Compositions and methods for organ-specific delivery of nucleic acids

Abstract

The present invention provides compositions that exhibit preferential targeting or delivery of nucleic acid compositions to specific organs. It also provides methods for treating diseases or disorders in patients, and methods for preparing lipid nanoparticles. [Solution] (A) A therapeutic agent, (B)(1) Selective organ-targeting compounds; (2) Cationic ionizable lipids; and (3) Phospholipids A composition comprising a lipid nanoparticle composition containing the following, wherein the nucleic acid is preferentially delivered to a target organ selected from the lungs, heart, brain, spleen, lymph nodes, bones, skeletal muscle, stomach, small intestine, large intestine, kidneys, bladder, breasts, testes, ovaries, uterus, spleen, thymus, brainstem, cerebellum, spinal cord, eyes, ears, tongue, or skin.

Description

[Technical Field]

[0001] This application claims priority under U.S. Provisional Application No. 62 / 726,741, filed on 4 September 2018, the entire contents of which are incorporated herein by reference.

[0002] 1. Field This disclosure relates, in general, to the field of molecular biology. More specifically, this disclosure relates to tissue-specific delivery of certain therapeutic agents, such as nucleic acids, proteins, or small molecule therapeutic agents, in lipid nanoparticles. [Background technology]

[0003] 2. Explanation of related technologies CRISPR / Cas (clustered regularly interspaced short palindromic repeat / CRISPR-related protein (Cas)) technology allows for precise, sequence-dependent editing and permanent alteration of the genome. Because CRISPR / Cas technology can target disease-causing mutations, it holds great promise for one-time genetic disease treatment. To date, successful editing has primarily been mediated by viral vectors. This requires cumbersome customization for each target and presents challenges to clinical translation due to concerns about immunogenicity, the creation of antibodies to prevent repeated administration, and rare but dangerous integration events. To expand the safe and effective applications of gene editing, achieving CRISPR / Cas editing via synthetic nanoparticles (NPs) is clearly necessary.

[0004] CRISPR / Cas enables sequence-specific DNA editing using RNA-guided CRISPR-associated protein 9 (Cas9) nuclease or its homolog, resulting in the formation of double-strand breaks (DSBs) in genomic DNA. Cas9 is guided by a programmable RNA called a single guide RNA (sgRNA). The Cas9 / sgRNA complex recognizes complementary genomic sequences containing a 3' protospacer adjacent motif (PAM) sequence. Following DNA breaks, the DSB repair pathway induces directional mutagenesis, i.e., insertions / deletions (indels) that delete target genes. For therapeutic practicality, transient Cas9 expression that limits off-target genomic changes is preferred. Since both the Cas9 protein and sgRNA must be present in the same cell, co-delivering Cas9 mRNA and sequence-targeted sgRNA to a single NP is an attractive method, especially for in vivo applications where tissue penetration and cell uptake are more difficult. While CRISPR / Cas editing using viruses, membrane deformation, ribonucleoprotein complex delivery, and hydrodynamic injection is functional, it has limitations that may hinder in vivo therapeutic use in clinical settings, including persistent Cas9 expression and off-target editing. Furthermore, these delivery systems generally lack selectivity for the specific organs where editing is required. For example, most lipid nanoparticles accumulate in the liver through biological processes, thus reducing the potency of the composition when delivered to the target organ.

[0005] Similarly, other therapeutic agents, such as protein and small molecule therapies, may benefit from organ-specific delivery. Many different types of compounds, such as chemotherapeutic agents, exhibit considerable cytotoxicity. Off-target effects can be reduced if these compounds can be better directed to deliver to the desired organ.

[0006] Therefore, there is still a need to develop novel lipid nanoparticles that exhibit preferential delivery to specific organs. [Overview of the project]

[0007] overview In several aspects, this disclosure provides lipid compositions that exhibit organ-specific delivery. These compositions may be used to deliver nucleic acid components to specific organs.

[0008] In some aspects, this disclosure is relevant. (A) Therapeutic agents, (B) (1) Selective organ-targeting compounds; (2) Cationic ionizable lipids; and (3) Phospholipids A lipid nanoparticle composition containing and The present invention provides a composition comprising nucleic acids that preferentially deliver nucleic acids to a target organ selected from the lungs, heart, brain, spleen, lymph nodes, bone, bone marrow, skeletal muscle, stomach, small intestine, large intestine, kidney, bladder, breast, liver, testes, ovaries, uterus, spleen, thymus, brainstem, cerebellum, spinal cord, eye, ear, tongue, or skin. In some embodiments, the target organ is selected from the lungs, heart, brain, spleen, lymph nodes, bone, bone marrow, skeletal muscle, stomach, small intestine, large intestine, kidney, bladder, breast, testes, ovaries, uterus, spleen, thymus, brainstem, cerebellum, spinal cord, eye, ear, tongue, or skin.

[0009] In some embodiments, the target organ is the lung, lymph node, or spleen. In some embodiments, the target organ is the lung. In other embodiments, the target organ is the spleen. In other embodiments, the target organ is the liver. In other embodiments, the target organ is the lymph node.

[0010] In some embodiments, the selectively organ-targeted compound is a permanent cationic lipid. In some embodiments, the permanent cationic lipid is present at a molar percentage of about 5% to about 20% based on the lipid nanoparticle composition. In some embodiments, a molar percentage of the permanent cationic lipid of about 12% to about 18% is present. In some embodiments, the molar percentage of the permanent cationic lipid is about 15%. In some embodiments, the permanent cationic lipid is present at a molar percentage of about 20% to about 65% based on the lipid nanoparticle composition. In some embodiments, a molar percentage of the permanent cationic lipid of about 40% to about 61% is present. In some embodiments, the molar percentage of the permanent cationic lipid is about 50%.

[0011] In some embodiments, the permanent cationic lipid contains a quaternary ammonium ion. In some embodiments, the permanent cationic lipid is further defined as TIFF2026082961000001.tif23128, wherein R1 and R2 are each independently alkyl (C8~C24) , alkenyl (C8~C24) , or a substituted form of any of the groups; R3, R3', and R3'' are each independently alkyl (C≦6) or substituted alkyl (C≦6) ; X - is a monovalent anion.

[0012] In some embodiments, R1 is alkenyl (C8~C24) or substituted alkenyl (C8~C24) . In some embodiments, R2 is alkenyl (C8~C24) or substituted alkenyl (C8~C24) . In other embodiments, R1 is alkyl (C8~C24) or substituted alkyl (C8~C24) . In other embodiments, R2 is alkyl (C8~C24) or substituted alkyl (C8~C24)In some embodiments, R1 and R2 are both the same. In some embodiments, R3, R3', and R3'' are each the same. In some embodiments, R3, R3', and R3'' are each methyl. In some embodiments, X - These are halide anions, for example, bromides or chlorides. In some embodiments, the permanent cationic lipid is It is further defined as TIFF2026082961000002.tif21128.

[0013] In other forms, permanent cationic lipids are, Further defined as TIFF2026082961000003.tif14128, During the ceremony, R4 and R4' are each independently alkyl (C6~C24) Alkenil (C6~C24) , or a substitution of either group; R4'' is alkyl (C≦24) Alkenil (C≦24) , or a substitution of either group; R4'''' is alkyl (C1~C8) Alkenil (C2~C8) , or a substitutional form of either group; and X2 is a monovalent anion.

[0014] In some embodiments, R4 is alkyl (C6~C24) or substituted alkyl (C6~C24) For example, octadecyl. In some embodiments, R4' is alkyl. (C6~C24) or substituted alkyl (C6~C24) For example, octadecyl. In some embodiments, R4'' is alkyl. (C≦24) or substituted alkyl (C≦24) In some embodiments, R4'' is alkyl. (C≦8) or substituted alkyl (C≦8) For example, methyl. In some embodiments, R4''' is alkyl (C1~C8) or substituted alkyl (C1~C8), for example, methyl. In some embodiments, X2 is a halide, for example, a chloride or bromide. In some embodiments, the permanent cationic lipid is It is further defined as TIFF2026082961000004.tif9128.

[0015] In some embodiments, permanent cationic lipids are Further defined as TIFF2026082961000005.tif24128, During the ceremony, R1 and R2 are each independently alkyl (C8~C24) Alkenil (C8~C24) , or a substitution of either group; R3, R3', and R3'' are each independently alkyl (C≦6) or substituted alkyl (C≦6) and; R4 is alkyl (C≦6) or substituted alkyl (C≦6) and X - It is a monovalent anion.

[0016] In some ways, R1 is alkenil (C8~C24) or substituted alkenyl (C8~C24) In some embodiments, R2 is an alkenil. (C8~C24) or substituted alkenyl (C8~C24) In another embodiment, R1 is alkyl. (C8~C24) or substituted alkyl (C8~C24) In another embodiment, R2 is alkyl. (C8~C24) or substituted alkyl (C8~C24) Therefore, in some ways, R1 and R2 are both the same.

[0017] In some embodiments, R3, R3', and R3'' are identical, for example, R3, R3', and R3'' are each methyl. In some embodiments, R4 is alkyl (C≦6) For example, ethyl. In some embodiments, X -These are halide anions, such as bromides or chlorides.

[0018] In some embodiments, permanent cationic lipids are It is further defined as TIFF2026082961000006.tif27128.

[0019] In other embodiments, the selective organ-targeting compound is a permanent anionic lipid. In some embodiments, the permanent anionic lipid is present in a molar percentage of about 5% to about 50% of the lipid nanoparticle composition. In some embodiments, the molar percentage of the permanent anionic lipid is about 10% to about 45%. In some embodiments, the molar percentage of the permanent anionic lipid is about 30%. In some embodiments, the permanent anionic lipid contains a phosphate group.

[0020] In some embodiments, permanent anionic lipids are Further defined as TIFF2026082961000007.tif24128, During the ceremony, R1 and R2 are each independently alkyl (C8~C24) Alkenil (C8~C24) , or a substitution of either group; R3 is hydrogen, alkyl (C≦6) , or substituted alkyl (C≦6) , or -Y1-R4, During the ceremony, Y1 is Alkanzil (C≦6) or substitute alkanediyl (C≦6) and R4 is acyloxy (C≦8~24) or substituted acyloxy (C≦8~24) That is the case.

[0021] In some ways, R1 is alkenil (C8~C24) or substituted alkenyl (C8~C24) In another embodiment, R2 is an alkenil. (C8~C24) or substituted alkenyl (C8~C24) In another embodiment, R1 is alkyl. (C8~C24)or substituted alkyl (C8~C24) In another embodiment, R2 is alkyl. (C8~C24) or substituted alkyl (C8~C24) Therefore, in some ways, R1 and R2 are both the same.

[0022] In some embodiments, R3 is hydrogen. In other embodiments, R3 is -Y1-R4, where in the formula, Y1 is Alkanzil (C≦6) or substitute alkanediyl (C≦6) and R4 is acyloxy (C≦8~24) or substituted acyloxy (C≦8~24) That is the case.

[0023] In some embodiments, Y1 is a substituted alkanediyl (C≦6) For example, 2-hydroxypropanediyl. In some embodiments, R4 is acyloxy (C≦8~24) For example, octadecenoate. In some embodiments, permanent anionic lipids are It is further defined as TIFF2026082961000008.tif103128.

[0024] In another embodiment, selective organ-targeting compounds are C6-C 24 This is diacylphosphotidylcholine. In some embodiments, diacylphosphotidylcholine is present in a molar percentage of about 5% to about 50% of the lipid nanoparticle composition. In some embodiments, diacylphosphotidylcholine is present in a molar percentage of about 10% to about 45%, for example, about 30%.

[0025] In some embodiments, the selective organ-targeting compound comprises at least two fatty acid chains, a quaternary amine, and an anionic phosphate group. In some embodiments, diacylphosphotidylcholine is Further defined as TIFF2026082961000009.tif24128, During the ceremony, R1 and R2 are each independently alkyl (C8~C24) Alkenil (C8~C24) , or a substitution of either group; R3, R3', and R3'' are each independently alkyl (C≦6) or substituted alkyl (C≦6) and X - It is a monovalent anion.

[0026] In some ways, R1 is alkenil (C8~C24) or substituted alkenyl (C8~C24) In some embodiments, R2 is an alkenil. (C8~C24) or substituted alkenyl (C8~C24) In another embodiment, R1 is alkyl. (C8~C24) or substituted alkyl (C8~C24) In another embodiment, R2 is alkyl. (C8~C24) or substituted alkyl (C8~C24) Therefore, in some ways, R1 and R2 are both the same.

[0027] In some embodiments, R3, R3', and R3'' are identical. In some embodiments, R3, R3', and R3'' are methyl. In some embodiments, X - It is a halide anion such as a bromide or chloride. In some embodiments, diacylphosphotidylcholine is It is further defined as TIFF2026082961000010.tif21128.

[0028] In some embodiments, cationic ionizable lipids are present at a molar percentage of about 5% to about 30% of the lipid nanoparticle composition. In some embodiments, cationic ionizable lipids are present at a molar percentage of about 7.5% to about 20%. In some embodiments, the molar percentage of cationic ionizable lipids is about 11.9%. In some embodiments, cationic ionizable lipids are present at a molar percentage of about 15% to about 30% of the lipid nanoparticle composition. In some embodiments, cationic ionizable lipids are present at a molar percentage of about 15% to about 25%. In some embodiments, the molar percentage of cationic ionizable lipids is about 20.3%.

[0029] In some embodiments, the cationic ionizable lipid contains an ammonium group that is positively charged at physiological pH and contains at least two hydrophobic groups. In some embodiments, the ammonium group is positively charged at pH about 6 to about 8. In some embodiments, the cationic ionizable lipid is a dendrimer or dendron. In some embodiments, the cationic ionizable lipid contains at least two C6-C24 alkyl or alkenyl groups. In some embodiments, the cationic ionizable lipid contains at least two C8-C24 alkyl groups.

[0030] In some embodiments, phospholipids are present in a molar percentage of about 8% to about 20% of the lipid nanoparticle composition. In some embodiments, the molar percentage of phospholipids is about 10% to about 14%. In some embodiments, the molar percentage of phospholipids is about 11.9%. In other embodiments, phospholipids are present in a molar percentage of about 20% to about 23% of the lipid nanoparticle composition. In some embodiments, the molar percentage of phospholipids is about 20% to about 21%. In some embodiments, the molar percentage of phospholipids is about 20.3%. In some embodiments, the phospholipid is further defined as 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine or 1,2-distearoyl-sn-glycero-3-phosphocholine. In some embodiments, the phospholipid is 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine.

[0031] In some embodiments, the composition further comprises a steroid. In some embodiments, the steroid is present in a molar percentage of about 39% to about 46% of the lipid nanoparticle composition. In some embodiments, the molar percentage of the steroid is about 40% to about 43%. In some embodiments, the molar percentage of the steroid is about 40.5%. In other embodiments, the steroid is present in a molar percentage of about 15% to about 39% of the lipid nanoparticle composition. In some embodiments, the molar percentage of the steroid is about 20% to about 27.5%. In some embodiments, the molar percentage of the steroid is about 23.8%. In some embodiments, the steroid is cholesterol.

[0032] In some embodiments, the composition further comprises a PEGylated lipid. In some embodiments, the PEGylated lipid is present at a molar percentage of from about 0.5% to about 10.0% based on the lipid nanoparticle composition. In some embodiments, there is a molar percentage of PEGylated lipid of from about 0.5% to about 5.0%. In other embodiments, there is a molar percentage of PEGylated lipid of from about 2.0% to about 2.8%. In some embodiments, the molar percentage of PEGylated lipid is about 2.4%. In other embodiments, the PEGylated lipid is present at a molar percentage of from about 3.9% to about 4.6% based on the lipid nanoparticle composition. In some embodiments, there is a molar percentage of PEGylated lipid of from about 4.0% to about 4.3%. In some embodiments, the molar percentage of PEGylated lipid is about 4.1%. In some embodiments, the PEGylated lipid comprises a PEG component of from about 1000 to about 10,000 daltons. In some embodiments, the PEG lipid is a PEGylated diacylglycerol. In some embodiments, the PEG lipid is further defined by the following formula: TIFF2026082961000011.tif18128, wherein R 12 and R 13 are each independently alkyl (C≦24) , alkenyl (C≦24) , or a substituted form of any of these groups; R e is hydrogen, alkyl (C≦8) , or substituted alkyl (C≦8) ; and x is from 1 to 250. In some embodiments, the PEG lipid is dimyristoyl-sn-glycerol or a compound of the following formula: TIFF2026082961000012.tif38128, wherein n1 is from 5 to 250; and n2 and n3 are each independently from 2 to 25.

[0033] In some embodiments, the composition comprises cholesterol and DMG-PEG. In some embodiments, the composition comprises DOPE. In other embodiments, the composition comprises DSPC. In some embodiments, the composition further comprises DLin-MC3-DMA. In other embodiments, the composition further comprises C12-200. In some embodiments, the composition further comprises 3A5-SC8, 3A3-SC8, 4A1-SC8, 4A3-SC8, 5A2-SC8, or 5A2-SC8 having five tails. In some embodiments, the composition further comprises 5A2-SC8. In some embodiments, the composition further comprises DOTAP. In some embodiments, the composition comprises cholesterol, DMG-PEG, DSPC, DLin-MC3-DMA, and DOTAP.

[0034] In some embodiments, therapeutic agents include small molecules such as anticancer agents, antifungal agents, psychiatric agents such as analgesics, agents that alter the level of consciousness such as anesthetics or hypnotics, nonsteroidal anti-inflammatory drugs (NSAIDS), anthelmintics, antiacids, anti-anginal agents, antiarrhythmic agents, anti-asthmatic agents, antibacterial agents, anti-benign prostatic agents, anticoagulants, antidepressants, antidiabetic agents, antiemetics, antiepileptic agents, antigout agents, antihypertensive agents, anti-inflammatory agents, antimalarial agents, anti-migraine agents, anti-muscarinic agents, anti-cancer agents, anti-obesity agents, anti-osteoporosis agents, anti-Parkinson's disease agents, antiproliferative agents, antiparasitic agents, antithyroid agents, antitussives, antitussives, anti-osteoporosis agents, anti-Parkinson's disease agents, antiproliferative agents, antiparasitic agents, antithyroid agents, antitussives, antitussives, antitussives, antiviral agents, anxiolytics, appetite suppressants, beta-blockers, cardiac inotropic agents, chemotherapeutic agents, and cognitive enhancers. The therapeutic agent is a small molecule selected from enhancers, contraceptives, corticosteroids, Cox-2 inhibitors, diuretics, erectile dysfunction treatments, expectorants, gastrointestinal agents, histamine receptor antagonists, immunosuppressants, keratolytic agents, lipid regulators, leukotriene inhibitors, macrolides, muscle relaxants, nerve blockers, nutritional supplements, opioid analgesics, protease inhibitors, or sedatives. In other embodiments, the therapeutic agent is a protein. In other embodiments, the therapeutic agent is a nucleic acid, for example, a therapeutic nucleic acid. In some embodiments, the nucleic acid is siRNA, miRNA, pri-miRNA, messenger RNA (mRNA), clustered and regularly arranged short palindromic sequence repeat (CRISPR)-associated nucleic acid, single guide RNA (sgRNA), CRISPRRNA (crRNA), transactivating crRNA (tracrRNA), plasmid DNA (pDNA), transfer RNA (tRNA), antisense oligonucleotide (ASO), guide RNA, double-stranded DNA (dsDNA), single-stranded DNA (ssDNA), single-stranded RNA (ssRNA), and double-stranded RNA (dsRNA). In some embodiments, the composition comprises a first nucleic acid and a second nucleic acid. In some embodiments, the first nucleic acid is messenger RNA. In some embodiments, the second nucleic acid is single guide RNA. In some embodiments, the first nucleic acid is messenger RNA (mRNA) and single guide RNA (sgRNA).In some embodiments, nucleic acids are present in a ratio of approximately 1:1 to approximately 1:100 of lipid nanoparticle composition. In some embodiments, the ratio is approximately 1:10 to approximately 1:60. In some embodiments, the ratio is approximately 1:40.

[0035] In some embodiments, the composition further comprises a protein. In some embodiments, the protein is a protein related to translation or transcription. In some embodiments, the protein is related to the CRISPR process. In some embodiments, the protein is a CRISPR-related protein. In some embodiments, the protein is Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csfl, Csf2, Csf3, Csf4, its homolog, or a modified version thereof. In some embodiments, the protein is Cas9. In some embodiments, the protein and nucleic acid are present in a molar ratio of about 1:1 to about 1:20. In some embodiments, the molar ratio is about 1:1 to about 1:10. In some embodiments, the molar ratio is about 1:3 to about 1:8.

[0036] In some embodiments, the composition has a negative zeta potential. In some embodiments, the zeta potential is between -0.25 mV and about -10 mV. In some embodiments, the zeta potential is between about -0.5 mV and about -2 mV. In some embodiments, the composition comprises both a protein and a nucleic acid. In some embodiments, the composition comprises a Cas9 protein and a single guide nucleic acid. In some embodiments, the composition comprises a Cas9 protein, a single guide nucleic acid, and donor DNA.

[0037] In another context, this disclosure is: (A) Compositions described herein, (B) Excipients and The present invention provides a pharmaceutical composition containing [the specified ingredient].

[0038] In some embodiments, the pharmaceutical composition is formulated for administration orally, intraadiposally, intraarterially, intraarterially, intraarticularly, intracranially, intradermally, intrafocally, intramuscularly, intranasal cavity, intraocularly, intrapericardially, intraperitoneally, intrapleurally, intraprostate, intrarectally, subarachnoidally, intratracheally, intratumorally, intraumbilical, intravaginally, intravenously, intravesicularly, intravitreously, liposomeally, topically, intramucosally, parenterally, intrarectally, subconjunctivally, subcutaneously, sublingually, locally, transbuccally, percutaneously, intravaginally, in cream, in lipid composition, via catheter, via lavage, via continuous infusion, via infusion, via inhalation, via injection, via local delivery, or via local perfusion. In some embodiments, the pharmaceutical composition is formulated for intravenous or intra-arterial injection. In some embodiments, the excipient is a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutically acceptable carrier is a solvent or solution. In some embodiments, pharmaceutical compositions are formulated as unit doses.

[0039] In yet another aspect, the present disclosure provides a method for regulating gene expression, comprising the step of delivering nucleic acid to cells, the method comprising contacting the cells with a composition or pharmaceutical composition described herein under conditions sufficient to cause uptake of nucleic acid into the cells.

[0040] In some embodiments, the cells are contacted in vitro or ex vivo. In some embodiments, the cells are contacted in vivo. In some embodiments, the regulation of gene expression is sufficient to treat a disease or disorder, such as cancer.

[0041] In yet another aspect, the present disclosure provides a method for treating a disease or disorder in a patient, comprising the step of administering to the patient a pharmaceutically effective amount of a composition or pharmaceutical composition described herein, wherein the composition or pharmaceutical composition comprises a therapeutic nucleic acid or protein for the disease or disorder.

[0042] In some embodiments, the disease or disorder is cancer. In some embodiments, the method further includes the step of administering one or more further cancer therapies to a patient. In some embodiments, the cancer therapy is a chemotherapeutic compound, surgery, radiotherapy, or immunotherapy. In some embodiments, the composition or pharmaceutical composition is administered to the patient once. In other embodiments, the composition or pharmaceutical composition is administered to the patient two or more times. In some embodiments, the patient is a mammal such as a human.

[0043] Furthermore, in other aspects, this disclosure relates to a method for preparing lipid nanoparticles, (A) A step of dissolving a permanent cationic lipid, a cationic ionizable lipid, and a phospholipid in a first solution to form a lipid solution, wherein the lipid solution is formed in an organic solvent; (B) A step of dissolving a therapeutic agent in a buffer to form a buffered therapeutic agent solution, wherein the buffer is a buffer with a pH of approximately 6.8 to approximately 7.6; and (C) A step of mixing a lipid solution with a buffered therapeutic agent solution to form lipid nanoparticles. This provides a method that includes [something].

[0044] In some embodiments, the organic solvent is a C1-C4 alcohol solvent such as ethanol. In some embodiments, the buffer is aqueous PBS buffer. In some embodiments, the encapsulation efficiency of the method exceeds 80%.

[0045] In another context, this disclosure is relevant. (A) Therapeutic agents, (B) (1) Cationic ionizable lipids; (2) Phospholipids; and (3) Selective organ-targeting compounds A lipid nanoparticle composition containing and The present invention provides a composition comprising an organ-targeting ligand that causes preferential delivery of the composition to organs other than the liver.

[0046] Furthermore, in yet another context, this disclosure is relevant. (A) Therapeutic agents, (B) (1) Cationic ionizable lipids; (2) Phospholipids; (3) Selective organ-targeting compounds; (4) Steroids; and (5) PEG lipids A lipid nanoparticle composition containing and The present invention provides a composition comprising an organ-targeting ligand that causes preferential delivery of the composition to organs other than the liver.

[0047] In another aspect, this disclosure relates to therapeutic agents and (A) Cationic ionizable lipids; (B) Phospholipids; and (C) Selective organ-targeting compounds A lipid nanoparticle composition containing and A composition comprising, having an apparent pK of about 8 to about 13 a The present invention provides a composition that has the following properties and primarily delivers nucleic acids to the lungs.

[0048] In another context, this disclosure relates to a therapeutic agent and (A) Cationic ionizable lipids; (B) Phospholipids; (C) Selective organ-targeting compounds; (D) Steroids; and (E)PEG lipids; A lipid nanoparticle composition containing and A composition comprising, having an apparent pK of about 8 to about 13 aProvided is a composition having the ability to primarily deliver nucleic acids to the lungs.

[0049] In yet another aspect, the present disclosure provides a composition comprising a therapeutic agent and (A) a cationic ionizable lipid; (B) a phospholipid; and (C) a selective organ targeting compound wherein the lipid nanoparticle composition has an apparent pK of about 3 to about 6 and primarily delivers nucleic acids to the spleen. Provided is a composition having the ability to primarily deliver nucleic acids to the spleen. a

[0050] In yet another aspect, the present disclosure provides a composition comprising a therapeutic agent and (A) a steroid; (B) a cationic ionizable lipid; (C) a phospholipid; (D) a PEG lipid; and (E) a selective organ targeting compound wherein the lipid nanoparticle composition has an apparent pK of about 3 to about 6 and primarily delivers nucleic acids to the spleen. Provided is a composition having the ability to primarily deliver nucleic acids to the spleen. a

[0051] In another aspect, the present disclosure provides a composition comprising a therapeutic agent and (A) a cationic ionizable lipid; (B) a phospholipid; and (C) C6-C 24 diacyl phosphatidylcholine wherein the lipid nanoparticle composition primarily delivers nucleic acids to the lymph nodes. Provided is a composition having the ability to primarily deliver nucleic acids to the lymph nodes.

[0052] In yet another aspect, the present disclosure provides a composition comprising a therapeutic agent and (A) a steroid; (B) a cationic ionizable lipid; (C) a phospholipid; (D) a PEG lipid; and (E) C6-C 24 ​Diacylphosphotidylcholine A lipid nanoparticle composition containing and The present invention provides a composition comprising the above, which primarily delivers nucleic acids to lymph nodes.

[0053] Furthermore, in yet another aspect, this disclosure relates to therapeutic agents and (A) Cationic ionizable lipids; (B) Phospholipids; and (C) Selective organ-targeting compounds A lipid nanoparticle composition containing and The present invention provides a composition comprising a substance such that the surface of the composition interacts with vitronectin, and the composition primarily delivers nucleic acids to the lungs.

[0054] In another aspect, this disclosure relates to therapeutic agents and (A) Cationic ionizable lipids; (B) Phospholipids; (C) Selective organ-targeting compounds; (D) Steroids; and (E)PEG lipids A lipid nanoparticle composition containing and The present invention provides a composition comprising a substance such that the surface of the composition interacts with vitronectin, and the composition primarily delivers nucleic acids to the lungs.

[0055] Furthermore, in yet another aspect, this disclosure relates to therapeutic agents and (A) Cationic ionizable lipids; (B) Phospholipids; and (C) Selective organ-targeting compounds A lipid nanoparticle composition containing and The present invention provides a composition comprising ApoH, wherein the surface of the composition interacts with ApoH, and the composition primarily delivers nucleic acids to the spleen.

[0056] In another context, this disclosure relates to a therapeutic agent and (A) Steroids; (B) Cationic ionizable lipids; (C) Phospholipids; (D)PEG lipids; and (E) Selective organ-targeting compounds A lipid nanoparticle composition containing and The present invention provides a composition comprising ApoH, wherein the surface of the composition interacts with ApoH, and the composition primarily delivers nucleic acids to the spleen.

[0057] In yet another aspect, the present disclosure provides a composition comprising a therapeutic agent and a lipid nanoparticle composition, wherein a targeted protein present on the surface of the composition is bound to a target protein substantially present in a target organ, and the target organ is not the liver.

[0058] In some embodiments, the targeting protein is selected from vitronectin or β2-glycoprotein I (ApoH). In some embodiments, the targeting protein is vitronectin and the target organ is the lung. In other embodiments, the targeting protein is ApoH and the target organ is the spleen.

[0059] In some embodiments, the lipid nanoparticle composition further comprises a selective organ-targeting compound that modifies the binding of proteins on the protein corona. In some embodiments, the selective organ-targeting compound is further selected from sugars, lipids, small molecule therapeutics, vitamins, or proteins. In some embodiments, the selective organ-targeting compound is a lipid, such as a permanently cationic lipid, a permanently anionic lipid, or phosphotidylcholine. In some embodiments, the lipid nanoparticle composition further comprises a cationic ionizable lipid. In some embodiments, the lipid nanoparticle composition further comprises a phospholipid. In some embodiments, the lipid nanoparticle composition further comprises a steroid. In some embodiments, the lipid nanoparticle composition further comprises a PEG lipid.

[0060] In another aspect, the present disclosure relates to a composition comprising a therapeutic agent and a lipid nanoparticle composition, wherein the lipid nanoparticle composition comprises a selective organ-targeting compound, and the selective organ-targeting compound has an apparent pK of about 3 to about 6.a The present invention provides a composition that yields a lipid nanoparticle composition containing [specific characteristic].

[0061] In another aspect, the present disclosure relates to a composition comprising a therapeutic agent and a lipid nanoparticle composition, wherein the lipid nanoparticle composition comprises a selective organ-targeting compound, and the selective organ-targeting compound has an apparent pK of about 8 to about 13. a The present invention provides a composition that yields a lipid nanoparticle composition containing [specific characteristic].

[0062] As used herein, "essentially absent" with respect to a specified component means that none of the specified components are intentionally formulated in the composition and / or are present only as contaminants or in trace amounts. The total amount of a specified component resulting from unintentional contamination of the composition is preferably less than 0.01%. Most preferably, the amount of a specified component cannot be detected using standard analytical methods.

[0063] As used herein and in the claims, “a” or “an” may mean one or more. As used herein and in the claims, when used with the word “comprising,” “a” or “an” may mean one or more. As used herein and in the claims, “another” or “further” may mean at least two or more.

[0064] As used herein and in the claims, the term “approximately” is used to indicate that a value includes inherent variations in the errors of the apparatus or method used to determine that value, or variations present among the objects being tested.

