Compositions and methods for organ-specific delivery of nucleic acids

Abstract

To provide: compositions which show preferential targeting or delivery of a nucleic acid composition to a particular organ; methods of treating a disease or disorder in a patient; and methods of preparing lipid nanoparticles.SOLUTION: A compositions comprises: (A) a therapeutic agent; and (B) a lipid nanoparticle composition comprising (1) a selective organ targeting compound, (2) an ionizable cationic lipid, and (3) a phospholipid. The composition preferentially delivers a nucleic acid to a target organ selected from the lungs, heart, brain, spleen, lymph nodes, bone, skeletal muscles, stomach, small intestine, large intestine, kidneys, bladder, breasts, liver, testes, ovaries, uterus, spleen, thymus, brainstem, cerebellum, spinal cord, eyes, ears, tongue and skin.SELECTED DRAWING: None

Description

[Technical Field]

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

[0002] 1. Field The present disclosure relates generally to the field of molecular biology. More specifically, the present 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. Description of Related Technology CRISPR / Cas (clustered regularly interspaced short palindromic repeats / CRISPR-associated proteins (Cas)) technology allows precise, sequence-dependent editing and permanent alterations of the genome. Because CRISPR / Cas technology can target disease-causing mutations, it holds great promise for one-time genetic disease treatments. To date, successful editing has primarily been mediated by viral vectors, which require laborious customization for each target and present challenges for clinical translation due to concerns about immunogenicity, the generation of antibodies that prevent repeated administration, and rare but dangerous integration events. To broaden the safe and effective application of gene editing, there is a clear need to achieve CRISPR / Cas editing via synthetic nanoparticles (NPs).

[0004] CRISPR / Cas enables sequence-specific DNA editing by the RNA-guided CRISPR-associated protein 9 (Cas9) nuclease or its homologs, 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 cleavage, DSB repair pathways result in directed mutagenesis, i.e., insertions / deletions (indels) that delete the target gene. For therapeutic applications, transient Cas9 expression is preferred to limit off-target genomic changes. Because both the Cas9 protein and sgRNA must be present in the same cell, co-delivery of Cas9 mRNA and sequence-targeting sgRNA in a single NP is an attractive method, especially for in vivo applications where tissue penetration and cellular uptake are more challenging. While CRISPR / Cas editing using viruses, membrane modification, ribonucleoprotein complex delivery, and hydrodynamic injection works, there are limitations that may prevent their in vivo therapeutic use in the clinic, including persistent Cas9 expression and off-target editing. Furthermore, these delivery systems generally lack selectivity for the specific organ where editing is required. For example, most lipid nanoparticles accumulate in the liver through biological processes, thus reducing the efficacy of the composition when delivered to the target organ.

[0005] Similarly, other therapeutics, such as protein and small molecule therapeutics, may benefit from organ-specific delivery. Many different types of compounds, such as chemotherapeutic agents, exhibit significant cytotoxicity. If these compounds could be better targeted for delivery to the desired organ, off-target effects would be reduced.

[0006] Therefore, there remains a need to develop new lipid nanoparticles that exhibit preferential delivery to specific organs. Summary of the Invention

[0007] overview In some aspects, the present disclosure provides lipid compositions that exhibit organ-specific delivery of the lipid compositions. These compositions can be used to deliver nucleic acid components to specific organs.

[0008] In some aspects, the present disclosure provides: (A) a therapeutic agent; (B) (1) Selective organ-targeting compounds; (2) cationic ionizable lipids; and (3) Phospholipids a lipid nanoparticle composition comprising wherein the composition preferentially delivers a nucleic acid to a target organ selected from the lung, heart, brain, spleen, lymph node, bone, bone marrow, skeletal muscle, stomach, small intestine, large intestine, kidney, bladder, breast, liver, testis, ovary, uterus, spleen, thymus, brainstem, cerebellum, spinal cord, eye, ear, tongue, or skin. In some embodiments, the target organ is selected from the lung, heart, brain, spleen, lymph node, bone, bone marrow, skeletal muscle, stomach, small intestine, large intestine, kidney, bladder, breast, testis, ovary, uterus, spleen, thymus, brainstem, cerebellum, spinal cord, eye, ear, tongue, or skin.

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

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

[0011] In some embodiments, the permanent cationic lipid comprises a quaternary ammonium ion. In some embodiments, the permanent cationic lipid comprises further defined as TIFF0007815196000001.tif23128, During the ceremony, R1 and R2 are each independently alkyl (C8~C24) , alkenyl (C8~C24) or a substituted form of either group; R3, R3', and R3'' are each independently alkyl (C≦6) or substituted alkyl (C≦6) and; 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 another embodiment, R 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 - is a halide anion, e.g., bromide or chloride. In some embodiments, the permanent cationic lipid is Further defined as TIFF0007815196000002.tif21128.

[0013] In other embodiments, the permanent cationic lipid is further defined as TIFF0007815196000003.tif14128, During the ceremony, R4 and R4' are each independently alkyl (C6~C24) , alkenyl (C6~C24) or a substituted form of either group; R4'' is alkyl (C≦24) , alkenyl (C≦24) or a substituted form of either group; R4''' is alkyl (C1~C8) , alkenyl (C2~C8) or a substituted form of either group; and X2 is a monovalent anion.

[0014] In some embodiments, R4 is alkyl (C6~C24) or substituted alkyl (C6~C24) In some embodiments, R4' is alkyl, for example, octadecyl. (C6~C24) or substituted alkyl (C6~C24) 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) In some embodiments, R4''' is alkyl. (C1~C8) or substituted alkyl (C1~C8)In some embodiments, X2 is a halide, e.g., chloride or bromide. In some embodiments, the permanent cationic lipid is Further defined as TIFF0007815196000004.tif9128.

[0015] In some embodiments, the permanent cationic lipid is further defined as TIFF0007815196000005.tif24128, During the ceremony, R1 and R2 are each independently alkyl (C8~C24) , alkenyl (C8~C24) or a substituted form 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 - is a monovalent anion.

[0016] 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 another embodiment, R 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.

[0017] In some embodiments, R3, R3', and R3" are each the same, e.g., R3, R3', and R3" are each methyl. In some embodiments, R4 is alkyl. (C≦6) In some embodiments, X is ethyl. -is a halide anion, for example, bromide or chloride.

[0018] In some embodiments, the permanent cationic lipid is Further defined as TIFF0007815196000006.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 the lipid nanoparticle composition of about 5% to about 50%. In some embodiments, the molar percentage of the permanent anionic lipid is present in a molar percentage of 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 comprises a phosphate group.

[0020] In some embodiments, the permanent anionic lipid is further defined as TIFF0007815196000007.tif24128, During the ceremony, R1 and R2 are each independently alkyl (C8~C24) , alkenyl (C8~C24) or a substituted form of either group; R3 is hydrogen, alkyl (C≦6) , or substituted alkyl (C≦6) or -Y1-R4, During the ceremony, Y1 is alkanediyl (C≦6) or substituted alkanediyl (C≦6) and R4 is acyloxy (C≦8~24) or substituted acyloxy (C≦8~24) is.

[0021] In some embodiments, R1 is alkenyl (C8~C24) or substituted alkenyl (C8~C24) In other embodiments, R2 is alkenyl (C8~C24) or substituted alkenyl (C8~C24) In another embodiment, R 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.

[0022] In some embodiments, R3 is hydrogen. In other embodiments, R3 is -Y1-R4, wherein: Y1 is alkanediyl (C≦6) or substituted alkanediyl (C≦6) and R4 is acyloxy (C≦8~24) or substituted acyloxy (C≦8~24) is.

[0023] In some embodiments, Y is substituted alkanediyl (C≦6) In some embodiments, R4 is acyloxy, for example, 2-hydroxypropanediyl. (C≦8~24) In some embodiments, the permanent anionic lipid is Further defined as TIFF0007815196000008.tif103128.

[0024] In other embodiments, the selective organ targeting compound is a C6-C 24 Diacylphos Fa Diacylcholine (diacyl phosph a In some embodiments, diacylphosphatidylcholine is Fa In some embodiments, the diacylphosphatidylcholine is present in a molar percentage of the lipid nanoparticle composition of about 5% to about 50%. In some embodiments, the diacylphosphatidylcholine is present in a molar percentage of the lipid nanoparticle composition of about 10% to about 45%, e.g., about 30%. Fa There is a mole percentage of tidylcholine.

[0025] In some embodiments, the selective organ targeting compound comprises at least two fatty acid chains, a quaternary amine, and an anionic phosphate group. Fa Tidylcholine is further defined as TIFF0007815196000009.tif24128, During the ceremony, R1 and R2 are each independently alkyl (C8~C24) , alkenyl (C8~C24) or a substituted form of either group; R3, R3', and R3'' are each independently alkyl (C≦6) or substituted alkyl (C≦6) and X - is a monovalent anion.

[0026] 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 another embodiment, R 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.

[0027] In some embodiments, R3, R3', and R3" are each the same. In some embodiments, R3, R3', and R3" are each methyl. In some embodiments, X - is a halide anion, such as bromide or chloride. Fa Tidylcholine is Further defined as TIFF0007815196000010.tif21128.

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

[0029] In some embodiments, the cationic ionizable lipid comprises 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 a pH of about 6 to about 8. In some embodiments, the cationic ionizable lipid is a dendrimer or dendron. In some embodiments, the cationic ionizable lipid comprises at least two C6-C24 alkyl or alkenyl groups. In some embodiments, the cationic ionizable lipid comprises at least two C8-C24 alkyl groups.

