mM NaCl
  • suitable carriers include, for example, water (nuclease-free water), buffered water, 0.4% saline, 0.3% glycine, etc., including glycoproteins such as albumin, lipoproteins, globulins, etc. for increasing stability.
  • carrier includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, etc.
  • pharmaceutically acceptable refers to molecular entities and compositions that do not produce an allergic or similar untoward reaction when administered to a human.
  • the pharmaceutically acceptable carrier is generally added after the particles are formed.
  • the particles can be diluted into a pharmaceutically acceptable carrier such as standard buffered saline.
  • the concentration of particles in the pharmaceutical preparation can vary widely, usually at or at least about 0.1-30% by weight, to about 10-90% by weight at most, and is selected mainly by fluid volume, viscosity, etc. according to the specific mode of administration selected.
  • the concentration can be increased to reduce the fluid load associated with the treatment. This may be particularly desirable in patients with congestive heart failure or severe hypertension associated with atherosclerosis.
  • the particles made of irritating lipids can be diluted to low concentrations, thereby alleviating inflammation at the administration site.
  • the pharmaceutical composition of the present invention can be sterilized by conventional, well-known sterilization techniques.
  • the aqueous solution can be packaged for standby use or filtered and lyophilized under aseptic conditions, and the lyophilized preparation is combined with a sterile aqueous solution before administration.
  • the composition can contain pharmaceutical auxiliary substances as needed to approach physiological conditions, such as pH adjustment and buffers, tension regulators, etc.
  • the particle suspension can include a lipid-protectant that protects lipids from free radicals and lipid-peroxidation damage during storage. Lipophilic free radical quenchers, such as alpha tocopherol and water-soluble ion-specific chelators, such as ferrioxamine are suitable.
  • therapeutic agents such as nucleic acids (e.g., mRNA) can be delivered to any cell grown in culture, regardless of plant or animal origin, vertebrate or invertebrate, and any tissue or type.
  • the cell is an animal cell, more preferably a mammalian cell, and most preferably a human cell.
  • the contact between cells and the polymer-lipid composition of the present invention occurs in a biocompatible culture medium when performed in vitro.
  • the particle concentration varies widely, depending on the specific application, but is generally between about 1 ⁇ mol and about 10 mmol.
  • Treatment of cells with the polymer-lipid composition is generally performed at physiological temperature (about 37° C.) for about 1-48 hours, preferably for a period of about 2-6 hours.
  • the polymer-lipid composition suspension is added to seeded cells at a cell density of about 10 3 to about 10 6 cells/ml, more preferably about 1x10 5 cells/ml, at a confluency of 60-80%.
  • the concentration of the suspension added to the cells is preferably about 0.01-0.4 ⁇ g/ml, more preferably about 0.2 ⁇ g/ml.
  • the delivery efficiency of the polymer-lipid compositions of the present invention can be optimized using the endosomal release parameter (ERP) assay.
  • ERP endosomal release parameter
  • administration can be by any means known in the art, for example, by injection, nasal drops, inhalation (e.g., atomization or dry powder formulations, inhaled intranasally or intratracheally), oral administration, transdermal administration, genital tract administration, or rectal administration.
  • Administration can be achieved by single or divided doses.
  • the pharmaceutical composition can be administered parenterally, i.e., intraarticularly, intravenously, intraperitoneally, subcutaneously, or intramuscularly.
  • the pharmaceutical composition is administered intravenously or intraperitoneally by push injection (see, e.g., U.S. Patent No. 5,286,634).
  • Intracellular nucleic acid delivery has also been discussed in Straubringer et al., Methods Enzymol., 101:512 (1983); Mannino et al., Biotechniques, 6:682 (1988); Nicolau et al., Crit. Rev. Ther. Drug Carrier Syst., 6:239 (1989); and Behr, Acc. Chem. Res., 26:274 (1993).
  • Other methods of administering lipid-based therapeutics are described, for example, in U.S. Pat. Nos. 3,993,754; 4,145,410; 4,235,871; 4,224,179; 4,522,803; and 4,588,578.
  • polymer-lipid compositions of the present invention can be administered by direct injection at the disease site or by injection at a site remote from the disease site (see, e.g., Culver, HUMAN GENE THERAPY, Mary Ann Liebert, Inc., Publishers, New York. Pages 70-71 (1994)).
  • Culver HUMAN GENE THERAPY
  • Mary Ann Liebert, Inc. Publishers, New York. Pages 70-71 (1994)
  • the disclosures of the above references are incorporated herein by reference in their entirety for all purposes.
  • compositions of the present invention may be in liquid form or solid form.
  • the compositions are in liquid form and can be delivered to the lungs via aerosolization, typically using various commercially available aerosol devices.
  • the compositions are in solid form and are suitable for inhalation to administer the compositions to the respiratory tract.
  • compositions can be delivered to the respiratory tract by suitable methods such as intranasal instillation, intratracheal instillation, and intratracheal injection.
  • suitable methods such as intranasal instillation, intratracheal instillation, and intratracheal injection.
  • the composition or nanoparticles are delivered intranasally, intrabronchially, or pulmonary.
  • a nebulizer or inhaler is used to deliver the composition or nanoparticles.
  • the composition is delivered to the lungs by a nebulized inhalation route.
  • Inhalation can be performed through the nose and/or mouth of an individual.
  • Administration can be performed by actively inhaling the composition or by administering the composition to an individual via a respirator.
  • Exemplary devices for delivering the composition to the respiratory tract include, but are not limited to, dry powder inhalers (DPI), pressurized metered dose inhalers (pMDI), nebulizers, and electric aerosol devices.
  • DPI dry powder inhalers
  • pMDI pressurized metered dose inhalers
  • nebulizers nebulizers
  • electric aerosol devices electric aerosol devices.
  • compositions of the present invention can be made into aerosol preparations (i.e., they can be "nebulized") alone or in combination with other suitable ingredients for administration by inhalation (e.g., intranasal or intratracheal) (see, Brigham et al., Am. J. Sci., 298: 278 (1989); and Patel et al., Advanced Materials, 31(8): e1805116 (2019)).
  • Aerosol preparations can be placed in a propellant that allows pressurization, such as dichlorodifluoromethane, propane, nitrogen, etc., or can be generated by a nebulizer.
  • composition of the present invention can be made into sprays individually or in combination with other suitable ingredients, so as to be used by nasal inhalation.
  • the pharmaceutical composition can be delivered by intranasal spray, inhalation, and/or other aerosol delivery vehicles.
  • the method for directly delivering nucleic acid compositions to the lungs by nasal aerosol spray has been described in, for example, U.S. Patent Nos. 5,756,353 and 5,804,212.
  • drug delivery using intranasal microparticle resins and lysophosphatidyl-glycerol compounds (U.S. Patent No. 5,725,871) is also well known in the pharmaceutical field.
  • transmucosal drug delivery using a polytetrafluoroethylene carrier matrix is described in U.S. Patent No. 5,780,045. The disclosure of the above patent is incorporated herein by reference as a whole for all purposes.
  • Formulations suitable for parenteral administration such as, for example, by intraarticular (in a joint), intravenous, intramuscular, intradermal, intraperitoneal, and subcutaneous routes include aqueous and nonaqueous, isotonic sterile injection solutions, which may contain antioxidants, buffers, bacteriostats, and solutes that make the formulation isotonic with the blood of the intended recipient, and aqueous and nonaqueous sterile suspensions that may include suspending agents, solubilizers, thickeners, stabilizers, and preservatives.
  • the composition is preferably administered, for example, by intravenous infusion, oral, topical, intraperitoneal, intravesical, or intrathecal administration.
  • Systemic delivery for in vivo therapy e.g., delivery of therapeutic nucleic acids through the body's circulatory system
  • To distant target cells such as PCT Publication Nos. WO 05/007196, WO 05/121348, WO 05/120152, and WO 04/002453 describe the use of nucleic acid-lipid particles to achieve such purposes, and the disclosure thereof is incorporated herein by reference for all purposes.
  • the present invention also provides a fully encapsulated polymer-lipid composition that protects nucleic acids from being degraded by nucleases in serum, is non-immunogenic, is very small in size, and is suitable for repeated administration.
  • polymer-lipid compositions are formulated using suitable pharmaceutical carriers.
  • suitable pharmaceutical carriers can be used in the compositions and methods of the present invention, and a variety of aqueous carriers can be used, for example, water, buffered water, physiological saline, 0.3% glycine, etc., and glycoproteins for enhancing stability, such as albumin, lipoprotein, globulin, etc., can also be used.
  • aqueous carriers for example, water, buffered water, physiological saline, 0.3% glycine, etc.
  • glycoproteins for enhancing stability such as albumin, lipoprotein, globulin, etc.
  • These compositions can be sterilized by conventional liposome sterilization techniques such as filtration.
  • compositions can contain pharmaceutical auxiliary substances as required to approach physiological conditions, such as pH adjustment and buffers, tension regulators, wetting agents, etc., such as sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc.
  • pharmaceutical auxiliary substances such as sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc.
  • the polymer-lipid composition disclosed herein can be delivered to an individual via oral administration.
