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WO2025228409A1 - Nanoparticule lipidique et son utilisation - Google Patents

Nanoparticule lipidique et son utilisation

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Publication number
WO2025228409A1
WO2025228409A1 PCT/CN2025/092313 CN2025092313W WO2025228409A1 WO 2025228409 A1 WO2025228409 A1 WO 2025228409A1 CN 2025092313 W CN2025092313 W CN 2025092313W WO 2025228409 A1 WO2025228409 A1 WO 2025228409A1
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WO
WIPO (PCT)
Prior art keywords
mol
lnps
lipids
peg
cells
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Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/CN2025/092313
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English (en)
Chinese (zh)
Inventor
李林鲜
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Shenzhen Shenxin Biotechnology Co Ltd
Original Assignee
Shenzhen Shenxin Biotechnology Co Ltd
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Publication of WO2025228409A1 publication Critical patent/WO2025228409A1/fr
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Classifications

    • 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
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/28—Steroids, e.g. cholesterol, bile acids or glycyrrhetinic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/88—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microencapsulation, e.g. using amphiphile liposome vesicle

Definitions

  • This invention belongs to the field of biotechnology and relates to a lipid nanoparticle and its application.
  • Nucleic acids such as mRNA and siRNA have emerged as a new class of drugs for the prevention and treatment of various diseases.
  • nucleic acids For nucleic acids to function effectively in vivo, safe, efficient, and stable delivery systems are needed to protect them from degradation and allow for cellular uptake and release.
  • Common delivery systems for nucleic acids include lipid nanoparticles (LNPs).
  • LNPs lipid nanoparticles
  • Lipid nanoparticles generally contain ionizable lipids, structural lipids, accessory lipids, and PEGylated lipids.
  • lipid nanoparticles accumulate significantly in the liver after intravenous injection, but their delivery efficiency to other organs (such as the lungs, kidneys, and spleen) is relatively low.
  • the spleen the largest immune organ in the body, accounts for 25% of the total lymphoid tissue and is the center of cellular and humoral immunity, exerting anti-tumor effects through multiple mechanisms. Therefore, achieving spleen-targeted delivery of nucleic acid drugs has significant application value.
  • lipid nanoparticles for tissue-specific mRNA delivery and CRISPR–Cas gene editing. Nature nanotechnology 15.4(2020):313-320.
  • SORT selective organ targeting
  • the SORT molecule is a negatively charged lipid (e.g., 18PA)
  • the lipid nanoparticles specifically deliver mRNA into the spleen for expression.
  • LNPs lipid nanoparticles
  • the LNPs comprise an active ingredient, ionizable lipids, auxiliary lipids, structural lipids, and polymeric lipids, wherein the polymeric lipids account for 0 mol% to 0.8 mol% of the total lipids present in the LNPs.
  • LNPs lipid nanoparticles
  • the LNPs comprise active ingredients, ionizable lipids, auxiliary lipids, structural lipids and polymeric lipids, and the particle size of the LNPs is 130 nm to 500 nm.
  • the particle size of the LNPs is 130 nm to 500 nm.
  • the polymeric lipids comprise 0 mol% to 0.8 mol% of the total lipids present in the LNPs.
  • the polymeric lipid is a PEG lipid.
  • the PEG lipid is selected from one or more of DMG-PEG, DSG-PEG and DPG-PEG.
  • the LNPs have particle sizes of 150nm–450nm, 150nm–400nm, 150nm–390nm, 150nm–380nm, 150nm–370nm, 150nm–360nm, 150nm–350nm, 150nm–340nm, 150nm–330nm, 150nm–320nm, 150nm–310nm, 150nm–300nm, 150nm–290nm, 150nm–280nm, 150nm–270nm, 150nm–260nm, 150nm– 250nm, 150nm ⁇ 240nm, 150nm ⁇ 230nm, 150nm ⁇ 210nm, 150nm ⁇ 200nm, 160nm ⁇ 320nm, 160nm ⁇ 310nm, 160nm ⁇ 300nm, 160nm ⁇ 290nm, 160nm ⁇ 2 80nm, 160nm ⁇ 270nm, 160nm ⁇ 260nm, 160nm ⁇ 250nm, 160nm ⁇ 240nm,
  • the polymer lipids account for 0.01 mol% to 0.8 mol%, 0.01 mol% to 0.7 mol%, 0.01 mol% to 0.6 mol%, 0.01 mol% to 0.5 mol%, 0.01 mol% to 0.4 mol%, 0.01 mol% to 0.3 mol%, 0.01 mol% to 0.2 mol%, 0.01 mol% to 0.1 mol%, and 0.05 mol% of the total lipids present in the LNPs.
  • the ionizable lipids account for 30 mol% to 60 mol% of the total lipids present in the LNPs
  • the accessory lipids account for 0 mol% to 30 mol% of the total lipids present in the LNPs
  • the structural lipids account for 10 mol% to 60 mol% of the total lipids present in the LNPs.
  • the assisting lipid is selected from one or more of the following: DSPC, DOPE, DOPC, and DOPS.
  • the structural lipid is cholesterol
  • the immune cells include one or more of the following: B cells, T cells, NK cells, DC cells, neutrophils, eosinophils, basophils, mast cells, monocytes, and macrophages.
  • the immune cells are B cells, T cells, or NK cells.
  • the active ingredient is a nucleic acid, protein, polypeptide, or small molecule.
  • the drug is used to prevent or treat diseases related to immune cells.
  • the drug is used to prevent or treat autoimmune diseases.
  • the active ingredient is a nucleic acid that inhibits an autoimmune response.
  • the drug is used to prevent or treat T-cell tumors or B-cell tumors.
  • the active ingredient is a nucleic acid that inhibits the growth of T-cell or B-cell tumors or kills T-cell or B-cell tumor cells.
  • the drug is used to prevent or treat tumors or cancer, and the active ingredient is a tumor-associated antigen or a nucleic acid encoding a tumor-associated antigen.
  • the drug is used to prevent or treat infectious diseases
  • the active ingredient is an infectious disease-related antigen or a nucleic acid encoding an infectious disease-related antigen.
  • the nucleic acid is mRNA.
  • LNPs lipid nanoparticles
  • the gene-edited immune cells are B cells, T cells, or NK cells.
  • the active ingredient is a nucleic acid required for gene editing.
  • the active ingredient is gRNA and nucleic acid encoding the Cas protein.
  • the active ingredient is gRNA, nucleic acid encoding the Cas protein, and donor DNA.
  • LNPs lipid nanoparticles
  • the modified immune cells are CAR-T cells, TCR-T cells, or CAR-NK cells.
  • the active ingredient is mRNA encoding CAR or TCR.
  • This disclosure also provides a method for increasing the content of an active ingredient in the spleen or immune cells, the method comprising the step of contacting LNPs as defined in any of the embodiments described above with the spleen or immune cells.
  • the active ingredient is mRNA.
  • the method is used to increase the amount of active ingredient delivered to the spleen or immune cells.
  • This disclosure also provides a method for increasing the ratio of active ingredients in the spleen and liver, the method comprising contacting LNPs as defined in any of the above embodiments with the spleen or immune cells.
  • the LNPs have a spleen or immune cell delivery preference, and the method is used to increase the spleen/liver delivery ratio of the active ingredient.
  • the active ingredient is mRNA.
  • the method is used to increase the expression of the mRNA in the spleen or immune cells, or the method is used to increase the spleen/liver delivery ratio of the mRNA.
  • the method is performed in vivo.
  • the method includes the step of administering the LNPs to the subject.
  • LNPs lipid nanoparticles
  • the active ingredient is mRNA.
  • This disclosure also provides a composition for preparing LNPs for the above-described uses.
  • the active ingredient is mRNA.
  • the expressions “comprising,” “including,” “containing,” and “having” are open-ended, meaning they include the listed elements, steps, or components but do not exclude other unlisted elements, steps, or components.
  • the expression “composed of” excludes any unspecified elements, steps, or components.
  • the expression “substantially composed of” means that the scope is limited to the specified elements, steps, or components, plus optional elements, steps, or components that do not significantly affect the essential and novel nature of the claimed subject matter. It should be understood that the expressions “substantially composed of” and “composed of” are encompassed within the meaning of the expression “including.”
  • the numerical ranges described herein should be understood to encompass any and all subranges contained therein.
  • the range “1 to 10” should be understood to include not only the explicitly stated values of 1 and 10, but also any single value within the range of 1 to 10 (e.g., 2, 3, 4, 5, 6, 7, 8, and 9) and subranges (e.g., 1 to 2, 1.5 to 2.5, 1 to 3, 1.5 to 3.5, 2.5 to 4, 3 to 4.5, etc.).
  • This principle also applies to ranges that use only one numerical value as their minimum or maximum value.
  • the terms “and/or,” “any combination thereof,” and their grammatical equivalents are used interchangeably. These terms can express any combination specifically.
  • the following phrases “A, B, and/or C” or “A, B, C, or any combination thereof” refer to any of the following: “A alone; B alone; C alone; A and B; B and C; A and C; and A, B, and C.”
  • nucleotide includes deoxyribonucleotides, ribonucleotides, deoxyribonucleotide derivatives, and ribonucleotide derivatives.
  • ribonucleotide is the building block of ribonucleic acid (RNA), consisting of one base, one pentose sugar, and one phosphate molecule. It refers to a nucleotide with a hydroxyl group at the 2' position of the ⁇ -D-ribofuranosyl group.
  • Deoxyribonucleotide is the building block of deoxyribonucleic acid (DNA), also consisting of one base, one pentose sugar, and one phosphate molecule. It refers to a nucleotide where the hydroxyl group at the 2' position of the ⁇ -D-ribofuranosyl group is replaced by hydrogen, and is a major chemical component of chromosomes.
  • Nucleotides are usually identified by a single letter representing the bases in them: "A” or “A nucleotide” refers to adenine deoxyribonucleotide or adenine ribonucleotide containing adenine; “C” or “C nucleotide” refers to cytosine deoxyribonucleotide or cytosine ribonucleotide containing cytosine; “G” or “G nucleotide” refers to guanine deoxyribonucleotide or guanine ribonucleotide containing guanine; “U” or “U nucleotide” refers to uracil ribonucleotide containing uracil; and “T” or “T nucleotide” refers to thymine deoxyribonucleotide containing thymine.
  • nucleic acid generally refers to any compound comprising a polymer of deoxyribonucleotides (deoxyribonucleic acid, or DNA) or a polymer of ribonucleotides (ribonucleic acid, or RNA), or a combination thereof.
  • nucleic acid as used herein also includes derivatives of nucleic acids.
  • derivatives of nucleic acids includes chemical derivatization of nucleic acids at the bases, sugars, or phosphates of nucleotides, as well as nucleic acids containing non-natural nucleotides and nucleotide analogs.
  • nucleic acids can be in the form of single-stranded or double-stranded linear or covalently closed circular molecules.
  • a polynucleotide may contain one or more segments (nucleic acid fragments) (e.g., segments 1, 2, 3, 4, 5, 6, 7, and 8).
  • a polynucleotide may contain a segment encoding a protein or polypeptide of interest.
  • a polynucleotide may contain a segment encoding a protein or polypeptide of interest as well as a regulatory segment (including, but not limited to, segments for transcriptional and translational regulation).
  • the regulatory segment comprises a polynucleotide corresponding to one or more of the following regulatory elements: a promoter, a 5' untranslated region (5'-UTR), a 3' untranslated region (3'-UTR), and a poly(A) tail.
  • promoter refers to a polynucleotide located upstream of the 5' end of the coding region of a gene. It contains a conserved sequence required for the specific binding of RNA polymerase and transcription initiation. It activates RNA polymerase, enabling it to bind accurately to the template DNA and possessing transcription initiation specificity. Promoters can originate from one or more of the following: viruses, bacteria, fungi, plants, insects, and animals.
  • promoters include the bacteriophage T7 promoter, bacteriophage T3 promoter, SP6 promoter, lac operon-promoter, tac promoter, SV40 late promoter, SV40 early promoter, RSV-LTR promoter, CMV IE promoter, SV40 early promoter, or SV40 late promoter and CMV IE promoter.
  • the term "5' untranslated region" or "5'-UTR” can refer to an RNA sequence in mRNA located upstream of the coding sequence that is not translated into protein.
  • the 5'-UTR in a gene typically begins at the transcription start site and ends with a nucleotide upstream of the translation start codon in the coding sequence.
  • the 5'-UTR can contain elements that control gene expression, such as ribosome binding sites, 5'-terminal oligopyrimidine bundles, and translation initiation signals such as the Kozak sequence.
  • mRNA can undergo post-transcriptional modification by adding a 5' cap. Therefore, the 5'-UTR in mature mRNA can also refer to the RNA sequence between the 5' cap and the start codon.
  • 3' untranslated region can refer to an RNA sequence in mRNA that is downstream of the coding sequence and is not translated into a protein.
  • the 3'-UTR in mRNA is located between the stop codon of the coding sequence and the poly(A) sequence, for example, starting from a nucleotide downstream of the stop codon and ending at a nucleotide upstream of the poly(A) sequence.
  • poly(A) nucleotide As used herein, the terms “poly(A) nucleotide,” “polyA,” “poly(A) sequence,” and “poly(A) tail” are used interchangeably. Naturally occurring poly(A) sequences typically consist of adenine ribonucleotides. Poly(A) sequences are usually located at the 3’ end of mRNA, such as the 3’ end (downstream) of a 3’-UTR.
  • the term "5'-cap structure” refers to a structure that is typically located at the 5' end of mature mRNA. In some embodiments, the 5'-cap structure is linked to the 5' end of the mRNA via a 5'-5'-triphosphate bond.
  • the 5'-cap structure is typically formed from modified (e.g., methylated) ribonucleotides, particularly guanine nucleotide derivatives.
  • m7GpppN (cap0, or “cap0”) is a cap structure formed by the interaction of the 5' phosphate group of hnRNA with the 5' phosphate group of m7GTP via guanylate transferase to form a 5',5'-phosphodiester bond, where N is the terminal 5' nucleotide of the nucleic acid carrying the 5'-cap structure.
  • the 5'-cap structure includes, but is not limited to, cap 0, cap 1 (a cap structure formed by further methylation of the 2'-OH of the first nucleotide glycosyl group of hnRNA on the basis of cap 0, or "cap1”), cap 2 (a cap structure formed by further methylation of the 2'-OH of the second nucleotide glycosyl group of hnRNA on the basis of cap 1, or "cap2”), cap 4, cap 0 analogue, cap 1 analogue, cap 2 analogue, or cap 4 analogue.
  • the term “expression” includes the transcription and/or translation of a nucleotide sequence. Therefore, expression can involve the production of transcripts and/or polypeptides.
  • transcription refers to the process of transcribing the genetic code in a DNA sequence into RNA (transcription).
  • in vitro transcription refers to the in vitro synthesis of RNA, particularly mRNA, in a cell-free system (e.g., in a suitable cell extract) (see, e.g., Pardi N., Muramatsu H., Weissman D., Karikó K. (2013). In: Rabinovich P. (eds) Synthetic Messenger RNA and Cell Metabolism Modulation.
  • transcripts are also called “transcription vectors,” which contain the regulatory sequences required for transcription.
  • transcription encompasses “in vitro transcription.”
  • peptide refers to a polymer containing two or more amino acids covalently linked by peptide bonds.
  • a “protein” may contain one or more polypeptides, wherein the polypeptides interact with each other in a covalent or non-covalent manner.
  • the term "plasmid” generally refers to a circular DNA molecule, but the term can also encompass linearized DNA molecules. Specifically, the term “plasmid” also encompasses molecules obtained by linearizing a circular plasmid, for example, by digesting the circular plasmid with a restriction enzyme, thereby converting the circular plasmid molecule into a linear molecule. Plasmids can replicate, i.e., amplify genetic information in cells independently of chromosomal DNA, and can be used for cloning, i.e., for amplifying genetic information in bacterial cells. In some embodiments, the DNA plasmid is a medium-copy or high-copy plasmid.
  • the DNA plasmid is a high-copy plasmid.
  • high-copy plasmids include, for example, pUC and pTZ plasmids or any other plasmid (e.g., pMB1, pCoIE1) containing a replication origin that supports high copy numbers.
  • an antigen generally refers to a substance that can be recognized by the immune system, preferably by the adaptive immune system, and is capable of triggering an antigen-specific immune response (e.g., the formation of antibodies and/or antigen-specific T cells).
  • an antigen can be or may contain a peptide or protein that can be presented to T cells by the MHC.
  • an antigen can be a translation product of the provided nucleic acid (e.g., RNA, RNA molecules, DNA as used herein).
  • fragments, variants, and derivatives of peptides or proteins derived from peptides or proteins containing at least one epitope or antigen can be understood as antigens.
  • treatment and the like are generally used to mean achieving a desired pharmacological and/or physiological effect. Therefore, the treatment of this application may relate to the treatment of a disease state, but may also relate to preventive treatment with regard to the complete or partial prevention of the disease or its symptoms. In some embodiments, the term “treatment” should be understood as being therapeutic in terms of partially or completely curing the disease and/or the adverse effects and/or symptoms attributable to the disease. Treatment can also be prophylactic or preventive treatment, i.e., measures taken to prevent disease, such as to prevent infection and/or the onset of disease.
  • the terms “subject” and “patient” are used interchangeably.
  • the subject is a mammal, such as a human, a non-human primate (e.g., apes, chimpanzees, monkeys, and orangutans), a domesticated animal (including dogs and cats, and livestock (e.g., horses, cattle, pigs, sheep, and goats)), or other mammals.
  • a mammal such as a human, a non-human primate (e.g., apes, chimpanzees, monkeys, and orangutans), a domesticated animal (including dogs and cats, and livestock (e.g., horses, cattle, pigs, sheep, and goats)), or other mammals.
  • Other mammals include, but are not limited to, mice, rats, guinea pigs, rabbits, hamsters, etc.
  • the subject is a human.
  • the subject is a mammal (e.g., a human) suffering from a genetic disease, rare disease, or infectious disease.
  • the subject is a mammal (e.g., a human) at risk of developing a genetic disease, rare disease, or infectious disease.
  • the term "administration" means the provision or administration of a drug to a subject by any effective route.
  • routes of administration include, but are not limited to, one or more of the following: injection (e.g., subcutaneous, intramuscular, intradermal, intraperitoneal, intrathecal, intravenous, intraventricular, or intravenous), oral, intraluminal bile duct, sublingual, rectal, transdermal, intranasal, vaginal, and inhalation.
  • microRNA refers to non-coding small RNAs. Generally, microRNAs are numbered according to the order of their discovery, with microRNAs having the same number indicating they originate from the same pre-miRNA. For example, miRNA-142-3p refers to the mature miRNA-142 derived from the 3' end arm of pre-miRNA-142, and miRNA-142-5p refers to the mature miRNA-142 derived from the 5' end arm of pre-miRNA-142.
  • miR-142 refers to miRNA-142-5p and/or miRNA-142-3p.
  • microRNA binding site refers to a polynucleotide, such as DNA or RNA, that is sufficiently complementary to all or part of a region of a miRNA to enable it to interact, link, or bind to the microRNA.
  • the polymeric lipid is PEG-DMG; in another embodiment, the LNPs contain polymeric lipids comprising 0.201 mol% to 0.595 mol% of the total lipids present in the LNPs, then the following is also an embodiment claimed by this invention: the LNPs contain DMG-PEG comprising 0.201 mol% to 0.595 mol% of the total lipids present in the LNPs.
  • LNPs Lipid Nanoparticles
  • LNPs Current lipid nanoparticles
  • the active ingredients encapsulated in LNPs are expected to be delivered to the spleen or immune cells, rather than the liver. Therefore, there is a need for LNPs capable of targeted delivery of the encapsulated active ingredients to the spleen or immune cells.
  • the inventors of this application unexpectedly discovered that adjusting (reducing) the polymer lipid content in LNPs can alter the delivery specificity of LNPs, enabling them to target the spleen or immune cells.
  • the spleen/liver delivery ratio of the active ingredient refers to the ratio of the amount of active ingredient delivered to the spleen to the amount delivered to the liver.
  • LNPs lipid nanoparticles
  • This disclosure provides lipid nanoparticles (LNPs) comprising ionizable lipids, accessory lipids, structural lipids, and polymeric lipids, wherein the polymeric lipids account for 0 mol% to 0.8 mol% of the total lipids present in the LNPs.
  • Polymer lipids are lipids that contain repeating subunits in their chemical structure. In some embodiments, polymer lipids are lipids that contain polymer components.
  • the lipids used to form the polymeric lipids include one or more of the following: 1,2-dimyristoyl-sn-glycerol (DMG), distearoyl-phosphatidyl-ethanolamine (DSPE), diacylglycerol (DAG), dialkyloxypropyl (DAA), phospholipids, ceramide (Cer), 1,2-distearoyl-rac-glycerol.
  • DMG 1,2-dimyristoyl-sn-glycerol
  • DSPE distearoyl-phosphatidyl-ethanolamine
  • DAG diacylglycerol
  • DAA dialkyloxypropyl
  • phospholipids ceramide
  • Cer 1,2-distearoyl-rac-glycerol.
  • Dipalmitoyl-rac-glycerol dipalmitoyl, dioleoyl, distearate, dipalmitoyl phosphatidylethanolamine (DPPE), 1,2-dimyristylpropyl-3-amine (DMA), phosphatidylethanolamine (PE), DOMG, disuccinic acid (DSA), 1,2-dinaphthyl-sn-glycerol-3-phosphate ethanolamine (DLPE), 1,2-dimyristoyl-sn-glycerol-3-phosphate ethanolamine (DMPE), PEG-1,2-dipalmitoyl-sn-glycerol-3-phosphate choline (DPPC), and 1,2-dipalmitoyl-rac-glycerol (dipalmitoyl-rac-glycero, DPG).
  • DPPE dipalmitoyl phosphatidylethanolamine
  • DMA 1,2-dimyristylpropyl-3-amine
  • PE phosphat
  • the polymer used to form the polymeric lipid includes one or two of the following: hydrophilic polymers and amphoteric polymers.
  • the polymer used to form the polymeric lipid is a hydrophilic polymer. In other embodiments, the polymer used to form the polymeric lipid is an amphoteric polymer.
  • the hydrophilic polymer includes one or more of the following: polyethylene glycol (PEG), poly(oxazolines) (POX), poly(glycerols) (PGs), poly(hydroxypropyl methacrylate) (PHPMA), poly(2-hydroxyethyl methacrylate) (PHEMA), poly(N-(2-hydroxypropyl)methacrylamide) (HPMA), and poly(vinylpyrrolidone) (PEG).
  • PEG polyethylene glycol
  • POX poly(oxazolines)
  • PGs poly(glycerols)
  • PPMA poly(hydroxypropyl methacrylate)
  • PHEMA poly(2-hydroxyethyl methacrylate)
  • HPMA poly(N-(2-hydroxypropyl)methacrylamide)
  • PEG poly(vinylpyrrolidone)
  • polymers are used: lpyrrolidone, PVP, poly(N,N-dimethylacrylamide) (PDMA), poly(N-acryloyl morpholine) (PAcM), polyamino acids, glycosaminoglycans (GAGs), heparin, hyaluronic acid (HA), polysialic acid (PSA), elastin-like polypeptides (ELPs), serum albumin, and CD47.
  • the hydrophilic polymer is polyethylene glycol (PEG).
  • the polymeric lipids in the LNPs described above include one or more of the following: polyethylene glycol-lipids (PEG lipids), polyoxazoline-lipids, polyglycerol-lipids, polyhydroxypropyl methacrylate-lipids, poly-2-hydroxyethyl methacrylate-lipids, poly-N-(2-hydroxypropyl)methacrylamide-lipids, polyvinylpyrrolidone-lipids, poly-N,N-dimethylacrylamide-lipids, poly-N-acryloylmorpholine-lipids, glycosaminoglycan-lipids, heparin-lipids, hyaluronic acid-lipids, polysialic acid-lipids, elastin-like lipids, serum albumin-lipids, and CD47-lipids.
  • PEG lipids polyethylene glycol-lipids
  • polyoxazoline-lipids polyglycerol-lipids
  • PEG lipids are conjugates containing polyethylene glycol and lipids
  • polyoxazoline-lipids refer to conjugates containing polyoxazoline and lipids
  • polyglycerol-lipids refer to conjugates containing polyglycerol and lipids; the same applies to other polymeric lipids.
  • the polymeric lipids of the above-mentioned LNPs include PEG lipids.
  • the polymeric lipids of the above-mentioned LNPs are PEG lipids.
  • the PEG lipid comprises one or more of the following: 1,2-dimylinol-sn-glycerol methoxy polyethylene glycol (PEG-DMG or DMG-PEG), 1,2-distearyl-sn-glycerol-3-phosphate ethanolamine-n-[amino(polyethylene glycol)] (PEG-DSPE), PEG-diacetylglycerol (PEG-DSG or DSG-PEG), PEG-dispalmitoyl, PEG-dioleyl, PEG-distearyl, PEG-diacylglycerol (PEG-DAG), PEG-dispalmitoylphosphatidylethanolamine (PEG-DPPE), PEG-1,2-dimylinolylpropyl-3-amine (PEG-c-DMA), dialkyloxy Propyl-PEG (dialkyloxypropyl-PEG, PEG-DAA), PEG-phospholipid
  • the PEG lipid is one or more of the following: DMG-PEG, DSG-PEG, and DPG-PEG.
  • the average molecular weight of PEG in the PEG lipid is about 2000 to 5000. In one optional specific example, the average molecular weight of PEG in the PEG lipid is about 2000. In one optional specific example, the polymer lipid is DMG-PEG 2000.
  • the amphoteric polymer includes one or more of the following: poly(carboxybetaine) (pCB), poly(sulfobetaine) (pSB), phosphobetaine-based polymers, and phosphorylcholine polymer.
  • the amphoteric polymer includes one or more of the following: poly(carboxybetaine acrylamide, pCBAA), poly(carboxybetaine methacrylate), poly(sulfobetaine methacrylate), poly(methacryloyloxyethyl phosphorylcholine), poly(vinyl-pyridinio propanesulfonate), poly(carboxybetaine)based on vinylimidazole, poly(sulfobetaine)based on vinylimidazole, and poly(sulfobetaine) based on vinypyridine.
  • the polymeric lipids include one or more of the following: polyhydroxybetaine-lipids, polysulfobetaine-lipids, phosphate betaine-based polymeric lipids, and phosphate choline polymeric lipids.
  • the polymeric lipids include one or more of the following: poly(carboxybetaine acrylamide)-lipids, poly(carboxybetaine methacrylate)-lipids, poly(sulfobetaine methacrylate)-lipids, poly(methacryloyloxyethyl phosphorylcholine)-lipids, poly(vinylpyridinylpropanesulfonate)-lipids, polyvinylimidazolyl betaine-lipids, polyvinylimidazolyl sulfobetaine-lipids, and polyvinylpyridinyl sulfobetaine-lipids.
  • the PEG lipid is a compound of formula (IV), or a salt thereof, or a stereoisomer thereof:
  • R3 is OR 0 ;
  • R ⁇ sub>0 ⁇ /sub> is hydrogen, an optionally substituted alkyl group, or an oxygen protecting group
  • g is an integer between 1 and 100;
  • L1 is an optionally substituted C1 - C10 alkylene group, wherein at least one methylene group of the optionally substituted C1 - C10 alkylene group is independently replaced by an optionally substituted carbocyclic group, an optionally substituted heterocyclic group, an optionally substituted aryl group, an optionally substituted heteroaryl group, -O-, -N( RM )-, -S-, -C(O)-, -C(O)N( RM )-, -NRMC (O)-, -C(O)O-, -OC(O)-, -OC(O)O-, -OC(O)O-, -OC (O)N( RM )-, -NRMC (O)O- or -NRMC(O)N( RM )-;
  • D represents a group obtained through click chemistry or a group that can be cleaved under physiological conditions
  • h 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;
  • Each L2 is independently a C1 - C6 alkylene group with a bond or optional substitution; optionally, one methylene group of the optionally substituted C1 - C6 alkylene group is optionally replaced by -O-, -N( RM )-, -S-, -C(O)-, -C(O)N( RM )-, -NRMC(O)-, -C ( O)O-, -OC(O)-, -OC(O)O-, -OC(O)N( RM )-, -NRMC (O)O- or -NRMC (O)N( RM )-;
  • Each R2 is independently a optionally substituted C1 - C30 alkyl, optionally substituted C1 - C30 alkenyl, or optionally substituted C1 - C30 alkynyl; optionally, one or more methylene groups of R2 are independently replaced by optionally substituted carbocyclic, optionally substituted heterocyclic, optionally substituted aryl, optionally substituted heteroaryl, -N( RM )-, -O-, -S-, -C(O)- , -C(O)N( RM )-, -NRMCC(O)-, -NRMCC(O)N( RM )-, -C(O)O-, -OC(O)-, -OC(O)O-, -OC(O)O-, -OC(O)O-, -OC(O)O-, -OC(O)O-, -OC(O)O-, -OC(O)N( RM
  • Each RM is independently hydrogen, optionally substituted alkyl or nitrogen protecting group
  • Ring B is an optionally substituted carbocyclic ring, an optionally substituted heterocyclic ring, an optionally substituted aryl group, or an optionally substituted heteroaryl group;
  • k 1 or 2.
  • the compound of formula (IV) is a PEG-OH lipid (i.e., R0 is H).
  • the PEG lipid is a compound of formula (V), or a salt thereof, or a stereoisomer thereof:
  • R3 is OR 0 ;
  • R ⁇ sub>0 ⁇ /sub> is hydrogen, an optionally substituted alkyl group, or an oxygen protecting group
  • g is an integer between 1 and 100;
  • R5 is an optionally substituted C10 - C40 alkyl, optionally substituted C10 - C40 alkenyl, or optionally substituted C10 - C40 alkynyl; optionally, one or more methylene groups of R5 are independently replaced by optionally substituted carbocyclic, optionally substituted heterocyclic, optionally substituted aryl, optionally substituted heteroaryl, -N( RM )-, -O-, -S-, -C(O)-, -C(O)N( RM )-, -NRMCC (O)-, -NRMCC(O)N( RM ) -, -C(O)O-, -OC(O)-, -OC(O)O-, -OC(O)O-, -OC(O)O-, -OC(O)O-, -OC(O)O-, -OC(O)N( RM ) -, -NRMCC(O)
  • Each RM is independently hydrogen, optionally substituted alkyl or nitrogen protecting group.
  • the compound of formula (V) is a PEG-OH lipid (i.e., R0 is H).
  • the polymer lipid may also be any one or more PEG lipids from Hoang Thi, Thai Thanh et al., Polymers, 12(2), 298(2020), the PEG lipids mentioned above are incorporated herein by reference.
  • LNPs lipid nanoparticles
  • PEG lipids account for 0 mol% to 0.8 mol% of the total lipids present in the LNPs.
  • LNPs lipid nanoparticle
  • the polymeric lipids account for 0 mol% to 0.8 mol% of the total lipids present in the LNPs
  • the ionizable lipids account for 30 mol% to 60 mol% of the total lipids present in the LNPs
  • the accessory lipids account for 0 mol% to 30 mol% of the total lipids present in the LNPs
  • the structural lipids account for 10 mol% to 60 mol% of the total lipids present in the LNPs.
  • LNPs lipid nanoparticle
  • the PEG lipids account for 0 mol% to 0.8 mol% of the total lipids present in the LNPs
  • the ionizable lipids account for 30 mol% to 60 mol% of the total lipids present in the LNPs
  • the accessory lipids account for 0 mol% to 30 mol% of the total lipids present in the LNPs
  • the structural lipids account for 10 mol% to 60 mol% of the total lipids present in the LNPs.
  • the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.0001 mol% to 0.8 mol% of the total lipids present in the LNPs.
  • the polymer lipids (e.g., PEG lipids) in the aforementioned LNPs account for 0.0001 mol%, 0.00011 mol%, 0.0002 mol%, 0.0003 mol%, 0.0004 mol%, 0.00045 mol%, 0.00049 mol%, 0.0005 mol%, 0.00051 mol%, 0.00055 mol%, 0.0006 mol%, 0.00065 mol%, 0.0007 mol%, 0.00075 mol%, 0.0008 mol%, 0.00085 mol%, 0.0009 mol%, 0.00095 mol%, 0.00099 mol%, 0.001 mol%, 0.0011 mol%, and 0.0015 mol% of the total lipids present in the LNPs.
  • the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.0001 mol%–0.79 mol%, 0.0001 mol%–0.75 mol%, 0.0001 mol%–0.7 mol%, 0.0001 mol%–0.65 mol%, 0.0001 mol%–0.6 mol%, 0.0001 mol%–0.55 mol%, 0.0001 mol%–0.5 mol%, and 0.0001 mol%–0.45 mol% of the total lipids present in the LNPs.
  • % 0.0001mol% ⁇ 0.4mol%, 0.0001mol% ⁇ 0.35mol%, 0.0001mol% ⁇ 0.3mol%, 0.0001mol% ⁇ 0.25mol%, 0.0001mol% ⁇ 0.2mol%, 0.0001mol% ⁇ 0.15mol%, 0.0001mol% ⁇ 0.1mol%, 0.0001mol% ⁇ 0.05mol%, 0.0001mol% ⁇ 0.01mol%, 0.0001mol% ⁇ 0.005mol% , 0.0001mol% ⁇ 0.001mol%, 0.0001mol% ⁇ 0.0005mol%, 0.0005mol% ⁇ 0.79mol%, 0.0005mol% ⁇ 0.75mol%, 0.0005mol% ⁇ 0.7m ol%, 0.0005mol% ⁇ 0.65mol%, 0.0005mol% ⁇ 0.6mol%, 0.0005mol% ⁇ 0.55mol%, 0.0005mol% ⁇ 0.5mol%, 0.0005mol% ⁇ 0.45mo l%, 0.0005mol% ⁇ 0.4mol%, 0.0005mol% ⁇ 0.35mol%, 0.0005mol
  • the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.01 mol%–0.8 mol%, 0.01 mol%–0.7 mol%, 0.01 mol%–0.6 mol%, 0.01 mol%–0.5 mol%, 0.01 mol%–0.4 mol%, 0.01 mol%–0.3 mol%, 0.01 mol%–0.2 mol%, and 0.01 mol%–0.1 mol% of the total lipids present in the LNPs.
  • the polymeric lipids (e.g., PEG lipids) in the LNPs account for 0.0001 mol% to 0.8 mol% of the total lipids present in the LNPs. In some embodiments, the polymeric lipids (e.g., PEG lipids) in the LNPs account for 0.005 mol% to 0.8 mol% of the total lipids present in the LNPs. In some embodiments, the polymeric lipids (e.g., PEG lipids) in the LNPs account for 0.01 mol% to 0.8 mol% of the total lipids present in the LNPs.
  • the polymeric lipids (e.g., PEG lipids) in the LNPs account for 0.05 mol% to 0.8 mol% of the total lipids present in the LNPs. In some embodiments, the polymeric lipids (e.g., PEG lipids) in the LNPs account for 0.1 mol% to 0.8 mol% of the total lipids present in the LNPs. In some embodiments, the polymeric lipids (e.g., PEG lipids) in the LNPs account for 0.2 mol% to 0.8 mol% of the total lipids present in the LNPs.
  • the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.3 mol% to 0.8 mol% of the total lipids present in the LNPs. In some embodiments, the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.4 mol% to 0.8 mol% of the total lipids present in the LNPs.
  • the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.0005 mol% to 0.7 mol% of the total lipids present in the LNPs. In some embodiments, the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.001 mol% to 0.7 mol% of the total lipids present in the LNPs. In some embodiments, the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.005 mol% to 0.7 mol% of the total lipids present in the LNPs.
  • the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.01 mol% to 0.7 mol% of the total lipids present in the LNPs. In some embodiments, the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.05 mol% to 0.7 mol% of the total lipids present in the LNPs. In some embodiments, the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.1 mol% to 0.7 mol% of the total lipids present in the LNPs.
  • the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.2 mol% to 0.7 mol% of the total lipids present in the LNPs. In some embodiments, the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.3 mol% to 0.7 mol% of the total lipids present in the LNPs. In some embodiments, the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.4 mol% to 0.7 mol% of the total lipids present in the LNPs.
  • the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.0005 mol% to 0.65 mol% of the total lipids present in the LNPs. In some embodiments, the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.001 mol% to 0.65 mol% of the total lipids present in the LNPs. In some embodiments, the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.005 mol% to 0.65 mol% of the total lipids present in the LNPs.
  • the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.01 mol% to 0.65 mol% of the total lipids present in the LNPs. In some embodiments, the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.05 mol% to 0.65 mol% of the total lipids present in the LNPs. In some embodiments, the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.1 mol% to 0.65 mol% of the total lipids present in the LNPs.
  • the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.2 mol% to 0.65 mol% of the total lipids present in the LNPs. In some embodiments, the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.3 mol% to 0.65 mol% of the total lipids present in the LNPs. In some embodiments, the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.4 mol% to 0.65 mol% of the total lipids present in the LNPs.
  • the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.0001 mol% to 0.6 mol% of the total lipids present in the LNPs. In some embodiments, the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.0005 mol% to 0.6 mol% of the total lipids present in the LNPs. In some embodiments, the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.001 mol% to 0.6 mol% of the total lipids present in the LNPs.