[0030] In some embodiments, the phospholipid is present at a molar percentage of the lipid nanoparticle composition of about 8% to about 20%. In some embodiments, the molar percentage of the phospholipid is about 10% to about 14%. In some embodiments, the molar percentage of the phospholipid is about 11.9%. In other embodiments, the phospholipid is present at a molar percentage of the lipid nanoparticle composition of about 20% to about 23%. In some embodiments, the molar percentage of the phospholipid is about 20% to about 21%. In some embodiments, the molar percentage of the phospholipid 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 at a molar percentage of the lipid nanoparticle composition of about 39% to about 46%. 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 at a molar percentage of the lipid nanoparticle composition of about 15% to about 39%. 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 about 0.5% to about 10.0% relative to the lipid nanoparticle composition. In some embodiments, the PEGylated lipid is present at a molar percentage of about 0.5% to about 5.0%. In other embodiments, the PEGylated lipid is present at a molar percentage of about 2.0% to about 2.8%. In some embodiments, the PEGylated lipid is present at a molar percentage of about 2.4% relative to the lipid nanoparticle composition. In some embodiments, the PEGylated lipid is present at a molar percentage of about 3.9% to about 4.6% relative to the lipid nanoparticle composition. In some embodiments, the PEGylated lipid is present at a molar percentage of about 4.0% to about 4.3%. In some embodiments, the PEGylated lipid is about 4.1%. In some embodiments, the PEGylated lipid comprises a PEG moiety of about 1000 to about 10,000 daltons. In some embodiments, the PEGylated lipid is a PEGylated diacylglycerol. In some embodiments, the PEGylated lipid has the formula: Further defined by TIFF0007815196000011.tif18128, During the ceremony, R 12 and R 13 are each independently an alkyl (C≦24) , alkenyl (C≦24) or substituted versions of any of these groups; R e is hydrogen, alkyl (C≦8) , or substituted alkyl (C≦8) and x is 1 to 250. In some embodiments, the PEG lipid is dimyristoyl-sn-glycerol or a PEG-lipid of the formula: TIFF0007815196000012.tif38128 compound, During the ceremony, n1 is 5 to 250; and n2 and n3 each independently represent 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 five-tailed 3A5-SC8, 3A3-SC8, 4A1-SC8, 4A3-SC8, 5A2-SC8, or six-tailed 5A2-SC8. 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, the therapeutic agent is a small molecule, e.g., an anti-cancer agent, an anti-fungal agent, a psychiatric agent, e.g., an analgesic, an agent that alters level of consciousness, e.g., an anesthetic or hypnotic, a non-steroidal anti-inflammatory drug (NSAIDS), an anthelmintic, an anti-acne agent, an anti-angina pectoris agent, an anti-arrhythmic agent, an anti-asthma agent, an antibacterial agent, an anti-benign prostatic hyperplasia agent, an anticoagulant, an antidepressant, an anti-diabetic agent, an anti-emetic agent, an anti-epileptic agent, an anti-gout agent, an antihypertensive agent, an anti-inflammatory agent, an anti-malarial agent, an anti-migraine agent, an anti-muscarinic agent, an anti-neoplastic agent, an anti-obesity agent, an anti-osteoporosis agent, an anti-Parkinson's agent, an anti-proliferative agent, an anti-protozoal agent, an anti-thyroid agent, an anti-tussive agent, an anti-urinary incontinence agent, an anti-viral agent, an anti-anxiety agent, an appetite suppressant, a beta-blocker, a cardiac inotropic agent, a chemotherapeutic agent, a cognition enhancer, a The therapeutic agent is a small molecule selected from an enhancer, a contraceptive, a corticosteroid, a Cox-2 inhibitor, a diuretic, an erectile dysfunction agent, an expectorant, a gastrointestinal agent, a histamine receptor antagonist, an immunosuppressant, a keratolytic agent, a lipid regulating agent, a leukotriene inhibitor, a macrolide, a muscle relaxant, a neuroleptic, a nutritional agent, an opioid analgesic, a protease inhibitor, or a sedative. In other embodiments, the therapeutic agent is a protein. In other embodiments, the therapeutic agent is a nucleic acid, e.g., a therapeutic nucleic acid. In some embodiments, the nucleic acid is siRNA, miRNA, pri-miRNA, messenger RNA (mRNA), clustered regularly interspaced short palindromic repeats (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, the nucleic acid is present in a ratio of lipid nanoparticle composition to nucleic acid of about 1:1 to about 1:100. In some embodiments, the ratio is about 1:10 to about 1:60. In some embodiments, the ratio is about 1:40.

[0035] In some embodiments, the composition further comprises a protein. In some embodiments, the protein is a protein associated with translation or transcription. In some embodiments, the protein is associated with CRISPR processing. In some embodiments, the protein is a CRISPR-associated protein. In some embodiments, the protein is Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Cas10, Csy1, Csy2, Csy3, Csel, 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, a homolog thereof, 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 aspect, the present disclosure provides a method for manufacturing a semiconductor device comprising: (A) a composition described herein; and (B) excipients and The present invention provides a pharmaceutical composition comprising:

[0038] In some embodiments, the pharmaceutical composition is formulated for administration orally, intraadiposally, intraarterially, intraarticularly, intracranially, intradermally, intralesionally, intramuscularly, intranasally, intraocularly, intrapericardially, intraperitoneally, intrapleurally, intraprostatically, intrarectally, intrathecally, intratracheally, intratumorally, intraumbilically, intravaginally, intravenously, intravesically, intravitreally, liposomally, topically, mucosally, parenterally, rectally, subconjunctivally, subcutaneously, sublingually, locally, transbuccally, transdermally, vaginally, in a cream, in a lipid composition, via a catheter, via irrigation, via continuous infusion, via infusion, via inhalation, via injection, via topical delivery, or via local perfusion. In some embodiments, the pharmaceutical composition is formulated for intravenous or intraarterial 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, the pharmaceutical composition is formulated as a unit dose.

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

[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, modulation of gene expression is sufficient to treat a disease or disorder, e.g., cancer.

[0041] In yet another aspect, the present disclosure provides a method of treating a disease or disorder in a patient, comprising administering to the patient in need of such treatment 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 comprises administering one or more additional cancer therapies to the patient. In some embodiments, the cancer therapy is a chemotherapeutic compound, surgery, radiation therapy, 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] In yet another aspect, the present disclosure provides a method for preparing lipid nanoparticles, comprising: (A) 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) dissolving the therapeutic agent in a buffer to form a buffered therapeutic agent solution, the buffer having a pH of about 6.8 to about 7.6; and (C) Mixing the lipid solution with a buffered therapeutic agent solution to form lipid nanoparticles. The present invention provides a method comprising:

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

[0045] In yet another aspect, the present disclosure provides a method for manufacturing a semiconductor device comprising: (A) a therapeutic agent; (B) (1) Cationic ionizable lipids; (2) phospholipids; and (3) Selective organ-targeting compounds a lipid nanoparticle composition comprising wherein the organ targeting ligand causes preferential delivery of the composition to an organ other than the liver.

[0046] In yet another aspect, the present disclosure provides a method for manufacturing a semiconductor device comprising: (A) a therapeutic agent; (B) (1) cationic ionizable lipids; (2) phospholipids; (3) selective organ-targeted compounds; (4) steroids; and (5) PEG lipids a lipid nanoparticle composition comprising wherein the organ targeting ligand causes preferential delivery of the composition to an organ other than the liver.

[0047] In yet another aspect, the present disclosure provides a method for treating a rheumatoid arthritis comprising administering to a subject a therapeutic agent and (A) cationic ionizable lipid; (B) phospholipids; and (C) Selective organ-targeting compounds a lipid nanoparticle composition comprising 1. A composition comprising: a and delivering nucleic acids primarily to the lungs.

[0048] In another aspect, the present disclosure provides a method for treating a rheumatoid arthritis comprising administering to a subject a therapeutic agent and (A) cationic ionizable lipid; (B) phospholipids; (C) Selective organ-targeted compounds; (D) steroids; and (E) PEG lipid; a lipid nanoparticle composition comprising 1. A composition comprising: aand delivering nucleic acids primarily to the lungs.

[0049] In yet another aspect, the present disclosure provides a method for treating a rheumatoid arthritis comprising administering to a subject a therapeutic agent and (A) cationic ionizable lipid; (B) phospholipids; and (C) Selective organ-targeting compounds a lipid nanoparticle composition comprising a composition comprising an apparent pK a and delivers nucleic acids primarily to the spleen.

[0050] In yet another aspect, the present disclosure provides a method for treating a rheumatoid arthritis comprising administering to a subject a therapeutic agent and (A) steroids; (B) cationic ionizable lipids; (C) phospholipids; (D) PEG-lipid; and (E) Selective organ-targeting compounds a lipid nanoparticle composition comprising 1. A composition comprising: a and delivers nucleic acids primarily to the spleen.

[0051] In another aspect, the present disclosure provides a method for treating a rheumatoid arthritis comprising administering to a subject a therapeutic agent and (A) cationic ionizable lipid; (B) phospholipids; and (C)C6~C 24 Diacylphos Fa Tidylcholine a lipid nanoparticle composition comprising wherein the composition primarily delivers nucleic acids to lymph nodes.

[0052] In yet another aspect, the present disclosure provides a method for treating a rheumatoid arthritis comprising administering to a subject a therapeutic agent and (A) steroids; (B) cationic ionizable lipids; (C) phospholipids; (D) PEG-lipid; and (E)C6~C 24 Diacylphos Fa Tidylcholine a lipid nanoparticle composition comprising wherein the composition primarily delivers nucleic acids to lymph nodes.

[0053] In yet another aspect, the present disclosure provides a method for treating a rheumatoid arthritis comprising administering to a subject a therapeutic agent and (A) cationic ionizable lipid; (B) phospholipids; and (C) Selective organ-targeting compounds a lipid nanoparticle composition comprising wherein the surface of the composition interacts with vitronectin, and the composition primarily delivers nucleic acids to the lung.

[0054] In yet another aspect, the present disclosure provides a method for treating a rheumatoid arthritis comprising administering to a subject a therapeutic agent and (A) cationic ionizable lipid; (B) phospholipids; (C) Selective organ-targeted compounds; (D) steroids; and (E) PEG lipid a lipid nanoparticle composition comprising wherein the surface of the composition interacts with vitronectin, and the composition primarily delivers nucleic acids to the lung.

[0055] In yet another aspect, the present disclosure provides a method for treating a rheumatoid arthritis comprising administering to a subject a therapeutic agent and (A) cationic ionizable lipid; (B) phospholipids; and (C) Selective organ-targeting compounds a lipid nanoparticle composition comprising wherein the surface of the composition interacts with Apo H, and the composition primarily delivers nucleic acids to the spleen.

[0056] In another aspect, the present disclosure provides a method for treating a rheumatoid arthritis comprising administering to a subject a therapeutic agent and (A) steroids; (B) cationic ionizable lipids; (C) phospholipids; (D) PEG-lipid; and (E) Selective organ-targeting compounds a lipid nanoparticle composition comprising wherein the surface of the composition interacts with Apo H, 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 targeting protein present in a protein corona on the surface of the composition binds 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 alters the binding of a protein on the protein corona. In some embodiments, the selective organ targeting compound is further selected from a sugar, a lipid, a small molecule therapeutic, a vitamin, or a protein. In some embodiments, the selective organ targeting compound is a lipid, e.g., a permanent cationic lipid, a permanent anionic lipid, or a phosphatase. Fa 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 provides 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 lipid nanoparticle composition according to the present invention comprises:

[0061] In yet another aspect, the present disclosure provides 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 lipid nanoparticle composition according to the present invention comprises:

[0062] As used herein, "essentially free" with respect to a named component is used herein to mean that none of the named components are intentionally formulated into the composition and / or are present only as contaminants or in trace amounts. The total amount of the named component resulting from unintentional contamination of the composition is preferably less than 0.01%. Most preferred are compositions in which the amount of the named component is undetectable using standard analytical methods.