  • the particles can be combined with excipients and in the form of ingestible tablets, buccal tablets, troche, capsules, pills, lozenges, elixirs, mouthwashes, suspensions, oral sprays, syrups, wafers, etc. (see, e.g., U.S. Patent Nos. 5,641,515, 5,580,579, and 5,792,451, the disclosure of which is incorporated herein by reference for all purposes).
  • These oral dosage forms may also include the following: adhesives, gelatin; excipients, lubricants, and/or flavorings.
  • the unit dosage form When the unit dosage form is a capsule, it may contain a lipid carrier in addition to the above-mentioned substances. A variety of other substances may exist as a coating or additionally modify the appearance of the dosage unit. Of course, any substance used in the preparation of any unit dose should be pharmaceutically pure and substantially nontoxic in the amount used.
  • these oral formulations can contain at least about 0.1% polymer-lipid composition or more, although the percentage of polymer-lipid composition can certainly be different, and can be conveniently between about 1% or 2% to about 60% or 70% or more of the total formulation weight or volume.
  • the amount of particles in each therapeutically effective composition that can be prepared is such that a suitable dose should be obtained in any given unit dose of the compound.
  • the technician in the field of preparing the pharmaceutical preparation should consider factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, and other pharmacological considerations, and as such, many dosages and treatment regimens may be desirable.
  • Formulations suitable for oral administration may consist of: (a) a lipid solution, such as a suspension in dilute (a) a therapeutic agent such as a nucleic acid (e.g., mRNA) in an effective amount of a packaged therapeutic agent such as a nucleic acid (e.g., mRNA) in a dispensing agent such as water, saline, or PEG400; (b) a capsule, sachet, or tablet, each containing a predetermined amount of a therapeutic agent such as a nucleic acid (e.g., mRNA), such as a liquid, solid, granular, or gelatin; (c) a suspension in an appropriate liquid; and (d) a suitable emulsion.
  • a therapeutic agent such as a nucleic acid (e.g., mRNA) in an effective amount of a packaged therapeutic agent such as a nucleic acid (e.g., mRNA) in a dispensing agent such as water, saline,
  • Tablet forms may include lactose, sucrose, mannose, sorbitol, calcium phosphate, corn starch, potato starch, microcrystalline cellulose, gelatin, colloidal silicon dioxide, talc, magnesium stearate, stearic acid, and other excipients, colorants, fillers, binders, diluents, buffers, wetting agents, preservatives, flavoring agents, dyes, disintegrants, and one or more of pharmaceutically compatible carriers.
  • Pastille forms can include a therapeutic agent such as a nucleic acid (e.g., mRNA) in a flavoring agent, such as sucrose, as well as pastilles containing the therapeutic agent in an inert matrix such as gelatin and glycerin or sucrose and acacia emulsions, gelatin, and similar pastilles containing in addition to the therapeutic agent carriers known in the art.
  • a therapeutic agent such as a nucleic acid (e.g., mRNA) in a flavoring agent, such as sucrose, as well as pastilles containing the therapeutic agent in an inert matrix such as gelatin and glycerin or sucrose and acacia emulsions, gelatin, and similar pastilles containing in addition to the therapeutic agent carriers known in the art.
  • the polymer-lipid composition can be incorporated into a wide range of topical dosage forms.
  • a suspension of the polymer-lipid composition containing nucleic acids can be formulated and applied as a gel, oil, emulsion, topical cream, suppository, paste, ointment, lotion, foam, mousse, etc.
  • the method of the present invention can be implemented in a variety of hosts.
  • Preferred hosts include mammalian species, such as primates (e.g., humans and chimpanzees and other non-human primates), canines, felines, equines, bovines, sheep, goats, rodents (e.g., rats and mice), lagomorphs, and pigs.
  • the amount of formulation administered will depend on the ratio of therapeutic agent (e.g., nucleic acid) to polymer-lipid component, the specific therapeutic agent (e.g., nucleic acid) used, the disease or functional disorder to be treated, the age, weight and condition of the patient, and the judgment of the clinician, but will generally be about 0.01 to about 50 mg/kg body weight, preferably about 0.1 to about 5 mg/kg body weight, or about 10 8 -10 10 particles/administration (e.g., nebulized inhalation).
  • therapeutic agent e.g., nucleic acid
  • polymer-lipid component e.g., polymer-lipid component
  • specific therapeutic agent e.g., nucleic acid
  • the pharmaceutical compositions described herein are formulated for administration by inhalation, orally, intraadipose, intraarterially, intraarticularly, intracranially, intradermally, intralesionally, intramuscularly, intranasally, intraocularly, intrapericardially, intraperitoneally, intrapleurally, intraprostatically, intrarectally, intrathecally, intratracheally, intratumorally, intraumbilically, intravaginally, intravenously, intravesically, intravitreally, topically, mucosally, parenterally, rectally, subconjunctivally, subcutaneously, sublingually, topically, buccally, transdermally, vaginally, in the form of creams, via catheter, via lavage, via continuous infusion, via infusion, via inhalation
  • the pharmaceutical composition is formulated for intravenous or intra-arterial injection.
  • compositions and methods of the present invention are used to treat a wide variety of cell types in vivo and in vitro.
  • Suitable cells include, for example, alveolar cells (including type I alveolar epithelial cells and type II alveolar epithelial cells), bronchial epithelial cells, goblet cells, ciliated cells, club cells, undifferentiated basal cells, pulmonary ionocytes, microfold cells (M cells), dendritic cells (DC cells), macrophages, T cells, B cells, NK cells, neutrophils, eosinophils, basophils, monocytes, hematopoietic precursor (stem) cells, fibroblasts, keratinocytes, hepatocytes, endothelial cells, skeletal and smooth muscle cells, osteoblasts, neurons, resting lymphocytes, terminally differentiated cells, slow or non-circulating primary cells, parenchymal cells, lymphoid cells, epithelial cells, osteocytes, etc.
  • active agents or therapeutic agents such as RNA (e.g., mRNA) are delivered to bronchial epithelial cells, ciliated cells, pulmonary ionocytes, microfold cells (M cells), dendritic cells (DC cells), T cells.
  • RNA e.g., mRNA
  • M cells microfold cells
  • DC cells dendritic cells
  • active agents or therapeutic agents such as RNA (e.g., mRNA) are delivered to cancer cells such as, for example, lung cancer cells, colon cancer cells, rectal cancer cells, anal cancer cells, bile duct cancer cells, small intestine cancer cells, gastric cancer cells, esophageal cancer cells, gallbladder cancer cells, liver cancer cells, pancreatic cancer cells, appendix cancer cells, breast cancer cells, ovarian cancer cells, cervical cancer cells, prostate cancer cells, renal cancer cells, central nervous system cancer cells, glioblastoma tumor cells, skin cancer cells, lymphoma cells, choriocarcinoma tumor cells, head and neck cancer cells, osteogenic sarcoma tumor cells, and blood cancer cells.
  • cancer cells such as, for example, lung cancer cells, colon cancer cells, rectal cancer cells, anal cancer cells, bile duct cancer cells, small intestine cancer cells, gastric cancer cells, esophageal cancer cells, gallbladder cancer cells, liver cancer cells, pancreatic cancer cells,
  • RNA e.g., mRNA
  • the methods and compositions can be used with a wide variety of vertebrates, including mammals, such as, for example, canines, felines, equines, bovines, sheep, goats, rodents (such as mice, rats and guinea pigs), lagomorphs, pigs, and primates (e.g., monkeys, chimpanzees and humans) cells.
  • mammals such as, for example, canines, felines, equines, bovines, sheep, goats, rodents (such as mice, rats and guinea pigs), lagomorphs, pigs, and primates (e.g., monkeys, chimpanzees and humans) cells.
  • tissue culture of cells may be required, this is well known in the art.
  • Freshney Culture of Animal Cells, a Manual of Basic Technique, 3rd ed., Wiley-Liss, New York (1994), Kuchler et al., Biochemical Methods in Cell Culture and Virology, Dowden, Hutchinson and Ross, Inc. (1977), and references cited therein provide a general description of cell culture.
  • General Guidance Cultured cell systems are most often in the form of cell monolayers, although cell suspensions are also used.
  • polymer-lipid composition of the present invention can be detected in a subject at about 1, 2, 3, 4, 5, 6, 7, 8 or more hours. In other embodiments, polymer-lipid composition of the present invention can be detected in a subject at about 8, 12, 24, 48, 60, 72 or 96 hours after applying particles, or about 6, 8, 10, 12, 14, 16, 18, 19, 22, 24, 25 or 28 days.
  • the presence of polymer-lipid composition particles can be detected in cells, tissues or other biological samples from a subject.
  • Polymer-lipid composition particles can, for example, by directly detecting particles, detecting therapeutic nucleic acids such as RNA (e.g., mRNA) sequences, detecting the target protein or polypeptide sequence encoded therein, or a combination thereof.
  • RNA e.g., mRNA
  • the polymer-lipid composition particles of the present invention can be detected using any known method in the art.
  • a marker can be coupled directly or indirectly to a component of the polymer-lipid composition particle using methods known in the art.