  • the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.005 mol% to 0.6 mol% of the total lipids present in the LNPs. In some embodiments, the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.01 mol% to 0.6 mol% of the total lipids present in the LNPs. In some embodiments, the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.05 mol% to 0.6 mol% of the total lipids present in the LNPs.
  • the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.1 mol% to 0.6 mol% of the total lipids present in the LNPs. In some embodiments, the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.2 mol% to 0.6 mol% of the total lipids present in the LNPs. In some embodiments, the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.3 mol% to 0.6 mol% of the total lipids present in the LNPs. In some embodiments, the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.4 mol% to 0.6 mol% of the total lipids present in the LNPs.
  • the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.0001 mol% to 0.5 mol% of the total lipids present in the LNPs. In some embodiments, the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.0005 mol% to 0.5 mol% of the total lipids present in the LNPs. In some embodiments, the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.001 mol% to 0.5 mol% of the total lipids present in the LNPs.
  • the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.005 mol% to 0.5 mol% of the total lipids present in the LNPs. In some embodiments, the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.01 mol% to 0.5 mol% of the total lipids present in the LNPs. In some embodiments, the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.05 mol% to 0.5 mol% of the total lipids present in the LNPs.
  • the polymeric lipids (e.g., PEG lipids) in the LNPs account for 0.06 mol% to 0.5 mol% of the total lipids present in the LNPs. In some embodiments, the polymeric lipids (e.g., PEG lipids) in the LNPs account for 0.07 mol% to 0.5 mol% of the total lipids present in the LNPs. In some embodiments, the polymeric lipids (e.g., PEG lipids) in the LNPs account for 0.08 mol% to 0.5 mol% of the total lipids present in the LNPs.
  • the polymeric lipids (e.g., PEG lipids) in the LNPs account for 0.09 mol% to 0.5 mol% of the total lipids present in the LNPs. In some embodiments, the polymeric lipids (e.g., PEG lipids) in the LNPs account for 0.1 mol% to 0.5 mol% of the total lipids present in the LNPs. In some embodiments, the polymeric lipids (e.g., PEG lipids) in the LNPs account for 0.2 mol% to 0.5 mol% of the total lipids present in the LNPs.
  • the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.3 mol% to 0.5 mol% of the total lipids present in the LNPs. In some embodiments, the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.4 mol% to 0.5 mol% of the total lipids present in the LNPs.
  • the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.0001 mol% to 0.4 mol% of the total lipids present in the LNPs. In some embodiments, the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.0005 mol% to 0.4 mol% of the total lipids present in the LNPs. In some embodiments, the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.001 mol% to 0.4 mol% of the total lipids present in the LNPs.
  • the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.005 mol% to 0.4 mol% of the total lipids present in the LNPs. In some embodiments, the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.01 mol% to 0.4 mol% of the total lipids present in the LNPs. In some embodiments, the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.05 mol% to 0.4 mol% of the total lipids present in the LNPs.
  • the polymeric lipids (e.g., PEG lipids) in the LNPs account for 0.06 mol% to 0.4 mol% of the total lipids present in the LNPs. In some embodiments, the polymeric lipids (e.g., PEG lipids) in the LNPs account for 0.07 mol% to 0.4 mol% of the total lipids present in the LNPs. In some embodiments, the polymeric lipids (e.g., PEG lipids) in the LNPs account for 0.08 mol% to 0.4 mol% of the total lipids present in the LNPs.
  • the polymeric lipids (e.g., PEG lipids) in the LNPs account for 0.09 mol% to 0.4 mol% of the total lipids present in the LNPs. In some embodiments, the polymeric lipids (e.g., PEG lipids) in the LNPs account for 0.1 mol% to 0.4 mol% of the total lipids present in the LNPs. In some embodiments, the polymeric lipids (e.g., PEG lipids) in the LNPs account for 0.2 mol% to 0.4 mol% of the total lipids present in the LNPs. In some embodiments, the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.3 mol% to 0.4 mol% of the total lipids present in the LNPs.
  • the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.0001 mol% to 0.35 mol% of the total lipids present in the LNPs. In some embodiments, the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.0005 mol% to 0.35 mol% of the total lipids present in the LNPs. In some embodiments, the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.001 mol% to 0.35 mol% of the total lipids present in the LNPs.
  • the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.005 mol% to 0.35 mol% of the total lipids present in the LNPs. In some embodiments, the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.01 mol% to 0.35 mol% of the total lipids present in the LNPs. In some embodiments, the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.05 mol% to 0.35 mol% of the total lipids present in the LNPs.
  • the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.1 mol% to 0.35 mol% of the total lipids present in the LNPs. In some embodiments, the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.2 mol% to 0.35 mol% of the total lipids present in the LNPs. In some embodiments, the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.3 mol% to 0.35 mol% of the total lipids present in the LNPs.
  • the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.0001 mol% to 0.3 mol% of the total lipids present in the LNPs. In some embodiments, the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.0005 mol% to 0.3 mol% of the total lipids present in the LNPs. In some embodiments, the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.001 mol% to 0.3 mol% of the total lipids present in the LNPs.
  • the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.005 mol% to 0.3 mol% of the total lipids present in the LNPs. In some embodiments, the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.01 mol% to 0.3 mol% of the total lipids present in the LNPs. In some embodiments, the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.05 mol% to 0.3 mol% of the total lipids present in the LNPs.
  • the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.06 mol% to 0.3 mol% of the total lipids present in the LNPs. In some embodiments, the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.07 mol% to 0.3 mol% of the total lipids present in the LNPs. In some embodiments, the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.08 mol% to 0.3 mol% of the total lipids present in the LNPs.
  • the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.09 mol% to 0.3 mol% of the total lipids present in the LNPs. In some embodiments, the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.1 mol% to 0.3 mol% of the total lipids present in the LNPs. In some embodiments, the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.15 mol% to 0.3 mol% of the total lipids present in the LNPs. In some embodiments, the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.2 mol% to 0.3 mol% of the total lipids present in the LNPs.
  • the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.0001 mol% to 0.25 mol% of the total lipids present in the LNPs. In some embodiments, the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.0005 mol% to 0.25 mol% of the total lipids present in the LNPs. In some embodiments, the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.001 mol% to 0.25 mol% of the total lipids present in the LNPs.
  • the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.005 mol% to 0.25 mol% of the total lipids present in the LNPs. In some embodiments, the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.01 mol% to 0.25 mol% of the total lipids present in the LNPs. In some embodiments, the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.05 mol% to 0.25 mol% of the total lipids present in the LNPs.
  • the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.1 mol% to 0.25 mol% of the total lipids present in the LNPs. In some embodiments, the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.15 mol% to 0.25 mol% of the total lipids present in the LNPs. In some embodiments, the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.2 mol% to 0.25 mol% of the total lipids present in the LNPs.
  • the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.0001 mol% to 0.2 mol% of the total lipids present in the LNPs. In some embodiments, the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.0005 mol% to 0.2 mol% of the total lipids present in the LNPs. In some embodiments, the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.001 mol% to 0.2 mol% of the total lipids present in the LNPs.
  • the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.005 mol% to 0.2 mol% of the total lipids present in the LNPs. In some embodiments, the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.01 mol% to 0.2 mol% of the total lipids present in the LNPs. In some embodiments, the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.05 mol% to 0.2 mol% of the total lipids present in the LNPs.
  • the polymeric lipids (e.g., PEG lipids) in the LNPs account for 0.06 mol% to 0.2 mol% of the total lipids present in the LNPs. In some embodiments, the polymeric lipids (e.g., PEG lipids) in the LNPs account for 0.07 mol% to 0.2 mol% of the total lipids present in the LNPs. In some embodiments, the polymeric lipids (e.g., PEG lipids) in the LNPs account for 0.08 mol% to 0.2 mol% of the total lipids present in the LNPs.
  • the polymeric lipids (e.g., PEG lipids) in the LNPs account for 0.09 mol% to 0.2 mol% of the total lipids present in the LNPs. In some embodiments, the polymeric lipids (e.g., PEG lipids) in the LNPs account for 0.1 mol% to 0.2 mol% of the total lipids present in the LNPs. In some embodiments, the polymeric lipids (e.g., PEG lipids) in the LNPs account for 0.15 mol% to 0.2 mol% of the total lipids present in the LNPs.
  • the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.0001 mol% to 0.15 mol% of the total lipids present in the LNPs. In some embodiments, the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.0005 mol% to 0.15 mol% of the total lipids present in the LNPs. In some embodiments, the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.001 mol% to 0.15 mol% of the total lipids present in the LNPs.
  • the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.005 mol% to 0.15 mol% of the total lipids present in the LNPs. In some embodiments, the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.01 mol% to 0.15 mol% of the total lipids present in the LNPs. In some embodiments, the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.05 mol% to 0.15 mol% of the total lipids present in the LNPs.
  • the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.06 mol% to 0.15 mol% of the total lipids present in the LNPs. In some embodiments, the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.07 mol% to 0.15 mol% of the total lipids present in the LNPs. In some embodiments, the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.08 mol% to 0.15 mol% of the total lipids present in the LNPs.
  • the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.09 mol% to 0.15 mol% of the total lipids present in the LNPs. In some embodiments, the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.1 mol% to 0.15 mol% of the total lipids present in the LNPs.
  • the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.06 mol% to 0.1 mol% of the total lipids present in the LNPs. In some embodiments, the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.07 mol% to 0.1 mol% of the total lipids present in the LNPs. In some embodiments, the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.08 mol% to 0.1 mol% of the total lipids present in the LNPs. In some embodiments, the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.09 mol% to 0.1 mol% of the total lipids present in the LNPs.
  • the proportion of polymeric lipids (e.g., PEG lipids) in the LNPs to the total lipids present in the LNPs is appropriately not one or more of the following: 0 mol%, 0.0001 mol%, 0.0005 mol%, 0.001 mol%, 0.005 mol%, 0.01 mol%, 0.05 mol%, 0.07 mol%, 0.1 mol%, 0.15 mol%, 0.2 mol%, 0.25 mol%, 0.3 mol%, 0.35 mol%, 0.4 mol%, 0.45 mol%, 0.5 mol%, 0.55 mol%, 0.6 mol%, 0.65 mol%, 0.7 mol%, 0.75 mol%, and 0.8 mol%.
  • PEG lipids polymeric lipids
  • the polymer lipids (e.g., PEG lipids) in the LNPs comprise 0 mol% to 0.8 mol% of the total lipids present in the LNPs, and are not 0 mol%, 0.0001 mol%, 0.0005 mol%, 0.001 mol%, 0.005 mol%, 0.01 mol%, 0.05 mol%, 0.07 mol%, 0.1 mol%, 0.15 mol%, 0.2 mol%, 0.25 mol%, 0.3 mol%, 0.35 mol%, 0.4 mol%, 0.45 mol%, 0.5 mol%, 0.55 mol%, 0.6 mol%, 0.65 mol%, 0.7 mol%, 0.75 mol%, and 0.8 mol%.
  • the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.0001 mol% to 0.749 mol% of the total lipids present in the LNPs and are not one or more of the following: 0.0001 mol%, 0.0005 mol%, 0.001 mol%, 0.005 mol%, 0.01 mol%, 0.05 mol%, 0.07 mol%, 0.1 mol%, 0.15 mol%, 0.2 mol%, 0.25 mol%, 0.3 mol%, 0.35 mol%, 0.4 mol%, 0.45 mol%, 0.5 mol%, 0.55 mol%, 0.6 mol%, 0.65 mol%, and 0.7 mol%.
  • the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.0001 mol% to 0.5 mol% of the total lipids present in the LNPs and are not one or more of the following: 0.0001 mol%, 0.0005 mol%, 0.001 mol%, 0.005 mol%, 0.01 mol%, 0.05 mol%, 0.07 mol%, 0.1 mol%, 0.15 mol%, 0.2 mol%, 0.25 mol%, 0.3 mol%, 0.35 mol%, 0.4 mol%, 0.45 mol%, and 0.5 mol%.
  • the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.0005 mol% to 0.4 mol% of the total lipids present in the LNPs and are not one or more of the following: 0.0005 mol%, 0.001 mol%, 0.005 mol%, 0.01 mol%, 0.05 mol%, 0.07 mol%, 0.1 mol%, 0.2 mol%, 0.25 mol%, 0.3 mol%, and 0.35 mol%.
  • the proportion of polymeric lipids (e.g., PEG lipids) in the LNPs to the total lipids present in the LNPs is not 0 mol%, 0.0001 mol%, 0.0005 mol%, 0.001 mol%, 0.005 mol%, 0.01 mol%, 0.05 mol%, 0.07 mol%, 0.1 mol%, 0.15 mol%, 0.2 mol%, 0.25 mol%, 0.3 mol%, 0.35 mol%, 0.4 mol%, 0.45 mol%, 0.5 mol%, 0.55 mol%, 0.6 mol%, 0.65 mol%, 0.7 mol%, 0.75 mol%, and 0.8 mol%.
  • PEG lipids polymeric lipids
  • the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0–0.8 mol% of the total lipids present in the LNPs, and are not 0 mol%, 0.0001 mol%, 0.0005 mol%, 0.001 mol%, 0.005 mol%, 0.01 mol%, 0.05 mol%, 0.07 mol%, 0.1 mol%, 0.15 mol%, 0.2 mol%, 0.25 mol%, 0.3 mol%, 0.35 mol%, 0.4 mol%, 0.45 mol%, 0.5 mol%, 0.6 mol%, 0.7 mol%, 0.75 mol%, and 0.8 mol%.
  • the polymer lipids (e.g., PEG lipids) in the LNPs comprise 0.0005 mol% to 0.8 mol% of the total lipids present in the LNPs, and are not 0.0005 mol%, 0.001 mol%, 0.005 mol%, 0.01 mol%, 0.05 mol%, 0.07 mol%, 0.1 mol%, 0.15 mol%, 0.2 mol%, 0.25 mol%, 0.3 mol%, 0.35 mol%, 0.4 mol%, 0.45 mol%, 0.5 mol%, 0.6 mol%, 0.7 mol%, 0.75 mol%, and 0.8 mol%.
  • the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.001 mol% to 0.8 mol% of the total lipids present in the LNPs, and are not 0.001 mol%, 0.005 mol%, 0.01 mol%, 0.05 mol%, 0.07 mol%, 0.1 mol%, 0.15 mol%, 0.2 mol%, 0.25 mol%, 0.3 mol%, 0.35 mol%, 0.4 mol%, 0.45 mol%, 0.5 mol%, 0.6 mol%, 0.7 mol%, 0.75 mol%, and 0.8 mol%.
  • the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.005 mol% to 0.8 mol% of the total lipids present in the LNPs, and are not 0.005 mol%, 0.01 mol%, 0.05 mol%, 0.07 mol%, 0.1 mol%, 0.15 mol%, 0.2 mol%, 0.25 mol%, 0.3 mol%, 0.35 mol%, 0.4 mol%, 0.45 mol%, 0.5 mol%, 0.6 mol%, 0.7 mol%, 0.75 mol%, and 0.8 mol%.
  • the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.01 mol% to 0.8 mol% of the total lipids present in the LNPs, and are not 0.01 mol%, 0.05 mol%, 0.07 mol%, 0.1 mol%, 0.15 mol%, 0.2 mol%, 0.25 mol%, 0.3 mol%, 0.35 mol%, 0.4 mol%, 0.45 mol%, 0.5 mol%, 0.6 mol%, 0.7 mol%, 0.75 mol%, and 0.8 mol%.
  • the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.05 mol% to 0.8 mol% of the total lipids present in the LNPs, and are not 0.05 mol%, 0.07 mol%, 0.1 mol%, 0.15 mol%, 0.2 mol%, 0.25 mol%, 0.3 mol%, 0.35 mol%, 0.4 mol%, 0.45 mol%, 0.5 mol%, 0.6 mol%, 0.7 mol%, 0.75 mol%, and 0.8 mol%.
  • the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.1 mol% to 0.8 mol% of the total lipids present in the LNPs, and not 0.1 mol%, 0.15 mol%, 0.2 mol%, 0.25 mol%, 0.3 mol%, 0.35 mol%, 0.4 mol%, 0.45 mol%, 0.5 mol%, 0.6 mol%, 0.7 mol%, 0.75 mol%, and 0.8 mol%.
  • the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.2 mol% to 0.8 mol% of the total lipids present in the LNPs, and not 0.2 mol%, 0.25 mol%, 0.3 mol%, 0.35 mol%, 0.4 mol%, 0.45 mol%, 0.5 mol%, 0.6 mol%, 0.7 mol%, 0.75 mol%, and 0.8 mol%.
  • the polymeric lipids (e.g., PEG lipids) in the aforementioned LNPs account for 0.3 mol% to 0.8 mol% of the total lipids present in the LNPs, and not 0.3 mol%, 0.35 mol%, 0.4 mol%, 0.45 mol%, 0.5 mol%, 0.6 mol%, 0.7 mol%, 0.75 mol%, and 0.8 mol%.
  • the polymeric lipids (e.g., PEG lipids) in the aforementioned LNPs account for 0.4 mol% to 0.8 mol% of the total lipids present in the LNPs, and not 0.4 mol%, 0.45 mol%, 0.5 mol%, 0.6 mol%, 0.7 mol%, 0.75 mol%, and 0.8 mol%.
  • the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.0005 mol% to 0.7 mol% of the total lipids present in the LNPs, and are not 0.0005 mol%, 0.001 mol%, 0.005 mol%, 0.01 mol%, 0.05 mol%, 0.07 mol%, 0.1 mol%, 0.15 mol%, 0.2 mol%, 0.25 mol%, 0.3 mol%, 0.35 mol%, 0.4 mol%, 0.45 mol%, 0.5 mol%, 0.6 mol%, and 0.7 mol%.
  • the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.001 mol% to 0.7 mol% of the total lipids present in the LNPs, and are not 0.001 mol%, 0.005 mol%, 0.01 mol%, 0.05 mol%, 0.07 mol%, 0.1 mol%, 0.15 mol%, 0.2 mol%, 0.25 mol%, 0.3 mol%, 0.35 mol%, 0.4 mol%, 0.45 mol%, 0.5 mol%, 0.6 mol%, and 0.7 mol%.
  • the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.005 mol% to 0.7 mol% of the total lipids present in the LNPs, and are not 0.005 mol%, 0.01 mol%, 0.05 mol%, 0.07 mol%, 0.1 mol%, 0.15 mol%, 0.2 mol%, 0.25 mol%, 0.3 mol%, 0.35 mol%, 0.4 mol%, 0.45 mol%, 0.5 mol%, 0.6 mol%, and 0.7 mol%.
  • the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.01 mol% to 0.7 mol% of the total lipids present in the LNPs, and are not 0.01 mol%, 0.05 mol%, 0.07 mol%, 0.1 mol%, 0.15 mol%, 0.2 mol%, 0.25 mol%, 0.3 mol%, 0.35 mol%, 0.4 mol%, 0.45 mol%, 0.5 mol%, 0.6 mol%, and 0.7 mol%.
  • the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.05 mol% to 0.7 mol% of the total lipids present in the LNPs, and not 0.05 mol%, 0.07 mol%, 0.1 mol%, 0.15 mol%, 0.2 mol%, 0.25 mol%, 0.3 mol%, 0.35 mol%, 0.4 mol%, 0.45 mol%, 0.5 mol%, 0.6 mol%, and 0.7 mol%.
  • the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.1 mol% to 0.7 mol% of the total lipids present in the LNPs, and not 0.1 mol%, 0.15 mol%, 0.2 mol%, 0.25 mol%, 0.3 mol%, 0.35 mol%, 0.4 mol%, 0.45 mol%, 0.5 mol%, 0.6 mol%, and 0.7 mol%.
  • the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.2 mol% to 0.7 mol% of the total lipids present in the LNPs, and not 0.2 mol%, 0.25 mol%, 0.3 mol%, 0.35 mol%, 0.4 mol%, 0.45 mol%, 0.5 mol%, 0.6 mol%, and 0.7 mol%.
  • the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.3 mol% to 0.7 mol% of the total lipids present in the LNPs, and not 0.3 mol%, 0.35 mol%, 0.4 mol%, 0.45 mol%, 0.5 mol%, 0.6 mol%, and 0.7 mol%.
  • the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.4 mol% to 0.7 mol% of the total lipids present in the LNPs, and not 0.4 mol%, 0.45 mol%, 0.5 mol%, 0.6 mol%, and 0.7 mol%.
  • the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.0001 mol% to 0.6 mol% of the total lipids present in the LNPs, and are not 0.0001 mol%, 0.0005 mol%, 0.001 mol%, 0.005 mol%, 0.01 mol%, 0.05 mol%, 0.07 mol%, 0.1 mol%, 0.15 mol%, 0.2 mol%, 0.25 mol%, 0.3 mol%, 0.35 mol%, 0.4 mol%, 0.45 mol%, 0.5 mol%, and 0.6 mol%.
  • the polymer lipids (e.g., PEG lipids) in the LNPs comprise 0.0005 mol% to 0.6 mol% of the total lipids present in the LNPs, and are not 0.0005 mol%, 0.001 mol%, 0.005 mol%, 0.01 mol%, 0.05 mol%, 0.07 mol%, 0.1 mol%, 0.15 mol%, 0.2 mol%, 0.25 mol%, 0.3 mol%, 0.35 mol%, 0.4 mol%, 0.45 mol%, 0.5 mol%, and 0.6 mol%.
  • the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.001 mol% to 0.6 mol% of the total lipids present in the LNPs, and are not 0.001 mol%, 0.005 mol%, 0.01 mol%, 0.05 mol%, 0.07 mol%, 0.1 mol%, 0.15 mol%, 0.2 mol%, 0.25 mol%, 0.3 mol%, 0.35 mol%, 0.4 mol%, 0.45 mol%, 0.5 mol%, and 0.6 mol%.
  • the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.005 mol% to 0.6 mol% of the total lipids present in the LNPs, and are not 0.005 mol%, 0.01 mol%, 0.05 mol%, 0.07 mol%, 0.1 mol%, 0.15 mol%, 0.2 mol%, 0.25 mol%, 0.3 mol%, 0.35 mol%, 0.4 mol%, 0.45 mol%, 0.5 mol%, and 0.6 mol%.
  • the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.01 mol% to 0.6 mol% of the total lipids present in the LNPs, and not 0.01 mol%, 0.05 mol%, 0.07 mol%, 0.1 mol%, 0.15 mol%, 0.2 mol%, 0.25 mol%, 0.3 mol%, 0.35 mol%, 0.4 mol%, 0.45 mol%, 0.5 mol%, and 0.6 mol%.
  • the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.05 mol% to 0.6 mol% of the total lipids present in the LNPs, and not 0.05 mol%, 0.07 mol%, 0.1 mol%, 0.15 mol%, 0.2 mol%, 0.25 mol%, 0.3 mol%, 0.35 mol%, 0.4 mol%, 0.45 mol%, 0.5 mol%, and 0.6 mol%.
  • the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.1 mol% to 0.6 mol% of the total lipids present in the LNPs, and not 0.1 mol%, 0.15 mol%, 0.2 mol%, 0.25 mol%, 0.3 mol%, 0.35 mol%, 0.4 mol%, 0.45 mol%, 0.5 mol%, and 0.6 mol%.
  • the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.2 mol% to 0.6 mol% of the total lipids present in the LNPs, and not 0.2 mol%, 0.25 mol%, 0.3 mol%, 0.35 mol%, 0.4 mol%, 0.45 mol%, 0.5 mol%, and 0.6 mol%.
  • the polymeric lipids (e.g., PEG lipids) in the aforementioned LNPs account for 0.3 mol% to 0.6 mol% of the total lipids present in the LNPs, and not 0.3 mol%, 0.35 mol%, 0.4 mol%, 0.45 mol%, 0.5 mol%, and 0.6 mol%.
  • the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.0001 mol% to 0.5 mol% of the total lipids present in the LNPs, and are not 0.0001 mol%, 0.0005 mol%, 0.001 mol%, 0.005 mol%, 0.01 mol%, 0.05 mol%, 0.07 mol%, 0.1 mol%, 0.15 mol%, 0.2 mol%, 0.25 mol%, 0.3 mol%, 0.35 mol%, 0.4 mol%, 0.45 mol%, and 0.5 mol%.
  • the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.0005 mol% to 0.5 mol% of the total lipids present in the LNPs, and are not 0.0005 mol%, 0.001 mol%, 0.005 mol%, 0.01 mol%, 0.05 mol%, 0.07 mol%, 0.1 mol%, 0.15 mol%, 0.2 mol%, 0.25 mol%, 0.3 mol%, 0.35 mol%, 0.4 mol%, 0.45 mol%, and 0.5 mol%.
  • the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.001 mol% to 0.5 mol% of the total lipids present in the LNPs, and are not 0.001 mol%, 0.005 mol%, 0.01 mol%, 0.05 mol%, 0.07 mol%, 0.1 mol%, 0.15 mol%, 0.2 mol%, 0.25 mol%, 0.3 mol%, 0.35 mol%, 0.4 mol%, 0.45 mol%, and 0.5 mol%.
  • the polymeric lipids (e.g., PEG lipids) in the aforementioned LNPs account for 0.005 mol% to 0.5 mol% of the total lipids present in the LNPs, and are not 0.005 mol%, 0.01 mol%, 0.05 mol%, 0.07 mol%, 0.1 mol%, 0.15 mol%, 0.2 mol%, 0.25 mol%, 0.3 mol%, 0.35 mol%, 0.4 mol%, 0.45 mol%, and 0.5 mol%.
  • the polymeric lipids (e.g., PEG lipids) in the aforementioned LNPs account for 0.01 mol% to 0.5 mol% of the total lipids present in the LNPs, and are not 0.01 mol%, 0.05 mol%, 0.07 mol%, 0.1 mol%, 0.15 mol%, 0.2 mol%, 0.25 mol%, 0.3 mol%, 0.35 mol%, 0.4 mol%, 0.45 mol%, and 0.5 mol%.
  • the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.05 mol% to 0.5 mol% of the total lipids present in the LNPs, and not 0.05 mol%, 0.07 mol%, 0.1 mol%, 0.15 mol%, 0.2 mol%, 0.25 mol%, 0.3 mol%, 0.35 mol%, 0.4 mol%, 0.45 mol%, and 0.5 mol%.
  • the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.1 mol% to 0.5 mol% of the total lipids present in the LNPs, and not 0.1 mol%, 0.15 mol%, 0.2 mol%, 0.25 mol%, 0.3 mol%, 0.35 mol%, 0.4 mol%, 0.45 mol%, and 0.5 mol%.
  • the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.2 mol% to 0.5 mol% of the total lipids present in the LNPs, and not 0.2 mol%, 0.25 mol%, 0.3 mol%, 0.35 mol%, 0.4 mol%, 0.45 mol%, and 0.5 mol%.
  • the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.3 mol% to 0.5 mol% of the total lipids present in the LNPs, and not 0.3 mol%, 0.35 mol%, 0.4 mol%, 0.45 mol%, and 0.5 mol%.
  • the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.4 mol% to 0.5 mol% of the total lipids present in the LNPs, and not 0.4 mol%, 0.45 mol%, and 0.5 mol%.
  • the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.0001 mol% to 0.45 mol% of the total lipids present in the LNPs, and are not 0.0001 mol%, 0.0005 mol%, 0.001 mol%, 0.005 mol%, 0.01 mol%, 0.05 mol%, 0.07 mol%, 0.1 mol%, 0.15 mol%, 0.2 mol%, 0.25 mol%, 0.3 mol%, 0.35 mol%, 0.4 mol%, and 0.45 mol%.
  • the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.0005 mol% to 0.45 mol% of the total lipids present in the LNPs, and are not 0.0005 mol%, 0.001 mol%, 0.005 mol%, 0.01 mol%, 0.05 mol%, 0.07 mol%, 0.1 mol%, 0.15 mol%, 0.2 mol%, 0.25 mol%, 0.3 mol%, 0.35 mol%, 0.4 mol%, and 0.45 mol%.
  • the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.001 mol% to 0.45 mol% of the total lipids present in the LNPs, and are not 0.001 mol%, 0.005 mol%, 0.01 mol%, 0.05 mol%, 0.07 mol%, 0.1 mol%, 0.15 mol%, 0.2 mol%, 0.25 mol%, 0.3 mol%, 0.35 mol%, 0.4 mol%, and 0.45 mol%.
  • the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.005 mol% to 0.45 mol% of the total lipids present in the LNPs, and not 0.005 mol%, 0.01 mol%, 0.05 mol%, 0.07 mol%, 0.1 mol%, 0.15 mol%, 0.2 mol%, 0.25 mol%, 0.3 mol%, 0.35 mol%, 0.4 mol%, and 0.45 mol%.
  • the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.01 mol% to 0.45 mol% of the total lipids present in the LNPs, and not 0.01 mol%, 0.05 mol%, 0.07 mol%, 0.1 mol%, 0.15 mol%, 0.2 mol%, 0.25 mol%, 0.3 mol%, 0.35 mol%, 0.4 mol%, and 0.45 mol%.
  • the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.05 mol% to 0.45 mol% of the total lipids present in the LNPs, and not 0.05 mol%, 0.07 mol%, 0.1 mol%, 0.15 mol%, 0.2 mol%, 0.25 mol%, 0.3 mol%, 0.35 mol%, 0.4 mol%, and 0.45 mol%.
  • the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.1 mol% to 0.45 mol% of the total lipids present in the LNPs, and not 0.1 mol%, 0.15 mol%, 0.2 mol%, 0.25 mol%, 0.3 mol%, 0.35 mol%, 0.4 mol%, and 0.45 mol%.
  • the polymeric lipids (e.g., PEG lipids) in the aforementioned LNPs account for 0.2 mol% to 0.45 mol% of the total lipids present in the LNPs, and not 0.2 mol%, 0.25 mol%, 0.3 mol%, 0.35 mol%, 0.4 mol%, and 0.45 mol%.
  • the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.0001 mol% to 0.4 mol% of the total lipids present in the LNPs, and are not 0.0001 mol%, 0.0005 mol%, 0.001 mol%, 0.005 mol%, 0.01 mol%, 0.05 mol%, 0.07 mol%, 0.1 mol%, 0.15 mol%, 0.2 mol%, 0.25 mol%, 0.3 mol%, 0.35 mol%, and 0.4 mol%.
  • the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.0005 mol% to 0.4 mol% of the total lipids present in the LNPs, and are not 0.0005 mol%, 0.001 mol%, 0.005 mol%, 0.01 mol%, 0.05 mol%, 0.07 mol%, 0.1 mol%, 0.15 mol%, 0.2 mol%, 0.25 mol%, 0.3 mol%, 0.35 mol%, and 0.4 mol%.
  • the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.001 mol% to 0.4 mol% of the total lipids present in the LNPs, and not 0.001 mol%, 0.005 mol%, 0.01 mol%, 0.05 mol%, 0.07 mol%, 0.1 mol%, 0.15 mol%, 0.2 mol%, 0.25 mol%, 0.3 mol%, 0.35 mol%, and 0.4 mol%.
  • the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.005 mol% to 0.4 mol% of the total lipids present in the LNPs, and not 0.005 mol%, 0.01 mol%, 0.05 mol%, 0.07 mol%, 0.1 mol%, 0.15 mol%, 0.2 mol%, 0.25 mol%, 0.3 mol%, 0.35 mol%, and 0.4 mol%.
  • the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.01 mol% to 0.4 mol% of the total lipids present in the LNPs, and not 0.01 mol%, 0.05 mol%, 0.07 mol%, 0.1 mol%, 0.15 mol%, 0.2 mol%, 0.25 mol%, 0.3 mol%, 0.35 mol%, and 0.4 mol%.
  • the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.05 mol% to 0.4 mol% of the total lipids present in the LNPs, and not 0.05 mol%, 0.07 mol%, 0.1 mol%, 0.15 mol%, 0.2 mol%, 0.25 mol%, 0.3 mol%, 0.35 mol%, and 0.4 mol%.
  • the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.1 mol% to 0.4 mol% of the total lipids present in the LNPs, and not 0.1 mol%, 0.15 mol%, 0.2 mol%, 0.25 mol%, 0.3 mol%, 0.35 mol%, and 0.4 mol%. In some embodiments, the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.2 mol% to 0.4 mol% of the total lipids present in the LNPs, and not 0.2 mol%, 0.25 mol%, 0.3 mol%, 0.35 mol%, and 0.4 mol%.
  • the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.3 mol% to 0.4 mol% of the total lipids present in the LNPs, and not 0.3 mol%, 0.35 mol%, and 0.4 mol%.
  • the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.0001 mol% to 0.35 mol% of the total lipids present in the LNPs, and are not 0.0001 mol%, 0.0005 mol%, 0.001 mol%, 0.005 mol%, 0.01 mol%, 0.05 mol%, 0.07 mol%, 0.1 mol%, 0.15 mol%, 0.2 mol%, 0.25 mol%, 0.3 mol%, and 0.35 mol%.
  • the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.0005 mol% to 0.35 mol% of the total lipids present in the LNPs, and not 0.0005 mol%, 0.001 mol%, 0.005 mol%, 0.01 mol%, 0.05 mol%, 0.07 mol%, 0.1 mol%, 0.15 mol%, 0.2 mol%, 0.25 mol%, 0.3 mol%, and 0.35 mol%.
  • the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.001 mol% to 0.35 mol% of the total lipids present in the LNPs, and not 0.001 mol%, 0.005 mol%, 0.01 mol%, 0.05 mol%, 0.07 mol%, 0.1 mol%, 0.15 mol%, 0.2 mol%, 0.25 mol%, 0.3 mol%, and 0.35 mol%.
  • the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.005 mol% to 0.35 mol% of the total lipids present in the LNPs, and not 0.005 mol%, 0.01 mol%, 0.05 mol%, 0.07 mol%, 0.1 mol%, 0.15 mol%, 0.2 mol%, 0.25 mol%, 0.3 mol%, and 0.35 mol%.
  • the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.01 mol% to 0.35 mol% of the total lipids present in the LNPs, and not 0.01 mol%, 0.05 mol%, 0.07 mol%, 0.1 mol%, 0.15 mol%, 0.2 mol%, 0.25 mol%, 0.3 mol%, and 0.35 mol%.
  • the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.05 mol% to 0.35 mol% of the total lipids present in the LNPs, and not 0.05 mol%, 0.07 mol%, 0.1 mol%, 0.15 mol%, 0.2 mol%, 0.25 mol%, 0.3 mol%, and 0.35 mol%.
  • the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.1 mol% to 0.35 mol% of the total lipids present in the LNPs, and not 0.1 mol%, 0.15 mol%, 0.2 mol%, 0.25 mol%, 0.3 mol%, and 0.35 mol%. In some embodiments, the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.2 mol% to 0.35 mol% of the total lipids present in the LNPs, and not 0.2 mol%, 0.25 mol%, 0.3 mol%, and 0.35 mol%.
  • the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.3 mol% to 0.35 mol% of the total lipids present in the LNPs, and not more than 0.3 mol% and 0.35 mol%.
  • the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.0001 mol% to 0.3 mol% of the total lipids present in the LNPs, and not 0.0001 mol%, 0.0005 mol%, 0.001 mol%, 0.005 mol%, 0.01 mol%, 0.05 mol%, 0.07 mol%, 0.1 mol%, 0.15 mol%, 0.2 mol%, 0.25 mol%, and 0.3 mol%.
  • the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.0005 mol% to 0.3 mol% of the total lipids present in the LNPs, and not 0.0005 mol%, 0.001 mol%, 0.005 mol%, 0.01 mol%, 0.05 mol%, 0.07 mol%, 0.1 mol%, 0.15 mol%, 0.2 mol%, 0.25 mol%, and 0.3 mol%.
  • the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.001 mol% to 0.3 mol% of the total lipids present in the LNPs, and not 0.001 mol%, 0.005 mol%, 0.01 mol%, 0.05 mol%, 0.07 mol%, 0.1 mol%, 0.15 mol%, 0.2 mol%, 0.25 mol%, and 0.3 mol%.
  • the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.005 mol% to 0.3 mol% of the total lipids present in the LNPs, and not 0.005 mol%, 0.01 mol%, 0.05 mol%, 0.07 mol%, 0.1 mol%, 0.15 mol%, 0.2 mol%, 0.25 mol%, and 0.3 mol%.
  • the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.01 mol% to 0.3 mol% of the total lipids present in the LNPs, and not 0.01 mol%, 0.05 mol%, 0.07 mol%, 0.1 mol%, 0.15 mol%, 0.2 mol%, 0.25 mol%, and 0.3 mol%.
  • the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.05 mol% to 0.3 mol% of the total lipids present in the LNPs, and not 0.05 mol%, 0.07 mol%, 0.1 mol%, 0.15 mol%, 0.2 mol%, 0.25 mol%, and 0.3 mol%.
  • the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.1 mol% to 0.3 mol% of the total lipids present in the LNPs, and not 0.1 mol%, 0.15 mol%, 0.2 mol%, 0.25 mol%, and 0.3 mol%.
  • the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.2 mol% to 0.3 mol% of the total lipids present in the LNPs, and not 0.2 mol%, 0.25 mol%, or 0.3 mol%.
  • the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.0001 mol% to 0.25 mol% of the total lipids present in the LNPs, and not 0.0001 mol%, 0.0005 mol%, 0.001 mol%, 0.005 mol%, 0.01 mol%, 0.05 mol%, 0.07 mol%, 0.1 mol%, 0.15 mol%, 0.2 mol%, and 0.25 mol%.