[0065] [Invention 1001] (A) Therapeutic agents, (B) (1) Selective organ-targeting compounds; (2) Cationic ionizable lipids; and (3) Phospholipids A lipid nanoparticle composition containing and A composition comprising the following, wherein nucleic acids are preferentially delivered to a target organ selected from the lungs, heart, brain, spleen, lymph nodes, bones, skeletal muscle, stomach, small intestine, large intestine, kidneys, bladder, breasts, testes, ovaries, uterus, spleen, thymus, brainstem, cerebellum, spinal cord, eyes, ears, tongue, or skin. [Invention 1002] A composition of the present invention 1001, wherein the target organ is the lung, lymph node, or spleen. [Invention 1003] A composition according to the present invention 1001 or 1002, wherein the selective organ-targeting compound is a permanently cationic lipid. [Invention 1004] The composition of the present invention 1003, wherein permanent cationic lipids are present in a molar percentage of approximately 5% to approximately 20% relative to the lipid nanoparticle composition. [Invention 1005] The composition of the present invention 1003, wherein permanent cationic lipids are present in a molar percentage of approximately 20% to approximately 65% ​​relative to the lipid nanoparticle composition. [Invention 1006] A composition according to any one of invention 1003 to 1005, wherein the permanent cationic lipid contains a quaternary ammonium ion. [Invention 1007] Permanent cationic lipids Further defined as TIFF2026082961000013.tif23128, During the ceremony, R1 and R2 are each independently alkyl (C8~C24) Alkenil (C8~C24) , or a substitution of either group; R3, R3', and R3'' are each independently alkyl (C≦6) or substituted alkyl (C≦6) and; X - It is a monovalent anion. A composition according to any of the present invention 1003 to 1006. [Invention 1008] Permanent cationic lipids The composition of the present invention 1007, further defined as TIFF2026082961000014.tif21128. [Invention 1009] Permanent cationic lipids Further defined as TIFF2026082961000015.tif14128, During the ceremony, R4 and R4' are each independently alkyl (C6~C24) Alkenil (C6~C24) , or a substitution of either group; R4'' is alkyl (C≦24) Alkenil (C≦24) , or a substitution of either group; R4'''' is alkyl (C1~C8) Alkenil (C2~C8) , or a substitutional form of either group; and X2 is a monovalent anion. A composition according to any of the present invention 1003 to 1006. [Invention 1010] Permanent cationic lipids The composition of the present invention 1009, further defined as TIFF2026082961000016.tif10128. [Invention 1011] Permanent cationic lipids Further defined as TIFF2026082961000017.tif24128, During the ceremony, R1 and R2 are each independently alkyl (C8~C24) Alkenil (C8~C24) , or a substitution of either group; R3, R3', and R3'' are each independently alkyl (C≦6) or substituted alkyl (C≦6) and; R4 is alkyl (C≦6) or substituted alkyl (C≦6) and X - It is a monovalent anion. A composition according to any of the present invention 1003 to 1006. [Invention 1012] Permanent cationic lipids A composition of the present invention 1011, further defined as TIFF2026082961000018.tif27128. [Invention 1013] A composition according to the present invention 1001 or 1002, wherein the selective organ-targeting compound is a permanent anionic lipid. [Invention 1014] The composition of the present invention 1013, wherein permanent anionic lipids are present in a molar percentage of approximately 5% to approximately 50% relative to the lipid nanoparticle composition. [Invention 1015] A composition according to the present invention 1013 or 1014, wherein the permanent anionic lipid contains a phosphate group. [Invention 1016] Permanent anionic lipids, Further defined as TIFF2026082961000019.tif24128, During the ceremony, R1 and R2 are each independently alkyl (C8~C24) Alkenil (C8~C24) , or a substitution of either group; R3 is hydrogen, alkyl (C≦6) , or substituted alkyl (C≦6) , or -Y1-R4, During the ceremony, Y1 is Arcanziel (C≦6) or substitute alkanediyl (C≦6) and R4 is acyloxy (C≦8~24) or substituted acyloxy (C≦8~24) That is, A composition according to any of invention 1013 to 1015. [Invention 1017] Permanent anionic lipids, The composition of the present invention 1016, further defined as TIFF2026082961000020.tif103128. [Invention 1018] Selective organ targeting compounds24 A composition of the present invention 1001 or 1002, wherein the composition is diacylphosphotidylcholine. [Invention 1019] The composition of the present invention 1018, wherein diacylphosphotidylcholine is present in a molar percentage of approximately 5% to approximately 50% relative to the lipid nanoparticle composition. [Invention 1020] A composition of the present invention 1018 or 1019, wherein the selective organ-targeting compound comprises at least two fatty acid chains, a quaternary amine, and an anionic phosphate group. [Invention 1021] Diacylphosphotidylcholine Further defined as TIFF2026082961000021.tif24128, During the ceremony, R1 and R2 are each independently alkyl (C8~C24) Alkenil (C8~C24) , or a substitution of either group; R3, R3', and R3'' are each independently alkyl (C≦6) or substituted alkyl (C≦6) and X - It is a monovalent anion. A composition according to any of invention 1018 to 1020. [Invention 1022] Diacylphosphotidylcholine A composition of the present invention 1021, further defined as TIFF2026082961000022.tif21128. [Invention 1023] A composition according to any one of the present invention 1001 to 1022, wherein cationic ionizable lipids are present in a molar percentage of approximately 5% to approximately 30% relative to the lipid nanoparticle composition. [Invention 1024] A composition according to any one of the present invention 1001 to 1022, wherein cationic ionizable lipids are present in a molar percentage of approximately 15% to approximately 30% relative to the lipid nanoparticle composition. [Invention 1025] A composition according to any one of the present invention 1001 to 1024, wherein the cationic ionizable lipid contains an ammonium group that is positively charged at physiological pH and contains at least two hydrophobic groups. [Invention 1026] Cationic ionizable lipids have at least two C6-C 24 A composition according to the present invention 1025, comprising an alkyl group or an alkenyl group. [Invention 1027] A composition according to any one of the present invention 1001 to 1026, wherein phospholipids are present in a molar percentage of approximately 8% to approximately 20% relative to the lipid nanoparticle composition. [Invention 1028] A composition according to any one of the present invention 1001 to 1026, wherein phospholipids are present in a molar percentage of approximately 20% to approximately 23% relative to the lipid nanoparticle composition. [Invention 1029] A composition of any of the present inventions 1001 to 1028, wherein the phospholipid is further defined as 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine or 1,2-distearoyl-sn-glycero-3-phosphocholine. [Invention 1030] A composition according to any one of the present invention 1001 to 1029, further comprising a steroid. [Invention 1031] The composition of the present invention 1030, wherein a steroid is present in a molar percentage of approximately 39% to approximately 46% relative to the lipid nanoparticle composition. [Invention 1032] The composition of the present invention 1031, wherein a steroid is present in a molar percentage of approximately 15% to approximately 39% relative to the lipid nanoparticle composition. [Invention 1033] A composition of the present invention 1031 or 1032, wherein the steroid is cholesterol. [Invention 1034] A composition according to any one of the present invention 1001 to 1033, further comprising PEGylated lipids. [Invention 1035] The composition of the present invention 1034, wherein PEGylated lipids are present in a molar percentage of approximately 0.5% to approximately 10.0% relative to the lipid nanoparticle composition. [Invention 1036] The composition of the present invention 1035, wherein PEGylated lipids are present in a molar percentage of approximately 3.9% to approximately 4.6% relative to the lipid nanoparticle composition. [Invention 1037] A composition according to the present invention 1035 or 1036, wherein the PEGylated lipid contains approximately 1,000 to approximately 10,000 daltons of PEG components. [Invention 1038] The composition of the present invention 1037, wherein the PEG lipid is PEGylated diacylglycerol. [Invention 1039] PEG lipids are expressed by the following formula: Further defined by TIFF2026082961000023.tif18128, During the ceremony, R 12 and R 13 Each of them independently, alkyl (C≦24) Alkenil (C≦24) , or a substitutional form of any of these groups; R e However, hydrogen, alkyl (C≦8) , or substituted alkyl (C≦8) and x is between 1 and 250. The composition of the present invention 1038. [Invention 1040] PEG lipids are dimyristoyl-sn-glycerol or the following formula: It is a compound of TIFF2026082961000024.tif38128, During the ceremony, n1 is between 5 and 250; and n2 and n3 are independently between 2 and 25. A composition according to invention 1035 or 1036. [Invention 1041] A composition according to any of the present invention 1001 to 1040, wherein the therapeutic agent is a low molecular weight. [Invention 1042] A composition according to any one of the present invention 1001 to 1040, wherein the therapeutic agent is a protein. [Invention 1043] A composition according to any one of the present invention 1001 to 1040, wherein the therapeutic agent is nucleic acid. [Invention 1044] A composition of the present invention 1043, wherein the nucleic acid is a therapeutic nucleic acid. [Invention 1045] The composition of Invention 1043, wherein the nucleic acid is siRNA, miRNA, pri-miRNA, messenger RNA (mRNA), clustered and regularly arranged short palindromic sequence repeat (CRISPR)-associated nucleic acid, single guide RNA (sgRNA), CRISPR-RNA (crRNA), trans-activated crRNA (tracrRNA), plasmid DNA (pDNA), transfer RNA (tRNA), antisense oligonucleotide (ASO), guide RNA, double-stranded DNA (dsDNA), single-stranded DNA (ssDNA), single-stranded RNA (ssRNA), and double-stranded RNA (dsRNA). [Invention 1046] A composition according to any one of the present invention 1043 to 1045, wherein nucleic acids are present in a ratio of approximately 1:1 to approximately 1:100 of the lipid nanoparticle composition. [Invention 1047] A composition according to any one of the present invention 1001 to 1046, further comprising a protein. [Invention 1048] A composition according to any one of the present invention 1001 to 1047, comprising both a protein and a nucleic acid. [Invention 1049] (A) A composition of any of the present invention 1001 to 1048, (B) Excipients and A pharmaceutical composition containing [the specified substance]. [Invention 1050] A method for regulating gene expression, comprising the step of delivering nucleic acids to cells, The method comprising the step of contacting cells with any of the compositions or pharmaceutical compositions of the present invention 1001 to 1049 under conditions sufficient to induce nucleic acid uptake into cells. [Invention 1051] A method for treating a disease or disorder in a patient, comprising the step of administering a pharmaceutically effective amount of any composition or pharmaceutical composition according to 1001 to 1049 of the present invention to a patient in need of treatment for the disease or disorder, The method wherein the composition or pharmaceutical composition comprises a nucleic acid for therapeutic purposes against a disease or disorder. [Invention 1052] A method for preparing lipid nanoparticles, comprising the following steps: (A) A step of dissolving a selective organ-targeting compound, a cationic ionizable lipid, and a phospholipid in a first solution to form a lipid solution, wherein the lipid solution is formed in an organic solvent; (B) A step of dissolving a therapeutic agent in a buffer to form a buffered therapeutic agent solution, wherein the buffer is a buffer with a pH of approximately 6.8 to approximately 7.6; and (C) A step of mixing a lipid solution with a buffered therapeutic agent solution to form lipid nanoparticles. [Invention 1053] (A) Therapeutic agents, (B) (1) Cationic ionizable lipids; (2) Phospholipids; and (3) Selective organ-targeting compounds A lipid nanoparticle composition containing and A composition comprising, A composition wherein an organ-targeting ligand causes preferential delivery of the composition to organs other than the liver. [Invention 1054] Treatment drugs, (A) Cationic ionizable lipids; (B) Phospholipids; and (C) Selective organ-targeting compounds A lipid nanoparticle composition containing and A composition comprising, Approximately 8 to 13 apparent pK a The composition having a property that primarily delivers nucleic acids to the lungs. [Invention 1055] Treatment drugs, (A) Cationic ionizable lipids; (B) Phospholipids; and (C) Selective organ-targeting compounds A lipid nanoparticle composition containing and A composition comprising, Apparent pK of approximately 3 to 6 a The composition having the above-mentioned properties and primarily delivering nucleic acids to the spleen. [Invention 1056] Treatment drugs, (A) Cationic ionizable lipids; (B) Phospholipids; and (C)C6~C 24 Diacylphosphotidylcholine A lipid nanoparticle composition containing and A composition comprising, The composition, which primarily delivers nucleic acids to lymph nodes. [Invention 1057] Treatment drugs, (A) Cationic ionizable lipids; (B) Phospholipids; and (C) Selective organ-targeting compounds A lipid nanoparticle composition containing and A composition comprising, A composition wherein the surface of the composition interacts with vitronectin, and the composition primarily delivers nucleic acids to the lungs. [Invention 1058] Treatment drugs, (A) Cationic ionizable lipids; (B) Phospholipids; and (C) Selective organ-targeting compounds A lipid nanoparticle composition containing and A composition comprising, A composition wherein the surface of the composition interacts with ApoH, and the composition primarily delivers nucleic acids to the spleen. [Invention 1059] A composition comprising a therapeutic agent and a lipid nanoparticle composition, wherein a protein corona on the surface of the composition binds to a protein substantially present in a target organ, and the target organ is not the liver. [Invention 1060] The composition of the present invention 1059, wherein the protein is vitronectin and the target organ is the lungs. [Invention 1061] A composition according to the present invention 1059, wherein the protein is ApoH and the target organ is the spleen. [Invention 1062] A composition according to any one of the present invention 1059 to 1061, further comprising a lipid nanoparticle composition and a selective organ-targeting compound that modifies the binding of proteins on a protein corona. [Invention 1063] A composition comprising a therapeutic agent and a lipid nanoparticle composition, wherein the lipid nanoparticle composition comprises a selective organ-targeting compound, and the selective organ-targeting compound has an apparent pK of about 3 to about 6. a The composition provides a lipid nanoparticle composition having the following properties. [Invention 1064] A composition comprising a therapeutic agent and a lipid nanoparticle composition, wherein the lipid nanoparticle composition comprises a selective organ-targeting compound, and the selective organ-targeting compound has an apparent pK of about 8 to about 13. a The composition provides a lipid nanoparticle composition having the following properties. Other purposes, features, and advantages of this disclosure will become apparent from the following detailed description. However, although certain aspects of this disclosure are shown, various changes and modifications within the intent and scope of this disclosure will be apparent to those skilled in the art from this detailed description, so the detailed description and specific examples should be understood to be given only as examples. [Brief explanation of the drawing]

[0066] The drawings form part of this specification and are included to further demonstrate certain aspects of this disclosure. This disclosure may be better understood by reference to one or more of these drawings in conjunction with the detailed descriptions of the particular embodiments presented herein. [Figure 1]Figures 1A-C: DOTAP mDLNP formulations mediated excellent mRNA delivery efficacy at low doses after IV injection and exhibited tissue-specific delivery characteristics at a given percentage of DOTAP. (Figure 1A) Schematic diagram of DOTAP mDLNP formation and DOTAP structure. The molar ratio of 5A2-SC8 / DOPE / Chol / DMG-PEG was fixed at 15 / 15 / 30 / 3 (named mDLNP). Then, to generate a series of DOTAP mDLNP formulations, only the DOTAP ratio was adjusted from 0 to 1200 and named DOTAPY. Y represents the percentage of DOTAP in total lipids. (Figure 1B) Ex vivo images of luciferase in major organs 6 hours after IV injection of 0.1 mg / kg dose of Luc mRNA (n=2). As the molar percentage of DOTAP increased, luciferase protein expression moved from the liver to the spleen, and then to the lungs. (Figure 1C) Quantitative data demonstrated that the DOTAP percentage was a factor in tissue-specific delivery. mDLNP (0%) was optimal for the liver, DOTAP 10-15 (similar among them) was optimal for the spleen, and DOTAP 50 was optimal for the lungs. Since luciferase expression was detected only in the liver, spleen, and lungs after IV injection, relative expression in each organ was calculated. Clearly, the higher the DOTAP (permanent cationic lipid) percentage in the formulation, the lower the luminescence in the liver, approaching 0 when >70%. However, the higher the DOTAP percentage, the higher the luminescence in the lungs, approaching 100% when >70%. DOTAP 5-20 showed high percentages in the spleen, with DOTAP 10 appearing to be the highest. [Figure 2]Figures 2A1-F2: Lipid structure determines mRNA expression profile after IV injection. Generally, quaternary lipids alter mRNA delivery from the liver to the spleen and then to the lungs as the percentage increases, and zwitterionic lipids, at high percentages, helped deliver mRNA to the spleen. However, tertiary amine lipids could not alter the mRNA expression organ, but instead improved delivery efficacy in the liver. To further confirm the delivery tendency of quaternary lipid mRNALNP, two more quaternary lipids, DDAB and EPC, were selected and subjected to in vivo mRNA delivery using the same improved strategy as DOTAP (Figures 2A1, 2B1). DDAB and EPC show significant structural differences between them and DOTAP, including three comparison phases: hydrophobic tail length, saturated and unsaturated bonds, and head chemical structure. Formulations with 5%, 15%, 40%, and 50% quaternary lipids were formed to detect size distribution and in vivo evaluation (0.1 mg / kg, 6h, n=2). (Figures 2A2, 2B2) Similar to DOTAP mDLNP, DDAB and EPC also showed similar mRNA delivery profiles. Low cation percentages (5%) delivered mRNA to the liver and spleen, and then more delivery to the spleen when increased to 15%. When increased to 40%, mRNA expression was hardly observed in the liver and spleen, but the lungs showed a high luciferase signal, which then decreased at 50%. These results are very similar to those of DOTAP mDLNP. This suggests that functionalized mDLNPs with quaternary lipids are a universal and generalizable strategy for tissue-targeted mRNA delivery. Next, similar to the DOTAP strategy, mRNA delivery was evaluated in vivo using representative zwitterionic lipids, DSPC and DOCPe (Figures 2C1, 2D1). The structures of DSPC and DOCPe lipids are in a common class of zwitterionic lipids. The size distribution of DSPC and DOCPe mDLNP formulations was tested by DLS before IV injection.In this specification, DSPC and DOCPe were compared in two stages in terms of structure: saturated vs. unsaturated hydrophobic tail and charge position in head groups (Figure 2C2, Figure 2D2). Interestingly, no similar mRNA expression profiles were observed, such as those seen with quaternary lipid preparations. Instead, both DSPC and DOCPe improved mRNA delivery to the spleen within a given range (less than 80% for DSPC and less than 50% for DOCPe), and no signal was detected in the lungs at any percentage (0.1 mg / kg, 6h, n=2). Inspired by these results, the same strategy was used to further test ionizable tertiary amine lipids, DODAP and C12-200. DODAP has the same structure as DOTAP except for the head (quaternary amine vs. tertiary amine), while C12-200, an effective lipidoid used for siRNA or mRNA delivery, has a completely different structure from DODAP. (Figures 2E1, 2F1) Similarly, the size distribution of both modified mDLNPs remained favorable at certain percentages (less than 80%). (Figures 2E2, 2F2) Surprisingly, DODAP and C12-200 were unable to alter the mRNA expression profile (different effect compared to quaternary or zwitterionic lipids). Instead, DODAP and C12-200 increased mRNA delivery to the liver. Supporting this, DODAP20 and C12-200 showed significantly better delivery efficacy than the original mDLNP formulations (0.1 mg / kg, 6h, n=2). As the percentage of DODAP or C12-200 increased (50% or 80%), luciferase signaling decreased significantly, but the liver remained the primary organ, rather than the spleen or lungs. [Figure 3]Figures 3A-C: Next, distribution assays and pKa detection were performed to confirm why various lipids can induce significant differences in mRNA expression in organs. Both in vivo distribution and pKa played a role in organ mRNA expression profiles. (Figure 3A) Organ distribution of Cy5.5-Luc mRNA preparations delivered by three modified mRNALNPs: DOTAP (quaternary lipid), DSPC (zwitterionic lipid), and DODAP (tertiary amine lipid). C57BL / 6 mice were intravenously injected with a dose of 0.5 mg / kg and imaged 6 hours after injection (n=2). DOTAP altered the organ distribution of mRNA compared to the original mRNALNP preparation (without DOTAP), and both DOTAP10 and DOTAP50 were able to deliver mRNA to the lungs, with DOTAP50 increasing it more. This may partially explain why DOTAP preparations mediated mRNA expression in the lungs at a high percentage. However, neither DSPC nor DODAP significantly altered mRNA distribution at 80% (DSPC) nor 50% (DODAP) percent. As shown in Figures 1 and 2, mRNA retention in the liver was also observed for DOTAP50 and DSPC80. The former was a lung-targeted NP, and the latter a spleen-targeted NP. Therefore, distribution was not the only factor explaining this mechanism. (Figure 3B) Next, the pKa of all tested and effective formulations, including the original mDLNP, DOTAP, DDAB, EPC, DSCP, DOCPe, DODAP, and formulations modified by C12-200, was measured. (Figure 3C) Finally, the relationship between pKa and tissue-specific mRNA delivery was plotted based on defined rules. Here, eight rules were designed for scoring, as shown in the table. Clearly, all liver-targeted therapies had narrow pKa ranges (approximately 6-7), and spleen-targeted therapies did not show a significant range, while lung-targeted delivery required a high pKa (>9.25). [Figure 4]Figures 4A - C: Liver gene editing and lung gene editing were achieved in both Td-Tomato and C57BL / 6 mice. (Figure 4A) The schematic diagram shows that when Cas9 mRNA and sgTom1 are co-delivered to Td-Tomato mice, td-tomato expression is activated. (Figure 4B) When treated with mDLNP and DOTAP50 formulations, Td-Tomato expression was induced in the liver and lung, respectively. Mice were injected intravenously with mDLNP and DOTAP50 formulations to co-deliver IVT Cas9 mRNA and modified sgTom1 (4 / 1, wt / wt) at a total dose of 2.5 mg / kg (50 μg each). Then, fluorescence in major organs was detected on day 10 after treatment. (Figure 4C) From the T7E1 assay, it was found that tissue-specific characteristics were further confirmed using in vivo PTEN editing. To achieve tissue-specific gene editing, C57 BL6 mice were injected intravenously with mDLNP, DODAP20, or DOTAP50. The total dose was 2.5 mg / kg (50 μg each). The weight ratio of IVT Cas9 mRNA to modified sgPTEN was 4 / 1, and the detection time was day 10 after treatment. [Figure 5] Figures 5A - C: Characterization of the DOTAP mDLNP formulation (n = 3). Size, PDI (Figure 5A), and zeta potential (Figure 5B) were detected by dynamic light scattering (DLS). (Figure 5C) Encapsulation efficiency (EE%) was tested by the Ribogreen RNA assay. [Figure 6]Figures 6A-C: DOTAP formulations showed excellent mRNA delivery efficiency and demonstrated delivery capability to these cargoes, e.g., proteins, which were not well tolerated in ethanol or acidic buffers. (Figure 6A) DOTAP mDLNPs mediated high Luc mRNA expression in Huh-7 and A549 cells, and it was found that 5%-50% DOTAP percentages were better for mRNA delivery, with 10% being the best. Luc mRNA expression and cell viability were tested 24 hours after transfection with a dose of mRNA of 50 ng / well (n=4). Here, DOTAP mDLNPs were formed in PBS rather than citrate buffer (10 mM, pH 4.0). (Figures 6B and 6C) Reduced volume percentage of ethanol did not affect characterization or mRNA delivery efficacy. To test the effect of ethanol on mRNA delivery, DOTAP25 was selected as a model, and four formulations were prepared using various volume ratios of ethanol:PBS (1:3, 1:5, 1:7.5, and 1:10). All four formulations showed similar EE, size, and PDI (Figure 6B) and demonstrated equal mRNA delivery efficacy in FaDu cells (50 ng / well mRNA, 24 h, n=4) (Figure 6C). Therefore, this formulation was optimized using 1XPBS (pH 7.4) instead of acidic buffer (10 mM pH 4.0), drastically reducing the ethanol percentage. This suggests that DOTAP formulations may be able to deliver cargo, such as proteins, that do not tolerate well under high ethanol concentrations or acidic buffers. [Figure 7] Quantitative data on the in vivo distribution in major organs. C57 BL6 mice were intravenously injected with various Cy5.5-Luc mRNA preparations at a dose of 0.5 mg / kg (n=2). After 6 hours, the heart, lungs, liver, spleen, and kidneys were isolated, imaged, and quantified. [Figure 8]Figures 8A - C: There was no significant difference in size distribution and Luc mRNA delivery efficacy for DOTAP10 formulations formed with PBS or citrate buffer, which was not applicable to DSPC50 and DODAP50. To test the influence of buffer on mRNA delivery efficacy in vivo, DOTAP10 (quaternary lipid), DSPC50 (zwitterionic lipid), and DODAP50 (tertiary amine lipid) were selected. Each Luc mRNA formulation was IV - injected into C57 BL6 mice at a dose of 0.1 mg / kg, and major organs were isolated and imaged 6 hours later (n = 2). (Figure 8A) There was little change in either size or delivery efficacy between DOTAP10 formed with PBS and DOTAP10 formed with citrate buffer (10 mM, pH 4.0). (Figures 8B and 8C) However, DSPC50 and DODAP50 formed with citrate buffer dramatically improved the mRNA delivery effect, but there was no significant difference in size distribution. DOTAP (or another permanently cationic lipid) may be added for LNP formation at neutral pH (e.g., 7.4 PBS buffer). [Figure 9] Western blot results of IVT Cas9 mRNA quality test delivered by mDLNP. To achieve tissue - specific gene editing, Cas9 mRNA and sgRNA were designed to be co - delivered. First, Cas9 mRNA was produced by IVT and analyzed by Western blot for quality testing. In this assay, Cas9 pDNA was delivered by Lipofectamine 2000 and commercially available Cas9 mRNA (TriLink). mDLNP was the positive control and mCherry mDLNP was the negative control. 293T cells were seeded in 12 - well plates the day before transfection, and Western blot was performed after treating the cells under each condition for 24 hours. IVT Cas9 mRNA worked significantly better than commercially available mRNA. Therefore, gene editing was performed in vivo using IVT Cas9 mRNA. [Figure 10]Figures 10A and B: sgRNA screening and optimization of weight ratio (Cas9 mRNA / sgRNA) in Td-Tomato mice. To achieve maximum gene editing in Td-Tomato mice, sgRNA sequences were screened and the weight ratio of Cas9 mRNA to sgRNA was optimized. (Figure 10A) Size distribution of Cas9 / sgTom1, Cas9 / sgTom2, and Cas9 / sgLoxP mDLNP formulations, as well as td-tomato expression in the liver. Mice were intravenously injected with the three types of mDLNPs at a total dose of 3 mg / kg (Cas9 / sgRNA, 4 / 1, wt / wt), and organs were imaged on day 7. Among the three candidates, sgTom1 appeared to be the leading candidate. (Figure 10B) SgTom1 was selected and further tested for liver gene editing with different weight ratios (Cas9 / sgRNA) of 2 / 1, 4 / 1, and 6 / 1. mDLNP was administered at a dose of 3 mg / kg for IV injection. td-tomato expression was detected on day 7. In this example, 4 / 1 worked better than 2 / 1 and 6 / 1. [Figure 11]Figures 11A-G: Characterization of Cas9 / sgRNA complexes and DOTNP lipid nanoparticles after encapsulation of the Cas9 / sgRNA complexes. Size (Figure 11A) and zeta potential (Figure 11B) of Cas9 / sgLUC complexes (mol / mol=1 / 1) in PBS (pH 7.4) and citrate buffer (pH 4.2). The Cas9 / sgLUC complex prepared in citrate buffer is very large (greater than 100 nm) and has a positively charged zeta potential, making encapsulation by lipid nanoparticles impossible. However, the Cas9 / sgLUC complex prepared in PBS is small (less than 20 nm) and has a negative charge, making encapsulation by lipid nanoparticles possible. Size (Figure 11C) and zeta potential (Figure 11D) of Cas9 / sgLUC complexes prepared with different Cas9 / sgRNA molar ratios (1 / 1, 1 / 3, and 1 / 5). Compared to the Cas9 / sgLUC complex (1 / 1, mol / mol), the size decreased and the negative charge increased as the molar ratio increased (1 / 3 and 1 / 5, mol / mol). This is beneficial for lipid nanoparticle encapsulation. Size (Figure 11E) and zeta potential (Figure 11F) of DOTNP10 lipid nanoparticle-encapsulated Cas9 / sgLUC complex (named DOTNP10-L) when prepared at different molar ratios (1 / 1, 1 / 3, 1 / 5). (Figure 11G) TEM image of DOTNP10-L (1 / 3, mol / mol). DOTAP lipid nanoparticles consist of five components: 5A2-SC8, cholesterol, DOPE, DMG-PEG, and DOTAP. The molar ratios of 5A2-SC8, cholesterol, DOPE, and DMG-PEG were fixed (15:15:30:5, mol / mol). DOTNPX refers to DOTNPs containing different molar percentages of DOTAP. Here, different sgRNAs were used, including sgLUC, sgGFP, sgTOM, and sgPTEN. To distinguish them, the first letter of each gene was added to the end of the DOTNP. For example, DOTNP10-L refers to the DOTNP10 lipid nanoparticle encapsulated Cas9 / sgLUC complex, and DOTNP10-G refers to the DOTNP10 lipid nanoparticle encapsulated Cas9 / sgGFP complex. [Figure 12]Figures 12A-F: DOTNP lipid nanoparticles were able to deliver Cas9 / sgRNA complexes to the nucleus, demonstrating efficient gene editing in vitro. (Figure 12A) Confocal images of Hela-Luc cells after incubation with DOTNP10-encapsulated Cas9-EGFP / sgLUC complex (1 / 3 mol / mol) for 1 hour, 3 hours, 6 hours, and 24 hours (using 9 nM sgRNA). Green: EGFP-fused Cas9 protein; Blue: Nuclei stained with Hoechst 33342. Red arrows indicate the process of DOTNP10 entering the nucleus. (Figure 12B) Indel percentages at the LUC locus after incubation with different molar ratios of DOTNP10-L for 3 days were analyzed by TIDE sequencing (using 24 nM sgRNA). DOTNP10 lipid nanoparticle-encapsulated Cas9 / sgGFP (DOTNP10-G) was used as a negative control. Here, two commercially available Cas9 proteins (GeneArt Cas9 and Truecut Cas9) were used. (Figure 12C) T7EI cleavage assay of Hela-Luc cells incubated with different formulations (using 24 nM sgRNA). 1. 100 bp DNA ladder; 2. PBS; 3. DOTNP10-G (1 / 3); 4. DOTNP10-L (1 / 1); 5. DOTNP10-L (1 / 3); 6. DOTNP10-L (1 / 5); 7. DOTNP10-L (1 / 3) were prepared in citrate buffer. Two commercially available Cas9 proteins (GeneArt Cas9 and Truecut Cas9) were used. Of these, the 1 / 3 molar ratio when using Truecut Cas9 protein showed the best gene editing. (Figure 12D) Fluorescence microscopy images of SKOV3-GFP cells incubated with DOTNP10-L and DOTNP10-G (using 24 nM sgRNA). Here, DOTNP10-L was used as a negative control. (Figure 12E) Flow cytometry analysis of SKOV3-GFP cells incubated with DOTNP10-L and DOTNP10-G. (Figure 12F) Mean fluorescence intensity of SKOV3-GFP cells incubated with DOTNP10-L and DOTNP10-G, as determined by flow cytometry. [Figure 13]Figures 13A and B: DOTNP demonstrated tissue-specific gene editing in vivo. (Figure 13A) Ex vivo images of tdTomato fluorescence in major organs 7 days after IV injection of different formulations (1.5 mg / kg sgRNA / mouse). DOTNP5-T refers to the DOTNP5 lipid nanoparticle encapsulated Cas9 / sgTom complex. DOTNP10-T refers to the DOTNP10 lipid nanoparticle encapsulated Cas9 / sgTom complex. DOTNP50-T refers to the DOTNP50 lipid nanoparticle encapsulated Cas9 / sgTom complex. In the DOTNP5-T treatment group, tdTomato fluorescence was observed only in the liver. In the DOTNP10-T group, slight fluorescence was observed in the lungs, and when the DOTAP dose was further increased to 50% (DOTNP50-T), most of the tdTomato fluorescence was observed in the lungs. (Figure 13B) T7EI cleavage assays of liver and lung organs after incubation with DOTNP5-P (DOTNP5 lipid nanoparticle encapsulated Cas9 / sgPTEN complex), DOTNP10-P (DOTNP10 lipid nanoparticle encapsulated Cas9 / sgPTEN complex), and DOTNP50-P (DOTNP50 lipid nanoparticle encapsulated Cas9 / sgPTEN complex) (2 mg / kg sgRNA / mouse). Results are consistent with those obtained by ex vivo imaging. After treatment with DOTNP5-P, gene editing was observed only in the liver. When incubated with DOTNP10-P, gene editing was obtained in both the liver and lungs. In contrast, in the DOTNP50-P treated group, most gene editing was observed in the lungs. [Figure 14] Figures 14A and B: (Figure 14A) shows the structure of each component, (Figure 14B) shows the molar ratio, weight ratio of total lipids to mRNA, size, and details of MC3 LNP and DOTAP-modified MC3 formulations, including PDI. [Figure 15]Figures 15A and B show that DLin-MC3-DMA (Figure 15A) and C12-200 (Figure 15B) were selected and evaluated using the DOTAP strategy at a dose of 0.1 mg / kg (6h, n=2). As the DOTAP percentage increased from 0 to 50%, MC3 and C12-200-based LNPs showed identical mRNA expression profiles, similar to mDLNPs where luciferase signaling migrated from the liver to the spleen and ultimately to the lungs. [Figure 16] Figures 16A and B: (Figure 16A) shows the structure of each component, (Figure 16B) shows the molar ratio, weight ratio of total lipids to mRNA, size, and PDI of the C12-200 LNP and DOTAP-modified C12-200 formulations. [Figure 17] Figures 17A and B: (Figure 17A) Further optimization of mRNALNP is shown. mRNALNP was modified using 5A2-SC8, the "fifth" lipid, as an important lipid in mRNALNP, to form four formulations with 10%–30% extra percentage. (Figure 17B) Exvivorciferase imaging and quantitative data showed that mRNA delivery efficacy was dramatically improved with an extra 15%–25% of 5A2-SC8, with the 20% formulation showing the highest signal (0.05 mg / kg, 6h, n=2). [Figure 18] The structures of 5A2-SC8, DOPE, cholesterol, and DMG-PEG are shown. mDLNP is an effective and safe mRNA delivery carrier for liver-targeted therapeutics developed in previous studies, composed of 5A2-SC8, DOPE, cholesterol, and DME-PEG in a molar ratio of 15 / 15 / 30 / 3. [Figure 19]Figures 19A-G: Selective Organ Targeting (SORT) demonstrates that lipid nanoparticles (LNPs) can be systematically and predictably manipulated to precisely edit cells in specific organs. (19A) The addition of auxiliary components (referred to as SORT lipids) to conventional LNPs systematically alters the in vivo delivery profile, mediating tissue-specific delivery as a function of the percentage and biophysical properties of the SORT lipids. This universal methodology successfully altered the orientation of multiple classes of nanoparticles. Here, we show bioluminescence images of mice intravenously injected with 0.1 mg / kg luciferase mRNA contained within lung-specific and spleen-specific DLin-MC3-DMA LNPs (Onpattro SNALP) and liver-enhanced 5A2-SC8 degradable dendrimer-based LNPs (DLNPs). Five-component SORT LNPs were created by incorporating SORT lipids into four-component 5A2-SC8, DLin-MC3-DMA, and C12-200 LNPs. (19B) 5A2-SC8 SORT LNP was formulated in the molar ratio of 5A2-SC8 / DOPE / Chol / DMG-PEG / SORT lipid = 15 / 15 / 30 / 3 / X (mol / mol). X was adjusted from 0 to 1200 to produce a series of LNPs using 0% to 100% SORT lipid (percentage of total lipids). Here, when permanent cationic lipids (DOTAP) were included, luciferase protein expression systematically shifted from the liver to the spleen to the lung as a function of DOTAP percentage (0.1 mg / kg Luc mRNA, 6h). (19C) Quantitative data demonstrated that the percentage of SORT lipid was a factor in tissue-specific delivery. 0% (mDLNP) was optimal for the liver. 5-15% was optimal for the spleen. 50% was optimal for the lung. (19D) It was demonstrated that fractional luciferase expression could be predictably adjusted from relative luciferase expression in each organ. (19E) The inclusion of anionic SORT lipids enabled selective mRNA delivery to the spleen. When 18PA lipids were introduced into mDLNPs up to 40%, luciferase expression was observed only in the spleen (0.1 mg / kg Luc mRNA, 6h).(19F) Ex vivo images of luminescence in major organs 6 hours after IV injection of DLin-MC3-DMA SORT LNP and 0.1 mg / kg dose of Luc mRNA. As the molar percentage of DOTAP increased, luciferase expression migrated from the liver to the lungs. 18PA mediated exclusive delivery of Luc mRNA to the spleen. The same trend was observed for modified C12-200 LNP (0.1 mg / kg, 6h). (19G) Details of selected SORT lipid formulations. [Figure 20]Figures 20A-C: (20A) Details of DOTAP and 18PA SORT LNPs, including molar ratio, molar percentage, total lipid to mRNA weight ratio, size, PDI, and zeta potential. (20B) LNPs were formulated using an improved ethanol dilution method. SORT lipids were included in the ethanol phase, and sgRNA / mRNA was encapsulated during LNP formation. (20C) Chemical structures of lipids used in standard mRNALNPs and DOTAP / 18PA SORT formulations are shown. To develop the SORT, a degradable dendrimer-based cationic ionizable lipid named 5A2-SC8 was the focus of the LNP, which could deliver siRNA / miRNA to extend survival in genetically engineered mouse models of MYC-driven hepatoma (Zhou et al., 2016; Zhang et al., 2018a; Zhang et al., 2018b) and toggle polyploidy in the liver. A molar composition of LNPs optimized for mRNA delivery aggregated in the liver and was named mDLNP (Cheng et al., 2018). This liver-targeted basic mRNA preparation, 5A2-SC8 / DOPE / cholesterol / DMG-PEG2000=15 / 15 / 30 / 3(mol), was prepared, and SORT lipids were added to prepare SORT LNPs (details in 20A). For further clarity, conventional four-component LNPs consist of cationic ionizable lipids (defined herein as containing amino groups with pKa<8), zwitterionic phospholipids (defined as lipids with an equal number of positive and negative charges), cholesterol, and poly(ethylene glycol) (PEG) lipids (most commonly PEG2000-DMG). SORT LNPs include a fifth lipid, e.g., a permanent cationic lipid (defined as having no pKa or being positively charged with pKa>8) or a permanent anionic lipid (defined as being negatively charged). [Figure 21]Figures 21A and B show the in vitro luciferase (Luc) mRNA delivery results of DOTAP-modified SORT mDLNPs in Huh-7 hepatocytes (Figure 21A) and A549 lung cells (Figure 21B) as a function of the incorporated DOTAP percentage. From the Luc mRNA delivery results, it was found that LNPs with a DOTAP percentage of 5% to 50% delivered the most mRNA in both Huh-7 hepatocytes and A549 lung cells. SORT LNPs containing 10% DOTAP were considerably more effective in vitro than previously reported basic mDLNPs. No apparent cytotoxicity was observed in any of the formulations, all were homogeneous (low PDI), and had diameters of 90 nm to 150 nm (Figure 20). From surface charge measurements, the zeta potential was close to 0 when DOTAP was less than 60%, indicating that DOTAP was encapsulated internally with the mRNA and not present on the LNP surface. The surface charge was positive only when the percentage exceeded 65% (Figure 20). This indicates that PEG lipid-coated SORT LNPs with a near-neutral surface charge and selective tissue tropism, characteristic of clinical translation, were discovered. The day before transfection, cells were seeded in 96-well plates at a density of 1 × 10⁴ cells / well. Luc mRNA expression and cell viability were measured 24 hours after treatment with a dose of 50 ng / well of Luc mRNA (n=4). [Figure 22]Figures 22A-C show the chemical structures of the lipids used in (22A) DLin-MC3-DMA SNALP (Jayaraman et al., 2012) and (22B) C12-200 LLNP (Love et al., 2010). Liver-targeted basic mRNA preparations were prepared using DLin-MC3-DMA / DSPC / cholesterol / DMG-PEG2000 = 50 / 10 / 38.5 / 1.5 (mol) and C12-200 / DOPE / cholesterol / DMG-PEG2000 = 35 / 16 / 46.5 / 2.5 (mol), and then SORT lipids were added to prepare SORT LNPs. (22C) Table and results of further SORT preparations using DLin-MC3-DMA and C12-200. For all DLin-MC3-DMA and C12-200LNP samples, the total lipid / mRNA weight ratio was 20 / 1 (wt / wt). [Figure 23] Figures 23A-C: Show that SORT relies on general biophysical properties rather than precise chemical structure. (23A) SORT lipids can be divided into specific groups based on defined biophysical properties. Permanently cationic SORT lipids (DDAB, EPC, and DOTAP) all yielded the same mRNA delivery profile (from liver to spleen to lung based on SORT lipid percentage) (0.1 mg / kg Luc mRNA, 6h). (23B) Anionic SORT lipids (14PA, 18BMP, 18PA) all yielded the same mRNA delivery profile (primarily to the spleen based on SORT lipid percentage). (23C) Cationic ionizable SORT lipids with tertiary amino groups (DODAP, C12-200) enhanced liver delivery with no luciferase expression in the lung (0.1 mg / kg Luc mRNA, 6h). [Figure 24]Figures 24A and B: To further enhance mRNA delivery to the liver, SORT was applied, and cationic ionizable lipids were used as SORT lipids. (24A) Schematic diagram of SORT. (24B) Using 5A2-SC8 as the SORT lipid, additional 5A2-SC8 was added to the basic mRNA mRNA delivery preparation (5A2-SC8 / DOPE / cholesterol / DMG-PEG2000 = 15 / 15 / 30 / 3(mol)) using the SORT method. Exvivorciferase imaging and quantitative data showed that mRNA delivery efficacy dramatically improved when an extra 15% to 25% of SORT lipid was added. Maximum expression occurred when 20% was incorporated (0.05 mg / kg, 6h, n=2). Therefore, a second-generation mRNA with increased efficacy was developed by SORT. [Figure 25] Figures 25A and B: Show the evaluation of the effects of zwitterionic SORT lipids. When zwitterionic SORT lipids were included in liver-targeted mRNA LNPs, expression shifted from the liver to the spleen as the incorporation of the SORT lipids increased. After IV injection, 80% DSPC and 50% DOCPe SORT LNPs delivered mRNA exclusively to the spleen. (25A) Schematic diagram of the SORT method. (25B) Ex vivo image of luminescence in major organs 6 hours after IV injection. DSPC and DOCPe, zwitterionic lipids with different structures, improved Luc mRNA delivery to the spleen as the percentage increased (0.1 mg / kg, 6h, n=2). [Figure 26]Figures 26A and B: SORT was evaluated as a potential strategy for “activating” inactive LNP formulations. (26A) Schematic diagram showing SORT lipids added to an inactive C1 formulation to test whether SORT could confer activity. (26B) Detailed information on C1 LNP (inactive LNP) and DOTAP (or DODAP) C1 SORT LNP, including lipid molar ratio, molar percentage, total lipid to mRNA weight ratio, size, and PDI. C1 LNPs were prepared using a method that allowed mRNA encapsulation and desirable biophysical properties (uniform <200 nm size). However, no protein was expressed after IV injection of C1 LNPs. Therefore, we questioned whether SORT could “activate” dead LNPs. DODAP and DOTAP SORT lipids were evaluated. DODAP@C1 LNP delivered mRNA to the spleen and liver, and DOTAP@C1 LNP delivered mRNA to the lungs and spleen (0.1 mg / kg, 6 h, n=2). Therefore, SORT can activate dead LNPs, leading to tissue selectivity. [Figure 27]Figures 27A and B: SORT alters the in vivo distribution of LNPs, revealing a correlation between relative apparent pKa and organ specificity. (27A) The in vivo distribution of SORT LNPs was tracked using fluorescent Cy5-labeled mRNA. Inclusion of DOTAP as a SORT lipid increased mRNA accumulation in the lungs. This partially explains the ability to deliver RNA to mouse lungs. 18PA increased uptake into the spleen. DODAP slightly increased hepatic accumulation and decreased splenic accumulation (0.5 mg / kg, 6h). Note that this data explains the location of SORT LNPs but not the ability to productively deliver mRNA into cells. (27B)67 The relative apparent pKa of all effective mRNA formulations was measured by TNS assay and plotted against in vivo delivery efficacy (functional delivery by mRNA translated into protein) in various organs. As expected, the pKa of all liver-targeted formulations was narrow (6-7). Surprisingly, high pKa (>9) was required for lung target delivery, while low pKa (<6) aided spleen delivery. Note that all SORT LNPs contain cationic ionizable lipids (for endosome evasion), along with a mixture of other charged and uncharged lipids (mediating tissue tropism in the population). [Figure 28] Figures 28A and B: Show the in vivo distribution of SORT LNPs using Cy5-labeled mRNA. Organ distribution of DSPC mDLNPs after IV injection. (28A) Schematic diagram of SORT. (28B) Cy5 fluorescence and quantitative data (0.5 mg / kg, 6h, n=2) of major organs treated with DSPC mDLNPs. [Figure 29]This paper demonstrates the measurement of the overall / apparent pKa of mRNA formulations using an improved TNS assay. A total of 67 successful NP formulations (high in vivo potency) were evaluated. Relative pKa was estimated compared to the base LNP formulation (without added SORT lipids) when 50% of the normalized signal occurred. The TNS assay has historically been used to measure LNPs with one cationic ionizable lipid and a neutral (non-ionizable) helper lipid to obtain values ​​that capture the ionization behavior of cationic lipids within self-assembled LNPs. Here, an improved method was used because SORT LNPs containing a high percentage (>40%) of permanent cationic lipids (e.g., DOTAP) contain cationic ionizable lipids but do not buffer the charge very well. Due to the complexity of SORT LNPs containing various charged lipids (not containing one cationic ionizable lipid as in conventional LNPs), we focused on the relative signal at 50%, which correlates with in vivo tissue-specific activity. [Figure 30] Figures 30A and B: Show that the presence of ionizable lipids (e.g., 5A2-SC8) was necessary to obtain efficacy. LNPs containing SORT lipids but not cationic ionizable lipids were inactive. (30A) Schematic diagram of SORT C2 LNP. (30B) Detailed view of C2 and SORT lipid C2 LNP. Exvivorciferase images showed that neither DODAP nor DOTAP enabled significant mRNA delivery of C2 LNPs. These results indicate that ionizable amino lipids are necessary for successful mRNA delivery (0.1 mg / kg, 6h, n=2). [Figure 31]Figures 31A-E: These figures demonstrate that tissue-specific gene editing was possible in Td-Tomato mice via Cre mRNA delivery when using SORT LNPs. (31A) The schematic diagram shows that Td-Tom expression is activated when Cre mRNA is delivered to Td-Tom transgenic mice. (31B) mRNA LNPs and 20% DODAP LNPs specifically induced Td-Tom fluorescence in the liver, while 50% DODAP LNPs selectively edited the lungs. Td-Tom fluorescence was detected in major organs two days after IV injection of LNPs loaded with Cre mRNA (0.3 mg / kg). (31C) 30% 18PA SORT LNPs induced gene editing in the spleen (note that liver background fluorescence was strong in PBS-injected mice). (31D) Effective tissue editing was further confirmed using confocal microscopy. Scale bars = 20 μm and 100 μm. (31E)FACS was used to quantify the percentage of TdTom+ cells within defined cell type populations of the liver, lung, and spleen (day 2, 0.3 mg / kg). [Figure 32] The B6.Cg-Gt(ROSA)26Sortm9(CAG-tdTomato)Hze / J(Ai9) mice exhibit some autofluorescence in the absorption region of TdTom. Furthermore, there are significant differences in TdTom autofluorescence between different organs (n=2). The liver and kidneys show the highest signals, while the spleen shows the lowest. This does not interfere with the detection of editing in most organs (excitation settings were appropriately adjusted to eliminate background), but it complicates the detection of splenic TdTom expression because the background in the spleen is considerably lower than in other organs. [Figure 33]Figures 33A-C: These figures show that CRISPR / Cas gene editing in the spleen was achieved in both Td-Tom transgenic mice and wild-type C57 / BL6 mice by co-delivering Cas9 mRNA and sgRNA. (33A) The schematic diagram shows that Td-Tom expression is activated when Cas9 mRNA and sgTom1 are co-delivered to Td-Tom mice. (33B) Td-Tom expression was induced in the spleen and liver by the spleen-targeted formulation 30% 18PA SORT LNP. Quantitative data showed that editing was greater in the spleen than in the liver. Td-Tom fluorescence was detected in major organs on day 2 after IV treatment with co-delivery of Cas9 mRNA and modified sgTom1 (2 / 1, wt / wt) at the full dose of 4 mg / kg. (33C) The T7E1 assay showed that specific PTEN editing in the spleen was achieved by co-delivering Cas9 mRNA (IVT) and sgPTEN. C57 / BL6 mice were intravenously injected with 30% 18PA SORT LNP at a total dose of 4 mg / kg (Cas9 mRNA / sgPTEN, 2 / 1, wt / wt). PTEN editing was detected on day 2. In this case, liver editing was not observed. This suggests that the spleen can be specifically edited. [Figure 34] Using DODAP-20 SORT LNP, a single dose of 0.3 mg / kg Cre mRNA administered to hepatocytes resulted in nearly 100% TdTom editing. As shown in the flow cytometry histogram, TdTom-control mice and TdTom+20% DODAP-treated mice were completely separated. After hepatic perfusion, the resected livers of mice treated with 20% DODAP SORT LNP were remarkably bright red compared to control livers. The livers were bright red due to complete activation of TdTom expression, even without fluorescence excitation. TdTom mice were injected with 0.3 mg / kg Cre mRNA and then sacrificed 2 days later (n=3). Hepatocytes were isolated by two-step collagenase perfusion, and TdTom fluorescence was analyzed by flow cytometry. [Figure 35]This paper describes a FACS gating strategy for analyzing TdTom+ expression in lung cells. Live and dead cells were distinguished using Ghost Red780. EpCam+ was used to define epithelial cells, CD45+ and CD31- to define immune cells, and CD45- and CD31+ to define endothelial cells. Gating for Td-Tom+ in cell types was drawn based on PBS-injected control mice. Td-Tom mice were injected with Cre mRNA preparations. Td-Tom+ was detected in the specified cell types by flow after 2 days (n=3). [Figure 36] We describe a FACS gating strategy for analyzing TdTom+ expression in splenocytes. Live and dead cells were distinguished using Ghost Red 780. CD44+ was used to identify immune cells, then CD3+ and CD11b- for T cells, CD3- and CD11b+ for macrophage cells, and CD19+ and CD11b- for B cells. Gating for Td-Tom+ in cell types was drawn based on PBS-injected control mice. Td-Tom mice were injected with Cre mRNA preparations. Td-Tom+ was detected in the given cell types by flow after 2 days (n=3). [Figure 37]Figures 37A-G: Tissue-specific CRISPR / Cas gene editing in Td-Tom transgenic mice and C57 / BL6 wild-type mice mediated by SORT LNPs, induced by simultaneous delivery of Cas9 mRNA and sgRNA and delivery of Cas9 RNPs. (37A) The schematic diagram shows that simultaneous delivery of Cas9 mRNA (or Cas9 protein) and sgTom1 to Td-Tom transgenic mice activates Td-Tom expression. (37B) mRNA LNPs and 20% DODAP LNPs specifically induced Td-Tom fluorescence in the liver, while 50% DODAP LNPs selectively edited the lungs. Td-Tom fluorescence was detected 10 days after IV injection of Cas9 mRNA and modified sgTom1 (4 / 1, wt / wt) at a total dose of 2.5 mg / kg. (37C) TdTom expression was confirmed by confocal imaging of tissue sections. Scale bars = 20 μm and 100 μm. (37D) To selectively edit the liver, lung, and spleen of C57 / BL6 mice, Cas9 mRNA and sgPTEN were co-delivered in SORT LNP (total dose of 2.5 mg / kg (Cas9 mRNA / sgPTEN, 4 / 1, wt / wt; measured 10 days after single injection)). Tissue-specific PTEN editing was achieved by the T7E1 assay. (37E) Successful PTEN editing was further confirmed by H&E sections and IHC. Clear cytoplasm showed lipid accumulation in H&E sections and PTEN disappearance in IHC images. Scale bar = 60 μm. (37F) Delivery of the Cas9 / sgTom1 ribonucleoprotein (RNP) complex in 7% DOTAP or 55% DOTAP SORT LNP specifically induced Td-Tom fluorescence in the liver and lung, respectively. Cas9 / sgTom1 RNP 1.5 mg / kg Td-Tom fluorescence was detected 7 days after IV injection of sgTom1. TdTom expression was confirmed by confocal imaging of tissue sections. Scale bars = 20 μm and 100 μm. (37G) Hepatotropic and pulmonary tropic SORT LNPs were also delivered to selectively edit the liver and lungs of C57 / BL6 mice, as well as Cas9 / sgPTEN RNPs (1.5 mg / kg sgPTEN; measured 7 days after a single injection).The T7E1 assay revealed that tissue-specific PTEN editing was achieved. [Figure 38] This shows that IVT Cas9 mRNA was evaluated by Western blotting. 293T cells were seeded in 12-well plates the day before transfection, and Western blotting was performed after treating the cells under each condition for 24 hours. Cas9 pDNA was delivered by lipofectamine 2000, and mRNA was delivered by mDLNP. [Figure 39] Figures 39A and B: Show that the weight ratio of IVT Cas9 mRNA to sgTom1 was optimized via a co-delivery strategy of Cas9 mRNA and sgRNA. (39A) The schematic diagram shows that Td-Tom expression is activated when Cas9 mRNA and sgTom1 are co-delivered to transgenic mice. (39B) Td-Tom fluorescence in major organs was imaged 7 days after IV injection. From this, it can be seen that a 2 / 1 Cas9 / sgTom1 (wt / wt) ratio is optimal. The total RNA dose was 1 mg / kg, and IVT Cas9 mRNA and modified sgTom1 were co-encapsulated with mDLNP. [Figure 40]Figures 40A - I: Illustrate the development of a modular approach for systemic nanoparticle delivery of CRISPR / Cas9 ribonucleoprotein (RNP) for tissue - specific genome editing. (40A) Addition of a permanent cationic co - component (e.g., DOTAP) to a conventional LNP formulation enabled encapsulation and protection of the Cas9 / sgRNA complex using a neutral buffer during nanoparticle formation. Precise adjustment of the DOTAP percentage mediated tissue - specific gene editing. (40B) Size distribution of Cas9 / sgLuc RNP prepared in PBS buffer (pH 7.4) and citrate buffer (pH 4.0). The increase in size is likely due to denaturization. (40C) Size distribution of 5A2 - DOT - 10 encapsulated Cas9 / sgLuc RNP prepared in PBS and citrate buffer. 5A2 - DOT - 10 prepared without RNP was used as a control. (40D) Size distribution of Cas9 / sgRNA RNP using Cas9 / sgLuc molar ratios of 1 / 1, 1 / 3, and 1 / 5. (40E) Size distribution of 5A2 - DOT - 10 encapsulated Cas9 / sgLuc using molar ratios of 1 / 1, 1 / 3, and 1 / 5. (40F) Zeta potential of Cas9 / sgRNA RNP showing decreasing charge. (40G) No significant difference in zeta potential was observed for 5A2 - DOT - 10 encapsulated Cas9 / sgLuc with different molar ratios. (40H) Time - dependent cellular uptake of 5A2 - DOT - 10 LNP - encapsulated EGFP - fused Cas9 / sgRNA showing cytoplasmic release and step - wise entry into the nucleus. (40I) Inhibition of 5A2 - DOT - 10 LNP uptake was studied using specific endocytosis inhibitors. AMI: macropinocytosis inhibitor; CMZ: inhibitor of clathrin - mediated endocytosis; GEN: inhibitor of caveola - mediated endocytosis; MβCD: lipid - raft - mediated endocytosis; 4°C: energy - mediated endocytosis. [Figure 41]Figures 41A-C: (41A) A table of 5A2-DOT-X LNPs is shown, indicating the molar ratios and molar percentages used to formulate 5A2-DOT-5 (5 mol% DOTAP), 5A2-DOT-10, 5A2-DOT-20, 5A2-DOT-30, 5A2-DOT-40, 5A2-DOT-50, and 5A2-DOT-60 (60 mol% DOTAP) LNPs. A total lipid / sgRNA ratio of 40:1 (wt.) was used for all LNPs. (41B) Gene editing in HeLa-Luc cells after treatment with different 5A2-DOT-X Cas9 / sgLuc RNP formulations was detected using the T7EI assay. (41C) Gene editing was analyzed using Sanger sequencing and ICE analysis. [Figure 42] Representative TEM images of 5A2-DOT-10 encapsulated Cas9 / sgLuc RNP complexes using a 1 / 3 molar ratio are shown. 5A2-DOT-10 Cas9 / sgLuc was prepared in PBS buffer at a total lipid concentration of 2 mg / mL. 3 μL of the nanoparticle solution was dropped onto a carbon TEM grid, deposited for 1 minute, and then blotted on filter paper. The TEM grid was then imaged using a transmission electron microscope (FEI Tecnai G2 Spirit Biotwin). [Figure 43] The images show confocal images of cellular uptake of PBS (control), free Cas9 / sgLuc complex (control), and 5A2-DOT-10 Cas9 / sgLuc in Hela-Luc cells 20 hours after treatment. The subcellular distribution of the Cas9 / sgRNA complex was tracked using the Cas9-EGFP fusion protein. The Cas9 / sgLuc complex did not show detectable green fluorescence beyond the background (PBS) within the cell. In contrast, a bright green signal was detected after treatment with 5A2-DOT-10. [Figure 44]Figures 44A-H: Demonstrates that gene editing occurs rapidly and effectively in vitro. (44A) T7EI cleavage assay of DNA isolated from HeLa-Luc cells treated with various nanoparticles and controls. Highly effective gene editing was mediated by 5A2-DOT-10 delivering Cas9 / sgLucRNP (1 / 3 and 1 / 5). Indels (%) at the Luc locus were quantified by ICE analysis. Note that gene editing was 0% for LNPs prepared using low pH citrate buffer (an established method currently in use). (44B) Fluorescence microscopy images of HeLa-GFP cells after treatment with various formulations. Scale bar = 100 μm. GFP fluorescence was significantly reduced by 5A2-DOT-10 Cas9 / sgGFP treatment. (44C) Flow cytometry analysis of HeLa-GFP cells after treatment with various formulations. Only in the 5A2-DOT-10 Cas9 / sgGFP group was the peak of GFP-positive cells completely shifted to the left. This indicates that almost all GFP-positive cells darkened. (44D) Time-dependent GFP fluorescence intensity of HeLa-GFP cells after various treatments. Permanent loss of GFP fluorescence was observed after 2 days following treatment with 5A2-DOT-10Cas9 / sgGFP. In contrast, ICE analysis of Sanger sequencing data showed that indels were maintained at over 90% after 2 days. (44E and 44F) Mean fluorescence intensity (%) of HeLa-GFP cells after treatment with Cas9 / sgGFP alone, Cas9 / sgGFP-loaded, 5A2-SC8, C12-200, DLin-MC3-DMA LNP formulations containing 10% supplemental DOTAP, conventional C12-200 and DLin-MC3-DMA LNP nanoformulations loaded with Cas9 / sgGFP, and RNAiMAX loaded with Cas9 / sgGFP. GFP fluorescence was significantly reduced after treatment with all three DOTAP-modified formulations. ICE analysis of Sanger sequencing data further confirmed that the highest gene editing efficiency was achieved using 5A2-DOT-10 LNP. Mean ± sem (n=3). Statistical significance was determined using a two-sided student t-test.†: t-value = 42.69, degrees of freedom (df) = 4 (P < 0.0001); ††: t-value = 16.75, degrees of freedom (df) = 4 (P < 0.0001); †††: t-value = 37.53, degrees of freedom (df) = 4 (P < 0.0001). A p-value of < 0.05 was considered statistically significant. (44G) 5A2-DOT-10 Cas9 / sgGFP LNPs were stored at 4°C for 2 months. Nanoparticle diameter and PDI were monitored over time. (44H) It was found that activity was not lost even when HeLa-GFP cells were regularly treated with the stored LNPs. This demonstrates the long-term stability and translational capacity of LNPs and RNPs. All cells in the above experiment were treated with 24nM sgRNA. [Figure 45] Figures 45A and B: Gene editing by different nano-formulations in Hela-GFP cells. (45A) Mean fluorescence intensity (%) of Hela-GFP cells after treatment with Cas9 / sgGFP alone, 5A2-DOT-10 Cas9 / sgLuc, and 5A2-DOT-10 Cas9 / sgGFP (40:1 total lipid / sgGFP weight ratio). (45B) Mean fluorescence intensity (%) of Hela-GFP cells after treatment with 5A2-DOT-10 Cas9 / sgGFP prepared at 10:1, 20:1, 30:1, and 40:1 total lipid / sgGFP weight ratios. [Figure 46]Figures 46A-K: A generalizable RNP delivery strategy (Figure 40A) is shown to be universal for cationic ionizable lipid nanoparticles (DLNP, LLNP, SNALP), other cationic lipids positively charged at pH 7.4, and other neutral buffers. (46A) Scheme of LNP formulations containing different ionizable lipids. (46B) Details of LNP formulations containing different ionizable lipids, including final molar ratios and percentages of each component, as well as the weight ratio of total lipids to sgRNA. (46C) Chemical structures of cationic ionizable lipids used in the formulations, including 5A2-SC8, C12-200, and Dlin-MC3-DMA. (46D) Mean fluorescence intensity (%) of HeLa-GFP cells after treatment with Cas9 / sgGFP RNPs encapsulated in 5A2-DOT-10, C12-200-DOT-10, and MC3-DOT-10. GFP fluorescence was significantly reduced after treatment with all three formulations. (46E) Scheme for the preparation of LNP formulations containing different permanent cationic lipids. (46F) Details of LNP formulations containing different permanent cationic lipids, including the final molar ratio and percentage of each component, as well as the weight ratio of total lipids to sgRNA. (46G) Chemical structures of the permanent cationic lipids used in the formulations, including DOTAP, DDAB, and EPC. (46H) Mean fluorescence intensity (%) of HeLa-GFP cells after treatment with Cas9 / sgGFP RNP encapsulated in 5A2-DOT-10, 5A2-DDAB-10, and 5A2-EPC-10. Other cationic lipids (DDAB and EPC) could also achieve efficient gene editing instead of DOTAP. (46I) Scheme for LNP formulations in different buffers. (46J) Mean fluorescence intensity (%) of HeLa-GFP cells after treatment with 5A2-DOT-10 formulated with different buffers, including PBS, Opti-MEM, HEPES, and citrate buffer. Neutral buffer was required for RNP encapsulation and delivery. Indels (%) at GFP loci in genomic DNA isolated from HeLa-GFP cells after treatment with 5A2-DOT-10 Cas9 / sgGFP LNP prepared with different (46K) buffers were measured using ICE analysis. All neutral buffers showed significant gene editing in the cells.This demonstrates the importance of neutral buffers in nanoparticle preparation. Note that Figures 44E and 44F are reproduced in Figure 45 above to facilitate understanding and to bring together relevant data. [Figure 47]Figures 47A-J: Demonstrates highly efficient multiple genome editing achieved in vivo. (47A) A schematic diagram shows how delivery of Cas9 / sgTOM RNPs activates Td-Tom expression in Td-Tomato transgenic mice. 5A2-DOT-X LNPs were injected locally (via intramuscular or intracerebral injection) and systemically (via IV injection through the tail vein) into Td-Tom mice. In vivo imaging of Td-Tom mice after intramuscular (1 mg / kg sgTom) (47B) or intracerebral (0.15 mg / kg sgTOM) (47D) injection of 5A2-DOT-10 Cas9 / sgTOM showed bright red fluorescence in the limb muscle or brain tissue (respectively). Successful CRISPR / Cas gene editing was further confirmed by confocal imaging of muscle (47C) and brain tissue sections (47E). Using 5A2-DOT-10 enabled higher gene editing efficiency than the positive control RNAiMAX previously used for local RNP injection. (47F) In vivo imaging of Td-Tom mice after intravenous (IV) injection of 5A2-DOT-X Cas9 / sgTOM LNP with different molar percentages of DOTAP. From Td-Tom fluorescence, which is the downstream readout of DNA editing, it was found that a low DOTAP percentage facilitated liver editing, while a high DOTAP percentage facilitated lung editing (1.5 mg / kg sgTOM, IV). (47G) Confocal imaging further confirmed the success of CRISPR / Cas gene editing. (47H) DNA isolated from liver and lung tissue after systemic IV treatment with 5A2-DOT-5, 5A2-DOT-10, 5A2-DOT-50, and 5A2-DOT-60 encapsulated Cas9 / sgPTEN was subjected to T7EI cleavage assays. Indel percentages were calculated and reported. (47I) td-Tom mice were IV administered 5A2-DOT-50 LNP (5A2-DOT-50-Pool) containing sgRNAs for six targets (sgTOM, sgP53, sgPTEN, sgEml4, sgALK, and sgRB1) at a total RNA dose of 2 mg / kg (0.33 mg / kg each sgRNA).Gene editing at the TOM locus was confirmed by in vivo imaging. (47J) Editing at five other loci was confirmed using the T7EI cleavage assay on lung tissue. [Figure 48]Figures 48A-H: 5A2-DOT-X LNPs demonstrate the simplification of complex mouse model construction. (48A) To create an in-situ liver-specific cancer model, adult C57BL / 6 mice were injected weekly with 5A2-DOT-5 LNP-encapsulated Cas9 / sgP53 / sgPTEN / sgRB1 RNPs (3 injections, 2.5 mg / kg total sgRNA, IV, n=4). Mice were sacrificed at 12, 15, and 20 weeks, and livers were collected for tumorigenesis analysis. (48B) T7EI cleavage results from genomic DNA extracted from livers confirmed that gene editing occurred at all three loci. (48C) Representative photograph of a mouse liver containing a tumor excised 20 weeks after injection. (48D) Progressive tumorigenesis was further confirmed by H&E and Ki67 staining. High expression of the tumor growth biomarker Ki67 was detected in the tumor lesions. Scale bar = 100 μm. (48E) To create an in-situ lung-specific cancer model, 5A2-DOT-50 LNP-encapsulated Cas9 / sgEml4 / sgAlk RNPs were injected once (2 mg / kg) or twice (1.5 mg / kg weekly over 2 weeks) into adult C57BL / 6 mice (IV, n=5). Mice were sacrificed at 10, 16, and 24 weeks, and lungs were collected for tumorigenesis analysis. (48F) T7EI cleavage results from genomic DNA extracted from lungs confirmed that gene editing occurred at the Eml4 and Alk loci. PCR amplicons of Eml4-Alk rearrangements were also detected in all lungs treated with 5A2-DOT-50 LNPs. (48G) Eml4-Alk rearrangements were further confirmed by subcloning and DNA sequencing. (Prediction = SEQ ID NO: 50; Clone 1 = SEQ ID NO: 51; Clone 2 = SEQ ID NO: 52; Clone 3 = SEQ ID NO: 53; Clone 4 = SEQ ID NO: 54; Clone 5 = SEQ ID NO: 55; Clone 6 = SEQ ID NO: 56; Clone 7 = SEQ ID NO: 57; Clone 8 = SEQ ID NO: 58). Progressive tumorigenesis was further confirmed by (48H)H&E and Ki67 staining. High expression of the tumor growth biomarker Ki67 was detected in the lung tumor lesions.Scale bar = 100 μm. [Figure 49] Figures 49A and B: Gene editing efficiency of unmodified sgRNA synthesized by in vitro transcription (IVT) compared with chemically modified and synthesized sgRNA (2'-methyl3'-phosphorothioate modification at the first and last three nucleotides). (49A) Relative luciferase activity in Hela-Luc-Cas9 cells after treatment with IVT sgRNA and chemically modified sgRNA encapsulated in nanoparticles. (49B) T7EI assay detecting gene editing efficiency of Cas9 / IVT sgRNA and Cas9 / chemically modified sgRNA encapsulated in nanoparticles. Clear cleavage bands of 536 bp and 184 bp were observed in the modified sgRNA treatment group. [Figure 50] This shows gene editing of the P53, PTEN, and RB1 genes in mouse liver after treatment with 5A2-DOT-5 LNP-encapsulated Cas9 / sgP53 / sgPTEN / sgRB1 RNP. The T7EI assay detected gene editing in liver genomic DNA at the PTEN, P53, and RB1 genomic loci after weekly treatment for two weeks. The PBS-treated group was used as a control. Breakdown bands were detected in 261 bp and 215 bp P53-targeted PCR amplicons. Breakdown bands were detected in 345 bp and 293 bp PTEN-targeted PCR amplicons. Breakdown bands were detected in 395 bp and 207 bp RB1-targeted PCR amplicons. [Figure 51]This document presents a T7EI assay detecting gene editing of the P53, PTEN, and RB1 genes in mouse livers treated with 5A2-DOT-5 LNP-encapsulated Cas9 / sgP53 / sgPTEN / sgRB1 RNPs. PBS-treated and 5A2-DOT-5-only (without Cas9 / sgRNA) treated groups were used as controls. T7EI results from genomic DNA extracted from tumors of mice treated with 5A2-DOT-5 LNP-encapsulated Cas9 / sgP53 / sgPTEN / sgRB1 RNPs for 20 weeks showed cleavage bands at all three genomic loci, demonstrating that knockout of these three genes induces tumorigenesis. [Figure 52] Representative images of resected tumors from mouse livers and mice treated for 15 weeks with 5A2-DOT-5 LNP-encapsulated Cas9 / sgP53 / sgPTEN / sgRB1 RNP are shown. [Figure 53] Figures 53A-C show H&E stained and Ki67 stained images of mouse livers after 15 and 20 weeks of treatment with 5A2-DOT-5 LNP alone (without Cas9 / sgRNA) (control) (53A), and tumors excised from mice treated for 20 weeks with 5A2-DOT-5 LNP-encapsulated Cas9 / sgP53 / sgPTEN / sgRB1 RNP (53B). No morphological changes were detected with treatment with 5A2-DOT-5 LNP alone. This suggests that nanovectors alone do not induce tumors. Scale bar: 100 μm. (53C) Magnified image of mouse liver tumor development after 20 weeks of treatment with 5A2-DOT-5 LNP-encapsulated Cas9 / sgP53 / sgPTEN / sgRB1 RNP. Scale bar: 500 μm. [Figure 54]Figures 54A-D: Show the occurrence of Eml4-Alk rearrangement in mouse lungs after 7 days of treatment with 5A2-DOT-50 LNP-encapsulated Cas9 / sgEml4 / sgAlk RNP (2 mg / kg total sgRNA). Eml4 editing (54A) and Alk editing (54B) were detected in genomic DNA extracted from mouse lungs by T7EI assay. (54C) PCR analysis was performed on genomic DNA extracted from mouse lungs to confirm Eml4-Alk inversion. (54D) PCR amplicons were subcloned. Sequences of six independent clones are listed, and representative chromatograms are shown in the upper panel. The chromatogram was exactly the same as predicted for the Eml4-Alk rearrangement (prediction = SEQ ID NO: 59; clone 1 = SEQ ID NO: 59; clone 2 = SEQ ID NO: 60; clone 4 = SEQ ID NO: 61; clone 5 = SEQ ID NO: 61; clone 6 = SEQ ID NO: 62). [Figure 55] H&E and Ki67 stained images of mouse livers treated with 5A2-DOT-50 LNP alone (without Cas9 / sgRNA) for 10 and 16 weeks are shown (LNP dose is equivalent to 1 mg / kg of total sgRNA). No morphological changes were detected in animals injected with 5A2-DOT-50 LNP alone. Scale bar: 100 μm. [Figure 56] This image shows magnified views of mouse lung tumor development after 24 weeks of treatment with 5A2-DOT-50 LNP-encapsulated Cas9 / sgEml4 / sgAlk RNP. Scale bar: 500 μm. Several tumor lesions (highlighted) were observed in both H&E stained and Ki67 stained images. [Figure 57] This study demonstrates that 5A2-DOT-10 LNP can efficiently deliver ovalbumin (OVA) protein to the cytoplasm of HeLa-Luc cells. Cells were treated with free rhodamine-labeled OVA protein and 5A2-DOT-10 LNP-encapsulated rhodamine-labeled OVA for 22 hours and then imaged using confocal microscopy. [Modes for carrying out the invention]