  • a wide variety of markers can be used, and the selection of the marker depends on the required sensitivity, the ease of conjugation with the polymer-lipid composition particle component, stability requirements, and available instruments and processing regulations.
  • Suitable labels include, but are not limited to, spectral labels such as fluorescent dyes (e.g., fluorescein and derivatives, such as fluorescein isothiocyanate (FITC) and Oregon Green TM ; rhodamine and derivatives, such as Texas Red, tetramethylrhodamine isothiocyanate (TRITC), etc., digoxigenin, biotin, phycoerythrin, AMCA, CyDyes TM , etc.; radioactive labels such as 3 H, 125 I, 35 S, 14 C, 32 P, 33 P, etc.; enzymes such as horseradish peroxidase, alkaline phosphatase, etc.; spectral colorimetric labels such as colloidal gold or colored glass or plastic beads such as polystyrene, polypropylene, latex, etc.
  • the labels can be detected by any means known in the art.
  • Nucleic acids are detected and quantified herein by any of a number of means known to those skilled in the art. Detection of nucleic acids can be performed by known methods such as Southern analysis, Northern analysis, gel electrophoresis, PCR, radiolabeling, scintillation counting, and affinity chromatography. Other analytical biochemical methods such as spectrophotometry, X-ray photography, electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), and high diffusion chromatography can also be used.
  • HPLC high performance liquid chromatography
  • TLC thin layer chromatography
  • the present disclosure provides a method for preventing and/or treating a disease or condition in a subject, the method comprising administering a pharmaceutically effective amount of a composition or pharmaceutical composition according to the present invention to a subject in need thereof in vivo, wherein the composition or pharmaceutical composition comprises an activating agent or therapeutic agent for the disease or condition.
  • 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.
  • the present invention provides the use of a composition or pharmaceutical composition according to the present invention in the preparation of a medicament for preventing and/or treating a disease in a subject, wherein the composition or pharmaceutical composition comprises the activating agent or therapeutic agent for the disease or condition.
  • the disease or condition is selected from immune system diseases, metabolic diseases, hereditary diseases, cancer, blood diseases, bacterial infections or viral infections.
  • the compositions are used for delivery to target organs and/or target tissues and/or target cells for treating human subjects.
  • the compositions described herein are used for delivery to the lungs or lung cells of a subject.
  • the compositions described herein are used for delivery to the liver or liver cells of a subject.
  • the compositions described herein are used for delivery to the spleen or spleen cells of a subject.
  • the compositions described herein are used for delivery to the muscles or muscle cells of a subject.
  • the present invention provides a method for delivering a composition described herein comprising an mRNA encoding a protein or polypeptide, the therapeutic composition being used to treat a lung disease.
  • the present invention can be used in a method for producing an mRNA encoding the cystic fibrosis transmembrane conductance regulator CFTR (CFTR mRNA).
  • the CFTR mRNA is delivered to the lungs of a subject in need of a therapeutic composition for treating cystic fibrosis.
  • the present invention can be used in a method for producing an mRNA encoding ⁇ 1-trypsin (A1AT mRNA).
  • the A1AT mRNA is delivered to the lungs and or liver of a subject in need of a therapeutic composition for ⁇ 1-trypsin deficiency.
  • the present invention can be used in a method for producing mRNA encoding the medium and heavy chains of axonal dynein (DNAI1 mRNA and DNAH5 mRNA).
  • DNAI1 mRNA or DNAH5 mRNA is delivered to the lungs of a subject in need of a therapeutic composition for treating primary ciliary dyskinesia.
  • the present invention provides a method for delivering a composition as described herein comprising mRNA encoding a protein or polypeptide, wherein the therapeutic composition is used to treat liver disease or metabolic disease.
  • Such proteins and polypeptides may include those associated with urea cycle disorders, lysosomal storage disorders, glycogen storage disorders, amino acid metabolism disorders, lipid metabolism or fibrosis disorders, methylmalonic acidemia, or any other metabolic disorder for which delivery or treatment of the liver or hepatocytes with enriched full-length mRNA provides a therapeutic benefit.
  • the invention provides a method for delivering a composition as described herein comprising an mRNA encoding a protein associated with a urea cycle disorder. In certain embodiments, the invention provides a method for delivering a composition as described herein comprising an mRNA encoding an ornithine transcarbamylase (OTC) protein.
  • OTC ornithine transcarbamylase
  • the invention provides a method for delivering a composition described herein comprising mRNA encoding a protein associated with a lysosomal storage disorder. In certain embodiments, the invention provides a method for delivering a composition described herein comprising mRNA encoding an alpha galactosidase protein.
  • the invention provides a method for delivering a composition as described herein comprising mRNA encoding a protein associated with glycogen storage disease. In certain embodiments, the invention provides a method for delivering a composition as described herein comprising mRNA encoding an acid alpha-glucosidase protein.
  • the invention provides a method for delivering a composition as described herein comprising mRNA encoding a protein related to amino acid metabolism. In certain embodiments, the invention provides a method for delivering a composition as described herein comprising mRNA encoding phenylalanine hydroxylase.
  • the present invention provides a method for delivering a composition as described herein comprising an mRNA encoding a protein associated with lipid metabolism or fibrotic diseases. In certain embodiments, the present invention provides a method for delivering a composition as described herein comprising an mRNA encoding an mTOR inhibitor.
  • the present invention provides a method for delivering a composition described herein comprising an mRNA encoding a protein associated with methylmalonic acidemia.
  • the present invention provides a method for delivering a composition described herein comprising an mRNA encoding a methylmalonyl-CoA mutant enzyme protein.
  • the therapeutic composition is used to deliver to or treat the liver or hepatocytes of a subject.
  • the invention provides a method for delivering a composition comprising a protein encoding ATP7B.
  • the present invention provides a method for delivering a composition as described herein comprising mRNA encoding porphobilinogen deaminase.
  • the present invention provides a method for delivering a composition as described herein comprising mRNA encoding human hemochromatosis (HFE) protein.
  • HFE human hemochromatosis
  • the therapeutic composition is used to deliver to or treat the cardiovascular system or cardiovascular cells of a subject.
  • the invention provides a method for delivering a composition described herein comprising mRNA encoding vascular endothelial growth factor A protein.
  • the therapeutic composition is used to deliver to or treat a subject's muscle or muscle cells.
  • the invention provides a method for delivering a composition as described herein comprising an mRNA encoding a dystrophin.
  • the invention provides a method for delivering a composition as described herein comprising an mRNA encoding a protein or polypeptide, the therapeutic composition being used to deliver to or treat a subject's myocardium or myocardial cells.
  • the invention provides a method for delivering a composition as described herein comprising an mRNA encoding a protein or polypeptide that modulates one or both of a potassium channel and a sodium channel in a muscle tissue or muscle cell.
  • the therapeutic composition is used to deliver to or treat the nervous system or nervous system cells of a subject.
  • the invention provides a method for delivering a composition described herein comprising an mRNA encoding a survival motor neuron 1 protein.
  • the therapeutic composition is for delivery to or treatment of the blood or bone marrow, or blood cells or bone marrow cells, of a subject.
  • the invention provides a method for delivering a composition described herein comprising mRNA encoding beta globin.
  • the therapeutic composition is used to deliver to or treat a kidney or renal cell of a subject.
  • the invention provides a method for delivering a composition described herein comprising mRNA encoding type IV collagen alpha 5 chain (COL4A5) protein.
  • the therapeutic composition is used to deliver to or treat an eye or ocular cell of a subject.
  • the invention provides a method for delivering a composition described herein comprising an mRNA encoding an ATP-binding cassette subfamily A member 4 (ABCA4) protein.
  • the invention provides a method for delivering a composition described herein comprising an mRNA encoding a retinoschizin protein.
  • the therapeutic composition is used to deliver a vaccine to a subject or a subject's cell or to treat with a vaccine.
  • the invention provides a method for delivering a composition described herein comprising mRNA encoding an antigen from an infectious source such as a virus.
  • the invention provides a method for preparing a composition described herein comprising delivering mRNA encoding a novel coronavirus (SARS-CoV-2) antigen.
  • SARS-CoV-2 novel coronavirus
  • the present invention provides mRNA for containing an antigen from an infectious source such as bacteria.
  • the present invention provides a method for preparing a composition as described herein comprising delivering an mRNA encoding a Mycobacterium tuberculosis antigen.
  • the present invention provides a method for preparing a composition as described herein comprising delivering an mRNA encoding a Pseudomonas aeruginosa antigen.
  • the present invention provides a method for preparing a composition as described herein comprising delivering an mRNA encoding a Klebsiella pneumoniae antigen.
  • the present invention provides a method for preparing a composition as described herein comprising delivering an mRNA encoding a Staphylococcus aureus antigen. In certain embodiments, the present invention provides a method for preparing a composition as described herein comprising delivering an mRNA encoding a Baumannii antigen. In certain embodiments, the present invention provides a method for preparing a composition as described herein comprising delivering an mRNA encoding a Baumannii antigen. In certain embodiments, the present invention provides a method for preparing a composition as described herein comprising delivering an mRNA encoding a Legionella pneumophila antigen.