  • the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.0005 mol% to 0.25 mol% of the total lipids present in the LNPs, and not 0.0005 mol%, 0.001 mol%, 0.005 mol%, 0.01 mol%, 0.05 mol%, 0.07 mol%, 0.1 mol%, 0.15 mol%, 0.2 mol%, and 0.25 mol%.
  • the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.001 mol% to 0.25 mol% of the total lipids present in the LNPs, and not 0.001 mol%, 0.005 mol%, 0.01 mol%, 0.05 mol%, 0.07 mol%, 0.1 mol%, 0.15 mol%, 0.2 mol%, and 0.25 mol%.
  • the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.005 mol% to 0.25 mol% of the total lipids present in the LNPs, and not 0.005 mol%, 0.01 mol%, 0.05 mol%, 0.07 mol%, 0.1 mol%, 0.15 mol%, 0.2 mol%, and 0.25 mol%.
  • the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.01 mol% to 0.25 mol% of the total lipids present in the LNPs, and not 0.01 mol%, 0.05 mol%, 0.07 mol%, 0.1 mol%, 0.15 mol%, 0.2 mol%, and 0.25 mol%.
  • the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.05 mol% to 0.25 mol% of the total lipids present in the LNPs, and not 0.05 mol%, 0.07 mol%, 0.1 mol%, 0.15 mol%, 0.2 mol%, and 0.25 mol%.
  • the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.1 mol% to 0.25 mol% of the total lipids present in the LNPs, and not 0.1 mol%, 0.15 mol%, 0.2 mol%, and 0.25 mol%.
  • the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.0001 mol% to 0.2 mol% of the total lipids present in the LNPs, and not 0.0001 mol%, 0.0005 mol%, 0.001 mol%, 0.005 mol%, 0.01 mol%, 0.05 mol%, 0.07 mol%, 0.1 mol%, 0.15 mol%, and 0.2 mol%.
  • the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.0005 mol% to 0.2 mol% of the total lipids present in the LNPs, and not 0.0005 mol%, 0.001 mol%, 0.005 mol%, 0.01 mol%, 0.05 mol%, 0.07 mol%, 0.1 mol%, 0.15 mol%, and 0.2 mol%.
  • the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.001 mol% to 0.2 mol% of the total lipids present in the LNPs, and not 0.001 mol%, 0.005 mol%, 0.01 mol%, 0.05 mol%, 0.07 mol%, 0.1 mol%, 0.15 mol%, and 0.2 mol%.
  • the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.005 mol% to 0.2 mol% of the total lipids present in the LNPs, and not 0.005 mol%, 0.01 mol%, 0.05 mol%, 0.07 mol%, 0.1 mol%, 0.15 mol%, and 0.2 mol%.
  • the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.01 mol% to 0.2 mol% of the total lipids present in the LNPs, and not 0.01 mol%, 0.05 mol%, 0.07 mol%, 0.1 mol%, 0.15 mol%, and 0.2 mol%.
  • the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.05 mol% to 0.2 mol% of the total lipids present in the LNPs, and not 0.05 mol%, 0.07 mol%, 0.1 mol%, 0.15 mol%, and 0.2 mol%. In some embodiments, the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.1 mol% to 0.2 mol% of the total lipids present in the LNPs, and not 0.1 mol%, 0.15 mol%, and 0.2 mol%.
  • the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.0001 mol% to 0.15 mol% of the total lipids present in the LNPs, and not 0.0001 mol%, 0.0005 mol%, 0.001 mol%, 0.005 mol%, 0.01 mol%, 0.05 mol%, 0.07 mol%, 0.1 mol%, and 0.15 mol%.
  • the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.0005 mol% to 0.15 mol% of the total lipids present in the LNPs, and not 0.0005 mol%, 0.001 mol%, 0.005 mol%, 0.01 mol%, 0.05 mol%, 0.07 mol%, 0.1 mol%, and 0.15 mol%.
  • the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.001 mol% to 0.15 mol% of the total lipids present in the LNPs, and not 0.001 mol%, 0.005 mol%, 0.01 mol%, 0.05 mol%, 0.07 mol%, 0.1 mol%, and 0.15 mol%.
  • the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.005 mol% to 0.15 mol% of the total lipids present in the LNPs, and not 0.005 mol%, 0.01 mol%, 0.05 mol%, 0.07 mol%, 0.1 mol%, and 0.15 mol%. In some embodiments, the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.01 mol% to 0.15 mol% of the total lipids present in the LNPs, and not 0.01 mol%, 0.05 mol%, 0.07 mol%, 0.1 mol%, and 0.15 mol%.
  • the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.05 mol% to 0.15 mol% of the total lipids present in the LNPs, and not 0.05 mol%, 0.07 mol%, 0.1 mol%, and 0.15 mol%. In some embodiments, the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.1 mol% to 0.15 mol% of the total lipids present in the LNPs, and not 0.1 mol% and 0.15 mol%.
  • the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.0001 mol% to 0.1 mol% of the total lipids present in the LNPs, and not 0.0001 mol%, 0.0005 mol%, 0.001 mol%, 0.005 mol%, 0.01 mol%, 0.05 mol%, 0.07 mol%, and 0.1 mol%.
  • the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.0005 mol% to 0.1 mol% of the total lipids present in the LNPs, and not 0.0005 mol%, 0.001 mol%, 0.005 mol%, 0.01 mol%, 0.05 mol%, 0.07 mol%, and 0.1 mol%.
  • the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.001 mol% to 0.1 mol% of the total lipids present in the LNPs, and not 0.001 mol%, 0.005 mol%, 0.01 mol%, 0.05 mol%, 0.07 mol%, and 0.1 mol%. In some embodiments, the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.005 mol% to 0.1 mol% of the total lipids present in the LNPs, and not 0.005 mol%, 0.01 mol%, 0.05 mol%, 0.07 mol%, and 0.1 mol%.
  • the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.01 mol% to 0.1 mol% of the total lipids present in the LNPs, and not 0.01 mol%, 0.05 mol%, 0.07 mol%, and 0.1 mol%. In some embodiments, the polymer lipids (e.g., PEG lipids) in the LNPs comprise 0.05 mol% to 0.1 mol% of the total lipids present in the LNPs, and not 0.05 mol%, 0.07 mol%, and 0.1 mol%.
  • the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.0001 mol% to 0.05 mol% of the total lipids present in the LNPs, and not 0.0001 mol%, 0.0005 mol%, 0.001 mol%, 0.005 mol%, 0.01 mol%, and 0.05 mol%.
  • the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.0005 mol% to 0.05 mol% of the total lipids present in the LNPs, and not 0.0005 mol%, 0.001 mol%, 0.005 mol%, 0.01 mol%, and 0.05 mol%.
  • the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.001 mol% to 0.05 mol% of the total lipids present in the LNPs, and not 0.001 mol%, 0.005 mol%, 0.01 mol%, and 0.05 mol%. In some embodiments, the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.005 mol% to 0.05 mol% of the total lipids present in the LNPs, and not 0.005 mol%, 0.01 mol%, or 0.05 mol%.
  • the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.01 mol% to 0.05 mol% of the total lipids present in the LNPs, and not 0.01 mol% or 0.05 mol%.
  • the proportion of polymeric lipids (e.g., PEG lipids) in the LNPs to the total lipids present in the LNPs is: greater than 0 and less than 0.0001 mol%, greater than 0.0001 mol% and less than 0.0005 mol%, greater than 0.0005 mol% and less than 0.001 mol%, greater than 0.001 mol% and less than 0.005 mol%, greater than 0.005 mol% and less than 0.01 mol%, greater than 0.01 mol% and less than 0.05 mol%, greater than 0.05 mol% and less than 0.07 mol%, greater than 0.07 mol% and less than 0.1 mol%, greater than 0.1 mol% and less than 0.05 mol%.
  • PEG lipids polymeric lipids
  • 0.15 mol% greater than 0.15 mol% and less than 0.2 mol%, greater than 0.2 mol% and less than 0.25 mol%, greater than 0.25 mol% and less than 0.3 mol%, greater than 0.3 mol% and less than 0.35 mol%, greater than 0.35 mol% and less than 0.4 mol%, greater than 0.4 mol% and less than 0.45 mol%, greater than 0.45 mol% and less than 0.5 mol%, greater than 0.5 mol% and less than 0.6 mol%, greater than 0.6 mol% and less than 0.7 mol%, greater than 0.7 mol% and less than 0.75 mol%, or greater than 0.75 mol% and less than 0.8 mol%.
  • the proportion of polymeric lipids (e.g., PEG lipids) in the LNPs to the total lipids present in the LNPs is: greater than 0.01% and less than 0.05%, greater than 0.05% and less than 0.07%, greater than 0.07% and less than 0.1%, greater than 0.1% and less than 0.15%, greater than 0.15% and less than 0.2%, greater than 0.2% and less than 0.25%, greater than 0.25% and less than 0.3%, greater than 0.3% and less than 0.35%, greater than 0.35% and less than 0.4%, greater than 0.4% and less than 0.45%, or greater than 0.45% and less than 0.5%.
  • PEG lipids polymeric lipids
  • the polymeric lipids (e.g., PEG lipids) in the aforementioned LNPs account for 0.00011 mol%–0.00049 mol%, 0.0051%–0.0099%, 0.011%–0.0499%, 0.00051 mol%–0.00099 mol%, 0.0011 mol%–0.0049 mol%, 0.051 mol%–0.0999 mol%, 0.101 mol%–0.149 mol%, 0.151 mol%–0.199 mol%, and 0.205 mol%–0.249 mol% of the total lipids present in the LNPs, respectively.
  • the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.011%–0.0499%, 0.051%–0.0999%, 0.101%–0.149%, 0.151%–0.199%, 0.205%–0.249%, 0.255%–0.299%, 0.305%–0.349%, 0.355%–0.399%, 0.405%–0.449%, or 0.455%–0.499% of the total lipids present in the LNPs.
  • the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.00015 mol% to 0.00045 mol% of the total lipids present in the LNPs. In some embodiments, the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.00055 mol% to 0.00095 mol% of the total lipids present in the LNPs. In some embodiments, the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.0015 mol% to 0.045 mol% of the total lipids present in the LNPs.
  • the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.055 mol% to 0.095 mol% of the total lipids present in the LNPs. In some embodiments, the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.11 mol% to 0.145 mol% of the total lipids present in the LNPs. In some embodiments, the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.155 mol% to 0.195 mol% of the total lipids present in the LNPs.
  • the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.21 mol% to 0.245 mol% of the total lipids present in the LNPs. In some embodiments, the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.255 mol% to 0.295 mol% of the total lipids present in the LNPs. In some embodiments, the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.31 mol% to 0.345 mol% of the total lipids present in the LNPs.
  • the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.355 mol% to 0.395 mol% of the total lipids present in the LNPs. In some embodiments, the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.41 mol% to 0.445 mol% of the total lipids present in the LNPs. In some embodiments, the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.455 mol% to 0.495 mol% of the total lipids present in the LNPs.
  • the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.51 mol% to 0.545 mol% of the total lipids present in the LNPs. In some embodiments, the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.551 mol% to 0.595 mol% of the total lipids present in the LNPs. In some embodiments, the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.61 mol% to 0.645 mol% of the total lipids present in the LNPs.
  • the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.655 mol% to 0.695 mol% of the total lipids present in the LNPs. In some embodiments, the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.71 mol% to 0.745 mol% of the total lipids present in the LNPs. In some embodiments, the polymeric lipids (e.g., PEG lipids) in the LNPs comprise 0.755 mol% to 0.795 mol% of the total lipids present in the LNPs.
  • ionizable lipid refers to a lipid that becomes positively charged when the pH drops below the pKa of its ionizable groups, but gradually becomes neutral at higher pH values. Below the pKa, the positively charged lipid can bind to negatively charged nucleic acids.
  • ionizable lipids include zwitterionic lipids.
  • the ionizable lipids of the above-mentioned LNPs include the following compound (I), its N-oxide, its salt or isomer:
  • R1 is selected from the group consisting of: C5 - C30 alkyl, C5 - C20 alkenyl, -R*YR”, -YR” and -R”'M'R’;
  • R2 and R3 are independently selected from the group consisting of: H, C1 - C14 alkyl, C2- C14 alkenyl, -R*YR", -YR" and -R*OR", or R2 and R3 together with the atoms to which they are attached form a heterocycle or carbide ring;
  • R4 is selected from the group consisting of: hydrogen, C3 - C6 carbocyclic rings, -( CH2 ) nQ , -( CH2 ) nCHQR , -( CH2 ) oC ( R10 ) 2 ( CH2 ) noQ , -CHQR, -CQ(R) 2 , and unsubstituted C1- C6 alkyl groups, wherein Q is selected from carbocyclic rings, heterocyclic rings, -OR, -O( CH2 ) nN (R) 2 , -C(O)OR, -OC(O)R, -CX3 , -CX2H , -CXH2 , -CN, -N(R) 2 , -C(O)N(R) 2 , -N(R)C(O)R, -N(R)S(O) 2R , -N(R)C(O)N(R) 2
  • Each R5 is independently selected from the group consisting of OH, C1 - C3 alkyl, C2 - C3 alkenyl, and H;
  • Each R6 is independently selected from the group consisting of OH, C1 - C3 alkyl, C2 - C3 alkenyl, and H;
  • M and M' are independently selected from -C(O)O-, -OC(O)-, -OC(O)-M”-C(O)O-, -C(O)N(R')-, -N(R')C(O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR')O-, -S(O) 2- , -SS-, aryl groups and heteroaryl groups, wherein M” is a bond, C1 - C13 alkylene or C2 - C13 alkenyl;
  • R 7 is selected from the group consisting of C1-3 alkyl, C2 - C3 alkenyl and H;
  • R8 is selected from the group consisting of C3-6 carbon rings and heterocycles
  • R9 is selected from the group consisting of: H, CN, NO2 , C1 - C6 alkyl, -OR, -S(O) 2R , -S(O) 2N (R) 2 , C2 - C6 alkenyl, C3 - C6 carbon ring and heterocycle;
  • R 10 is selected from the group consisting of H, C1 - C3 alkyl and C2 -C3 alkenyl groups;
  • Each R is independently selected from the group consisting of: C1 - C3 alkyl, C2 - C3 alkenyl, ( CH2 ) qOR **, and H.
  • each q is independently selected from 1, 2, and 3;
  • Each R' is independently selected from the group consisting of: C1 - C18 alkyl, C2 - C18 alkenyl, -R*YR”, -YR”, H and Furthermore, R11 is selected from the group consisting of C1 - C12 alkylene and C2 - C12 alkenylene, and R12 and R13 are each independently selected from the group consisting of C1 - C12 alkylene and C2 - C12 alkenylene.
  • Each R is independently selected from the group consisting of C3- C15 alkyl and C3- C15 alkenyl groups ;
  • Each R”' is independently selected from the group consisting of C3 - C15 alkylene and C3 - C15 alkenylene groups;
  • Each R* is independently selected from the group consisting of non-existent C1 - C12 alkylene and C2 - C12 alkenyl groups;
  • Each R** is independently selected from the group consisting of non-existent C1 - C12 alkyl and C2 -C12 alkenyl groups;
  • Each Y is independently a C3 - C6 carbon ring
  • Each X is independently selected from the following groups: F, Cl, Br, and I;
  • n is selected from 5, 6, 7, 8, 9, 10, 11, 12 and 13; and when R4 is -( CH2 ) nQ , -( CH2 ) nCHQR , –CHQR or -CQ(R) 2 , then (i) when n is 1, 2, 3, 4 or 5, Q is not -N(R) 2 ; or (ii) when n is 1 or 2, Q is not a 5, 6 or 7-membered heterocyclic alkyl group.
  • the ionizable lipids of the above-mentioned LNPs include the following compound (II), its N-oxide, its salt or isomer:
  • R1 , R2 , R3 , R5 , R6 , M, and R7 are as described above.
  • R N is H or C1 - C3 alkyl
  • Xa and Xb are each independently O or S;
  • R 14 is selected from H, halogen, -OH, R b , -N(R b ) 2 , -CN, -N 3 , -C(O)OH, -C(O)OR b , -OC(O)R b , -OR b , -SR b , -S(O)R b , -S(O)OR b , -S(O) 2 OR b , -NO 2 , -S(O) 2 N(R b ) 2 , -N(R b )S(O) 2 R b , -NH(CH 2 ) t1 N(R b ) 2 , -NH(CH 2 ) p1 O(CH 2 ) q1 N(R b ) 2 , -NH(CH 2 ) s1 OR b , -N((CH 2 ) S OR b ) 2 , -N(R b
  • Each Rb is independently selected from the group consisting of C1 - C3 alkyl, C2 - C3 alkenyl and H;
  • u is 5, 6, 7, 8, 9, 10, 11, 12 or 13;
  • w is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;
  • r is 0 or 1;
  • t1 can be 1, 2, 3, 4, or 5;
  • p1 is 1, 2, 5, 4 or 5;
  • q1 is 1, 2, 5, 4, or 5;
  • s1 can be 1, 2, 3, 4 or 5.
  • the ionizable lipids of the above-mentioned LNPs include the following compound (III), its N-oxide, its salt or isomer:
  • G1 and G2 are each independently an unsubstituted C1 - C12 alkylene or C2 - C12 alkenylene;
  • G3 is a C1 - C24 alkylene group, a C2 - C24 alkenyl group, a C3 - C8 cycloalkylene group, or a C3 - C8 cycloalkenyl group;
  • Ra is an H or C1 - C12 hydrocarbon group
  • R15 and R16 are each independently C6 - C24 alkyl or C6 - C24 alkenyl
  • R 19 is a C1 - C12 hydrocarbon group
  • R 18 is an H or C1 - C6 hydrocarbon group
  • x 0, 1, or 2.
  • the ionizable lipids of the above-mentioned LNPs include or are the following compounds (VI), their N-oxides, their salts, or isomers thereof:
  • A1 , A2 , and A3 are one of the following:
  • A1 is H, C1 - C5 hydrocarbon group or C1 - C5 heterohydrocarbon group
  • A2 is H, C1 - C5 hydrocarbon group or C1 - C5 heterohydrocarbon group
  • A3 is C1 - C5 alkylene group or bond
  • A1 is a C1 - C5 alkylene group or a C1 - C5 heteroalkylene group
  • A2 is a C1 - C5 alkylene group or a C1 - C5 heteroalkylene group
  • A3 is a C1 - C5 alkylene group or a bond.
  • A1 is a C1 - C5 alkylene group or a C1 - C5 heteroalkylene group
  • A2 is H, a C1 - C5 hydrocarbon group or a C1 - C5 heteroalkylene group
  • A3 is a C1 - C5 alkylene group.
  • A1 and A3 together with the nitrogen atom they are connected to form a nitrogen-containing heterocycle.
  • A1 is a C1 - C5 alkylene group or a C1 - C5 heteroalkylene group
  • A2 is a C1 - C5 alkylene group or a C1 - C5 heteroalkylene group
  • A3 is a C1 - C5 alkylene group
  • A1 , A2 and A3 together with the nitrogen atom they are connected to form a nitrogen-containing spirocyclic heterocycle, fused heterocycle or bridged heterocycle
  • A4 is a C1 - C5 alkylene group or bond
  • A5 , A6 , A7 and A8 are each independently a C1 - C18 alkylene group, a C1 - C18 heteroalkylene group or bond;
  • R 22 are each independently an H or C1 - C8 hydrocarbon group
  • Each R20 and each R21 is independently a C1 - C24 hydrocarbon group or a C1 - C24 heterohydrocarbon group containing O or S;
  • Each A9 is a C1 - C8 alkylene group or bond
  • Each A10 and each A11 is independently a C1 - C8 hydrocarbon group
  • the hydrocarbon group is alkyl, alkenyl, or ynyl
  • the alkylene group is an alkylene group, an alkenylene group, or an alkynylene group
  • the heteroalkyl group is a heteroalkyl, heteroalkenyl, or heteroynyl group
  • the heteroalkyl group is a heteroalkyl group, a heteroalkenyl group, or a heteroyne group.
  • the ionizable lipids of the above-mentioned LNPs are one or more of the following: SM-102, AL-0315, MC3, and
  • the ionizable lipids of the above-mentioned LNPs do not include SM-102, AL-0315, Dlin-DMA, Dlin-D-DMA, DLin-MC3-DMA (i.e., MC3), DODMA, DSDMA, DLen-DMA, ⁇ -DLen-DMA, Dlin-K-DMA, Dlin-KC2-DMA (i.e., Dlin-C2K-DMA, XTC2 or C2K), Dlin-K-C3-DMA, Dlin-K-C4-DMA, ⁇ -Dlen-K-C2K-DMA, Dlin-M-C2-DMA (i.e., MC2), Dlin-MP-DMA (i.e., 1-B11), DODAP or DOTAP.
  • the ionizable lipids in the aforementioned LNPs account for 25 mol% to 75 mol% of the total lipids present in the LNPs.
  • the ionizable lipids in the aforementioned LNPs account for 30 mol%–65 mol%, 35 mol%–65 mol%, 40 mol%–65 mol%, 45 mol%–65 mol%, 46 mol%–65 mol%, 47 mol%–65 mol%, 48 mol%–65 mol%, 49 mol%–65 mol%, 50 mol%–65 mol%, 51 mol%–65 mol%, 52 mol%–65 mol%, 53 mol%–65 mol%, 54 mol%–65 mol%, 55 mol%–65 mol%, 56 mol%–65 mol%, 57 mol%–65 mol%, 30 mol%–60 mol%, 35 mol%–60 mol%, 40 mol%–60 mol%, 45 mol%–60 mol%, and 46 mol%–60 mol% of the total lipids present in the LNPs.
  • the ionizable lipids in the LNPs comprise 32 mol% to 59.5 mol% of the total lipids present in the LNPs.
  • the ionizable lipids in the LNPs comprise 35 mol% to 59.5 mol% of the total lipids present in the LNPs.
  • the ionizable lipids in the LNPs comprise 40 mol% to 56 mol% of the total lipids present in the LNPs.
  • the ionizable lipids in the LNPs comprise 45 mol% to 55 mol% of the total lipids present in the LNPs.
  • the ionizable lipids in the LNPs comprise 48 mol% to 55 mol% of the total lipids present in the LNPs.
  • the ionizable lipids in the LNPs comprise 49 mol% to 52 mol% of the total lipids present in the LNPs.
  • the ionizable lipids in the LNPs comprise 49.5 mol% to 51.5 mol% of the total lipids present in the LNPs.
  • the accessory lipids of the aforementioned LNPs include phospholipids.
  • Phospholipids are typically semi-synthetic, but can also be of natural origin or chemically modified.
  • the accessory lipids of the aforementioned LNPs are phospholipids.
  • the phospholipids of the LNPs include one or more of the following: DSPC (distearylphosphatidylcholine), DOPE (dioleoylphosphatidylethanolamine), DOPC (dioleoyllecithin), DOPS (dioleoylphosphatidylserine), DSPG (1,2-octacosanyl-sn-glycerol-3-phosphate-(1’-rac-glycerol)), DPPG (dispalmitoylphosphatidylglycerol), DPPC (dispalmitoylphosphatidylcholine), DGTS (1,2-dispalmitoyl-sn-glycerol-3-O-4'-(N,N,N-trimethyl)homoserine), and lysophospholipids.
  • the accessory lipids of the LNPs are selected from one or more of the following: DSPC, DOPE, DOPC,
  • the auxiliary lipids of the above-mentioned LNPs are DSPC and/or DOPE.
  • the auxiliary lipid of the above-mentioned LNPs is DSPC.
  • the accessory lipids (e.g., DSPC) in the LNPs comprise 0 mol% to 45 mol% of the total lipids present in the LNPs.
  • the accessory lipids (e.g., DSPC) in the LNPs comprise 0 mol% to 30 mol% of the total lipids present in the LNPs.
  • the proportion of accessory lipids (e.g., DSPC) in the LNPs is greater than 0 and less than 30 mol% of the total lipids present in the LNPs.
  • the accessory lipids (e.g., DSPC) in the aforementioned LNPs account for 0.5 mol%–29.5 mol%, 0.5 mol%–27.5 mol%, 1.5 mol%–27.5 mol%, 2.5 mol%–27.5 mol%, 4.5 mol%–27.5 mol%, 5.5 mol%–27.5 mol%, 6.5 mol%–27.5 mol%, 7.5 mol%–27.5 mol%, and 8 mol%–27.5 mol% of the total lipids present in the LNPs, respectively.
  • the accessory lipids (e.g., DSPC) in the LNPs comprise 7.5 mol% to 25 mol% of the total lipids present in the LNPs.
  • the accessory lipids (e.g., DSPC) in the LNPs comprise 8 mol% to 20 mol% of the total lipids present in the LNPs.
  • the accessory lipids (e.g., DSPC) in the LNPs comprise 10 mol% to 18 mol% of the total lipids present in the LNPs.
  • the accessory lipids (e.g., DSPC) in the LNPs comprise 12 mol% to 16 mol% of the total lipids present in the LNPs.
  • the structural lipids of the LNPs include sterols. In one optional specific example, the structural lipids of the LNPs are sterols. In some embodiments, the sterols of the LNPs include one or more of the following: phytosterols, 20 ⁇ -hydroxycholesterol, cholesterol, cholesterol esters, sterol hormones, sterol vitamins, bile acids, cholesterol, ergosterol, ⁇ -sitosterol, and oxidized cholesterol derivatives. In some embodiments, the structural lipids of the LNPs include at least one of cholesterol, cholesterol esters, sterol hormones, sterol vitamins, and bile acids.
  • the structural lipids of the LNPs described above are not phytosterols or their derivatives.
  • the structural lipids of the aforementioned LNPs are cholesterol.
  • the structural lipids of the aforementioned LNPs are high-purity cholesterol, particularly injectable high-purity cholesterol, such as CHO-HP (produced by AVT).
  • the structural lipids of the LNPs mentioned above are 20 ⁇ -hydroxycholesterol.
  • the structural lipids (e.g., cholesterol) in the LNPs comprise 0 mol% to 60 mol% of the total lipids present in the LNPs.
  • examples include 0 mol%, 1 mol%, 5 mol%, 8 mol%, 10 mol%, 12 mol%, 14 mol%, 15 mol%, 16 mol%, 18 mol%, 20 mol%, 22 mol%, 24 mol%, 25 mol%, 27 mol%, 27.5 mol%, 28 mol%, 28.5 mol%, 29 mol%, 29.5 mol%, 30 mol%, 30.5 mol%, 31 mol%, 31.5 mol%, 32 mol%, 32.5 mol%, 33 mol%, 33.5 mol%, 34 mol%, 34.5 mol%, 35 mol%, and 35.5 mol%.
  • the structural lipids (e.g., cholesterol) in the aforementioned LNPs account for 10 mol%–60 mol%, 11 mol%–60 mol%, 13 mol%–60 mol%, 15 mol%–60 mol%, 17 mol%–60 mol%, 19 mol%–60 mol%, 21 mol%–60 mol%, 23 mol%–60 mol%, 25 mol%–60 mol%, 26 mol%–60 mol%, 26.5 mol%–60 mol%, 27 mol%–60 mol%, 27.5 mol%–60 mol%, 28 mol%–60 mol%, 28.5 mol%–60 mol%, 29 mol%–60 mol%, 29.5 mol%–60 mol%, 30 mol%–60 mol%, 30.5 mol%–60 mol%, 31 mol%–60 mol%, and 31.5 mol%–60 mol% of the total lipids present in the LNP
  • the structural lipids (e.g., cholesterol) in the LNPs comprise 15 mol% to 50 mol% of the total lipids present in the LNPs.
  • the structural lipids (e.g., cholesterol) in the LNPs comprise 27 mol% to 42 mol% of the total lipids present in the LNPs.
  • the structural lipids (e.g., cholesterol) in the LNPs comprise 30 mol% to 40 mol% of the total lipids present in the LNPs.
  • the LNPs described above comprise ionizable lipids, accessory lipids (e.g., DSPC), structural lipids (e.g., cholesterol), and polymeric lipids (e.g., PEG lipids), and do not contain stearic acid (SA) and/or its analogues and derivatives.
  • accessory lipids e.g., DSPC
  • structural lipids e.g., cholesterol
  • polymeric lipids e.g., PEG lipids
  • SA stearic acid
  • the total lipids present in the above-mentioned LNPs consist of ionizable lipids, accessory lipids (e.g., DSPC), structural lipids (e.g., cholesterol), and polymeric lipids (e.g., PEG lipids).
  • accessory lipids e.g., DSPC
  • structural lipids e.g., cholesterol
  • polymeric lipids e.g., PEG lipids
  • the LNPs comprise ionizable lipids, accessory lipids (e.g., DSPC), structural lipids (e.g., cholesterol), and polymeric lipids (e.g., PEG lipids), wherein the polymeric lipids (e.g., PEG lipids) account for 0.0001 mol%–0.8 mol%, 0.01 mol%–0.8 mol%, 0.05 mol%–0.8 mol%, 0.1 mol%–0.8 mol%, 0.2 mol%–0.8 mol%, 0.3 mol%–0.8 mol%, 0.4 mol%–0.8 mol%, 0.5 mol%–0.8 mol%, 0.05 mol%–0.8 mol%, 0.1 mol%–0.8 mol%, 0.2 mol%–0.8 mol%, 0.3 mol%–0.8 mol%, 0.4 mol%–0.8 mol%, 0.0001 mol% of the total lipids present in the LNPs.
  • accessory lipids e.g.
  • the LNPs comprise: ionizable lipids, accessory lipids (e.g., DSPC), structural lipids (e.g., non-phytosterols (e.g., cholesterol)), and polymeric lipids (e.g., PEG lipids), wherein the ionizable lipids account for 30 mol% to 60 mol% of the total lipids present in the LNPs, the accessory lipids (e.g., DSPC) account for 0 mol% to 30 mol% of the total lipids present in the LNPs, the structural lipids (e.g., non-phytosterols (e.g., cholesterol)) account for 10 mol% to 60 mol% of the total lipids present in the LNPs, and the polymeric lipids (e.g., PEG lipids) account for 0 mol% to 0.8 mol% of the total lipids present in the LNPs.
  • accessory lipids e.g
  • the LNPs comprise: ionizable lipids, accessory lipids (e.g., DSPC), structural lipids (e.g., non-phytosterols (e.g., cholesterol)), and polymeric lipids (e.g., PEG lipids), wherein the ionizable lipids account for 30 mol% to 60 mol% of the total lipids present in the LNPs, the accessory lipids (e.g., DSPC) account for 0 mol% to 30 mol% of the total lipids present in the LNPs, the structural lipids (e.g., non-phytosterols (e.g., cholesterol)) account for 10 mol% to 60 mol% of the total lipids present in the LNPs, and the polymeric lipids (e.g., PEG lipids) account for 0.0001 mol% to 0.8 mol%, 0.001 mol% to 0.8 mol%, 0.01 mol
  • the LNPs comprise: ionizable lipids, accessory lipids (e.g., DSPC), structural lipids (e.g., non-phytosterols (e.g., cholesterol)), and polymeric lipids (e.g., PEG lipids), wherein the ionizable lipids account for 30 mol% to 60 mol% of the total lipids present in the LNPs, the accessory lipids (e.g., DSPC) account for 0 mol% to 30 mol% of the total lipids present in the LNPs, the structural lipids (e.g., non-phytosterols (e.g., cholesterol)) account for 10 mol% to 60 mol% of the total lipids present in the LNPs, and the polymeric lipids (e.g., PEG lipids) account for...
  • accessory lipids e.g., DSPC
  • structural lipids e.g., non-phytosterol
  • PEG lipids account for 0.0001 mol%–0.8 mol% of the total lipids present in LNPs, 0.001 mol%–0.8 mol%, 0.01 mol%–0.8 mol%, 0.01 mol%–0.8 mol%, 0.01 mol%–0.65 mol%, 0.01 mol%–0.6 mol%, 0.01 mol%–0.55 mol%, 0.01 mol%–0.5 mol%, 0.01 mol%–0.4 mol%, 0 .01mol% ⁇ 0.3mol%, 0.01mol% ⁇ 0.2mol%, 0.01mol% ⁇ 0.15mol%, 0.05mol% ⁇ 0.8mol%, 0.05mol% ⁇ 0.8mol%, 0.05mol% ⁇ 0.65 mol%, 0.05mol% ⁇ 0.6mol%, 0.05mol% ⁇ 0.55mol%, 0.05mol% ⁇ 0.5mol%, 0.05mol% ⁇ 0.4mol%, 0.05mol% ⁇ 0.3mol%, 0.05mol% ⁇ 0.2mol%, 0.05mol% ⁇ 0.2mol%, 0.05mol% ⁇ 0.55mol%
  • the aforementioned LNPs comprise: ionizable lipids, accessory lipids (e.g., DSPC), structural lipids (e.g., non-phytosterols (e.g., cholesterol)), and polymeric lipids (e.g., PEG lipids), wherein the ionizable lipids account for 30 mol% to 60 mol% of the total lipids present in the LNPs, the accessory lipids (e.g., DSPC) account for 0 mol% to 30 mol% of the total lipids present in the LNPs, the structural lipids (e.g., non-phytosterols (e.g., cholesterol)) account for 10 mol% to 60 mol% of the total lipids present in the LNPs, and the polymeric lipids (e.g., PEG lipids) account for 0.1 mol% to 0.6 mol% of the total lipids present in the LNPs, and are not 0.1 mol
  • the LNPs comprise: ionizable lipids, accessory lipids (e.g., DSPC), structural lipids (e.g., non-phytosterols (e.g., cholesterol)), and polymeric lipids (e.g., PEG lipids), wherein the ionizable lipids account for 45 mol% to 55 mol% of the total lipids present in the LNPs, the accessory lipids (e.g., DSPC) account for 8 mol% to 25 mol% of the total lipids present in the LNPs, the structural lipids (e.g., non-phytosterols (e.g., cholesterol)) account for 20 mol% to 45 mol% of the total lipids present in the LNPs, and the polymeric lipids (e.g., PEG lipids) account for 0.0001 mol% to 0.8 mol%, 0.001 mol% to 0.8 mol%, 0.01 mol%
  • the LNPs comprise: ionizable lipids, accessory lipids (e.g., DSPC), structural lipids (e.g., non-phytosterols (e.g., cholesterol)), and polymeric lipids (e.g., PEG lipids), wherein the ionizable lipids account for 45 mol% to 55 mol% of the total lipids present in the LNPs, the accessory lipids (e.g., DSPC) account for 8 mol% to 25 mol% of the total lipids present in the LNPs, and the structural lipids (e.g., non-phytosterols (e.g., cholesterol)) account for 20 mol% to 45 mol% of the total lipids present in the LNPs.
  • accessory lipids e.g., DSPC
  • structural lipids e.g., non-phytosterols (e.g., cholesterol)
  • polymeric lipids e.g.,
  • polymer lipids account for 0.0001 mol%–0.8 mol% of the total lipids present in LNPs, 0.001 mol%–0.8 mol%, 0.01 mol%–0.8 mol%, 0.01 mol%–0.8 mol%, 0.01 mol%–0.65 mol%, 0.01 mol%–0.6 mol%, 0.01 mol%–0.55 mol%, 0.01 mol%–0.5 mol%, 0 .01mol% ⁇ 0.4mol%, 0.01mol% ⁇ 0.3mol%, 0.01mol% ⁇ 0.2mol%, 0.01mol% ⁇ 0.15mol%, 0.05mol% ⁇ 0.8mol%, 0.05mol% ⁇ 0.8mol%, 0.05mol% ⁇ 0.65mol%, 0.05mol% ⁇ 0.6mol%, 0.05mol% ⁇ 0.55mol%, 0.05mol% ⁇ 0.4mol% , 0.05mol% ⁇ 0.3mol%,
  • the aforementioned LNPs include: ionizable lipids, accessory lipids (e.g., DSPC), structural lipids (e.g., non-phytosterols (e.g., cholesterol)), and polymeric lipids (e.g., PEG lipids), wherein ionizable lipids account for 45 mol% to 55 mol% of the total lipids present in the LNPs, accessory lipids (e.g., DSPC) account for 8 mol% to 25 mol% of the total lipids present in the LNPs, structural lipids (e.g., non-phytosterols (e.g., cholesterol)) account for 20 mol% to 45 mol% of the total lipids present in the LNPs, and polymeric lipids (e.g., PEG lipids) account for 0.01 mol% to 0.6 mol% of the total lipids present in the LNPs, and not 0.01 mol%, 0.05
  • the LNPs comprise: ionizable lipids, accessory lipids (e.g., DSPC), structural lipids (e.g., non-phytosterols (e.g., cholesterol)), and polymeric lipids (e.g., PEG lipids), wherein the ionizable lipids account for 47 mol% to 55 mol% of the total lipids present in the LNPs, the accessory lipids (e.g., DSPC) account for 10 mol% to 18 mol% of the total lipids present in the LNPs, the structural lipids (e.g., non-phytosterols (e.g., cholesterol)) account for 27 mol% to 42 mol% of the total lipids present in the LNPs, and the polymeric lipids (e.g., PEG lipids) account for 0.0001 mol% to 0.8 mol%, 0.001 mol% to 0.8 mol%, 0.01 mol%
  • the LNPs comprise: ionizable lipids, accessory lipids (e.g., DSPC), structural lipids (e.g., non-phytosterols (e.g., cholesterol)), and polymeric lipids (e.g., PEG lipids), wherein the ionizable lipids account for 47 mol% to 55 mol% of the total lipids present in the LNPs, the accessory lipids (e.g., DSPC) account for 10 mol% to 18 mol% of the total lipids present in the LNPs, and the structural lipids (e.g., non-phytosterols (e.g., cholesterol)) account for 27 mol% to 42 mol% of the total lipids present in the LNPs.