[0067] Exemplary Description Lipid nanoparticles (LNPs) comprising 1) permanent cationic lipids, 2) cationic ionizable lipids, and 3) phospholipids are described herein and may optionally contain cholesterol and / or lipid PEG. The inclusion of permanent cationic lipids helps direct LNPs to specific organs such as the lungs, lymph nodes, or spleen. The data presented herein demonstrate that this effect is universal and that the components are modular, showing that each category can be replaced by any cationic ionizable lipid, 5A2-SC8, DOTAP, and DOPE. In some embodiments, formulations containing cholesterol and / or lipid PEG are also possible, but formulations without cholesterol or / or lipid PEG are also feasible. These carriers can deliver mRNA, sgRNA, and proteins to specific organs in vivo, thus solving a major challenge.

[0068] A. Chemical definition When used in the context of chemical groups: "Hydrogen" means -H; "Hydroxy" means -OH; "Oxo" means =O; "Carbonyl" means -C(=O)-; "Carboxylate" means -C(=O)OH (also written as -COOH or -CO2H); "Halo" independently means -F, -Cl, -Br or -I; "Amino" means -NH2; "Hydroxyamino" means -NHOH; "Nitro" means -NO2; "Imino" means -NH; "Cyano" means -CN; "Isocyanate" means -N=C=O; "Azide" means -N3; ​​In a monovalent context, "phosphate" means -OP(O)(OH)2 or its deprotonated form; In a divalent context, "phosphate" means -OP(O)(OH)O- or its deprotonated form; "Mercapto" means -SH; "Thio" means =S; "Sulfonyl" means -S(O)2-; "hydroxysulfonyl" means -S(O)2OH; "sulfonamide" means -S(O)2NH2; and "sulfinyl" means -S(O)-.

[0069] In the context of chemical formulas, the symbol "-" represents a single bond, "=" represents a double bond, and "≡" represents a triple bond. The symbol "----" represents any bond, and if present, it is either a single or double bond. Symbols: TIFF2026082961000025.tif5128 represents either a single bond or a double bond. Therefore, for example, formula: TIFF2026082961000026.tif10128 is, Includes TIFF2026082961000027.tif12128. It is understood that such ring atoms do not form part of two or more double bonds. Furthermore, note that the covalent bond symbol "-" does not indicate any preferred stereochemistry when linking one or two stereogenic atoms. Rather, it encompasses all stereoisomers and mixtures thereof. Symbol: TIFF2026082961000028.tif5128 is drawn perpendicularly across the join. TIFF2026082961000029.tif8140 shows the bond points of 140 groups. To help readers clearly identify the bond points, note that bond points are typically identified only in this manner for larger groups. Symbol: TIFF2026082961000030.tif5128 represents a single bond where the group attached to the thick end of the wedge is "outside the page". Symbol: TIFF2026082961000031.tif5128 represents a single bond where the group attached to the thick end of the wedge is "inside the page". Symbol: TIFF2026082961000032.tif5128 represents a single bond where the geometry around the double bond (e.g., either E or Z) is undefined. Therefore, both options, and combinations thereof, are intended. Any undefined valence on an atom in the structure shown in this application implies a hydrogen atom bonded to that atom. A bold dot on a carbon atom indicates that the hydrogen bonded to that carbon is oriented out of the plane of the paper.

[0070] The base "R" is, for example, in the formula: In TIFF2026082961000033.tif13128, if R is depicted as a “floating group” on a ring system, then R can replace any hydrogen atom bonded to any of the ring atoms, including the depicted, implied, or explicitly defined hydrogen, as long as a stable structure is formed. For example, if the group “R” is in formula: As shown in TIFF2026082961000034.tif16128, if depicted as a “floating group” on a fused ring system, R can, unless otherwise specified, replace any hydrogen bonded to any of the ring atoms of the fused ring. Substitutable hydrogens include depicted hydrogens (e.g., the hydrogen bonded to nitrogen in the above formula), implied hydrogens (e.g., the hydrogen in the above formula that is not shown but is understood to exist), explicitly defined hydrogens, and any hydrogen whose existence depends on the identity of the ring atoms (e.g., the hydrogen bonded to group X if X is equal to -CH-). In the depicted example, R can exist in either a five-membered or six-membered ring of the fused ring system. In the above formula, the subscript “y” immediately following the parenthesized group “R” represents a numerical variable. Unless otherwise specified, this variable can be 0, 1, 2, or any integer greater than 2, limited only by the maximum number of substitutetable hydrogen atoms in the ring or ring system.

[0071] For chemical groups and compound classes, the number of carbon atoms in a group or class is indicated as follows: "Cn" defines the exact number (n) of carbon atoms in the group / class. "C≦n" defines the maximum number (n) of carbon atoms that can be in the group / class, and the minimum number is as small as possible for the group / class in question, for example, the group "alkenyl". (C≦8) " or class "Alken (C≦8) It is understood that the minimum number of carbon atoms in "alkoxy" is 2. (C≦10) Compare this to "alkyl". "Cn-n'" defines both the minimum (n) and maximum (n') number of carbon atoms in the group. Therefore, "alkyl (C2~10) " indicates an alkyl group having 2 to 10 carbon atoms. These carbon number indicators may be before or after the chemical group or class they modify, and may or may not be enclosed in parentheses without indicating any change in meaning. Thus, "C5 olefin", "C5-olefin", "olefin" (C5) " and "olefin C5All of the terms are synonymous.

[0072] When the term "saturated" is used to modify a compound or chemical group, it means that the compound or chemical group is free of carbon-carbon double bonds and carbon-carbon triple bonds, except as described below. When the term is used to modify an atom, it means that the atom is not part of any double or triple bonds. In the case of substitutional forms of saturated groups, one or more carbon-oxygen double bonds or carbon-nitrogen double bonds may be present. If such bonds are present, carbon-carbon double bonds that may arise as part of keto-enol tautomerism or imine / enamine tautomerism are not excluded. When the term "saturated" is used to modify a solution of a substance, it means that the substance can no longer dissolve in the solution.

[0073] The term "aliphatic," when used without the modifier "substituted," indicates that a compound or chemical group thus modified is acyclic or cyclic but non-aromatic hydrocarbon compound or group. In aliphatic compounds / groups, carbon atoms can be linked together in a linear, branched, or non-aromatic ring (alicyclic). Aliphatic compounds / groups can be saturated (alkane / alkyl) linked by single carbon-carbon bonds, or unsaturated (alkene / alkenyl) linked by one or more carbon-carbon double bonds or one or more carbon-carbon triple bonds (alkyne / alkynyl).

[0074] When the term "aromatic" is used to modify a compound or chemical group atom, it refers to a compound or chemical group that contains a planar unsaturated ring of atoms stabilized by the interaction of ring-forming bonds.

[0075] The term "alkyl," when used without the modifier "substituted," refers to a monovalent saturated aliphatic group that has a carbon atom as a bond site, a linear or branched acyclic structure, and contains no atoms other than carbon and hydrogen. Examples include the groups -CH3(Me), -CH2CH3(Et), -CH2CH2CH3(n-Pr or propyl), and -CH(CH3)2(i-Pr). i Pr or isopropyl), -CH2CH2CH2CH3(n-Bu), -CH(CH3)CH2CH3(sec-butyl), -CH2CH(CH3)2(isobutyl), -C(CH3)3(tert-butyl, t-butyl, t-Bu or tBu), and -CH2C(CH3)3(neo-pentyl) are non-restrictive examples of alkyl groups. The term "alkanediyl," when used without the modifier "substituted," refers to a divalent saturated aliphatic group having one or two saturated carbon atoms as bonding sites, a linear or branched acyclic structure, no carbon-carbon double or triple bonds, and no atoms other than carbon and hydrogen. The groups -CH2- (methylene), -CH2CH2-, -CH2C(CH3)2CH2-, and -CH2CH2CH2- are non-restrictive examples of alkanediyl groups. "Alkane" refers to a class of compounds having the formula HR, where R is an alkyl group as defined above. When any of these terms is used with the modifier "substituted," one or more hydrogen atoms are independently replaced by -OH, -F, -Cl, -Br, -I, -NH2, -NO2, -CO2H, -CO2CH3, -CN, -SH, -OCH3, -OCH2CH3, -C(O)CH3, -NHCH3, -NHCH2CH3, -N(CH3)2, -C(O)NH2, -C(O)NHCH3, -C(O)N(CH3)2, -OC(O)CH3, -NHC(O)CH3, -S(O)2OH, or -S(O)2NH2. The following groups are non-limiting examples of substituted alkyl groups: -CH2OH, -CH2Cl, -CF3, -CH2CN, -CH2C(O)OH, -CH2C(O)OCH3, -CH2C(O)NH2, -CH2C(O)CH3, -CH2OCH3, -CH2OC(O)CH3, -CH2NH2, -CH2N(CH3)2, and -CH2CH2Cl. The term "haloalkyl" is a subset of substituted alkyl groups in which the hydrogen atom substitution is limited to halo (i.e., -F, -Cl, -Br, or -I) so that no other atoms other than carbon, hydrogen, and halogen are present. The group -CH2Cl is a non-limiting example of a haloalkyl group. The term "fluoroalkyl" is a subset of substituted alkyl groups in which the hydrogen atom substitution is limited to fluoro so that no other atoms other than carbon, hydrogen, and fluorine are present. The groups -CH2F, -CF3, and -CH2CF3 are non-limiting examples of fluoroalkyl groups.