  • the present invention provides a method for delivering a therapeutic composition encoding a cancer-associated antigen of a subject or an mRNA of an antigen identified from a subject's cancer cells. In certain embodiments, the present invention provides a method for preparing a composition described herein comprising delivering an mRNA encoding an antigen determined from a subject's own cancer cells, i.e., providing a personalized cancer vaccine. In certain embodiments, the present invention provides a method for preparing a composition described herein comprising delivering an mRNA encoding an antigen expressed from a mutant KRAS gene.
  • the present invention provides a method for delivering a composition described herein comprising mRNA encoding an antibody.
  • the antibody may be a bispecific antibody.
  • the antibody may be part of a fusion protein.
  • the two independent mRNA-loaded polymer-lipid compositions (mRNA-PoLixNano) in the method comprise mRNA encoding an antibody light chain and a heavy chain.
  • the mRNA-PoLixNano composition of the present invention may include a combination of different PoLixNano containing different lipid compositions and mRNA encapsulating an antibody light chain or heavy chain.
  • the present invention provides a method for delivering a composition described herein comprising mRNA encoding an antibody against OX40. In certain embodiments, the present invention provides a method for delivering a composition comprising mRNA encoding an antibody against VEGF. In certain embodiments, the present invention provides a method for delivering a composition as described herein comprising mRNA encoding an antibody to tissue necrosis factor alpha. In certain embodiments, the present invention provides a method for delivering a composition as described herein comprising mRNA encoding an antibody to CD3. In certain embodiments, the present invention provides a method for delivering a composition as described herein comprising mRNA encoding an antibody to CD19.
  • the present invention provides a method for delivering a composition described herein comprising an mRNA encoding an immunomodulator. In certain embodiments, the present invention provides a method for delivering a composition described herein comprising an mRNA encoding interleukin 2 (IL-2). In certain embodiments, the present invention provides a method for delivering a composition described herein comprising an mRNA encoding interleukin 12 (IL-12). In certain embodiments, the present invention provides a method for delivering a composition described herein comprising an mRNA encoding granulocyte-macrophage colony stimulating factor (GM-CSF).
  • GM-CSF granulocyte-macrophage colony stimulating factor
  • the present invention provides a method for delivering a composition described herein comprising an mRNA encoding interleukin 23 (IL-23). In certain embodiments, the present invention provides a method for delivering a composition described herein comprising an mRNA encoding C-C domain chemokine ligand 28 (CCL28). In certain embodiments, the present invention provides a method for delivering a composition described herein comprising an mRNA encoding interleukin 36 ⁇ (IL-36 ⁇ ). In certain embodiments, the present invention provides a method for delivering a composition described herein comprising an mRNA encoding thymic stromal lymphopoietin or a derivative thereof. In certain embodiments, the invention provides a method for delivering a composition described herein comprising mRNA encoding one or more constitutively active variants of the stimulator of interferon genes (STING) protein.
  • STING interferon genes
  • the invention provides a method for delivering a composition as described herein comprising an mRNA encoding a nuclease. In certain embodiments, the invention provides a method for delivering a composition as described herein comprising an mRNA encoding a DNA endonuclease protein (such as a Cas 9 protein) guided by RNA. In certain embodiments, the invention provides a method for delivering a composition as described herein comprising an mRNA encoding a meganuclease protein. In certain embodiments, the invention provides a method for delivering a composition as described herein comprising an mRNA encoding a transcription activator-like effector nuclease protein. In certain embodiments, the invention provides a method for delivering a composition as described herein comprising an mRNA encoding a zinc finger nuclease protein.
  • the formulations described herein can be used to deliver any mRNA, in particular, in vitro transcribed mRNA (IVT-mRNA) encoding a specific antigen or therapeutic protein.
  • IVT-mRNA in vitro transcribed mRNA
  • the formulations described herein can also be used to deliver any modified or unmodified mRNA, or mRNA with a naturally occurring sequence or codon optimization.
  • the mRNA delivered by the formulations described herein can (but not exclusively) encode firefly luciferase (Fluc) and the new coronavirus SARS-CoV-2 spike protein receptor binding domain RBD antigen.
  • the mRNA encoding firefly luciferase (Fluc-mRNA) and encoding the RBD antigen (RBD-mRNA) of the spike protein of the novel coronavirus SARS-CoV-2 described herein can be synthesized by constructing a recombinant DNA plasmid comprising a T7 promoter, 5'UTR, ORF (Fluc or RBD), 3'UTR, and a 3' poly (A) tail of about 100 nucleotides in length; preparing a linearized DNA template by enzyme digestion or PCR amplification; performing in vitro transcription with the linearized DNA template, and then adding a 5' cap structure (Cap 1) to prepare Fluc-mRNA and RBD-mRNA. After purification, the IVT-mRNA band size and purity are determined by gel electrophoresis.
  • aqueous buffer 50 mM citrate buffer, pH 4.0
  • Fluc-mRNA or RBD-mRNA
  • 12.68 mg/mL citric acid and 8.77 mg/mL sodium citrate were mixed in a volume ratio of 1:1, and the pH of the mixture was about 4.0.
  • Fluc-mRNA (or RBD-mRNA) was diluted to 0.17 mg/mL with the above buffer.
  • the lipid-dissolved ethanol solution was fully mixed with the citrate buffer containing mRNA using a microfluidic mixing device (NanoFac series, Sichuan Shuogen Technology Co., Ltd.) (the volume ratio of aqueous phase to organic phase was 3:1) at a total flow rate of 12 mL/min.
  • a microfluidic mixing device NaFac series, Sichuan Shuogen Technology Co., Ltd.
  • the volume ratio of aqueous phase to organic phase was 3:1
  • the solution was mixed, it was dialyzed with 1 ⁇ PBS (pH 7.4) at 4°C (dialysis bag molecular weight cutoff MWCO: 8000-14000) for 20 hours, filtered with a 0.22 ⁇ m filter membrane, and stored at 2-8°C.
  • the final concentration of Fluc-mRNA and RBD-mRNA encapsulated in the prepared LNP preparation was about 0.12 mg/mL.
  • Preparation of aqueous phase Prepare 50 mM citrate buffer by the method described in "Formulation Example #1", use this buffer to prepare Fluc-mRNA (or RBD-mRNA) and T904 solutions into 0.34 mg/mL and 8 mg/mL stock solutions, respectively, then mix the above Fluc-mRNA (or RBD-mRNA) stock solution and T904 stock solution in a volume ratio of 1:1 and set aside.
  • Preparation of organic phase 50 mg/mL of Dlin-MC3-DMA in ethanol, 50 mg/mL of DSPC in ethanol, 50 mg/mL of cholesterol in ethanol and 50 mg/mL of DMG-PEG2K in ethanol were mixed in a molar ratio of 50:14:35:1, and diluted with ethanol to a total lipid concentration of 15.30 mg/mL.
  • the aqueous solution and the organic solution were fully mixed using a microfluidic mixing device (NanoFac series, Sichuan Shuogeng Technology Co., Ltd.) (the volume ratio of aqueous phase to organic phase was 3:1, and the total flow rate was 12 mL/min).
  • the mixed solution was collected, the collected solution was dialyzed (dialysis bag MWCO: 8000-14000) for 20 h in a 4°C environment using 1 ⁇ PBS (pH 7.4), filtered with a 0.22 ⁇ m filter membrane, and stored at 2-8°C.
  • the final concentration of Fluc-mRNA (or RBD-mRNA) encapsulated in the prepared PoLixNano preparation was approximately 0.10 mg/mL.
  • T904 was diluted to 75 mg/mL with 1 ⁇ PBS solution, filtered through a 0.22 ⁇ m filter membrane, and set aside.
  • the LNP solution prepared and purified by the method described in "Formulation Example #1" was mixed with the above T904 solution at a volume ratio of 4:1. After standing at room temperature for 20 minutes, it was filtered with a 0.22 ⁇ m filter membrane and stored at 2-8°C. The final concentration of Fluc-mRNA (or RBD-mRNA) encapsulated in the prepared LNP+T904 formulation was about 0.10 mg/mL.
  • T904 was diluted to 15 mg/mL with 1 ⁇ PBS, filtered through a 0.22 ⁇ m filter membrane, and set aside.
  • the PoLixNano solution prepared and purified in the above "Formulation Example #2" and the above T904 were mixed thoroughly at a volume ratio of 4:1. After incubation at room temperature for 20 minutes, the mixture was filtered through a 0.22 ⁇ m filter membrane and stored at 2-8°C. The final concentration of Fluc-mRNA (or RBD-mRNA) encapsulated in the prepared PoLixNano+T904 formulation was about 0.08 mg/mL.
  • PoLixNano solution prepared and purified in the above "Formulation Example #2” and 50% sucrose solution were mixed thoroughly at a volume ratio of 4:1. After incubation at room temperature for 20 minutes, the mixture was filtered through a 0.22 ⁇ m filter membrane and stored at 2-8°C. The final concentration of Fluc-mRNA encapsulated in the prepared PoLixNano + 10% sucrose formulation was about 0.08 mg/mL.