  • accessory lipids e.g., DSPC
  • structural lipids e.g., non-phytosterols (e.g., cholesterol)
  • polymeric lipids e.g.,
  • polymer lipids account for 0.0001 mol%–0.8 mol% of the total lipids present in LNPs, 0.001 mol%–0.8 mol%, 0.01 mol%–0.8 mol%, 0.01 mol%–0.8 mol%, 0.01 mol%–0.65 mol%, 0.01 mol%–0.6 mol%, 0.01 mol%–0.55 mol%, 0.01 mol%–0.5 mol%, 0 .01mol% ⁇ 0.4mol%, 0.01mol% ⁇ 0.3mol%, 0.01mol% ⁇ 0.2mol%, 0.01mol% ⁇ 0.15mol%, 0.05mol% ⁇ 0.8mol%, 0.05mol% ⁇ 0.8mol%, 0.05mol% ⁇ 0.65mol%, 0.05mol% ⁇ 0.6mol%, 0.05mol% ⁇ 0.55mol%, 0.05mol% ⁇ 0.4mol% , 0.05mol% ⁇ 0.3mol%,
  • the aforementioned LNPs include: ionizable lipids, accessory lipids (e.g., DSPC), structural lipids (e.g., non-phytosterols (e.g., cholesterol)), and polymeric lipids (e.g., PEG lipids), wherein ionizable lipids account for 47 mol% to 55 mol% of the total lipids present in the LNPs, accessory lipids (e.g., DSPC) account for 10 mol% to 18 mol% of the total lipids present in the LNPs, structural lipids (e.g., non-phytosterols (e.g., cholesterol)) account for 27 mol% to 42 mol% of the total lipids present in the LNPs, and polymeric lipids (e.g., PEG lipids) account for 0.01 mol% to 0.6 mol% of the total lipids present in the LNPs, and not 0.01 mol%, 0.05
  • LNPs comprise: ionizable lipids, accessory lipids (e.g., DSPC), structural lipids (e.g., non-phytosterols (e.g., cholesterol)), and polymeric lipids (e.g., PEG lipids), wherein the ionizable lipids account for 30 mol% to 60 mol% of the total lipids present in the LNPs, the accessory lipids (e.g., DSPC) account for 0 mol% to 30 mol% of the total lipids present in the LNPs, the structural lipids (e.g., non-phytosterols (e.g., cholesterol)) account for 10 mol% to 60 mol% of the total lipids present in the LNPs, and the polymeric lipids (e.g., PEG lipids) account for 0.05 mol% to 0.8 mol%, 0.05 mol% to 0.7 mol%, 0.05 mol% to
  • LNPs comprise: ionizable lipids, accessory lipids (e.g., DSPC), structural lipids (e.g., non-phytosterols (e.g., cholesterol)), and polymeric lipids (e.g., PEG lipids), wherein the ionizable lipids constitute 30 mol% to 60 mol% of the total lipids present in the LNPs, the accessory lipids (e.g., DSPC) constitute 0 mol% to 30 mol% of the total lipids present in the LNPs, the structural lipids (e.g., non-phytosterols (e.g., cholesterol)) constitute 10 mol% to 60 mol% of the total lipids present in the LNPs, and the polymeric lipids (e.g., PEG lipids) constitute 10 mol% to 60 mol% of the total lipids present in the LNPs.
  • the ionizable lipids constitute 30 mol% to
  • the percentages are 0.05 mol% to 0.8 mol%, 0.05 mol% to 0.7 mol%, 0.05 mol% to 0.6 mol%, 0.1 mol% to 0.6 mol%, or 0.2 mol% to 0.5 mol%, and are not 0.05 mol%, 0.07 mol%, 0.1 mol%, 0.15 mol%, 0.2 mol%, 0.25 mol%, 0.3 mol%, 0.35 mol%, 0.4 mol%, 0.45 mol%, 0.5 mol%, 0.6 mol%, 0.7 mol%, 0.75 mol%, and 0.8 mol%.
  • LNPs comprise: ionizable lipids, accessory lipids (e.g., DSPC), structural lipids (e.g., non-phytosterols (e.g., cholesterol)), and polymeric lipids (e.g., PEG lipids), wherein the ionizable lipids account for 30 mol% to 60 mol% of the total lipids present in the LNPs, the accessory lipids (e.g., DSPC) account for 0 mol% to 30 mol% of the total lipids present in the LNPs, the structural lipids (e.g., non-phytosterols (e.g., cholesterol)) account for 10 mol% to 60 mol% of the total lipids present in the LNPs, and the polymeric lipids (e.g., PEG lipids) account for 0.05 mol% to 0.8 mol% of the total lipids present in the LNPs.
  • accessory lipids e.g.
  • LNPs comprise: ionizable lipids, accessory lipids (e.g., DSPC), structural lipids (e.g., non-phytosterols (e.g., cholesterol)), and polymeric lipids (e.g., PEG lipids), wherein the ionizable lipids comprise 30 mol% to 60 mol% of the total lipids present in the LNPs, the accessory lipids (e.g., DSPC) comprise 0 mol% to 30 mol% of the total lipids present in the LNPs, the structural lipids (e.g., non-phytosterols (e.g., cholesterol)) comprise 10 mol% to 60 mol% of the total lipids present in the LNPs, and the polymeric lipids (e.g., PEG lipids) comprise 0.05 mol% to 0.8 mol% of the total lipids present in the LNPs, and not 0.05 mol%, 0.07
  • the LNPs comprise: ionizable lipids, accessory lipids (e.g., DSPC), structural lipids (e.g., non-phytosterols (e.g., cholesterol)), and polymeric lipids (e.g., PEG lipids), wherein the ionizable lipids account for 30 mol% to 60 mol% of the total lipids present in the LNPs, the accessory lipids (e.g., DSPC) account for 0 mol% to 30 mol% of the total lipids present in the LNPs, the structural lipids (e.g., non-phytosterols (e.g., cholesterol)) account for 10 mol% to 60 mol% of the total lipids present in the LNPs, and the polymeric lipids (e.g., PEG lipids) account for 0.1 mol% to 0.6 mol% of the total lipids present in the LNPs.
  • accessory lipids e.g
  • LNPs comprise: ionizable lipids, accessory lipids (e.g., DSPC), structural lipids (e.g., non-phytosterols (e.g., cholesterol)), and polymeric lipids (e.g., PEG lipids), wherein the ionizable lipids comprise 30 mol% to 60 mol% of the total lipids present in the LNPs, the accessory lipids (e.g., DSPC) comprise 0 mol% to 30 mol% of the total lipids present in the LNPs, the structural lipids (e.g., non-phytosterols (e.g., cholesterol)) comprise 10 mol% to 60 mol% of the total lipids present in the LNPs, and the polymeric lipids (e.g., PEG lipids) comprise 0.1 mol% to 0.6 mol% of the total lipids present in the LNPs, and not 0.1 mol%, 0.15
  • LNPs comprise: ionizable lipids, accessory lipids (e.g., DSPC), structural lipids (e.g., non-phytosterols (e.g., cholesterol)), and polymeric lipids (e.g., PEG lipids), wherein the ionizable lipids account for 30 mol% to 60 mol% of the total lipids present in the LNPs, the accessory lipids (e.g., DSPC) account for 0 mol% to 30 mol% of the total lipids present in the LNPs, the structural lipids (e.g., non-phytosterols (e.g., cholesterol)) account for 10 mol% to 60 mol% of the total lipids present in the LNPs, and the polymeric lipids (e.g., PEG lipids) account for 0.2 mol% to 0.5 mol% of the total lipids present in the LNPs.
  • accessory lipids e.g.
  • LNPs comprise: ionizable lipids, accessory lipids (e.g., DSPC), structural lipids (e.g., non-phytosterols (e.g., cholesterol)), and polymeric lipids (e.g., PEG lipids), wherein the ionizable lipids comprise 30 mol% to 60 mol% of the total lipids present in the LNPs, the accessory lipids (e.g., DSPC) comprise 0 mol% to 30 mol% of the total lipids present in the LNPs, the structural lipids (e.g., non-phytosterols (e.g., cholesterol)) comprise 10 mol% to 60 mol% of the total lipids present in the LNPs, and the polymeric lipids (e.g., PEG lipids) comprise 0.2 mol% to 0.5 mol% of the total lipids present in the LNPs, and not 0.2 mol%, 0.25
  • LNPs comprise: ionizable lipids, accessory lipids (e.g., DSPC), structural lipids (e.g., non-phytosterols (e.g., cholesterol)), and polymeric lipids (e.g., PEG lipids), wherein the ionizable lipids account for 45 mol% to 55 mol% of the total lipids present in the LNPs, the accessory lipids (e.g., DSPC) account for 8 mol% to 25 mol% of the total lipids present in the LNPs, the structural lipids (e.g., non-phytosterols (e.g., cholesterol)) account for 20 mol% to 45 mol% of the total lipids present in the LNPs, and the polymeric lipids (e.g., PEG lipids) account for 0.05 mol% to 0.8 mol%, 0.05 mol% to 0.7 mol%, 0.05 mol% to 0.6
  • LNPs comprise: ionizable lipids, accessory lipids (e.g., DSPC), structural lipids (e.g., non-phytosterols (e.g., cholesterol)), and polymeric lipids (e.g., PEG lipids), wherein the ionizable lipids constitute 45 mol% to 55 mol% of the total lipids present in the LNPs, the accessory lipids (e.g., DSPC) constitute 8 mol% to 25 mol% of the total lipids present in the LNPs, the structural lipids (e.g., non-phytosterols (e.g., cholesterol)) constitute 20 mol% to 45 mol% of the total lipids present in the LNPs, and the polymeric lipids (e.g., PEG lipids) constitute 20 mol% to 45 mol% of the total lipids present in the LNPs.
  • the ionizable lipids constitute 45 mol% to 55
  • the percentages are 0.05 mol% to 0.8 mol%, 0.05 mol% to 0.7 mol%, 0.05 mol% to 0.6 mol%, 0.1 mol% to 0.6 mol%, or 0.2 mol% to 0.5 mol%, and are not 0.05 mol%, 0.07 mol%, 0.1 mol%, 0.15 mol%, 0.2 mol%, 0.25 mol%, 0.3 mol%, 0.35 mol%, 0.4 mol%, 0.45 mol%, 0.5 mol%, 0.6 mol%, 0.7 mol%, 0.75 mol%, and 0.8 mol%.
  • LNPs comprise: ionizable lipids, accessory lipids (e.g., DSPC), structural lipids (e.g., non-phytosterols (e.g., cholesterol)), and polymeric lipids (e.g., PEG lipids), wherein the ionizable lipids account for 45 mol% to 55 mol% of the total lipids present in the LNPs, the accessory lipids (e.g., DSPC) account for 8 mol% to 25 mol% of the total lipids present in the LNPs, the structural lipids (e.g., non-phytosterols (e.g., cholesterol)) account for 20 mol% to 45 mol% of the total lipids present in the LNPs, and the polymeric lipids (e.g., PEG lipids) account for 0.05 mol% to 0.8 mol% of the total lipids present in the LNPs.
  • accessory lipids e.g.,
  • LNPs comprise: ionizable lipids, accessory lipids (e.g., DSPC), structural lipids (e.g., non-phytosterols (e.g., cholesterol)), and polymeric lipids (e.g., PEG lipids), wherein the ionizable lipids constitute 45 mol% to 55 mol% of the total lipids present in the LNPs, the accessory lipids (e.g., DSPC) constitute 8 mol% to 25 mol% of the total lipids present in the LNPs, the structural lipids (e.g., non-phytosterols (e.g., cholesterol)) constitute 20 mol% to 45 mol% of the total lipids present in the LNPs, and the polymeric lipids (e.g., PEG lipids) constitute 0.05 mol% to 0.8 mol% of the total lipids present in the LNPs, and not 0.05 mol%, 0.07 mol
  • LNPs comprise: ionizable lipids, accessory lipids (e.g., DSPC), structural lipids (e.g., non-phytosterols (e.g., cholesterol)), and polymeric lipids (e.g., PEG lipids), wherein the ionizable lipids account for 45 mol% to 55 mol% of the total lipids present in the LNPs, the accessory lipids (e.g., DSPC) account for 8 mol% to 25 mol% of the total lipids present in the LNPs, the structural lipids (e.g., non-phytosterols (e.g., cholesterol)) account for 20 mol% to 45 mol% of the total lipids present in the LNPs, and the polymeric lipids (e.g., PEG lipids) account for 0.1 mol% to 0.6 mol% of the total lipids present in the LNPs.
  • accessory lipids e.g.,
  • LNPs comprise: ionizable lipids, accessory lipids (e.g., DSPC), structural lipids (e.g., non-phytosterols (e.g., cholesterol)), and polymeric lipids (e.g., PEG lipids), wherein the ionizable lipids comprise 45 mol% to 55 mol% of the total lipids present in the LNPs, the accessory lipids (e.g., DSPC) comprise 8 mol% to 25 mol% of the total lipids present in the LNPs, the structural lipids (e.g., non-phytosterols (e.g., cholesterol)) comprise 20 mol% to 45 mol% of the total lipids present in the LNPs, and the polymeric lipids (e.g., PEG lipids) comprise 0.1 mol% to 0.6 mol% of the total lipids present in the LNPs, and not 0.1 mol%, 0.15 mol
  • LNPs comprise: ionizable lipids, accessory lipids (e.g., DSPC), structural lipids (e.g., non-phytosterols (e.g., cholesterol)), and polymeric lipids (e.g., PEG lipids), wherein the ionizable lipids account for 45 mol% to 55 mol% of the total lipids present in the LNPs, the accessory lipids (e.g., DSPC) account for 8 mol% to 25 mol% of the total lipids present in the LNPs, the structural lipids (e.g., non-phytosterols (e.g., cholesterol)) account for 20 mol% to 45 mol% of the total lipids present in the LNPs, and the polymeric lipids (e.g., PEG lipids) account for 0.2 mol% to 0.5 mol% of the total lipids present in the LNPs.
  • accessory lipids e.g.,
  • LNPs comprise: ionizable lipids, accessory lipids (e.g., DSPC), structural lipids (e.g., non-phytosterols (e.g., cholesterol)), and polymeric lipids (e.g., PEG lipids), wherein the ionizable lipids comprise 45 mol% to 55 mol% of the total lipids present in the LNPs, the accessory lipids (e.g., DSPC) comprise 8 mol% to 25 mol% of the total lipids present in the LNPs, the structural lipids (e.g., non-phytosterols (e.g., cholesterol)) comprise 20 mol% to 45 mol% of the total lipids present in the LNPs, and the polymeric lipids (e.g., PEG lipids) comprise 0.2 mol% to 0.5 mol% of the total lipids present in the LNPs, and not 0.2 mol%, 0.25 mol
  • the LNPs comprise: ionizable lipids, accessory lipids (e.g., DSPC), structural lipids (e.g., non-phytosterols (e.g., cholesterol)), and polymeric lipids (e.g., PEG lipids), wherein the ionizable lipids account for 47 mol% to 55 mol% of the total lipids present in the LNPs, the accessory lipids (e.g., DSPC) account for 10 mol% to 18 mol% of the total lipids present in the LNPs, the structural lipids (e.g., non-phytosterols (e.g., cholesterol)) account for 27 mol% to 42 mol% of the total lipids present in the LNPs, and the polymeric lipids (e.g., PEG lipids) account for 0.05 mol% to 0.8 mol%, 0.05 mol% to 0.7 mol%, 0.05 mol% to
  • LNPs comprise: ionizable lipids, accessory lipids (e.g., DSPC), structural lipids (e.g., non-phytosterols (e.g., cholesterol)), and polymeric lipids (e.g., PEG lipids), wherein the ionizable lipids account for 47 mol% to 55 mol% of the total lipids present in the LNPs, the accessory lipids (e.g., DSPC) account for 10 mol% to 18 mol% of the total lipids present in the LNPs, the structural lipids (e.g., non-phytosterols (e.g., cholesterol)) account for 27 mol% to 42 mol% of the total lipids present in the LNPs, and the polymeric lipids (e.g., PEG lipids) account for [a certain percentage] of the total lipids present in the LNPs.
  • accessory lipids e.g., DSPC
  • the content of the substance is 0.05 mol% to 0.8 mol%, 0.05 mol% to 0.7 mol%, 0.05 mol% to 0.6 mol%, 0.1 mol% to 0.6 mol%, or 0.2 mol% to 0.5 mol%, and is not 0.05 mol%, 0.07 mol%, 0.1 mol%, 0.15 mol%, 0.2 mol%, 0.25 mol%, 0.3 mol%, 0.35 mol%, 0.4 mol%, 0.45 mol%, 0.5 mol%, 0.6 mol%, 0.7 mol%, 0.75 mol%, and 0.8 mol%.
  • LNPs comprise: ionizable lipids, accessory lipids (e.g., DSPC), structural lipids (e.g., non-phytosterols (e.g., cholesterol)), and polymeric lipids (e.g., PEG lipids), wherein the ionizable lipids account for 47 mol% to 55 mol% of the total lipids present in the LNPs, the accessory lipids (e.g., DSPC) account for 10 mol% to 18 mol% of the total lipids present in the LNPs, the structural lipids (e.g., non-phytosterols (e.g., cholesterol)) account for 27 mol% to 42 mol% of the total lipids present in the LNPs, and the polymeric lipids (e.g., PEG lipids) account for 0.1 mol% to 0.6 mol% of the total lipids present in the LNPs.
  • accessory lipids e.g.,
  • LNPs comprise: ionizable lipids, accessory lipids (e.g., DSPC), structural lipids (e.g., non-phytosterols (e.g., cholesterol)), and polymeric lipids (e.g., PEG lipids), wherein the ionizable lipids comprise 47 mol% to 55 mol% of the total lipids present in the LNPs, the accessory lipids (e.g., DSPC) comprise 10 mol% to 18 mol% of the total lipids present in the LNPs, the structural lipids (e.g., non-phytosterols (e.g., cholesterol)) comprise 27 mol% to 42 mol% of the total lipids present in the LNPs, and the polymeric lipids (e.g., PEG lipids) comprise 0.1 mol% to 0.6 mol% of the total lipids present in the LNPs, and not 0.1 mol%, 0.15 mol
  • LNPs comprise: ionizable lipids, accessory lipids (e.g., DSPC), structural lipids (e.g., non-phytosterols (e.g., cholesterol)), and polymeric lipids (e.g., PEG lipids), wherein the ionizable lipids account for 47 mol% to 55 mol% of the total lipids present in the LNPs, the accessory lipids (e.g., DSPC) account for 10 mol% to 18 mol% of the total lipids present in the LNPs, the structural lipids (e.g., non-phytosterols (e.g., cholesterol)) account for 27 mol% to 42 mol% of the total lipids present in the LNPs, and the polymeric lipids (e.g., PEG lipids) account for 0.2 mol% to 0.5 mol% of the total lipids present in the LNPs.
  • accessory lipids e.g.,
  • LNPs comprise: ionizable lipids, accessory lipids (e.g., DSPC), structural lipids (e.g., non-phytosterols (e.g., cholesterol)), and polymeric lipids (e.g., PEG lipids), wherein the ionizable lipids comprise 47 mol% to 55 mol% of the total lipids present in the LNPs, the accessory lipids (e.g., DSPC) comprise 10 mol% to 18 mol% of the total lipids present in the LNPs, the structural lipids (e.g., non-phytosterols (e.g., cholesterol)) comprise 27 mol% to 42 mol% of the total lipids present in the LNPs, and the polymeric lipids (e.g., PEG lipids) comprise 0.2 mol% to 0.5 mol% of the total lipids present in the LNPs, and not 0.2 mol%, 0.25 mol
  • the particle size of the aforementioned LNPs is 120 nm to 1000 nm.
  • Examples include 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, 150 nm, 151 nm, 152 nm, 153 nm, 154 nm, 155 nm, 156 nm, 157 nm, 158 nm, 159 nm, 160 nm, 161 nm, 162 nm, 163 nm, 164 nm, 165 nm, 166 nm, 167 nm, 168 nm, 169 nm, 170 nm, 171 nm, 172 nm, 173 nm, 174 nm, 175 nm, 176 nm, 177 nm, 178 nm, 179 nm, 180 nm, 181 nm, and so on.
  • the particle sizes of the aforementioned LNPs are 120nm–800nm, 120nm–760nm, 120nm–700nm, 120nm–650nm, 120nm–600nm, 120nm–550nm, 120nm–500nm, 120nm–450nm, 120nm–400nm, 120nm–350nm, 120nm–300nm, 120nm–299.5nm, 125nm–299.5nm, 130nm–299.5nm, and 135nm–299.5nm.
  • the particle size of the aforementioned LNPs is 120nm–450nm, 135nm–450nm, 150nm–450nm, 160nm–450nm, 170nm–450nm, 180nm–450nm, 190nm–450nm, 200nm–450nm, 210nm–450nm, 220nm–450nm, 230nm–450nm, 240nm–450nm, 250nm–450nm, 120nm–400nm, 135nm–400nm, 150nm–400nm, 160nm–400nm, 170nm–400nm, or 180nm.
  • ⁇ 400nm 190nm ⁇ 400nm, 200nm ⁇ 400nm, 210nm ⁇ 400nm, 220nm ⁇ 450nm, 230nm ⁇ 400nm, 240nm ⁇ 400nm, 250nm ⁇ 400nm, 120nm ⁇ 380nm, 135nm ⁇ 380nm, 150nm ⁇ 380nm, 160nm ⁇ 380nm, 170nm ⁇ 380nm, 180nm ⁇ 380nm, 190nm ⁇ 380nm, 200nm ⁇ 380nm, 210nm ⁇ 380nm, 220nm ⁇ 380nm, 120nm ⁇ 360nm, 135nm ⁇ 360nm, 150nm ⁇ 360nm, 160nm ⁇ 360nm, 170nm ⁇ 360nm, 180nm ⁇ 360nm, 190nm ⁇ 360nm, 200nm ⁇ 360nm, 210nm ⁇ 360nm, 220nm ⁇ 360nm, 120nm ⁇ 340nm, 135nm ⁇ 340n
  • the particle size of the above-mentioned LNPs is 140nm-450nm, 140nm-400nm, 140nm-390nm, 140nm-380nm, 140nm-370nm, 140nm-360nm, 140nm-350nm, 140nm-340nm, 140nm-330nm, 140nm-320nm, 140nm-310nm, 140nm-300nm, 140nm-290nm, 140nm-280nm, 140nm-270nm, 140nm-260nm, 140nm-250nm, 140nm-240nm, 140nm-230nm, 140nm-230nm, 140nm-210nm, or 140nm-200nm.
  • the particle size of the aforementioned LNPs is 150nm–450nm, 150nm–400nm, 150nm–390nm, 150nm–380nm, 150nm–370nm, 150nm–360nm, 150nm–350nm, 150nm–340nm, 150nm–330nm, 150nm–320nm, 150nm–310nm, 150nm–300nm, 150nm–290nm, 150nm–280nm, 150nm–270nm, 150nm–260nm, 150nm– 250nm, 150nm ⁇ 240nm, 150nm ⁇ 230nm, 150nm ⁇ 210nm, 150nm ⁇ 200nm, 160nm ⁇ 320nm, 160nm ⁇ 310nm, 160nm ⁇ 300nm, 160nm ⁇ 290nm, 160nm ⁇ 2 80nm, 160nm ⁇ 270nm, 160nm ⁇ 260nm, 160nm ⁇ 250nm, 160nm ⁇ 2 80nm
  • the particle sizes of the aforementioned LNPs are 151nm–229nm, 161nm–229nm, 171nm–229nm, 181nm–229nm, 191nm–229nm, 201nm–229nm, 211nm–229nm, 221nm–229nm, 151nm–219nm, 161nm–219nm, 171nm–219nm, 181nm–219nm, 19 1nm ⁇ 219nm, 201nm ⁇ 219nm, 211nm ⁇ 219nm, 151nm ⁇ 209nm, 161nm ⁇ 209nm, 171nm ⁇ 209nm, 181nm ⁇ 209nm, 191nm ⁇ 209nm, 201nm ⁇ 209nm, 151nm ⁇ 199nm, 161nm ⁇ 199nm, 171nm ⁇ 199nm, 181nm ⁇
  • the particle size of the LNPs is 135 nm to 420 nm. In some embodiments, the particle size of the LNPs is 135 nm to 400 nm. In some embodiments, the particle size of the LNPs is 135 nm to 380 nm. In some embodiments, the particle size of the LNPs is 135 nm to 350 nm. In some embodiments, the particle size of the LNPs is 135 nm to 320 nm. In some embodiments, the particle size of the LNPs is 135 nm to 300 nm. In some embodiments, the particle size of the LNPs is 135 nm to 290 nm.
  • the particle size of the LNPs is 135 nm to 280 nm. In some embodiments, the particle size of the LNPs is 135 nm to 270 nm. In some embodiments, the particle size of the LNPs is 135 nm to 260 nm. In some embodiments, the particle size of the LNPs is 135 nm to 250 nm. In some embodiments, the particle size of the LNPs is 135 nm to 240 nm. In some embodiments, the particle size of the LNPs is 135 nm to 230 nm. In some embodiments, the particle size of the LNPs is 135 nm to 220 nm.
  • the particle size of the LNPs is 135 nm to 200 nm. In some embodiments, the particle size of the LNPs is 135 nm to 190 nm. In some embodiments, the particle size of the LNPs is 135 nm to 180 nm.
  • the particle size of the LNPs is 150 nm to 420 nm. In some embodiments, the particle size of the LNPs is 150 nm to 400 nm. In some embodiments, the particle size of the LNPs is 150 nm to 380 nm. In some embodiments, the particle size of the LNPs is 150 nm to 350 nm. In some embodiments, the particle size of the LNPs is 150 nm to 320 nm. In some embodiments, the particle size of the LNPs is 150 nm to 300 nm. In some embodiments, the particle size of the LNPs is 150 nm to 290 nm.
  • the particle size of the LNPs is 150 nm to 280 nm. In some embodiments, the particle size of the LNPs is 150 nm to 270 nm. In some embodiments, the particle size of the LNPs is 150 nm to 260 nm. In some embodiments, the particle size of the LNPs is 150 nm to 250 nm. In some embodiments, the particle size of the LNPs is 150 nm to 240 nm. In some embodiments, the particle size of the LNPs is 150 nm to 230 nm. In some embodiments, the particle size of the LNPs is 150 nm to 220 nm.
  • the particle size of the LNPs is 150 nm to 210 nm. In some embodiments, the particle size of the LNPs is 150 nm to 200 nm. In some embodiments, the particle size of the LNPs is 150 nm to 190 nm.
  • the particle size of the LNPs is 160 nm to 420 nm. In some embodiments, the particle size of the LNPs is 160 nm to 400 nm. In some embodiments, the particle size of the LNPs is 160 nm to 380 nm. In some embodiments, the particle size of the LNPs is 160 nm to 350 nm. In some embodiments, the particle size of the LNPs is 160 nm to 320 nm. In some embodiments, the particle size of the LNPs is 160 nm to 300 nm. In some embodiments, the particle size of the LNPs is 160 nm to 290 nm.
  • the particle size of the LNPs is 160 nm to 280 nm. In some embodiments, the particle size of the LNPs is 160 nm to 270 nm. In some embodiments, the particle size of the LNPs is 160 nm to 260 nm. In some embodiments, the particle size of the LNPs is 160 nm to 250 nm. In some embodiments, the particle size of the LNPs is 160 nm to 240 nm. In some embodiments, the particle size of the LNPs is 160 nm to 230 nm. In some embodiments, the particle size of the LNPs is 160 nm to 220 nm.
  • the particle size of the LNPs is 160 nm to 210 nm. In some embodiments, the particle size of the LNPs is 160 nm to 200 nm. In some embodiments, the particle size of the LNPs is 160 nm to 190 nm.
  • the particle size of the LNPs is 170 nm to 420 nm. In some embodiments, the particle size of the LNPs is 170 nm to 400 nm. In some embodiments, the particle size of the LNPs is 170 nm to 380 nm. In some embodiments, the particle size of the LNPs is 170 nm to 350 nm. In some embodiments, the particle size of the LNPs is 170 nm to 320 nm. In some embodiments, the particle size of the LNPs is 170 nm to 300 nm. In some embodiments, the particle size of the LNPs is 170 nm to 280 nm.
  • the particle size of the LNPs is 170 nm to 260 nm. In some embodiments, the particle size of the LNPs is 170 nm to 250 nm. In some embodiments, the particle size of the LNPs is 170 nm to 240 nm. In some embodiments, the particle size of the LNPs is 170 nm to 230 nm. In some embodiments, the particle size of the LNPs is 170 nm to 220 nm. In some embodiments, the particle size of the LNPs is 170 nm to 220 nm. In some embodiments, the particle size of the LNPs is 170 nm to 210 nm. In some embodiments, the particle size of the LNPs is 170 nm to 200 nm. In some embodiments, the particle size of the LNPs is 170 nm to 190 nm.
  • the particle size of the LNPs is 180 nm to 420 nm. In some embodiments, the particle size of the LNPs is 180 nm to 400 nm. In some embodiments, the particle size of the LNPs is 180 nm to 380 nm. In some embodiments, the particle size of the LNPs is 180 nm to 350 nm. In some embodiments, the particle size of the LNPs is 180 nm to 320 nm. In some embodiments, the particle size of the LNPs is 180 nm to 300 nm. In some embodiments, the particle size of the LNPs is 180 nm to 280 nm.
  • the particle size of the LNPs is 180 nm to 260 nm. In some embodiments, the particle size of the LNPs is 180 nm to 250 nm. In some embodiments, the particle size of the LNPs is 180 nm to 240 nm. In some embodiments, the particle size of the LNPs is 180 nm to 230 nm. In some embodiments, the particle size of the LNPs is 180 nm to 220 nm. In some embodiments, the particle size of the LNPs is 180 nm to 220 nm. In some embodiments, the particle size of the LNPs is 180 nm to 210 nm. In some embodiments, the particle size of the LNPs is 180 nm to 200 nm. In some embodiments, the particle size of the LNPs is 180 nm to 190 nm.
  • the particle size of the LNPs is not necessarily one or more of the following: 150nm, 175nm, 200nm, 225nm, 250nm, 275nm, 300nm, 350nm, 400nm, 450nm, 500nm, 550nm, 600nm, 650nm, 700nm, 750nm, 800nm, 850nm, 900nm, 950nm, and 1000nm.
  • the particle size of the LNPs is 120nm to 500nm and is not one or more of the following: 150nm, 175nm, 200nm, 225nm, 250nm, 275nm, 300nm, 350nm, 400nm, 450nm, and 500nm.
  • the particle size of the LNPs is 120 nm to 300 nm and is not one or more of the following: 150 nm, 175 nm, 200 nm, 225 nm, 250 nm, 275 nm, and 300 nm.
  • the particle size of the LNPs is 120 nm to 280 nm and is not one or more of the following: 150 nm, 175 nm, 200 nm, 225 nm, 250 nm, and 275 nm.
  • the particle size of the LNPs is 150 nm to 250 nm and is not one or more of the following: 150 nm, 175 nm, 200 nm, 225 nm, and 250 nm.
  • the particle size of the LNPs is 150 nm to 230 nm and is not one or more of the following: 150 nm, 175 nm, 200 nm, and 225 nm.
  • the particle size of the LNPs is not 150nm, 175nm, 200nm, 225nm, 250nm, 275nm, 300nm, 350nm, 400nm, 450nm, 500nm, 550nm, 600nm, 650nm, 700nm, 750nm, 800nm, 850nm, 900nm, 950nm, or 1000nm.
  • the particle size of the LNPs is 120 nm to 300 nm and is not 150 nm, 175 nm, 200 nm, 225 nm, 250 nm, 275 nm, or 300 nm. In some embodiments, the particle size of the LNPs is 120 nm to 290 nm and is not 150 nm, 175 nm, 200 nm, 225 nm, 250 nm, or 275 nm. In some embodiments, the particle size of the LNPs is 120 nm to 280 nm and is not 150 nm, 175 nm, 200 nm, 225 nm, 250 nm, or 275 nm.
  • the particle size of the LNPs is 120 nm to 270 nm and is not 150 nm, 175 nm, 200 nm, 225 nm, or 250 nm. In some embodiments, the particle size of the LNPs is 120 nm to 260 nm and is not 150 nm, 175 nm, 200 nm, 225 nm, or 250 nm. In some embodiments, the particle size of the LNPs is 120 nm to 250 nm and is not 150 nm, 175 nm, 200 nm, 225 nm, or 250 nm.
  • the particle size of the LNPs is 120 nm to 240 nm and is not 150 nm, 175 nm, 200 nm, or 225 nm. In some embodiments, the particle size of the LNPs is 120 nm to 230 nm and is not 150 nm, 175 nm, 200 nm, or 225 nm. In some embodiments, the particle size of the LNPs is 120 nm to 220 nm and is not 150 nm, 175 nm, or 200 nm. In some embodiments, the particle size of the LNPs is 120 nm to 210 nm and not 150 nm, 175 nm, or 200 nm.
  • the particle size of the LNPs is 120 nm to 200 nm and not 150 nm, 175 nm, or 200 nm. In some embodiments, the particle size of the LNPs is 120 nm to 190 nm and not 150 nm or 175 nm. In some embodiments, the particle size of the LNPs is 120 nm to 180 nm and not 150 nm or 175 nm.
  • the particle size of the LNPs is 135 nm to 300 nm and is not 150 nm, 175 nm, 200 nm, 225 nm, 250 nm, 275 nm, or 300 nm. In some embodiments, the particle size of the LNPs is 135 nm to 290 nm and is not 150 nm, 175 nm, 200 nm, 225 nm, 250 nm, or 275 nm. In some embodiments, the particle size of the LNPs is 135 nm to 280 nm and is not 150 nm, 175 nm, 200 nm, 225 nm, 250 nm, or 275 nm.
  • the particle size of the LNPs is 135 nm to 270 nm and is not 150 nm, 175 nm, 200 nm, 225 nm, or 250 nm. In some embodiments, the particle size of the LNPs is 135 nm to 260 nm and is not 150 nm, 175 nm, 200 nm, 225 nm, or 250 nm. In some embodiments, the particle size of the LNPs is 135 nm to 250 nm and is not 150 nm, 175 nm, 200 nm, 225 nm, or 250 nm.
  • the particle size of the LNPs is 135 nm to 240 nm and is not 150 nm, 175 nm, 200 nm, or 225 nm. In some embodiments, the particle size of the LNPs is 135 nm to 230 nm and is not 150 nm, 175 nm, 200 nm, or 225 nm. In some embodiments, the particle size of the LNPs is 135 nm to 220 nm and is not 150 nm, 175 nm, or 200 nm. In some embodiments, the particle size of the LNPs is 135 nm to 200 nm and not 150 nm, 175 nm, or 200 nm.
  • the particle size of the LNPs is 135 nm to 190 nm and not 150 nm or 175 nm. In some embodiments, the particle size of the LNPs is 135 nm to 180 nm and not 150 nm or 175 nm.
  • the particle size of the LNPs is 140 nm to 500 nm and is not 140 nm, 145 nm, 150 nm, 175 nm, 200 nm, 225 nm, 250 nm, 275 nm, 300 nm, 350 nm, 400 nm, 450 nm, or 500 nm. In some embodiments, the particle size of the LNPs is 140 nm to 450 nm and is not 140 nm, 145 nm, 150 nm, 175 nm, 200 nm, 225 nm, 250 nm, 275 nm, 300 nm, 350 nm, 400 nm, or 450 nm.
  • the particle size of the LNPs is 140 nm to 400 nm and is not 140 nm, 145 nm, 150 nm, 175 nm, 200 nm, 225 nm, 250 nm, 275 nm, 300 nm, 350 nm, or 400 nm. In some embodiments, the particle size of the LNPs is 140 nm to 390 nm and is not 140 nm, 145 nm, 150 nm, 175 nm, 200 nm, 225 nm, 250 nm, 275 nm, 300 nm, or 350 nm.
  • the particle size of the LNPs is 140 nm to 350 nm and is not 140 nm, 145 nm, 150 nm, 175 nm, 200 nm, 225 nm, 250 nm, 275 nm, 300 nm, or 350 nm. In some embodiments, the particle size of the LNPs is 140 nm to 300 nm, and not 140 nm, 145 nm, 150 nm, 175 nm, 200 nm, 225 nm, 250 nm, 275 nm, or 300 nm.
  • the particle size of the LNPs is 140 nm to 290 nm, and not 140 nm, 145 nm, 150 nm, 175 nm, 200 nm, 225 nm, 250 nm, or 275 nm. In some embodiments, the particle size of the LNPs is 140 nm to 280 nm, and not 140 nm, 145 nm, 150 nm, 175 nm, 200 nm, 225 nm, 250 nm, or 275 nm.