[0076] The term "cycloalkyl," when used without the modifier "substituted," refers to a monovalent saturated aliphatic group having a carbon atom as a bond site, where the carbon atom forms part of one or more non-aromatic ring structures, lacking a carbon-carbon double or triple bond, and containing no atoms other than carbon and hydrogen. Non-restrictive examples include -CH(CH2)2 (cyclopropyl), cyclobutyl, cyclopentyl, or cyclohexyl (Cy). The term "cycloalkanediyl," when used without the modifier "substituted," refers to a divalent saturated aliphatic group having two carbon atoms as bond sites, lacking a carbon-carbon double or triple bond, and containing no atoms other than carbon and hydrogen. Group: TIFF2026082961000035.tif8128 is a non-restrictive example of a cycloalkanediyl group. "Cycloalkane" refers to a class of compounds having the formula HR, where R is a cycloalkyl as the term is defined above. When any of these terms is used with the modifier "substituted", one or more hydrogen atoms are independently replaced by -OH, -F, -Cl, -Br, -I, -NH2, -NO2, -CO2H, -CO2CH3, -CN, -SH, -OCH3, -OCH2CH3, -C(O)CH3, -NHCH3, -NHCH2CH3, -N(CH3)2, -C(O)NH2, -C(O)NHCH3, -C(O)N(CH3)2, -OC(O)CH3, -NHC(O)CH3, -S(O)2OH, or -S(O)2NH2.

[0077] The term "alkenyl," when used without the modifier "substituted," refers to a monovalent unsaturated aliphatic group having a carbon atom as a bond site, a linear or branched acyclic structure, at least one non-aromatic carbon-carbon double bond, no carbon-carbon triple bond, and no atoms other than carbon and hydrogen. Non-restrictive examples include -CH=CH2 (vinyl), -CH=CHCH3, -CH=CHCH2CH3, -CH2CH=CH2 (allyl), -CH2CH=CHCH3, and -CH=CHCH=CH2. The term "alkenediyl," when used without the modifier "substituted," refers to a divalent unsaturated aliphatic group having two carbon atoms as a bond site, a linear or branched chain, a linear or branched acyclic structure, at least one non-aromatic carbon-carbon double bond, no carbon-carbon triple bond, and no atoms other than carbon and hydrogen. The groups -CH=CH-, -CH=C(CH3)CH2-, -CH=CHCH2-, and -CH2CH=CHCH2- are non-restrictive examples of alkenediyl groups. Although alkenediyl groups are aliphatic, it is known that when linked at both ends, these groups are not excluded from forming part of an aromatic structure. The terms "alkene" and "olefin" are synonymous, and R refers to a class of compounds having the formula HR, where R is an alkenyl as defined above. Similarly, the terms "terminal alkene" and "α-olefin" are synonymous, referring to an alkene having only one carbon-carbon double bond, the bond being part of a vinyl group at the end of the molecule. When any of these terms is used with the modifier "substituted," one or more hydrogen atoms are independently replaced by -OH, -F, -Cl, -Br, -I, -NH2, -NO2, -CO2H, -CO2CH3, -CN, -SH, -OCH3, -OCH2CH3, -C(O)CH3, -NHCH3, -NHCH2CH3, -N(CH3)2, -C(O)NH2, -C(O)NHCH3, -C(O)N(CH3)2, -OC(O)CH3, -NHC(O)CH3, -S(O)2OH, or -S(O)2NH2. The groups -CH=CHF, -CH=CHCl, and -CH=CHBr are non-exclusive examples of substituted alkenyl groups.

[0078] The term "alkynyl," when used without the modifier "substituted," refers to a monovalent unsaturated aliphatic group having a carbon atom as a bonding site, a linear or branched acyclic structure, at least one carbon-carbon triple bond, and no atoms other than carbon and hydrogen. As used herein, the term alkynyl does not exclude the presence of one or more non-aromatic carbon-carbon double bonds. The groups -C≡CH, -C≡CCH3, and -CH2C≡CCH3 are non-exclusive examples of alkynyl groups. "Alkyne" refers to a class of compounds having the formula HR, where R is alkynyl. When any of these terms is used with the modifier "substituted," one or more hydrogen atoms are independently replaced by -OH, -F, -Cl, -Br, -I, -NH2, -NO2, -CO2H, -CO2CH3, -CN, -SH, -OCH3, -OCH2CH3, -C(O)CH3, -NHCH3, -NHCH2CH3, -N(CH3)2, -C(O)NH2, -C(O)NHCH3, -C(O)N(CH3)2, -OC(O)CH3, -NHC(O)CH3, -S(O)2OH, or -S(O)2NH2.

[0079] The term "aryl," when used without the modifier "substituted," refers to a monovalent unsaturated aromatic group having an aromatic carbon atom as a bonding site, the carbon atom forming part of one or more six-membered aromatic ring structures, all ring atoms being carbon, and the group consisting of no atoms other than carbon and hydrogen. If two or more rings are present, the rings may or may not be condensed. As used herein, the term does not exclude the presence of one or more alkyl or aralkyl groups (as permitted by the carbon number limitation) bonded to the first aromatic ring or any further aromatic rings present. Non-exclusive examples of aryl groups include monovalent groups derived from phenyl (Ph), methylphenyl, (dimethyl)phenyl, -C6H4CH2CH3 (ethylphenyl), naphthyl, and biphenyl. The term "arendiyl," when used without the modifier "substituted," refers to a divalent aromatic group having two aromatic carbon atoms as bonding sites, the carbon atoms forming part of one or more six-membered aromatic ring structures, all ring atoms being carbon, and the monovalent group consisting of no atoms other than carbon and hydrogen. As used herein, this term does not exclude the presence of one or more alkyl, aryl, or aralkyl groups (with carbon number limitations permitted) bonded to the first aromatic ring or any further aromatic rings present. If two or more rings are present, the rings may or may not be fused. Unfused rings may be linked via one or more of the following: covalent bonds, alkanediyl, or alkenediyl groups (with carbon number limitations permitted). Non-limiting examples of arendiyl groups include: TIFF2026082961000036.tif32140 is one example.

[0080] "Arene" refers to a class of compounds having the formula HR, where R is an aryl as defined above. Benzene and toluene are non-restrictive examples of arenes. When any of these terms is used with the modifier "substituted", one or more hydrogen atoms are independently replaced by -OH, -F, -Cl, -Br, -I, -NH2, -NO2, -CO2H, -CO2CH3, -CN, -SH, -OCH3, -OCH2CH3, -C(O)CH3, -NHCH3, -NHCH2CH3, -N(CH3)2, -C(O)NH2, -C(O)NHCH3, -C(O)N(CH3)2, -OC(O)CH3, -NHC(O)CH3, -S(O)2OH, or -S(O)2NH2.

[0081] The term "aralkyl," when used without the modifier "substituted," refers to a monovalent group-alkanediyl-aryl, where the terms alkanediyl and aryl are used in a manner consistent with the aforementioned definitions. Non-restrictive examples include phenylmethyl(benzyl, Bn) and 2-phenylethyl. When the term aralkyl is used with the modifier "substituted," one or more hydrogen atoms from the alkanediyl and / or aryl group are independently replaced by -OH, -F, -Cl, -Br, -I, -NH2, -NO2, -CO2H, -CO2CH3, -CN, -SH, -OCH3, -OCH2CH3, -C(O)CH3, -NHCH3, -NHCH2CH3, -N(CH3)2, -C(O)NH2, -C(O)NHCH3, -C(O)N(CH3)2, -OC(O)CH3, -NHC(O)CH3, -S(O)2OH, or -S(O)2NH2. Non-restrictive examples of substituted aralkyls include (3-chlorophenyl)methyl and 2-chloro-2-phenyl-ethyl.

[0082] The term "heteroaryl," when used without the modifier "substituted," refers to a monovalent aromatic group having an aromatic carbon or nitrogen atom as a bonding site, the carbon or nitrogen atom forming part of one or more aromatic ring structures, at least one of the ring atoms being nitrogen, oxygen, or sulfur, and the heteroaryl group consisting of atoms other than carbon, hydrogen, aromatic nitrogen, aromatic oxygen, and aromatic sulfur. A heteroaryl ring may contain one, two, three, or four ring atoms selected from nitrogen, oxygen, and sulfur. If two or more rings are present, the rings may or may not be condensed. As used herein, the term does not exclude the presence of one or more alkyl, aryl, and / or aralkyl groups (as permitted by the carbon number restriction) bonded to an aromatic ring or aromatic ring system. Non-exclusive examples of heteroaryl groups include furanyl, imidazolyl, indolyl, indazolyl (Im), isoxazolyl, methylpyridinyl, oxazolyl, phenylpyridinyl, pyridyl, pyrrolyl, pyrimidinyl, pyrazinyl, quinolyl, quinazolyl, quinoxalinyl, triazinyl, tetrazolyl, thiazolyl, thienyl, and triazolyl. The term "N-heteroaryl" refers to a heteroaryl group having a nitrogen atom as a bond site. The term "heterearenediyl," when used without the modifier "substituted," refers to a divalent aromatic group having two aromatic carbon atoms, two aromatic nitrogen atoms, or one aromatic carbon atom and one aromatic nitrogen atom as two bond sites, wherein the atoms form part of one or more aromatic ring structures, at least one of the ring atoms is nitrogen, oxygen, or sulfur, and the divalent group consists of no atoms other than carbon, hydrogen, aromatic nitrogen, aromatic oxygen, and aromatic sulfur. If two or more rings are present, the rings may or may not be fused. Unfused rings may be linked via one or more of the following: covalent bonds, alkanediyl, or alkenediyl groups (with carbon number limitations permitted). As used herein, this term does not exclude the presence of one or more alkyl, aryl, and / or aralkyl groups (with carbon number limitations permitted) linked to an aromatic ring or aromatic ring system.Non-restrictive examples of heteroarenediyl groups include: TIFF2026082961000037.tif15128 is an example. "Heterearene" refers to a class of compounds having the formula HR, where R is a heteroaryl. Pyridine and quinoline are non-exclusive examples of heteroarenes. When these terms are used with the modifier "substituted", one or more hydrogen atoms are independently substituted by -OH, -F, -Cl, -Br, -I, -NH2, -NO2, -CO2H, -CO2CH3, -CN, -SH, -OCH3, -OCH2CH3, -C(O)CH3, -NHCH3, -NHCH2CH3, -N(CH3)2, -C(O)NH2, -C(O)NHCH3, -C(O)N(CH3)2, -OC(O)CH3, -NHC(O)CH3, -S(O)2OH, or -S(O)2NH2.

[0083] The term "heterocycloalkyl," when used without the modifier "substituted," refers to a monovalent non-aromatic group having a carbon or nitrogen atom as a bonding site, the carbon or nitrogen atom forming part of one or more non-aromatic ring structures, at least one of the ring atoms being nitrogen, oxygen, or sulfur, and the heterocycloalkyl group consisting of atoms other than carbon, hydrogen, nitrogen, oxygen, and sulfur. A heterocycloalkyl ring may contain one, two, three, or four ring atoms selected from nitrogen, oxygen, or sulfur. If two or more rings are present, the rings may or may not be condensed. As used herein, the term does not exclude the presence of one or more alkyl groups (as permitted by the carbon number restriction) bonded to the ring or ring system. Similarly, the term does not exclude the presence of one or more double bonds in the ring or ring system, provided that the resulting group remains non-aromatic. Non-limiting examples of heterocycloalkyl groups include azilidinyl, azetidinyl, pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, tetrahydrofuranil, tetrahydrothiofuranil, tetrahydropyranil, pyranyl, oxyranil, and oxetanil. The term "N-heterocycloalkyl" refers to a heterocycloalkyl group having a nitrogen atom as a bond site. N-pyrrolidinyl is an example of such a group. The term "heterocycloalkanediyl," when used without the modifier "substituted," refers to a divalent ring group having two carbon atoms, two nitrogen atoms, or one carbon atom and one nitrogen atom as two bond sites, the atoms forming part of one or more ring structures, at least one of the ring atoms being nitrogen, oxygen, or sulfur, and the divalent group consisting of no atoms other than carbon, hydrogen, nitrogen, oxygen, and sulfur. If two or more rings are present, the rings may or may not be condensed. The non-condensed rings may be linked via one or more of the following: a covalent bond, an alkanediyl group, or an alkenediyl group (with carbon number limitations permitted). As used herein, this term does not exclude the presence of one or more alkyl groups (with carbon number limitations permitted) linked to the ring or ring system.Similarly, this term does not exclude the presence of one or more double bonds in a ring or ring system, provided that the resulting group remains non-aromatic. Non-restrictive examples of heterocycloalkanediyl groups include: TIFF2026082961000038.tif14128 is an example. When these terms are used with the modifier "substituted", one or more hydrogen atoms are independently replaced by -OH, -F, -Cl, -Br, -I, -NH2, -NO2, -CO2H, -CO2CH3, -CN, -SH, -OCH3, -OCH2CH3, -C(O)CH3, -NHCH3, -NHCH2CH3, -N(CH3)2, -C(O)NH2, -C(O)NHCH3, -C(O)N(CH3)2, -OC(O)CH3, -NHC(O)CH3, -S(O)2OH, or -S(O)2NH2.

[0084] The term "acyl," when used without the modifier "substituted," refers to the group -C(O)R, where R is hydrogen, alkyl, cycloalkyl, alkenyl, aryl, aralkyl, or heteroaryl, as those terms are defined above. The groups -CHO, -C(O)CH3 (acetyl, Ac), -C(O)CH2CH3, -C(O)CH2CH2CH3, -C(O)CH(CH3)2, -C(O)CH(CH2)2, -C(O)C6H5, -C(O)C6H4CH3, -C(O)CH2C6H5, and -C(O) (imidazolyl) are non-restrictive examples of acyl groups. "Thioacyl" is defined in a similar manner, except that the oxygen atom of the group -C(O)R is replaced by a sulfur atom, and is -C(S)R. The term "aldehyde" corresponds to the alkanes, as defined above, in which at least one hydrogen atom is replaced by an -CHO group. When any of these terms is used with the modifier "substituted", one or more hydrogen atoms (including, if any, hydrogen atoms directly bonded to the carbon atoms of the carbonyl or thiocarbonyl group) are independently replaced by -OH, -F, -Cl, -Br, -I, -NH2, -NO2, -CO2H, -CO2CH3, -CN, -SH, -OCH3, -OCH2CH3, -C(O)CH3, -NHCH3, -NHCH2CH3, -N(CH3)2, -C(O)NH2, -C(O)NHCH3, -C(O)N(CH3)2, -OC(O)CH3, -NHC(O)CH3, -S(O)2OH, or -S(O)2NH2. The groups -C(O)CH2CF3, -CO2H (carboxyl), -CO2CH3 (methylcarboxyl), -CO2CH2CH3, -C(O)NH2 (carbamoyl), and -CON(CH3)2 are non-restrictive examples of substituted acyl groups.

[0085] The term "alkoxy," when used without the modifier "substituted," refers to a group -OR where R is an alkyl group as defined above. Non-restrictive examples include -OCH3 (methoxy), -OCH2CH3 (ethoxy), -OCH2CH2CH3, -OCH(CH3)2 (isopropoxy), -OC(CH3)3 (tert-butoxy), -OCH(CH2)2, -O-cyclopentyl, and -O-cyclohexyl. The terms "cycloalkoxy," "alkenyloxy," "alkynyloxy," "aryloxy," "aralkoxy," "heteroaryloxy," "heterocycloalkoxy," and "acyloxy," when used without the modifier "substituted," refer to a group defined as -OR where R is cycloalkyl, alkenyl, alkynyl, aryl, aralkyl, heteroaryl, heterocycloalkyl, and acyl, respectively. The term "alkoxydiyl" refers to a divalent group -O-alkanediyl-, -O-alkanediyl-O-, or -alkanediyl-O-alkanediyl-. The terms "alkylthio" and "acylthio" refer to a group -SR where R is alkyl and acyl, respectively, when used without the modifier "substituted". The term "alcohol" corresponds to an alkane as defined above, in which at least one hydrogen atom is replaced by a hydroxyl group. The term "ether" corresponds to an alkane as defined above, in which at least one hydrogen atom is replaced by an alkoxy group. When any of these terms is used with the modifier "substituted," one or more hydrogen atoms are independently replaced by -OH, -F, -Cl, -Br, -I, -NH2, -NO2, -CO2H, -CO2CH3, -CN, -SH, -OCH3, -OCH2CH3, -C(O)CH3, -NHCH3, -NHCH2CH3, -N(CH3)2, -C(O)NH2, -C(O)NHCH3, -C(O)N(CH3)2, -OC(O)CH3, -NHC(O)CH3, -S(O)2OH, or -S(O)2NH2.

[0086] The term "alkylamino," when used without the modifier "substituted," refers to a group -NHR where R is an alkyl as defined above. Non-restrictive examples include -NHCH3 and -NHCH2CH3. The term "dialkylamino," when used without the modifier "substituted," refers to a group -NRR' where R and R' may be the same or different alkyl groups, or R and R' together may represent an alkanediyl. Non-restrictive examples of dialkylamino groups include -N(CH3)2 and -N(CH3)(CH2CH3). The terms "cycloalkylamino," "alkenylamino," "alkynylamino," "arylamino," "aralkylamino," "heteroarylamino," "heterocycloalkylamino," "alkoxyamino," and "alkylsulfonylamino," when used without the modifier "substituted," refer to a group defined as -NHR where R is cycloalkyl, alkenyl, alkynyl, aryl, aralkyl, heteroaryl, heterocycloalkyl, alkoxy, and alkylsulfonyl, respectively. An unrestricted example of an arylamino group is -NHC6H5. The term "alkylaminodiyl" refers to a divalent group -NH-alkanediyl-, -NH-alkanediyl-NH-, or -alkanediyl-NH-alkanediyl-. The term "amide" (acylamino), when used without the modifier "substituted," refers to a group -NHR where R is an acyl as defined above. An unrestricted example of an amide group is -NHC(O)CH3. The term "alkylimino," when used without the modifier "substituted," refers to a divalent group =NR where R is an alkyl as defined above.When any of these terms are used with the modifier "substituted," one or more hydrogen atoms bonded to a carbon atom are independently replaced by -OH, -F, -Cl, -Br, -I, -NH2, -NO2, -CO2H, -CO2CH3, -CN, -SH, -OCH3, -OCH2CH3, -C(O)CH3, -NHCH3, -NHCH2CH3, -N(CH3)2, -C(O)NH2, -C(O)NHCH3, -C(O)N(CH3)2, -OC(O)CH3, -NHC(O)CH3, -S(O)2OH, or -S(O)2NH2. The groups NHC(O)OCH3 and -NHC(O)NHCH3 are non-exclusive examples of substituted amide groups.

[0087] The use of the words “a” or “an,” when used in conjunction with the term “comprising” in the claims and / or herein, may mean “one,” but also coincide with the meanings of “one or more,” “at least one,” and “one or more.”

[0088] Throughout this application, the term “approximately” is used to indicate that a value includes inherent variations in the errors of the apparatus or method used to determine that value, or variations that exist among the objects being tested.

[0089] As used in this application, the term "average molecular weight" refers to the relationship between the number of moles of each polymer species and the molar mass of that species. In particular, each polymer molecule may have different levels of polymerization and therefore different molar masses. The average molecular weight can be used to represent the molecular weight of multiple polymer molecules. The average molecular weight is typically synonymous with the average molar mass. In particular, there are three main types of average molecular weight: number-average molar mass, weight-average molar mass, and Z-average molar mass. In the context of this application, unless otherwise specified, the average molecular weight refers to either the number-average molar mass or the weight-average molar mass of the formula. In some embodiments, the average molecular weight is the number-average molar mass. In some embodiments, the average molecular weight may be used to describe the PEG components present in lipids.

[0090] The terms "comprise," "have," and "include" are unrestricted linking verbs. There are no restrictions on any form or tense of one or more of these verbs, such as "comprises," "comprising," "has," "having," "includes," and "including." For example, any way of "comprises," "has," or "includes" one or more stages is not limited to having only one or more of those stages, but also encompasses other stages not listed.

[0091] Where the term “effective” is used herein and / or in the claims, it means sufficient to achieve the desired, expected, or intended result. Where “effective dose,” “therapeutic effective dose,” or “pharmaceutical effective dose” is used in the context of treating a patient or subject with the compound, it means an amount of the compound sufficient to perform such treatment for the disease when administered to the subject or patient for the purpose of treating the disease.

[0092] When used herein, "IC" 50 The term "inhibitory dose" refers to the inhibitory dose that represents 50% of the maximum response obtained. This quantitative measure indicates how much of a particular drug or other substance (inhibitor) is needed to inhibit a given biological, biochemical, or chemical process (or component of the process, i.e., enzyme, cell, cell receptor, or microorganism) by half.

[0093] An "isomer" of the first compound is a different compound in which each molecule contains the same constituent atoms as the first compound, but the three-dimensional arrangement of those atoms is different.

[0094] As used herein, the terms “patient” or “subject” refer to a living mammal, such as a human, monkey, cattle, sheep, goat, dog, cat, mouse, rat, guinea pig, or their transgenic species. In certain embodiments, the patient or subject is a primate. Non-limiting examples of human subjects include adults, adolescents, infants, and fetuses.

[0095] As used herein, “pharmaceutically acceptable” means a compound, material, composition, and / or dosage form that, within the bounds of sound medical judgment, is suitable for use in contact with human and animal tissues, organs, and / or bodily fluids without excessive toxicity, irritation, allergic response, or other problems or complications, in proportion to a reasonable risk-benefit ratio.

[0096] "Pharmacologically acceptable salt" means a salt of the compound of the Disclosure that is pharmaceutically acceptable and possesses the desired pharmacological activity, as defined above. Such salts include inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, and phosphoric acid; or 1,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, 2-naphthalenesulfonic acid, 3-phenylpropionic acid, 4,4'-methylenebis(3-hydroxy-2-ene-1-carboxylic acid), 4-methylbicyclo[2.2.2]octa-2-ene-1-carboxylic acid, acetic acid, aliphatic mono- and dicarboxylic acids, aliphatic sulfuric acid, aromatic sulfuric acid, benzenesulfonic acid, benzoic acid, camphorsulfonic acid, carbonic acid, cinnamic acid, citric acid, cyclopentanepropionic acid, ethanesulfonic acid, fumaric acid, and glucoheptonic acid. This includes acid addition salts formed with organic acids such as gluconic acid, glutamic acid, glycolic acid, heptanoic acid, hexanoic acid, hydroxynaphthoic acid, lactic acid, lauryl sulfate, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, muconic acid, o-(4-hydroxybenzoyl)benzoic acid, oxalic acid, p-chlorobenzenesulfonic acid, phenyl-substituted alkanic acid, propionic acid, p-toluenesulfonic acid, pyruvic acid, salicylic acid, stearic acid, succinic acid, tartaric acid, tertiary butylacetic acid, and trimethylacetic acid. Pharmaceutically acceptable salts also include base addition salts that can be formed when the present acidic protons are reactable with inorganic or organic bases. Acceptable inorganic bases include sodium hydroxide, sodium carbonate, potassium hydroxide, aluminum hydroxide, and calcium hydroxide. Acceptable organic bases include ethanolamine, diethanolamine, triethanolamine, tromethamine, and N-methylglucamine. It should be understood that any specific anion or cation forming part of any salt of this disclosure is irrelevant insofar as the salt as a whole is pharmacokinetically acceptable. Further examples of pharmacokinetically acceptable salts and their preparation and use are presented in Handbook of Pharmaceutical Salts: Properties, and Use (PH Stahl & CG Wermuth eds., Verlag Helvetica Chimica Acta, 2002).

[0097] As used herein, the term “pharmaceutically acceptable carrier” means a pharmaceutically acceptable material, composition, or medium, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulation material, that is involved in supporting or transporting a chemical substance.

[0098] "Prevention" or "prevention" includes (1) inhibiting the onset of the disease in subjects or patients who are at high risk of and / or predisposed to the disease but have not yet experienced or presented any or all of the pathological or total symptoms of the disease, and / or (2) delaying the onset of any or all of the pathological or total symptoms of the disease in subjects or patients who are at high risk of and / or predisposed to the disease but have not yet experienced or presented any or all of the pathological or total symptoms of the disease.

[0099] A "repeating unit" is the simplest structural entity of a particular material, such as an organic, inorganic, or organometallic material, or a polymer skeleton and / or polymer. In the case of a polymer chain, repeating units are linked together continuously along the chain, like beads on a necklace. For example, polyethylene-[-CH2CH2-] n In -, the repeating unit is -CH2CH2-. The subscript "n" indicates the degree of polymerization, i.e., the number of repeating units linked together. If the value of "n" remains undefined, or if "n" is absent, it simply specifies the repetition of the expression in parentheses, not just the polymericity of the material. The concept of repeating units applies equally where the linkage between repeating units extends three-dimensionally, such as in metal-organic skeletons, modified polymers, and thermosetting polymers. In the context of dendrimers, repeating units can also be described as branched units, inner layers, or generations. Similarly, terminal groups can also be described as surface groups.

[0100] A "stereoisomer" or "optical isomer" is an isomer of a given compound in which the same atom is bonded to the same other atom, but the three-dimensional arrangement of those atoms differs. "Enantiomers" are stereoisomers of a given compound that are mirror images of each other, like left and right hands. "Diastereomers" are stereoisomers of a given compound that are not enantiomers. A chiral molecule contains a chiral center, also called a stereocenter or stereogenic center, which is any point in a molecule carrying a group such that the exchange of any two groups leads to a stereoisomer, but it is not necessarily an atom. In organic compounds, the chiral center is typically a carbon, phosphorus, or sulfur atom, but other atoms can also be stereocenters in organic and inorganic compounds. A molecule can have multiple stereocenters and produce many stereoisomers. In a compound whose stereoisomerism is due to a tetrahedral stereogenic center (e.g., tetrahedral carbon), the hypothetically possible total number of stereoisomers is 2 n n is the number of tetrahedral stereocenters, not exceeding . Molecules with symmetry often have fewer stereoisomers than the maximum possible number. A 50:50 mixture of enantiomers is called a racemic mixture. Alternatively, a mixture of enantiomers can be enantiomerically concentrated such that one enantiomer is present in greater quantities than 50%. Typically, enantiomers and / or diastereomers can be divided or separated using techniques known in the art. For any stereocenter or axis of chirality whose stereochemistry is not defined, it is intended that that stereocenter or axis of chirality may exist as a mixture of R and S types, including its R type, S type, or a mixture of racemic and non-racemic types. As used herein, the phrase "substantially free of other stereoisomers" means that the composition contains ≤15%, more preferably ≤10%, even more preferably ≤5%, or most preferably ≤1% of another stereoisomer.

[0101] "Treatment" or "doing treatment" includes (1) inhibiting the disease in a subject or patient experiencing or presenting the pathophysiology or overall symptoms of the disease (e.g., stopping the further development of the pathophysiology and / or overall symptoms), (2) improving the disease in a subject or patient experiencing or presenting the pathophysiology or overall symptoms of the disease (e.g., reversing the pathophysiology and / or overall symptoms), and / or (3) making any measurable reduction of the disease in a subject or patient experiencing or presenting the pathophysiology or overall symptoms of the disease.

[0102] The definitions set forth herein supersede any conflicting definitions in any reference incorporated herein by reference. However, the fact that certain terms are defined should not be interpreted as indicating that any undefined terms are unclear. Rather, all terms used are intended to describe this disclosure in a manner that would enable those skilled in the art to understand and practice the scope of this disclosure.

[0103] B. Cationic ionizable lipids In some aspects of this disclosure, compositions are provided that contain a compound comprising a lipophilic component and a cationic component, wherein the cationic component is ionizable. In some embodiments, the cationic ionizable lipid contains one or more groups that are protonated at physiological pH but can be deprotonated at pH levels greater than 8, 9, 10, 11, or 12. The ionizable cationic group may contain one or more protonable amines that can form a cationic group at physiological pH. The cationic ionizable lipid compound also contains one or more lipid components, e.g., C6-C6 24 The compounds may further contain two or more fatty acids having alkyl or alkenyl carbon groups. These lipid groups may be attached via ester bonds, or further added by Michael addition to sulfur atoms. In some embodiments, these compounds may be dendrimers, dendrons, polymers, or combinations thereof.

[0104] In some embodiments, these cationic ionizable lipids are dendrimers, which are polymers exhibiting regular dendritic branching, formed by sequentially or generationally adding branched layers to or from a core, and characterized by a core, at least one internal branched layer, and a surface branched layer. (See Petar R. Dvornic and Donald A. Tomalia in Chem. in Britain, 641-645, August 1994). In other embodiments, the term “dendrimer” as used herein is intended to include, but is not limited to, a molecular structure having an internal core, an internal layer (or “generation”) of repeating units regularly bonded to this internal core, and an external surface of terminal groups bonded to the outermost generation. A “dendron” is a species of dendrimer having branches emanating from a foci that are directly or through linkages to the core to form or potentially form a larger dendrimer. In some embodiments, the dendrimer structure has radioactive repeating groups from a central core, with each branch also serving as each repeating unit. In some embodiments, the dendrimers described herein may be described as small molecules, medium-sized molecules, lipids, or lipid-like materials. These terms may be used to describe compounds described herein that have a dendron-like appearance (e.g., molecules emitting from a single focal point).

[0105] Dendrimers are polymers, but they are preferred over conventional polymers because they have a controllable structure, a single molecular weight, numerous controllable surface functional groups, and traditionally adopt a spherical structure after reaching a certain generation. Dendrimers can be prepared by sequential reactions of each repeating unit to produce monodisperse, dendritic, and / or generational polymer structures. Each dendrimer consists of a central core molecule having dendritic wedges bonded to one or more functional sites on its central core. The dendrimer surface layer can have various functional groups, including anionic, cationic, hydrophilic, or lipophilic groups, arranged on it by the assembly monomers used during preparation.

[0106] The physical properties of dendrimers can be modified by altering the functional groups and / or chemical properties of the core and repeating units, as well as the surface or terminal groups. Some of the properties that can be altered include, but are not limited to, solubility, toxicity, immunogenicity, and biological adhesion ability. Dendrimers are often described by the number of repeating units in their generation or branches. A dendrimer consisting only of the core molecule is called generation 0, while each continuous repeating unit along all branches, down to the terminal or surface groups, is designated as first generation, second generation, etc. In some embodiments, half-generations are possible, arising only from the first condensation reaction with an amine, rather than a second condensation reaction with a thiol.

[0107] The preparation of dendrimers requires a certain level of synthetic control, achieved through a series of stepwise reactions involving the construction of a dendrimer by successive groups. Dendrimer synthesis can be convergent or divergent. During various dendrimer synthesis processes, molecules are assembled from core to periphery through a stepwise process of attaching one generation to the previous generation, followed by altering the functional groups for the next step in the reaction. The transformation of functional groups is necessary to prevent uncontrolled polymerization, which results in highly branched molecules known as hyperbranched polymers, rather than monodisperse. As dendrimer repeating units continue to react, steric effects lead to the formation of spherical or spherical molecules until steric overcrowding prevents complete reaction in certain generations, destroying the monodispersity of the molecule. Thus, in some embodiments, dendrimers of G1-G10 generations are particularly intended. In some embodiments, the dendrimer contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 repeating units, or any range that can be derived therefrom. In some embodiments, the dendrimers used herein are G0, G1, G2, or G3. However, the number of possible generations (11, 12, 13, 14, 15, 20, or 25, etc.) can be increased by reducing the spacing units in the branched polymer.

[0108] Furthermore, dendrimers possess two main chemical environments: one created by specific surface groups during terminal formation, and another within the dendritic structure that can be shielded from the bulk medium and surface groups by its higher-order structure. Due to these different chemical environments, dendrimers have found numerous diverse potential applications, including therapeutic uses.

[0109] In some aspects, dendrimers that can be used in the compositions of the present invention are assembled using the differential reactivity of acrylate and methacrylate groups with amines and thiols. The dendrimers include secondary or tertiary amines and thioethers formed by the reaction of acrylate groups with primary or secondary amines and methacrylates with mercapto groups. Furthermore, the repeating units of the dendrimer may contain groups that are biodegradable under physiological conditions. In some embodiments, these repeating units may contain one or more initial (germinal) diethers, esters, amides, or disulfide groups. In some embodiments, the core molecule is a monoamine that allows unidirectional dendritic polymerization only. In other embodiments, the core molecule is a polyamine having multiple distinct dendritic branches, each of which may contain one or more repeating units. The dendrimer may be formed by removing one or more hydrogen atoms from this core. In some embodiments, these hydrogen atoms are on heteroatoms such as nitrogen atoms. In some embodiments, the terminal groups are lipophilic groups such as long-chain alkyl or alkenyl groups. In other embodiments, the terminal group is a long-chain haloalkyl or haloalkenyl group. In other embodiments, the terminal group is an aliphatic or aromatic group containing an ionizable group such as an amine (-NH2) or a carboxylic acid (-CO2H). In yet another embodiment, the terminal group is an aliphatic or aromatic group containing one or more hydrogen bond donors such as a hydroxyl group, an amide group, or an ester.

[0110] The cationic ionizable lipids of this disclosure contain one or more asymmetrically substituted carbon or nitrogen atoms and can be isolated in an optically active form or a racemic form. Therefore, unless a specific stereochemical or isomeric form is specifically indicated, all chiral, diastereomer, racemic, epimeric, and geometric isomeric forms of the chemical formula are intended. Cationic ionizable lipids may arise as racemic compounds and racemic mixtures, single enantiomers, diastereomer mixtures, and individual diastereomers. In some embodiments, a single diastereomer is obtained. The chiral centers of the cationic ionizable lipids of this disclosure may have an S configuration or an R configuration. Furthermore, it is intended that one or more cationic ionizable lipids may exist as structural isomers. In some embodiments, compounds have the same formula but different bonding characteristics with the core nitrogen atom. While we do not wish to be bound by any theory, it is reasonable to assume that such cationic ionizable lipids exist because the starting monomer first reacts with a primary amine, and then statistically reacts with any of the secondary amines present. Therefore, the structural isomers may represent a mixture of a completely reacted primary amine and then a reacted secondary amine.

[0111] The chemical formulas used to represent cationic ionizable lipids in this disclosure will typically represent only one of several different tautomers. For example, many types of ketone groups are known to exist in equilibrium with the corresponding enol group. Similarly, many types of imine groups exist in equilibrium with the enamine group. Regardless of which tautomer is depicted for a given formula, and regardless of which one is most common, all tautomers of a given chemical formula are intended.

[0112] The cationic ionizable lipids of this disclosure may also have advantages over compounds known in the prior art, whether used in the indications described herein or otherwise, such as being more effective, less toxic, longer-acting, potent, producing fewer side effects, being easily absorbed, and / or having a good pharmacokinetic profile (e.g., high oral bioavailability and / or low clearance), and / or having other useful pharmacological, physical, or chemical properties compared to compounds known in the prior art.

[0113] Furthermore, the atoms constituting the cationic ionizable lipids of this disclosure are intended to include all isotopic forms of such atoms. When used herein, isotopes include atoms with the same atomic number but different mass numbers. As a general example, but not limited to, isotopes of hydrogen include tritium and deuterium, and isotopes of carbon include 13 C and 14 C is included.

[0114] It should be recognized that certain anions or cations forming part of any salt form of cationic ionizable lipids provided herein are irrelevant as long as the salt as a whole is pharmacologically acceptable. Further examples of pharmaceutically acceptable salts and their preparation and use are presented in Handbook of Pharmaceutical Salts: Properties, and Use (2002), which is incorporated herein by reference.

[0115] In some embodiments, cationic ionizable lipids are present in amounts of approximately 20 to approximately 23. In some embodiments, the mole percentage is approximately 20, 20.5, 21, 21.5, 22, 22.5 to approximately 23, or any range that can be derived within that range. In other embodiments, the mole percentage is approximately 7.5 to approximately 20. In some embodiments, the mole percentage is approximately 7.5, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 to approximately 20, or any range that can be derived within that range.

[0116] C. Selective organ targeting (SORT) compounds In some aspects, the present disclosure includes one or more selective organ targeting (SORT) compounds that lead to the selective delivery of the composition to a specific organ. These compounds may be lipids, small molecule therapeutic agents, sugars, vitamins, or proteins.

[0117] In some embodiments, the selective organ targeting (SORT) compound is present in the composition in a molar ratio of about 2%, 4%, 5%, 10%, 15%, 20%, 22%, 24%, 26%, 28%, 30%, 32%, 34%, 36%, 38%, 40%, 45%, 50%, 55%, 60%, 65% to about 70%, or any range within which this can be derived. In some embodiments, the SORT compound may be present in an amount of about 5% to about 40%, about 10% to about 40%, about 20% to about 35%, about 25% to about 35%, or about 28% to about 34%.

[0118] In some embodiments, the SORT compound may be a lipid. Lipids are defined as C6-C6 24 It is a small molecule having two or more alkyl or alkenyl chains. A small molecule therapeutic agent is a compound containing fewer than 100 non-hydrogen atoms and less than 2,000 daltons by weight. A sugar is a molecule with the molecular formula C n H 2n O nA molecule containing or a combination of multiple molecules of the formula, where n is 3 to 7. A protein is a sequence of amino acids containing at least three amino acid residues. Proteins that do not have a regular tertiary structure are sometimes called peptides. Proteins may also include intact proteins that have a tertiary structure. Vitamins are macronutrients, including vitamins A, B1, B2, B3, B5, B6, B7, B9, and B5. 12 It consists of one or more compounds selected from vitamin C, vitamin D, vitamin E, and vitamin K.