[0063] As used herein in the specification and claims, "a" or "an" may mean one or more. As used herein in the specification and claims, when used with the word "comprising," the words "a" or "an" may mean one or more. As used herein in the specification and claims, "another" or "further" may mean at least a second or more.

[0064] As used in this specification and claims, the term "about" is used to indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value, or the variation that exists among test subjects.

[0065] [The present invention 1001] (A) a therapeutic agent; (B) (1) Selective organ-targeting compounds; (2) cationic ionizable lipids; and (3) Phospholipids a lipid nanoparticle composition comprising 1. A composition comprising: a nucleic acid that preferentially delivers a nucleic acid to a target organ selected from lung, heart, brain, spleen, lymph node, bone, skeletal muscle, stomach, small intestine, large intestine, kidney, bladder, breast, testis, ovary, uterus, spleen, thymus, brainstem, cerebellum, spinal cord, eye, ear, tongue, or skin. [The present invention 1002] 1001. The composition of claim 1001, wherein the target organ is a lung, a lymph node, or a spleen. [The present invention 1003] The composition of any one of claims 1001 to 1002, wherein the selective organ targeting compound is a permanent cationic lipid. [The present invention 1004] 1003. The composition of the present invention, wherein the permanent cationic lipid is present in a molar percentage of the lipid nanoparticle composition of about 5% to about 20%. [The present invention 1005] 1003. The composition of the present invention, wherein the permanent cationic lipid is present in a molar percentage of the lipid nanoparticle composition of about 20% to about 65%. [The present invention 1006] 6. The composition of any one of claims 1003 to 1005, wherein the permanent cationic lipid comprises a quaternary ammonium ion. [The present invention 1007] The permanent cationic lipid is further defined as TIFF0007815196000013.tif23128, During the ceremony, R1 and R2 are each independently alkyl (C8~C24) , alkenyl (C8~C24) or a substituted form of either group; R3, R3', and R3'' are each independently alkyl (C≦6) or substituted alkyl (C≦6) and; X - is a monovalent anion, The composition of any one of 1003 to 1006 of the present invention. [The present invention 1008] The permanent cationic lipid is The composition of the present invention 1007, further defined as TIFF0007815196000014.tif21128. [The present invention 1009] The permanent cationic lipid is further defined as TIFF0007815196000015.tif14128, During the ceremony, R4 and R4' are each independently alkyl (C6~C24) , alkenyl (C6~C24) or a substituted form of either group; R4'' is alkyl (C≦24) , alkenyl (C≦24) or a substituted form of either group; R4''' is alkyl (C1~C8) , alkenyl (C2~C8) or a substituted form of either group; and X2 is a monovalent anion; The composition of any one of 1003 to 1006 of the present invention. [The present invention 1010] The permanent cationic lipid is The composition of the present invention 1009, further defined as TIFF0007815196000016.tif10128. [The present invention 1011] The permanent cationic lipid is further defined as TIFF0007815196000017.tif24128, During the ceremony, R1 and R2 are each independently alkyl (C8~C24) , alkenyl (C8~C24) or a substituted form 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 - is a monovalent anion, The composition of any one of 1003 to 1006 of the present invention. [The present invention 1012] The permanent cationic lipid is The composition of the present invention 1011, further defined as TIFF0007815196000018.tif27128. [The present invention 1013] The composition of any one of claims 1001 to 1002, wherein the selective organ targeting compound is a permanent anionic lipid. [The present invention 1014] The composition of the present invention 1013, wherein the permanent anionic lipid is present in a molar percentage of the lipid nanoparticle composition of about 5% to about 50%. [The present invention 1015] 1015. The composition of claim 1013 or 1014, wherein the permanent anionic lipid comprises a phosphate group. [The present invention 1016] The permanent anionic lipid is further defined as TIFF0007815196000019.tif24128, During the ceremony, R1 and R2 are each independently alkyl (C8~C24) , alkenyl (C8~C24) or a substituted form of either group; R3 is hydrogen, alkyl (C≦6) , or substituted alkyl (C≦6) or -Y1-R4, During the ceremony, Y1 is alkanediyl (C≦6) or substituted alkanediyl (C≦6) and R4 is acyloxy (C≦8~24) or substituted acyloxy (C≦8~24) That is, Any of the compositions 1013 to 1015 of the present invention. [The present invention 1017] The permanent anionic lipid is The composition of the present invention 1016, further defined as TIFF0007815196000020.tif103128. [The present invention 1018] Selective organ-targeting compounds target C6-C 24 Diacylphos Fa Diacylcholine (diacyl phosph a The composition of the present invention 1001 or 1002, wherein the compound is tidylcholine. [The present invention 1019] Diacylphos Fa 1018. The composition of claim 1018, wherein the tidylcholine is present in a molar percentage of the lipid nanoparticle composition of about 5% to about 50%. [The present invention 1020] The composition of any one of claims 1018 to 1019, wherein the selective organ targeting compound comprises at least two fatty acid chains, a quaternary amine, and an anionic phosphate group. [The present invention 1021] Diacylphos Fa Tidylcholine is further defined as TIFF0007815196000021.tif24128, During the ceremony, R1 and R2 are each independently alkyl (C8~C24) , alkenyl (C8~C24) or a substituted form of either group; R3, R3', and R3'' are each independently alkyl (C≦6) or substituted alkyl (C≦6) and X - is a monovalent anion, Any of the compositions 1018 to 1020 of the present invention. [The present invention 1022] Diacylphos Fa Tidylcholine is The composition of the present invention 1021, further defined as TIFF0007815196000022.tif21128. [The present invention 1023] 1023. Any of the compositions of claims 1001 to 1022, wherein the cationic ionizable lipid is present in a molar percentage of the lipid nanoparticle composition of about 5% to about 30%. [The present invention 1024] Any of the compositions of claims 1001 to 1022, wherein the cationic ionizable lipid is present in a molar percentage of the lipid nanoparticle composition of about 15% to about 30%. [The present invention 1025] The composition of any of claims 1001 to 1024, wherein the cationic ionizable lipid comprises an ammonium group that is positively charged at physiological pH and contains at least two hydrophobic groups. [The present invention 1026] The cationic ionizable lipid has at least two C6-C 24 The composition of the present invention 1025, comprising an alkyl or alkenyl group. [The present invention 1027] 1027. The composition of any one of claims 1001 to 1026, wherein the phospholipid is present in a molar percentage of the lipid nanoparticle composition of about 8% to about 20%. [The present invention 1028] Any of the compositions of claims 1001 to 1026, wherein the phospholipid is present in a molar percentage of the lipid nanoparticle composition of about 20% to about 23%. [The present invention 1029] The composition of any of claims 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. [The present invention 1030] The composition of any one of claims 1001 to 1029, further comprising a steroid. [The present invention 1031] The composition of the present invention 1030, wherein the steroid is present in a molar percentage of the lipid nanoparticle composition of about 39% to about 46%. [The present invention 1032] 1031. The composition of claim 1031, wherein the steroid is present in a molar percentage of the lipid nanoparticle composition of about 15% to about 39%. [The present invention 1033] The composition of any one of claims 1031 to 1032, wherein the steroid is cholesterol. [The present invention 1034] The composition of any one of claims 1001 to 1033, further comprising a PEGylated lipid. [This invention 1035] The composition of the present invention 1034, wherein the PEGylated lipid is present in a molar percentage of the lipid nanoparticle composition of about 0.5% to about 10.0%. [The present invention 1036] The composition of the present invention 1035, wherein the PEGylated lipid is present in a molar percentage of the lipid nanoparticle composition of about 3.9% to about 4.6%. [This invention 1037] 1035 or 1036, wherein the PEGylated lipid comprises a PEG moiety of about 1000 to about 10,000 daltons. [The present invention 1038] 1037. The composition of claim 1037, wherein the PEG lipid is a PEGylated diacylglycerol. [This invention 1039] The PEG lipid has the formula: Further defined by TIFF0007815196000023.tif18128, During the ceremony, 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 But hydrogen, alkyl (C≦8) , or substituted alkyl (C≦8) and x is between 1 and 250, Composition of the present invention 1038. [The present invention 1040] The PEG lipid may be dimyristoyl-sn-glycerol or the following formula: TIFF0007815196000024.tif38128 compound, During the ceremony, n1 is 5 to 250; and n2 and n3 each independently represent 2 to 25. The composition of the present invention 1035 or 1036. [The present invention 1041] The composition of any one of claims 1001 to 1040, wherein the therapeutic agent is a small molecule. [The present invention 1042] The composition of any one of claims 1001 to 1040, wherein the therapeutic agent is a protein. [This invention 1043] The composition of any one of claims 1001 to 1040, wherein the therapeutic agent is a nucleic acid. [This invention 1044] The composition of claim 1043, wherein the nucleic acid is a therapeutic nucleic acid. [This invention 1045] The composition of the present invention 1043, wherein the nucleic acid is siRNA, miRNA, pri-miRNA, messenger RNA (mRNA), clustered regularly interspaced short palindromic repeats (CRISPR)-associated nucleic acid, single guide RNA (sgRNA), CRISPR-RNA (crRNA), trans-activating 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). [The present invention 1046] 1046. The composition of any of claims 1043 to 1045, wherein the nucleic acid is present in a ratio of lipid nanoparticle composition to nucleic acid of about 1:1 to about 1:100. [This invention 1047] The composition of any one of claims 1001 to 1046, further comprising a protein. [This invention 1048] The composition of any one of claims 1001 to 1047, comprising both a protein and a nucleic acid. [This invention 1049] (A) any one of compositions 1001 to 1048 of the present invention; (B) excipients and 10. A pharmaceutical composition comprising: [The present invention 1050] 1. A method for regulating gene expression, comprising delivering a nucleic acid to a cell, The method comprising the step of contacting a cell with the composition or pharmaceutical composition of any of claims 1001 to 1049 under conditions sufficient to cause uptake of the nucleic acid into the cell. [This invention 1051] A method of treating a disease or disorder in a patient, comprising administering to a patient in need of such treatment a pharmaceutically effective amount of any one of the compositions or pharmaceutical compositions of the present inventions 1001 to 1049, The method, wherein the composition or pharmaceutical composition comprises a therapeutic nucleic acid for a disease or disorder. [This invention 1052] 1. A method for preparing lipid nanoparticles, comprising the steps of: (A) 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) dissolving the therapeutic agent in a buffer to form a buffered therapeutic agent solution, the buffer having a pH of about 6.8 to about 7.6; and (C) Mixing the lipid solution with a buffered therapeutic agent solution to form lipid nanoparticles. [This invention 1053] (A) a therapeutic agent; (B) (1) cationic ionizable lipids; (2) phospholipids; and (3) Selective organ-targeting compounds a lipid nanoparticle composition comprising A composition comprising: The composition, wherein the organ targeting ligand causes preferential delivery of the composition to an organ other than the liver. [This invention 1054] A therapeutic agent; (A) cationic ionizable lipid; (B) phospholipids; and (C) Selective organ-targeting compounds a lipid nanoparticle composition comprising A composition comprising: Apparent pK of about 8 to about 13 a and delivers nucleic acids primarily to the lungs. [This invention 1055] A therapeutic agent; (A) cationic ionizable lipid; (B) phospholipids; and (C) Selective organ-targeting compounds a lipid nanoparticle composition comprising A composition comprising: Apparent pK of about 3 to about 6 a and delivers the nucleic acid primarily to the spleen. [The present invention 1056] A therapeutic agent; (A) cationic ionizable lipid; (B) phospholipids; and (C)C6~C 24 Diacylphos Fa Tidylcholine a lipid nanoparticle composition comprising A composition comprising: The composition primarily delivers nucleic acids to lymph nodes. [This invention 1057] A therapeutic agent; (A) cationic ionizable lipid; (B) phospholipids; and (C) Selective organ-targeting compounds a lipid nanoparticle composition comprising A composition comprising: A composition wherein the surface of the composition interacts with vitronectin, and the composition primarily delivers nucleic acids to the lungs. [This invention 1058] A therapeutic agent; (A) cationic ionizable lipid; (B) phospholipids; and (C) Selective organ-targeting compounds a lipid nanoparticle composition comprising A composition comprising: The composition, wherein the surface of the composition interacts with ApoH, and the composition primarily delivers the nucleic acid to the spleen. [This 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. [The present invention 1060] 1059. The composition of claim 1059, wherein the protein is vitronectin and the target organ is the lung. [This invention 1061] The composition of claim 1059, wherein the protein is ApoH and the target organ is the spleen. [This invention 1062] The composition of any of claims 1059 to 1061, wherein the lipid nanoparticle composition further comprises a selective organ targeting compound that alters the binding of a protein on the protein corona. [The present 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 present 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: Other objects, features, and advantages of the present disclosure will become apparent from the following detailed description. It should be understood, however, that while particular embodiments of the present disclosure are set forth, the detailed description and specific examples are given by way of illustration only, since various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from this detailed description. [Brief explanation of the drawings]