  • PoLixNano formulation prepared from three-component lipids consisting of ionizable/cationic lipids, neutral lipids, and cholesterol (i.e., PoLixNano without a PEG-lipid component):
  • Preparation of aqueous phase Use 50 mM citrate buffer to dilute Fluc-mRNA (or RBD-mRNA) and Poloxamer 407 solutions to 0.34 mg/mL and 13.33 mg/mL solutions, respectively, and then mix the Fluc-mRNA (or RBD-mRNA) solution and Poloxamer 407 solution in a volume ratio of 1:1 for standby use.
  • Preparation of organic phase 50 mg/mL of Dlin-MC3-DMA in ethanol, 50 mg/mL of DOPE in ethanol, and 50 mg/mL of cholesterol in ethanol were mixed at a molar ratio of 40:32:28 and diluted with ethanol to a total lipid concentration of 18.96 mg/mL.
  • the aqueous phase solution and the organic phase solution were fully mixed using a microfluidic mixing device (NanoFac series, Sichuan Shuogeng Technology Co., Ltd.) (the volume ratio of aqueous phase: organic phase was 3:1, and the total flow rate was 12 mL/min).
  • a microfluidic mixing device NanoFac series, Sichuan Shuogeng Technology Co., Ltd.
  • the volume ratio of aqueous phase: organic phase was 3:1, and the total flow rate was 12 mL/min.
  • the mixed solution was collected, it was dialyzed (dialysis bag MWCO: 8000-14000) for 20 h in a 4°C environment using 1 ⁇ PBS (pH 7.4), filtered with a 0.22 ⁇ m filter membrane, and stored at 2-8°C.
  • PoLixNano formulation prepared from three-component lipids consisting of cholesterol-derived cationic lipids, neutral lipids, and PEG-lipids:
  • Fluc-mRNA (or RBD-mRNA) and T904 solutions were diluted to 0.34 mg/mL and 8 mg/mL solutions, respectively, and then the Fluc-mRNA (or RBD-mRNA) solution and the T904 solution were mixed evenly at a volume ratio of 1:1 for standby use.
  • the aqueous phase solution and the organic phase solution were fully mixed using a microfluidic mixing device (NanoFac series, Sichuan Shuogeng Technology Co., Ltd.) (the volume ratio of aqueous phase: organic phase was 3:1, and the total flow rate was 12 mL/min).
  • a microfluidic mixing device NanoFac series, Sichuan Shuogeng Technology Co., Ltd.
  • the volume ratio of aqueous phase: organic phase was 3:1, and the total flow rate was 12 mL/min.
  • the mixed solution was collected, it was dialyzed (dialysis bag MWCO: 8000-14000) for 20 h in a 4°C environment using 1 ⁇ PBS (pH 7.4), filtered with a 0.22 ⁇ m filter membrane, and stored at 2-8°C.
  • PoLixNano formulation prepared from three-component lipids consisting of ionizable/cationic lipids, cholesterol, and PEG-lipids (i.e., PoLixNano without a phospholipid component):
  • Preparation of aqueous phase Use 50 mM citrate buffer to dilute Fluc-mRNA (or RBD-mRNA) and T904 solution to 0.34 mg/mL and 8 mg/mL solution, respectively, and then mix the Fluc-mRNA (or RBD-mRNA) solution and T904 solution in a volume ratio of 1:1 for standby use.
  • Preparation of organic phase 50 mg/mL ethanol solution of CKK-E12, 50 mg/mL ethanol solution of DOTAP, 50 mg/mL ethanol solution of cholesterol and 50 mg/mL ethanol solution of DMG-PEG2K were mixed at a molar ratio of 30:39:30:1 and diluted with ethanol to a total lipid concentration of 5.86 mg/mL.
  • the aqueous phase solution and the organic phase solution were fully mixed using a microfluidic mixing device (NanoFac series, Sichuan Shuogeng Technology Co., Ltd.) (the volume ratio of aqueous phase: organic phase was 3:1, and the total flow rate was 12 mL/min).
  • a microfluidic mixing device NanoFac series, Sichuan Shuogeng Technology Co., Ltd.
  • the volume ratio of aqueous phase: organic phase was 3:1, and the total flow rate was 12 mL/min.
  • the mixed solution was collected, it was dialyzed (dialysis bag MWCO: 8000-14000) for 20 h in a 4°C environment using 1 ⁇ PBS (pH 7.4), filtered with a 0.22 ⁇ m filter membrane, and stored at 2-8°C.
  • the particle size of the LNP and PoLixNano preparations was determined by a laser particle size analyzer. PDI, potential information and encapsulation efficiency. Encapsulation of nucleic acids was determined using the Quant-iT RiboGreen RNA assay. The RiboGreen assay was essentially as described in the article published by Heyes et al., Journal of Controlled Released, 2005, 107:276-287. The above physicochemical characterization results and the lipid components and amphiphilic block copolymer compositions of the formulations are summarized in Table 1.
  • This example illustrates exemplary methods of administering relevant formulations loaded with mRNA and methods for analyzing proteins expressed by the delivered mRNA in target tissues in vivo.
  • mice were housed in a specific pathogen-free (SPF) animal room with a 12-hour/12-hour light/dark cycle and free access to food and water. All experimental animals had at least 7 days of acclimatization time before the start of the experiment.
  • SPF pathogen-free
  • a high-pressure MicroSprayer needle (PennCentury, USA) was used to spray the mRNA dose of 2.5 ⁇ g mRNA/50 ⁇ L of PoLixNano or LNP formulation into the trachea of a single mouse. Unless otherwise specified, this dose was used in all experiments of the same type.
  • mice were administered PoLixNano or LNP formulations at an mRNA dose of 2.0 ⁇ g mRNA/40 ⁇ L.
  • the solution was applied as a drop to the nostril of a single animal during isoflurane (Isothesia) inhalation anesthesia, and the complex formulation was naturally inhaled. Unless otherwise specified, all experiments of the same type used this dose.
  • the PoLixNano formulation or LNP formulation with a dose of 25 ⁇ g mRNA/mouse was placed in a nebulizer system (Aerogen, Dangan, Ireland) with a nebulization rate of 30 ⁇ L/10 s and a nebulization time of 15 min. Unless otherwise specified, this dose was used in all experiments of the same type.
  • the skin surface of the outer thigh muscle of Balb/c mice was disinfected with 75% alcohol cotton balls, and the muscle was quickly pierced with a syringe to inject PoLixNano or LNP preparations with a mRNA dose of 5 ⁇ g mRNA/100 ⁇ L. Unless otherwise specified, all experiments of the same type used this dose.
  • IVIS in vivo imaging device In order to detect the expression of Luciferase reporter gene in mice administered with Fluc-mRNA preparation, IVIS in vivo imaging device was used for investigation. First, anesthesia was performed using an isoflurane anesthesia device at 1-3% (usually 2.5%). D-luciferin solution (3 mg D-luciferin dissolved in 100 ⁇ L PBS) was administered to each animal via intraperitoneal injection (i.p.) at a dose of 150 mg/kg. Allow the luciferin to distribute for 10 minutes. The animal was placed in an isoflurane chamber until anesthetized. The anesthetized animal was placed in the dorsal recumbency of the IVIS imaging chamber with the head placed in the ventilator.
  • Bioluminescence imaging was performed using the IVIS in vivo imaging system (Perkin Elmer, USA).
  • the instrument settings were as follows: the camera height was at D level, F/Stop was f1, the pixel combination (binning) was 8 ⁇ 8, and the exposure time was automatic.
  • the animals were killed by cervical dislocation, and the lungs, liver, or spleen were quickly removed, and the isolated organs were imaged again for bioluminescence.
  • the images were analyzed by measuring the radiance in the set region of interest (ROI) using Living Image software (PerkinElmer, USA). The values are shown as total radiance [p/s].
  • PoLixNano formulations containing different concentrations of T904 and T704 were prepared using the method described in the above "Formulation Example #2" using different lipid ratios.
  • PolixNano nanoparticles formed a multi-chamber structure ( Figure 1A). Studies have shown that the chamber structure in this type of nanoparticle can increase the transfection efficiency of mRNA (Cheng MHY, Cullis PR, et al. Induction of Bleb Structures in Lipid Nanoparticle Formulations of mRNA Leads to Improved Transfection Potency. Adv Mater. 2023, 35 (31): e2303370).
  • the PoLixNano preparation containing 6 mg/mL T704 can be efficiently taken up by human bronchial epithelial cells 16HBE, dendritic cells DC2.4 and mouse bone marrow-derived dendritic cells (BMDC) cells, effectively express Fluc-mRNA, and is non-toxic to cells (Figure 1B and C).
  • the PoLixNano preparation has a stronger ability to penetrate the mucus layer ( Figure 1D).
  • the PoLixNano preparation can more effectively stimulate the differentiation and maturation of BMDC cells and better mediate subsequent adaptive immune responses (Figure 1E).
  • Example 6 Gene transfection effects of different preparations 6 hours after intratracheal spray (i.t.) administration
  • LNPs were prepared using the method described in Formulation Example #1, and PoLixNano formulations containing 0.025 mg/mL T904, 3 mg/mL T904, 0.025 mg/mL T704, and 6 mg/mL T704 were prepared using the method described in Formulation Example #2.