[0119] 1. Permanent cationic lipids In some aspects, this disclosure provides one or more types of lipids having one or more hydrophobic components and permanent cationic groups. Permanently cationic lipids may contain groups that have a positive charge regardless of pH. One of the permanent cationic groups that can be used in permanently cationic lipids is a quaternary ammonium group. These permanently cationic lipids are expressed by the following formula: It includes a structure like the one described in TIFF2026082961000039.tif13128, During the ceremony, At least one of Y1, Y2, and Y3 is X2N + Under the condition R3R4R5, Y1, Y2, or Y3 are each independently X1C(O)R1 or X2N + R3R4R5 is; R1 is C1~C 24 Alkyl, C1-C 24 Substitutive alkyl, C1~C 24 Alkenyl, C1~C 24 It is a substituted alkenyl; X1 is O or NR a And, During the ceremony, R a is hydrogen, C1-C4 alkyl, or C1-C4 substituted alkyl; X2 is a C1-C6 alkanediyl or a C1-C6 substituted alkanediyl; R3, R4, and R5 are each independent of C1~C 24 Alkyl, C1-C 24 Substitutive alkyl, C1~C 24 Alkenyl, C1~C 24 It is a substituted alkenyl; A1 is X2N in the compound. + It is an anion with a charge equal to the number of R3R4R5 groups.

[0120] In another embodiment, the permanent cationic lipid is given by the following formula: Further defined by TIFF2026082961000040.tif13128, During the ceremony, At least one of R6~R9 is C8~C 24 Under the condition that R6~R9 are independent of each other, C1~C 24 Alkyl, C1-C 24 Substitutive alkyl, C1~C 24 Alkenyl, C1~C 24 It is a substituted alkenyl; and A2 is a monovalent anion.

[0121] In another embodiment, the permanent cationic lipid is given by the following formula: Further defined by TIFF2026082961000041.tif24128, During the ceremony, R1 and R2 are each independently alkyl (C8~C24) Alkenil (C8~C24) , or a substitution of either group; R3, R3', and R3'' are each independently alkyl (C≦6) or substituted alkyl (C≦6) and; R4 is alkyl (C≦6) or substituted alkyl (C≦6) and X - It is a monovalent anion.

[0122] In some embodiments, the permanent cationic lipid is present in an amount of about 4 to about 16 moles relative to the total lipid composition. The composition may contain about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 moles, or any range that can be derived therefrom. In other embodiments, the composition may contain about 18 to about 66 moles relative to the total lipid composition. In some embodiments, the composition may contain about 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, or 66 moles, or any range that can be derived therefrom.

[0123] 2. Permanent anionic lipids In some aspects, this disclosure provides one or more types of lipids having one or more hydrophobic components and permanent anionic groups. One of the anionic groups that may be used in permanent anionic lipids is a phosphate group. The phosphate group may be a compound that is deprotonated at pH levels lower than 8, 9, 10, 11, 12, 13, or 14 and has a negative charge. The hydrophobic components are one or more C6-C6 24 The group may be an alkyl group or an alkenyl group. The compound may have one hydrophobic group, two hydrophobic groups, or three hydrophobic groups.

[0124] In some embodiments, the permanent anionic lipid is given by the following formula: It has the structure of TIFF2026082961000042.tif24128, During the ceremony, R1 and R2 are each independently alkyl (C8~C24) Alkenil (C8~C24) , or a substitution of either group; R3 is hydrogen, alkyl (C≦6) , or substituted alkyl (C≦6) , or -Y1-R4, During the ceremony, Y1 is Alkanzil (C≦6) or substitute alkanediyl (C≦6) and R4 is acyloxy(C≦8~24) or substituted acyloxy (C≦8~24) That is the case.

[0125] 3. Phosphotidylcholine In some aspects, the present disclosure provides one or more lipids comprising one or more hydrophobic components, a cationic amine group, and a negatively charged phosphate group. The cationic amine group may be a quaternary amine in which three methyl groups are bonded to a nitrogen atom. The hydrophobic component is one or more C6-C6 24 It may be an alkyl group or an alkenyl group. The compound may have one hydrophobic group, two hydrophobic groups, or three hydrophobic groups. In some embodiments, the phosphotidylcholine compound is Further defined as TIFF2026082961000043.tif24128, During the ceremony, R1 and R2 are each independently alkyl (C8~C24) Alkenil (C8~C24) , or a substitution of either group; R3, R3', and R3'' are each independently alkyl (C≦6) or substituted alkyl (C≦6) and X - It is a monovalent anion.

[0126] D. Further lipids in lipid nanoparticles In some aspects of this disclosure, a composition containing one or more types of lipids is mixed with a cationic ionizable lipid to produce a composition. In some embodiments, the cationic ionizable lipid is mixed with one, two, three, four, or five different types of lipids. The cationic ionizable lipid is intended to be able to be mixed with multiple different lipids of one type. In some embodiments, the cationic ionizable lipid composition comprises at least a steroid or steroid derivative, a PEG lipid, and a phospholipid.

[0127] In some embodiments, lipid nanoparticles are preferentially delivered to a target organ. In some embodiments, the target organ is selected from the lungs, heart, brain, spleen, bone marrow, bone, skeletal muscle, stomach, small intestine, large intestine, kidney, bladder, breast, liver, testes, ovaries, uterus, spleen, thymus, brainstem, cerebellum, spinal cord, eye, ear, tongue, or skin. Alternatively, the composition may be preferentially delivered to a target organ system such as the nervous system, cardiovascular system, or respiratory system, or to a portion of one of these organ systems. As used herein, the term “preferentially delivered” is used to refer to a composition in which at least 25% of the administered amount is delivered to a target organ or organ system. The term is also used to refer to a composition in which at least 25%, 50%, or at least 75% of the administered amount is delivered to a target organ or organ system.

[0128] 1. Steroids and steroid derivatives In some aspects of this disclosure, a composition is prepared by mixing a cationic ionizable lipid with one or more steroids or steroid derivatives. In some aspects, the steroid or steroid derivative includes any steroid or steroid derivative. As used herein, in some aspects, the term “steroid” refers to a class of compounds having a tetracyclic 17-carbon ring structure, which may further include one or more substitutions, including alkyl groups, alkoxy groups, hydroxyl groups, oxo groups, acyl groups, or double bonds between two or more carbon atoms. In one aspect, the ring structure of the steroid includes three condensed cyclohexyl rings and a condensed cyclopentyl ring, as shown in the following formula: TIFF2026082961000044.tif17128. In some embodiments, the steroid derivative comprises the above-mentioned ring structure having one or more non-alkyl substitutions. In some embodiments, the steroid or steroid derivative has the formula, It is a sterol, as further defined in TIFF2026082961000045.tif18128.

[0129] In some aspects of this disclosure, the steroid or steroid derivative is cholestane or cholestane derivative. In cholestane, the ring structure is given by formula: Further defined by TIFF2026082961000046.tif35128. As described above, cholestane derivatives include one or more non-alkyl substitutions in the above ring system. In some embodiments, cholestane or cholestane derivatives are cholestene or cholestene derivatives or sterol or sterol derivatives. In other embodiments, cholestane or cholestane derivatives are both cholestene and sterol or derivatives thereof.

[0130] In some embodiments, the composition may further comprise a molar percentage of steroids to the total lipid composition of about 40 to about 46. In some embodiments, the molar percentage is about 40, 41, 42, 43, 44, 45, ~ about 46 or any range that can be derived therein. In other embodiments, the molar percentage of steroids to the total lipid composition is about 15 to about 40. In some embodiments, the molar percentage is 15, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38 or 40 or any range that can be derived therein.

[0131] 2. PEG or PEGylated lipids In some aspects of this disclosure, polymers are mixed with one or more PEGylated lipids (or PEG lipids) to create lipid compositions. In some embodiments, this disclosure includes the use of any lipid to which a PEG group is attached. In some embodiments, the PEG lipid is a diglyceride that also includes a PEG chain attached to a glycerol group. In other embodiments, the PEG lipid is a compound that includes one or more C6-C24 long-chain alkyl or alkenyl groups or C6-C24 fatty acid groups attached to a linker group in the PEG chain. Some non-limiting examples of PEG lipids include PEG-modified phosphatidylethanolamine and phosphatidic acid, PEG-ceramide conjugate-PEG-modified dialkylamine and PEG-modified 1,2-diacyloxypropane-3-amine, PEG-modified diacylglycerol and dialkylglycerol. In some embodiments, PEG-modified diastearoylphosphatidylethanolamine or PEG-modified dimyristoyl-sn-glycerol. In some embodiments, the PEG modification is determined by the molecular weight of the PEG component of the lipid. In some embodiments, PEG modifications have a molecular weight of approximately 100 to approximately 15,000. In some embodiments, the molecular weight is approximately 200 to approximately 500, approximately 400 to approximately 5,000, approximately 500 to approximately 3,000, or approximately 1,200 to approximately 3,000. The molecular weights of PEG modifications are approximately 100, 200, 400, 500, 600, 800, 1,000, 1,250, 1,500, 1,750, 2,000, 2,250, 2,500, 2,750, 3,000, 3,500, 4,000, 4,500, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 12,500 to approximately 15,000. Some non-limiting examples of lipids that may be used in this disclosure are taught in U.S. Patent No. 5,820,873, WO 2010 / 141069, or U.S. Patent No. 8,450,298, which are incorporated herein by reference.

[0132] In another context, PEG lipids have the following formula: TIFF2026082961000047.tif18128 formula, R12 and R 13 Each is independently alkyl (C≦24) Alkenil (C≦24) , or a substitution of any of these groups; R e hydrogen, alkyl (C≦8) Alternatively, substituted alkyl groups (C≦8) And x is 1 to 250. In some embodiments, R e This includes alkyl groups such as methyl. (C≦8) That is. R 12 and R 13 Each is independently alkyl (C≦4~20) In some embodiments, x is 5 to 250. In one embodiment, x is 5 to 125, or x is 100 to 250. In some embodiments, the PEG lipid is 1,2-dimyristoyl-sn-glycerol, methoxypolyethylene glycol.

[0133] In another context, PEG lipids have the following formula: In formula TIFF2026082961000048.tif38128, n1 is an integer between 1 and 100, and n2 and n3 are each independently selected from integers between 1 and 29. In some embodiments, n1 is 5, 10, 15, 20, 25, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100, or any range that can be derived from these. In some embodiments, n1 is approximately 30 to approximately 50. In some embodiments, n2 is 5 to approximately 23. In some embodiments, n2 is 11 to approximately 17. In some embodiments, n3 is 5 to approximately 23. In some embodiments, n3 is between 11 and approximately 17.

[0134] In some embodiments, the composition may further include a molar percentage of PEG lipids to the total lipid composition of about 4.0 to about 4.6. In some embodiments, the molar percentage is about 4.0, 4.1, 4.2, 4.3, 4.4, 4.5 to about 4.6, or any range that can be derived therein. In other embodiments, the molar percentage is about 1.5 to about 4.0. In some embodiments, the molar percentage is about 1.5, 1.75, 2, 2.25, 2.5, 2.75, 3, 3.25, 3.5, 3.75 to about 4.0, or any range that can be derived therein.

[0135] 3. Phospholipids In some aspects of this disclosure, the polymer is mixed with one or more phospholipids to produce a composition. In some embodiments, any lipid also containing a phosphate group. In some embodiments, the phospholipid has a structure containing one or two long-chain C6-C24 alkyl or alkenyl groups, glycerol or sphingosine, one or two phosphate groups and optionally an organic low molecule. In some embodiments, the organic low molecule is an amino acid, sugar, or amino-substituted alkoxy group, such as choline or ethanolamine. In some embodiments, the phospholipid is phosphatidylcholine. In some embodiments, the phospholipid is distearoylphosphatidylcholine or dioleoylphosphatidylethanolamine.

[0136] In some embodiments, the composition may further contain a molar percentage of phospholipids to the total lipid composition of about 20 to about 23. In some embodiments, the molar percentage is about 20, 20.5, 21, 21.5, 22, 22.5 to about 23, or any range that can be derived therein. In other embodiments, the molar percentage is about 7.5 to about 20. In some embodiments, the molar percentage is about 7.5, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 to about 20, or any range that can be derived therein.

[0137] E. Therapeutic agents 1. Nucleic acids In some aspects of this disclosure, the lipid composition comprises one or more nucleic acids. In some embodiments, the lipid composition comprises one or more nucleic acids present in a weight ratio of about 5:1 to about 1:100 of the lipid composition. In some embodiments, the weight ratio of nucleic acids to the lipid composition is about 5:1, 2.5:1, 1:1, 1:5, 1:10, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, 1:50, 1:60, 1:70, 1:80, 1:90, or 1:100, or any range that can be derived therefrom. In some embodiments, the weight ratio is about 1:40. Furthermore, it should be clear that this disclosure is not limited to the specific nucleic acids disclosed herein. However, since those skilled in the art can readily identify relevant congeners in various other sources of nucleic acids, including nucleic acids from non-human species (e.g., mice, rats, rabbits, dogs, monkeys, gibbons, chimpanzees, apes, baboons, cattle, pigs, horses, sheep, cattle, and other species), this disclosure is not limited to any particular source, sequence, or type of nucleic acid. The nucleic acids used in this disclosure are intended to include sequences based on naturally occurring sequences. Considering the degeneracy of the genetic code, these sequences have at least about 50%, generally at least about 60%, more generally about 70%, most commonly about 80%, preferably at least about 90%, and most preferably about 95% of nucleotides that are identical to the nucleotide sequence of the natural sequence. In another embodiment, the nucleic acid is a complementary sequence to the natural sequence, or complementary to 75%, 80%, 85%, 90%, 95%, and 100%. Longer polynucleotides encoding 250, 500, 1000, 1212, 1500, 2000, 2500, 3000 or more are intended in this invention.

[0138] The nucleic acids used herein may be derived from genomic DNA, that is, they may be cloned directly from the genome of a particular organism. However, in a preferred embodiment, the nucleic acids will include complementary DNA (cDNA). cDNA with introns derived from natural introns or from other genes is also intended; such manipulated molecules are sometimes called “minigenes.” At least these and other nucleic acids of this disclosure can be used, for example, as molecular weight standards in gel electrophoresis.

[0139] The term "cDNA" is intended to refer to DNA prepared using messenger RNA (mRNA) as a template. In contrast to genomic DNA, or DNA polymerized from unprocessed or partially processed RNA templates, the advantage of using cDNA is that it primarily contains the coding sequence for the corresponding protein. Complete or partial genomic sequences may be preferable, such as when non-coding regions are required for optimal expression, or when non-coding regions, such as introns, are targeted in antisense strategies.

[0140] In some embodiments, nucleic acids contain one or more antisense segments that inhibit the expression of a gene or gene product. Antisense methodologies take advantage of the fact that nucleic acids tend to pair with "complementary" sequences. Complementarity means that polynucleotides can base-pair according to the standard Watson-Crick complementarity rules. That is, larger purines will base-pair with smaller pyrimidines to form combinations such as guanine paired with cytosine (G:C), adenine paired with thymine in the case of DNA (A:T), or adenine paired with uracil in the case of RNA (A:U). Including less common bases such as inosine, 5-methylcytosine, 6-methyladenine, hypoxanthine, and others in the hybridizing sequence does not interfere with pairing.

[0141] Targeting double-stranded (ds) DNA with polynucleotides will result in triple helix formation; targeting RNA will result in double helix formation. When introduced into target cells, antisense polynucleotides specifically bind to the target polynucleotide, interfering with transcription, RNA processing, transport, translation, and / or stability. Antisense RNA constructs, or DNA encoding such antisense RNA, can be used in host cells, either in vitro or in vivo, in host animals, including human subjects, to inhibit gene transcription, translation, or both.

[0142] Antisense constructs can be designed to bind to promoters and other regulatory regions, exons, introns, or even exon-intron boundaries of genes. The most effective antisense constructs are intended to contain regions complementary to intron / exon splice junctions. Therefore, a preferred embodiment is proposed to contain antisense constructs complementary to regions within 50–200 base pairs of intron-exon splice junctions. It has been observed that several exon sequences can be included in the construct without significantly affecting its target selectivity. The amount of exon material included will vary depending on the specific exon and intron sequences used. Whether too much exon DNA is included can be easily tested by simply testing the construct in vitro to determine whether normal cellular function is affected or whether the expression of related genes with complementary sequences is affected.

[0143] As described above, "complementary" or "antisense" refers to polynucleotide sequences that are substantially complementary throughout their entire length and have very few base mismatches. For example, a 15-base sequence can be called complementary if it has a complementary nucleotide at position 13 or 14. Naturally, a perfectly complementary sequence would be one that is perfectly complementary throughout its entire length and has no base mismatches. Other sequences with lower degrees of homology are also conceivable. For example, antisense constructs (e.g., ribozymes; see below) can be designed that have limited highly homologous regions and also include non-homologous regions. These molecules have less than 50% homology but will bind to the target sequence under appropriate conditions.

[0144] 2. Modified nucleic acid bases In some embodiments, the nucleic acids of the present disclosure comprise one or more modified nucleosides containing a modified sugar moiety. Such compounds comprising one or more sugar-modified nucleosides may possess desirable properties, such as increased nuclease stability or increased binding affinity to a target nucleic acid, compared to oligonucleotides comprising only nucleosides containing a natural sugar moiety. In some embodiments, the modified sugar moiety is a substituted sugar moiety. In some embodiments, the modified sugar moiety is a sugar substitute. Such a sugar substitute may comprise one or more substitutions corresponding to the substitution of the substituted sugar moiety.

[0145] In some embodiments, the modified sugar moiety is a substituted sugar moiety containing one or more non-crosslinked sugar substituents, including but not limited to substituents at the 2' and / or 5' positions. Examples of suitable sugar substituents at the 2' position include, but are not limited to, 2'-F, 2'-OCH3 ("OMe" or "O-methyl"), and 2'-O(CH2)2OCH3 ("MOE"). In certain embodiments, the sugar substituent at the 2' position may be allyl, amino, azide, thio, O-allyl, or O-C1~C 10 Alkyl, O--C1~C 10Substituted alkyl groups; selected from OCF3, O(CH2)2SCH3, O(CH2)2--O--N(Rm)(Rn), and O--CH2--C(=O)--N(Rm)(Rn), where each Rm and Rn is independently H or substituted or unsubstituted C1-C 10 It is alkyl. Examples of sugar substituents at the 5' position include, but are not limited to, 5'-methyl (R or S); 5'-vinyl; and 5'-methoxy. In some embodiments, the substituted sugar comprises two or more non-crosslinked sugar substituents, e.g., TF-5'-methyl sugar moiety (see, for example, further 5',2'-bis-substituted sugar moieties and nucleosides, see PCT international application WO 2008 / 101157).

[0146] Nucleosides containing a 2'-substituted sugar moiety are called 2'-substituted nucleosides. In some embodiments, 2'-substituted nucleosides are halo, allyl, amino, azide, SH, CN, OCN, CF3, OCF3, O, S, or N(R) m )-alkyl; O, S, or N(R m )-alkenyl; O, S or N(R m )-Alkynyl; O-Alkyrenyl-O-alkyl, Alkynyl, Alkal, Aralkyl, O-Alkal, O-Aralkyl, O(CH2)2SCH3, O(CH2)2--O--N(R m )(R n ) or O--CH2--C(=O)--N(R m )(R n ) includes a 2'-substituted selected from, each R m and R n These are independently H, an amino protecting group, or substituted or unsubstituted C1-C 10 These are alkyl groups. These 2'-substituents can be further substituted with one or more substituents independently selected from hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro (NO2), thiol, thioalkoxy (S-alkyl), halogen, alkyl, aryl, alkenyl, and alkynyl groups.

[0147] In some embodiments, 2'-substituted nucleosides are F, NH2, N3, OCF3, O--CH3, O(CH2)3NH2, CH2-CH=CH2, O--CH2-CH=CH2, OCH2CH2OCH3, O(CH2)2SCH3, O--(CH2)2--O--N(R m )(R n ), O(CH2)2O(CH2)2N(CH3)2, and N-substituted acetamides (O--CH2--C(=O)--N(R m )(R n ) includes a 2'-substituted selected from, each R m and R n These are independently H, an amino protecting group, or substituted or unsubstituted C1-C 10 It is alkyl.

[0148] In some embodiments, the 2'-substituted nucleoside comprises a sugar moiety containing a 2'- substituent selected from F, OCF3, O--CH3, OCH2CH2OCH3, O(CH2)2SCH3, O(CH2)2--O--N(CH3)2, --O(CH2)2O(CH2)2N(CH3)2, and O--CH2--C(=O)--N(H)CH3.

[0149] In some embodiments, the 2'-substituted nucleoside comprises a sugar moiety containing a 2'-substituent selected from F, O--CH3, and OCH2CH2OCH3.

[0150] Certain modified sugar moieties include bridging sugar substituents that form a second ring resulting in a bicyclic sugar moiety. In some such embodiments, the bicyclic sugar moiety includes bridging between 4' and 2' furanose ring atoms. An example of such a 4'-2' sugar substituent is --[C(R a )(R b )] n --, --[C(R a )(R b )] n --O--, --C(R a R b )--N(R)--O--or, --C(R a R b)--O--N(R)--; 4'-CH2-2', 4'-(CH2)2-2', 4'-(CH2)--O-2' (LNA); 4'-(CH2)--S-2'; 4'-(CH2)2--O-2' (ENA); 4'-CH(CH3)--O-2' (cEt) and 4'-CH(CH2OCH3)--O-2', and their analogues (see, e.g., U.S. Patent No. 7,399,845); 4'-C(CH3)(CH3)--O-2' and its analogues (see, e.g., WO 2009 / 006478); 4'-CH2--N(OCH3)-2' and its analogues (see, e.g., WO 2008 / 150729); 4'-CH2--O--N(CH3)-2' (For example, see US2004 / 0171570, published on September 2, 2004); Examples include, but are not limited to, 4'-CH2--O--N(R)-2' and 4'-CH2--N(R)--O-2'-, where each R independently represents H, a protecting group, or C1-C 12 Alkyl; 4'-CH2--N(R)--O-2', where R is H, C1~C 12 Alkyl or protecting groups (see U.S. Patent No. 7,427,672); 4'-CH2--C(H)(CH3)-2' (see, for example, Chattopadhyaya et al., J. Org. Chem., 2009, 74, 118-134); and 4'-CH2--C(=CH2)-2' and its analogues (see PCT international application WO 2008 / 154401).

[0151] In some embodiments, such 4'-2' bridges are independent of --[C(R a )(R b )] n --, --C(R a )=C(R b )--,--C(R a )=N--, ​​--C(=NR a )--, --C(=O)--, --C(=S)--, --O--, --Si(R a )2--,--S(=O) x --, and --N(R a) -- comprising 1 to 4 linking groups independently selected from; in the formula, x is 0, 1, or 2; n is 1, 2, 3, or 4; Each R a and R b These are independently H, protecting group, hydroxyl, C1-C 12 Alkyl, substituted C1-C 12 Alkyl, C2~C 12 Alkenyl, substituted C2-C 12 Alkenyl, C2~C 12 Alkinyl, substituted C2-C 12 Alkinyl, C5~C 20 Aryl, substituted C5~C 20 Aryl, heterocyclic group, substituted heterocyclic group, heteroaryl, substituted heteroaryl, C5-C7 alicyclic group, substituted C5-C7 alicyclic group, halogen, OJ1, NJ1J2, SJ1, N3, COOJ1, acyl (C(=O)--H), substituted acyl, CN, sulfonyl (S(=O)2-J1), or sulfoxyl (S(=O)-J1); and Each J1 and J2 is independently H, C1~C 12 Alkyl, substituted C1-C 12 Alkyl, C2~C 12 Alkenyl, substituted C2-C 12 Alkenyl, C2~C 12 Alkinyl, substituted C2-C 12 Alkinyl, C5~C 20 Aryl, substituted C5~C 20 Aryl, acyl (C(=O)--H), substituted acyl, heterocyclic group, substituted heterocyclic group, C1~C 12 Aminoalkyl, substituted C1-C 12 It is an aminoalkyl group or a protecting group.

[0152] Nucleosides containing a bicyclic sugar moiety are called bicyclic nucleosides or BNAs. Bicyclic nucleosides include (A) α-L-methyleneoxy(4'-CH2--O-2')BNA, (B) β-D-methyleneoxy(4'-CH2--O-2')BNA (also called locked nucleic acid or LNA), (C) ethyleneoxy(4'-(CH2)2--O-2')BNA, (D) aminooxy(4'-CH2--O--N(R)-2')BNA, (E) oxyamino(4'-CH2--N(R)--O-2')BNA, (F) methyl(methyleneoxy)(4'-CH(CH3)--O-2')BNA (also called restricted ethyl or cEt), (G) methylene-thio(4'-CH2--S-2')BNA, (H) This includes, but is not limited to, methylene-amino(4'-CH2-N(R)-2')BNA, (I) methyl carboncyclic(4'-CH2--CH(CH3)-2')BNA, (J) propylene carboncyclic(4'-(CH2)3-2')BNA, and (K) methoxy(ethylene oxy)(4'-CH(CH2OMe)-O-2')BNA (also called restricted MOE or cMOE).

[0153] Further bicyclic sugar moieties are known in the art, e.g.: Singh et al., Chem. Commun., 1998, 4, 455-456; Koshkin et al., Tetrahedron, 1998, 54, 3607-3630; Wahlestedt et al., Proc. Natl. Acad. Sci. USA, 2000, 97, 5633-5638; Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222; Singh et al., J. Org. Chem., 1998, 63, 10035-10039; Srivastava et al., J. Am. Chem. Soc., 129(26) 8362-8379 (Jul. 4, 2007); Elayadi et al., Curr. Opinion Invens. Drugs, 2001, 2, 5561; Braasch et al., Chem. Biol., 2001, 8, 1-7; Orum et al., Curr. Opinion Mol. Ther., 2001, 3, 239-243; U.S. Patent Nos. 7,053,207, 6,268,490, 6,770,748, 6,794,499, 7,034,133, 6,525,191, 6,670,461, and 7,399,845; WO 2004 / 106356, WO 1994 / 14226, WO 2005 / 021570, and WO 2007 / 134181; U.S. Patent Application Publications 2004 / 0171570, 2007 / 0287831, and 2008 / 0039618; U.S. Patent Applications 12 / 129,154, 60 / 989,574, 61 / 026,995, 61 / 026,998, 61 / 056,564, 61 / 086,231, 61 / 097,787, and 61 / 099,844; Also, PCT international application numbers PCT / US2008 / 064591, PCT / US2008 / 066154, and PCT / US2008 / 068922.

[0154] In some embodiments, bicyclic sugar moieties, and nucleosides incorporating such bicyclic sugar moieties, are further defined by their isomer configuration. For example, nucleosides containing a 4'-2'-methylene-oxy bridge can be in either an α-L or β-D configuration. To date, α-L-methyleneoxy(4'-CH2--O-2') bicyclic nucleosides have been incorporated into antisense oligonucleotides exhibiting antisense activity (Frieden et al., Nucleic Acids Research, 2003, 21, 6365-6372).

[0155] In some embodiments, the substituted sugar moiety comprises one or more non-crosslinked sugar substituents and one or more crosslinked sugar substituents (e.g., 5'-substituted and 4'-2'-crosslinked sugars; PCT international application WO 2007 / 134181, where LNA is substituted with, for example, a 5'-methyl or 5'-vinyl group).

[0156] In some embodiments, the modified sugar moiety is a sugar substitute. In some such embodiments, the oxygen atom of the natural sugar is substituted with, for example, a sulfur, carbon, or nitrogen atom. In some such embodiments, such modified sugar moiety also includes bridging and / or non-bridging substituents as described above. For example, certain sugar substitutes include a 4'-sulfur atom and substitution at the 2'-position (see, e.g., published U.S. Patent Application No. 2005 / 0130923) and / or the 5' position. Further examples include carbocyclic bicyclic nucleosides having a 4'-2' bridge (see, e.g., Freier et al., Nucleic Acids Research, 1997, 25(22), 4429-4443 and Albaek et al., J. Org. Chem., 2006, 71, 7731-7740).

[0157] In some embodiments, the sugar substitute contains a ring other than a 5-atom. For example, in some embodiments, the sugar substitute contains a 6-membered tetrahydropyran. Such tetrahydropyrans may be further modified or substituted. Nucleosides containing such modified tetrahydropyrans include, but are not limited to, hexitol nucleic acid (HNA), anitol nucleic acid (ANA), manitol nucleic acid (MNA) (see Leumann, C J. Bioorg. & Med. Chem. (2002) 10:841-854), and fluoroHNA (F-HNA).

[0158] In some embodiments, modified THP nucleosides of formula VII are provided, where q1, q2, q3, q4, q5, q6, and q7 are each H. In certain embodiments, at least one of q1, q2, q3, q4, q5, q6, and q7 is non-H. In some embodiments, at least one of q1, q2, q3, q4, q5, q6, and q7 is methyl. In some embodiments, THP nucleosides of formula VII are provided, where one of R1 and R2 is F. In certain embodiments, R1 is fluoro and R2 is H, R1 is methoxy and R2 is H, and R1 is methoxyethoxy and R2 is H.

[0159] Many other bicyclo and tricyclosaccharide substitute ring systems are also known in the art and can be used to modify nucleosides for incorporation into antisense compounds (see, for example, review: Leumann, J. C, Bioorganic & Medicinal Chemistry, 2002, 10, 841-854).

[0160] Non-limitingly, combinations of modifications are also provided, such as 2'-F-5'-methyl-substituted nucleosides (see PCT international application WO 2008 / 101157 for other disclosed 5',2'-bis-substituted nucleosides), substitution of the ribosyl ring oxygen atom at S and further substitution at the 2' position (see U.S. Patent Application No. 2005 / 0130923), or 5' substitution of bicyclic nucleic acids (see PCT international application WO 2007 / 134181, where 4'-CH2--O-2' bicyclic nucleosides are further substituted with a 5'-methyl or 5'-vinyl group at the 5' position). The synthesis and preparation of carbocyclic and bicyclic nucleosides are also described along with their oligomerization and biochemical studies (see, e.g., Srivastava et al., 2007).

[0161] In some embodiments, the disclosure provides oligonucleotides comprising modified nucleosides. These modified nucleotides may comprise modified sugars, modified nucleic acid bases, and / or modified bonds. The specific modifications are selected such that the resulting oligonucleotide possesses desired characteristics. In some embodiments, the oligonucleotide comprises one or more RNA-like nucleosides. In some embodiments, the oligonucleotide comprises one or more DNA-like nucleotides.

[0162] In some embodiments, the nucleosides of the Disclosure comprise one or more unmodified nucleic acid bases. In certain embodiments, the nucleosides of the Disclosure comprise one or more modified nucleic acid bases.

[0163] In some embodiments, modified nucleic acid bases include universal bases, hydrophobic bases, promiscuous bases, size-extended bases, and fluorinated bases as defined herein, 2-aminopropyladenine, 5-propynyluracil, 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6, and O-6 substituted purines; 5-propynylcytosine; 5-Hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynylCH3)uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azouracil, cytosine and thymine, 5-uracil (pseuducracil), 4-thiouracil, 8-halo, 8-amino, 8- Selected from thiols, 8-thioalkyls, 8-hydroxyls and other 8-substituted adenines and guanines, 5-halos, particularly 5-bromos, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine, 3-deazaguanine and 3-deazaadenine, universal bases, hydrophobic bases, indiscriminate bases, size-expanding bases and fluorinated bases as defined herein. Further modified nucleic acid bases include tricyclic pyrimidines such as phenoxazinecytidine ([5,4-b][1,4]benzoxazine-2(3H)-one) and phenothiazinecytidine (1H-pyrimido[5,4-b][1,4]benzothiadin-2(3H)-one), substituted phenoxazinecytidines (e.g., 9-(2-aminoethoxy)-H-pyrimido[5,4-13][1,4]benzoxazine-2(3H)-one), and carbazolecytidine ( 2G-clamps such as H-pyrimido[4,5-b]indole-2-one and pyridoindolecytidine (H-pyrimido[3',2':4,5]pyrrolo[2,3-d]pyrimidine-2-one) are included. Modified nucleic acid bases may also include those in which a purine or pyrimidine base is substituted with another heterocycle, such as 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine, and 2-pyridone. Further nucleic acid bases include those disclosed in U.S. Patent No. 3,687,808, The Concise Encyclopedia Of Polymer Science And Engineering, Kroschwitz, JI, Ed., John Wiley & Sons, 1990, 858-859; those disclosed by Englisch et al., 1991; and those disclosed by Sanghvi, YS, 1993.

[0164] Representative U.S. patents teaching the above-mentioned modified nucleic acid bases and several preparations of other modified nucleic acid bases include, but are not limited to, U.S. Patent Nos. 3,687,808; 4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121; 5,596,091; This includes Nos. 5,614,617; 5,645,985; 5,681,941; 5,750,692; 5,763,588; 5,830,653 and 6,005,096, each of which is incorporated herein by reference in its entirety.

[0165] In some embodiments, the disclosure provides oligonucleotides comprising bound nucleosides. In such embodiments, nucleosides may be linked together using any internucleoside linkage. Two main classes of internucleoside linkages are defined by the presence or absence of a phosphorus atom. Typical phosphorus-containing internucleoside links include, but are not limited to, phosphodiesters (P=O), phosphotriesters, methylphosphonates, phosphoramidates, and phosphorothioates (P=S). Typical non-phosphorus-containing internucleoside linking groups include, but are not limited to, methylenemethylimino (--CH2--N(CH3)--O--CH2--), thiodiesters (--O--C(O)--S--), thionocarbamates (--O--C(O)(NH)--S--); siloxanes (--O--Si(H)2--O--); and N,N'-dimethylhydrazine (--CH2--N(CH3)--N(CH3)--). Compared to natural phosphodiester links, modified links can be used to alter, typically increase, the nuclease resistance of oligonucleotides. In some embodiments, internucleoside links with chiral atoms can be prepared as racemic mixtures or as distinct enantiomers. Typical chiral links include, but are not limited to, alkylphosphonates and phosphorothioates. Methods for preparing phosphorus-containing and non-phosphorus-containing nucleoside bonds are well known to those skilled in the art.

[0166] Oligonucleotides described herein contain one or more chiral centers and thus produce enantiomers, diastereomers, and other stereoisomer configurations that can be defined with respect to absolute stereochemistry as (R) or (S), α or β in the case of sugar anomers, or (D) or (L) in the case of amino acids, etc. Antisense compounds provided herein include all such possible isomers, as well as their racemic and optically pure forms.

[0167] Neutral nucleoside interbonding includes, non-limitingly, phosphotriesters, methylphosphonates, MMI (3'-CH2--N(CH3)--O-5'), amide-3 (3'-CH2--C(=O)--N(H)-5'), amide-4 (3'-CH2--N(H)--C(=O)-5'), formacetal (3'-O--CH2--O-5'), and thioformacetal (3'-S--CH2--O-5'). Further neutral nucleoside bonds include nonionic bonds containing siloxanes (dialkylsiloxanes), carboxylate esters, carboxamides, sulfides, sulfonate esters, and amides (see, for example: Carbohydrate Modifications in Antisense Research; YS Sanghvi and PD Cook, Eds., ACS Symposium Series 580; Chapters 3 and 4, 40-65). Further neutral nucleoside bonds include nonionic bonds containing mixed N, O, S, and CH2 components.

[0168] Further modifications can also be made at other positions on the oligonucleotide, particularly at the 3' position of the sugar on the 3' terminal nucleotide and at the 5' position of the 5' terminal nucleotide. For example, one further modification of a ligand-binding oligonucleotide of this disclosure involves chemically linking one or more additional non-ligand moieties or conjugates to the oligonucleotide, which enhance the activity, cell distribution, or cell uptake of the oligonucleotide. Such portions include lipid portions such as cholesterol (Letsinger et al., 1989), cholic acid (Manoharan et al., 1994), thioethers, such as hexyl-5-tritylthiol (Manoharan et al., 1992; Manoharan et al., 1993), thiocholesterol (Oberhauser et al., 1992), aliphatic chains, such as dodecanediol or undecyl residues (Saison-Behmoaras et al., 1991; Kabanov et al., 1990; Svinarchuk et al., 1993), phospholipids, such as di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., 1995; Shea et al., This includes, but is not limited to, polyamines or polyethylene glycol chains (Manoharan et al., 1995), adamantane acetate (Manoharan et al., 1995), palmityl moieties (Mishra et al., 1995), or octadecylamine or hexylamino-carbonyl-oxycholesterol moieties (Crooke et al., 1996).

[0169] Representative U.S. patents teaching the preparation of such oligonucleotide conjugates include U.S. Patent Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; and 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; This includes, but is not limited to, Nos. 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941, each of which is incorporated herein by reference.

[0170] 3. Protein In some embodiments, the composition may further contain one or more types of proteins. Some proteins may include enzymes such as nuclease enzymes. The compositions described herein may contain one or more types of CRISPR-related proteins (e.g., CRISPR enzymes) including Cas proteins. Non-exclusive examples of Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csfl, Csf2, Csf3, Csf4, their homologs, or modified versions thereof. These enzymes are well known. For example, the amino acid sequence of the S. pyrogenes Cas9 protein can be found in the SwissProt database under accession number Q99ZW2.

[0171] The proteins in the compositions described herein may be Cas9 (e.g., derived from S. pyrogenes or S. pneumonia). CRISPR enzymes can induce cleavage of one or both strands within the target sequence and / or the complementary strand of the target sequence at the location of the target sequence. CRISPR enzymes can be mutated from their corresponding wild-type enzymes such that the mutated CRISPR enzyme loses the ability to cleave one or both strands of a target polynucleotide containing the target sequence. For example, a substitution of aspartic acid to alanine (D10A) in the RuvCI catalytic domain of Cas9 derived from S. pyrogenes converts Cas9 from a nuclease that cleaves both strands to a nickase (single-strand cleavage). In some embodiments, the Cas9 nickase may be used in conjunction with guide sequences, e.g., two guide sequences targeting the sense and antisense strands of a DNA target. This combination allows for the creation of breaks in both strands, which can then be used to induce either NHEJ or HDR.