  • the particle size of the LNPs is 140 nm to 270 nm and is not 140 nm, 145 nm, 150 nm, 175 nm, 200 nm, 225 nm, or 250 nm. In some embodiments, the particle size of the LNPs is 140 nm to 260 nm and is not 140 nm, 145 nm, 150 nm, 175 nm, 200 nm, 225 nm, or 250 nm.
  • the particle size of the LNPs is 140 nm to 250 nm and is not 140 nm, 145 nm, 150 nm, 175 nm, 200 nm, 225 nm, or 250 nm. In some embodiments, the particle size of the LNPs is 140 nm to 240 nm and is not 140 nm, 145 nm, 150 nm, 175 nm, 200 nm, or 225 nm. In some embodiments, the particle size of the LNPs is 140 nm to 230 nm and is not 140 nm, 145 nm, 150 nm, 175 nm, 200 nm, or 225 nm.
  • the particle size of the LNPs is 140 nm to 220 nm and is not 140 nm, 145 nm, 150 nm, 175 nm, or 200 nm. In some embodiments, the particle size of the LNPs is 140 nm to 200 nm and is not 140 nm, 145 nm, 150 nm, 175 nm, or 200 nm. In some embodiments, the particle size of the LNPs is 140 nm to 190 nm and is not 140 nm, 145 nm, 150 nm, or 175 nm. In some embodiments, the particle size of the LNPs is 140 nm to 180 nm and is not 140 nm, 145 nm, 150 nm, or 175 nm.
  • the particle size of the LNPs is 150nm to 500nm and is not 150nm, 175nm, 200nm, 225nm, 250nm, 275nm, 300nm, 350nm, 400nm, 450nm and 500nm.
  • the particle size of the LNPs is 150 nm to 450 nm and is not 150 nm, 175 nm, 200 nm, 225 nm, 250 nm, 275 nm, 300 nm, 350 nm, 400 nm, or 450 nm. In some embodiments, the particle size of the LNPs is 150 nm to 400 nm and is not 150 nm, 175 nm, 200 nm, 225 nm, 250 nm, 275 nm, 300 nm, 350 nm, or 400 nm.
  • the particle size of the LNPs is 150 nm to 350 nm and is not 150 nm, 175 nm, 200 nm, 225 nm, 250 nm, 275 nm, 300 nm, or 350 nm. In some embodiments, the particle size of the LNPs is 150 nm to 320 nm and is not 150 nm, 175 nm, 200 nm, 225 nm, 250 nm, 275 nm, or 300 nm.
  • the particle size of the LNPs is 150 nm to 300 nm and is not 150 nm, 175 nm, 200 nm, 225 nm, 250 nm, 275 nm, or 300 nm. In some embodiments, the particle size of the LNPs is 150 nm to 290 nm and is not 150 nm, 175 nm, 200 nm, 225 nm, 250 nm, or 275 nm. In some embodiments, the particle size of the LNPs is 150 nm to 280 nm and is not 150 nm, 175 nm, 200 nm, 225 nm, 250 nm, or 275 nm.
  • the particle size of the LNPs is 150 nm to 270 nm, and not 150 nm, 175 nm, 200 nm, 225 nm, or 250 nm. In some embodiments, the particle size of the LNPs is 150 nm to 260 nm, and not 150 nm, 175 nm, 200 nm, 225 nm, or 250 nm. In some embodiments, the particle size of the LNPs is 150 nm to 250 nm, and not 150 nm, 175 nm, 200 nm, 225 nm, or 250 nm.
  • the particle size of the LNPs is 150 nm to 240 nm, and not 150 nm, 175 nm, 200 nm, or 225 nm. In some embodiments, the particle size of the LNPs is 150 nm to 230 nm, and not 150 nm, 175 nm, 200 nm, or 225 nm. In some embodiments, the particle size of the LNPs is 150 nm to 220 nm and is not 150 nm, 175 nm, or 200 nm. In some embodiments, the particle size of the LNPs is 150 nm to 210 nm and is not 150 nm, 175 nm, or 200 nm.
  • the particle size of the LNPs is 150 nm to 200 nm and is not 150 nm, 175 nm, or 200 nm. In some embodiments, the particle size of the LNPs is 150 nm to 190 nm and is not 150 nm or 175 nm. In some embodiments, the particle size of the LNPs is 150 nm to 180 nm and is not 150 nm or 175 nm.
  • the particle size of the LNPs is 160 nm to 450 nm and is not 175 nm, 200 nm, 225 nm, 250 nm, 275 nm, 300 nm, 350 nm, 400 nm, or 450 nm. In some embodiments, the particle size of the LNPs is 160 nm to 400 nm and is not 175 nm, 200 nm, 225 nm, 250 nm, 275 nm, 300 nm, 350 nm, or 400 nm.
  • the particle size of the LNPs is 160 nm to 350 nm and is not 175 nm, 200 nm, 225 nm, 250 nm, 275 nm, 300 nm, or 350 nm. In some embodiments, the particle size of the LNPs is 160 nm to 320 nm and is not 175 nm, 200 nm, 225 nm, 250 nm, 275 nm, or 300 nm. In some embodiments, the particle size of the LNPs is 160 nm to 300 nm and not 175 nm, 200 nm, 225 nm, 250 nm, 275 nm, or 300 nm.
  • the particle size of the LNPs is 160 nm to 290 nm and not 175 nm, 200 nm, 225 nm, 250 nm, or 275 nm. In some embodiments, the particle size of the LNPs is 160 nm to 280 nm and not 175 nm, 200 nm, 225 nm, 250 nm, or 275 nm. In some embodiments, the particle size of the LNPs is 160 nm to 270 nm and not 175 nm, 200 nm, 225 nm, or 250 nm.
  • the particle size of the LNPs is 160 nm to 260 nm and not 175 nm, 200 nm, 225 nm, or 250 nm. In some embodiments, the particle size of the LNPs is 160 nm to 250 nm and not 175 nm, 200 nm, 225 nm, or 250 nm. In some embodiments, the particle size of the LNPs is 160 nm to 240 nm and not 175 nm, 200 nm, or 225 nm. In some embodiments, the particle size of the LNPs is 160 nm to 230 nm and not 175 nm, 200 nm, or 225 nm.
  • the particle size of the LNPs is 160 nm to 220 nm and not 175 nm or 200 nm. In some embodiments, the particle size of the LNPs is 160 nm to 210 nm and not 175 nm or 200 nm. In some embodiments, the particle size of the LNPs is 160 nm to 200 nm and not 175 nm or 200 nm. In some embodiments, the particle size of the LNPs is 160 nm to 190 nm and not 175 nm. In some embodiments, the particle size of the LNPs is 160 nm to 180 nm and not 175 nm.
  • the particle size of the LNPs is 170 nm to 450 nm and is not 175 nm, 200 nm, 225 nm, 250 nm, 275 nm, 300 nm, 350 nm, 400 nm, or 450 nm. In some embodiments, the particle size of the LNPs is 170 nm to 400 nm and is not 175 nm, 200 nm, 225 nm, 250 nm, 275 nm, 300 nm, 350 nm, or 400 nm.
  • the particle size of the LNPs is 170 nm to 350 nm and is not 175 nm, 200 nm, 225 nm, 250 nm, 275 nm, 300 nm, or 350 nm. In some embodiments, the particle size of the LNPs is 170 nm to 320 nm and is not 175 nm, 200 nm, 225 nm, 250 nm, 275 nm, or 300 nm. In some embodiments, the particle size of the LNPs is 170 nm to 300 nm and not 175 nm, 200 nm, 225 nm, 250 nm, 275 nm, or 300 nm.
  • the particle size of the LNPs is 170 nm to 290 nm and not 175 nm, 200 nm, 225 nm, 250 nm, or 275 nm. In some embodiments, the particle size of the LNPs is 170 nm to 280 nm and not 175 nm, 200 nm, 225 nm, 250 nm, or 275 nm. In some embodiments, the particle size of the LNPs is 170 nm to 270 nm and not 175 nm, 200 nm, 225 nm, or 250 nm.
  • the particle size of the LNPs is 170 nm to 260 nm and not 175 nm, 200 nm, 225 nm, or 250 nm. In some embodiments, the particle size of the LNPs is 170 nm to 250 nm and not 175 nm, 200 nm, 225 nm, or 250 nm. In some embodiments, the particle size of the LNPs is 170 nm to 240 nm and not 175 nm, 200 nm, or 225 nm. In some embodiments, the particle size of the LNPs is 170 nm to 230 nm and not 175 nm, 200 nm, or 225 nm.
  • the particle size of the LNPs is 170 nm to 220 nm and not 175 nm or 200 nm. In some embodiments, the particle size of the LNPs is 170 nm to 210 nm and not 175 nm or 200 nm. In some embodiments, the particle size of the LNPs is 170 nm to 200 nm and not 175 nm or 200 nm. In some embodiments, the particle size of the LNPs is 170 nm to 190 nm and not 175 nm. In some embodiments, the particle size of the LNPs is 170 nm to 180 nm and not 175 nm.
  • the particle size of the LNPs is greater than 140 nm and less than 145 nm or greater than 145 nm and less than 150 nm.
  • the particle size of the LNPs is: greater than 120 nm and less than 150 nm, greater than 150 nm and less than 175 nm, greater than 175 nm and less than 200 nm, greater than 200 nm and less than 225 nm, greater than 225 nm and less than 250 nm, greater than 250 nm and less than 275 nm, greater than 275 nm and less than 300 nm, greater than 300 nm and less than 350 nm, greater than 350 nm and less than 400 nm, greater than 400 nm.
  • nm and less than 450nm greater than 450nm and less than 500nm, greater than 500nm and less than 550nm, greater than 550nm and less than 600nm, greater than 600nm and less than 650nm, greater than 650nm and less than 700nm, greater than 700nm and less than 750nm, greater than 750nm and less than 800nm, greater than 800nm and less than 850nm, greater than 850nm and less than 900nm, or greater than 900nm and less than 950nm.
  • the particle size of the LNPs is 140.2 nm to 144.9 nm or 145.5 nm to 149.9 nm.
  • the particle size of the LNPs is: 120.5nm ⁇ 149.9nm, 150.5nm ⁇ 174.9nm, 175.5nm ⁇ 199.9nm, 200.5nm ⁇ 224.5nm, 225.5nm ⁇ 249.9nm, 250.5nm ⁇ 274.9nm, or 275.5nm ⁇ 299.9nm.
  • the particle size of the LNPs is: 125nm ⁇ 149nm, 152nm ⁇ 174nm, 178nm ⁇ 199nm, 201nm ⁇ 224nm, 226nm ⁇ 245nm, 252nm ⁇ 272nm or 276nm ⁇ 299nm.
  • the particle size of the LNPs is: 125nm-145nm, 155nm-170nm, 180nm-195nm, 205nm-220nm, 230nm-245nm, 255nm-270nm, or 280nm-295nm.
  • the particle size of the LNPs is greater than 140 nm and less than 145 nm.
  • the particle size of the LNPs is greater than 145 nm and less than 150 nm.
  • the particle size of the LNPs is greater than 120 nm and less than 150 nm.
  • the particle size of the LNPs is greater than 150 nm and less than 175 nm.
  • the particle size of the LNPs is greater than 175 nm and less than 200 nm.
  • the particle size of the LNPs is greater than 200 nm and less than 225 nm.
  • the particle size of the LNPs is greater than 225 nm and less than 250 nm.
  • the particle size of the LNPs is greater than 250 nm and less than 275 nm.
  • the particle size of the LNPs is greater than 275 nm and less than 300 nm.
  • the particle size of the LNPs is greater than 300 nm and less than 350 nm.
  • the particle size of the LNPs is greater than 350 nm and less than 400 nm.
  • the particle size of the LNPs is greater than 400 nm and less than 450 nm.
  • the particle size of the LNPs is greater than 141 nm to 144 nm.
  • the particle size of the LNPs is greater than 146 nm to 149 nm.
  • the particle size of the LNPs is greater than 151 nm to 154 nm.
  • the particle size of the LNPs is 125 nm to 145 nm.
  • the particle size of the LNPs is 155 nm to 170 nm.
  • the particle size of the LNPs is 180 nm to 195 nm.
  • the particle size of the LNPs is 205 nm to 220 nm.
  • the particle size of the LNPs is 230 nm to 245 nm.
  • the particle size of the LNPs is 255 nm to 270 nm.
  • the particle size of the LNPs is 280 nm to 295 nm.
  • the particle size of the LNPs is 305 nm to 345 nm.
  • the particle size of the LNPs is 355 nm to 395 nm.
  • the particle size of the LNPs is 405 nm to 445 nm.
  • the LNPs comprise ionizable lipids, accessory lipids (e.g., DSPC), structural lipids (e.g., cholesterol), and PEG lipids, wherein the polymer lipids (e.g., PEG lipids) account for 0 mol% to 0.8 mol%, 0.0001 mol% to 0.8 mol%, 0.01 mol% to 0.8 mol%, 0.05 mol% to 0.8 mol%, 0.1 mol% to 0.8 mol%, and 0.2 mol% to 0.
  • accessory lipids e.g., DSPC
  • structural lipids e.g., cholesterol
  • PEG lipids e.g., PEG lipids
  • the LNPs have particle sizes of 140 nm–500 nm, 140 nm–450 nm, 140 nm–400 nm, 140 nm–390 nm, 140 n
  • the LNPs are available in concentrations of 1%, 0.0001 mol%–0.1 mol%, 0.01 mol%–0.1 mol%, 0.05 mol%, 0.05 mol% or 0.01 mol%–0.05 mol%. 140nm ⁇ 360nm, 140nm ⁇ 350nm, 140nm ⁇ 340nm, 140nm ⁇ 330nm, 140nm ⁇ 320nm, 140nm ⁇ 310nm, 140nm ⁇ 300nm, 140n m ⁇ 290nm, 140nm ⁇ 280nm, 140nm ⁇ 270nm, 140nm ⁇ 260nm, 140nm ⁇ 250nm, 140nm ⁇ 240nm, 140nm ⁇ 230nm, 140nm ⁇ 21 0nm, 140nm ⁇ 200nm, 150nm ⁇ 500nm, 150nm ⁇ 450nm, 150nm ⁇ 400nm, 150nm ⁇ 390nm, 150nm ⁇ 380nm, 150nm ⁇ 370nm, 150nm ⁇ 360nm, 150nm ⁇ 350nm
  • the LNPs comprise ionizable lipids, accessory lipids (e.g., DSPC), structural lipids (e.g., cholesterol), and PEG lipids, wherein the polymeric lipids (e.g., PEG lipids) account for 0.0001 mol%–0.8 mol%, 0.001 mol%–0.8 mol%, 0.01 mol%–0.8 mol%, 0.01 mol%–0.01 mol%–0.7 mol%, 0.01 mol%–0.6 mol%, 0.01 mol%–0.5 mol%, 0.01 mol%–0.4 mol%, 0.01 mol%–0.3 mol%, and 0.01 mol%–0.2 mol% of the total lipids present in the LNPs.
  • accessory lipids e.g., DSPC
  • structural lipids e.g., cholesterol
  • PEG lipids e.g., PEG lipids
  • the LNPs comprise ionizable lipids, accessory lipids (e.g., DSPC), structural lipids (e.g., cholesterol), and PEG lipids, wherein the polymeric lipids (e.g., PEG lipids) account for 0.01 mol%–0.8 mol%, 0.01 mol%–0.6 mol%, 0.01 mol%–0.5 mol%, 0.01 mol%–0.3 mol%, 0.01 mol%–0.2 mol%, 0.01 mol%–0.15 mol%, 0.05 mol%–0.8 mol%, 0.05 mol%–0.6 mol%, 0.05 mol%–0.55 mol%, and 0% of the total lipids present in the LNPs.
  • accessory lipids e.g., DSPC
  • structural lipids e.g., cholesterol
  • PEG lipids e.g., PEG lipids
  • the particle sizes of the above LNPs are 140 nm to 400 nm, ranging from 0.05 mol% to 0.5 mol%, 0.05 mol% to 0.4 mol%, 0.05 mol% to 0.2 mol%, 0.05 mol% to 0.15 mol%, 0.1 mol% to 0.8 mol%, 0.1 mol% to 0.7 mol%, 0.1 mol% to 0.6 mol%, 0.1 mol% to 0.5 mol%, 0.1 mol% to 0.4 mol%, 0.15 mol% to 0.55 mol%, 0.15 mol% to 0.4 mol%, 0.2 mol% to 0.5 mol%, or 0.2 mol% to 0.4 mol%.
  • LNPs comprise: ionizable lipids, accessory lipids (e.g., DSPC), structural lipids (e.g., non-phytosterols (e.g., cholesterol)), and polymeric lipids (e.g., PEG lipids), wherein the ionizable lipids account for 30 mol% to 60 mol% of the total lipids present in the LNPs, the accessory lipids (e.g., DSPC) account for 0 mol% to 30 mol% of the total lipids present in the LNPs, and the structural lipids (e.g., non-phytosterols (e.g., cholesterol)) account for 10 mol% to 60 mol% of the total lipids present in the LNPs.
  • accessory lipids e.g., DSPC
  • structural lipids e.g., non-phytosterols (e.g., cholesterol)
  • polymeric lipids e.g.,
  • 0 mol% of total lipids present in LNPs 0 mol%–0.8 mol%, 0.0001 mol%–0.8 mol%, 0.01 mol%–0.8 mol%, 0.05 mol%–0.8 mol%, 0.1 mol%–0.8 mol%, 0.2 mol%–0.8 mol%, 0.3 mol%–0.8 mol%, 0.4 mol%–0.8 mol%, 0.5 mol%–0.8 mol%, 0.05 mol%.
  • the LNPs have particle sizes of 140 nm to 500 nm and 140 nm to 450 nm, respectively, and are available in the following concentrations: 0.15 mol%, 0.01 mol% to 0.15 mol%, 0.05 mol% to 0.15 mol%, 0.1 mol% to 0.15 mol%, 0.0001 mol% to 0.005 mol%, 0.01 mol
  • LNPs comprise: ionizable lipids, accessory lipids (e.g., DSPC), structural lipids (e.g., non-phytosterols (e.g., cholesterol)), and polymeric lipids (e.g., PEG lipids), wherein the ionizable lipids account for 30 mol% to 60 mol% of the total lipids present in the LNPs, the accessory lipids (e.g., DSPC) account for 0 mol% to 30 mol% of the total lipids present in the LNPs, the structural lipids (e.g., non-phytosterols (e.g., cholesterol)) account for 10 mol% to 60 mol% of the total lipids present in the LNPs, and the polymeric lipids (e.g., PEG lipids) account for 0.0001 mol% to 0.8 mol%, 0.001 mol% to 0.8 mol%, and 0.001
  • LNPs comprise: ionizable lipids, accessory lipids (e.g., DSPC), structural lipids (e.g., non-phytosterols (e.g., cholesterol)), and polymeric lipids (e.g., PEG lipids), wherein the ionizable lipids account for 30 mol% to 60 mol% of the total lipids present in the LNPs, the accessory lipids (e.g., DSPC) account for 0 mol% to 30 mol% of the total lipids present in the LNPs, the structural lipids (e.g., non-phytosterols (e.g., cholesterol)) account for 10 mol% to 60 mol% of the total lipids present in the LNPs, and the polymeric lipids (e.g., PEG lipids) account for 0.01 mol% to 0.8 mol%, 0.01 mol% to 0.6 mol%, 0.01 mol% to
  • the LNPs have particle sizes of 0.2 mol% to 0.5 mol% or 0.2 mol% to 0.4 mol%, and particle sizes of 140 nm to 400 nm, 140 nm to 390 nm, 140 nm to 380 nm, 140 nm to 370 nm, 140 nm to 360 nm, 140 nm to 350 nm, 140 nm to 340 nm
  • 140nm ⁇ 200nm, 150nm ⁇ 400nm 150nm ⁇ 350nm, 150nm ⁇ 300nm, 150nm ⁇ 250nm, 150nm ⁇ 200nm, 160nm ⁇ 350nm, 160nm ⁇ 300nm, 160nm ⁇ 250nm, 160nm ⁇ 200nm, 180nm ⁇ 320nm, 180nm ⁇ 300nm, 180nm ⁇ 250nm, 180nm ⁇ 200nm, 190nm ⁇ 320nm, 190nm ⁇ 300nm, 190nm ⁇ 250nm, 190nm ⁇ 200nm, 200nm ⁇ 320nm, 200nm ⁇ 300nm, 200nm ⁇ 250nm or 200nm ⁇ 230nm.
  • LNPs comprise: ionizable lipids, accessory lipids (e.g., DSPC), structural lipids (e.g., non-phytosterols (e.g., cholesterol)), and polymeric lipids (e.g., PEG lipids), wherein the ionizable lipids constitute 45 mol% to 55 mol% of the total lipids present in the LNPs, the accessory lipids (e.g., DSPC) constitute 8 mol% to 25 mol% of the total lipids present in the LNPs, the structural lipids (e.g., non-phytosterols (e.g., cholesterol)) constitute 20 mol% to 45 mol% of the total lipids present in the LNPs, and the polymeric lipids (e.g., PEG lipids) constitute 0.0001 mol% to 0.8 mol%, 0.001 mol% to 0.8 mol%, 0.01 mol% to 0.8 mol
  • LNPs comprise: ionizable lipids, accessory lipids (e.g., DSPC), structural lipids (e.g., non-phytosterols (e.g., cholesterol)), and polymeric lipids (e.g., PEG lipids), wherein the ionizable lipids account for 45 mol% to 55 mol% of the total lipids present in the LNPs, the accessory lipids (e.g., DSPC) account for 8 mol% to 25 mol% of the total lipids present in the LNPs, the structural lipids (e.g., non-phytosterols (e.g., cholesterol)) account for 20 mol% to 45 mol% of the total lipids present in the LNPs, and the polymeric lipids (e.g., PEG lipids) account for 0.01 mol% to 0.8 mol%, 0.01 mol% to 0.6 mol%, 0.01 mol% to 0.3
  • the LNPs have particle sizes of 0.2 mol% to 0.5 mol% or 0.2 mol% to 0.4 mol%, and particle sizes of 140 nm to 400 nm, 140 nm to 390 nm, 140 nm to 380 nm, 140 nm to 370 nm, 140 nm to 360 nm, 140 nm to 350 nm, 140 nm to 340 nm
  • 140nm ⁇ 200nm, 150nm ⁇ 400nm 150nm ⁇ 350nm, 150nm ⁇ 300nm, 150nm ⁇ 250nm, 150nm ⁇ 200nm, 160nm ⁇ 350nm, 160nm ⁇ 300nm, 160nm ⁇ 250nm, 160nm ⁇ 200nm, 180nm ⁇ 320nm, 180nm ⁇ 300nm, 180nm ⁇ 250nm, 180nm ⁇ 200nm, 190nm ⁇ 320nm, 190nm ⁇ 300nm, 190nm ⁇ 250nm, 190nm ⁇ 200nm, 200nm ⁇ 320nm, 200nm ⁇ 300nm, 200nm ⁇ 250nm or 200nm ⁇ 230nm.
  • LNPs comprise: ionizable lipids, accessory lipids (e.g., DSPC), structural lipids (e.g., non-phytosterols (e.g., cholesterol)), and polymeric lipids (e.g., PEG lipids), wherein the ionizable lipids account for 47 mol% to 55 mol% of the total lipids present in the LNPs, the accessory lipids (e.g., DSPC) account for 10 mol% to 18 mol% of the total lipids present in the LNPs, the structural lipids (e.g., non-phytosterols (e.g., cholesterol)) account for 27 mol% to 42 mol% of the total lipids present in the LNPs, and the polymeric lipids (e.g., PEG lipids) account for 0.0001 mol% to 0.8 mol%, 0.001 mol% to 0.8 mol%, 0.01 mol% to
  • LNPs comprise: ionizable lipids, accessory lipids (e.g., DSPC), structural lipids (e.g., non-phytosterols (e.g., cholesterol)), and polymeric lipids (e.g., PEG lipids), wherein the ionizable lipids account for 47 mol% to 55 mol% of the total lipids present in the LNPs, the accessory lipids (e.g., DSPC) account for 10 mol% to 18 mol% of the total lipids present in the LNPs, the structural lipids (e.g., non-phytosterols (e.g., cholesterol)) account for 27 mol% to 42 mol% of the total lipids present in the LNPs, and the polymeric lipids (e.g., PEG lipids) account for 0.01 mol% to 0.8 mol%, 0.01 mol% to 0.6 mol%, 0.01 mol% to 0.3
  • the LNPs have particle sizes of 0.2 mol% to 0.5 mol% or 0.2 mol% to 0.4 mol%, and particle sizes of 140 nm to 400 nm, 140 nm to 390 nm, 140 nm to 380 nm, 140 nm to 370 nm, 140 nm to 360 nm, 140 nm to 350 nm, 140 nm to 340
  • 140nm ⁇ 200nm, 150nm ⁇ 400nm 150nm ⁇ 350nm, 150nm ⁇ 300nm, 150nm ⁇ 250nm, 150nm ⁇ 200nm, 160nm ⁇ 350nm, 160nm ⁇ 300nm, 160nm ⁇ 250nm, 160nm ⁇ 200nm, 180nm ⁇ 320nm, 180nm ⁇ 300nm, 180nm ⁇ 250nm, 180nm ⁇ 200nm, 190nm ⁇ 320nm, 190nm ⁇ 300nm, 190nm ⁇ 250nm, 190nm ⁇ 200nm, 200nm ⁇ 320nm, 200nm ⁇ 300nm, 200nm ⁇ 250nm or 200nm ⁇ 230nm.
  • the spleen is the largest lymphoid organ, containing a large number of immune cells, such as B cells (e.g., plasmablasts, plasma cells, lymphoplasmacytic cells, memory B cells, B-2 cells, regulatory B cells, etc.), T cells (e.g., cytotoxic T cells, helper T cells, regulatory T cells, naive T cells, effector T cells, memory T cells, etc.), dendritic cells, macrophages, monocytes, NK cells (e.g., immature NK cells, activated NK cells), and NKT cells.
  • B cells e.g., plasmablasts, plasma cells, lymphoplasmacytic cells, memory B cells, B-2 cells, regulatory B cells, etc.
  • T cells e.g., cytotoxic T cells, helper T cells, regulatory T cells, naive T cells, effector T cells, memory T cells, etc.
  • dendritic cells e.g., macrophages, monocytes, NK cells (
  • the aforementioned LNPs possess spleen or immune cell targeting properties. They can increase the content of active ingredients in the spleen or immune cells, increase the spleen/liver delivery ratio of active ingredients, and decrease the content of active ingredients in the liver. They can be used to prepare drugs or delivery vehicles that target the spleen or immune cells (e.g., drugs or delivery vehicles targeting the spleen or immune cells administered in vivo), thereby enabling their use in the prevention or treatment of diseases. For example, they can be used to prevent or treat diseases related to immune cells (e.g., autoimmune diseases, T-cell tumors, B-cell tumors, etc.), tumors or cancers, infectious diseases, etc.
  • diseases related to immune cells e.g., autoimmune diseases, T-cell tumors, B-cell tumors, etc.
  • the aforementioned LNPs possess spleen or immune cell targeting properties. They can increase the content of active ingredients in the spleen or immune cells, increase the spleen/liver delivery ratio of active ingredients, and decrease the content of active ingredients in the liver. They can be used to prepare drugs or delivery carriers that target the spleen or immune cells in vivo, thereby enabling the prevention or treatment of diseases. For example, they can be used to prevent or treat diseases related to immune cells (e.g., autoimmune diseases, T-cell tumors, B-cell tumors), tumors or cancers, and infectious diseases.
  • the delivery effect of the aforementioned LNPs on the active substance can be determined using methods known in the art. For example, the delivery effect of the aforementioned LNPs on the active substance can be determined by detecting the degree to which the aforementioned LNPs encapsulating mRNA encoding a reporter protein express the mRNA in vivo.
  • this disclosure also provides another type of lipid nanoparticle (LNPs) comprising ionizable lipids, auxiliary lipids, structural lipids and polymeric lipids, wherein the particle size of the LNPs is 120 nm to 1000 nm.
  • LNPs lipid nanoparticle
  • the particle size of the LNPs is as described in the section on lipid nanoparticles.
  • the particle size of the LNPs is 120nm–500nm, 130nm–500nm, 140nm–500nm, 140nm–450nm, 140nm–400nm, 140nm–390nm, 140nm–380nm, 140nm–370nm, 140nm–360nm, 140nm–350nm, 140nm–340nm, 140nm–330nm, 140nm–320nm, 140nm–310nm, 140nm– 300nm, 140nm ⁇ 290nm, 140nm ⁇ 280nm, 140nm ⁇ 270nm, 140nm ⁇ 260nm, 140nm ⁇ 250nm, 140nm ⁇ 240nm, 140nm ⁇ 230nm, 140n m ⁇ 210nm, 140nm ⁇ 200nm, 150nm ⁇ 500nm, 150nm ⁇ 450nm, 150nm ⁇ 400n
  • the particle size of the LNPs is 130 nm to 500 nm. In some embodiments, the particle size of the LNPs is 140 nm to 500 nm. In some embodiments, the particle size of the LNPs is 140 nm to 450 nm. In some embodiments, the particle size of the LNPs is 140 nm to 400 nm. In some embodiments, the particle size of the LNPs is 140 nm to 380 nm. In some embodiments, the particle size of the LNPs is 140 nm to 350 nm. In some embodiments, the particle size of the LNPs is 140 nm to 300 nm.
  • the particle size of the LNPs is 140 nm to 280 nm. In some embodiments, the particle size of the LNPs is 140 nm to 270 nm. In some embodiments, the particle size of the LNPs is 140 nm to 260 nm. In some embodiments, the particle size of the LNPs is 140 nm to 200 nm.
  • the particle size of the LNPs is 150 nm to 450 nm.
  • the particle size of the LNPs is 150 nm to 400 nm.
  • the particle size of the LNPs is 150 nm to 350 nm.
  • the particle size of the LNPs is 150 nm to 300 nm.
  • the particle size of the LNPs is 150 nm to 280 nm.
  • the particle size of the LNPs is 150 nm to 270 nm.
  • the particle size of the LNPs is 150 nm to 260 nm.
  • the particle size of the LNPs is 150 nm to 200 nm.
  • the types, contents, and ratios of the ionizable lipids, auxiliary lipids, structural lipids, and polymeric lipids in the above-mentioned LNPs are as described in the section on lipid nanoparticles.
  • the LNPs comprise ionizable lipids, accessory lipids (e.g., DSPC), structural lipids (e.g., non-phytosterols (e.g., cholesterol)), and polymeric lipids (e.g., PEG lipids), wherein the polymeric lipids comprise 0 mol% to 0.8 mol% of the total lipids present in the LNPs, and the particle size of the LNPs is as described in the section on lipid nanoparticles.
  • accessory lipids e.g., DSPC
  • structural lipids e.g., non-phytosterols (e.g., cholesterol)
  • polymeric lipids e.g., PEG lipids
  • the LNPs comprise ionizable lipids, accessory lipids (e.g., DSPC), structural lipids (e.g., non-phytosterols (e.g., cholesterol)), and PEG lipids (e.g., DMG-PEG), wherein the PEG lipids comprise 0 mol% to 0.8 mol% of the total lipids present in the LNPs, and the particle size of the LNPs is as described in the section on lipid nanoparticles.
  • accessory lipids e.g., DSPC
  • structural lipids e.g., non-phytosterols (e.g., cholesterol)
  • PEG lipids e.g., DMG-PEG
  • the LNPs comprise ionizable lipids, accessory lipids (e.g., DSPC), structural lipids (e.g., non-phytosterols (e.g., cholesterol)), and polymeric lipids (e.g., PEG lipids), wherein the ionizable lipids account for 30 mol% to 60 mol% of the total lipids present in the LNPs, the accessory lipids (e.g., DSPC) account for 0 mol% to 30 mol% of the total lipids present in the LNPs, the structural lipids (e.g., non-phytosterols (e.g., cholesterol)) account for 10 mol% to 60 mol% of the total lipids present in the LNPs, and the polymeric lipids (e.g., PEG lipids) account for 0 mol% to 0.8 mol% of the total lipids present in the LNPs, and the particle size of the L
  • the LNPs comprise ionizable lipids, accessory lipids (e.g., DSPC), structural lipids (e.g., non-phytosterols (e.g., cholesterol)), and PEG lipids (e.g., DMG-PEG), wherein the ionizable lipids account for 30 mol% to 60 mol% of the total lipids present in the LNPs, the accessory lipids (e.g., DSPC) account for 0 mol% to 30 mol% of the total lipids present in the LNPs, the structural lipids (e.g., non-phytosterols (e.g., cholesterol)) account for 10 mol% to 60 mol% of the total lipids present in the LNPs, and the PEG lipids account for 0 mol% to 0.8 mol% of the total lipids present in the LNPs.
  • the particle size of the LNPs is as described in the section on lipid nanop
  • the LNPs comprise ionizable lipids, accessory lipids (e.g., DSPC), structural lipids (e.g., non-phytosterols (e.g., cholesterol)), and polymeric lipids (e.g., PEG lipids), wherein the polymeric lipids comprise 0.1 mol% to 0.7 mol%, 0.1 mol% to 0.6 mol%, or 0.2 mol% to 0.5 mol% of the total lipids present in the LNPs, and the particle size of the LNPs is as described in the section on lipid nanoparticles.
  • accessory lipids e.g., DSPC
  • structural lipids e.g., non-phytosterols (e.g., cholesterol)
  • polymeric lipids e.g., PEG lipids
  • the LNPs comprise ionizable lipids, accessory lipids (e.g., DSPC), structural lipids (e.g., non-phytosterols (e.g., cholesterol)), and PEG lipids (e.g., DMG-PEG), wherein the PEG lipids comprise 0.1 mol% to 0.7 mol%, 0.1 mol% to 0.6 mol%, or 0.2 mol% to 0.5 mol% of the total lipids present in the LNPs, and the particle size of the LNPs is as described in the section on lipid nanoparticles.
  • accessory lipids e.g., DSPC
  • structural lipids e.g., non-phytosterols (e.g., cholesterol)
  • PEG lipids e.g., DMG-PEG
  • the PEG lipids comprise 0.1 mol% to 0.7 mol%, 0.1 mol% to 0.6 mol%, or 0.2 mol% to
  • the LNPs comprise ionizable lipids, accessory lipids (e.g., DSPC), structural lipids (e.g., non-phytosterols (e.g., cholesterol)), and polymeric lipids (e.g., PEG lipids), wherein the ionizable lipids account for 30 mol% to 60 mol% of the total lipids present in the LNPs, the accessory lipids (e.g., DSPC) account for 0 mol% to 30 mol% of the total lipids present in the LNPs, the structural lipids (e.g., non-phytosterols (e.g., cholesterol)) account for 10 mol% to 60 mol% of the total lipids present in the LNPs, and the polymeric lipids (e.g., PEG lipids)..
  • accessory lipids e.g., DSPC
  • structural lipids e.g., non-phytosterols (e
  • the LNPs comprise 0.01 mol%–0.5 mol%, 0.01 mol%–0.2 mol%, 0.01 mol%–0.3 mol%, 0.01 mol%–0.15 mol%, 0.05 mol%–0.5 mol%, 0.05 mol%–0.2 mol%, 0.05 mol%–0.15 mol%, 0.1 mol%–0.7 mol%, 0.1 mol%–0.6 mol%, or 0.2 mol%–0.5 mol% of the total lipids present in the LNPs, and the particle size of the LNPs is as described in the section on lipid nanoparticles.
  • the LNPs comprise ionizable lipids, accessory lipids (e.g., DSPC), structural lipids (e.g., non-phytosterols (e.g., cholesterol)), and PEG lipids (e.g., DMG-PEG), wherein the ionizable lipids account for 30 mol% to 60 mol% of the total lipids present in the LNPs, the accessory lipids (e.g., DSPC) account for 0 mol% to 30 mol% of the total lipids present in the LNPs, the structural lipids (e.g., non-phytosterols (e.g., cholesterol)) account for 10 mol% to 60 mol% of the total lipids present in the LNPs, and the PEG lipids account for [missing information - likely a percentage of the total lipids present in the LNPs].
  • accessory lipids e.g., DSPC
  • structural lipids e.
  • the total lipids containing Ps are 0.01 mol%–0.5 mol%, 0.01 mol%–0.2 mol%, 0.01 mol%–0.3 mol%, 0.01 mol%–0.15 mol%, 0.05 mol%–0.5 mol%, 0.05 mol%–0.2 mol%, 0.05 mol%–0.15 mol%, 0.1 mol%–0.7 mol%, 0.1 mol%–0.6 mol%, or 0.2 mol%–0.5 mol%, and the particle size of LNPs is as described in the section on lipid nanoparticles.
  • the LNPs comprise ionizable lipids, accessory lipids (e.g., DSPC), structural lipids (e.g., non-phytosterols (e.g., cholesterol)), and polymeric lipids (e.g., PEG lipids), wherein the ionizable lipids account for 45 mol% to 55 mol% of the total lipids present in the LNPs, the accessory lipids (e.g., DSPC) account for 8 mol% to 25 mol% of the total lipids present in the LNPs, the structural lipids (e.g., non-phytosterols (e.g., cholesterol)) account for 20 mol% to 45 mol% of the total lipids present in the LNPs, and the polymeric lipids (e.g., PEG lipids)..
  • accessory lipids e.g., DSPC
  • structural lipids e.g., non-phytosterols (e.