[0066] The drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure. The present disclosure may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein. [Figure 1] Figure 1A-C: DOTAP mDLNP formulations mediated excellent mRNA delivery efficacy at low doses after IV injection and demonstrated 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). The DOTAP ratio alone was then adjusted from 0 to 1200 to generate a series of DOTAP mDLNP formulations, named DOTAPY. Y represents the percentage of DOTAP in the total lipid. (Figure 1B) Ex vivo images of luciferase activity in major organs 6 hours after IV injection of a 0.1 mg / kg dose of Luc mRNA (n=2). As the molar percentage of DOTAP increased, luciferase protein expression shifted from the liver to the spleen and then to the lungs. (Figure 1C) Quantitative data demonstrated that DOTAP percentage was a factor in tissue-specific delivery. mDLNP (0%) was optimal for the liver, DOTAP10-15 (similar between them) for the spleen, and DOTAP50 for the lung. Because luciferase expression was detected only in the liver, spleen, and lung after IV injection, relative expression in each organ was calculated. Clearly, the higher the percentage of DOTAP (a permanent cationic lipid) in the formulation, the less luminescence there was in the liver, approaching 0 at >70%. However, the higher the percentage of DOTAP, the more luminescence there was in the lung, approaching 100% at >70%. DOTAP5-20 showed a higher percentage in the spleen, with DOTAP10 appearing to be the highest. [Figure 2]Figures 2A1-F2: Lipid structure determines mRNA expression profiles after IV injection. Generally, increasing percentages of quaternary lipids shifted mRNA delivery from the liver to the spleen and then to the lungs, while zwitterionic lipids helped deliver mRNA to the spleen at higher percentages. However, tertiary amine lipids failed to alter mRNA expression in the organs where it was expressed, instead improving delivery efficacy in the liver. To further confirm the delivery trends of quaternary lipid mDLNPs, two other quaternary lipids, DDAB and EPC, were selected for in vivo mRNA delivery using the same improved strategy as DOTAP (Figures 2A1 and 2B1). DDAB and EPC exhibit significant structural differences between themselves and DOTAP at three comparative stages: hydrophobic tail length, saturated and unsaturated bonds, and headgroup chemical structure. To detect size distribution and in vivo evaluation, formulations with 5%, 15%, 40%, and 50% quaternary lipid were formed (0.1 mg / kg, 6 h, n = 2). Similar to DOTAP mDLNPs, DDAB and EPC also showed similar mRNA delivery profiles (Figures 2A2 and 2B2). A low cation percentage (5%) delivered mRNA to the liver and spleen, followed by increased delivery to the spleen when the percentage was increased to 15%. When the percentage was increased to 40%, mRNA expression was barely observed in the liver and spleen, while the lung showed high luciferase signals, which then decreased at 50%. These results were very similar to those of DOTAP mDLNPs. This suggests that mDLNPs functionalized with quaternary lipids are a universal and generalizable strategy for tissue-targeted mRNA delivery. Next, similar to the DOTAP strategy, we evaluated mRNA delivery in vivo using representative zwitterionic lipids, DSPC and DOCPe (Figures 2C1 and 2D1). The structures of the DSPC and DOCPe lipids are in the general class of zwitterionic lipids. The size distribution of DSPC and DOCPe mDLNP formulations was examined by DLS prior to IV injection.Herein, DSPC and DOCPe showed a two-level comparison in terms of structure: saturated versus unsaturated hydrophobic tail and charge position versus head groups (Figures 2C2 and 2D2). Interestingly, similar mRNA expression profiles were not observed as with quaternary lipid formulations. 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), with no signal observed in the lung at any percentage (0.1 mg / kg, 6 h, n = 2). Inspired by these results, we further tested the ionizable tertiary amine lipids, DODAP and C12-200, using the same strategy. DODAP has the same structure as DOTAP except for the head group (quaternary amine versus tertiary amine), while C12-200 is an effective lipidoid used for siRNA or mRNA delivery, but has a completely different structure from DODAP. (Figures 2E1, 2F1) Similarly, the size distribution of both modified mDLNPs remained favorable at a certain percentage (below 80%). (Figures 2E2, 2F2) Surprisingly, DODAP and C12-200 failed to alter the mRNA expression profile (different effects compared to quaternary or zwitterionic lipids). Instead, DODAP and C12-200 increased mRNA delivery to the liver. Supporting this, DODAP and C12-200 demonstrated significantly better delivery efficacy than the original mDLNP formulation (0.1 mg / kg, 6 h, n = 2). As the percentage of DODAP or C12-200 increased (to 50% or 80%), the luciferase signal significantly decreased, but the liver, not the spleen or lung, remained the primary organ. [Figure 3]Figure 3A-C: Next, we performed distribution assays and pKa detection to confirm why various lipids can induce such large differences in mRNA expression in organs. Both biodistribution and pKa played a role in organ mRNA expression profiles. (Figure 3A) Organ distribution of Cy5.5-Luc mRNA formulations delivered by three modified mDLNPs: DOTAP (quaternary lipid), DSPC (zwitterionic lipid), and DODAP (tertiary amine lipid). C57BL / 6 mice were injected IV at 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 mDLNP formulation (without DOTAP). Both DOTAP10 and DOTAP50 were able to deliver mRNA to the lungs, with DOTAP50 demonstrating a greater increase. This may partially explain why the DOTAP formulation mediated a higher percentage of mRNA expression in the lungs. However, both DSPC and DODAP failed to significantly alter mRNA distribution at either 80% (DSPC) or 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, while the latter was a spleen-targeted NP. Therefore, distribution was not the only factor explaining this mechanism. (Figure 3B) Next, we measured the pKa of all tested and effective formulations, including the original mDLNP, DOTAP, DDAB, EPC, DSCP, DOCPe, DODAP, and formulations modified with C12-200. (Figure 3C) Finally, we plotted the relationship between pKa and tissue-specific mRNA delivery based on the defined rules. Here, we designed eight rules for scoring, as shown in the table. Apparently, the pKa of all liver-targeted formulations was narrow (approximately 6–7), and there was no significant range for spleen-targeted formulations, whereas lung-targeted delivery required a high pKa (>9.25). [Figure 4]Figure 4A-C: Liver and lung gene editing was achieved in both Td-Tomato and C57BL / 6 mice. (Figure 4A) Schematic diagram shows that co-delivery of Cas9 mRNA and sgTom1 in Td-Tomato mice activates td-Tomato expression. (Figure 4B) Treatment with mDLNP and DOTAP50 formulations induced Td-Tomato expression in the liver and lung, respectively. To co-deliver Cas9 mRNA and modified sgTom1 (4 / 1, wt / wt) at a total dose of 2.5 mg / kg (50 μg each), mice were intravenously injected with mDLNP and DOTAP50 formulations. Fluorescence was then detected in major organs on day 10 of treatment. (Figure 4C) T7E1 assays demonstrated further tissue-specific characteristics using in vivo PTEN editing. To achieve tissue-specific gene editing, C57BL6 mice were intravenously injected 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 10 days after treatment. [Figure 5] Figure 5A-C: Characterization of DOTAP mDLNP formulations (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 Ribogreen RNA assay. [Figure 6]Figure 6A-C: DOTAP formulations demonstrated excellent mRNA delivery efficiency and demonstrated delivery capacity for cargoes, e.g., proteins, that were not well tolerated in ethanol or acidic buffer. (Figure 6A) DOTAP mDLNPs mediated high Luc mRNA expression in Huh-7 and A549 cells. A DOTAP percentage of 5% to 50% was found to be better for mRNA delivery, with 10% being the best. Luc mRNA expression and cell viability were tested 24 h after transfection with a 50 ng / well dose of mRNA (n=4). Here, DOTAP mDLNPs were formed in PBS rather than citrate buffer (10 mM, pH 4.0). (Figures 6B and 6C) A reduced volume percentage of ethanol did not affect characterization and mRNA delivery efficacy. To test the effect of ethanol on mRNA delivery, we selected DOTAP25 as a model and formed four formulations using various volume ratios of ethanol:PBS (1:3, 1:5, 1:7.5, and 1:10). All four formulations exhibited similar EE, size, and PDI (Figure 6B) and demonstrated equivalent mRNA delivery efficacy in FaDu cells (50 ng / well mRNA, 24 h, n = 4) (Figure 6C). Therefore, we optimized this formulation using 1X PBS (pH 7.4) instead of acidic buffer (10 mM pH 4.0) to dramatically reduce the ethanol percentage. This suggests that DOTAP formulations have the potential to deliver cargoes, such as proteins, that are poorly tolerated in both high ethanol concentrations and acidic buffers. [Figure 7] Quantitative biodistribution data in major organs. C57 BL6 mice were injected IV with various Cy5.5-Luc mRNA formulations at a dose of 0.5 mg / kg (n=2). Six hours later, the heart, lungs, liver, spleen, and kidneys were isolated, imaged, and quantified. [Figure 8]Figure 8A-C: There was no significant difference in size distribution and Luc mRNA delivery efficacy for DOTAP10 formulations formed with PBS or citrate buffer, but this did not apply to DSPC50 and DODAP50. To test the effect of buffer on mRNA delivery efficacy in vivo, we selected DOTAP10 (quaternary lipid), DSPC50 (zwitterionic lipid), and DODAP50 (tertiary amine lipid). C57 BL6 mice were intravenously injected with each Luc mRNA formulation at a dose of 0.1 mg / kg, and major organs were isolated and imaged 6 hours later (n=2). (Figure 8A) Both size and delivery efficacy were largely unchanged 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 mRNA delivery efficacy, but there was no significant difference in size distribution. DOTAP (or another permanent cationic lipid) may be added for LNP formation at neutral pH (e.g., 7.4 PBS buffer). [Figure 9] Western blot results of quality testing of IVT Cas9 mRNA delivered by mDLNP. To achieve tissue-specific gene editing, we designed the co-delivery of Cas9 mRNA and sgRNA. First, Cas9 mRNA was generated via IVT and analyzed by Western blot for quality testing. In this assay, Cas9 pDNA was delivered using Lipofectamine 2000 and a commercially available Cas9 mRNA (TriLink). mDLNP served as a positive control, and mCherry mDLNP served as a negative control. 