  • T904-mRNA preparation (or T704-mRNA preparation): Mix 4 ⁇ Tyrode buffer and 0.24 mg/mL Fluc-mRNA in a volume ratio of 1:1, and then mix the mixture with 6 mg/mL T904 solution (or T704 solution) in a volume ratio of 1:1. Incubate at room temperature for 20 minutes to obtain T904-mRNA preparation (or T704-mRNA preparation) with a Fluc-mRNA dose of 3 ⁇ g/50 ⁇ L.
  • Fluc-mRNA was diluted to 3 ⁇ g/50 ⁇ L with PBS.
  • Example 7 Intratracheal spraying of PoLixNano formulations containing different types of amphiphilic polymers (i.t.) Transfection effect 6h after administration
  • PoLixNano formulations containing different types of amphiphilic polymers as shown in FIG3 were prepared using the method described in the above “Formulation Example #2”.
  • the concentration of the amphiphilic polymer in FIG3 is the final concentration in the PoLixNano particles.
  • the results are shown in Figure 3.
  • the PoLixNano preparations containing different concentrations of T904 and T704 can significantly increase the luciferase level produced by Fluc-mRNA in living mice and lungs, especially the PoLixNano preparations containing 40 mg/mL T904 and 6 mg/mL T704 significantly improved the transfection efficiency of mRNA in the lungs ( Figure 3A and B).
  • the intratracheal spray (i.t.) administration of PoLixNano preparations prepared by replacing T704 or T904 with Tween 80, other Poloxamers or Poloxamines can still efficiently transfect Fluc-mRNA in the lungs ( Figure 3C and D).
  • Example 8 PoLixNano preparations prepared with different N/P ratios and lipid molar ratios were administered intratracheally Transfection effect 6 hours after intravenous (i.t.) administration
  • PoLixNano formulations Using the method described in the above "Formulation Example #2", keeping the molar ratio of each lipid component unchanged, PoLixNano formulations with N/P ratios of 2, 4, 5, 6, 7, 8, 9, 10, 12 and 15 were prepared. Using the method described in the above “Formulation Example #2", adjusting N/P to 8, PoLixNano formulations containing lipids in different molar ratios were prepared, wherein the molar ratio of DMG-PEG2000 was between 0.1 and 5, the molar ratio of DSPC was between 8 and 50.1, the molar ratio of Dlin-MC3-DMA was between 25 and 70, and the molar ratio of cholesterol was between 20.2 and 46.5.
  • PoLixNano preparations prepared with different lipid molar ratios had different gene transfection abilities in living mice and lungs and were significantly higher than the LNP control group, especially PoLixNano preparations (DMG-PEG2000 molar ratio between 0.3 and 1, DSPC molar ratio between 10 and 30.1, Dlin-MC3-DMA molar ratio between 35 and 65, and cholesterol molar ratio between 21 and 46.5) all had the potential for efficient transfection of Fluc-mRNA ( Figures 4B and C).
  • Example 9 PoLixNano formulations prepared with different types of lipids were administered via intratracheal spray (i.t.) Transfection effect after 6 hours
  • the results are shown in Figure 5.
  • the PoLixNano preparations prepared with different lipids can significantly increase the luciferase level produced by Fluc-mRNA in living mice and lungs.
  • the PoLixNano preparation groups prepared with Dlin-MC3-DMA, DSPC, cholesterol and DMG-PEG2000 the PoLixNano prepared by replacing lipids with different structures (except SM-102) has a comparable level of luciferase produced by Fluc-mRNA in the lungs, and the PoLixNano prepared by SM-102 significantly improves the transfection efficiency of Fluc-mRNA in the lungs, which indicates that the PoLixNano preparations prepared by replacing lipids with different structures have the potential to achieve comparable or even more efficient mRNA transfection through respiratory administration.
  • Example 10 Preparation of Poloxamers, Poloxamines and Lipids Transfection effect of PoLixNano formulation 6 hours after i.n. administration
  • PoLixNano formulations Using the method described in the above "Formulation Example #5", the molar ratio of Dlin-MC3-DMA, DSPC, cholesterol and DMG-PEG2000 was adjusted to 49:20.1:30:0.9, and N/P was 8. PoLixNano + 10% sucrose formulations containing different types of poloxamers, poloxamines and lipids were prepared as shown in Figure 6. The concentrations of poloxamers and poloxamines were Final concentration within PoLixNano particles.
  • the PoLixNano preparations could efficiently transfect Fluc-mRNA in the lungs, especially the PoLixNano preparations (DMG-PEG2000 molar ratio between 0.5 and 2, DSPC molar ratio between 8 and 20.1, Dlin-MC3-DMA molar ratio between 47.9 and 56.4, and cholesterol molar ratio between 30 and 34.7 ( Figure 6B)).
  • Example 11 Physically mixed poloxamers, poloxamines, polypeptides and sucrose to obtain The preparation can further promote gene transfection of Fluc-mRNA in the lungs
  • LNP was prepared by the method described in "Formulation Example #1"; the corresponding LNP physical mixed preparation in Figure 7 was prepared by the method described in "Formulation Example #3".
  • LNP formulations The LNP control formulations without Poloxamer and Poloxamine were prepared by the methods described in the above "Formulation Example #6", “Formulation Example #7” and “Formulation Example #8", respectively.
  • the LNP formulation lacking the DSPC component was prepared by adjusting the lipid components and molar ratio to 8 N/P using the method described in the above "Formulation Example #1".
  • Preparation of PoLixNano preparations The methods described in the above-mentioned "Preparation Example #6", “Preparation Example #7” and “Preparation Example #8” were respectively used to adjust the lipid components and their molar ratios of the method described in "Preparation Example #2” (lacking DSPC component group), with N/P being 8, to prepare the corresponding PoLixNano preparations.
  • PoLixNano (GL67) and PoLixNano (CKK-E12) preparations were able to significantly increase the level of luciferase produced by Fluc-mRNA in the lungs after 6 hours of intratracheal spray (i.t.) administration ( Figures 8A and B).
  • PoLixNano preparations without DMG-PEG2000 or DSPC effectively increased the transfection efficiency of Fluc-mRNA in the liver, lungs and spleen after 6 hours of intratracheal spray (i.t.) administration.
  • PoLixNano preparations without DMG-PEG2000 or DSPC significantly increased the luciferase produced by Fluc-mRNA in the lungs via the nasal route (i.n.) ( Figures 8C and D).
  • Example 13 Analysis of the physicochemical properties of LNP and PoLixNano preparations before and after atomization and the Transfection effects of Poloxamers, Poloxamines and lipid-based PoLixNano formulations after 6 hours
  • the PoLixNano preparation or LNP control preparation was atomized using the atomization device shown in FIG9A.
  • the morphology of the LNP nanoparticles and the PoLixNano nanoparticles before and after atomization was observed using a transmission electron microscope (TEM).
  • TEM transmission electron microscope
  • the LNP preparation nanoparticles before atomization were uniform in size and intact in structure, while the LNP preparation nanoparticles after atomization were aggregated, broken and deformed, showing irregular morphology (FIG9B).
  • poloxamer 188 When poloxamer 188 was added to the above components at a final concentration of 10 mg/mL, the particle size and structural shape of the prepared PolixNano preparation particles did not change significantly before and after atomization (Table 2 and FIG9B), indicating that poloxamer 188 can protect the PolixNano nanoparticles during the atomization process, reduce the impact of the mechanical force of the atomization device on the nanoparticles, ensure the integrity of their morphological structure, and thus ensure the transfection efficiency of the mRNA loaded in the PolixNano nanoparticles.
  • Example 14 Administration of PoLixNano formulations containing different lipid ratios and doses by aerosol inhalation Transfection effect after h
  • Preparation of LNP preparation Using the method described in the above "Formulation Example #1", the molar ratio of Dlin-MC3-DMA (or SM-102), DSPC, cholesterol and DMG-PEG2000 was adjusted to 48.5:25:25:1.5, and N/P was 8.
  • Preparation of PoLixNano preparation Using the method described in the above-mentioned "Preparation Example #2", the molar ratio of Dlin-MC3-DMA, DSPC, cholesterol and DMG-PEG2000 was adjusted, wherein the molar ratio of DMG-PEG2000 was between 0.5 and 3, the molar ratio of DSPC was between 14 and 40, the molar ratio of Dlin-MC3-DMA was between 39 and 63, and the molar ratio of cholesterol was between 17.5 and 35.
  • the level of luciferase produced by Fluc-mRNA in the 50 ⁇ g/mouse group was higher than that in the 25 ⁇ g/mouse group and lower than that in the 150 ⁇ g/mouse group, indicating that the expression of Fluc-mRNA in the PoLixNano preparation administered by nebulized inhalation was dose-dependent (Figure 10C).
  • bronchial epithelial cells CD326 +
  • DC cells CD11c +
  • endothelial cells CD31 +
  • type I alveolar cells Podoplanin +
  • macrophages F4/80 +
  • the expression trend of luciferase produced by Fluc-mRNA and FLuc-circRNA at different times of administration of PoLixNano preparations by intratracheal spray was further analyzed. The results showed that luciferase with comparable expression levels was detected in the lungs 6h, 12h and 24h after administration, but the luciferase signal detected in the liver and spleen 24h after administration was significantly lower than that at 12h.