[0172] In some embodiments, the Disclosure provides compounds containing one or more therapeutic proteins. The therapeutic proteins that may be included in the composition include a wide range of molecules, such as cytokines, chemokines, interleukins, interferons, growth factors, coagulation factors, anticoagulants, blood factors, bone morphogenetic proteins, immunoglobulins, and enzymes. Some non-exclusive examples of specific therapeutic proteins include erythropoietin (EPO), granulocyte colony-stimulating factor (G-CSF), α-galactosidase A, α-L-idulonidase, thyrotropin α, N-acetylgalactosamine-4-sulfatase (rhASB), dorunase alpha, tissue plasminogen activator (TPA), activase, glucocerebrosidase, interferon (IF) β-1a, interferon β-1b, interferon γ, interferon α, TNF-α, IL-1 to IL-36, human growth hormone (rHGH), human insulin (BHI), human chorionic gonadotropin α, darbepoetin α, follicle-stimulating hormone (FSH), and factor VIII.

[0173] 4. Small molecule therapeutic agents In several aspects, this disclosure provides compositions comprising therapeutic agents. These therapeutic agents include low molecular weight compounds such as 7-methoxypteridine, 7-methylpteridine, abacavir, abafungin, abarelix, acebutol, acenaphthene, acetaminophen, acetanilide, acetazolamide, acetohexamide, acitretin, acribastine, adenine, adenosine, alatrofloxacin, albendazole, albuterol, alclofenac, aldesleukin, alemtuzumab, alfuzosin, alitretinoin, alobarbital, allopurinol, all-trans retinoic acid (ATRA), aloxyprine, alprazolam, alprenolol, altretamine, amiphostine, amiloride, aminoglutethimide, aminopyrine, and amiodarone HCl. l, amitriptyline, amlodipine, amobarbital, amodiaquin, amoxapine, amphetamine, amphotericin, amphotericin B, ampicillin, amprenavir, amsacrin, amyl nitrate, amirobarbiton, anastrozole, anrinone, anthracene, anthracycline, aprobarbital, arsenic trioxide, asparaginase, aspirin, astemizole, atenolol, atorvastatin, atobakon, atrazine, atropine, atropine azathioprine, auranofin, azacitidine, azapropazon, azathioprine, azintamide, azithromycin, aztreonam, baclofen, barbiton, BCG live, beclamide, beclomethasone, bendroflumethiazide, benezepril, benidipine, benorylate, bemperidol, bentazepam, benzamide, benzanthracene, benzathine penicillin, benzhexol HCl, benznidazole, benzodiazepine, benzoic acid, befenium hydroxynaphthoate, betamethasone, bevacizumab (avastin), bexarotene, bezafibrate, bicalutamide, bifonazole, biperiden, bisacodyl, bisanthren, bleomycin, bleomycin, bortezomib, brinzolamide, bromazepam, bromocriptine mesylate, bromperidol, brotizolam, budesonide, bumetanide, bupropion,Busulfan, Butarvital, Butamben, Butenafine HCl, Butobarbiton, Butobarbiton (Butetal), Butoconazole, Butoconazole nitrate, Butylparaben, Caffeine, Calcifediol, Calciprotriene, Calcitriol, Calsterone, Cambendazole, Camphor, Camptothecin, Camptothecin analog, Candesartan, Capecitabine, Capsaicin, Captopril, Carbamazepine, Carbimazole, Carbofuran, Carboplatin, Carbromal, Carimazole (car imazole), carmustine, cephamandol, cefazolin, cefixime, ceftazidime, cefuroxime axetil, celecoxib, cefradin, cerivastatin, cetuzimab, cetuximab, chlorambucil, chloramphenicol, chlordiazepoxide, chlormethiazole, chloroquine, chlorothiazide, chlorpheniramine, chlorproguanil HCl, chlorpromazine, chlorpropamide, chlorprothixene, chlorpyrifos, chlortetracycline, chlorthalidon, chlorzoxazone, cholecalci Ferrol, Chrysene, Cilostazol, Cimetidine, Cinnarizine, Synoxacin, Ciprofibrate, Ciprofloxacin HCl, Cisapride, Cisplatin, Citalopram, Cladribine, Clarithromycin, Clemastine Fumarate, Cryoquinol, Clobazam, Clofarabine, Clofazimine, Clofibrate, Clomifene Citrate, Clomipramine, Clonazepam, Clopidogrel, Clotiazepam, Clotrimazole, Cloxacillin, Clozapine, Cocaine, Codeine, Colchicine, Colistin, Conjugated S Trogen, corticosterone, cortisone, cortisone acetate, cyclidine, cyclobarbital, cyclobenzaprine, cyclobutane-spirobarbitrate, cycloethane-spirobarbitrate, cycloheptane-spirobarbitrate, cyclohexane-spirobarbitrate, cyclopentane-spirobarbitrate, cyclophosphamide, cyclopropane-spirobarbitrate, cycloserine, cyclosporine, cyproheptadine, cyproheptadine HCl, cytarabine, cytosine, dacarbazine, dactinomycin, danazol, danthron,Dantrolene sodium, dapsone, darbepoetin α, dalodipine, daunorubicin, decoquinate, dehydroepiandrosterone, delavirdin, demeclocycline, denileukin, deoxycorticosterone, deoxymethasone, dexamethasone, dexamfetamine, dexchlorpheniramine, dexfenfluramine, dexrazoxane, dextropropoxifen, diamorphine, diatrizoic acid, diazepam, diazoxide, dichlorophene, dichlorprop, diclofenac, dicumarol, didanosine, diflunisal, digitox Syn, digoxin, dihydrocodeine, dihydroechiline, dihydroergotamine mesylate, diiodohydroxyquinoline, diltiazem HCl, diloxanide phlofluate, dimenhydrinate, dimorpholamine, dinitramide, diosgenin, diphenoxylate HCl, diphenyl, dipyridamole, dilithromycin, disopyramide, disulfiram, diuron, docetaxel, domperidone, donepezil, doxazosin, doxazosin HCl, doxorubicin (neutral), doxorubicin HCl, doxycycline, dromostanolone propionate, do Loperidol, Diphylline, Echinocandin, Econazole, Econazole Nitrate, Efavirenz, Ellipticin, Enalapril, Enlimomab, Enoximon, Epinephrine, Epipodophyllotoxin derivatives, Epirubicin, Epoetin alfa, Eposartan, Echirenin, Echirin, Ergocalciferol, Ergotamine Tartrate, Erlotinib, Erythromycin, Estradiol, Estramustine, Estriol, Estrone, Ethacrine, Ethambutol, Ethinamate, Ethio Namide, etopropazine HCl, ethyl-4-aminobenzoate (benzocaine), ethylparaben, ethinylestradiol, etodolac, etomidate, etoposide, etretinate, exemestane, felbamate, felodipine, fenbendazole, fenbuconazole, fenbufen, fenchlorfos, fenclofenac, fenfluramine, fenofibrate, fenoldepam, fenoprofen calcium, phenoxycarb, fenpiclonil, fentanyl,Fenticonazole, fexofenadine, filgrastim, finasteride, flecamide acetate, floxuridine, fludarabine, fluconazole, flucytosine, fludioxonil, fludrocortisone, fludrocortisone acetate, flufenamic acid, flunanisone, flunarizine HCl, flunisolide, flunitrazepam, fluocortone, fluomethron, fluorene, fluorouracil, fluoxetine HCl, fluoxymesterone, flupentixol decanoate, flupentixol decanoate, flu Lurazepam, flurbiprofen, fluticasone propionate, fluvastatin, folic acid, fosenopril, fosphenytoin sodium, flovatriptan, furosemide, fulvestrant, furazolidone, gabapentin, G-BHC (Linden), gefitinib, gemcitabine, gemfibrozil, gemtuzumab, graphenin, glibenclamide, gliclazide, glimepiride, glipizide, glutetimide, glibrid, glyceryl trinitrate (nitroglycerin), goserelin acetate, glepafloxacin, griseofulvin, guaifenesin, guana acetate Benz, guanine, halofantrin HCl, haloperidol, hydrochlorothiazide, heptabarbital, heroin, hesperetin, hexachlorobenzene, hexetal, histreline acetate, hydrocortisone, hydroflumethiazide, hydroxyurea, hyoscyamine, hypoxanthine, ibritumomab, ibuprofen, idarubicin, idobutal, ifosfamide, ihydroequilenin, imatinib mesylate, imipenem, indapamide, indinavir, indomethacin, indoprofen, interph Interferon α-2a, Interferon α-2b, Iodamide, Iopanoic acid, Iprodione, Irbesartan, Irinotecan, Isabuconazole, Isocarboxazide, Isoconazole, Isoguanine, Isoniazid, Isopropyl barbiturate, Isoproturone, Isosorbide dinitrate, Isosorbide mononitrate, Isradipine, Itraconazole, Itraconazole (Itra), Ivermectin, Ketoconazole, Ketoprofen, Ketrolac, Kerin, Labetalol, Lamivudine, Lamotrigine, Lanatoside C, Lansoprazole,L-DOPA, leflunomide, lenalidomide, letrozole, leucovorin, leuprolide acetate, levamizole, levofloxacin, lidocaine, linuron, lisinopril, lomefloxacin, lomustine, loperamide, loratadine, lorazepam, lomefloxacin, lormetazepam, losartan mesylate, lovastatin, lithlide maleate, maprotiline HCl, mazindol, mebendazole, meclizine HCl, meclofenamic acid, medazepam, medigoxin, medroxyprogesterone acetate, mefenamic acid, mefloquine HCl, vinegar Megestrol acid, melphalan, mepenzolate bromide, meprobamate, meptazinol, mercaptopurine, mesalazine, mesna, mesolidazine, mestranol, methadone, methacarone, methocarbamol, methoin, methotrexate, methoxsalen, methosuximide, meticlothiazide, methylphenidate, methylphenobarbiton, methyl-p-hydroxybenzoate, methylprednisolone, methyltestosterone, metiprilon, methyserzide maleate, metoclopramide, metrazone, metoprolol, metronidazole Mianserin HCl, miconazole, midazolam, mifepristone, miglitol, minocycline, minoxidil, mitomycin C, mitotane, mitoxantrone, mofetil mycophenolate, morindone, montelukast, morphine, moxifloxacin HCl, nabumetone, nadolol, nalbuffine, nalidixic acid, nandrolone, naphthacene, naphthalene, naproxen, naratriptan HCl, natamycin, nelarabine, nelfinavir, nevirapine, nicardipine HCl, nicotinamide, nicotinic acid, nicumaron, Nifedipine, nilutamide, nimodipine, nimorazole, nisoldipine, nitrazepam, nitrofurantoin, nitrofurazone, nizatidine, nofetumomab, norethisterone, norfloxacin, norgestrel, nortriptyline HCl, nystatin, estradiol, ofloxacin, olanzapine, omeprazole, omoconazole, ondansetron HCl, oprelbequine, ornidazole, oxaliplatin, oxamnicine, oxantelembonate, oxaprozin, oxatomide, Oxazepam, oxcarbazepine, oxfendazole, oxiconazole, oxprenolol, oxyfenbutazone, oxyfencycline HCl, paclitaxel, parifermin, pamidronate, p-aminosalicylic acid, pantoprazole, paramethadione, paroxetine HCl, pegademase, pegaspargase, pegfilgrastim, pemetrexed disodium, penicillamine, pentaerythritol trinitrate, pentazocine, pentobarbital, pentobarbiton, pentostatin, pentoxifylline, Perphenazine, perphenazine pimozide, perylene, phenasemide, phenacetin, phenanthrene, phenindione, phenobarbital, phenolbarbitone, phenolphthalein, phenoxybenzamine, phenoxybenzamine HCl, phenoxymethylpenicillin, fensuccinate, phenylbutazone, phenytoin, pindolol, pioglitazone, pipoproman, piroxicam, pizotifen maleate, platinum compounds, plicamycin, polyene, polymyxin B, porfimer sodium, pos Conazole (Posa), pramipexole, prasterone, pravastatin, praziquantel, prazosin, prazosin HCl, prednisolone, prednisone, primidone, probarbital, probenecid, probucol, procarbazine, prochlorperazine, progesterone, proguanyl HCl, promethazine, propofol, propoxul, propranolol, propylparaben, propylthiouracil, prostaglandin, pseudoephedrine, pteridine-2-methylthiol, pteridine-2-thiol, pteridine-4-methylthiol Chil-thiol, pteridine-4-thiol, pteridine-7-methyl-thiol, pteridine-7-thiol, pyrantelembonate, pyratinamide, pyrene, pyridostigmine, pyrimethamine, quetiapine, quinacrine, quinapril, quinidine, quinidine sulfate, quinine, quinine sulfate, rabeprazole sodium, ranitidine HCl, rasburicase, ravconazole, repaglinide, reposal, reserpine, retinoid, rifabutin, rifampicin, rifapentin, rimexolone, risperidone,Ritonavir, rituximab, rizatriptan benzoate, lofecoxib, ropinirole HCl, rosiglitazone, saccharin, salbutamol, salicylamide, salicylic acid, saquinavir, salglamostim, secobarbital, secobarbital, sertaconazole, certindol, sertraline HCl, simvastatin, sirolimus, sorafenib, sparfloxacin, spiramycin, spironolactone, stanolon, stanozolol, stabudine, stilbestrol, streptozocin, strychnine, sulconazole, sulconazole nitrate Nazole, sulfacetamide, sulfadiazine, sulfamerazine, sulfamethoxazole, sulfanilamide, sulfatiazole, sulindac, sulfabenzuamide, sulfacetamide, sulfadiazine, sulfadoxine, sulfafurazole, sulfamerazine, sulfamethoxazole, sulfapyridine, sulfasalazine, flufinpyrazone, sulpiride, sultiam, sumatriptan succinate, sunitinib maleate, tacrine, tacrolimus, tarbutal, tamoxifen citrate, tamrosin (t amulosin, targretin, taxane, tazarotene, telmisartan, temazepam, temozolomide, teniposide, tenoxicam, terazosin, terazosin HCl, terbinafine HCl, terbutaline sulfate, terconazole, terfenadine, testolactone, testosterone, tetracycline, tetrahydrocannabinol, tetroxoprim, thalidomide, thebaine, theobromine, theophylline, thiabendazole, thiamphenicol, thioguanine, thioridazine, thiotepa, totoin, thymine, thiagabine H Cl, tiborone, ticlopidine, tinidazole, thioconazole, tyrofiban, tizanidine HCl, trazamide, tolbutamide, tolcapone, topiramate, topotecan, toremifene, tositumomab, tramadol, trastuzumab, trazodone HCl, tretinoin, triamcinolone, triamterene, triazolam, triazole, triflupromazine, trimethoprim, trimipramine maleate, triphenylene, troglitazone, tromethamine, tropicamide, trovafloxacin, cibamate, ubidecarenone (coenzyme Q10),Undecenoic acid, uracil, uracil mustard, uric acid, valproic acid, barrubicin, valsartan, vancomycin, venlafaxine HCl, vigabatrin, vinbarbital, vinblastine, vincristine, vinorelbine, voriconazole, xanthine, zafirlukast, zidovudine, diloton, zoledronate, zoledronic acid, zolmitriptan, zolpidem, and zopiclone may also be used.

[0174] F. Kit This disclosure also provides kits. Any of the components disclosed herein can be combined in the form of a kit. In some embodiments, the kit comprises the compositions described above or in the claims.

[0175] A kit generally includes at least one vial, test tube, flask, bottle, syringe, or other container in which the components can be arranged and, preferably, appropriately dispensed. If the kit contains two or more components, the kit also generally includes a second, third, or other additional container in which the additional components can be arranged separately. However, various combinations of components can be included in the container. In some embodiments, all of the lipid nanoparticle components are combined in a single container. In other embodiments, some or all of the lipid nanoparticle components are provided in separate containers.

[0176] The kits of this disclosure also typically include packaging for sealing and containing various containers for commercial sale. Such packaging may include cardboard or injection-molded or blow-molded plastic packaging in which the desired containers are held. The kits may also include instructions for utilizing the kit components. The instructions may include viable variations. [Examples]

[0177] F. Examples The following embodiments are included to demonstrate preferred embodiments of the Disclosure. Those skilled in the art will understand that the techniques disclosed in the following embodiments represent techniques discovered by the inventors to function well in the practice of the Disclosure, and can therefore be considered to constitute a preferred mode for that practice. However, those skilled in the art should recognize that, in light of the Disclosure, many modifications can be made in the particular embodiments disclosed without departing from the spirit and scope of the Invention, and similar or comparable results can still be obtained.

[0178] Example 1: Preparation of DOTAP-modified lipid nanoparticles Lipid nanoparticles (LNPs) are the most effective carrier class for in vivo nucleic acid delivery. Historically, effective LNPs consist of four components: cationic ionizable lipids, zwitterionic phospholipids, cholesterol, and lipid poly(ethylene glycol) (PEG). However, these LNPs do not target organs or tissues, delivering nucleic acids only systemically. LNPs typically deliver RNA only to the liver. Therefore, novel LNP formulations are being sought in attempts to provide targeted nucleic acid delivery.

[0179] Four classical types of lipids were mixed in a 15:15:30:3 molar ratio with or without the addition of permanently cationic lipids. Briefly, LNPs were prepared by mixing 5A2-SC8 (cationic and ionizable), DOPE (zwitterionic), cholesterol, DMG-PEG, and DOTAP (permanently cationic) in the ratios shown in Table 0.1.

[0180] (Table 0.1) Molar ratio and molar percentage of lipids in modified LNPs TIFF2026082961000049.tif96170

[0181] To prepare the mDLNP formulation, 5A2-SC8, DOPE, cholesterol, and DMG-PEG were dissolved in ethanol in a predetermined molar ratio (15:15:30:3). The mRNA was then dissolved in citrate buffer (10 mM, pH 4.0). Next, the mRNA was diluted with the lipid solution by mixing it with the lipid solution in a volume ratio of 3:1 (mRNA:lipid, v / v) to achieve a weight ratio of 40:1 (total lipids:mRNA). This solution was then incubated at room temperature for 10 minutes. To form the DOTAP-modified mDLNP formulation, the mRNA was dissolved in 1×PBS or citrate buffer (10 mM, pH 4.0) and rapidly mixed with ethanol containing 5A2-SC8, DOPE, cholesterol, DMG-PEG, and DOTAP to fix the weight ratio of 40:1 (total lipids:mRNA) and the volume ratio of 3:1 (mRNA:lipid). Each formulation was named DOTAPX, as shown in Table 1. In the formula, X represents the molar percentage of DOTAP in total lipids.

[0182] Example 2: Characterization of mDLNP formulations modified with DOTAP To characterize various mDLNP formulations, the size, polydispersity index, and zeta potential of each formulation were examined three times by dynamic light scattering. The size and polydispersity index are shown in Figure 5A. From this, it can be seen that all formulations fall within the size range of approximately 90 nm to 160 nm, regardless of DOTAP concentration, while the polydispersity index, which indicates the relative uniformity of size, varies from approximately 0.1 to 0.3. The zeta potential of each formulation is shown in Figure 5B. From this, it can be seen that the zeta potential generally increases with DOTAP concentration.

[0183] Next, encapsulation efficiency was tested using the Ribogreen RNA assay (Zhao et al., 2016). Briefly, when mRNA was dissolved in acidic buffer (10 mM citrate, pH 4), the mRNA was encapsulated in DOTAP-free mDLNP with approximately 85% efficiency (Figure 5C). A low pH is required to protonate the ionizable amines in cationic ionizable lipids (e.g., 5A2-SC8, C12-200, DLin-MC3-DMA) and enable electrostatic complex formation with negatively charged mRNA. For all other formulations shown in this figure, mixing was performed using mRNA dissolved in PBS at pH 7.4. Clearly, encapsulation efficiency was low when using low concentrations of DOTAP, but increased to >80% when the molar percentage of DOTAP exceeded 25% (Figure 5C). The encapsulation efficiency was approximately 80% to 95% for all formulations using DOTAP at molar percentages exceeding 25%. Therefore, the potential of using a neutral pH PBS mixture is a characteristic feature of the permanent cationic lipid strategy. This strategy enables tissue-specific delivery and encapsulation of large amounts of Cas9 protein. The addition of permanent cationic lipids enables LNP formation at neutral pH. These encapsulation results were obtained when PBS was used as the buffer. When an acidic buffer was used (e.g., citrate buffer (10 mM, pH 4.0)), the encapsulation efficiency was high (>90%) for all formulations of 0-100% DOTAP.

[0184] Finally, pKa was determined using the 2-(p-toluidino)-6-naphthalenesulfonic acid (TNS) assay (Figure 3B) (Zhao et al., 2016). Based on defined rules, the relationship between pKa and tissue-specific mRNA delivery was plotted. Here, eight rules were designed for scoring, as shown in the table. Clearly, liver-targeted agents had a narrow pKa range (approximately 6-7), spleen-targeted agents had no significant range, while lung-targeted delivery required a high pKa (>9.25). Considering distribution and pKa detection together, we concluded that the internal charge of NPs is one factor influencing mRNA distribution, and the overall / apparent LNP pKa is another factor determining the mRNA-mediated protein expression profile in organs.

[0185] Example 3: Efficacy of mDLNP modified with permanent cationic lipids for mRNA delivery To investigate the delivery efficacy of LNPs containing permanent cationic lipids in vitro, luciferase-encoding mRNA was loaded into DOTAP-modified mRNA LNPs, and Huh-7 hepatocytes and A549 adenocarcinoma human alveolar basal epithelial cells were transfected with 50 ng / well mRNA. After culturing these cells for 24 hours, luciferase expression and cell viability were examined. As shown in Figure 6A, 5%–50% DOTAP percentages were better for mRNA delivery and expression in Huh-7 hepatocytes in vitro, with 10% DOTAP appearing to show the best luciferase delivery and expression (Figure 6A). It should be noted that in vivo delivery characteristics may differ. Generally, due to further in vivo barriers, organ distribution, and cellular specificity of SORT LNPs, these studies may not be useful in predicting in vivo activity or tissue tropism. Furthermore, cell viability was examined, and among the mDLNPs that showed robust luciferase expression, 10% DOTAP was found to result in high viability (Figure 6A). Similar results were observed when the same transfection was performed using the A549 lung cancer cell line. Cells transfected with the DOTAP10 formulation showed nearly twice the fluorescence of those transfected with other formulations and maintained high cell viability (Figure 6A). DOTAP SORT LNPs were formed in PBS (pH 7.4) rather than citrate buffer (10 mM, pH 4.0), but they can be formed in either buffer system. This is characteristic as it allows for the encapsulation and delivery of cargo that is unstable in both ethanol and acidic buffers, such as proteins.

[0186] To investigate the effect of ethanol concentration in the formulation, DOTAP25 was selected and prepared using various ethanol:PBS ratios (1:3, 1:5, 1:7.5, and 1:10) (Figure 6B). All four formulations showed similar encapsulation efficiency, size, and PDI (Figure 2B). mRNA delivery efficiency was also measured by transfecting FaDu hypopharyngeal cancer cells with 50 ng / well mRNA present in each formulation. The delivery efficiency of each formulation was similar and had little effect on cell viability (Figure 6C). Therefore, these formulations appear to be applicable to a number of cell types, including liver, lung, and pharynx. Furthermore, this formulation was also successfully modified to use 1×PBS (pH 7.4) instead of acidic buffer (pH 4.0). These data show that the percentage of ethanol can be drastically reduced. This suggests that DOTAP formulations may be capable of delivering cargo, such as proteins, that are considerably sensitive to high ethanol concentrations and acidic buffers.

[0187] Next, to test the ability of these mDLNPs to deliver mRNA in vivo, mice were injected with 0.1 mg / kg doses of Luc mRNA, as present in each formulation. Figure 1B shows ex vivo images of luciferase in major organs 6 hours after IV injection of each formulation. Interestingly, as the molar percentage of DOTAP increased, luciferase expression shifted from the liver to the spleen, and then to the lungs, demonstrating organ-specific delivery. Quantifying these data revealed that the DOTAP percentage is a factor in tissue-targeted delivery, with mDLNP (0% DOTAP) being best for liver delivery, 5–15% DOTAP being best for the spleen, and DOTAP 50 (50%) being best for lung delivery (Figure 1B). Assuming that luciferase expression was detected only in the liver, spleen, and lungs after IV injection, the percentage of luciferase expressed in each organ can be calculated (Figure 1C). These data clearly show that as the molar percentage of DOTAP in the formulation increases, delivery to the liver and expression in the liver decrease, with near-zero expression observed in the liver when the DOTAP percentage exceeds 70% (Figures 1B, 1C). However, the higher the DOTAP percentage, the greater the luminescence observed in lung tissue, with near-100% luminescence observed in the lungs when the DOTAP percentage exceeds 80% (Figure 1C). While DOTAP 5 and 30 molar percentages showed high percentage luminescence in spleen tissue, DOTAP 10 showed the highest relative luminescence in the spleen compared to other tissues (Figure 1C). These results suggest that lipid concentrations can be adjusted to match specific tissue delivery after injection.

[0188] To further investigate the organ-specific biodistribution of specific DOTAP formulations, C57BL / 6 mice (n=2) were injected with PBS or liver-targeted NPs (mDLNPs), spleen-targeted NPs (DOTAP10), and lung-targeted NPs (DOTAP50) at doses of 0.5 mg / kg Cy5-Luc mRNA (dye-labeled mRNA tracking RNA LNPs). Major organs were collected and imaged 6 hours after injection (Figure 3A). The organ distribution of the formulations varied with DOTAP dose, with accumulation in the liver gradually shifting to the lungs as the DOTAP percentage increased, although NPs remained in the liver regardless of the DOTAP percentage (Figures 7 and 8). Considering this data together with the data in Figure 1, it is clear that organ distribution is insufficient to analyze tissue-targeted delivery efficacy (mRNA translation to protein). Furthermore, given the similar size distribution and EE among these formulations, zeta potential and pKa may play a role in tissue-targeted mRNA expression.

[0189] In an attempt to understand whether the effect of adding DOTAP to mDLNPs is limited, or whether the distribution shown above is universal for mDLNPs formulated with permanent cationic lipids, we prepared mDLNPs containing another popular cationic lipid, didodecyldimethylammonium bromide (DDAB) (Figure 2A1). DDAB has 18 carbon atoms and two hydrophobic tails without unsaturated bonds, giving it a head that is completely different from DOTAP (Figure 1C). DDAB5, DDAB15, DDAB40, and DDAB50 formulations were selected for in vivo delivery (0.1 mg / kg, 6h, n=2). Similar to the DOTAP formulations above, there was little difference in size distribution (Figure 2A1), but the DDAB percentage in the NPs changed tenfold (5% to 50%). In vivo voluciferase expression showed a tendency for luminescence to move from the liver to the spleen and then to the lungs as the DDAB percentage increased, similar to DOTAP NPs (Figure 2A2).

[0190] mDLNPs were formed using a third permanent cationic lipid with a head 1,2-dimiristoyl-sn-glycero-3-ethyl phosphocholinchloride that has a structure similar to DOTAP but with a shorter 14-carbon hydrophobic tail ((14:0)EPC) (Figure 2B1). Similar to the DDAB strategy, (14:0)EPC5, (14:0)EPC15, (14:0)EPC40, and (14:0)EPC50 formulations were prepared and their size distribution (Figure 2B1) and in vivo Luc mRNA delivery (0.1 mg / kg, 6h, n=2) were examined (Figure 2B2). Similar to the mDLNPs analyzed above, the particle size was generally uniform (Figure 2B1), and as expected, luminescence migrated from the liver to the spleen and then to the lungs as the (14:0)EPC molar percentage increased (Figure 2B2). Considering all of this data, including different hydrophobic tails, saturated and unsaturated bonds, and different heads, it appears that mDLNPs formulated with cationic lipids are universal for tissue-targeted mRNA delivery.

[0191] In an attempt to understand whether the effect of DOTAP addition to LNPs is specific to permanent cationic lipids, we investigated the effect of adding zwitterionic lipids instead of permanent cationic lipids to mDLNP formulations. Two representative zwitterionic lipids with different chemical structures, phosplolipids: DSPC and DOCPe, were tested. Furthermore, this was also tested to determine whether the addition of zwitterionic lipids (instead of permanent cationic lipids) affects tissue-specific delivery efficacy. Figures 2C1 and 2D1 show the chemical structures of DSPC and DOCPe lipids (zwitterionic lipids). There are differences in the position of the positively charged and negatively charged functional heads and the hydrophobic domain (saturated vs. unsaturated). From this, it is suggested that the observed effect is general / universal to zwitterionic lipids. mDLNPs formulated with DSPC or DOCPe were similar (Figures 2C1, 2D1). Interestingly, including zwitterionic lipids in the five-component modified DLNP did not alter the protein expression profile from liver to lung, as did with DOTAP and other permanent cationic lipids. Instead, both DSPC and DOCPe improved mRNA delivery to the spleen within a given range (less than 80% for DSPC and less than 50% for DOCPe). No protein expression occurred in the lung at any percentage (0.1 mg / kg, 6h, n=2) (Figure 2C2, 2D2). Therefore, while the inclusion of further zwitterionic lipids may aid splenic delivery, it cannot modulate the liver-to-spleen-to-lung delivery efficacy as it does with the inclusion of permanent cationic lipids.

[0192] In an attempt to understand whether the effect of adding DOTAP to LNPs is specific to permanent cationic lipids, we investigated the effect of adding cationic ionizable lipids instead of permanent cationic lipids to mRNA DLNP formulations. Two representative cationic ionizable lipids with different chemical structures, C12-200 and DODAP, were tested. DODAP has the same structure as DOTAP except for the head (quaternary amine vs. tertiary amine). C12-200 is an effective lipidoid used for siRNA or mRNA delivery, containing an ionizable tertiary amine (also not a quaternary amine), and has a completely different structure from DODAP. (Figures 2E1, 2F1) Similarly, the size distribution of both modified mRNAs remained uniform at a certain percentage (less than 80%). (Figures 2E1, 2F1) Surprisingly, including cationic ionizable lipids in the five-component modified mRNA did not alter the protein expression profile from liver to spleen to lung, as with DOTAP and other permanent cationic lipids. Instead, the inclusion of cationic ionizable lipids in DLNPs increased mRNA delivery efficacy to the liver. These showed significantly better delivery efficacy than the original mDLNP (0.1 mg / kg, 6h, n=2) without additional cationic ionizable lipids (5A2-SC8 only). As the percentage of DODAP or C12-200 increased (50% or 80%), luciferase signaling decreased significantly, but the liver remained the primary organ rather than the spleen or lungs. Therefore, we concluded that the organ-specific effect may be attributable to the inclusion of a specific ratio of permanent cationic lipids. Furthermore, these data suggest that permanent cationic lipids produce a different effect than cationic ionizable lipids. Moreover, these data suggest that these trends are universal with respect to lipid classes.

[0193] Example 4: CRISPR / Cas9 gene editing using an improved mDLNP that simultaneously delivers Cas9 mRNA and sgRNA. First, to determine which sgRNA would be most effective in later experiments, we compared three sgRNAs that target Td-Tomato mice. These sgRNAs were sgTom1, sgTom2, and sgLoxP. We delivered and expressed sgTom1 and sgLoxP as shown in Figure 10A, and obtained similar results. They performed better than sgTom2 in inducing TdTomato (Figure 10A). Considering the weak PAM of sgLoxP (NAG), we ultimately selected sgTom1 for further experiments.

[0194] Given that tissue-specific mRNA (luc mRNA) delivery was demonstrated using DOTAP NPs, and that NPs modified by DDAB and EPC showed similar delivery tendencies, the next step was to use DOTAP-modified mDLNPs to simultaneously deliver Cas9 mRNA / sgRNA, with the goal of achieving tissue-specific gene editing. To investigate in vivo simultaneous delivery, genetically modified mice containing a homozygous Rosa26 promoter Lox-Stop-Lox tdTomato (tdTO) cassette present in all cells were used (Figure 4A). Simultaneous delivery of Cas9-mRNA and DOTAP-modified mDLNPs containing sgRNA for LoxP or Tom resulted in deletion of the Stop cassette and induction of tdTO expression (Figure 4B). Mice were intravenously injected with mDLNPs and DOTAP50 formulations to simultaneously deliver IVT Cas9 mRNA and modified sgTom1 (4 / 1, wt / wt) at a total dose of 2.5 mg / kg (50 ug each). Subsequently, fluorescence in major organs was detected 10 days after treatment (Figure 4B). Liver-specific and lung-specific CRISPR / Cas gene editing was achieved. Spleen-specific editing was also achieved. However, due to very strong background red autofluorescence, spleen editing could not be quantified using this TdTomato reporter mouse.

[0195] To further investigate tissue-specific editing, PTEN was selected as the endogenous target. C57BL / 6 mice were intravenously injected with mDLNP, DODAP20, or DOTAP50 to achieve tissue-specific gene editing. The total dose was 2.5 mg / kg (50 ug each), the weight ratio of IVT Cas9 mRNA to modified sgPTEN was 4 / 1, and the detection time was 10 days post-treatment. PTEN-targeted sgRNA was used. T7E1 assays revealed further confirmation of tissue-specific characteristics by in vivo PTEN editing (Figure 4C).

[0196] Example 5: CRISPR / Cas9 gene editing using a modified mDLNP that delivers Cas9 protein / sgRNA ribonucleoprotein (RNP) Building on the discovery of the inclusion of a permanent cationic lipid (e.g., DOTAP) in conventional LNP formulations containing cationic ionizable lipids, zwitterionic lipids, cholesterol, and PEGylated lipids, we investigated whether this formulation methodology could also deliver other cargoes sensitive to ethanol and / or low pH acidic buffered aqueous solutions. A key element of the DOTAP strategy is that formulations can be prepared using neutral pH PBS. Therefore, we also investigated whether this methodology could encapsulate and deliver large proteins such as Cas9 for gene editing applications. Thus, DOTNP lipid nanoparticles consist of modules containing five components: cationic ionizable lipids (e.g., 5A2-SC8), zwitterionic lipids (e.g., DOPE), cholesterol, DMG-PEG, and a permanent cationic lipid (e.g., DOTAP). The molar ratios of 5A2-SC8, cholesterol, DOPE, and DMG-PEG were fixed (15:15:30:5, mol / mol). DOTNPX refers to DOTNP containing different molar percentages of DOTAP.

[0197] We investigated whether the initially characterized Cas9 / sgRNA complexes were sensitive to acidic pH. The size (diameter) (Figure 11A) and zeta potential (Figure 11B) of Cas9 / sgLUC complexes (mol / mol = 1 / 1) were measured in PBS (pH 7.4) and citrate buffer (pH 4.2). The Cas9 / sgLUC complexes prepared in citrate buffer were large (greater than 100 nm) and positively charged. Due to these two characteristics (larger size than typical effective LNPs) and positive charge (unsuitable for complexing with positively charged lipids), effective encapsulation by lipid nanoparticles is impossible. However, the Cas9 / sgLUC complexes prepared in PBS were small (less than 20 nm) and negatively charged. Therefore, they can be encapsulated by lipid nanoparticles when formulated at a neutral pH. Next, Cas9 / sgRNA complexes with different Cas9 protein:sgRNA molar ratios were prepared and characterized. Size (Figure 11C) and zeta potential (Figure 11D) of Cas9 / sgLUC complexes prepared with different Cas9 / sgRNA molar ratios (1 / 1, 1 / 3, and 1 / 5). Compared to the Cas9 / sgLUC complex (1 / 1, mol / mol), larger molar ratios (1 / 3 and 1 / 5, mol / mol) resulted in smaller size and larger negative charge. This may be beneficial for lipid nanoparticle encapsulation. These compositions were prepared and the DOTNP lipid nanoparticles after encapsulating the Cas9 / sgRNA complex were characterized. Size (Figure 11E) and zeta potential (Figure 11F) of DOTNP10 lipid nanoparticle-encapsulated Cas9 / sgLUC complexes (named DOTNP10-L) when prepared with different molar ratios (1 / 1, 1 / 3, and 1 / 5). This data demonstrates the encapsulation of Cas9 / sgRNA RNPs into monodisperse LNPs. Figure 11G shows a TEM image of DOTNP10-L(1 / 3, mol / mol)LNP containing encapsulated RNPs. Following this initial study, different sgRNAs, including sgLUC, sgGFP, sgTOM, and sgPTEN, were used. To distinguish them, the first letter of each gene was added to the end of the DOTNP.For example, DOTNP10-L refers to the DOTNP10 lipid nanoparticle encapsulated Cas9 / sgLUC complex. DOTNP10-G refers to the DOTNP10 lipid nanoparticle encapsulated Cas9 / sgGFP complex.