293T cells were seeded in 12-well plates the day before transfection, and Western blot analysis was performed after treating the cells with each condition for 24 hours. IVT Cas9 mRNA performed significantly better than the commercially available mRNA. Therefore, we performed in vivo gene editing using IVT Cas9 mRNA. [Figure 10]Figures 10A and 10B: sgRNA screening and weight ratio (Cas9 mRNA / sgRNA) optimization 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 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 leader. (Figure 10B) SgTom1 was selected and further tested for liver gene editing using different weight ratios (Cas9 / sgRNA) of 2 / 1, 4 / 1, and 6 / 1. mDLNP was used at a dose of 3 mg / kg for IV injection. td-tomato expression was detected on day 7. In this example, 4 / 1 performed better than 2 / 1 and 6 / 1. [Figure 11]Figure 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 complexes prepared in citrate buffer are very large (larger than 100 nm) and have a positive zeta potential, making them impossible to encapsulate in lipid nanoparticles. However, the Cas9 / sgLUC complexes prepared in PBS are small (smaller than 20 nm) and negatively charged, making them encapsulable in lipid nanoparticles. Size (Figure 11C) and zeta potential (Figure 11D) of Cas9 / sgLUC complexes prepared at different Cas9 / sgRNA molar ratios (1 / 1, 1 / 3, and 1 / 5). Compared with the Cas9 / sgLUC complex (1 / 1, mol / mol), larger molar ratios (1 / 3 and 1 / 5, mol / mol) resulted in smaller size and more negative charge, which is beneficial for lipid nanoparticle encapsulation. Figure 11E shows the size (Figure 11F) and zeta potential (Figure 11F) of DOTNP10 lipid nanoparticle-encapsulated Cas9 / sgLUC complexes (named DOTNP10-L) prepared at different molar ratios (1 / 1, 1 / 3, 1 / 5). Figure 11G shows a TEM image of DOTNP10-L (1 / 3, mol / mol). DOTAP lipid nanoparticles consist of five components, including 5A2-SC8, cholesterol, DOPE, DMG-PEG, and DOTAP. The molar ratio of 5A2-SC8, cholesterol, DOPE, and DMG-PEG was fixed (15:15:30:5, mol / mol). DOTNPX refers to DOTNP with different molar percentages of DOTAP. Here, we used different sgRNAs, including sgLUC, sgGFP, sgTOM, and sgPTEN. To distinguish between these, we added the first letter of each gene to the end of 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]Figure 12A-F: DOTNP lipid nanoparticles were able to deliver the Cas9 / sgRNA complex 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 complexes (1 / 3, mol / mol) for 1, 3, 6, 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) The percentage of indels at the LUC locus after 3 days of incubation with different molar ratios of DOTNP10-L was 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 (24 nM sgRNA was used). 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, a 1 / 3 molar ratio showed the best gene editing when using Truecut Cas9 protein. (Figure 12D) Fluorescence microscopy images of SKOV3-GFP cells incubated with DOTNP10-L and DOTNP10-G (24 nM sgRNA was used). Here, DOTNP10-L was used as a negative control. (FIG. 12E) Flow cytometry analysis of SKOV3-GFP cells incubated with DOTNP10-L and DOTNP10-G. (FIG. 12F) Mean fluorescence intensity by flow cytometry of SKOV3-GFP cells incubated with DOTNP10-L and DOTNP10-G. [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 DOTNP5 lipid nanoparticle-encapsulated Cas9 / sgTom complexes. DOTNP10-T refers to DOTNP10 lipid nanoparticle-encapsulated Cas9 / sgTom complexes. DOTNP50-T refers to DOTNP50 lipid nanoparticle-encapsulated Cas9 / sgTom complexes. 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. When the DOTNP dose was further increased to 50% (DOTNP50-T), most of the tdTomato fluorescence was observed in the lungs. (Figure 13B) T7EI cleavage assay 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). The 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 observed in both the liver and lung. In contrast, in the DOTNP50-P-treated group, most of the gene editing was observed in the lung. [Figure 14] Figures 14A and B: Details of the MC3 LNP and DOTAP-modified MC3 formulations are shown, including (Figure 14A) the structure of each component, (Figure 14B) the molar ratio, total lipid to mRNA weight ratio, size, and 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 (6 h, n = 2). As the DOTAP percentage increased from 0 to 50%, MC3- and C12-200-based LNPs showed identical mRNA expression profiles as mDLNPs, with luciferase signals migrating from the liver to the spleen and finally to the lungs. [Figure 16] Figures 16A and B: Details of C12-200 LNP and DOTAP-modified C12-200 formulations are shown, including (Figure 16A) the structure of each component, (Figure 16B) the molar ratio, total lipid to mRNA weight ratio, size, and PDI. [Figure 17] Figures 17A and B: (Figure 17A) Further mDLNP optimization is shown. mDLNPs were modified using the "fifth" lipid, 5A2-SC8, as the key lipid in mDLNPs to form four formulations with an extra percentage of 10% to 30%. (Figure 17B) Ex vivo luciferase images and quantitative data showed that using an extra 15% to 25% of 5A2-SC8 dramatically improved mRNA delivery efficacy, with a 20% signal producing the highest signal (0.05 mg / kg, 6 h, n=2). [Figure 18] The structures of 5A2-SC8, DOPE, cholesterol, and DMG-PEG are shown. mDLNPs, composed of 5A2-SC8, DOPE, cholesterol, and DMG-PEG in a molar ratio of 15 / 15 / 30 / 3, are effective and safe mRNA delivery carriers for liver-targeting therapeutic agents, as previously developed. [Figure 19]Figures 19A–G show that selective organ targeting (SORT) can be used to systematically and predictably engineer lipid nanoparticles (LNPs) for precise cell editing in specific organs. (19A) The addition of auxiliary components (termed SORT lipids) to conventional LNPs systematically alters the in vivo delivery profile, mediating tissue-specific delivery as a function of the percentage of SORT lipid and biophysical properties. This universal methodology has been successfully used to target multiple classes of nanoparticles. Here, we show bioluminescence images of mice intravenously injected with 0.1 mg / kg luciferase mRNA inside 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 including SORT lipids in four-component 5A2-SC8, DLin-MC3-DMA, and C12-200 LNPs. (19B) 5A2-SC8 SORT LNPs were formulated with a 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 create a series of LNPs using 0% to 100% SORT lipid (percentage of total lipid). Here, the inclusion of a permanent cationic lipid (DOTAP) systematically shifted luciferase protein expression from the liver to the spleen to the lung as a function of DOTAP percentage (0.1 mg / kg Luc mRNA, 6 h). (19C) Quantitative data demonstrated that the SORT lipid percentage was a factor in tissue-specific delivery. 0% (mDLNP) was optimal for the liver; 5-15% for the spleen; and 50% for the lung. (19D) Relative luciferase expression in each organ demonstrated predictable modulation of fractional expression. (19E) Inclusion of anionic SORT lipids enabled selective mRNA delivery to the spleen. When 18PA lipid was incorporated into mDLNPs up to 40%, luciferase expression was observed only in the spleen (0.1 mg / kg Luc mRNA, 6 h).(19F) Ex vivo imaging of luminescence in major organs 6 hours after IV injection of DLin-MC3-DMA SORT LNPs and Luc mRNA at a dose of 0.1 mg / kg. As the molar percentage of DOTAP increased, luciferase expression shifted from the liver to the lung. 18PA mediated exclusive delivery of Luc mRNA to the spleen. The same trend was observed for modified C12-200 LNPs (0.1 mg / kg, 6 h). (19G) Details of selected SORT lipid formulations. [Figure 20]Figures 20A-C: (20A) Details of DOTAP and 18PA SORT LNPs are shown, including molar ratios, molar percentages, total lipid to mRNA weight ratios, size, PDI, and zeta potential. (20B) LNPs were formulated using an improved ethanol dilution method. SORT lipids were included in the ethanol phase to encapsulate sgRNA / mRNA during LNP formation. (20C) Chemical structures of the lipids used in standard mDLNP and DOTAP / 18PA SORT formulations are shown. To develop SORT, a degradable dendrimer-based cationic ionizable lipid named 5A2-SC8 was the focus of our LNPs capable of delivering siRNA / miRNA to extend survival in genetically engineered mouse models of MYC-driven liver cancer (Zhou et al., 2016; Zhang et al., 2018a; Zhang et al., 2018b) and liver toggle polyploidy. LNP molar compositions optimized for mRNA delivery were designated mDLNPs (Cheng et al., 2018). This liver-targeting basic mRNA formulation was prepared with a molar ratio of 5A2-SC8 / DOPE / cholesterol / DMG-PEG2000 = 15 / 15 / 30 / 3 (mol), and SORT lipids were added to prepare SORT LNPs (detailed in 20A). For clarity, traditional four-component LNPs consist of a cationic ionizable lipid (defined herein as containing an amino group with a pKa < 8), a zwitterionic phospholipid (defined as a lipid with an equal number of positive and negative charges), cholesterol, and a poly(ethylene glycol) (PEG) lipid (most commonly PEG2000-DMG). SORT LNPs contain a fifth lipid, e.g., a permanent cationic lipid (defined as having no pKa or a positive charge with a pKa > 8) or a permanent anionic lipid (defined as being negatively charged). [Figure 21]Figures 21A and 21B show in vitro luciferase (Luc) mRNA delivery results of DOTAP-modified SORT mDLNPs in (Figure 21A) Huh-7 liver cells and (Figure 21B) A549 lung cells as a function of DOTAP percentage incorporation. Luc mRNA delivery results indicated that LNPs with DOTAP percentages between 5% and 50% delivered the most mRNA in both Huh-7 liver cells and A549 lung cells. SORT LNPs containing 10% DOTAP were significantly more effective in vitro than the previously reported base mDLNPs. No obvious cytotoxicity was observed in any of the formulations, and all were uniform (low PDI) and ranged in diameter from 90 nm to 150 nm (Figure 20). Surface charge measurements revealed that DOTAP was encapsulated internally with the mRNA and not present on the LNP surface, as the zeta potential was close to zero when DOTAP was less than 60%. Only in excess of 65% of cases did the surface charge become positive (Figure 20). This indicates that PEG-lipid-coated SORT LNPs possess a near-neutral surface charge and selective tissue tropism, attributes suitable for clinical translation. The day before transfection, cells were seeded into 96-well plates at a density of 1 x 104 cells / well. Luc mRNA expression and cell viability were measured 24 hours after treatment with 50 ng / well of Luc mRNA (n = 4). [Figure 22]Figures 22A-C: Chemical structures of lipids used in (22A) DLin-MC3-DMA SNALP (Jayaraman et al., 2012) and (22B) C12-200 LLNP (Love et al., 2010) are shown. Liver-targeting basic mRNA formulations were prepared with 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), followed by the addition of SORT lipids to prepare SORT LNP. (22C) Table and results of additional SORT formulations using DLin-MC3-DMA and C12-200. The total lipid / mRNA weight ratio was 20 / 1 (wt / wt) for all DLin-MC3-DMA and C12-200 LNPs. [Figure 23] Figures 23A-C show that SORT relies on general biophysical properties and not precise chemical structure. (23A) SORT lipids can be divided into specific groups with defined biophysical properties. Permanently cationic SORT lipids (DDAB, EPC, and DOTAP) all produced the same mRNA delivery profile (liver to spleen to lung based on SORT lipid percentage) (0.1 mg / kg Luc mRNA, 6 h). (23B) Anionic SORT lipids (14PA, 18BMP, 18PA) all produced the same mRNA delivery profile (exclusively spleen based on SORT lipid percentage). (23C) A cationic, ionizable SORT lipid with a tertiary amino group (DODAP, C12-200) enhanced liver delivery (0.1 mg / kg Luc mRNA, 6 h) with no luciferase expression in the lung. [Figure 24]Figures 24A and B: To further enhance mDLNP liver delivery, SORT was further applied to utilize cationic ionizable lipids as the SORT lipid. (24A) Schematic diagram of SORT. (24B) Using 5A2-SC8 as the SORT lipid, additional 5A2-SC8 was added to the basic mRNA mDLNP formulation (5A2-SC8 / DOPE / cholesterol / DMG-PEG2000 = 15 / 15 / 30 / 3 (mol)) using the SORT method. Ex vivo luciferase imaging and quantitative data showed that mRNA