  • the PoLixNano preparation encapsulating Fluc-circRNA was administered by intratracheal spray (i.t.). The expression level of FLuc-circRNA was the highest at 6h after administration, and the expression level of Fluc-circRNA gradually decreased over time. A trace amount of luciferase signal could still be detected on the ninth day of administration.
  • PoLixNano preparation can mediate efficient transfection of mRNA and circRNA in mice and achieve sustained expression to a certain extent via intratracheal spray (i.t.) administration.
  • Preparation of LNP preparations Using the method described in the above-mentioned "Formulation Example #1", the molar ratio of Dlin-MC3-DMA, DSPC, cholesterol and DMG-PEG2000 was adjusted to 50:14:35:1 (intratracheal spray (i.t.)), 49:20.1:30:0.9 (nasal route (i.n.)) or 46:23:30:1 (atomized inhalation), and N/P was 8.
  • Preparation of PoLixNano preparations Use the methods described in the above "Preparation Example #2” and “Preparation Example #4" to prepare PoLixNano preparations containing different Poloxamer, Poloxamine or Tween 80 corresponding to the LNP preparations.
  • luciferase signals were detected in the lungs, liver and spleen of the LNP preparation group; luciferase signals were specifically detected in the lung tissue of the PoLixNano preparation containing 3mg/mL 188 or 3mg/mL T304, and the PoLixNano preparation containing 3mg/mL 338 was first prepared and then physically mixed with 1mg/mL 338.
  • Preparation of LNP preparation Using the method described in the above "Preparation Example #1", the molar ratio of Dlin-MC3-DMA, DSPC, cholesterol and DMG-PEG2000 was adjusted to 49:20.1:30:0.9, and N/P was 8.
  • the expression levels of IL-4, IL-6, IL-17 and TNF- ⁇ in the PoLixNano preparation group containing 3 mg/mL T904 were comparable to those in the PBS group (i.t.), and were significantly lower than those in the LNP preparation group (i.t.) ( Figure 13C).
  • the liver and kidney function indicators in the serum of the PoLixNano preparation group containing 3 mg/mL T904 after i.t. administration were normal, and no specific IgG antibodies induced by the T904 component and lipid component were detected in the serum.
  • PoLixNano formulation can promote C57black 6 after 6 hours of administration via the respiratory route Gene transfection in the lungs of C57BL/6 mice and Sprague-Dawley (SD) rats
  • PoLixNano formulations Using the method described in the above "Formulation Example #2", prepare the corresponding PoLixNano formulations containing 3 mg/mL T904 (intratracheal spray (i.t.) or nasal route (i.n.)) and 10 mg/mL 188 (nebulized inhalation) of the LNP formulation.
  • Example 19 PoLixNano formulation encapsulated RBD-mRNA vaccine via intratracheal spray route (i.t.) Induces a long-lasting and highly effective immune response in mice after vaccination
  • the mRNA encoding the RBD antigen of the SARS-CoV-2 spike protein (RBD-mRNA) was used as a vaccine model, and the immune response mediated by the RBD-mRNA/PoLixNano preparation in mice was investigated via intratracheal spray (i.t.).
  • LNP formulation Using the method described in the above "Formulation Example #1", the molar ratio of Dlin-MC3-DMA, DSPC, cholesterol and DMG-PEG2000 was adjusted to 50:14:35:1, and N/P was 8. LNP formulation encapsulating RBD-mRNA vaccine formulation (hereinafter referred to as "LNP”) was prepared.
  • PoLixNano preparation Using the method described in the above-mentioned "Preparation Example #2", the molar ratio of Dlin-MC3-DMA, DSPC, cholesterol and DMG-PEG2000 was adjusted to 50:14:35:1, N/P was 8, and the PoLixNano preparation containing T904 at a final concentration of 3.0 mg/mL was prepared to encapsulate RBD-mRNA vaccine preparation (hereinafter referred to as "PoLixNano").
  • the immunization dose was 2.5 ⁇ g RBD-mRNA/mouse.
  • the first vaccination time was counted as "day 0", and the same vaccination route, the same preparation and the same dose were used for booster immunization on day 21.
  • Serum, bronchoalveolar lavage fluid (BALF), nasal lavage fluid (NLF), pulmonary lymphocytes and splenocytes were collected from mice 28 days after the first immunization and the humoral immune response, mucosal immune response and adaptive immune response were detected.
  • mice inoculated with LNP preparations in different ways under the same conditions were used as controls, and mouse samples inoculated with PBS solution under the same conditions (hereinafter referred to as "PBS" group) were used as negative controls.
  • PBS mouse samples inoculated with PBS solution under the same conditions
  • the immunization dose was 3 ⁇ g RBD-mRNA/mouse, and the first vaccination time was counted as "Day 0", and booster immunization was performed on Day 21 using the same vaccination route, the same preparation and the same dose.
  • Serum was collected from mice 14 days, 28 days and 280 days after the first immunization, and bronchoalveolar lavage fluid (BALF) and nasal lavage fluid (NLF) were collected from mice on Day 28 to detect the ability of PoLixNano preparation to induce humoral immune response and mucosal immune response in mice, and mediastinal lymph node cells and splenocytes were collected from mice.
  • BALF bronchoalveolar lavage fluid
  • NVF nasal lavage fluid
  • the IgG antibody titer of RBD antigen in the mouse serum samples collected 14 days, 28 days and 280 days after the first immunization was determined by enzyme linked immunosorbent assay (ELISA).
  • ELISA enzyme linked immunosorbent assay
  • ELISA was used to measure the IgG antibody titer of RBD antigen in the bronchoalveolar lavage fluid (BALF) and nasal lavage fluid (NLF) samples collected from mice 28 days after the first immunization.
  • the IgG antibody titer produced in the BALF and NLF of mice induced by intratracheal spray (it) was significantly higher than that of the LNP-it group.
  • the results of Figure 15B show that the sIgA antibody titer of RBD antigen in the BALF and NLF samples collected 28 days and 280 days after the first immunization was measured by ELISA.
  • the sIgA antibody titer produced in the BALF and NLF of mice induced by intratracheal spray (it) PoLixNano was significantly higher than that of the LNP-it group.
  • the serum gradient dilution samples and BALF gradient dilution samples collected 28 days after the first immunization were mixed with pseudovirus and added to hACE-293T cells for infection for 24 hours, and the pseudovirus neutralization titer was calculated by detecting the relative luciferase unit (RLU).
  • the neutralizing antibody titers in the serum and BALF of mice in the PoLixNano preparation group were significantly higher than those in the LNP preparation group using different vaccination methods, indicating that the PoLixNano preparation group can significantly improve the level of neutralizing antibodies.
  • the enzyme-linked immunospot technique (ELISpot) was used to detect the production of IgG and sIgA antibody-secreting cells (ASC) in the mediastinal lymph node cells (Mediastinal lymphocytes) and spleen cells (Splenocytes) of mice 28 days after immunization.
  • the IgG antibody produced by PoLixNano in the mediastinal lymph node cells and spleen cells was significantly higher than that in the LNP preparation group using different vaccination methods.
  • Example 20 PoLixNano formulation encapsulated RBD-mRNA vaccine via intratracheal spray route (i.t.) inoculation induces cellular immune response, innate trained immune response and long-term adaptation in mice sexual Immune Response
  • the mRNA encoding the RBD antigen of the spike protein of the new coronavirus (RBD-mRNA) was used as a vaccine model to investigate the immune response mediated by the RBD-mRNA/PoLixNano preparation in mice via intratracheal spray (i.t.).
  • the preparation methods of the LNP preparation and PoLixNano preparation, as well as the immunization dose and method, were the same as those in Example 19.
  • BALF samples were collected from mice 28 days, 35 days, and 280 days after the first immunization. Pulmonary lymphocytes and spleen cells were also collected from mice.
  • the enzyme-linked immunospot technique was used to analyze the ability of the splenocytes and pulmonary lymphocytes of mice to produce cytokines interferon- ⁇ (IFN- ⁇ ), interleukin-4 (IL-4), and interleukin-17 (IL-17) 28 days after the first immunization.
  • IFN- ⁇ interferon- ⁇
  • IL-4 interleukin-4
  • IL-17 interleukin-17
  • LNP preparation groups with different inoculation methods induced the production of CD4+IFN- ⁇ + cells and CD8+IFN- ⁇ + cells Similar to the PBS group.
  • the above results show that compared with the LNP preparation, the PoLixNano preparation can significantly increase the IFN- ⁇ secretion level of lung CD4+T cells and CD8+T cells, and the PoLixNano preparation can improve the immune response of CD4+ and CD8+T cells.
  • the results of Figure 16C show that the tissue-resident memory T cell (Trm) subpopulation, effector memory T cell (Tem) subpopulation, and central memory T cell (Tcm) subpopulation in mice 28 days and 280 days after the first immunization were analyzed by flow cytometry.
  • Inoculation of PoLixNano preparations via intratracheal spray (it) can induce mice to produce more MHC+IM cells.