[0198] (Table 7) Characterization of DOTAP10, DSPC50, and DODAP50 formulations formed with PBS and citrate buffer, including size, PDI, and encapsulation efficacy. TIFF2026082961000050.tif45151

[0199] (Table 8) All primers used in this study are listed, including the length of the PCR product and its purpose (Cas9=SEQ ID NO:3~4; Ca9 Seq-1=SEQ ID NO:5; Ca9 Seq-2=SEQ ID NO:6; Ca9 Seq-3=SEQ ID NO:7; Ca9 Seq-4=SEQ ID NO:8; Ca9 Seq-5=SEQ ID NO:9; Ca9 Seq-6=SEQ ID NO:10; Ca9 Seq-7=SEQ ID NO:11; PTEN=SEQ ID NO:14~15; IVT sgTom1=SEQ ID NO:44~45; IVT sgTom2=SEQ ID NO:46~47; IVT sgLoxP=SEQ ID NO:48~49). TIFF2026082961000051.tif124170

[0200] A series of experiments were conducted to investigate whether DOTNP lipid nanoparticles can deliver Cas9 / sgRNA RNP complexes to the nucleus in vitro and mediate efficient gene editing. First, DOTNPs containing Cas9 / sgRNA RNPs tagged with green fluorescent EGFP were tracked by confocal microscopy (Figure 12A). Images of Hela-Luc cells (using 9nM sgRNA) after incubation of DOTNP10-encapsulated Cas9-EGFP / sgLUC complexes (1 / 3 mol / mol) for 1, 3, 6, and 24 hours showed that DOTNPs translocated into the cells and Cas9 RNPs were transported to the nucleus. Green: EGFP-fused Cas9 protein; Blue: Nuclei stained with Hoechst33342. Red arrows indicate the process of DOTNP10 entering the nucleus. (Figure 12B)

[0201] Next, we investigated whether DOTNP lipid nanoparticles could deliver Cas9 / sgRNA RNP complexes and whether these complexes could cleave target luciferase DNA. The percentage of DNA indels (insertions and deletions) at the LUC locus after incubation with different molar ratios of DOTNP10-L for 3 days was quantified using the TIDE assay (using 24 nM sgRNA). DOTNP10 lipid nanoparticle-encapsulated Cas9 / sgGFP (DOTNP10-G) was used as a negative control. Two commercially available Cas9 proteins (GeneArt Cas9 and Truecut Cas9) were used here (Figure 12B). Next, to demonstrate DNA editing, T7EI cleavage assays (24 nM sgRNA) were performed on Hela-Luc cells incubated with different formulations (Figure 12C). Of the conditions tested, a 1 / 3 molar ratio using Truecut Cas9 protein showed the best gene editing. Finally, GFP editing was tested using fluorescence microscopy (Figure 12D). Images of SKOV3-GFP cells incubated with DOTNP10-L (control, not targeting GFP) and DOTNP10-G (targeting GFP) (24 nM sgRNA). Editing on the target was demonstrated by loss of GFP protein expression. Finally, flow cytometry was used to analyze SKOV3-GFP cells incubated with DOTNP10-L and DOTNP10-G (Figure 12E). (Figure 12F) Mean fluorescence intensity of SKOV3-GFP cells incubated with DOTNP10-L and DOTNP10-G, as determined by flow cytometry, indicated CRSPR / Cas-mediated GFP editing.

[0202] Next, we investigated whether DOTNP lipid nanoparticles could deliver Cas9 / sgRNA RNP complexes in vivo to achieve CRISPR / Cas-mediated gene editing. As before, we used a genetically modified TdTomato mouse model. 1.5 mg / kg of sgRNA was delivered per mouse using the following formulations: DOTNP5-T refers to the DOTNP5 LNP-encapsulated Cas9 / sgTom complex; DOTNP10-T refers to the DOTNP10 LNP-encapsulated Cas9 / sgTom complex; and DOTNP50-T refers to the DOTNP50 LNP-encapsulated Cas9 / sgTom complex. After IV injection of these formulations, tdTomato fluorescence was quantified ex vivo in major organs 7 days after injection (Figure 13A). In the DOTNP5-T treated group, tdTomato fluorescence was detected only in the liver. In the DOTNP10-T group, slight fluorescence was observed in the lungs, and when the DOTAP dose was further increased to 50% (DOTNP50-T), almost all of the tdTomato fluorescence was observed in the lungs. Therefore, similar to the mRNA delivery experiments summarized above, the DOTAP methodology also enables tissue-specific gene editing of the Cas9 / sgRNA ribonucleoprotein (RNP) complex. To further investigate delivery, LNPs containing sgRNA against PTEN were delivered. Using T7EI cleavage assays of liver and lung organs, IV injection of DOTNP5-P (DOTNP5 LNP-encapsulated Cas9 / sgPTEN complex), DOTNP10-P (DOTNP10 LNP-encapsulated Cas9 / sgPTEN complex), and DOTNP50-P (DOTNP50 LNP-encapsulated Cas9 / sgPTEN complex) (2 mg / kg sgRNA / mouse) was confirmed to mediate gene editing (Figure 13B). The results are consistent with those obtained by ex vivo imaging. Gene editing was observed only in the liver after treatment with DOTNP5-P. When incubated with DOTNP10-P, gene editing was observed in both the liver and lungs. In contrast, in the DOTNP50-P treated group, most of the gene editing was observed in the lungs.

[0203] The data presented herein demonstrate that lipid nanoparticles can be prepared using a wide variety of compositions to specifically target payloads to particular tissues. In particular, lipid nanoparticles containing low concentrations of permanent cationic lipids (≤10%) are effective in delivering nucleic acids to the liver, LNPs containing less than 30% permanent cationic lipids are effective in delivering nucleic acids to the spleen, and LNPs containing more than 30% permanent cationic lipids effectively deliver nucleic acids to the lungs. These findings appear to be universal, with head, saturation, and tail length having little effect.

[0204] Example 6: Adding another lipid to four known lipid compositions alters the delivery target. Next, we explored the generalizability of an approach (methodology) that includes a "fifth" lipid in the established four-component LNP.

[0205] To investigate whether the inclusion of a permanent cationic lipid (e.g., DOTAP) can alter the tissue specificity of other cationic ionizable lipids, two well-known and well-established cationic ionizable lipid LNP systems were selected. DLin-MC3-DMA was selected and formulated with DSPC, cholesterol, and PEG-DMG. The same molar compositions as Patisiran / Onpattro (Alnylam Pharmaceuticals) were produced, and 15% or 50% DOTAP (an extra fifth lipid in the Onpattro 4 lipid formulation) was added (Figure 14). DLin-MC3-DMA LNPs are considered the "gold standard" for siRNA and mRNA delivery. To date, they have only been shown to be delivered to the liver after IV administration. As shown in Figure 15A, DOTAP altered the organ mRNA expression profile of DLin-MC3-DMA-based LNPs (0.1 mg / kg luciferase mRNA, 6 hours). As the percentage of DOTAP increased, the luciferase signal moved from the liver to the spleen and finally to the lungs. This was exactly the same phenomenon as with 5A2-SC8 mDLNP. To further investigate the universality of this approach, the inventors included DOTAP in C12-200 LNP (Figure 16). DLin-MC3-DMA is a lipid with one dimethylamine head and two tails, considered to be a stable nucleic acid lipid nanoparticle (SNALP), while C12-200 is a representative "lipidoid" that can be formulated into lipid-like LNPs. All three are cationic ionizable lipids. The results were identical to those with 5A2-SC8 and DLin-MC3-DMA, but when 15% or 50% DOTAP was included in C12-200 LNP, luciferase protein expression after mRNA delivery shifted from the liver to the spleen to the lungs (Figure 15B). Thus, the fifth lipid methodology (e.g., adding permanent cationic lipids) is generalizable to other cationic ionizable lipid LNPs.

[0206] Example 7: Adding another lipid to four known lipid compositions improves delivery. Furthermore, we explored the generalizability of the approach (methodology) by asking whether additional cationic ionizable lipids improve liver delivery.

[0207] In general, to investigate whether cationic ionizable lipids promote hepatic delivery, an additional 5A2-SC8 cationic ionizable lipid was included as a "fifth" lipid in LNPs containing 5A2-SC8, DOPE, cholesterol, and PEG-DMG in appropriate ratios. Extra 5A2-SC8 was included in 10–30 percent increments (Figures 17A and 18). To avoid luminescence saturation, low mRNA doses of 0.05 mg / kg were tested (IV, 6 hours). As shown in Figure 17B, both ex vivo images and quantitative data demonstrated that an increased 15–25% extra 5A2-SC8 helped improve mRNA delivery efficacy in the liver. 5A2-SC8^20 (5A2-SC8 LNP + 20% extra 5A2-SC8) increased luciferase by 2–3 times compared to the original mDLNP formulation.

[0208] Example 8: Research related to selective organ-targeting compositions This disclosure describes a strategy called selective organ targeting (SORT) that enables the systematic engineering of nanoparticles to deliver diverse cargoes, including mRNA, Cas9 mRNA / sgRNA, and Cas9 ribonucleoprotein (RNP) complexes, precisely to the lungs, spleen, and liver of mice after intravenous (IV) administration (Figure 19A). Conventional LNPs consist of cationic ionizable lipids, zwitterionic phospholipids, cholesterol, and poly(ethylene glycol) (PEG) lipids. This disclosure shows that the addition of auxiliary components (referred to as SORT compounds or selective organ targeting compounds) precisely alters the in vivo RNA delivery profile and mediates tissue-specific gene delivery and editing as a function of the percentage of added SORT lipids and their biophysical properties. This disclosure provides theoretical evidence for tissue-specific delivery, establishes the applicability of this methodology to various nanoparticle systems, and provides a method for designing LNPs for editing therapeutically relevant cells.

[0209] Effective intracellular delivery materials have traditionally relied on an optimal balance of ionizable amines (pKa 6.0-6.5) for binding to and releasing RNA, and nanoparticles to stabilize hydrophobicity (Kanasty et al., 2013; Jayaraman et al., 2012; Nelson et al., 2013; Hao et al., 2015). While not bound by any theory, it is thought that internal and / or external charges may be factors that modulate tissue tropism. Intravenous administration of the developed SORT LNP enabled high levels of tissue-specific gene editing. SORT is compatible with various methods of locating gene editing mechanisms, including mRNA, Cas9 mRNA / sgRNA, and Cas9 RNP (systemic RNP delivery). Lung-targeted SORT LNP edited 40% of epithelial cells and 65% of endothelial cells. Spleen-targeted SORT LNP edited 13% of B cells and 10% of T cells. Enhanced liver-targeted SORT LNP edited 93% of hepatocytes after a single low-dose injection.

[0210] A. Discovery and Development of SORT To investigate the hypothesis that tissue-specific delivery can be mediated by regulating internal charge, a strategy was devised to add a fifth lipid to already established LNP compositions, and its efficacy in hepatocytes was verified. The principle was to prepare effective LNP formulations without disrupting the core four-component ratio typically used to mediate RNA encapsulation and endosomal evasion (Wittrup et al., 2015; Cheng et al., 2018).

[0211] The effects of adding permanent cationic lipids (defined as having no pKa or being positively charged with pKa > 8) to a degradable dendrimer-ionizable (pKa < 8) cationic lipid named 5A2-SC8, used in mDLNPs, were investigated (Zhou et al., 2016; Zhang et al., 2018a; Zhang et al., 2018b). This effectively delivered fumarylacetoacetate hydrolase (FAH) mRNA to hepatocytes and extended survival time in FAH knockout mice (Cheng et al., 2018). This initial basic mRNA LNP formulation consisted of 5A2-SC8, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), cholesterol, DMG-PEG (15 / 15 / 30 / 3, mol / mol), and mRNA (5A2-SC8 / mRNA, 20 / 1, wt / wt) (Figure 20). Subsequently, a series of LNPs were formed by systematically increasing the percentage of additional permanent cationic lipids from 5 to 100% relative to the total lipids (Figures 19B and 20). First, the well-known quaternary aminolipid 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) was selected as the SORT lipid to be added to the LNP formulation. Thus, the DOTAP-modified SORT formulation contained five lipid components. A series of titrated formulations were prepared by fixing 5A2-SC8 / DOPE / Chol / DMG-PEG to 15 / 15 / 30 / 3 (mol / mol) and adding DOTAP in molar ratios from 0 to 1200 (Figure 20).

[0212] Next, the effects of SORT modification were evaluated by intravenous (IV) delivery of luciferase (Luc) mRNA at a dose of 0.1 mg / kg. As the molar percentage of DOTAP increased, the resulting luciferase protein expression successively moved from the liver to the spleen and then to the lungs. This demonstrated a clear and precise organ-specific delivery tendency with a threshold enabling exclusive lung delivery (Figure 19B). The DOTAP percentage was a key factor modulating tissue specificity. The basic LNP (0% DOTAP) was optimal for liver delivery, as it had been previously optimized for hepatocyte delivery and was therefore expected (Cheng et al., 2018). With the addition of 10-15% DOTAP, the resulting SORT LNP was able to deliver mRNA to cells in the spleen. Further increases in the permanent cationic SORT lipid revealed that 50% DOTAP was optimal for lung delivery (Figure 19C). It is noteworthy that 50% DOTAP SORT LNPs were effective in delivering mRNA to the lungs in vivo, but not as effective as in vitro delivery (Figure 21). Furthermore, 50% DOTAP SORT LNPs possess a neutral zeta potential surface charge (-0.52 mV) (Figure 20). This indicates that tissue tropism is not due to MPS uptake associated with a positive charge. When calculating relative expression in each organ, using DOTAP as the SORT lipid completely shifted delivery from the liver to the lungs (Figure 19D). Thus, considering that >99% of modern IV nanomedicines have been estimated to be sequestrated by MPS (Wilhelm et al., 2016; Gustafson et al., 2015), these novel SORT nanoparticles overcome a long-standing challenge in nanomedicines.

[0213] While not bound by any particular theory, the elucidation of the functional role of permanently cationic SORT lipids suggests that the inclusion of other lipids may also alter tissue tropism. To explore this potential, negatively charged 1,2-dioleoil-sn-glycero-3-phosphate (18PA) was incorporated as a SORT lipid, similar to DOTAP (Figure 20). With 10-40% 18PA incorporation, SORT LNPs mediated completely selective delivery to the spleen, resulting in no luciferase expression in other organs (Figure 19E). Therefore, negatively charged SORT lipids enable clear delivery to the spleen. These results indicate that the percentage of SORT lipids can be adjusted to match specific tissue mRNA delivery via IV injection.

[0214] B.SORT is generalizable to other LNP types and lipid classes. Next, to test whether SORT is universal, we explored whether the SORT methodology could be applied to other classes of established four-component LNPs. First, we formulated DLin-MC3-DMA with DSPC, cholesterol, and PEG-DMG using the same molar composition as the FDA-approved Onpattro (Patisiran) (30), which is considered the "gold standard" for siRNA and mRNA delivery (Figure 22). To date, these have only been shown to be delivered to the liver after IV administration, and this was confirmed here as well (Figure 19F). As expected, the addition of DOTAP to DLin-MC3-DMA LNP altered the protein expression profile of the Onpattro formulation. As the SORT lipid percentage increased, the luciferase signaling moved from the liver to the spleen to the lungs. This was exactly the same phenomenon as with the 5A2-SC8 DLNP that we tested earlier. To further study the universality of this approach, we incorporated DOTAP into C12-200 LNP (Figures 19G and 22). C12-200 LNP has also been well-documented for RNA delivery to the liver (Kove et al., 2010; Kauffman et al., 2015). Similar to the results obtained with 5A2-SC8 and DLin-MC3-DMA LNP, the inclusion of 15% or 50% DOTAP in C12-200 LNP shifted luciferase protein expression after mRNA delivery from the liver to the spleen to the lungs (Figures 19G and 22). Furthermore, the inclusion of 18PA as a SORT lipid replicated the results obtained with 5A2-SC8, mediating exclusive Luc mRNA delivery to the spleen for both DLin-MC3-DMA SNALP and C12-200 LNP (Figures 19F-G). DLin-MC3-DMA is a lipid with one dimethylamine head and two tails that forms stable nucleic acid lipid nanoparticles (SNALPs), whereas C12-200 is a representative lipidoid that forms lipid-like LNPs (LLNPs). Therefore, the SORT methodology has been shown to be generalizable to other classes of cationic ionizable lipid LNPs. This allows for the easy modification of existing liver-targeted LNPs to deliver mRNA to the spleen or lungs.Specifically, using SORT technology may allow for the rapid redevelopment of FDA-approved Onpattro to treat diseases in the lungs and spleen.

[0215] To understand whether the observed tissue tropism profiles are specific to the exact chemical structure or generalizable to defined chemical classes, we evaluated multiple permanent cationic, anionic, zwitterionic, and cationically ionizable SORT lipids (Figure 23). First, 5A2-SC8 LNPs were prepared with two additional permanent cationic lipids: didodecyldimethylammonium bromide (DDAB) and 1,2-dimyristoyl-sn-glycero-3-ethyl phosphocholinchloride (EPC). Although all of these lipids contain quaternary amino groups, they exhibit significant chemical differences (e.g., saturation) in the polar head region, linker region, and hydrophobic domain. LNPs containing 5%, 15%, 40%, 50%, and 100% DDAB or EPC were formulated and characterized. The in vivo luciferase expression profile was consistent with that of DOTAP LNP, and luminescence activity systematically shifted from the liver to the spleen and then to the lungs as the DDAB or EPC percentage increased (0.1 mg / kg, 6h). When the percentage increased to 40%, high luciferase signaling was observed exclusively in the lungs (Figure 23A). As representative anionic lipids with different structures compared to 18PA, 1,2-dimiristoyl-sn-glycero-3-phosphate (14PA) and sn-(3-oleoyl-2-hydroxy)-glycerol-1-phospho-sn-3'-(1',2'-dioleoyl)-glycerol (18BMP) were constructed. All anionic SORT lipids facilitated exclusive delivery to the spleen (Figure 23B). This flexibility opens the way for balancing multiple factors, including efficacy, selectivity, and tolerability, by optimizing the SORT compounds.

[0216] Inspired by these findings, other cationic ionizable lipids were added to established formulations. As expected, the addition of DODAP or C12-200 to 5A2-SC8 LNP did not significantly alter tissue tropism, but surprisingly, liver delivery increased >10-fold at 20% uptake (Figure 23C). Adding additional 5A2-SC8 as a SORT lipid to already established 5A2-SC8 LNP dramatically improved liver mRNA delivery, increasing 10-fold at an extremely low dose of 0.05 mg / kg. 7 photon / sec / cm 2 The results obtained were obtained. Therefore, SORT offers a novel strategy to further improve the liver-targeted LNP system (Figure 24). The effects of using zwitterionic lipids (DOCPe and DSPC) as SORT lipids were also evaluated. Tissue tropism was found to move from the liver to the spleen, but it was not selective compared to the use of cationic or anionic SORT lipids (Figure 25).

[0217] To test the limitations of the SORT methodology, we investigated whether SORT could "activate" inactive formulations. Indeed, adding DODAP or DOTAP to completely inactive formulations resulted in tissue-specific delivery to the spleen and lungs (Figure 26). These results suggest that SORT is a modular and universal strategy for achieving tissue-targeted delivery.

[0218] C.SORT mediates organ-specific delivery of protein corona, LNP in vivo distribution, and apparent pK a Change Mechanistic experiments were conducted to explore how and why mRNA delivery to different organs is controlled by including excess lipids in defined categories. It is self-evident that LNPs delivered to cells in the lung must be biodistributed (accumulated) in the lung. Cy5-labeled mRNA was delivered to track the in vivo distribution of 5A2-SC8 LNPs containing SORT lipids oriented to the lung (DOTAP quaternary aminolipid), spleen (18PA anionic lipid and DSPC zwitterionic lipid), and liver (DODAP ionizable tertiary aminolipid) (Figures 27A and 28). All LNPs were intravenously injected at a dose of 0.5 mg / kg Cy5-labeled mRNA, and imaging was performed 6 hours later. As shown in Figure 27A, DOTAP altered the biodistribution, with lung accumulation progressively increasing as a function of DOTAP percentage. 18PA uptake increased splenic uptake. DODAP slightly increased hepatic accumulation and decreased splenic accumulation. Interestingly, while lung-specific and spleen-specific SORT LNPs showed no protein expression in the liver, these LNPs still accumulated in the liver. This suggests that organ-specific distribution is necessary for organ-specific efficacy, but it is not the only factor explaining the mechanism of tissue-targeted delivery.

[0219] While not bound by any theory, it is thought that changes in the in vivo distribution and activity within defined cell populations may be due to changes in the protein corona. Binding of specific proteins to the protein corona results in a functionally active biological identity. Quantitative mass spectrometry revealed that the addition of SORT molecules drastically altered both the most closely bound specific protein and the overall protein corona composition. While not bound by any theory, lung-specific SORT LNPs appear to have selectively and most abundantly bound to vitronectin. Vitronectin can interact with positively charged lipids and binds to αvβ3 integrin, which is highly expressed on the surface of lung endothelial and epithelial cells. This endogenous targeting mechanism directly compares to adenoviruses that utilize αvβ5 integrin to target bronchial epithelium. Spleen-specific SORT LNPs have been shown to interact with negatively charged lipids and most closely bound to β2-glycoprotein I, which may play a role in splenic localization with immune cell populations in the spleen. It is noteworthy that a complex mixture of bound proteins may also play a role. This ensemble effect is also considered a potential mechanism for targeting multiple cell types and one way to further enhance specificity to certain cell types within an organ using alternative SORT molecules. Apolipoprotein E has been previously shown to bind to DLin-MC3-DMA Onpattro LNP, and its potency is lost in ApoE knockout animals. Therefore, there is strong evidence that ApoE is required in hepatocytes, presumably for receptor-mediated targeting and uptake by the LDL receptor, and that the described protein corona mechanism can control cell specificity and potency. Thus, it is encouraging to find that mDLNP also strongly binds to ApoE, which provides further evidence for these hepatocyte potencies and strengthens the validity of the protein corona assay. Liver-enhancing SORT LNP retains ApoE binding, but is also abundant in albumin.This suggests that cell types may have proliferated within the liver. These data cumulatively demonstrate that the chemical structure of SORT molecules can direct specific protein coronas that alter organ tropism and cell specificity. While not bound by any theory, it is thought that the identity of SORT molecules can control protein corona identity. This suggests that SORT molecules can contain sugars, lipids, small molecule therapeutics, vitamins, small molecules, hydrophilic molecules, hydrophobic molecules, amphiphilic molecules, peptides, proteins, and more.

[0220] Apparent / overall pK is a parameter that correlates LNP with functional activity. a Since this has been proven, the apparent / overall pK a We investigated this. For example, delivery to hepatocytes requires approximately 6.4 pK a This has been shown to be optimal (Jayaraman et al., 2012). Using the TNS assay, apparent pK was determined for all effective in vivo formulations (67 types of LNPs). a The following was analyzed (Figures 27B and 29, Table 1). Since SORT involves the inclusion of further charged lipids, the resulting TNS titration curve captures the ionization behavior of more complex mixed species LNPs. Therefore, instead, the relative pK at which 50% of the normalized signal occurred was used. a The relative pK was estimated for tissue orientation. a When plotted, SORT LNP is defined as the apparent pK a The range was divided (Figure 27B). As expected, all effective liver-targeted drugs' pK a The range was very narrow and well-established, within 6-7 (Jayaraman et al., 2012). All lung-targeted drugs have high pK a It was located in the range (>9). Conversely, splenotropic SORT LNP was low pK a The range was divided into (2-6). These results support the idea that 6-7 is optimal for delivery to the liver, but high pK a This mediates lung delivery, low pK aThe discovery that it mediates spleen delivery has been revealed. It should be noted that all SORT LNPs still contain cationic ionizable lipids. Cationic ionizable lipids are considered useful for endosome evasion because they have the ability to acquire charge (Wittrup et al., 2015). A control experiment was conducted. It was confirmed that the presence of cationic ionizable lipids is necessary to obtain efficacy (Figure 30). Therefore, SORT requires specific microspecies pK to be effective at the desired molar ratio. a While enabling the retention of molecules with pK, including SORT lipids results in apparent pK a The mechanism is modified. While not bound by any theory, a two-part mechanism is thought to play a role. SORT LNP selectively binds to specific proteins in the serum, enabling receptor-mediated efficacy in cells within the lungs or spleen. This is very similar to how lipoprotein particles (e.g., LDL) transport cholesterol in nature. This controlled and predictable endogenous targeting mechanism allows SORT LNP to reach non-hepatic targets. The second part involves the physiological and chemical properties (e.g., overall / apparent pK) that enable the SORT molecule to achieve hepatic efficacy. a 6.4) This involves techniques to alter the properties of non-liver-targeted SORT LNPs so that they no longer possess this property. This provides precision. It should also be noted that other more complex factors, such as differences in cell-specific endocytotic transport, may also play a role. Considering the results, the internal charge of LNP nanostructures mediates their biodistribution and apparent pK a This suggests a correlation with protein expression profiles in specific organs. This unique value could be used to continue developing other organ-specific nanoparticles.

[0221] (Table 1) Details of mDLNP formulations (SORT LNP) modified by DDAB, EPC, 14PA, 18BMP, DODAP, C12-200, 5A2-SC8, DSPC, and DOCPe, including the molar ratio and molar percentage of each component, the weight ratio of total lipids to mRNA, size, and PDI. TIFF2026082961000052.tif177170 a X represents DDAB, EPC, 14PA, 18BMP, DODAP, C12-200, 5A2-SC8, DSPC, and DOCPe.

[0222] D.SORT enables lung-specific, liver-specific, and spleen-specific gene editing after IV administration. Assuming that SORT LNPs have the ability to target specific organs, these findings were then applied to tissue-specific gene editing via IV injection. CRISPR / Cas (clustered and regularly arranged short palindromic sequence repeats / CRISPR-related protein (Cas)) technology can precisely and sequentially edit genomes and has rapidly developed for use in a wide variety of application areas, including the potential correction of disease-causing mutations (Jinek et al., 2012; Cong et al., 2013; Mali et al., 2013; Hendel et al., 2015; Yin et al., 2016; Yin et al., 2017; Wang et al., 2018; Amoasii et al., 2018). Gene editing can be achieved by local injection (Zuris et al., 2015; Sun et al., 2015; Chew et al., 2016; Staahl et al., 2017). However, many serious genetic disorders are caused by mutations in cells deep within organs, and correcting specific cells is necessary to cure the disease. Such corrections may be best achieved by systemic administration. Recently, simultaneous IV delivery of Cas9 mRNA and sgRNA has been reported as a safe and effective strategy for gene editing (Miller et al., 2017; Yin et al., 2017; Finn et al., 2018). However, to date, there have been no reports of LNPs rationally engineered to edit cells in organs other than the liver.

[0223] To investigate and quantify the ability of SORT LNPs to mediate organ-specific gene editing, we used genetically modified tdTomato (tdTom) reporter mice containing a LoxP adjacent stop cassette (Tabebordbar et al., 2016) that blocks the expression of the tdTom protein (Staahl et al., 2017). When the stop cassette is deleted, tdTom fluorescence is turned on, allowing for the detection of gene-edited cells (Figure 31A). Cre recombinase mRNA (Cre mRNA) was first delivered to activate tdTom in edited cells. Fluorescent tissue was readily visible in selected organs treated with liver-selective, lung-selective, and spleen-selective SORT LNPs (Figure 31B). It should be noted that separate controls had to be used for each experiment because these mice have some background organ fluorescence. Background organ fluorescence in the spleen was weakest compared to the other organs (Figures 31C and 32). Consequently, detecting spleen specificity in the tdTom mouse model is difficult. Subsequently, when endogenous PTEN was edited, the T7E1 assay showed that spleen-specific SORT LNPs clearly cleaved DNA only in the spleen (Figure 33C), but not in the liver or lung. Nevertheless, tdTom-positive cells were readily identified by confocal imaging of tissue sections (Figure 31D).

[0224] E.SORT enables high levels of editing in specific, therapy-relevant cell populations. Gene editing of specific cell types in the liver, lungs, and spleen was quantified using flow cytometry of single cells extracted from edited organs (Figure 31E). Liver-specific SORT (20% DODAP) 5A2-SC8 LNP edited approximately 93% of all hepatocytes in the liver after a single injection of 0.3 mg / kg Cre mRNA (Figures 31E and 34). This is the highest level of hepatocyte gene editing reported to date. Lung-specific SORT (50% DODAP) 5A2-SC8 LNP edited approximately 40% of all epithelial cells, approximately 65% ​​of all endothelial cells, and approximately 20% of immune cells in the lungs at the same dose (Figures 31E and 35). Given that epithelial cells are the primary target for correcting CFTR mutations that cause cystic fibrosis, these results establish lung-specific SORT LNP as a compelling delivery system that can be readily applied to correct CFTR mutations. Finally, spleen-specific SORT(30%18PA)5A2-SC8 LNP edited approximately 13% of total B cells, approximately 10% of total T cells, and approximately 20% of total macrophages (Figures 31E and 36). Due to improved selectivity compared to previous studies, spleen-specific SORT LNP may be applicable to treat non-Hodgkin B-cell lymphoma and other immunodisorders. The initial focus was on the quantification of a single low-dose injection, but higher levels of editing may be achievable by administering higher doses or multiple injections.

[0225] F.SORT enables tissue-specific gene editing through simultaneous intravenous delivery of Cas9 mRNA / sgRNA and delivery of Cas9 RNPs. Next, the ability of SORT LNPs to achieve tissue-specific CRISPR / Cas gene editing was investigated by co-delivering Cas9 mRNA and sgRNA via intravenous injection (IV) within a single nanoparticle (Figures 37A, 38, and 39, Table 2). Liver-targeted SORT LNPs and lung-targeted SORT LNPs were injected at a dose of 2.5 mg / kg total RNA (4:1 mRNA:sgRNA, wt:wt) and gene editing was quantified 10 days after a single IV injection. As shown in Figure 37B, potent tdTom fluorescence was observed in the liver of both basic LNP-treated mice and 20% DODAP SORT LNP-treated mice, and potent fluorescence was observed in the lungs of 50% DODAP SORT LNP-treated mice. All results were consistent with Luc mRNA delivery results. Because splenic immune cells are rapidly replaced in mice (Kamath et al., 2000), the Cas9 / sgRNA weight ratio was optimized to 2 / 1 (Figure 39), and spleen editing was tested two days after injection. Taking background autofluorescence into account, bright tdTom fluorescence was observed in the spleens of 30% 18PA-treated mice. Clear T7E1 cleavage bands were detected exclusively in DNA isolated from the spleen (no liver or lung editing was observed) (Figure 33). Subsequently, fluorescence was confirmed by imaging tissue sections with a confocal microscope (Figure 37C).

[0226] Next, we explored direct delivery of Cas9 RNPs, the most challenging strategy for synthetic carriers. Using permanently cationic SORT lipids, we were able to encapsulate the Cas9 protein / sgtdTom complex with controlled tissue tropism. Intravenous injection of 7% DOTAP SORT LNPs edited the liver, while 55% DOTAP SORT LNPs exclusively edited the lungs (Figure 37F). These data demonstrate that the described methodology enables liver, lung, and spleen-specific CRISPR / Cas gene editing.

[0227] The ability of tissue-specific LNPs to edit endogenous targets was tested in go beyond reporter mice. PTEN was selected because it is a well-established tumor suppressor expressed in most cells. Wild-type C57BL / 6 mice were injected with SORT LNPs (2.5 mg / kg total RNA) co-loaded with Cas9 mRNA and sgPTEN. Insertion and deletion (indel) development was quantified 10 days after a single IV injection. As shown in Figure 37D, clear DNA break bands were observed in specific tissues by the T7E1 assay. This demonstrated that both basic LNPs and 20% DODAP SORT LNPs mediated effective PTEN editing in the liver, but not at all in the lungs or spleen. Notably, 50% DODAP SORT LNPs showed PTEN editing exclusively in the lungs. To further confirm PTEN editing, H&E staining and immunohistochemistry (IHC) were performed on tissue sections. As shown in Figure 37E, cells in the tissue sections clearly exhibited clear cytoplasm, a known phenotype of PTEN loss due to lipid accumulation (Xue et al., 2014). Furthermore, negative staining for PTEN was observed in both liver and lung IHC sections. This provided clear evidence of PTEN editing. Spleen-specific 18PA SORT LNP editing was difficult to identify in the tdTom mouse model, but clear spleen PTEN editing was observed in wild-type mice using an optimized weight ratio of Cas9 / sgPTEN (2 / 1) and detection time (2 days). T7E1 assay performed on 18PA SORT LNP-injected mice did not reveal DNA editing in either the liver or lung (Figure 33). Finally, endogenous PTEN editing was investigated by applying SORT to Cas9 RNP. As before, 7% and 55% DOTAP SORT LNPs containing Cas9 protein / sgPTEN enabled liver and lung-specific editing, respectively (Figure 37G). These results, targeting endogenous genes, demonstrate that rationally guided, tissue-selective gene editing was achieved by synthetic carriers.

[0228] G. Materials and Methods I. Materials 5A2-SC8 (Zhou et al., 2016), DLin-MC3-DMA (Jayaraman et al., 2012), and C12-200 (Love et al., 2010) were synthesized and purified according to the following published protocols. 1,2-Dioleoyl-3-trimethylammonium-propane (DOTAP), Dimethyldioctadecylammonium (DDAB), 1,2-Dimyristoyl-sn-glycero-3-ethylphosphocholine (EPC), 1,2-Dioleoyl-sn-glycero-3-phosphate (sodium salt) (18PA), 1,2-Dimyristoyl-sn-glycero-3-phosphate (sodium salt) (14PA), sn-(3-oleoyl-2-hydroxy)-glycerol-1-phospho-sn-3'-(1',2'-Dioleoyl)-glycerol (ammonium salt) (18:1 Hemi BMP, 18BMP), 1,2-dioleoyl-3-dimethylammonium-propane (DODAP), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 2-((2,3-bis(oleoyloxy)propyl)dimethylammonio)ethylethyl phosphate (DOCPe), and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) were purchased from Avanti Polar Lipids. Cholesterol was purchased from Sigma-Aldrich. 1,2-Dimiristoyl-sn-glycerol-methoxy(poly(ethylene glycol)MW 2000)(DMG-PEG2000) was purchased from NOF America Corporation. Cas9 protein was purchased from Thermo Fisher. The ONE-Glo + Tox Luciferase Reporter assay kit was purchased from Promega Corporation. Pur-A-Lyzer Midi Dialysis Kits (WMCO, 3.5kDa) were purchased from Sigma-Aldrich. 4',6-Diamidino-2-phenylindole dihydrochloride (DAPI) was purchased from Thermo Fisher Scientific. Cas9 mRNA was produced by in vitro translation (IVT).Cy5-labeled firefly luciferase mRNA (Cy5-Luc mRNA), unlabeled firefly luciferase mRNA (Luc mRNA), and mCherry mRNA were purchased from TriLink BioTechnologies. D-luciferin (sodium salt) was purchased from Gold Biotechnology. Modified sgTom1 and sgPTEN (Table 2) were purchased from Synthego.

[0229] (Table 2) Relative apparent pK of SORT LNPs measured by TNS assay a value TIFF2026082961000053.tif114170

[0230] II. Nanoparticle Formation RNA-loaded LNP preparations were formed using the ethanol dilution method (Zhou et al., 2016). A liver-targeted mRNA preparation (mDLNP) was developed and reported in a previous paper (Cheng et al., 2018). As previously described (Jayaraman et al., 2012; Love et al., 2010), basic formulations were prepared. Unless otherwise noted, total lipids in the specified molar ratios were dissolved in ethanol, and RNA was dissolved in 10 mM citrate buffer (pH 4.0). The two solutions were rapidly mixed by volume in a 3:1 aqueous solution:ethanol ratio (3:1, aqueous solution:ethanol, vol:vol) to satisfy a final weight ratio of 40:1 (total lipids:mRNA), and then incubated at room temperature for 10 minutes. To prepare SORT LNP formulations containing anionic SORT lipids (e.g., 18PA, 14PA, and 18BMP), the anionic lipids were first dissolved in tetrahydrofuran (THF), then mixed with the other lipid components in ethanol, and finally, the formulation was obtained using mRNA buffer (10 mM, pH 3.0) as described above. All formulations were named based on the additional lipids. DOTAP Taking mDLNP as an example, the internal molar ratio of mDLNP was fixed with 5A2-SC8 / DOPE / cholesterol / DMG-PEG in a ratio of 15 / 15 / 30 / 3, as reported in a published paper (Cheng et al., 2018). As an additional lipid, DOTAP was dissolved in the above ethanol-lipid mixture in a specified amount such that the molar ratio of 5A2-SC8 / DOPE / cholesterol / DMG-PEG / DOTAP was equal to 15 / 15 / 30 / 3 / X, and rapidly mixed with the mRNA aqueous solution according to the standard protocol above to finally obtain SORT LNP. This was named Y%DOTAP, where Y represents the molar percentage of DOTAP in the total lipids. Formulations containing other additional lipids were formed using the same method (Figure 20 and Table 3). For Cas9 / sgRNA ribonucleoprotein (RNP) encapsulation, 1×PBS was used in the formulation, and the molar ratio of Cas9 to sgRNA was fixed at 1:3.For in vitro assays and size detection, after SORT LNP formation, fresh LNP preparations were diluted with 1×PBS to 0.5 ng / μL mRNA (final ethanol concentration <5%). For in vivo experiments, the preparations were dialyzed in 1×PBS for 2 hours (Pur-A-Lyzer Midi Dialysis Kits, WMCO 3.5kDa, Sigma-Aldrich), and for intravenous (IV) injection, they were diluted with PBS to 15 μL / g.

[0231] (Table 3) sgRNA sequences TIFF2026082961000054.tif34170

[0232] III. Characterization of mRNA preparations The size distribution and polydispersity index (PDI) were measured using dynamic light scattering (DLS, Malvern MicroV model; He-Ne laser, λ=632nm), and the zeta potential was measured after dilution with 1×PBS. The apparent pK of mRNA formulations was also measured. a To measure fluorescence intensity, we used the 2-(p-toluidino)-6-naphthalenesulfonic acid (TNS) assay with some modifications (Cheng et al., 2018; McLaughlin and Harary, 1976; Bailey and Cullis, 1994; Heyes et al., 2005). mRNA preparations (60 μM total lipids) and TNS probes (2 μM) were incubated for 5 minutes with a series of buffers containing 10 mM HEPES, 10 mM MES (4-morpholine ethanesulfonic acid), 10 mM ammonium acetate, and 130 mM NaCl (pH range 2.5–11). The average fluorescence intensity of each well (black-bottom 96-well plate) was measured by a Tecan plate reader using λ. Ex =321nm and λ Em Measurements were taken at 445 nm, and the data was normalized to a pH of 2.5. Typically, apparent pK a This was defined as the pH at which the fluorescence value is halved. This method is used for the overall / apparent pK of most LNPs. aWhile useful for estimating the relative pK, it could not be used for SORT LNPs containing >40% permanent cationic lipids because these LNPs are always charged. Therefore, instead, the relative pK at which 50% of the normalized signal was obtained was used, compared to the basic LNP formulation (without added SORT lipids). a This was estimated. This alternative calculation is pK for most LNPs. a Without altering the parameters, we were able to estimate permanently cationic SORT LNPs, which were consistent with experimental results for tissue-selective RNA delivery. Therefore, it can be suggested that a standard TNS assay is used when LNPs contain one type of cationic ionizable lipid, while an alternative 50% normalized signaling method is used for systems such as SORTs that contain a complex mixture of multiple lipids with various charge states.