delivery efficacy dramatically improved when an extra 15% to 25% SORT lipid was added. Maximum expression occurred when 20% was incorporated (0.05 mg / kg, 6 h, n = 2). Therefore, a second-generation mDLNP with increased efficacy was developed by SORT. [Figure 25] Figures 25A and B show the effect of zwitterionic SORT lipids was evaluated. When zwitterionic SORT lipids were included in liver-targeted mDLNPs, expression shifted from the liver to the spleen as SORT lipid incorporation increased. After IV injection, 80% DSPC and 50% DOCPe SORT LNPs delivered mRNA exclusively to the spleen. (25A) Schematic of the SORT method. (25B) Ex vivo images of luminescence in major organs 6 hours after IV injection. Zwitterionic lipids with different structures, DSPC and DOCPe, improved Luc mRNA delivery to the spleen as the percentage increased (0.1 mg / kg, 6 h, n=2). [Figure 26]Figures 26A and B: SORT was evaluated as a potential strategy to "activate" inactive LNP formulations. (26A) Schematic of adding SORT lipids to an inactive C1 formulation to test whether SORT could confer activity. (26B) Detailed information on C1 LNPs (inactive LNPs) and DOTAP (or DODAP) C1 SORT LNPs, including lipid molar ratio, molar percentage, total lipid-to-mRNA weight ratio, size, and PDI. C1 LNPs were prepared using a method that enabled mRNA encapsulation and favorable biophysical properties (uniform size <200 nm). However, no protein was expressed after IV injection of C1 LNPs. Therefore, we asked whether SORT could "activate" dead LNPs. DODAP and DOTAP SORT lipids were evaluated. DODAP@C1 LNPs delivered mRNA to the spleen and liver, and DOTAP@C1 LNPs delivered mRNA to the lungs and spleen (0.1 mg / kg, 6 h, n = 2). Thus, SORT can activate dead LNPs, resulting in tissue selectivity. [Figure 27]Figures 27A and B show that SORT alters LNP biodistribution, revealing a correlation between relative apparent pKa and organ specificity. (27A) Fluorescent Cy5-labeled mRNA was used to track the biodistribution of SORT LNPs. Inclusion of DODAP as a SORT lipid increased mRNA accumulation in the lung, partially explaining their ability to deliver RNA to mouse lungs. 18PA increased spleen uptake. DODAP slightly increased liver accumulation and decreased spleen accumulation (0.5 mg / kg, 6 h). Note that this data describes the location of SORT LNPs, not their ability to productively deliver mRNA into cells. (27B) The relative apparent pKa of all 67 effective mRNA formulations was measured by TNS assay and plotted against their in vivo delivery efficacy (functional delivery of mRNA translated into protein) in various organs. As expected, the pKa of all liver-targeting formulations was narrow (6–7). Surprisingly, a high pKa (>9) was required for lung-targeted delivery, while a low pKa (<6) favored spleen delivery. Note that all SORT LNPs contain a cationic ionizable lipid (for endosomal escape) along with a mixture of other charged and uncharged lipids (which collectively mediate tissue tropism). [Figure 28] Figures 28A and B: Cy5-labeled mRNA was used to track the biodistribution of SORT LNPs. Organ distribution of DSPC mDLNPs after IV injection. (28A) Schematic of SORT. (28B) Cy5 fluorescence and quantification data of major organs treated with DSPC mDLNPs (0.5 mg / kg, 6 h, n=2). [Figure 29]We demonstrate the use of an improved TNS assay to measure the overall / apparent pKa of mRNA formulations. A total of 67 successful NP formulations (high in vivo potency) were evaluated. Relative pKa was estimated by comparing it to the base LNP formulation (no added SORT lipids) at which 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 the cationic lipids within the self-assembled LNP. Here, we used an improved method because SORT LNPs with a high percentage (>40%) of permanent cationic lipids (e.g., DOTAP) contain cationic ionizable lipids but do not buffer charge significantly. Due to the complexity of SORT LNPs containing various charged lipids (rather than a single 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: Demonstrate that the inclusion of an ionizable lipid (e.g., 5A2-SC8) was necessary for efficacy. LNPs containing SORT lipids but without cationic ionizable lipids were inactive. (30A) Schematic of SORT C2 LNP. (30B) Details of C2 and SORT lipid C2 LNP. Ex vivo luciferase images showed that neither DODAP nor DOTAP enabled significant mRNA delivery with C2 LNP. These results demonstrate the requirement of an ionizable amino lipid for successful mRNA delivery (0.1 mg / kg, 6 h, n=2). [Figure 31]Figures 31A-E: SORT LNPs enable tissue-specific gene editing in Td-Tomato mice via Cre mRNA delivery. (31A) Schematic diagram showing activation of Td-Tom expression by Cre mRNA delivery in Td-Tom transgenic mice. (31B) mDLNPs and 20% DODAP LNPs specifically induced Td-Tom fluorescence in the liver, while 50% DODAP LNPs selectively edited the lung. Td-Tom fluorescence in major organs was detected 2 days after IV injection of Cre mRNA-loaded LNPs (0.3 mg / kg). (31C) 30% 18PA SORT LNPs induced gene editing in the spleen (note the strong liver background fluorescence 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 in liver, lung, and spleen (day 2, 0.3 mg / kg). [Figure 32] We show that B6.Cg-Gt(ROSA)26Sortm9(CAG-tdTomato)Hze / J(Ai9) mice exhibit some autofluorescence in the TdTom absorption region. Furthermore, there is a large difference in TdTom autofluorescence between different organs (n=2). The liver and kidneys exhibit the highest signal, while the spleen exhibits the lowest. While this does not interfere with the detection of editing in most organs (excitation settings were appropriately adjusted to eliminate background), it complicates the detection of splenic TdTom expression because background is significantly lower in the spleen than in the other organs. [Figure 33]Figures 33A-C: CRISPR / Cas gene editing in the spleen was achieved in both Td-Tom transgenic and wild-type C57 / BL6 mice by co-delivery of Cas9 mRNA and sgRNA. (33A) Schematic diagram showing that co-delivery of Cas9 mRNA and sgTom1 in Td-Tom mice activates Td-Tom expression. (33B) Td-Tom expression was induced in the spleen and liver by the spleen-targeting formulation 30% 18PA SORT LNP. Quantitative data showed that editing was more prevalent in the spleen than in the liver. Td-Tom fluorescence was detected in major organs on day 2 after IV co-delivery of Cas9 mRNA and modified sgTom1 (2 / 1, wt / wt) at a total dose of 4 mg / kg. (33C) T7E1 assay demonstrated that co-delivery of Cas9 mRNA (IVT) and sgPTEN resulted in specific PTEN editing in the spleen. C57 / BL6 mice were injected IV with 30% 18PA SORT LNPs 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, suggesting that spleen-specific editing is possible. [Figure 34] Using DODAP-20 SORT LNPs, we demonstrate that administration of a single 0.3 mg / kg Cre mRNA dose to hepatocytes achieved nearly 100% TdTom editing. Flow cytometry histograms show complete separation between TdTom-control and TdTom+20% DODAP-treated mice. After liver perfusion, the excised livers of mice treated with 20% DODAP SORT LNPs were strikingly bright red compared to control livers. Even without fluorescence excitation, the livers were bright red due to fully activated TdTom expression. 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]The FACS gating strategy for analyzing TdTom+ expression in lung cells is described. Ghost Red 780 was used to distinguish live from dead cells. EpCam+ was used to define epithelial cells, CD45+ and CD31- were used to define immune cells, and CD45- and CD31+ were used to define endothelial cells. Td-Tom+ cell type gates were drawn based on PBS-injected control mice. Td-Tom mice were injected with Cre mRNA preparations. Td-Tom+ was detected in the indicated cell types by flow cytometry after 2 days (n=3). [Figure 36] The FACS gating strategy for analyzing TdTom+ expression in splenocytes is described. Ghost Red 780 was used to distinguish live from dead cells. CD44+ was used to identify immune cells, followed by CD3+ and CD11b- for T cells, CD3- and CD11b+ for macrophages, and CD19+ and CD11b- for B cells. Td-Tom+ cell types were gated based on PBS-injected control mice. Td-Tom mice were injected with a Cre mRNA preparation. Td-Tomato+ was detected in the indicated cell types by flow cytometry after 2 days (n=3). [Figure 37]Figures 37A-G: SORT LNP-mediated tissue-specific CRISPR / Cas gene editing in Td-Tom transgenic and C57 / BL6 wild-type mice via co-delivery of Cas9 mRNA and sgRNA and delivery of Cas9 RNP. (37A) Schematic diagram showing that co-delivery of Cas9 mRNA (or Cas9 protein) and sgTom1 in Td-Tom transgenic mice activates Td-Tom expression. (37B) mDLNP and 20% DODAP LNP specifically induced Td-Tom fluorescence in the liver, while 50% DODAP LNP selectively edited the lung. 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) Confocal imaging of tissue sections confirmed tdTom expression. Scale bars = 20 μm and 100 μm. (37D) Cas9 mRNA and sgPTEN were co-delivered in SORT LNPs to selectively edit the liver, lung, and spleen of C57 / BL6 mice (2.5 mg / kg total dose (Cas9 mRNA / sgPTEN, 4 / 1, wt / wt; measured 10 days after a single injection). T7E1 assays demonstrated that tissue-specific PTEN editing was achieved. (37E) Successful PTEN editing was further confirmed by H&E sections and IHC. Clear cytoplasm indicated lipid accumulation in H&E sections, and PTEN loss in IHC images. Scale bar = 60 μm. (37F) Delivery of Cas9 / sgTom1 ribonucleoprotein (RNP) complexes in 7% DOTAP or 55% DOTAP SORT LNPs specifically induced Td-Tom fluorescence in the liver and lung, respectively. Cas9 / sgTom1 RNPs at 1.5 mg / kg Td-Tom fluorescence was detected 7 days after IV injection of a dose of sgTom1. Confocal imaging of tissue sections confirmed tdTom expression. Scale bars = 20 μm and 100 μm. (37G) Liver- and lung-tropic SORT LNPs also delivered Cas9 / sgPTEN RNPs to selectively edit the liver and lung of C57 / BL6 mice (1.5 mg / kg sgPTEN; measured 7 days after a single injection).T7E1 assays demonstrated that tissue-specific PTEN editing was achieved. [Figure 38] IVT Cas9 mRNA was assessed by Western blot. 293T cells were seeded in 12-well plates the day before transfection and treated with each condition for 24 hours before Western blot analysis. Cas9 pDNA was delivered using Lipofectamine 2000, and mRNA was delivered using mDLNPs. [Figure 39] Figures 39A and B show the optimization of the weight ratio of IVT Cas9 mRNA to sgTom1 via the Cas9 mRNA and sgRNA co-delivery strategy. (39A) Schematic diagram shows that co-delivery of Cas9 mRNA and sgTom1 in transgenic mice activates Td-Tom expression. (39B) Td-Tom fluorescence was imaged in major organs 7 days after IV injection, demonstrating 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 by mDLNPs. [Figure 40]Figures 40A-I show that a modular approach for systemic nanoparticle delivery of CRISPR / Cas9 ribonucleoproteins (RNPs) for tissue-specific genome editing has been developed. (40A) Adding a permanent cationic auxiliary component (e.g., DOTAP) to conventional LNP formulations allowed for 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 RNPs prepared in PBS buffer (pH 7.4) and citrate buffer (pH 4.0). The increase in size is likely due to denaturation. (40C) Size distribution of 5A2-DOT-10-encapsulated Cas9 / sgLuc RNPs prepared in PBS and citrate buffer. 