  • intratracheal spray (it) mRNA/PoLixNano formulation can induce a higher level of innate trained immune response in mice.
  • Example 21 RBD-mRNA vaccine encapsulated by PoLixNano formulation via intratracheal spray route Evaluation of protection effect of challenge after (i.t.) vaccination
  • the mRNA encoding the RBD antigen of the spike protein of the new coronavirus (RBD-mRNA) was used as a vaccine model to investigate the immune response mediated by the RBD-mRNA/PoLixNano preparation in mice via intratracheal spray (i.t.).
  • the preparation methods of the LNP preparation and PoLixNano preparation, as well as the immunization dose and method, were the same as those in Example 19.
  • the mRNA PoLixNano vaccine can completely protect balb/c mice from challenge with lethal SARS-CoV-2 strains after immunization with the intratracheal spray route (IT).
  • Mice were immunized by intratracheal spray route (IT) on days 0 and 21. 28 days after the initial immunization, the mice were challenged with a lethal dose of the original strain of SARS-CoV-2 and the Omicron variant strain by nasal drops, and specific tissues were collected at designated time points after the challenge to detect viral load and lung pathology.
  • the "gold standard" LNP preparation is difficult to mediate the mRNA vaccine to produce an efficient antigen-specific immune response in the respiratory mucosa, and the mRNA/LNP vaccine inoculated by the respiratory route is extremely limited in its ability to induce antigen-specific humoral immune responses and cellular immunity.
  • the PoLixNano preparation administered by the respiratory route can induce the mRNA vaccine to produce a strong, balanced and lasting multiple antigen-specific immune response, including mucosal immune response, humoral immune response and cellular immune response.
  • the mRNA/PoLixNano vaccine inoculated by the respiratory route is expected to achieve the ideal effect of providing strong immune protection to the vaccine recipients and blocking the spread of pathogens.
  • Example 22 RBD-mRNA vaccine encapsulated in PoLixNano formulation was administered via nasal route (i.n.) Post-immune response evaluation
  • LNP formulation Using the method described in the above "Formulation Example #1", the molar ratio of Dlin-MC3-DMA, DSPC, cholesterol and DMG-PEG2000 was adjusted to 50:14:35:1, and N/P was 8. The vaccine formulation of LNP formulation encapsulating RBD-mRNA was prepared.
  • the immunization dose was 2.0 ⁇ g Fluc-mRNA/mouse, and the expression of the reporter gene in mice was investigated by IVIS live imaging technology at 6h, 12h, 24h, 48h and 72h.
  • the reporter gene was highly expressed in mice at 12h and 24h, but no longer expressed after 72h (Figure 18A).
  • the immunization dose was 2.0 ⁇ g RBD-mRNA/mouse, and the first vaccination time was counted as "Day 0". On the 21st day, booster immunization was performed using the same vaccination route, the same preparation and the same dose.
  • the ELISA method was used to determine the RBD antigen-specific IgG in the serum of mice vaccinated with PoLixNano preparations on days 14, 21, 28, 35, 42 and 49. After booster immunization, the antibody level was significantly increased, and the LNP preparations injected via nasal injection did not induce the body to produce IgG ( Figure 18B).
  • the levels of sIgA antibodies produced in the BALF, NLF and saliva of mice vaccinated with PoLixNano preparations were significantly higher than those of the LNP group.
  • the enzyme-linked immunospot technique (ELISpot) was further used to analyze the production of cytokine interferon- ⁇ (IFN- ⁇ ) by the lung lymphocytes (Splenocytes) of mice.
  • IFN- ⁇ cytokine interferon- ⁇
  • Splenocytes lung lymphocytes
  • IL-4 Interleukin-4
  • IL-17 interleukin-17
  • the mouse lung lymphocytes obtained on the 28th day were stimulated by the RBD overlapping peptide library (covering the overlapping peptide library of the new crown RBD protein, a single polypeptide length of 14 amino acids, and adjacent polypeptides overlapped 9 amino acids) to secrete IFN- ⁇ , IL-4, and IL-17.
  • the ability is far superior to the control group of the LNP preparation inoculated via the nasal route.
  • Figure 18D shows that the resident memory T cells (Trm) and effector memory T cells (Tem) subpopulations of the lung tissue of immunized mice (inoculated on days 0 and 21) were analyzed by flow cytometry.
  • Preparation of LNP preparations Using the method described in the above "Formulation Example #1", the molar ratio of Dlin-MC3-DMA, DSPC, cholesterol and DMG-PEG2000 was adjusted to 50:10:38.5:1.5, and N/P was 6; using the methods described in the above "Formulation Example #6", “Formulation Example #7” and “Formulation Example #8", LNP control preparations without Poloxamer and Poloxamine were prepared respectively. Using the method described in the above “Formulation Example #1”, the lipid components and molar ratios were adjusted, and N/P was 8 to prepare LNP preparations lacking DMG-PEG2000 components as shown in Figure 8. Vaccine preparations encapsulating mRNA in LNP preparations were prepared.
  • the inoculation dose was 2.5 ⁇ g Fluc-mRNA/mouse.
  • IVIS In vivo imaging technology
  • mice 6 hours after administration Mouse samples of LNP preparations inoculated in the same way under the same conditions were used as controls.
  • the intramuscular administration route (im) Administration of PoLixNano can induce higher luciferase expression in living mice and isolated organs ( Figure 19A).
  • DMG-PEG2000 is missing in the lipid component
  • administration of PoLixNano via intramuscular administration (im) can induce higher expression of luciferase in living mice and isolated organs ( Figure 19B).
  • the ELISA method was used to determine the RBD antigen-specific IgG antibody titer in the serum samples of mice administered with PoLixNano preparations via intramuscular administration (im) 14 days, 21 days, 28 days, and 35 days after the first immunization.
  • the antigen-specific humoral immune response ability induced by the LNP preparation (LNP-im) via intramuscular injection was significantly improved.
  • the ELISpot method was further used to analyze the ability of mouse spleen cells (Splenocytes) to produce cytokines, interleukin-4 (IL-4) and interleukin-17 (IL-17).
  • the PoLixNano preparation was administered by intramuscular injection (im).
  • the number of cells secreting IL-4 and IL-17 in mouse spleen cells obtained after stimulation with the RBD overlapping peptide library was significantly higher than that in the LNP control preparation group administered by intramuscular injection.

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Abstract

La présente invention concerne une composition destinée à améliorer l'administration d'un principe actif ou d'un agent thérapeutique, tel qu'un acide nucléique thérapeutique. La composition comprend : (A) le principe actif ou l'agent thérapeutique, le principe actif ou l'agent thérapeutique comprenant de préférence un acide nucléique ; (B) un copolymère séquencé amphiphile ; (C) un lipide cationique ; et (D) un lipide non cationique. La présente invention concerne en outre un procédé de préparation, d'administration et d'utilisation de la composition et l'utilisation de la composition pour le traitement et/ou la prévention de maladies ou d'états associés.
PCT/CN2023/128795 2022-11-03 2023-10-31 Séquence de blocage, kit correspondant et procédé pour son utilisation WO2024094027A1 (fr)

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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20190336452A1 (en) * 2016-11-08 2019-11-07 Modernatx, Inc. Stabilized formulations of lipid nanoparticles
US20200022921A1 (en) * 2018-07-23 2020-01-23 Translate Bio, Inc. Dry powder formulations for messenger rna
US20200069599A1 (en) * 2016-06-14 2020-03-05 Modernatx, Inc. Stabilized formulations of lipid nanoparticles
US20210046192A1 (en) * 2019-07-23 2021-02-18 Translate Bio, Inc. Stable compositions of mrna-loaded lipid nanoparticles and processes of making
US20220023204A1 (en) * 2020-04-20 2022-01-27 Board Of Regents, The University Of Texas System Biologically active dry powder compositions and method of their manufacture and use
WO2022192239A1 (fr) * 2021-03-08 2022-09-15 Board Of Regents, The University Of Texas System Formulations de poudre sèche de nanoparticules lipidiques d'acide nucléique

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20200069599A1 (en) * 2016-06-14 2020-03-05 Modernatx, Inc. Stabilized formulations of lipid nanoparticles
US20190336452A1 (en) * 2016-11-08 2019-11-07 Modernatx, Inc. Stabilized formulations of lipid nanoparticles
US20200022921A1 (en) * 2018-07-23 2020-01-23 Translate Bio, Inc. Dry powder formulations for messenger rna
US20210046192A1 (en) * 2019-07-23 2021-02-18 Translate Bio, Inc. Stable compositions of mrna-loaded lipid nanoparticles and processes of making
CN114401748A (zh) * 2019-07-23 2022-04-26 川斯勒佰尔公司 负载mRNA的脂质纳米颗粒的稳定组合物及制备方法
US20220023204A1 (en) * 2020-04-20 2022-01-27 Board Of Regents, The University Of Texas System Biologically active dry powder compositions and method of their manufacture and use
WO2022192239A1 (fr) * 2021-03-08 2022-09-15 Board Of Regents, The University Of Texas System Formulations de poudre sèche de nanoparticules lipidiques d'acide nucléique

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