[0233] IV. In vitro luciferase expression and cell viability studies Huh-7 or A549 cells were transfected into a white 96-well plate in 1x10⁶ layers the day before transfection. 4 Cells were seeded at a cell / well density. The medium was replaced with 150 μL of fresh DMEM medium (5% FBS), and then 50 μL of Luc mRNA preparation was added, fixing 25 ng mRNA per well. After incubation for a further 24 hours, mRNA expression and cytotoxicity were detected using the ONE-Glo+Tox kit according to Promega's standard protocol.

[0234] V. Animal experiments All animal experiments were approved by the Institution Animal Care and Use Committees of The University of Texas Southwestern Medical Center and complied with local, state, and federal regulations where applicable. C57BL / 6 mice were obtained from the UTSW Mouse Breeding Core Facility. B6.Cg-Gt(ROSA)26Sor tm9(CAG-tdTomato)Hze / J mice (also known as Ai9 or Ai9(RCL-tdT) mice) were obtained from Jackson Laboratory (007909) and mated to maintain homozygous expression of a Cre reporter allele with a loxP-adjacent STOP cassette that inhibits the transcription of the red fluorescent tdTomato protein driven by the CAG promoter. After Cre-mediated recombination, Ai9 mice express tdTomato fluorescence. Ai9 mice are congenic on the C57BL / 6J genetic background.

[0235] VI. In vivo Luc mRNA delivery and in vivo distribution C57BL / 6 mice weighing 18-20g were intravenously injected with various Luc mRNA preparations at doses of 0.1 mg / kg or 0.05 mg / kg. n=2-4 mice / group. Six hours later, D-luciferin (150 mg / kg) was injected intraperitoneally (IP) into the mice, and imaging was performed using the IVIS Lumina system (Perkin Elmer). For in vivo distribution, C57BL / 6 mice were intravenously injected with a Cy5-Luc mRNA preparation at a dose of 0.5 mg / kg. Ex vivo imaging (Cy5 channel) was performed six hours after injection.

[0236] VII.mRNA synthesis Optimized Cre recombinase mRNA and Cas9 mRNA were generated by in vitro transcription (IVT). Briefly, NLS-Cre and Cas9 fragments were prepared by PCR programs using pCAG-CreERT2 and pSpCas9(BB)-2A-GFP(PX458) as PCR templates, respectively. These fragments were then cloned into pCS2+MT vectors containing optimized 5'(3') untranslated regions (UTRs) and poly(A) sequences. IVT reactions were performed according to standard protocols, except that typical UTPs were replaced with N1-methylpsoiduridine-5'-triphosphate. Finally, the mRNAs were capped (Cap-1) with vaccinia capping enzyme and 2'-O-methyltransferase (NEB). Table 4 shows the primers used herein.

[0237] (Table 4) Primers including PCR product length and purpose TIFF2026082961000055.tif116170

[0238] The code sequences for NLS-Cre and Cas9 are as follows: TIFF2026082961000056.tif17145TIFF2026082961000057.tif221146TIFF2026082961000058.tif209146

[0239] VIII. Western blot IVT Cas9 mRNA quality was analyzed by Western blotting. The day before transfection, 293T cells were placed in 12-well plates at a rate of 1 × 10⁶ 5Cells were seeded at a density of cells / well. In a total volume of 600 μL, the cells were further treated for 24 hours with various formulations including mCherry mDLNP (0.5 μg mRNA / well), mCherry mDLNP (1.0 μg mRNA / well), IVT Cas9 mDLNP (0.5 μg mRNA / well), IVT Cas9 mDLNP (1.0 μg mRNA / well), and lipofectamine 2000 / Cas9 pDNA (0.5 μg pDNA / well). After washing three times with 1× PBS, 100 μL of lysis buffer (50 mM Tris HCl, pH 7.4 containing 150 mM NaCl, 1 mM EDTA, and 1% TRITON X-100) and 1 μL of protein inhibitor cocktail (100×, Thermo Fisher) were added to each well, and the wells were shaken at RT for 20 minutes. Cell lysates were collected and placed in 1.6 mL tubes, then centrifuged at 4°C for 10 minutes (13,000 g). The supernatant was collected and placed in a new tube, and stored at -80°C if not used immediately. Before performing Western blotting, protein concentrations were measured using a BCA assay kit (ThermoFisher). 15 micrograms of total protein were loaded and separated on a 4-20% polyacrylamide gel (ThermoFisher). The separated proteins were then transferred to a polyvinylidene membrane (BioRad) and blocked with 5% BSA (dissolved in PBST) at RT for 1 hour. Primary antibody was applied overnight at 4°C. After washing four times with PBST, the membrane was incubated with secondary antibody at RT for 1 hour, then imaged with ECL substrate, and washed four times with PBST (ThermoFisher).

[0240] Gene editing (Cre mRNA) in the IX.Td-Tomato mouse model As described above, a Cre mRNA preparation was prepared and administered via IV injection (0.3 mg / kg Cre mRNA). Two days later, the mice (n=4 per group) were sacrificed, and their major organs were imaged using the IVIS Lumina system (Perkin Elmer).

[0241] X. Cell isolation and staining for flow cytometry Td-Tomato cells in each organ's cell type + To test the cells, they were treated with Cre mRNA preparation (0.3 mg / kg), isolated and stained two days later, and then analyzed by flow cytometry.

[0242] For hepatocyte isolation, a two-step collagenase perfusion was performed as previously described (Cheng et al., 2018). Briefly, mice were anesthetized with isofluorane and fixed. Perfusion was initiated for 7-10 minutes using liver perfusion medium (Thermo Fisher Scientific, 17701038), followed by a further 7-10 minutes of liver digestion medium (Thermo Fisher Scientific, 17703034). The liver was collected in a plate containing 10 mL of liver digestion medium, dissected, and hepatocytes were dissociated. The dissociated hepatocytes were then collected and washed twice with hepatocyte wash medium (Thermo Fisher Scientific, 17704024) and once with 1×PBS. After further isolation by filtration and slow (50 × g) centrifugation, hepatocytes were analyzed using a FACS Aria II SORP machine (BD Biosciences).

[0243] For the isolation and staining of spleen cell types, the removed spleen was finely chopped with a sterile blade and homogenized in 250 μL of 1× digestion medium (45 units / μL collagenase I, 25 units / μL DNase I, and 30 units / μL hyaluronidase). The spleen solution was transferred to a 15 mL tube containing 5–10 mL of 1× digestion medium. The spleen solution was then filtered through a 70 μm filter and washed once with 1× PBS. A cell pellet was obtained by centrifugation at 300 × g for 5 minutes. The supernatant was removed, and the cell pellet was resuspended in 2 mL of 1× RBC lysis buffer (BioLegend, 420301) and incubated on ice for 5 minutes. After incubation, 4 mL of cell staining buffer (BioLegend) was added to stop RBC lysis. The solution was centrifuged at 300 × g for 5 minutes to obtain the cell pellet. Single cells were resuspended in cell staining buffer and added to a flow tube containing antibody (total volume 100 μL). The cells were incubated with the antibody at 4°C in the dark for 20 minutes. The stained cells were washed twice with 1 mL of 1× PBS and then resuspended in 500 μL of 1× PBS for flow cytometry analysis. The antibodies used were Pacific Blue anti-mouse CD45 (BioLegend, 103126), Alexa Fluor 488 anti-mouse / human CD11b (BioLegend, 101217), Alexa Fluor 647 anti-mouse CD19 (BioLegend, 115522), and PerCP-Cyanine 5.5 anti-mouse CD3e (145-2C11) (Tonbo Biosciences, 65-0031). Live cells were identified using Ghost Dye Red 780 (Tonbo Biosciences, 13-0865-T500).

[0244] For the isolation and staining of lung cell types, isolated lungs were finely dissected with a sterile blade and then transferred to 15 mL tubes containing 10 mL of 2× digestion medium (90 units / μL collagenase I, 50 units / μL DNase I, and 60 units / μL hyaluronidase), and incubated at 37°C for 1 hour with shaking. After incubation, the remaining lung tissue was homogenized. The following steps were similar to the spleen protocol described above. The antibodies used were Pacific Blue anti-mouse CD45 (BioLegend, 103126), Alexa Fluor 488 anti-mouse CD31 (BioLegend, 102414), and Alexa Fluor 647 anti-mouse CD326 (Ep-CAM) (BioLegend, 118212). Live cells were identified using Ghost Dye Red 780 (Tonbo Biosciences, 13-0865-T500).

[0245] Gene editing (Cas9 mRNA / sgRNA and Cas9 / sgRNA RNP) in the XI.Td-Tomato mouse model. To evaluate in vivo gene editing, Td-Tom mice of equivalent weight and sex were selected. Cas9 mRNA and sgRNA were co-delivered to tdTomato (td-Tom) mice. Cas9 mRNA / sgTom1 (4 / 1, wt / wt) was co-delivered using various formulations at a total RNA dose equivalent to 2.5 mg / kg. Major organs were removed 10 days after IV injection and imaged using the IVIS Lumina system. For the spleen-targeted formulation, the total RNA dose was 4 mg / kg, the weight ratio of Cas9 mRNA to sgTom1 was 2 / 1, and the detection time was 2 days. For RNP delivery, the molar ratio of Cas9 protein to sgRNA was fixed at 1:3, the injection dose was 1.5 mg / kg RNA, and the detection time was 7 days after injection (n=2-4 mice / group). To confirm Td-Tom expression, tissue sections were further prepared and imaged using confocal microscopy. In short, tissue blocks were embedded in an optimal cutting temperature compound (OCT) (Sakura Finetek), and frozen sections were prepared using a cryostat (Leica Biosystems) (8 μm). The mounted tissue slices were stained with 4,6-diamidino-2-phenylindole (DAPI, Vector Laboratories) and then imaged using a confocal microscope with a Zeiss LSM 700.

[0246] Gene editing (Cas9 mRNA / sgPTEN and Cas9 / sgRNA RNP) in XII.C57BL / 6 mice PTEN was selected to investigate endogenous gene editing in vivo. Various carriers were intravenously injected into wild-type C57BL / 6 mice by co-delivering Cas9 mRNA and modified sgPTEN (4 / 1, mRNA / sgRNA, wt / wt) at a total dose of 2.5 mg / kg (n=2-4 mice / group). Tissue was collected 10 days later, and genomic DNA was extracted using the PureLink Genomic DNA Mini Kit (ThermoFisher). For the spleen-targeted formulation, the total RNA dose was 4 mg / kg, Cas9 mRNA / sgTom1 was 2 / 1 (wt / wt), and detection time was 2 days after injection. For RNP delivery, the molar ratio of Cas9 protein to sgRNA was fixed at 1:3, the injection dose was 1.5 mg / kg RNA, and detection time was 7 days after injection (n=2-4 mice / group). After obtaining PTEN PCR products, a T7E1 assay (NEB) was performed to confirm gene editing efficacy according to a standard protocol. Furthermore, PTEN editing was evaluated on tissue sections by H&E staining and immunohistochemistry (IHC). Briefly, the Molecular Pathology Core at UTSW embedded paraformaldehyde (PFA) fixed tissue in paraffin, prepared sections, and stained them with H&E. 4 μm sections were prepared using a standard method and detected for IHC using the Elite ABC Kit and DAB Substrate (Vector Laboratories).

[0247] Example 9: Formulation using a neutral buffer solution Cas9 RNPs were observed to denature in acidic buffers, resulting in an increase in their hydrodynamic size from 10 nm to 150 nm (Figure 40B). Therefore, RNP encapsulation in monodisperse nanoparticles is difficult, if not impossible. These studies focus on lipid nanoparticles (LNPs) because they are the most effective class of RNA delivery carriers in preclinical models and in humans (Wood, 2018) (Wang et al., 2017; Doudna & Charpentier, 2014; Hajj & Whitehead, 2017; Sander & Joung, 2014). Of the four LNP components [cationic ionizable lipids, zwitterionic phospholipids, cholesterol, and poly(ethylene glycol) (PEG) lipids], pK a Cationic ionizable lipids with a charge of approximately 6.4 bind to negatively charged RNA at a mixed pH (e.g., pH 4, where the amine is protonated), lose their charge at neutral pH, are taken up by the cell, then fuse with the endosomal membrane, and regain their charge when the intraendosomal pH decreases to release the cargo into the cytoplasm, making them useful for activity. However, this characteristic hinders the effective encapsulation of cargo at neutral pH because cationic ionizable lipids are uncharged at neutral pH. To overcome this challenge, adding a fifth component, specifically a cationic lipid that is positively charged at neutral pH, makes it possible to encapsulate RNA and protein using a neutral buffer (instead of an acidic buffer), thus preserving the tertiary structure and stability of RNP (Figure 40A).

[0248] To evaluate this strategy, 5A2-SC8 was selected as the cationic ionizable lipid because 5A2-SC8 LNPs safely deliver short siRNA / miRNAs and long mRNAs to mice with impaired liver function, including those with MYC-driven hepatitis (Zhou et al., 2016; Zhang et al., 2018a; Zhang et al., 2018b) or fumarylacetoacetate hydrolase (FAH) gene knockout (Cheng et al., 2018). Indeed, when a permanent cationic lipid (e.g., DOTAP) was introduced into the conventional four-component 5A2-SC8 LNP formulation, controlled self-assembly occurred by mixing an ethanol solution of the lipids with a PBS solution of the RNPs (1 / 3, v / v). The incorporation of 5–60 mol% of DOTAP relative to the total lipids was evaluated (Figure 41). From this, it was revealed that high levels of gene editing and the formation of stable RNP-loaded nanoparticles with a size of <200 nm occurred in vitro at 10-20% concentrations (Figure 42). First, using a reporter luciferase-targeted sgRNA (sgLuc), the size of LNPs with 10 mol% DOTAP incorporation (5A2-DOT-10:5A2-SC8 / DOPE / Chol / DMG-PEG / DOTAP=15 / 15 / 30 / 3 / 7 (mol / mol)) prepared in PBS buffer was observed. This was slightly larger than nanoparticles without RNP loading. The same LNPs prepared using a low pH buffer did not change in size. This means that the RNP was not encapsulated (Figure 40C). To determine the optimal molar ratio of Cas9 protein to sgRNA, Cas9 / sgRNA complexes of 1 / 1, 1 / 3, and 1 / 5 (mol / mol) were prepared. This resulted in a decrease in RNP size (Figure 40D) and an increase in negative charge (Figure 40F). This RNP ratio did not change the size or zeta potential of the resulting LNPs after encapsulation (Figures 1E, 1G). The surface charge of all LNPs was neutral. This indicates not only that encapsulation was successful, but also that it is useful in minimizing in vivo uptake by the immunomononuclear phagocyte (MPS) system.To further investigate whether 5A2-DOT-10 can successfully mediate RNP delivery to the nucleus, we tracked LNPs containing encapsulated fluorescent EGFP-fused Cas9 proteins. Only free RNPs could not enter the cell, as no green fluorescence was detected above the background (Figure 43). After 3 hours of treatment with 5A2-DOT-10, bright green fluorescence was observed in the cytoplasm of the cells. Subsequently, due to the presence of a nuclear localization signal for Cas9, the EGFP-fused Cas9 protein was observed to gradually enter the nucleus within 6 hours (Figure 40H). Endocytosis is energy-dependent and primarily dependent on lipid rafts, as treatment with MβCD, an inhibitor of lipid raft-based endocytosis, significantly inhibited the uptake of nanoparticles into cells (Figure 40I).

[0249] To quantify gene editing efficacy, HeLa-Luc and HeLa-GFP reporter cells were used. When various Cas9 / sgLuc ratios were examined, gene editing was high at 1 / 3 and 1 / 5 (Figure 44A). T7 endonuclease I (T7EI) assay results demonstrated that most target DNA bands (720 bp) were cleaved into two cleavage bands (536 bp and 184 bp). No cleavage bands were observed in the control group. To test the hypothesis that a neutral pH buffer is necessary to encapsulate RNPs while protecting Cas9, the gene editing efficiency of 5A2-DOT-10 prepared with pH 4 citrate buffer was also evaluated. No cleavage bands were observed (Figure 44A). Furthermore, negative results were confirmed by Sanger sequencing. This provides additional evidence that conventional acid-based formulation methods do not produce effective NPs. When switching to GFP-expressing cells, 5A2-DOT-10-encapsulated Cas9 / sgGFP induced indels in GFP DNA, knocking out almost all GFP expression. The control group showed similar fluorescence intensity to PBS-treated cells (Figure 44B). This was confirmed by flow cytometry (Figures 44C and 45). Permanent gene editing was evident by indistinct GFP loss in growing cells, which was confirmed by Sanger sequencing. Inference of CRISPR Edits (ICE) analysis revealed that 95% of the indels were formed (Figure 44D). Turning to clinical translation, the stability of RNP-loaded 5A2-DOT-10 was monitored at 4°C for 2 months. The LNPs remained uniform in size (PDI < 0.2) (Figure 44G). Continuous testing of 5A2-DOT-10 nanoparticles revealed that the gene editing activity remained constant even after 60 days of storage (Figure 44H).

[0250] To efficiently deliver RNPs, the strategy of adding permanent cationic lipids to the classic four-component LNP is not limited to dendrimer-based ionizable lipids such as 5A2-SC8. To demonstrate this, we added auxiliary DOTAP to nanoformulations prepared using other classes of ionizable materials: the well-known DLin-MC3-DMA lipid (Wood, 2018) used in FDA-approved Onpattro, and the C12-200 lipid (Figure 46A-B). Although these have considerably different chemical structures compared to 5A2-SC8 (Figure 46C), all DOTAP-modified nanoparticles were able to efficiently edit cells, while previously established C12-200 or MC3 formulations without DOTAP showed lower editing efficiency (Figure 44E). 5A2-DOT-10 also achieved higher editing efficiency than the positive control RNAiMAX. Since 5A2-DOT-10 LNP was more effective than MC3-DOT-10 and C12-200-DOT-10, all subsequent experiments were performed using 5A2-SC8. In addition to DOTAP, other cationic lipids, including DDAB and EPC, were also introduced into the LNP formulation (Figures 46E-G). These results were similar for all three cationic lipids with different chemical structures (Figure 46H). These results indicate that this strategy is universal for cationic ionizable lipid nanoparticles (DLNP, LLNP, SNALP) and other cationic lipids that are positively charged at pH 7.4. This methodology allows for the preparation of FDA-approved Onpattro formulations capable of delivering RNP, thus opening up various directions for the treatment of human diseases that can be clinically translated.

[0251] The key to successful RNP delivery is replacing standard acidic buffer with PBS buffer to maintain protein stability. To test the compatibility of this methodology with other neutral buffers, LNPs were formulated in PBS, Opti-MEM medium, and HEPES. Formulations prepared in citrate buffer (pH 4) were used as a control (Figure 46I). Significant and equivalent gene editing (>90%) was achieved using LNPs prepared under all three neutral buffer conditions, but not with acidic buffer (Figure 44F). ICE analysis of sequencing results was consistent with those shown by flow cytometry (Figure 46K).

[0252] To investigate in vivo gene editing, a 5A2-DOT-10 encapsulated Cas9 / sgTOM complex was delivered to a Td-Tomato mouse model (Figure 47A). In successfully edited cells from these mice, CRISPR-mediated Lox-Stop-Lox cassette deletion activated downstream tdTom expression. 5A2-DOT-10 LNPs loaded with Cas9 / sgTOM RNPs were injected intramuscularly into the left limb of mice at a dose of 1 mg / kg sgTOM. RNAiMAX complexed with Cas9 / sgTOM RNPs was used for comparison because it had been previously used for direct injection gene editing (Zuris et al., 2015). Stronger Td-Tom fluorescence was observed in muscles treated with 5A2-DOT-10 than in mice treated with RNAiMAX (Figure 47B). Imaging of tissue sections further confirmed gene editing in the 5A2-DOT-10 treated group, which produced bright red fluorescence (Figure 47C). 5A2-DOT-10 was injected into the brains of Td-Tom mice (0.15 mg / kg sgTOM). In this case, a bright red signal was observed near the injection site, confirming editing in the mouse brains (Figures 47D-E).

[0253] The improved stability and potency of 5A2-DOT-10 enabled successful and systematic gene editing in the evaluated tissues. To investigate this RNP delivery strategy, LNPs with various DOTAP molar percentages (5-60%) were prepared, and the RNPs were delivered to Td-Tom mice IV (1.5 mg / kg sgTOM). Seven days after injection of 5A2-DOT-5, Td-Tom fluorescence was observed primarily in the liver. As the incorporated DOTAP percentage increased from 5 to 60%, fluorescence (CRISPR-guided gene editing) gradually shifted from the liver to the lungs. 5A2-DOT-60 enabled editing primarily in the lungs (Figure 47F). These results suggest that deep tissues can be edited tissue-specifically by adjusting the internal lipid component chemistry and molar ratio. Tissue-specific editing was further confirmed by confocal imaging of tissue sections (Figure 47G). Next, we evaluated the editing of the endogenous target Pten by systemic injection of LNP-encapsulated Cas9 / sgPTEN RNPs into wild-type C57BL / 6 mice. Distinct T7EI cleavage bands were detected only in the livers of 5A2-DOT-5 treated mice and in the lungs of 5A2-DOT-50 and 5A2-DOT-60 treated mice (Figure 47H).

[0254] To evaluate whether multiple genes could be edited simultaneously in vivo, Cas9 protein and six different sgRNAs were encapsulated in 5A2-DOT-50. sgTOM, sgP53, sgPTEN, sgEml4, sgALK, and sgRB1 were loaded onto the Cas9 protein and encapsulated. Td-Tom mice were then treated with 5A2-DOT-50 (pooled) by tail vein injection (0.33 mg / kg of each sgRNA). Bright Td-Tom fluorescence was detected in the lungs after one week, indicating that TOM had been gene-edited (Figure 47I). Clear T7EI cleavage bands were observed at all five other genomic loci. This demonstrated that 5A2-DOT-50 can simultaneously and effectively edit multiple genes at low doses (Figure 47J). Quantitative analysis revealed that the target editing efficiency reached 22% in the lungs (Figures 47 and 48). To enhance sgRNA stability and reproducibility, sgRNAs with terminal modifications on the first and last three nucleotides were used herein (Figure 49) (Finn et al., 2018; Hendel et al., 2015). Reports have shown that precise modifications to additional nucleotides can increase in vivo gene editing by 2–4 times compared to end-modified sgRNAs (Finn et al., 2018; Yin et al., 2017). This suggests that further optimization of the sgRNA may be possible to increase the editing efficiency reported herein. Nevertheless, the high efficacy and tissue specificity of 5A2-DOT-50 allowed for the simultaneous editing of six targets in the lung with a single injection.

[0255] Traditionally, animal models are created through genetic engineering or transgenesis of embryonic stem cells, which is time-consuming and expensive. Direct mutation of tumor-related and other disease-related genes in adult mice using CRISPR / Cas is a feasible approach for rapid model creation. This is only achievable with expensive lentiviruses that must be manipulated target by target and via hydrodynamic injection into the liver (Xue et al., 2014; Maddalo et al., 2014). Typically, multiple gene mutations are required to create functional cancer models, so the development of inexpensive and effective non-viral nanoparticle-based approaches for multiplexing is highly desirable. 5A2-DOT-X LNPs are potent, can simultaneously edit multiple targets, can be repeatedly administered, and provide tissue specificity, thus opening the way for the creation of a wide variety of animal models.

[0256] Using 5A2-DOT-5, three tumor suppressor genes (P53, PTEN, and RB1) were selectively knocked out simultaneously in the liver. These genes have been identified in many human cancers, including liver cancer. C57BL / 6 mice were treated with weekly IV injections of 2.5 mg / kg total sgRNA for 3 weeks, and gene editing efficiency in the mouse liver was detected (Figure 48A). After 2, 12, 15, and 20 weeks of treatment, distinct cleavage bands were observed at all three gene loci by the T7EI assay (Figures 48B, 50, and 51). As time progressed, these cleavage bands became considerably brighter, indicating tumor growth. When mice were sacrificed at 15 and 20 weeks, visible tumors were found in the liver, along with several metastatic tumors in the peritoneal cavity (Figures 48C and 52). Tumor development was also detected at various time points using H&E staining and IHC staining targeting the tumor growth biomarker Ki67 (Figures 48D and 53).

[0257] To create a challenging lung cancer mouse model, we focused on the Eml4-Alk chromosomal rearrangement, a compound mutation found in many solid human tumors, particularly non-small cell lung cancer (Maddalo et al., 2014; Blasco et al., 2014). The Eml4-Alk fusion protein resulting from the rearrangement between Eml4 and Alk promotes cancer development. Utilizing the high efficacy and lung-targeting specificity of 5A2-DOT-50, we evaluated the tumorigenetic process by injecting either a single IV dose (2 mg / kg total sgRNA) or two IV doses (1.5 mg / kg total sgRNA weekly) (Figure 48E). Indel development was detectable from lung DNA extracted from mice in both groups at all time points examined (Figures 48F and 54). A clear gene rearrangement band was detected in the lungs o...

Claims

1. (A) Therapeutic agents, (B) (1) Selective organ-targeting compounds; (2) Cationic ionizable lipids; and (3) Phospholipids A lipid nanoparticle composition containing and A composition comprising the following, wherein nucleic acids are preferentially delivered to a target organ selected from the lungs, heart, brain, spleen, lymph nodes, bones, skeletal muscle, stomach, small intestine, large intestine, kidneys, bladder, breasts, testes, ovaries, uterus, spleen, thymus, brainstem, cerebellum, spinal cord, eyes, ears, tongue, or skin.

2. The composition according to claim 1, wherein the target organ is the lung, lymph node, or spleen.

3. The composition according to claim 1 or 2, wherein the selective organ-targeting compound is a permanently cationic lipid.

4. The composition according to claim 3, wherein permanent cationic lipids are present in a molar percentage of about 5% to about 20% relative to the lipid nanoparticle composition.

5. The composition according to claim 3, wherein permanent cationic lipids are present in a molar percentage of about 20% to about 65% of the lipid nanoparticle composition.

6. The composition according to any one of claims 3 to 5, wherein the permanent cationic lipid contains a quaternary ammonium ion.

7. Permanent cationic lipids Further defined as, During the ceremony, R 1 and R 2 Each of them independently, alkyl (C8～C24) Alkenil (C8～C24) , or a substitution of either group; R 3 、R 3 ', and R 3 '' are each independently alkyl (C≦6) or substituted alkyl (C≦6) ; X - It is a monovalent anion. The composition according to any one of claims 3 to 6.

8. Permanent cationic lipids The composition according to claim 7, further defined as:

9. Permanent cationic lipids Further defined as, During the ceremony, R 4 and R 4 Each of them independently, alkyl (C6～C24) Alkenil (C6～C24) , or a substitution of either group; R 4 '' is alkyl (C≦24) Alkenil (C≦24) , or a substitution of either group; R 4 ''' is alkyl (C1～C8) Alkenil (C2～C8) , or a substitutional form of either group; and X 2 It is a monovalent anion. The composition according to any one of claims 3 to 6.

10. Permanent cationic lipids The composition according to claim 9, further defined as:

11. Permanent cationic lipids Further defined as, During the ceremony, R 1 and R 2 Each of them independently, alkyl (C8～C24) Alkenil (C8～C24) , or a substitution of either group; R 3 , R 3 ', and R 3 Each of the '' independently becomes alkyl (C≦6) or substituted alkyl (C≦6) and; R 4 However, alkyl (C≦6) or substituted alkyl (C≦6) and X - It is a monovalent anion. The composition according to any one of claims 3 to 6.

12. Permanent cationic lipids The composition according to claim 11, further defined as:

13. The composition according to claim 1 or 2, wherein the selective organ-targeting compound is a permanent anionic lipid.

14. The composition according to claim 13, wherein permanent anionic lipids are present in a molar percentage of about 5% to about 50% relative to the lipid nanoparticle composition.

15. The composition according to claim 13 or 14, wherein the permanent anionic lipid contains a phosphate group.

16. Permanent anionic lipids, Further defined as, During the ceremony, R 1 and R 2 Each of them independently, alkyl (C8～C24) Alkenil (C8～C24) , or a substitution of either group; R 3 However, hydrogen, alkyl (C≦6) , or substituted alkyl (C≦6) , or -Y 1 -R 4 And, During the ceremony, Y 1 But, Arcanziel (C≦6) or substitute alkanediyl (C≦6) and R 4 However, acyloxy (C≦8～24) or substituted acyloxy (C≦8～24) That is, The composition according to any one of claims 13 to 15.

17. Permanent anionic lipids, The composition according to claim 16, further defined as:

18. Selective organ-targeting compounds are C 6 ~C 24 The composition according to claim 1 or 2, wherein the composition is diacylphosphotidylcholine.

19. The composition according to claim 18, wherein diacylphosphotidylcholine is present in a molar percentage of about 5% to about 50% relative to the lipid nanoparticle composition.

20. The composition according to claim 18 or 19, wherein the selective organ-targeting compound comprises at least two fatty acid chains, a quaternary amine, and an anionic phosphate group.

21. Diacylphosphotidylcholine Further defined as, During the ceremony, R 1 and R 2 Each of them independently, alkyl (C8～C24) Alkenil (C8～C24) , or a substitution of either group; R 3 , R 3 ', and R 3 Each of the '' independently becomes alkyl (C≦6) or substituted alkyl (C≦6) and X - It is a monovalent anion. The composition according to any one of claims 18 to 20.

22. Diacylphosphotidylcholine The composition according to claim 21, further defined as:

23. The composition according to any one of claims 1 to 22, wherein cationic ionizable lipids are present in a molar percentage of about 5% to about 30% relative to the lipid nanoparticle composition.

24. The composition according to any one of claims 1 to 22, wherein cationic ionizable lipids are present in a molar percentage of about 15% to about 30% relative to the lipid nanoparticle composition.

25. The composition according to any one of claims 1 to 24, wherein the cationic ionizable lipid contains an ammonium group that is positively charged at physiological pH and contains at least two hydrophobic groups.

26. Cationic ionizable lipids have at least two C 6 ~C 24 The composition according to claim 25, comprising an alkyl group or an alkenyl group.

27. The composition according to any one of claims 1 to 26, wherein phospholipids are present in a molar percentage of about 8% to about 20% relative to the lipid nanoparticle composition.

28. The composition according to any one of claims 1 to 26, wherein phospholipids are present in a molar percentage of about 20% to about 23% relative to the lipid nanoparticle composition.

29. The composition according to any one of claims 1 to 28, wherein the phospholipid is further defined as 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine or 1,2-distearoyl-sn-glycero-3-phosphocholine.

30. A composition according to any one of claims 1 to 29, further comprising a steroid.

31. The composition according to claim 30, wherein the steroid is present in a molar percentage of approximately 39% to approximately 46% of the lipid nanoparticle composition.

32. The composition according to claim 31, wherein the steroid is present in a molar percentage of about 15% to about 39% relative to the lipid nanoparticle composition.

33. The composition according to claim 31 or 32, wherein the steroid is cholesterol.

34. A composition according to any one of claims 1 to 33, further comprising PEGylated lipids.

35. The composition according to claim 34, wherein PEGylated lipids are present in a molar percentage of about 0.5% to about 10.0% relative to the lipid nanoparticle composition.

36. The composition according to claim 35, wherein PEGylated lipids are present in a molar percentage of approximately 3.9% to approximately 4.6% relative to the lipid nanoparticle composition.

37. The composition according to claim 35 or 36, wherein the PEGylated lipid contains PEG components in an amount of about 1,000 to about 10,000 daltons.

38. The composition according to claim 37, wherein the PEG lipid is PEGylated diacylglycerol.

39. PEG lipids are expressed by the following formula: Further defined by, During the ceremony, R 12 and R 13 Each of them independently, alkyl (C≦24) Alkenil (C≦24) , or a substitutional form of any of these groups; R e However, hydrogen, alkyl (C≦8) , or substituted alkyl (C≦8) and x is between 1 and 250. The composition according to claim 38.

40. PEG lipids are dimyristoyl-sn-glycerol or the following formula: It is a compound of, During the ceremony, n 1 is 5 to 250; and n 2 and n 3 Each of them is independent and ranges from 2 to 25. The composition according to claim 35 or 36.

41. The composition according to any one of claims 1 to 40, wherein the therapeutic agent is a low molecular weight agent.

42. The composition according to any one of claims 1 to 40, wherein the therapeutic agent is a protein.

43. The composition according to any one of claims 1 to 40, wherein the therapeutic agent is a nucleic acid.

44. The composition according to claim 43, wherein the nucleic acid is a therapeutic nucleic acid.

45. The composition according to claim 43, wherein the nucleic acid is siRNA, miRNA, pri-miRNA, messenger RNA (mRNA), clustered and regularly arranged short palindromic sequence repeat (CRISPR)-associated nucleic acid, single guide RNA (sgRNA), CRISPR-RNA (crRNA), transactivating crRNA (tracrRNA), plasmid DNA (pDNA), transfer RNA (tRNA), antisense oligonucleotide (ASO), guide RNA, double-stranded DNA (dsDNA), single-stranded DNA (ssDNA), single-stranded RNA (ssRNA), and double-stranded RNA (dsRNA).

46. The composition according to any one of claims 43 to 45, wherein nucleic acids are present in a ratio of approximately 1:1 to approximately 1:100 of the lipid nanoparticle composition.

47. A composition according to any one of claims 1 to 46, further comprising a protein.

48. A composition according to any one of claims 1 to 47, comprising both a protein and a nucleic acid.

49. (A) A composition according to any one of claims 1 to 48, (B) Excipients and A pharmaceutical composition containing [the specified substance].

50. A method for regulating gene expression, comprising the step of delivering nucleic acids to cells, The method comprising the step of contacting cells with the composition or pharmaceutical composition according to any one of claims 1 to 49 under conditions sufficient to induce nucleic acid uptake into cells.

51. A method for treating a disease or disorder in a patient, comprising the step of administering to the patient, who is in need of treatment for a disease or disorder, a pharmaceutically effective amount of the composition or pharmaceutical composition according to any one of claims 1 to 49, The method wherein the composition or pharmaceutical composition comprises a nucleic acid for therapeutic purposes against a disease or disorder.

52. A method for preparing lipid nanoparticles, comprising the following steps: (A) A step of dissolving a selective organ-targeting compound, a cationic ionizable lipid, and a phospholipid in a first solution to form a lipid solution, wherein the lipid solution is formed in an organic solvent; (B) A step of dissolving a therapeutic agent in a buffer to form a buffered therapeutic agent solution, wherein the buffer is a buffer with a pH of approximately 6.8 to approximately 7.6; and (C) A step of mixing a lipid solution with a buffered therapeutic agent solution to form lipid nanoparticles.

53. (A) Therapeutic agents, (B) (1) Cationic ionizable lipids; (2) Phospholipids; and (3) Selective organ-targeting compounds A lipid nanoparticle composition containing and A composition comprising, A composition wherein an organ-targeting ligand causes preferential delivery of the composition to organs other than the liver.

54. Treatment drugs, (A) Cationic ionizable lipids; (B) Phospholipids; and (C) Selective organ-targeting compounds A lipid nanoparticle composition containing and A composition comprising, Approximately 8 to 13 apparent pK a The composition having a property that primarily delivers nucleic acids to the lungs.

55. Treatment drugs, (A) Cationic ionizable lipids; (B) Phospholipids; and (C) Selective organ-targeting compounds A lipid nanoparticle composition containing and A composition comprising, Apparent pK of approximately 3 to 6 a The composition having the above-mentioned properties and primarily delivering nucleic acids to the spleen.

56. Treatment drugs, (A) Cationic ionizable lipids; (B) Phospholipids; and (C)C 6 ~C 24 Diacylphosphotidylcholine A lipid nanoparticle composition containing and A composition comprising, The composition, which primarily delivers nucleic acids to lymph nodes.

57. Treatment drugs, (A) Cationic ionizable lipids; (B) Phospholipids; and (C) Selective organ-targeting compounds A lipid nanoparticle composition containing and A composition comprising, A composition wherein the surface of the composition interacts with vitronectin, and the composition primarily delivers nucleic acids to the lungs.

58. Treatment drugs, (A) Cationic ionizable lipids; (B) Phospholipids; and (C) Selective organ-targeting compounds A lipid nanoparticle composition containing and A composition comprising, A composition wherein the surface of the composition interacts with ApoH, and the composition primarily delivers nucleic acids to the spleen.

59. A composition comprising a therapeutic agent and a lipid nanoparticle composition, wherein a protein corona on the surface of the composition binds to a protein substantially present in a target organ, and the target organ is not the liver.

60. The composition according to claim 59, wherein the protein is vitronectin and the target organ is the lung.

61. The composition according to claim 59, wherein the protein is ApoH and the target organ is the spleen.

62. The composition according to any one of claims 59 to 61, wherein the lipid nanoparticle composition further comprises a selective organ-targeting compound that modifies the binding of proteins on a protein corona.

63. A composition comprising a therapeutic agent and a lipid nanoparticle composition, wherein the lipid nanoparticle composition comprises a selective organ-targeting compound, and the selective organ-targeting compound has an apparent pK of about 3 to about 6. a The composition provides a lipid nanoparticle composition having the following properties.

64. A composition comprising a therapeutic agent and a lipid nanoparticle composition, wherein the lipid nanoparticle composition comprises a selective organ-targeting compound, and the selective organ-targeting compound has an apparent pK of about 8 to about 13. a The composition provides a lipid nanoparticle composition having the following properties.

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