5A2-DOT-10 prepared without RNPs was used as a control. (40D) Size distribution of Cas9 / sgRNA RNPs 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 RNPs showing decreasing charge. (40G) No significant difference in zeta potential was observed for 5A2-DOT-10-encapsulated Cas9 / sgLuc at different molar ratios. (40H) Time-dependent cellular uptake of 5A2-DOT-10 LNP-encapsulated EGFP-fused Cas9 / sgRNA, showing cytoplasmic release and gradual 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 caveolae-mediated endocytosis; MβCD: lipid raft-mediated endocytosis; 4°C: energy-mediated endocytosis. [Figure 41]Figure 41A-C: (41A) A table of 5A2-DOT-X LNPs showing 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 a T7EI assay. (41C) Gene editing was analyzed using Sanger sequencing and ICE analysis. [Figure 42] A representative TEM image of 5A2-DOT-10-encapsulated Cas9 / sgLuc RNP complexes using a 1 / 3 molar ratio is shown. 5A2-DOT-10 Cas9 / sgLuc was prepared in PBS buffer at a total lipid concentration of 2 mg / mL. 3 μL of nanoparticle solution was dropped onto a carbon TEM grid and allowed to settle for 1 minute before blotting with filter paper. The TEM grid was then imaged using a transmission electron microscope (FEI Tecnai G2 Spirit Biotwin). [Figure 43] Confocal images are shown showing the cellular uptake of PBS (control), free Cas9 / sgLuc complex (control), and 5A2-DOT-10 Cas9 / sgLuc in Hela-Luc cells 20 hours after treatment. A Cas9-EGFP fusion protein was used to track the subcellular distribution of the Cas9 / sgRNA complex. The Cas9 / sgLuc complex showed no detectable green fluorescence within the cells above background (PBS). In contrast, a bright green signal was detected after treatment with 5A2-DOT-10. [Figure 44]Figures 44A-H: Gene editing is demonstrated to occur rapidly and efficiently in vitro. (44A) T7EI cleavage assay of DNA isolated from HeLa-Luc cells treated with various nanoparticles and a control. 5A2-DOT-10 delivering Cas9 / sgLuc RNP (1 / 3 and 1 / 5) mediated highly effective gene editing. The percentage of indels at the Luc locus was quantified by ICE analysis. Note that LNPs prepared using low-pH citrate buffer (currently used established method) resulted in 0% gene editing. (44B) Fluorescence microscopy images of HeLa-GFP cells after treatment with various formulations. Scale bar = 100 μm. 5A2-DOT-10 Cas9 / sgGFP treatment significantly reduced GFP fluorescence. (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 nearly all GFP-positive cells became dark. (44D) Time-dependent GFP fluorescence intensity of HeLa-GFP cells after various treatments. Permanent GFP fluorescence loss was observed after 2 days of 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, and DLin-MC3-DMA LNP formulations containing 10% supplemental DOTAP, conventional Cas9 / sgGFP-loaded C12-200 and DLin-MC3-DMA LNP nanoformulations, and Cas9 / sgGFP-loaded RNAiMAX. 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 with 5A2-DOT-10 LNP. Mean ± sem (n = 3). Statistical significance was determined using a two-tailed Student's t-test.†: t-value = 42.69, df = 4 (P < 0.0001); ††: t-value = 16.75, df = 4 (P < 0.0001); †††: t-value = 37.53, df = 4 (P < 0.0001). A P value < 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) Periodic treatment of HeLa-GFP cells with stored LNPs showed no loss of activity, demonstrating long-term LNP and RNP stability and translational competence. All cells in the above experiment were treated with 24 nM sgRNA. [Figure 45] Figures 45A and B show gene editing of different nanoformulations in Hela-GFP cells. (45A) Mean fluorescence intensity (%) of Hela-GFP cells after treatment with Cas9 / sgGFP only, 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 with 10:1, 20:1, 30:1, and 40:1 total lipid / sgGFP weight ratios. [Figure 46]Figures 46A-K show that the generalizable RNP delivery strategy (Figure 40A) is universal for cationic ionizable lipid nanoparticles (DLNP, LLNP, SNALP), as well as other cationic lipids that are 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 the final molar ratio and percentage of each component and the weight ratio of total lipid 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 RNP 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 of 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 and the weight ratio of total lipid 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) instead of DOTAP could also achieve efficient gene editing. (46I) Scheme of LNP formulation 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. A neutral buffer was required for RNP encapsulation and delivery. (46K) ICE analysis was used to measure the percentage of indels at the GFP locus in genomic DNA isolated from HeLa-GFP cells after treatment with 5A2-DOT-10 Cas9 / sgGFP LNPs prepared using different buffers. All neutral buffers demonstrated significant gene editing in the cells.This demonstrates the importance of a neutral buffer in nanoparticle preparation. Note that Figures 44E and 44F have been reproduced above in Figure 45 to collect and combine relevant data for ease of understanding. [Figure 47]Figures 47A-J: Highly efficient multiplexed genome editing was achieved in vivo. (47A) Schematic diagram shows how delivery of Cas9 / sgTOM RNPs activates Td-Tomato expression in Td-Tomato transgenic mice. 5A2-DOT-X LNPs were injected locally (via intramuscular or intracerebral injection) and systemically (via intravenous 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 limb muscle or brain tissue (respectively). Successful CRISPR / Cas gene editing was further confirmed by confocal imaging of (47C) muscle and (47E) brain tissue sections. 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 LNPs containing different molar percentages of DOTAP. Td-Tom fluorescence, a downstream readout of DNA editing, revealed that a low DOTAP percentage facilitated liver editing, whereas a high DOTAP percentage facilitated lung editing (1.5 mg / kg sgTOM, IV). (47G) Successful CRISPR / Cas gene editing was further confirmed by confocal imaging. (47H) T7EI cleavage assays were performed on 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. Indels (%) were calculated and reported. (47I) 5A2-DOT-50 LNPs (5A2-DOT-50-Pool) containing sgRNAs against six targets (sgTOM, sgP53, sgPTEN, sgEml4, sgALK, and sgRB1) were administered IV to td-Tom mice 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 a T7EI cleavage assay on lung tissue. [Figure 48]Figures 48A-H: 5A2-DOT-X LNPs simplify the creation of complex mouse models. (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 RNP (three injections, 2.5 mg / kg total sgRNA, IV, n=4). After 12, 15, and 20 weeks, mice were sacrificed, and livers were collected for tumor development analysis. (48B) T7EI digestion results from genomic DNA extracted from the liver confirmed that gene editing had occurred at all three loci. (48C) Representative photograph of a tumor-bearing mouse liver resected 20 weeks after injection. (48D) H&E and Ki67 staining further confirmed progressive tumor formation. High expression of the tumor proliferation biomarker Ki67 was detected in tumor lesions. Scale bar = 100 μm. (48E) To generate an in situ lung-specific cancer model, adult C57BL / 6 mice were injected once (2 mg / kg) or twice (1.5 mg / kg weekly for 2 weeks) with 5A2-DOT-50 LNP-encapsulated Cas9 / sgEml4 / sgAlk RNP (IV, n = 5). After 10, 16, and 24 weeks, mice were sacrificed, and lungs were collected for tumor development analysis. (48F) T7EI digestion results from genomic DNA extracted from the lungs confirmed that gene editing had occurred at the Eml4 and Alk loci. PCR amplicons for the Eml4-Alk rearrangement were also detected in all 5A2-DOT-50 LNP-treated lungs. (48G) The Eml4-Alk rearrangement was further confirmed by subcloning and DNA sequencing. (Predicted = 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). (48H) H&E and Ki67 staining further confirmed aggressive tumor formation. High tumor proliferation biomarker Ki67 expression was detected in lung tumor lesions.Scale bar = 100 μm. [Figure 49] Figures 49A and B show the gene editing efficiency of unmodified sgRNA synthesized by in vitro transcription (IVT) compared to chemically modified and synthesized sgRNA (2'-methyl 3'-phosphorothioate modifications 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 the gene editing efficiency of Cas9 / IVT sgRNA and Cas9 / chemically modified sgRNA encapsulated in nanoparticles. Cleavage bands of 536 bp and 184 bp were clearly observed in the modified sgRNA treatment group. [Figure 50] Figure 1 shows gene editing of the P53, PTEN, and RB1 genes in mouse livers after treatment with 5A2-DOT-5 LNP-encapsulated Cas9 / sgP53 / sgPTEN / sgRB1 RNP. T7EI assay detected gene editing of liver genomic DNA at the PTEN, P53, and RB1 genomic loci after weekly treatment for two weeks. The PBS-treated group served as a control. Cleavage bands were detected in the 261-bp and 215-bp P53-targeted PCR amplicons. Cleavage bands were detected in the 345-bp and 293-bp PTEN-targeted PCR amplicons. Cleavage bands were detected in the 395-bp and 207-bp RB1-targeted PCR amplicons. [Figure 51]Figure 1 shows a T7EI assay detecting gene editing of the P53, PTEN, and RB1 genes in mouse livers after treatment with 5A2-DOT-5 LNP-encapsulated Cas9 / sgP53 / sgPTEN / sgRB1 RNPs. PBS-treated and 5A2-DOT-5 only (no Cas9 / sgRNA)-treated groups served as controls. T7EI results for genomic DNA extracted from tumors in 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 photographs of mouse liver and resected tumors excised from mice in the group treated with 5A2-DOT-5 LNP-encapsulated Cas9 / sgP53 / sgPTEN / sgRB1 RNPs for 15 weeks are shown. [Figure 53] Figures 53A-C: H&E and Ki67 staining images of mouse livers (53A) after 15 and 20 weeks of treatment with 5A2-DOT-5 LNPs alone (no Cas9 / sgRNA) (control) and tumors (53B) excised from mice treated with 5A2-DOT-5 LNP-encapsulated Cas9 / sgP53 / sgPTEN / sgRB1 RNPs for 20 weeks. No morphological changes were detected after treatment with 5A2-DOT-5 LNPs alone, suggesting that the nanovector alone does not induce tumors. Scale bar: 100 μm. (53C) Magnified images of mouse liver tumor development after 20 weeks of treatment with 5A2-DOT-5 LNP-encapsulated Cas9 / sgP53 / sgPTEN / sgRB1 RNPs. 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) To confirm the Eml4-Alk inversion, PCR analysis was performed on genomic DNA extracted from mouse lungs. (54D) PCR amplicons were subcloned. Sequences of six independent clones are listed, and a representative chromatogram is shown in the upper panel. The chromatogram was exactly as expected for the Eml4-Alk rearrangement (expected = 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 staining images of mouse livers treated with 5A2-DOT-50 LNPs alone (no Cas9 / sgRNA) for 10 and 16 weeks are shown (LNP dose equivalent to 1 mg / kg total sgRNA). No morphological changes were detected in animals injected with 5A2-DOT-50 LNPs alone. Scale bar: 100 μm. [Figure 56] Magnified images of mouse lung tumor development after 24 weeks of treatment with 5A2-DOT-50 LNP-encapsulated Cas9 / sgEml4 / sgAlk RNPs are shown. Scale bar: 500 μm. Several tumor lesions (highlighted) were observed in both H&E and Ki67 stained images. [Figure 57] We demonstrate that 5A2-DOT-10 LNPs can efficiently deliver ovalbumin (OVA) protein into 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 by confocal microscopy. DETAILED DESCRIPTION OF THE INVENTION