  • the LNPs comprise 0.01 mol%–0.5 mol%, 0.01 mol%–0.2 mol%, 0.01 mol%–0.3 mol%, 0.01 mol%–0.15 mol%, 0.05 mol%–0.5 mol%, 0.05 mol%–0.2 mol%, 0.05 mol%–0.15 mol%, 0.1 mol%–0.7 mol%, 0.1 mol%–0.6 mol%, or 0.2 mol%–0.5 mol% of the total lipids present in the LNPs, and the particle size of the LNPs is as described in the section on lipid nanoparticles.
  • the LNPs comprise ionizable lipids, accessory lipids (e.g., DSPC), structural lipids (e.g., non-phytosterols (e.g., cholesterol)), and PEG lipids (e.g., DMG-PEG), wherein the ionizable lipids account for 45 mol% to 55 mol% of the total lipids present in the LNPs, the accessory lipids (e.g., DSPC) account for 8 mol% to 25 mol% of the total lipids present in the LNPs, the structural lipids (e.g., non-phytosterols (e.g., cholesterol)) account for 20 mol% to 45 mol% of the total lipids present in the LNPs, and the PEG lipids account for [missing information - likely a percentage].
  • accessory lipids e.g., DSPC
  • structural lipids e.g., non-phytosterols (e.
  • the total lipids containing Ps are 0.01 mol%–0.5 mol%, 0.01 mol%–0.2 mol%, 0.01 mol%–0.3 mol%, 0.01 mol%–0.15 mol%, 0.05 mol%–0.5 mol%, 0.05 mol%–0.2 mol%, 0.05 mol%–0.15 mol%, 0.1 mol%–0.7 mol%, 0.1 mol%–0.6 mol%, or 0.2 mol%–0.5 mol%, and the particle size of LNPs is as described in the section on lipid nanoparticles.
  • the LNPs comprise: ionizable lipids, accessory lipids (e.g., DSPC), structural lipids (e.g., non-phytosterols (e.g., cholesterol)), and polymeric lipids (e.g., PEG lipids), wherein the ionizable lipids account for 47 mol% to 55 mol% of the total lipids present in the LNPs, the accessory lipids (e.g., DSPC) account for 10 mol% to 18 mol% of the total lipids present in the LNPs, the structural lipids (e.g., non-phytosterols (e.g., cholesterol)) account for 27 mol% to 42 mol% of the total lipids present in the LNPs, and the polymeric lipids (e.g., PEG lipids) account for...
  • accessory lipids e.g., DSPC
  • structural lipids e.g., non-phytosterols
  • the LNPs comprise 0.01 mol%–0.5 mol%, 0.01 mol%–0.2 mol%, 0.01 mol%–0.3 mol%, 0.01 mol%–0.15 mol%, 0.05 mol%–0.5 mol%, 0.05 mol%–0.2 mol%, 0.05 mol%–0.15 mol%, 0.1 mol%–0.7 mol%, 0.1 mol%–0.6 mol%, or 0.2 mol%–0.5 mol% of the total lipids present in the LNPs, and the particle size of the LNPs is as described in the section on lipid nanoparticles.
  • the LNPs comprise: ionizable lipids, accessory lipids (e.g., DSPC), structural lipids (e.g., non-phytosterols (e.g., cholesterol)), and PEG lipids, wherein the ionizable lipids account for 47 mol% to 55 mol% of the total lipids present in the LNPs, the accessory lipids (e.g., DSPC) account for 10 mol% to 18 mol% of the total lipids present in the LNPs, the structural lipids (e.g., non-phytosterols (e.g., cholesterol)) account for 27 mol% to 42 mol% of the total lipids present in the LNPs, and the PEG lipids account for [missing information - likely a percentage] of the total lipids present in the LNPs.
  • accessory lipids e.g., DSPC
  • structural lipids e.g., non-phytosterols
  • the total lipid content is 0.01 mol%–0.5 mol%, 0.01 mol%–0.2 mol%, 0.01 mol%–0.3 mol%, 0.01 mol%–0.15 mol%, 0.05 mol%–0.5 mol%, 0.05 mol%–0.2 mol%, 0.05 mol%–0.15 mol%, 0.1 mol%–0.7 mol%, 0.1 mol%–0.6 mol%, or 0.2 mol%–0.5 mol%, and the particle size of LNPs is as described in the section on lipid nanoparticles.
  • the LNPs of any of the above embodiments further comprise an active ingredient.
  • the active ingredient is bound to and/or encapsulated within the LNPs.
  • the active ingredient encapsulated in and/or bound to the LNPs is delivered along with the LNPs to the spleen or immune cells.
  • the active ingredient contained in the LNPs described above is the active ingredient that needs to be delivered to the spleen.
  • the LNPs described above encapsulate the active ingredient that needs to be delivered to the spleen.
  • the active ingredient is delivered to the spleen by being carried by the LNPs.
  • the spleen is the largest lymphoid organ and contains a large number of immune cells; the active ingredient delivered to the spleen can be taken up by immune cells and delivered to them. Therefore, in some embodiments, the active ingredient is delivered to immune cells.
  • the active ingredient is delivered to one or more of the following immune cells: B cells, T cells, dendritic cells, macrophages, monocytes, NK cells (e.g., immature NK cells, activated NK cells), and NKT cells.
  • the B cells to which the active ingredient is delivered are selected from one or more of the following: plasmablasts, plasma cells, lymphoplasmacytic cells, memory B cells, B-2 cells, and regulatory B cells.
  • the T cells to which the active ingredient is delivered are selected from one or more of the following: cytotoxic T cells, helper T cells, regulatory T cells, naive T cells, effector T cells, and memory T cells.
  • the immune cells to which the active ingredient is delivered are primate immune cells. In one optional specific example, the active ingredient is delivered to human immune cells.
  • LNPs The specific functions and types of active ingredients contained in the aforementioned LNPs can be determined based on the application scenario and its principles (e.g., the efficacy of a specific drug and the principle by which it achieves its efficacy). These can be substances that exert their effects directly after being delivered to the spleen or immune cells by LNPs (e.g., antisense oligonucleotides, tRNA, rRNA, snRNAs, gRNA, siRNA, microRNA, microRNA binding sites, non-nucleic acid small molecule drugs, chemotherapy drugs, peptides or proteins, etc.).
  • LNPs e.g., antisense oligonucleotides, tRNA, rRNA, snRNAs, gRNA, siRNA, microRNA, microRNA binding sites, non-nucleic acid small molecule drugs, chemotherapy drugs, peptides or proteins, etc.
  • LNPs e.g., nucleic acids encoding peptides or proteins of interest (e.g., mRNA), dsRNA, shRNA, etc.).
  • the active ingredients contained in the above-mentioned LNPs include one or more of the following: nucleic acids, non-nucleic acid small molecules, chemotherapeutic drugs, peptides, and proteins.
  • the active ingredient contained in the LNPs described above comprises nucleic acids.
  • nucleic acids By encapsulating and/or binding nucleic acids within the LNPs, they are made less susceptible to degradation after entering the body and before reaching the spleen, thereby enabling them to function in the spleen.
  • the type of nucleic acid contained in the active ingredient of the LNPs described above is not particularly limited.
  • the nucleic acid contained in the active ingredient is RNA, such as mRNA, microRNA, microRNA binding sites, tRNA, dsRNA, siRNA, snRNA, shRNA, sgRNA, antisense oligonucleotides, rRNA, etc.
  • the nucleic acid contained in the active ingredient is DNA, such as ssDNA, expression vectors, etc. In some embodiments, the nucleic acid contained in the active ingredient is a DNA-RNA hybrid strand.
  • the nucleic acids contained in the active ingredients of the above LNPs can all be RNA (e.g., a mixture of sgRNA and Cas9 mRNA), all be DNA, all be DNA-RNA hybrid strands, or be a mixture of at least two of DNA, RNA, and DNA-RNA hybrid strands (e.g., a mixture of sgRNA and a plasmid expressing Cas9 as the active molecule).
  • the active ingredients contained in the above-mentioned LNPs are nucleic acids, non-nucleic acid small molecules, chemotherapeutic drugs, peptides, or proteins.
  • the active ingredient in the LNPs described above is a mixture of nucleic acids and non-nucleic acid small molecule compounds, or a conjugate of nucleic acids and non-nucleic acid small molecule compounds.
  • the active ingredient in the LNPs described above is a mixture of nucleic acids and peptides or proteins, such as sgRNA and Cas9.
  • the active ingredient in the LNPs described above is a mixture of non-nucleic acid small molecule compounds and peptides or proteins.
  • the active ingredient in the LNPs described above is a conjugate of non-nucleic acid small molecule compounds and peptides or proteins (e.g., antibody-drug conjugates).
  • the LNPs described above contain an active ingredient for increasing the amount of the target protein.
  • the active ingredient e.g., a polypeptide or protein of interest, mRNA encoding the polypeptide or protein of interest, or a vector (e.g., a plasmid) expressing the polypeptide or protein of interest
  • the amount of the active ingredient delivered to the spleen or immune cells e.g., the content, activity, or expression level of the active ingredient in the spleen
  • the amount of the active ingredient delivered to the spleen or immune cells can be increased, thereby increasing the amount of the target protein in immune cells, the spleen, or other tissues or organs, which is beneficial for the prevention or treatment of diseases.
  • the polypeptide or protein of interest refers to a polypeptide or protein that has preventive or therapeutic effects and is active in treatment or pharmacology, and is needed or beneficial in the spleen.
  • the active ingredients contained in the aforementioned LNPs are used to activate or enhance immune cell activity.
  • the active ingredient in the aforementioned LNPs contains a substance that activates or enhances immune cell activity.
  • Examples include one or more of the following polypeptides or proteins, or nucleic acids (e.g., mRNA or expression vectors) encoding one or more of the following polypeptides or proteins: antigens, cytokines, chemokines or chemokine receptors, co-stimulatory factors, T cell receptors (TcRs), chimeric antigen receptors (CARs), recruitment factors, transcription factors, effector molecules, MHC molecules, and enzymes.
  • nucleic acids e.g., mRNA or expression vectors
  • the active ingredient of the above-mentioned LNPs comprises or stimulates the activation or enhances the activity of immune cells, or a nucleic acid (e.g., mRNA or expression vector) encoding the cytokine.
  • the cytokines that stimulate the activation or enhance the activity of immune cells include one or more of the following: IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-12, IL-13, IL-15, IL-17, IL-18, IL-21, IL-23, TNF- ⁇ , TNF- ⁇ , IFN- ⁇ , and IFN- ⁇ .
  • the active ingredient of the LNPs comprises either a cytokine that promotes T cell activation or differentiation, or a nucleic acid (e.g., mRNA or expression vector) encoding a cytokine that promotes T cell activation or differentiation.
  • the cytokine that promotes T cell activation or differentiation includes one or more of the following: IL-2, IL-4, IL-12, IL-13, IL-15, and IFN- ⁇ .
  • the active ingredient of the LNPs comprises a cytokine that promotes Th2 responses, or a nucleic acid (e.g., mRNA or expression vector) encoding such a cytokine.
  • the cytokine that promotes Th2 responses includes one or two of the following: IL-4 and IL-10.
  • the active ingredients contained in the LNPs described above include either cytokines that promote B cell activation or nucleic acids (e.g., mRNA or expression vectors) encoding cytokines that promote B cell activation.
  • the cytokines that promote B cell activation include one or more of the following: IL-4, IL-5, IL-6, IL-10, IL-13, and IFN.
  • the active ingredients of the LNPs described above comprise either pro-inflammatory cytokines or nucleic acids (e.g., mRNA or expression vectors) encoding pro-inflammatory cytokines.
  • Pro-inflammatory cytokines can be used in treatments requiring stimulation of an inflammatory response (e.g., in cancer treatment, pro-inflammatory cytokines can enhance anti-tumor immunity).
  • the pro-inflammatory cytokines include one or more of the following: IL-1, IL-6, IL-17, IL-18, IL-23, TNF- ⁇ , IFN- ⁇ , IFN- ⁇ , and interferon- ⁇ .
  • the active ingredients of the aforementioned LNPs include either chemokines or chemokine receptors, or nucleic acids (e.g., mRNA or expression vectors) encoding chemokines or chemokine receptors.
  • Chemokines participate in regulating inflammatory and immune responses, controlling the movement of inflammatory cells (including granulocytes and monocytes) and regulating the movement of various immune cells (including lymphocytes, natural killer cells, and dendritic cells). Furthermore, chemokines influence the proliferation and invasive characteristics of cancer cells.
  • the active ingredients contained in the LNPs include either a chemokine or chemokine receptor that acts on regulatory T cells, or a nucleic acid (e.g., mRNA or expression vector) encoding the chemokine or chemokine receptor.
  • the chemokine or chemokine receptor that acts on regulatory T cells includes one or more of the following: CCL22, CCL28, CCR4, and CCR10.
  • the active ingredients contained in the LNPs include either a chemokine or chemokine receptor that acts on cytotoxic T cells, or a nucleic acid (e.g., mRNA or expression vector) encoding the chemokine or chemokine receptor.
  • the chemokine or chemokine receptor that acts on cytotoxic T cells includes one or more of the following: CXCL9, CXCL10, CXCL11, and CXCR3.
  • the active ingredients contained in the LNPs include either a chemokine or chemokine receptor that acts on natural killer cells, or a nucleic acid (e.g., mRNA or expression vector) encoding the chemokine or chemokine receptor.
  • the chemokine or chemokine receptor that acts on natural killer cells includes one or more of the following: CXCL9, CXCL10, CXCL11, CCL3, CCL4, CCL5, CCL2, CCL8, CCL12, CCL13, CCL19, CCL21, CX3CL1, CXCR3, CCR1, CCR5, CCR2, and CX3CR1.
  • the active ingredient contained in the LNPs includes either a chemokine or chemokine receptor that acts on immature dendritic cells, or a nucleic acid (e.g., mRNA or expression vector) encoding the chemokine or chemokine receptor.
  • the chemokine or chemokine receptor that acts on immature dendritic cells includes one or more of the following: CCL3, CCL4, CCL5, CCL2, CCL7, CCL8, CCL22, CCL1, CCL17, CXCL12, CCR1, CCR2, CCR4, CCR5, CCR6, CCR8, and CXCR4.
  • the active ingredients contained in the LNPs include either a chemokine or chemokine receptor that acts on mature dendritic cells, or a nucleic acid (e.g., mRNA or expression vector) encoding the chemokine or chemokine receptor.
  • the chemokine or chemokine receptor that acts on mature dendritic cells includes one or more of the following: CCL19, CCL21, CXCL12, CCR7, and CXCR4.
  • the active ingredients of the LNPs described above include or represent chemokines or chemokine receptors that act on tumor-associated macrophages, or nucleic acids (e.g., mRNA or expression vectors) encoding such chemokines or chemokine receptors.
  • the chemokines or chemokine receptors that act on tumor-associated macrophages include one or more of the following: CCL2, CCL7, CCL8, CCL3, CCL4, CCL5, CXCL12, CCR2, CCR5, and CXCR4.
  • the active ingredients contained in the LNPs described above include either recruitment factors that stimulate or enhance immune cell activation or activity, or nucleic acids (e.g., mRNA or expression vectors) encoding recruitment factors.
  • a "recruitment factor” refers to any protein that promotes the recruitment of immune cells to a desired location (e.g., a tumor site or a site of inflammation).
  • recruitment factors that stimulate or enhance immune cell activation or activity include one or more of the following: CXCR3, CXCR5, CCR5, CCL5, CXCL10, CXCL12, CXCL16, and IFN- ⁇ .
  • the active ingredient contained in the LNPs described above for activating or enhancing immune cell activity includes or is a pathogenic antigen, RNA encoding a pathogenic antigen (e.g., mRNA), or DNA expressing a pathogenic antigen (e.g., an expression vector).
  • a pathogenic antigen e.g., mRNA
  • DNA expressing a pathogenic antigen e.g., an expression vector.
  • the LNPs described above are used to deliver the active ingredient (pathogenic antigen, RNA encoding a pathogenic antigen, or expression vector expressing a pathogenic antigen)
  • the amount of pathogenic antigen, RNA encoding a pathogenic antigen, or expression vector expressing a pathogenic antigen delivered to the spleen increases, thereby increasing the amount of pathogenic antigen in the spleen.
  • immune cells e.g., T cells, B cells, dendritic cells, etc.
  • pathogenic antigen e.g., IL-2, IL-2, etc.
  • target proteins e.g., cytokines, specific antibodies, etc.
  • the pathogenic antigen is derived from a pathogenic organism capable of evoking an immune response in a subject (e.g., a mammalian subject, further, such as a human).
  • a pathogenic organism includes or is one or more of the following: bacteria, viruses, fungi, and protozoa (e.g., single-celled organisms, multicellular organisms).
  • the pathogenic antigen comprises or is a surface antigen, an immunogenic fragment, a variant, or a variant of an immunogenic fragment, such as a protein located on the surface of a virus, bacteria, or protozoan, its immunogenic fragment (e.g., the outer portion of a surface antigen), a variant, or a variant of an immunogenic fragment.
  • the pathogenic antigen comprises or is derived from a polypeptide or protein of a pathogen associated with an infectious disease.
  • the pathogenic antigen is selected from, but not limited to, the group consisting of pathogen-derived antigens described on pages 21 to 35 of WO2018/078053A1, pathogen-derived antigens described on page 57, paragraph 3 to page 63, paragraph 2 of WO2019/077001A1, pathogen-derived antigens described on page 32, line 26 to page 34, line 27 of WO2013/120628A1, and antigens described on page 34, line 29 to page 59, line 5 of WO2013/120628A1.
  • the active ingredient in the LNPs described above for activating or enhancing immune cells includes an antigen receptor (e.g., a chimeric antigen receptor or a T-cell antigen receptor) or a nucleic acid (e.g., mRNA or an expression vector) encoding the antigen receptor.
  • an antigen receptor e.g., a chimeric antigen receptor or a T-cell antigen receptor
  • a nucleic acid e.g., mRNA or an expression vector
  • Using the LNPs described above to deliver the antigen receptor or the nucleic acid encoding the antigen receptor can increase the amount of antigen receptor or the nucleic acid encoding the antigen receptor delivered to the spleen, thereby increasing the amount of antigen receptor-immune cells in the spleen, and consequently enhancing the drug's specificity and killing power against tumor cells.
  • the active ingredients contained in the above-mentioned LNPs include or are: chimeric antigen receptors or T-cell antigen receptors, RNA (e.g., mRNA) encoding chimeric antigen receptors or T-cell antigen receptors, or DNA (e.g., expression vectors) expressing chimeric antigen receptors or T-cell antigen receptors.
  • RNA e.g., mRNA
  • DNA e.g., expression vectors
  • Chimeric antigen receptors are artificial transmembrane receptors that enable immune effector cells (such as T cells, natural killer cells, or other immune cells) containing CARs to have specificity with their ligands. CARs activate effector cells after the CAR specifically recognizes and binds to its ligand.
  • a typical CAR generally contains a tumor antigen-binding domain (e.g., scFv), an extracellular hinge domain, a transmembrane domain, and intracellular immune receptor tyrosine activation motifs (e.g., CD3 ⁇ , CD28, CD137 (also known as 4-lBB), CD134 (also known as OX40), and CD278 (also known as ICOS). Since tumor antigens are generally only present on the surface of tumor cells and not expressed in normal tissues, CARs can direct the action of CAR-T cells, enabling CAR-T cells to kill only tumor cells.
  • T-cell receptors used in therapy are generally specific receptors on the surface of T cells that can recognize and bind to tumor antigens. These TcRs specifically recognize and bind to tumor antigens presented by the major histocompatibility complex (MHC) on the surface of antigen-presenting cells (APCs), forming a TcR-antigen-MHC complex, thereby inducing T-cell activation and exerting adaptive immune effector functions.
  • MHC major histocompatibility complex
  • APCs antigen-presenting cells
  • the chimeric antigen receptor is capable of recognizing one or more of the following tumor antigens: CD19, CD20, CD22, CD30, CD33, CD123, CD138, CD171, CEA, EGFR, HER2, mesothelin, and PSMA.
  • the TcR is able to recognize one or more of the following tumor antigens: CD19, CD20, CD22, CD30, CD33, CD123, CD138, CD171, CEA, EGFR, HER2, mesothelin, and PSMA.
  • the tumor antigen is selected from, but not limited to, the group consisting of tumor antigens described on pages 47-51 of WO2018/078053A1.
  • the LNPs described above contain an active ingredient for reducing the amount of the target protein.
  • These LNPs are spleen-targeting, and using them to deliver the active ingredient (e.g., gene knockout, gene silencing, or antibody) can increase the amount of the active ingredient delivered to the spleen, thereby enhancing the effect of the active ingredient and subsequently reducing (i.e., lowering, inhibiting, or downregulating) the expression of the target protein in the spleen or other tissues or organs.
  • the active ingredients contained in the aforementioned LNPs are used to suppress immune cell activation or activity.
  • the active ingredients By delivering the active ingredients through LNPs, the amount of these active ingredients in immune cells is increased, thereby enhancing the effect of inhibiting or downgrading immune cell activation or activity.
  • an increase in the amount of active ingredient delivered to the spleen can enhance the effect of the active molecule on the target mRNA (interfering with the expression of the target mRNA), thereby reducing the production of proteins corresponding to the target mRNA (e.g., cytokines that activate immune cells), thus inhibiting immune cell activation or reducing immune cell activity, and reducing the production of target proteins (e.g., cytokines) by immune cells (e.g., T cells, B cells, dendritic cells).
  • proteins corresponding to the target mRNA e.g., cytokines that activate immune cells
  • target proteins e.g., cytokines
  • an increase in the amount of active ingredient delivered to the spleen e.g., nucleic acids encoding self-antigens (e.g., 1-methylpseuuridine-modified mRNA)
  • the LNPs described above contain an active ingredient for reducing the amount of the target protein that is an active ingredient for suppressing an immune response. In some embodiments, the LNPs described above contain an active ingredient for reducing the amount of the target protein that is an active ingredient for suppressing a lymphocyte immune response.
  • the LNPs described above contain an active ingredient for reducing the amount of the target protein that suppresses an immune response through one or more of the following mechanisms: B cell activity regulation, B cell depletion, antigen tolerance, inhibition of antibody production, regulation of B reg differentiation, T effector cell regulation, induction of effector T cell anergy (e.g., induction of effector T cell migration, inhibition of cytokine or cytokine receptor expression), and effector T cell depletion.
  • B cell activity regulation B cell depletion
  • antigen tolerance inhibition of antibody production
  • regulation of B reg differentiation T effector cell regulation
  • induction of effector T cell anergy e.g., induction of effector T cell migration, inhibition of cytokine or cytokine receptor expression
  • effector T cell depletion e.g., induction of effector T cell migration, inhibition of cytokine or cytokine receptor expression
  • the target protein inhibited by the active component of the above-mentioned LNPs is selected from one or more of the following: transcription factors (e.g., FoxP3, T-bet, RoRgt, STAT3, AhR, NFkB), cytoplasmic proteins (e.g., Mcl-1, HDAC10 histone deacetylase, asparagine endopeptidase (AEP), SOCS1, SOCS2, PPARg, GILZ, AMKa1, AMKa2, SHP-1, SHP-2, CAMKK2, IDO1, IDO2, TDO), transmembrane proteins (e.g., cell surface receptors, such as antibodies, T cell receptors, immune checkpoint inhibitors), secreted proteins (e.g., cytokines, chemokines), intracellular signaling proteins, and enzymes (e.g., AMPKa1, AMPKa2, HDAC10, AEP, SHP-1, SHP-2, CAMKK2, IDO1, IDO2, TDO), transme
  • the LNPs contain an active ingredient (e.g., siRNA, microRNA, etc.) for inhibiting the expression of the target protein in immune cells. In some embodiments, the LNPs contain an active ingredient (e.g., siRNA, microRNA, etc.) for inhibiting the expression of the target protein in T cells. In some embodiments, the LNPs contain an active ingredient (e.g., siRNA, microRNA, etc.) for inhibiting the expression of the target protein in B cells. In some embodiments, the LNPs contain an active ingredient (e.g., siRNA, microRNA, etc.) for inhibiting the expression of the target protein in dendritic cells.
  • an active ingredient e.g., siRNA, microRNA, etc.
  • the LNPs described above contain an active ingredient for reducing the amount of the target protein, including one or more of the following: nucleic acids encoding antibodies (e.g., mRNA); microRNA binding sites; microRNAs; antagonists (e.g., antibodies against self-antigens); small (short) interfering RNA (siRNA) (including short-chain RNA and dicer-substrate RNA); RNA interference (RNAi) molecules; antisense RNA; ribozymes; small hairpin RNAs (shRNAs); locked nucleic acids (LNAs); and molecules associated with CRISPR/Cas9 technology.
  • nucleic acids encoding antibodies (e.g., mRNA); microRNA binding sites; microRNAs; antagonists (e.g., antibodies against self-antigens); small (short) interfering RNA (siRNA) (including short-chain RNA and dicer-substrate RNA); RNA interference (RNAi) molecules; antisense RNA;
  • siRNAs inhibit gene expression by posttranscriptionally degrading mRNA, thereby preventing translation.
  • Naturally occurring and synthetic siRNAs, and their therapeutic uses e.g., delivery via nanoparticles
  • have been described in the art e.g., Hamilton and Balcombe (1999) Science 286:950-952; Elbashir et al. (2001) Nature 411:494-498; Shen et al. (2012) Cancer Gene Therap. 19:367-373; Wittrup et al. (2015) Nat. Rev. Genet. 16:543-552.
  • the siRNAs described above are incorporated herein by reference.
  • microRNAs are small non-coding RNA molecules (typically containing about 22 nucleotides) that play a role in the posttranscriptional regulation of RNA silencing and gene expression. MicroRNAs suppress gene expression by base pairing with complementary sequences within the mRNA molecule. miRNAs and their functions have been described in this field (e.g., Ambros (2004) Nature 431:350-355; Bartel (2004) Cell 116:281-297; Bartel (2009) Cell 136:215-233; Fabian et al. (2010) Ann. Rev. Biochem. 79:351-379). The microRNAs described in the above literature are incorporated herein by reference.
  • suitable non-restricted examples of microRNAs include one or more of the following: Let-7d-5p, miR-7, miR-10a, miR-10b, miR-15, miR-18a, miR-20a, miR-20b, miR-21, miR-26a, miR-34a, miR-96, miR-99a, miR-100, miR-124, miR-125a, miR-126, miR-142-3p, miR-146, miR-150, miR-155, miR-181a, and miR-210.
  • the LNPs described above contain an active ingredient for reducing the amount of the target protein, or an antagonist (e.g., an antibody) of a substance (e.g., a cytokine, chemokine, transcription factor, antigen receptor, etc.) for activating or enhancing the activity of immune cells in one of the embodiments described above.
  • an antagonist e.g., an antibody
  • a substance e.g., a cytokine, chemokine, transcription factor, antigen receptor, etc.
  • the LNPs described above contain an active ingredient for reducing the amount of the target protein, including one or more of the following: an antibody for IL-10 (e.g., for the treatment of inflammatory bowel disease, rheumatoid arthritis, and other autoimmune diseases), an antibody for IL-11 (e.g., for the treatment of inflammatory bowel disease and other autoimmune diseases), an antibody for IFN- ⁇ (e.g., for the treatment of multiple sclerosis and other autoimmune diseases), an antibody for IL-5 (e.g., for the treatment of allergies), an antibody for IL-6 (e.g., for the treatment of rheumatoid arthritis and other autoimmune diseases), an antibody for IL-12 (e.g., for autoimmune therapy), an antibody for IL-13 (e.g., for the treatment of allergies), an antibody for IL-17 (e.g., for the treatment of plaque psoriasis and other autoimmune diseases), an antibody for IL-18 (e.g., for the target protein, including one or
  • the active ingredient in the LNPs described above for reducing the amount of the target protein includes an antibody against an autoantibody, RNA (e.g., mRNA) encoding an antibody against an autoantibody, or DNA (e.g., an expression vector) expressing an antibody against an autoantibody.
  • RNA e.g., mRNA
  • DNA e.g., an expression vector
  • the active ingredients of the above-mentioned LNPs do not contain the reporter gene-encoded protein (e.g., luciferase, hEPO, EPO, eGFP, GFP, mCherry, mOX40L, OX40L, Cre, etc.), the reporter gene, or the RNA corresponding to the reporter gene.
  • the reporter gene-encoded protein e.g., luciferase, hEPO, EPO, eGFP, GFP, mCherry, mOX40L, OX40L, Cre, etc.
  • the active ingredient of the above-mentioned LNPs includes nucleic acids.
  • the active ingredient of the above-mentioned LNPs contains RNA.
  • the RNA contained in the active ingredient comprises RNA for expressing a polypeptide or protein of interest, wherein the polypeptide or protein of interest includes, but is not limited to, one or more of the following: antigens, antibodies against autoantigens, antagonists of substances for activating or enhancing immune cell activity, cytokines, chemokines or chemokine receptors, co-stimulatory factors, T cell receptors, chimeric antigen receptors, recruitment factors, transcription factors, effector molecules, MHC molecules, and enzymes.
  • the RNA contained in the active ingredient for expressing the polypeptide or protein of interest comprises one or more of the following: stem-loop, chain-terminating nucleoside, polyadenylation signal, 5'-UTR, 3'-UTR, and poly(A) tail.
  • the active ingredient of the above-mentioned LNPs contains mRNA for expressing the polypeptide or protein of interest.
  • the mRNA comprising the active ingredient of the LNPs for expressing the polypeptide or protein of interest comprises one or more of the following: a cap structure, a stem-loop, a polyadenylation signal, a 5'-UTR, a 3'-UTR, and a poly(A) tail.
  • the mRNA comprising the active ingredient for expressing the polypeptide or protein of interest comprises a cap structure, a 5'-UTR, a 3'-UTR, and a poly(A) tail.
  • the cap structure is located at the 5' end of the 5'-UTR, also referred to as a "5'-cap structure".
  • the cap structure is selected from at least one of m7GpppG , m27,3′ -OGpppG , m7Gppp (5')N1, and m7Gppp (m2′ -O )N1; wherein " m7G " represents 7-methylguanosine cap nucleotide, "ppp” represents the triphosphate bond between the 5' carbon of the cap nucleotide and the first nucleotide of the primary RNA transcript, N1 is the most 5' nucleotide, "G” represents guanine nucleotide, "7” represents the methyl group at the 7-position of guanine, and " m2′-O " represents the methyl group at the 2′-O position of the nucleotide.
  • the cap structure is m7Gp
  • the RNA length is at least 1000 nt, 1200 nt, 1500 nt, 1550 nt, 1600 nt, 1650 nt, 1700 nt, 1750 nt, 1800 nt, 1850 nt, 1900 nt, 1950 nt, 2000 nt, 2050 nt, 2100 nt, 2150 nt, 2200 nt, 2250 nt, 2300 nt, 2350 nt, 2400 nt, 2450 nt, 2500 nt, or 2550 nt.
  • the RNA length is 1000nt–8000nt, 1500nt–7000nt, 1500nt–6500nt, 1500nt–6000nt, 1500nt–5500nt, 1500nt–5000nt, 1500nt–4500nt, 1500nt–4000nt, 1500nt–3500nt, 1500nt–3000nt, 1600nt–2500nt, 1700nt–2400nt, 1800nt–2400nt, or 1900nt–2400nt.
  • regulatory elements can be incorporated into the nucleic acid used to express the polypeptide or protein of interest.
  • the expression of the nucleic acid can be suppressed or not expressed in organs, tissues, or cells containing microRNA that specifically binds to the microRNA binding site. Therefore, in some embodiments, the nucleic acid (e.g., mRNA) included in the active ingredient for expressing the polypeptide or protein of interest contains regulatory elements (e.g., microRNA binding sites, transcription factor binding sites, etc.).
  • the active ingredient of the LNPs contains a nucleic acid (e.g., mRNA) for expressing a polypeptide or protein of interest, which includes a microRNA binding site.
  • a nucleic acid e.g., mRNA
  • the active ingredient of the LNPs described above contains mRNA for expressing the polypeptide or protein of interest, and the mRNA contains a microRNA binding site.
  • the specific location of the microRNA binding site in the nucleic acid is well known to those skilled in the art, for example, it is located in the 3’-UTR and/or 5’-UTR.
  • the microRNA binding site in the nucleic acid (e.g., mRNA) contained in the active ingredient is capable of binding to microRNA expressed in the liver.
  • the microRNAs expressed in the liver include, but are not limited to, one or more of the following: miR-107, miR-122-3p, miR-122-5p, miR-1228-3p, miR-1228-5p, miR-1249, miR-129-5p, miR-1303, miR-151a-3p, miR-151a-5p, miR-152, miR-194-3p, miR-194-5p, miR-199a-3p, miR-199a-5p, miR-199b-3p, miR-199b-5p, miR-296-5p, miR-557, miR-581, miR-939-3p, and miR-939-5p.
  • the microRNA binding site in the nucleic acid (e.g., mRNA) contained in the active ingredient can bind to microRNAs expressed in the lungs.
  • MicroRNAs expressed in the lungs include, but are not limited to, one or more of the following: let-7a-2-3p, let-7a-3p, let-7a-5p, miR-126-3p, miR-126-5p, miR-127-3p, miR-127-5p, miR-130a-3p, miR-130a-5p, miR-130b-3p, miR-130b-5p, miR-133a, miR-1 33b,miR-134,miR-18a-3p,miR-18a-5p,miR-18b-3p,miR-18b-5p,miR-24-l-5p,miR-24-2-5p,miR -24-3p,miR-296-3p,miR-296-5p,miR-32-3p,miR-337-3p,miR-
  • the microRNA binding sites in the nucleic acids (e.g., mRNA) contained in the active ingredient can bind microRNAs expressed in the heart.
  • MicroRNAs expressed in the heart include, but are not limited to, one or more of the following: miR-1, miR-133a, miR-133b, miR-149-3p, miR-149-5p, miR-186-3p, miR-186-5p, miR-208a, miR-208b, miR-210, miR-296-3p, miR-320, miR-451a, miR-451b, miR-499a-3p, miR-499a-5p, miR-499b-3p, miR-499b-5p, miR-744-3p, miR-744-5p, miR-92b-3p, and miR-92b-5p.
  • the microRNA binding site in the nucleic acid (e.g., mRNA) contained in the active ingredient is capable of binding to microRNAs expressed in the nervous system.
  • MicroRNAs expressed in the nervous system include, but are not limited to, one or more of the following: miR-124-5p, miR-125a-3p, miR-125a-5p, miR-125b-1-3p, miR-125b-2-3p, miR-125b-5p, miR-1271-3p, miR-1271-5p, miR-128, miR-132-5p, miR-135a-3p, miR-135a-5p, miR-135b...
  • the microRNA binding sites in the nucleic acids (e.g., mRNA) contained in the active ingredient are capable of binding to microRNAs expressed in neurons and/or microRNAs expressed in glial cells.
  • MicroRNAs expressed in neurons include, but are not limited to, one or more of the following: miR-132-3p, miR-132-3p, miR-148b-3p, miR-148b-5p, miR-151a-3p, miR-151a-5p, miR-212-3p, miR-212-5p, miR-320b, miR-320e, miR-323a-3p, miR-323a-5p, miR-324-5p, miR-325, miR-326, miR-328, and miR-922.
  • MicroRNAs expressed in glial cells include, but are not limited to, one or more of the following: miR-1250, miR-219-1-3p, miR-219-2-3p, miR-219-5p, miR-23a-3p, miR-23a-5p, miR-3065-3p, miR-3065-5p, miR-30e-3p, miR-30e-5p, miR-32-5p, miR-338-5p, and miR-657.
  • the microRNA binding sites in the nucleic acids (e.g., mRNA) contained in the active ingredient can bind microRNAs expressed in the pancreas.
  • MicroRNAs expressed in the pancreas include, but are not limited to, one or more of the following: miR-105-3p, miR-105-5p, miR-184, miR-195-3p, miR-195-5p, miR-196a-3p, miR-196a-5p, miR-214-3p, miR-214-5p, miR-216a-3p, miR-216a-5p, miR-30a-3p, miR-33a-3p, miR-33a-5p, miR-375, miR-7-1-3p, miR-7-2-3p, miR-493-3p, miR-493-5p, and miR-944.
  • the microRNA binding site in the nucleic acid (e.g., mRNA) contained in the active ingredient can bind to microRNAs expressed in the kidney.
  • MicroRNAs expressed in the kidney include, but are not limited to, one or more of the following: miR-122-3p, miR-145-5p, miR-17-5p, miR-192-3p, miR-192-5p, miR-194-3p, miR-194-5p, miR-20a-3p, miR-20a-5p, miR-204-3p, miR-204-5p, miR-210, miR-216a-3p.
  • the microRNA binding sites in the nucleic acids (e.g., mRNA) contained in the active ingredient can bind microRNAs including those expressed in muscle.
  • MicroRNAs expressed in muscle include, but are not limited to, one or more of the following: let-7g-3p, let-7g-5p, miR-1, miR-1286, miR-133a, miR-133b, miR-140-3p, miR-143-3p, miR-143-5p, miR-145-3p, miR-145-5p, miR-188-3p, miR-188-5p, miR-206, miR-208a, miR-208b, miR-25-3p, and miR-25-5p.
  • the microRNA binding site in the nucleic acid (e.g., mRNA) contained in the active ingredient is capable of binding to microRNAs expressed in endothelial cells.
  • MicroRNAs expressed in endothelial cells include, but are not limited to, one or more of the following: let-7b-3p, let-7b-5p, miR-100-3p, miR-100-5p, miR-101-3p, miR-101-5p, miR-126-3p, miR-126-5p, miR-1236-3p, miR-1236-5p, miR-130a.
  • the microRNA binding site in the nucleic acid (e.g., mRNA) contained in the active ingredient can bind to microRNAs expressed in epithelial cells.
  • MicroRNAs expressed in epithelial cells include, but are not limited to, one or more of the following: let-7b-3p, let-7b-5p, miR-1246, miR-200a-3p, miR-200a-5p, miR-200b-3p, miR-200b-5p, miR-200c-3p, miR-200c-5p, miR-338-3p, miR-429, miR-451a, miR-451b, miR-494, and miR-802.
  • microRNAs expressed in respiratory ciliated epithelial cells include, but are not limited to, one or more of the following: miR-34a, miR-34b-5p, miR-34c-5p, miR-449a, miR-449b-3p, and miR-449b-5p.
  • MicroRNAs expressed in lung epithelial cells include, but are not limited to, one or more of the following: the let-7 family, miR-133a, miR-133b, and miR-126.
  • MicroRNAs expressed in renal tubular epithelial cells include, but are not limited to, one or more of the following: miR-382-3p and miR-382-5p.
  • MicroRNAs expressed in corneal epithelial cells include, but are not limited to, miR-762.
  • the microRNA binding site in the nucleic acid (e.g., mRNA) contained in the active ingredient can bind microRNAs expressed in embryonic stem cells.
  • MicroRNAs expressed in embryonic stem cells include, but are not limited to, one or more of the following: let-7a-2-3p, let-a-3p, let-7a-5p, let7d-3p, let-7d-5p, miR-103a-2-3p, miR-103a-5p, miR-106b-3p, miR-106b-5p, miR-1246, miR-1275, miR-138-1-3p, miR-138-2-3p, miR-138-5p, miR-154 -3p,miR-154-5p,miR-200c-3p,miR-200c-5p,miR-290,miR-301a-3p,miR-301a-5p,miR-302a-3p,miR-302a-5p,mi R-302b-3p,miR-302b
  • the microRNAs expressed in embryonic stem cells include, but are not limited to, one or more mentioned in Morin RD et al., Genome Res., 2008., 18., 610-621, Goff LA et al., PLoS One., 2009., 4., e7192, and Bar M et al., Stem cells., 2008., 26., 2496-2505.
  • microRNAs are abnormally overexpressed in certain abnormal cells (such as cancer cells), while others are underexpressed in other abnormal cells.
  • cells, tissues, or diseases exhibiting differential microRNA expression include: cancer cells (WO2008/154098, US2013/0059015, US2013/0042333, WO2011/157294), cancer stem cells (US2012/0053224), pancreatic cancer and related diseases (US2009/0131348, US2011/0171646, US2010/0286232, US8).
  • the nucleic acid (e.g., mRNA) contained in the active ingredient includes a microRNA binding site.
  • the expression of the corresponding protein which was suppressed due to the microRNA binding to the endogenous microRNA binding site, is released.
  • the microRNA binding site can be removed from the nucleic acid (e.g., mRNA) used to express the polypeptide or protein of interest (the protein suppressed by microRNA), thus compensating for the expression of the protein suppressed by microRNA through nucleic acid expression.
  • the nucleic acid contained in the active ingredient includes microRNA.
  • the exogenous microRNA compensates for the deficiency of endogenous microRNA, thus repairing the cellular abnormalities caused by the deficiency of endogenous microRNA.
  • the nucleic acid (e.g., mRNA) contained in the active ingredient of the aforementioned LNPs can be in a naturally occurring form (i.e., without additional artificially modified nucleotides) or in a chemically modified form. Therefore, in some embodiments, the nucleic acid (e.g., mRNA) contained in the active ingredient does not contain modified nucleotides. In other embodiments, the nucleic acid (e.g., mRNA) contained in the active ingredient contains modified nucleotides.
  • the modified nucleoside includes at least one of modified uridine, modified cytidine, modified adenosine, and modified guanosine.
  • the nucleoside of the nucleic acid (e.g., mRNA) contained in the active ingredient comprises or is modified with uridine. In some embodiments, 0.1% to 100% of the uridine in the nucleic acid (e.g., mRNA) contained in the active ingredient is modified.
  • nucleic acid e.g., mRNA
  • 80% to 100% of the uridine in the nucleic acid (e.g., mRNA) contained in the above-mentioned active ingredient is modified. In some embodiments, 100% of the uridine in the nucleic acid (e.g., mRNA) contained in the above-mentioned active ingredient is modified.
  • Exemplary modified uridines include pseudouridine ( ⁇ ), N1-methylpseudouridine, pyridine-4-ketoribonucleoside, 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s2U), 4-thio-uridine (s4U), 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uridine (ho5U), 5-aminoallyl-uridine, 5-halo-uridine (e.g., 5-iodo-uridine or 5-bromo-uridine), and 3-methyl-uridine.
  • pseudouridine ⁇
  • N1-methylpseudouridine pyridine-4-ketoribonucleoside
  • 5-aza-uridine 6-aza-uridine
  • 2-thio-5-aza-uridine 2-thio-uridine
  • s2U 2-thio-uridine
  • s4U 4-thio-pseudouridine
  • the modified uridine in the nucleic acid (e.g., mRNA) contained in the active ingredient is of one type.
  • the nucleic acid (e.g., mRNA) contained in the active ingredient contains multiple modified uridines, all of which are N1-methylpseudouridine.
  • the modified uridine in the nucleic acid (e.g., mRNA) contained in the active ingredient is of multiple types.
  • the nucleic acid (e.g., mRNA) contained in the active ingredient contains multiple modified uridines, which are selected from at least two of the exemplary modified uridines described above (e.g., pseudouridine and N1-methylpseudouridine).
  • the modified nucleosides in the nucleic acids (e.g., mRNA) contained in the active ingredient include or are modified cytidines. In some embodiments, 0.1% to 100% of the cytidines in the nucleic acids (e.g., mRNA) contained in the active ingredient are modified.
  • nucleic acids e.g., mRNA
  • 80% to 100% of the cytidine in the nucleic acid (e.g., mRNA) contained in the above-mentioned active ingredient is modified. In some embodiments, 100% of the cytidine in the nucleic acid (e.g., mRNA) contained in the above-mentioned active ingredient is modified.
  • Exemplary modified cytidines include 5-aza-cytidine, 6-aza-cytidine, pseudoisocytidine, 3-methylcytidine (m3C), N4-acetylcytidine (ac4C), 5-formylcytidine (f5C), N4-methylcytidine (m4C), 5-methylcytidine (m5C), 5-halo-cytidine (e.g., 5-iodocytidine), 5-hydroxymethylcytidine (hm5C), 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine (s2C), 2-thio-5-methylcytidine, 4-thio-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, 4-thio-1-methyl-1-deazo-pseudoisoc
  • the modified cytidine in the nucleic acid (e.g., mRNA) contained in the active ingredient is of one type.
  • the nucleic acid (e.g., mRNA) contained in the active ingredient contains multiple modified cytidines, all of which are 5-aza-cytidine.
  • the modified cytidine in the nucleic acid (e.g., mRNA) contained in the active ingredient is of multiple types.
  • the nucleic acid (e.g., mRNA) contained in the active ingredient contains multiple modified cytidines, which are selected from at least two of the exemplary modified cytidines described above (e.g., 5-aza-cytidine and 6-aza-cytidine).
  • the modified nucleosides in the nucleic acids (e.g., mRNA) contained in the active ingredient include or are modified adenosine. In some embodiments, 0.1% to 100% of the adenosine in the nucleic acids (e.g., mRNA) contained in the active ingredient is modified.
  • nucleic acids e.g., mRNA
  • 80% to 100% of the adenosine in the nucleic acid (e.g., mRNA) contained in the above-mentioned active ingredient is modified. In some embodiments, 100% of the adenosine in the nucleic acid (e.g., mRNA) contained in the above-mentioned active ingredient is modified.
  • Exemplary modified adenosines include 2-amino-purine, 2,6-diamino-purine, 2-amino-6-halo-purine (e.g., 2-amino-6-chloro-purine), 6-halo-purine (e.g., 6-chloro-purine), 2-amino-6-methyl-purine, 8-azido-adenosine, 7-deadenine, 7-deadenine-8-aza-adenosine, 7-deadenine-2-amino-purine, 7-deadenine-8-aza-2-amino-purine, 7-deadenine-2,6-diamino-purine, 7-deadenine-8-aza-2,6-diamino-purine, 1-methyl -Adenosine (m1A), 2-methyl-adenine (m2A), N6-methyl-adenosine (m6A), 2-methylthio-N6-methyl-adeno
  • the modified adenosine in the nucleic acid (e.g., mRNA) contained in the active ingredient is of one type.
  • the nucleic acid (e.g., mRNA) contained in the active ingredient contains multiple modified adenosines, all of which are 2-aminopurines.
  • the modified adenosine in the nucleic acid (e.g., mRNA) contained in the active ingredient is of multiple types.
  • the nucleic acid (e.g., mRNA) contained in the active ingredient contains multiple modified adenosines, which are selected from at least two of the exemplary modified adenosines described above (e.g., 2-aminopurine and 2,6-diaminopurine).
  • modified adenosines which are selected from at least two of the exemplary modified adenosines described above (e.g., 2-aminopurine and 2,6-diaminopurine).
  • the modified nucleoside in the nucleic acid (e.g., mRNA) contained in the above-mentioned active ingredient is a modified guanosine. In some embodiments, 0.1% to 100% of the guanosine in the nucleic acid (e.g., mRNA) contained in the above-mentioned active ingredient is modified.
  • nucleic acid e.g., mRNA
  • 80% to 100% of the guanosine in the nucleic acid (e.g., mRNA) contained in the above-mentioned active ingredient is modified. In some embodiments, 100% of the guanosine in the nucleic acid (e.g., mRNA) contained in the above-mentioned active ingredient is modified.
  • Exemplary modified guanosines include inosine (I), 1-methyl-inosine (m1I), wyosine (imG), methyl- ⁇ -glucoside (mimG), 4-demethyl- ⁇ -glucoside (imG-14), iso- ⁇ -glucoside (imG2), hysteroside (yW), peroxyhysteroside (o2yW), hydroxyhysteroside (OHyW), undermodified hydroxyhysteroside (OHyW*), 7-denitro-guanosine, queuosine (Q), and epoxyqueuosine (o Q), galactosyl-guanosine (galQ), mannosyl-guanosine (manQ), 7-cyano-7-deazo-guanosine (preQ0), 7-aminomethyl-7-deazo-guanosine (preQ1), archaenoside (G+), 7-deazo-8-aza-guanosine, 6-thio-guanosine,
  • the modified guanosine in the nucleic acid (e.g., mRNA) contained in the active ingredient is of one type.
  • the nucleic acid (e.g., mRNA) contained in the active ingredient contains multiple modified guanosines, all of which are inosine.
  • the modified guanosine in the nucleic acid (e.g., mRNA) contained in the active ingredient is of multiple types.
  • the nucleic acid (e.g., mRNA) contained in the active ingredient contains multiple modified guanosines, which are selected from at least two of the exemplary modified guanosines described above (e.g., inosine and 1-methyl-inosine).
  • the modified nucleotides of the nucleic acid (e.g., mRNA) contained in the above-mentioned active ingredient include nucleotides containing isotopes.
  • the nucleic acid (e.g., mRNA) contained in the active ingredient comprises a nucleotide containing a hydrogen isotope.
  • the hydrogen isotope is not limited to deuterium or tritium.
  • the nucleic acid (e.g., mRNA) contained in the active ingredient also comprises or contains nucleotides containing isotopes of elements other than hydrogen, including but not limited to carbon, oxygen, nitrogen, and phosphorus.
  • the LNPs described above do not contain an active ingredient.
  • the active ingredient can be added when they are used to prepare a carrier, drug, or pharmaceutical composition targeting the spleen.
  • the above-mentioned LNPs can increase the content of the active ingredient in the spleen, increase the content of the active ingredient in immune cells, increase the spleen/liver delivery ratio of the active ingredient, and/or decrease the content of the active ingredient in the liver.
  • the above-mentioned LNP when the PEG lipid content of the above-mentioned LNP is in the range of 0 to 0.8 mol%, compared with other LNPs whose PEG lipid content is greater than the maximum value (0.8 mol%) of the PEG lipid content range of the above-mentioned LNP, the above-mentioned LNP can increase the content of active ingredient in the spleen, increase the content of active ingredient in immune cells, increase the spleen/liver delivery ratio of active ingredient, and/or decrease the content of active ingredient in the liver.
  • the aforementioned LNPs can reduce liver side effects.
  • the aforementioned LNPs have at least the following advantages: the aforementioned LNPs can increase the content of the bound or encapsulated active ingredient in the spleen, increase the ratio of the content of the bound or encapsulated active ingredient in the spleen to that in the liver, and/or decrease the content of the bound or encapsulated active ingredient in the liver.
  • compositions for preparing LNPs III. Compositions for preparing LNPs
  • this disclosure also provides a composition for preparing LNPs, which is used to prepare LNPs according to any of the above embodiments.
  • compositions for preparing LNPs are for preparing LNPs according to any of the above embodiments. Therefore, it is understood that the components of the above compositions correspond to the components of the LNPs to be prepared.
  • the LNPs to be prepared contain 48 mol% ionizable lipids, 10 mol% accessory lipids, 41.9 mol% structural lipids, and 0.1 mol% polymeric lipids
  • the above compositions correspondingly contain 48 mol% ionizable lipids, 10 mol% accessory lipids, 41.9 mol% structural lipids, and 0.1 mol% polymeric lipids.
  • the LNPs to be prepared contain 48 mol% SM-102, 10 mol% DEPC, 41.9 mol% cholesterol, and 0.1 mol% DMG-PEG
  • the above compositions correspondingly contain 48 mol% SM-102, 10 mol% DEPC, 41.9 mol% cholesterol, and 0.1 mol% DMG-PEG.
  • the above composition also contains a solvent (e.g., ethanol).
  • a solvent e.g., ethanol
  • composition for preparing LNPs can be a mixture of components that have been prepared in proportion, or it can be a reagent that has been packaged separately in a predetermined proportion and mixed only when used.
  • composition for preparing LNPs can be used to conveniently prepare the aforementioned LNPs.
  • this disclosure also provides a method for preparing LNPs according to any of the above embodiments, the method comprising the steps of preparing LNPs using the composition for preparing LNPs according to any of the above embodiments.
  • the above preparation method uses the composition for preparing LNPs described above to prepare LNPs using microfluidic technology.
  • composition comprising LNPs of any of the above embodiments is also disclosed.
  • the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable means approved for use in animals and/or humans by a regulatory authority (e.g., the China Food and Drug Administration, the U.S. Food and Drug Administration (FDA)) or a recognized pharmacopoeia (e.g., the Chinese Pharmacopoeia, the European Pharmacopoeia).
  • the pharmaceutically acceptable carrier comprises a diluent, sweetener, flavoring agent, wetting agent, adjuvant, flow aid, preservative, dye/coloring agent, surfactant, dispersant, suspending agent, stabilizer, isotonic agent, solvent, or emulsifier.
  • this disclosure also provides the following applications of the above-described LNPs, the above-described pharmaceutical compositions, and the above-described compositions for preparing LNPs: 1. Application in the preparation of spleen-targeting delivery vectors; 2. Application in the preparation of spleen-targeting drugs; 3. Application in the preparation of immune cell-targeting delivery vectors; 4.
  • LNPs and compositions for preparing LNPs in the preparation of gene-edited immune cells 5.
  • Application of LNPs and compositions for preparing LNPs in the preparation of modified immune cells 6.
  • Application in the preparation of drugs for targeting immune cells 7.
  • Method for increasing the content of the active ingredient in the spleen 8.
  • Method for decreasing the content of the active ingredient in the liver 9.
  • Method for increasing the content of the active ingredient in immune cells 11.
  • Method for increasing the expression of nucleic acids encoding polypeptides or proteins of interest in immune cells 12.
  • Method for enhancing or weakening the activation or activity of immune cells and 13.
  • the types, contents, and ratios of ionizable lipids, accessory lipids, structural lipids, and polymeric lipids in the LNPs and compositions used in the above applications are as described in the LNPs section above.
  • the LNPs or compositions used in the above applications may contain ionizable lipids, accessory lipids, structural lipids, and polymeric lipids, wherein the molar percentage of polymeric lipids is 0 mol% to 0.8 mol%.
  • this disclosure also provides the use of LNPs of any of the above embodiments, pharmaceutical compositions containing LNPs of any of the above embodiments, or compositions for preparing LNPs of any of the above embodiments in the preparation of spleen-targeting delivery carriers.
  • the LNPs in the above applications contain active ingredients.
  • the active ingredient contained in the LNPs in the above applications is the active ingredient that needs to be delivered to the spleen.
  • the LNPs in the above applications contain the active ingredients that need to be delivered to the spleen as described above.
  • this disclosure also provides a delivery carrier that includes LNPs of any of the above embodiments.
  • the LNPs of the delivery vector described above contain an active ingredient.
  • the active ingredient contained in the LNPs of the delivery carrier described above is the active ingredient that needs to be delivered to the spleen.
  • the LNPs of the above-described delivery carrier contain the active ingredients that need to be delivered to the spleen as described above.
  • the above-mentioned delivery carriers have at least the following advantages: by utilizing the spleen-targeting characteristics of the above-mentioned LNPs, the above-mentioned delivery carriers can more effectively deliver active ingredients (e.g., active ingredients that need to be delivered to the spleen) to the spleen, thereby enabling the active ingredients to exert their effects better.
  • active ingredients e.g., active ingredients that need to be delivered to the spleen
  • this disclosure also provides the use of LNPs of any of the above embodiments, pharmaceutical compositions containing LNPs of any of the above embodiments, compositions for preparing LNPs of any of the above embodiments, or delivery carriers of any of the above embodiments in the preparation of medicaments targeting the spleen.
  • the LNPs or delivery carriers used in the above applications contain an active ingredient.
  • the LNPs or delivery carriers used in the above applications contain an active ingredient that needs to be delivered to the spleen.
  • the active ingredient that needs to be delivered to the spleen is as described above.
  • the drugs used in the above applications target immune cells in the spleen. Immune cells are as described above.
  • the drug used in the above applications also contains other active ingredients besides the active ingredient that needs to be delivered to the spleen.
  • this disclosure also provides a medicament or pharmaceutical composition targeting the spleen, the medicament or pharmaceutical composition comprising LNPs of any of the above embodiments containing an active ingredient or a delivery carrier of any of the above embodiments.
  • the active ingredient of the above-described drug or pharmaceutical composition comprises an active ingredient that needs to be delivered to the spleen.
  • the active ingredient that needs to be delivered to the spleen is as described above.
  • the above-mentioned drugs or drug compositions contain the above-mentioned LNPs or delivery carriers, and the above-mentioned drugs or drug compositions also have the corresponding advantages of the above-mentioned LNPs or delivery carriers, such as targeting the spleen.
  • the drug is used for the treatment and/or prevention of a disease.
  • this disclosure also provides the use of LNPs of any of the above embodiments, pharmaceutical compositions containing LNPs of any of the above embodiments, or compositions for preparing LNPs of any of the above embodiments in the preparation of delivery vectors for targeting immune cells.
  • the immune cells used in the above applications are as described above.
  • the LNPs in the above applications contain active ingredients.
  • the active ingredients contained in the LNPs in the above applications are active ingredients that need to be delivered to the immune system (e.g., active molecules involved in immune regulation).
  • the active ingredients contained in the LNPs in the above applications also include other active ingredients besides those that need to be delivered to immune cells.
  • this disclosure also provides a delivery carrier for targeting immune cells, the delivery carrier comprising LNPs of any of the above embodiments containing an active ingredient.
  • the delivery vector described above contains the LNPs described above, and the delivery vector also has the corresponding advantages of the LNPs described above, such as targeting immune cells.
  • this disclosure also provides the use of LNPs of any of the above embodiments, pharmaceutical compositions containing LNPs of any of the above embodiments, or compositions for preparing LNPs of any of the above embodiments in the preparation of gene-edited immune cells.
  • the LNPs described above contain an active ingredient.
  • the active ingredient of the LNPs described above contains nucleic acids required for gene editing.
  • the nucleic acids required for gene editing are those based on CRISPR/CAS systems, zinc finger nuclease systems, or TALEN systems.
  • the nucleic acids required for gene editing include sgRNA and nucleic acids (e.g., mRNA or plasmid) encoding Cas proteins (e.g., Cas9).
  • the nucleic acids required for gene editing include sgRNA, nucleic acids encoding Cas proteins (e.g., Cas9) (e.g., mRNA or plasmid), and donor DNA.
  • Cas proteins e.g., Cas9
  • donor DNA e.g., mRNA or plasmid
  • the gene-edited immune cells include one or more of the following: B cells, T cells, NK cells, DC cells, neutrophils, eosinophils, basophils, mast cells, monocytes, and macrophages; in some embodiments, the gene-edited immune cells are B cells, T cells, or NK cells.
  • the LNPs described above are loaded with nucleic acids required for gene editing.
  • the gene-edited immune cells are B cells, and the LNPs are loaded with the nucleic acids required for gene editing.
  • the LNPs described above are loaded with nucleic acids required for gene editing based on CRISPR/CAS, zinc finger nuclease, or TALEN systems.
  • LNPs are loaded with sgRNA and nucleic acids (e.g., mRNA or plasmids) encoding Cas proteins (e.g., Cas9).
  • nucleic acids e.g., mRNA or plasmids
  • Cas proteins e.g., Cas9
  • the LNPs described above are loaded with sgRNA, nucleic acid (e.g., mRNA or plasmid) encoding a Cas protein (e.g., Cas9), and donor DNA.
  • nucleic acid e.g., mRNA or plasmid
  • Cas protein e.g., Cas9
  • This invention also provides a method for preparing gene-edited immune cells, the method comprising the following steps:
  • Gene-edited immune cells are prepared by contacting LNPs loaded with nucleic acids required for gene editing with immune cells according to any of the above embodiments.
  • the immune cells that come into contact with LNPs in the above method include one or more of the following: B cells, T cells, NK cells, DC cells, neutrophils, eosinophils, basophils, mast cells, monocytes, and macrophages.
  • the immune cells that come into contact with LNPs in the above method are B cells, T cells, or NK cells.
  • the above methods are performed in vitro. In some embodiments, the above methods include contacting LNPs loaded with nucleic acids required for gene editing, according to any of the above embodiments, with isolated immune cells to prepare gene-edited immune cells.
  • the above methods are performed in vivo.
  • the above methods include administering LNPs loaded with the nucleic acids required for gene editing according to any of the above embodiments into an organism (e.g., in a human or animal body) to contact them with immune cells in the body to prepare gene-edited immune cells.
  • the present invention also provides a gene-edited immune cell, which is prepared by the method for preparing gene-edited immune cells according to any of the above embodiments.
  • the present invention also provides the use of LNPs of any of the above embodiments, a pharmaceutical composition comprising LNPs of any of the above embodiments, or a composition for preparing LNPs of any of the above embodiments in the preparation of modified immune cells;
  • the LNPs described above contain an active ingredient.
  • the active ingredient of the above-mentioned LNPs contains a nucleic acid encoding a CAR or TCR.
  • the active ingredient of the LNPs described above contains a nucleic acid encoding a therapeutic protein.
  • the modified immune cells include one or more of the following: B cells, T cells, NK cells, DC cells, neutrophils, eosinophils, basophils, mast cells, monocytes, and macrophages.
  • the modified immune cells are B cells, T cells, or NK cells.
  • the modified immune cells are CAR-T cells, TCR-T cells, or CAR-NK cells, and the LNPs are loaded with nucleic acids encoding CAR or TCR.
  • the nucleic acid encoding the CAR or TCR is mRNA or DNA (e.g., plasmid) encoding the CAR or TCR.
  • the modified immune cells are B cells, and the LNPs described above are loaded with nucleic acids encoding therapeutic proteins.
  • the therapeutic protein is an antibody.
  • the nucleic acid encoding the therapeutic protein is mRNA or DNA (e.g., a plasmid).
  • the present invention also provides a method for preparing modified immune cells, the method comprising the step of contacting immune cells with LNPs loaded with nucleic acids encoding CAR or TCR according to any of the above embodiments, a pharmaceutical composition containing LNPs according to any of the above embodiments, or a composition for preparing LNPs according to any of the above embodiments.
  • the immune cells that come into contact with LNPs in the above method include one or more of the following: B cells, T cells, NK cells, DC cells, neutrophils, eosinophils, basophils, mast cells, monocytes, and macrophages.
  • the immune cells that come into contact with LNPs in the above method are B cells, T cells, or NK cells.
  • the above methods are performed in vitro. In some embodiments, the above methods include contacting LNPs loaded with nucleic acids encoding CAR or TCR from any of the above embodiments with isolated immune cells to prepare modified immune cells.
  • the above methods are performed in vivo.
  • the above methods include administering LNPs loaded with nucleic acids encoding CAR or TCR according to any of the above embodiments into the body (e.g., in a human or animal body) to contact them with immune cells in the body to prepare modified immune cells.
  • the present invention also provides a modified immune cell, which is prepared by the method for preparing modified immune cells according to any of the above embodiments.
  • this disclosure also provides the use of LNPs of any of the above embodiments, pharmaceutical compositions containing LNPs of any of the above embodiments, compositions for preparing LNPs of any of the above embodiments, or delivery carriers of any of the above embodiments in the preparation of drugs targeting immune cells.
  • the immune cells used in the above applications are as described above.
  • the LNPs or delivery carriers used in the above applications contain an active ingredient.
  • the active ingredient contained in the LNPs or delivery vectors used in the above applications is an active ingredient that needs to be delivered to immune cells (e.g., an active molecule involved in immune regulation).
  • the active ingredients contained in the LNPs or delivery vectors used in the above applications are nucleic acids, peptides, proteins, and small molecules.
  • the active ingredients contained in the LNPs or delivery carriers used in the above applications also include other active ingredients besides those that need to be delivered to immune cells.
  • this disclosure also provides a drug or pharmaceutical composition that targets immune cells, the drug or pharmaceutical composition comprising LNPs of any of the above embodiments containing an active ingredient or a delivery carrier of any of the above embodiments.
  • the above-mentioned drugs or drug compositions contain the above-mentioned LNPs or delivery carriers, and the above-mentioned drugs or drug compositions also have the corresponding advantages of the above-mentioned LNPs or delivery carriers, such as targeting immune cells.
  • the drug is used for the treatment and/or prevention of disease.
  • diseases are selected from the following groups: rare diseases, infectious diseases, cancer, genetic diseases, autoimmune diseases, diabetes, neurodegenerative diseases, cardiovascular diseases, renal vascular diseases, and metabolic diseases.
  • cancer includes one or more of the following: lung cancer, stomach cancer, liver cancer, esophageal cancer, colon cancer, pancreatic cancer, brain cancer, lymphoma, leukemia, and prostate cancer.
  • Genetic disorders include one or more of the following: hemophilia, thalassemia, and Gaucher's disease.
  • Rare diseases include one or more of the following: osteogenesis imperfecta (OI), Wilson's disease, spinal muscular atrophy (SMA), Huntington's disease, Rett syndrome, amyotrophic lateral sclerosis (ALS), Duchenne type muscular dystrophy, Friedrichs' ataxia, methylmalonic acidemia (MMA), cystic fibrosis (CF), glycogen storage disease 1a (GSD1a), and glycogen storage disease 3a (GSD3).
  • OFI osteogenesis imperfecta
  • SMA spinal muscular atrophy
  • ALS amyotrophic lateral sclerosis
  • ALS amyotrophic lateral sclerosis
  • MMA methylmalonic acidemia
  • CF cystic fibrosis
  • GSD3a glycogen storage disease 3a
  • the active ingredient in the LNPs described above is a nucleic acid, which includes one or two of the following: RNA and DNA (e.g., plasmids).
  • the active ingredient in the LNPs described above is a nucleic acid, which includes mRNA or RNAi-based nucleic acid.
  • the RNAi-based nucleic acid is selected from one or more of the following: siRNA, antisense oligonucleotides (ASO), and miRNA.
  • the RNA contained in the LNPs is not limited to the above-mentioned mRNA, siRNA, miRNA, and ASO, and may also be other RNAs.
  • the nucleic acid encoding the protein (antibody) is mRNA or DNA (e.g., plasmids).
  • the drug is used to treat and/or prevent diseases related to immune cells.
  • the drug is used to treat and/or prevent B-cell-related diseases.
  • the drug is used to treat B-cell tumors, wherein the aforementioned LNPs contain an active ingredient for treating B-cell tumors, the active ingredient comprising one or more of the following: nucleic acids, peptides, proteins, and small molecules.
  • the active ingredient has a B-cell tumor-killing effect.
  • the drug is used to treat and/or prevent T-cell-related diseases.
  • the drug is used to treat T-cell tumors, wherein the LNPs contain an active ingredient for treating T-cell tumors, the active ingredient comprising one or more of the following: nucleic acids, peptides, proteins, and small molecules.
  • the active ingredient has a T-cell tumor-killing effect.
  • the drug is used to treat and/or prevent NK cell-related diseases.
  • the drug is used to treat NK cell tumors, wherein the LNPs contain an active ingredient for treating NK cell tumors, the active ingredient comprising one or more of the following: nucleic acids, peptides, proteins, and small molecules.
  • the active ingredient has an NK cell tumor-killing effect.
  • the drug is used to treat and/or prevent autoimmune diseases.
  • the active ingredient in the LNPs described above is a nucleic acid, peptide, protein, or small molecule that inhibits an autoimmune response.
  • the active ingredient in the LNPs described above is a nucleic acid encoding a protein (e.g., an antibody) that can inhibit an autoimmune response.
  • the nucleic acid encoding a protein (e.g., an antibody) that can inhibit an autoimmune response is mRNA.
  • the drug is used to treat and/or prevent tumors (cancer).
  • the active ingredient in the LNPs described above is a nucleic acid, peptide, protein, or small molecule that inhibits tumor growth or kills tumor cells.
  • the active ingredient in the LNPs described above is a nucleic acid encoding a CAR or TCR.
  • the active ingredient in the LNPs is mRNA encoding a CAR or TCR.
  • the drug treats a disease through gene editing.
  • the disease treated by gene editing is a genetic disease or a rare disease.
  • the active ingredient in the LNPs described above is a nucleic acid required for gene editing.
  • the active ingredient in the LNPs described above is a nucleic acid required for gene editing based on a CRISPR/CAS system, a zinc finger nuclease system, or a TALEN system.
  • the active ingredient in the LNPs described above is sgRNA and a nucleic acid encoding a Cas protein.
  • the active ingredient in the LNPs described above is sgRNA, a nucleic acid encoding a Cas protein, and donor DNA.
  • the drug is used as a vaccine for treating and/or preventing infectious diseases.
  • the active ingredient in the LNPs described above is an antigen (e.g., a protein, peptide, or nucleic acid) associated with an infectious disease.
  • the active ingredient in the LNPs described above is a nucleic acid encoding an infectious disease-associated antigen.
  • the active ingredient in the LNPs described above is mRNA encoding an infectious disease-associated antigen.
  • the drug is used as a vaccine for treating and/or preventing tumors (cancer).
  • the active ingredient in the LNPs described above is a tumor (cancer)-associated antigen (e.g., a protein, peptide, or nucleic acid).
  • the active ingredient in the LNPs described above is a nucleic acid encoding a tumor (cancer)-associated antigen.
  • the active ingredient in the LNPs described above is mRNA encoding a tumor (cancer)-associated antigen.
  • this disclosure provides a method for increasing the content of active ingredients in the spleen.
  • the method includes the steps of contacting the spleen with LNPs of any of the above embodiments, a pharmaceutical composition containing LNPs of any of the above embodiments, a delivery carrier of any of the above embodiments, a spleen-targeting drug of any of the above embodiments, or a pharmaceutical composition of any of the above embodiments.
  • the active ingredient in the above-described method for increasing the content of the active ingredient in the spleen is as described in LNPs above.
  • the method can increase the amount of active ingredient delivered to the spleen.
  • the above-described method for increasing the content of the active ingredient in the spleen can increase the content of the delivered active ingredient in the spleen by at least 7%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, and 190%.
  • % 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 310%, 320%, 330%, 340%, 350%, 360%, 370%, 380%, 390%, 400%, 410%, 420%, 430%, 440%, 450%, 460%, 470%, 480%, 490%, 500%, 510%, 520%, 530%, 540%, 550%, 560%, 570%, 580% 590%, 600%, 610%, 620%, 630%, 640%, 650%, 660%, 670%, 680%, 690%, 700%, 710%, 720%, 730%, 740%, 750%, 760%, 770%, 780%, 790%, 800%, 810%, 820%, 830%, 840%, 850%, 860%, 870%, 880%, 890%, 900%, 910%, 920%, 930%, 940%, 950%,
  • the methods described above for increasing the content of the active ingredient in the spleen are performed in vivo and/or in vitro.
  • the above-described method for increasing the content of the active ingredient in the spleen is performed in vitro, the spleen being spleen cells, and also includes the step of reinfusing the spleen cells into the subject.
  • the above-described method for increasing the content of the active ingredient in the spleen is performed in vivo, and the steps of the method include: administering to a subject LNPs containing the active ingredient of any of the above embodiments, a pharmaceutical composition containing LNPs of any of the above embodiments, a delivery carrier of any of the above embodiments, a spleen-targeting drug of any of the above embodiments, or a pharmaceutical composition of any of the above embodiments.
  • the method described above for increasing the content of the active ingredient in the spleen is used to regulate the activity of immune cells (e.g., T cells or B cells).
  • immune cells e.g., T cells or B cells. This method increases the amount of the active ingredient delivered to the spleen, thereby increasing the amount of the active ingredient delivered to immune cells, and thus making it more effective for the active ingredient to regulate the activity of immune cells.
  • this disclosure provides a method for reducing the content of active ingredients in the liver.
  • the method includes the steps of administering to a subject LNPs of any of the above embodiments, a pharmaceutical composition containing LNPs of any of the above embodiments, a delivery carrier of any of the above embodiments, a spleen-targeting drug of any of the above embodiments, or a pharmaceutical composition of any of the above embodiments.
  • the active ingredient in the above-described method for reducing the content of the active ingredient in the liver is as described in the LNPs section above.
  • the above-described method of reducing the content of the active ingredient in the liver can reduce the content of the active ingredient delivered to the liver by at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 100%, 110%, or 120%.
  • this disclosure provides a method for increasing the content ratio of active ingredients in the spleen and liver.
  • the method includes the following steps: administering to a subject LNPs of any of the above embodiments, a pharmaceutical composition containing LNPs of any of the above embodiments, a delivery carrier of any of the above embodiments, a spleen-targeting drug of any of the above embodiments, or a pharmaceutical composition of any of the above embodiments.
  • the active ingredient in the above-described method for increasing the ratio of active ingredient content in the spleen to the liver is as described in the LNPs section above.
  • the ratio of active ingredient content in the spleen to the liver refers to the ratio of the content of the active ingredient in the spleen to the content of the active ingredient in the liver.
  • the LNPs have a spleen or immune cell delivery preference, which can increase the spleen/liver delivery ratio of the active ingredient.
  • the ratio of the active ingredient content in the spleen to the liver is increased by at least 1, 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 times.
  • the above-described methods for increasing the ratio of active ingredients in the spleen to the liver are performed in vivo and/or in vitro.
  • the method for increasing the ratio of active ingredients in the spleen to the liver is performed in vitro, with the spleen being spleen cells, and also includes the step of reinfusing the spleen cells into the subject.
  • the method for increasing the ratio of active ingredient content in the spleen to liver is performed in vivo, and the steps of the method include: administering to a subject LNPs containing the active ingredient of any of the above embodiments, a pharmaceutical composition containing LNPs of any of the above embodiments, a delivery carrier of any of the above embodiments, a spleen-targeting drug of any of the above embodiments, or a pharmaceutical composition of any of the above embodiments.
  • the LNPs used in the above-described method for increasing the ratio of active ingredients in the spleen to the liver have a spleen or immune cell delivery preference, which can improve the spleen/liver delivery ratio of the active ingredients.
  • the spleen is the largest immune organ, storing a large number of immune cells (such as B cells), and the aforementioned LNPs have the characteristic of targeting the spleen.
  • the LNPs accumulate in the spleen after entering the body. This increases the contact between the LNPs and immune cells, thereby enhancing the uptake of LNPs by immune cells and ultimately increasing the content of the active ingredient in immune cells.
  • this disclosure also provides a method for increasing the content of an active ingredient in immune cells, the method comprising the steps of: contacting immune cells with LNPs of any of the above embodiments, a pharmaceutical composition comprising LNPs of any of the above embodiments, a delivery carrier of any of the above embodiments, a drug targeting immune cells of any of the above embodiments, or a pharmaceutical composition targeting immune cells of any of the above embodiments.
  • the active ingredient in the above-described method for increasing the content of the active ingredient in immune cells is as described in the LNPs section above.
  • the above-described method for increasing the content of the active ingredient in immune cells can increase the content of the delivered active ingredient in immune cells by at least 7%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, and 180% respectively.
  • % 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 310%, 320%, 330%, 340%, 350%, 360%, 370%, 380%, 390%, 400%, 410%, 420%, 430%, 440%, 450%, 460%, 470%, 480%, 490%, 500%, 510%, 520%, 530%, 540%, 550%, 560%, 570%, 5 80%, 590%, 600%, 610%, 620%, 630%, 640%, 650%, 660%, 670%, 680%, 690%, 700%, 710%, 720%, 730%, 740%, 750%, 760%, 770%, 780%, 790%, 800%, 810%, 820%, 830%, 840%, 850%, 860%, 870%, 880%, 890%, 900%, 910%, 920%, 930%, 94
  • the methods described above for increasing the content of the active ingredient in immune cells are performed in vivo and/or in vitro.
  • the methods described above for increasing the content of the active ingredient in immune cells are performed in vitro.
  • the method for increasing the content of the active ingredient in immune cells is performed in vivo, and the steps of the method include: administering to a subject LNPs containing the active ingredient of any of the above embodiments, a pharmaceutical composition containing LNPs of any of the above embodiments, a delivery carrier of any of the above embodiments, a drug targeting immune cells of any of the above embodiments, or a pharmaceutical composition targeting immune cells of any of the above embodiments.
  • the above-described method for increasing the content of the active ingredient in immune cells includes the following steps:
  • Immune cells were extracted from the subject.
  • the subject's immune cells are brought into in vitro contact with LNPs of any of the above embodiments containing an active ingredient, a pharmaceutical composition containing LNPs of any of the above embodiments, a delivery carrier of any of the above embodiments, a drug targeting immune cells of any of the above embodiments, or a pharmaceutical composition targeting immune cells of any of the above embodiments; and
  • the immune cells that have been exposed to the virus are then reinfused into the subject.
  • the contact time is at least 10 min, 15 min, 30 min, 1 h, 1.5 h, 2 h, 3 h, 5 h, 6 h, 7 h, 8 h, 9 h, 10 h, 12 h, 18 h, or 24 h.
  • the methods described above for increasing the content of the active ingredient in the immune system are used to regulate the activity of immune cells (e.g., T cells or B cells).
  • immune cells e.g., T cells or B cells.
  • This method can increase the amount of the active ingredient delivered to the spleen, thereby increasing the amount of the active ingredient delivered to immune cells, and thus making it more effective for the active ingredient to regulate the activity of immune cells.
  • the spleen is the largest immune organ, storing a large number of immune cells (such as B cells), and the aforementioned LNPs are characterized by targeting the spleen.
  • the LNPs containing the nucleic acids encoding the peptides or proteins of interest accumulate in the spleen.
  • This increases the contact between the LNPs containing the nucleic acids encoding the peptides or proteins of interest and immune cells, thereby enhancing the uptake of LNPs by immune cells, increasing the content of the nucleic acids encoding the peptides or proteins of interest in immune cells, and consequently increasing the expression level of the nucleic acids encoding the peptides or proteins of interest in immune cells.
  • this disclosure also provides a method for improving the expression of nucleic acids encoding polypeptides or proteins of interest in immune cells, the method comprising the steps of: contacting immune cells with LNPs of any of the above embodiments, a pharmaceutical composition comprising LNPs of any of the above embodiments, a delivery carrier of any of the above embodiments, a drug of any of the above embodiments targeting immune cells, or a pharmaceutical composition of any of the above embodiments targeting immune cells, wherein the LNPs, delivery carrier, drug, or pharmaceutical composition comprises nucleic acids encoding polypeptides or proteins of interest.
  • the nucleic acid encoding the polypeptide or protein of interest in the above-described method for improving the expression of the nucleic acid encoding the polypeptide or protein of interest in immune cells is as described above.
  • the immune cells used in the above-described method for enhancing the expression of nucleic acids encoding peptides or proteins of interest in immune cells are as described above. In some embodiments, the immune cells used in the above-described method for enhancing the expression of nucleic acids encoding peptides or proteins of interest in immune cells are selected from one or more of the following: B cells, T cells, dendritic cells, macrophages, monocytes, NK cells, and NKT cells.
  • the above-described methods for increasing or decreasing the expression of nucleic acids encoding peptides or proteins of interest in immune cells are capable of increasing the expression level of nucleic acids encoding peptides or proteins of interest in immune cells by at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 100%, 110%, 120%, 130%, 140%, and 150%.
  • % 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 310%, 320%, 330%, 340%, 350%, 360%, 370%, 380%, 390%, 400%, 410%, 420%, 430%, 440%, 450%, 460%, 470%, 480%, 490%, 500%, 510%, 520%, 530%, 540%, 550%, 5 60%, 570%, 580%, 590%, 600%, 610%, 620%, 630%, 640%, 650%, 660%, 670%, 680%, 690%, 700%, 710%, 720%, 730%, 740%, 750%, 760%, 770%, 780%, 790%, 800%, 810%, 820%, 830%, 840%, 850%, 860%, 870%, 880%, 890%, 900%, 910%, 92
  • the methods described above for increasing or decreasing the expression of nucleic acids encoding polypeptides or proteins of interest in immune cells are performed in vivo and/or in vitro.
  • the steps of the above-described methods for increasing or decreasing the expression of nucleic acids encoding polypeptides or proteins of interest in immune cells include: contacting immune cells in vitro with LNPs of any of the above embodiments, a pharmaceutical composition comprising LNPs of any of the above embodiments, a delivery vector of any of the above embodiments, a spleen-targeting drug of any of the above embodiments, or a pharmaceutical composition of any of the above embodiments.
  • the steps of the above-described methods for increasing or decreasing the expression of nucleic acids encoding polypeptides or proteins of interest in immune cells include: administering to a subject LNPs of any of the above embodiments, a pharmaceutical composition comprising LNPs of any of the above embodiments, a delivery vector of any of the above embodiments, a spleen-targeting drug of any of the above embodiments, or a pharmaceutical composition of any of the above embodiments.
  • the methods described above for increasing or decreasing the expression of nucleic acids encoding polypeptides or proteins of interest in immune cells include the following steps:
  • Immune cells were extracted from the subject.
  • the subject's immune cells are brought into in vitro contact with LNPs of any of the above embodiments containing the active ingredient, a pharmaceutical composition containing LNPs of any of the above embodiments, a delivery carrier of any of the above embodiments, a spleen-targeting drug of any of the above embodiments, or a pharmaceutical composition of any of the above embodiments; and
  • the immune cells that have been exposed to the virus are then reinfused into the subject.
  • the in vitro contact time in any of the above methods is at least 10 min, 15 min, 30 min, 1 h, 1.5 h, 2 h, 3 h, 5 h, 6 h, 7 h, 8 h, 9 h, 10 h, 12 h, 18 h, or 24 h.
  • the spleen is the largest immune organ, storing a large number of immune cells (such as B cells), and the aforementioned LNPs have the characteristic of targeting the spleen. Therefore, when using these LNPs to deliver active ingredients that regulate the activation or activity of immune cells, the LNPs containing these active ingredients accumulate in the spleen. This increases the contact between the LNPs containing these active ingredients and immune cells, thereby increasing the uptake of LNPs by immune cells, increasing the content of the active ingredients regulating the activation or activity of immune cells in immune cells, and thus enhancing or weakening the activation or activity of immune cells.
  • this disclosure provides a method for enhancing or weakening the activation or activity of immune cells, the method comprising: contacting immune cells with LNPs of any of the above embodiments, a pharmaceutical composition comprising LNPs of any of the above embodiments, a delivery carrier of any of the above embodiments, a drug of any of the above embodiments targeting spleen or immune cells, or a pharmaceutical composition of any of the above embodiments targeting spleen or immune cells, wherein the LNPs, delivery carrier, drug, or pharmaceutical composition comprises an active ingredient that modulates the activation or activity of immune cells.
  • the immune cells used in the methods for enhancing or weakening the activation or activity of immune cells described above are as described in the preceding embodiments. In some embodiments, the immune cells used in the methods for enhancing or weakening the activation or activity of immune cells described above are selected from one or more of the following: B cells, T cells, dendritic cells, macrophages, monocytes, NK cells, and NKT cells.
  • the methods described above for enhancing or weakening the activation or activity of immune cells are methods for regulating the activation or activity of T cells.
  • the methods described above for enhancing or weakening the activation or activity of immune cells are methods for regulating the activation or activity of B cells.
  • the methods described above for enhancing or weakening the activation or activity of immune cells are methods for enhancing the activation or activity of immune cells. These methods increase the levels of active components delivered to immune cells (e.g., cytokines IFN- ⁇ , IFN- ⁇ / ⁇ , IL-6, etc.), thereby enhancing the activation or activity of immune cells.
  • active components delivered to immune cells e.g., cytokines IFN- ⁇ , IFN- ⁇ / ⁇ , IL-6, etc.
  • the methods described above for enhancing or weakening the activation or activity of immune cells are methods for enhancing the activation or activity of immune cells, wherein the active ingredient in the method is an active molecule for disease prevention or treatment based on CAR-T cell therapy.
  • This method increases the amount of the active ingredient (e.g., nucleic acid expressing a CAR that specifically recognizes tumor antigens) delivered to T cells, thereby increasing the amount of receptors specifically recognizing tumor antigens expressed by the subject's T cells, enabling the subject's T cells to more effectively recognize and kill tumor cells.
  • the methods described above for enhancing or weakening the activation or activity of immune cells include methods for enhancing the activation or activity of immune cells in which the active ingredient in the LNPs comprises a nucleic acid (e.g., a nucleic acid expressing a CAR that specifically recognizes a tumor antigen). This method enhances the activation or activity of immune cells by increasing the amount, activity, or expression level of the nucleic acid delivered to the immune cells.
  • a nucleic acid e.g., a nucleic acid expressing a CAR that specifically recognizes a tumor antigen
  • the methods described above for enhancing or weakening the activation or activity of immune cells are methods for weakening the activation or activity of immune cells. This method weakens the activation or activity of immune cells and prevents excessive immune responses by increasing the content of active ingredients (e.g., nucleic acids expressing Treg cell-inducing factors) delivered to immune cells, thereby increasing the number of Treg cells.
  • active ingredients e.g., nucleic acids expressing Treg cell-inducing factors
  • the methods described above for enhancing or weakening the activation or activity of immune cells are methods for weakening the activation or activity of immune cells, wherein the active components of the LNPs in the method include nucleic acids (e.g., nucleic acids expressing IL-2, TGF- ⁇ , IL-10, etc.). This method weakens the activation or activity of immune cells by increasing the amount, activity, or expression level of nucleic acids delivered to the immune cells.
  • nucleic acids e.g., nucleic acids expressing IL-2, TGF- ⁇ , IL-10, etc.
  • the above-described methods for enhancing or weakening the activation or activity of immune cells deliver the active ingredient to immune cells at least by 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 2 70%, 280%, 290%, 300%, 310%, 320%, 330%, 340%, 350%, 360%, 370%, 380%, 390%, 400%, 410%, 420%, 430%, 440%, 450%, 460%, 470%, 480%, 490%, 500%, 510%, 520%, 530%, 540%, 550%, 560%, 570%, 580%, 590%, 600%
  • the methods described above for enhancing or weakening the activation or activity of immune cells enhance the uptake of the active ingredient by immune cells.
  • the active ingredient in the methods described above for enhancing or weakening the activation or activity of immune cells is a nucleic acid, and the method enhances the uptake of the nucleic acid by immune cells.
  • detectable labels can be used to assess the uptake amount.
  • fluorescently labeled LNPs can be used, and the location of such LNPs in or on immune cells can be tracked after different exposure periods for assessment.
  • the methods described above for enhancing or reducing the activation or activity of immune cells are performed in vivo and/or in vitro.
  • the steps of the above-described method for enhancing or reducing the activation or activity of immune cells include: contacting immune cells in vitro with LNPs of any of the above embodiments containing an active ingredient that regulates the activation or activity of immune cells, a pharmaceutical composition containing LNPs of any of the above embodiments, a delivery carrier of any of the above embodiments, a drug of any of the above embodiments targeting the spleen or immune cells, or a pharmaceutical composition of any of the above embodiments targeting the spleen or immune cells.
  • the above-described methods for enhancing or reducing the activation or activity of immune cells include the following steps:
  • Immune cells were extracted from the subject.
  • the subject's immune cells are contacted in vitro with LNPs of any of the above embodiments containing an active ingredient that regulates the activation or activity of immune cells, a pharmaceutical composition containing LNPs of any of the above embodiments, a delivery carrier of any of the above embodiments, a spleen-targeting drug of any of the above embodiments, or a pharmaceutical composition of any of the above embodiments; and
  • the immune cells that have been exposed to the virus are then reinfused into the subject.
  • the steps of the above-described methods for enhancing or reducing the activation or activity of immune cells include: administering to a subject LNPs of any of the above embodiments containing an active ingredient that modulates the activation or activity of immune cells, a pharmaceutical composition containing LNPs of any of the above embodiments, a delivery carrier of any of the above embodiments, a pharmaceutical medicament targeting the spleen or immune cells of any of the above embodiments, or a pharmaceutical composition targeting the spleen or immune cells of any of the above embodiments.
  • the in vitro contact time of any of the above methods is at least 10 min, 15 min, 30 min, 1 h, 1.5 h, 2 h, 3 h, 5 h, 6 h, 7 h, 8 h, 9 h, 10 h, 12 h, 18 h, or 24 h.
  • the spleen is the largest immune organ, storing a large number of immune cells (such as B cells), and the aforementioned LNPs are characterized by targeting the spleen. Therefore, when using these LNPs to deliver active ingredients (such as antigens of interest (e.g., pathogenic antigens or autoantigens), nucleic acids encoding antigens of interest, reagents for reducing or decreasing the activity of autoantigens of interest in vivo), the LNPs containing the active ingredients accumulate in the spleen. This increases the contact between the LNPs containing the active ingredients and immune cells, thereby increasing the uptake of LNPs by immune cells, increasing the content of the active ingredients in immune cells, and consequently enhancing or reducing the immune response to the antigens of interest.
  • active ingredients such as antigens of interest (e.g., pathogenic antigens or autoantigens), nucleic acids encoding antigens of interest, reagents for reducing or decreasing the activity of autoantigens
  • this disclosure provides a method for enhancing or reducing an immune response to an antigen of interest, the method comprising the steps of: contacting immune cells with LNPs of any of the above embodiments, a pharmaceutical composition comprising LNPs of any of the above embodiments, a delivery carrier of any of the above embodiments, a drug of any of the above embodiments targeting the spleen or immune cells, or a pharmaceutical composition of any of the above embodiments targeting the spleen or immune cells, wherein the LNPs, delivery carrier, drug, or pharmaceutical composition comprises the antigen of interest, a nucleic acid encoding the antigen of interest, a reagent for reducing the antigen of interest in vivo, or a reagent for reducing the activity of the antigen of interest.
  • the active ingredient of the LNPs described above includes an antigen of interest, a nucleic acid encoding the antigen of interest, a reagent for reducing the antigen of interest in vivo, or a reagent for reducing the activity of the antigen of interest.
  • the antigen of interest is as described above in the section on lipid nanoparticles.
  • the antigen of interest is a cancer antigen or an infectious disease antigen.
  • the antigen of interest is one or more of the following: bacterial antigens, viral antigens, fungal antigens, protozoan antigens, and parasitic antigens.
  • the immune response is an antigen-specific antibody response.
  • the immune response is an antigen-specific T-cell response.
  • the methods described above for enhancing or reducing the immune response to an antigen of interest are methods for enhancing the immune response to an antigen of interest.
  • This method enhances the body's immune system's immune response to the antigen of interest by increasing the amount of the antigen of interest (e.g., cancer antigen or infectious disease antigen) in immune cells, thereby enabling the secretion of more cytokines or antibodies to kill tumor cells or pathogens, thereby preventing or treating the disease.
  • the antigen of interest e.g., cancer antigen or infectious disease antigen
  • the methods described above for increasing or decreasing the immune response to an antigen of interest are methods for decreasing the immune response to an antigen of interest.
  • This method increases the amount of the antigen of interest (e.g., cancer antigen or infectious disease antigen) in the spleen, thereby increasing the body's immune tolerance to the antigen of interest, which in turn reduces cytokines or antibodies, thereby preventing or treating the disease.
  • the antigen of interest e.g., cancer antigen or infectious disease antigen
  • the immune response induced by the above-described methods for enhancing or reducing the immune response to the antigen of interest is enhanced or reduced by at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, respectively.
  • the methods described above for enhancing or reducing the immune response to the antigen of interest are performed in vivo and/or in vitro.
  • the steps of the above-described method for enhancing or reducing an immune response to an antigen of interest include: contacting immune cells in vitro with LNPs of any of the above embodiments containing an active ingredient (e.g., an antigen of interest), a pharmaceutical composition containing LNPs of any of the above embodiments, a delivery carrier of any of the above embodiments, a drug of any of the above embodiments targeting the spleen or immune cells, or a pharmaceutical composition of any of the above embodiments targeting the spleen or immune cells.
  • an active ingredient e.g., an antigen of interest
  • a pharmaceutical composition containing LNPs of any of the above embodiments e.g., a pharmaceutical composition containing LNPs of any of the above embodiments
  • a delivery carrier of any of the above embodiments e.g., a drug of any of the above embodiments targeting the spleen or immune cells
  • a drug of any of the above embodiments targeting the spleen or immune cells e.g., an
  • the methods for enhancing or reducing the immune response to the antigen of interest include the following steps:
  • Immune cells were extracted from the subject.
  • the subject's immune cells are contacted in vitro with LNPs of any of the above embodiments containing an active ingredient (e.g., an antigen of interest), a pharmaceutical composition containing LNPs of any of the above embodiments, a delivery carrier of any of the above embodiments, a drug targeting the spleen or immune cells of any of the above embodiments, or a pharmaceutical composition targeting the spleen or immune cells of any of the above embodiments; and
  • an active ingredient e.g., an antigen of interest
  • the immune cells that have been exposed to the virus are then reinfused into the subject.
  • the in vitro contact time in any of the above methods is at least 10 min, 15 min, 30 min, 1 h, 1.5 h, 2 h, 3 h, 5 h, 6 h, 7 h, 8 h, 9 h, 10 h, 12 h, 18 h, or 24 h.
  • the steps of the above-described method for enhancing or reducing an immune response to an antigen of interest include: administering to a subject LNPs of any of the above embodiments containing an active ingredient (e.g., an antigen of interest), a pharmaceutical composition containing LNPs of any of the above embodiments, a delivery carrier of any of the above embodiments, a pharmaceutical composition of any of the above embodiments targeting the spleen or immune cells, or a pharmaceutical composition of any of the above embodiments targeting the spleen or immune cells.
  • an active ingredient e.g., an antigen of interest
  • the active ingredient in the method or application of any of the above embodiments comprises or is a nucleic acid.
  • the active ingredient in any of the above embodiments in the method or application comprises or is mRNA.
  • the active ingredient in any of the methods or applications described above comprises or is mRNA containing a modified nucleoside.
  • the active ingredient in any of the above embodiments in the method or application comprises or is 100% modified uridine mRNA.
  • the content of the active ingredient delivered to the spleen, liver, or immune cells in any of the above embodiments can be a direct measurement result (e.g., when the active ingredient is a polypeptide or protein, the content of the active ingredient can be directly measured using ELISA), or it can be an indirect measurement result (e.g., measuring the activity or expression level of the active ingredient).
  • the active ingredient is mRNA
  • the content of the active ingredient can be indirectly measured by measuring the content of the polypeptide or protein expressed by the active ingredient using ELISA.
  • the LNPs applied in any of the above methods or applications are one of the group consisting of LNPs selected from the above embodiments.
  • the LNPs applied in any of the above methods or applications are two, three, three or more selected from the group consisting of LNPs described in the above embodiments.
  • two LNPs are applied, which encapsulate different active ingredients (e.g., nucleic acids).
  • adjusting (reducing) the polymer lipid content of LNPs can enable them to target the spleen or immune cells (i.e., compared to before the polymer lipid content was adjusted (reduced), the content of the active ingredient delivered by the adjusted (reduced) LNPs in the spleen and/or its spleen/liver delivery ratio is increased). Therefore, using the above-mentioned LNPs can deliver active ingredients with disease prevention or treatment effects to the spleen or immune cells, which is more conducive to the prevention and treatment of diseases related to the spleen or immune cells.
  • this disclosure also provides a method for preventing or treating a disease, the steps of which include: contacting the immune cells of a subject with an LNP comprising an active ingredient of any of the above embodiments, a pharmaceutical composition comprising an LNP comprising any of the above embodiments, a delivery carrier of any of the above embodiments, or a spleen-targeting drug of any of the above embodiments.
  • This disclosure also provides a method for preventing or treating a disease, the steps of which include: administering to a subject in need LNPs of any of the above embodiments, a pharmaceutical composition comprising LNPs of any of the above embodiments, a delivery carrier of any of the above embodiments, a spleen-targeting drug of any of the above embodiments, gene-edited immune cells of any of the above embodiments, or modified immune cells of any of the above embodiments.
  • LNPs comprising the active ingredient of any of the above embodiments
  • pharmaceutical compositions comprising LNPs comprising any of the above embodiments
  • delivery carriers comprising any of the above embodiments
  • spleen-targeting drugs comprising any of the above embodiments
  • pharmaceutical compositions comprising any of the above embodiments deliver an active ingredient having a preventive or therapeutic effect to the spleen or immune cells, thereby achieving a preventive or therapeutic effect.
  • the LNPs of any of the above embodiments containing the active ingredient, the pharmaceutical composition containing the LNPs of any of the above embodiments, the delivery carrier of any of the above embodiments, the spleen-targeting drug of any of the above embodiments, or the pharmaceutical composition of any of the above embodiments are introduced into the immune cells of a subject (e.g., in vivo or in vitro) to modify the immune cells, thereby obtaining modified immune cells with preventive or therapeutic effects.
  • the disease is a disease related to immune cells.
  • the present invention also provides a method for treating and/or preventing diseases related to immune cells, the method comprising the steps of: administering (e.g., intravenous injection) to a subject in need LNPs containing an active ingredient according to any of the above embodiments, a pharmaceutical composition containing LNPs according to any of the above embodiments, gene-edited immune cells prepared by a method for preparing gene-edited immune cells according to any of the above embodiments, and modified immune cells prepared by a method for preparing modified immune cells according to any of the above embodiments.
  • administering e.g., intravenous injection
  • a pharmaceutical composition containing LNPs according to any of the above embodiments e.g., gene-edited immune cells prepared by a method for preparing gene-edited immune cells according to any of the above embodiments
  • modified immune cells prepared by a method for preparing modified immune cells according to any of the above embodiments.
  • diseases associated with immune cells are B-cell tumors, T-cell tumors, or NK-cell tumors.
  • the present invention also provides a method for treating and/or preventing B-cell tumors, the method comprising the steps of: administering (e.g., intravenous injection) to a subject in need of LNPs containing an active ingredient according to any of the above embodiments, a pharmaceutical composition containing LNPs according to any of the above embodiments, gene-edited B cells prepared by a method for preparing gene-edited immune cells according to any of the above embodiments, and modified B cells prepared by a method for preparing modified immune cells according to any of the above embodiments.
  • administering e.g., intravenous injection
  • a pharmaceutical composition containing LNPs according to any of the above embodiments e.g., gene-edited B cells prepared by a method for preparing gene-edited immune cells according to any of the above embodiments
  • modified B cells prepared by a method for preparing modified immune cells according to any of the above embodiments.
  • the method of treating and/or preventing B-cell tumors includes: contacting ex vivo B cells of a subject with an LNP containing an active ingredient or a pharmaceutical composition of any of the above embodiments; and reinfusing the contacted ex vivo B cells into the subject.
  • the present invention provides a method for treating and/or preventing T-cell tumors, the method comprising administering (e.g., intravenous injection) to a subject in need LNPs containing an active ingredient according to any of the above embodiments, a pharmaceutical composition containing LNPs according to any of the above embodiments, gene-edited T cells prepared by a method for preparing gene-edited immune cells according to any of the above embodiments, and modified T cells prepared by a method for preparing modified immune cells according to any of the above embodiments.
  • administering e.g., intravenous injection
  • a pharmaceutical composition containing LNPs according to any of the above embodiments e.g., gene-edited T cells prepared by a method for preparing gene-edited immune cells according to any of the above embodiments
  • modified T cells prepared by a method for preparing modified immune cells according to any of the above embodiments.
  • the method of treating and/or preventing T-cell tumors includes: contacting ex vivo T cells of a subject with LNPs containing an active ingredient according to any of the above embodiments, or a pharmaceutical composition containing LNPs according to any of the above embodiments; and reinfusing the contacted ex vivo T cells into the subject.
  • the present invention also provides a method for treating and/or preventing NK cell tumors, the method comprising administering (e.g., intravenous injection) to a subject in need LNPs containing an active ingredient according to any of the above embodiments, a pharmaceutical composition containing LNPs according to any of the above embodiments, gene-edited NK cells prepared by a method for preparing gene-edited immune cells according to any of the above embodiments, and modified NK cells prepared by a method for preparing modified immune cells according to any of the above embodiments.
  • administering e.g., intravenous injection
  • a pharmaceutical composition containing LNPs according to any of the above embodiments e.g., gene-edited NK cells prepared by a method for preparing gene-edited immune cells according to any of the above embodiments
  • modified NK cells prepared by a method for preparing modified immune cells according to any of the above embodiments.
  • the method of treating and/or preventing NK cell tumors includes: contacting ex vivo NK cells of a subject with LNPs containing an active ingredient according to any of the above embodiments, or a pharmaceutical composition containing LNPs according to any of the above embodiments; and reinfusing the contacted ex vivo NK cells into the subject.
  • the present invention provides a method for treating and/or preventing autoimmune diseases, the method comprising administering (e.g., intravenous injection) to a subject in need LNPs containing an active ingredient according to any of the above embodiments, a pharmaceutical composition containing LNPs according to any of the above embodiments, gene-edited immune cells prepared by a method for preparing gene-edited immune cells according to any of the above embodiments, and modified immune cells prepared by a method for preparing modified immune cells according to any of the above embodiments.
  • administering e.g., intravenous injection
  • a pharmaceutical composition containing LNPs according to any of the above embodiments e.g., gene-edited immune cells prepared by a method for preparing gene-edited immune cells according to any of the above embodiments
  • modified immune cells prepared by a method for preparing modified immune cells according to any of the above embodiments.
  • the present invention provides a method for treating and/or preventing autoimmune diseases, the method comprising administering (e.g., intravenous injection) to a subject in need LNPs comprising an active ingredient capable of preventing or treating autoimmune diseases according to any of the above embodiments, a pharmaceutical composition comprising LNPs according to any of the above embodiments, gene-edited immune cells capable of preventing or treating autoimmune diseases prepared by a method for preparing gene-edited immune cells according to any of the above embodiments, and modified immune cells capable of preventing or treating autoimmune diseases prepared by a method for preparing modified immune cells according to any of the above embodiments.
  • administering e.g., intravenous injection
  • LNPs comprising an active ingredient capable of preventing or treating autoimmune diseases according to any of the above embodiments
  • a pharmaceutical composition comprising LNPs according to any of the above embodiments, gene-edited immune cells capable of preventing or treating autoimmune diseases prepared by a method for preparing gene-edited immune cells according to any of
  • the present invention provides a method for treating and/or preventing tumors (cancer), the method comprising administering (e.g., intravenous injection) to a subject in need LNPs containing an active ingredient according to any of the above embodiments, a pharmaceutical composition containing LNPs according to any of the above embodiments, and modified T cells obtained by a method for preparing modified immune cells according to any of the above embodiments.
  • administering e.g., intravenous injection
  • the method of treating and/or preventing tumors includes: contacting ex vivo T cells of a subject with an active ingredient of any of the above embodiments, or a pharmaceutical composition containing LNPs of any of the above embodiments; and reinfusing the contacted ex vivo T cells into the subject.
  • the present invention provides a method for treating and/or preventing hereditary diseases or rare diseases, the method comprising administering (e.g., intravenous injection) to a subject in need LNPs containing an active ingredient according to any of the above embodiments, a pharmaceutical composition containing LNPs according to any of the above embodiments, or gene-edited immune cells prepared by a method for preparing gene-edited immune cells according to any of the above embodiments.
  • the method of treating and/or preventing hereditary diseases or rare diseases includes: contacting ex vivo immune cells of a subject with an active ingredient, or a pharmaceutical composition containing an active ingredient from any of the above embodiments; and reinfusing the contacted ex vivo immune cells into the subject.
  • the active ingredient of the LNPs is a nucleic acid required for gene editing.
  • the active ingredient of the LNPs is a nucleic acid required for gene editing based on a CRISPR/CAS system, a zinc finger nuclease system, or a TALEN system.
  • the active ingredient of the LNPs is sgRNA and a nucleic acid encoding a Cas protein.
  • the active ingredient of the LNPs is sgRNA, a nucleic acid encoding a Cas protein, and donor DNA.
  • the present invention provides a method for treating and/or preventing infectious diseases, the method comprising administering (e.g., intravenous injection) to a subject in need of LNPs comprising an active ingredient, or a pharmaceutical composition comprising LNPs comprising any of the above embodiments.
  • the active ingredient contained in the LNPs is an antigen (e.g., a protein, polypeptide, or nucleic acid) associated with an infectious disease.
  • the active ingredient contained in the LNPs is a nucleic acid encoding an antigen associated with an infectious disease.
  • the active ingredient contained in the LNPs is mRNA encoding an antigen associated with an infectious disease.
  • the present invention provides a method for treating and/or preventing tumors (cancer), the method comprising administering (e.g., intravenous injection) to a subject in need of LNPs comprising an active ingredient, or a pharmaceutical composition comprising LNPs comprising any of the above embodiments.
  • the active ingredient contained in the LNPs is a tumor (cancer)-associated antigen (e.g., a protein, polypeptide, or nucleic acid).
  • the active ingredient contained in the LNPs is a nucleic acid encoding a tumor (cancer)-associated antigen.
  • the active ingredient contained in the LNPs is mRNA encoding a tumor (cancer)-associated antigen.
  • the active ingredient in the methods for preventing or treating the aforementioned diseases is as described above. It is understood that the disease in the methods for preventing or treating the aforementioned diseases corresponds to the active ingredient.
  • the active ingredient in the prevention or treatment of the aforementioned diseases comprises or is a nucleic acid.
  • the active ingredient in the prevention or treatment of the aforementioned diseases comprises or is mRNA.
  • the active ingredient in the prevention or treatment of the above-mentioned diseases comprises or is mRNA containing modified nucleosides.
  • the active ingredient in the prevention or treatment of the aforementioned diseases comprises or is 100% modified uridine mRNA.
  • the subjects to whom the aforementioned disease prevention or treatment methods are administered are mammals, such as humans, non-human primates (e.g., apes, chimpanzees, monkeys, and orangutans).
  • the subject is a human.
  • the prevention or treatment methods for the aforementioned diseases are applied once, twice, three times, four times, or more.
  • the methods of prevention or treatment of the above-mentioned diseases are administered by one or more of the following: nasal, tracheal, or injectable (e.g., intravenous, intraocular, intravitreal, intramuscular, intradermal, intracardiac, intranodal, intratumoral, intraperitoneal, and subcutaneous).
  • injectable e.g., intravenous, intraocular, intravitreal, intramuscular, intradermal, intracardiac, intranodal, intratumoral, intraperitoneal, and subcutaneous.
  • the methods of prevention or treatment of the above-mentioned diseases are one or more of the following: intravenous injection, intramuscular injection, subcutaneous injection, intranodal injection, and intratumoral injection.
  • the prevention or treatment of the above-mentioned diseases is administered via intravenous or intramuscular injection.
  • the prevention or treatment of the above-mentioned diseases is administered via intravenous or intramuscular injection.
  • the prevention or treatment of the above-mentioned diseases includes the following steps:
  • Immune cells were extracted from the subject.
  • the LNPs of any of the above embodiments containing the active ingredient, the pharmaceutical composition containing the LNPs of any of the above embodiments, the delivery carrier of any of the above embodiments, the spleen-targeting drug of any of the above embodiments, or the pharmaceutical composition of any of the above embodiments are contacted in vitro with the immune cells of the subject.
  • the LNPs used for the prevention or treatment of the above-mentioned diseases are one of the LNPs selected from the group consisting of the LNPs described in the above implementations.
  • the LNPs used in the prevention or treatment of the above-mentioned diseases are two, three, three or more selected from the group consisting of LNPs described in the above embodiments.
  • two LNPs are used, which encapsulate different active ingredients (e.g., nucleic acids).
  • DLin-MC3-DMA (MC3), DSPC, cholesterol, and DMG-PEG2000 (catalog number: O02005) were dissolved in anhydrous ethanol to prepare solutions with concentrations of 20 mg/mL, 10 mg/mL, 20 mg/mL, and 25 mg/mL, respectively.
  • Dialysis The product from step (2) was loaded into a dialysis bag and placed in Tris Buffer-8% (m/V) sucrose solution for displacement to remove residual ethanol, unassembled lipids and other components. Dialysis was performed for 2 hours at room temperature in the dark with magnetic stirring (the dialysis solution was changed every 1 hour).
  • step (3) Pass the product of step (3) through a 0.22 ⁇ m microporous membrane for sterilization, and then dispense it.
  • LNP formulations Various lipid nanoparticle formulations (LNP formulations) encapsulating Fluc mRNA were prepared.
  • the mass ratio of Fluc mRNA to lipid in each LNP formulation was 1:10, the concentration of mRNA was 0.2 mg/mL, the encapsulation efficiency was above 80%, and the particle size and particle size distribution index are shown in Table 1.
  • mice Female BALB/c mice aged 6–8 weeks were selected and housed in an SPF-grade enclosure. Animal experiments were conducted strictly in accordance with national health agency guidelines and animal ethics requirements.
  • mice Six hours after injection of the LNP formulation, mice were injected with 200 ⁇ L of D-Luciferin luciferase inhibitor substrate (catalog number: 122799; manufacturer: Perkin Elmer). Following substrate injection, mice were anesthetized by isoflurane inhalation, and the injection time of the luciferase inhibitor substrate was recorded. Ten minutes after substrate injection, liver and spleen were harvested, and the signal distribution and expression intensity in the isolated liver and spleen were observed. The results are shown in Table 2. The unit of average photon count in Table 2 is p/s/ cm2 /sr. The spleen/liver delivery ratio in Table 2 is the average ratio of the average photon count of the spleen to the average photon count of the liver in multiple mice.
  • the spleen/liver delivery ratio of LNP increases (for example, the spleen/liver delivery ratio of LNP with a DMG-PEG2000 concentration of 0.1% is 206 times that of LNP with a DMG-PEG2000 concentration of 1%), indicating that LNP with reduced polymer lipid content is more likely to target the spleen, which is beneficial for the targeted delivery of active substances to the spleen.
  • Example 1 with (abbreviated as 011), DSPC, cholesterol and DMG-PEG2000 were used to prepare lipid nanoparticle formulations for encapsulating Fluc mRNA.
  • the mass ratio of Fluc mRNA to Lipid in the prepared lipid nanoparticle formulations was 1:10, the concentration of mRNA was 0.1 mg/mL, the encapsulation efficiency was above 80%, and the particle size and particle size distribution index are shown in Table 3.
  • the spleen/liver delivery ratio of LNP increases (for example, the spleen/liver delivery ratio of LNP with a DMG-PEG2000 concentration of 0.1% is 359 times that of LNP with a DMG-PEG2000 concentration of 1%), indicating that LNP with reduced polymer lipid content is more likely to target the spleen, which is beneficial for the targeted delivery of active substances to the spleen.
  • Example 1 with (abbreviated as 012), DSPC, cholesterol and DMG-PEG2000 were used to prepare lipid nanoparticle formulations for encapsulating Fluc mRNA.
  • the mass ratio of Fluc mRNA to Lipid in the prepared lipid nanoparticle formulations was 1:10, the concentration of mRNA was 0.1 mg/mL, the encapsulation efficiency was above 80%, and the particle size and particle size distribution index are shown in Table 4.
  • the spleen/liver delivery ratio of LNP increases (for example, the spleen/liver delivery ratio of LNP with a DMG-PEG2000 concentration of 0.1% is 153 times that of LNP with a DMG-PEG2000 concentration of 1%), indicating that LNP with reduced polymer lipid content is more likely to target the spleen, which is beneficial for the targeted delivery of active substances to the spleen.

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Abstract

L'invention concerne une nanoparticule lipidique, qui comprend un composant actif, un lipide ionisable, un lipide auxiliaire, un lipide structural et un lipide polymère. Le lipide polymère représente de 0 à 0,8 % en moles du lipide total existant dans la nanoparticule lipidique. La réduction de la teneur en lipide polymère dans la nanoparticule lipidique peut modifier la spécificité d'administration de la nanoparticule lipidique, de telle sorte que la nanoparticule lipidique peut cibler la rate ou les cellules immunitaires.
PCT/CN2025/092313 2024-04-30 2025-04-30 Nanoparticule lipidique et son utilisation Pending WO2025228409A1 (fr)

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CNPCT/CN2024/090904 2024-04-30
CN2024090904 2024-04-30
CNPCT/CN2024/122758 2024-09-30
CN2024122758 2024-09-30

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WO2025228409A1 true WO2025228409A1 (fr) 2025-11-06

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