[0067] Description of Exemplary Embodiments Described herein are lipid nanoparticles (LNPs) composed of 1) a permanent cationic lipid, 2) a cationic ionizable lipid, and 3) a phospholipid, which may optionally contain cholesterol and / or lipid PEG. The inclusion of a permanent cationic lipid helps target the LNP to specific organs, such as the lung, lymph nodes, or spleen. The data presented herein demonstrate that this effect is universal and the components are modular, with each category showing that 5A2-SC8 can be replaced with any cationic ionizable lipid, DOTAP can be replaced with any cationic lipid, and DOPE can be replaced with any phospholipid. In some embodiments, cholesterol and lipid PEG are also included, although formulations without cholesterol or lipid PEG are 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)-; "carboxy" means -C(=O)OH (also written as -COOH or -COH); "halo" means, independently, -F, -Cl, -Br, or -I; "amino" means -NH; "hydroxyamino" means -NHOH; "nitro" means -NO; imino means -NH; "cyano" means -CN; "isocyanato" means -N=C=O; "azido" means -N; in the monovalent context, "phosphate" means -OP(O)(OH) or its deprotonated form; in the 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; "sulfonamido" means -S(O)2NH2; and "sulfinyl" means -S(O)-.

[0069] In the context of chemical formulas, the symbol "-" denotes a single bond, "=" denotes a double bond, and "≡" denotes a triple bond. The symbol "----" represents any bond, which, if present, is either a single or double bond. Symbols: TIFF0007815196000025.tif5128 indicates a single or double bond. Thus, for example, the formula: TIFF0007815196000026.tif10128 is TIFF0007815196000027.tif12128. It is understood that such ring atoms do not form part of more than one double bond. Furthermore, it should be noted 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: TIFF0007815196000028.tif5128 when drawn perpendicularly across the bond TIFF0007815196000029.tif8140 indicates the point of attachment of the group. Note that points of attachment are typically only identified in this manner for larger groups to aid the reader in clearly identifying the point of attachment. Symbol: TIFF0007815196000030.tif5128 represents a single bond in which the group attached to the thick end of the wedge is "off the page." Symbol: TIFF0007815196000031.tif5128 represents a single bond in which the group attached to the thick end of the wedge is "into the page." Symbol: TIFF0007815196000032.tif5128 refers to 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 contemplated. Any undefined valence on an atom of a 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 group "R" may, for example, be of the formula: In TIFF0007815196000033.tif13128, if depicted as a "floating group" on a ring system, R may replace any hydrogen atom attached to any ring atom, including drawn, implied, or explicitly defined hydrogens, so long as a stable structure is formed. When depicted as a "floating group" on a fused ring system, as in TIFF0007815196000034.tif16128, R may replace any hydrogen bonded to any ring atom of any of the fused rings, unless otherwise specified. Substitutable hydrogens include drawn hydrogens (e.g., hydrogens bonded to nitrogen in the formula above), implied hydrogens (e.g., hydrogens in the formula above that are not shown but are understood to be present), explicitly defined hydrogens, and any hydrogens whose presence depends on the identity of the ring atom (e.g., hydrogens bonded to group X when X is equal to -CH-), so long as a stable structure is formed. In the depicted example, R may be present on either the 5-membered or 6-membered ring of the fused ring system. In the formula above, 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 substitutable hydrogen atoms in the ring or ring system.

[0071] In the case of chemical groups and compound classes, the number of carbon atoms in the 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, the minimum number being as small as possible for the group / class in question, e.g., the group "alkenyl (C≦8) " or class "Alkene (C≦8) " is understood to be the minimum number of carbon atoms in "alkoxy" which refers to an alkoxy group having 1 to 10 carbon atoms. (C≦10) "Cn-n'" defines both the minimum (n) and maximum (n') number of carbon atoms in the group. (C2~10) " denotes an alkyl group having 2 to 10 carbon atoms. These carbon number indicators may precede or follow the chemical group or class that 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 "Olefins C5The terms " and " are all synonymous.

[0072] The term "saturated," when used to modify a compound or chemical group, means that the compound or chemical group has no carbon-carbon double bonds and no carbon-carbon triple bonds, except as noted 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 substituted forms of saturated groups, one or more carbon-oxygen or carbon-nitrogen double bonds may be present. If such bonds are present, carbon-carbon double bonds that may occur as part of keto-enol 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 is no longer soluble in the solution.

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

[0074] The term "aromatic," when used to modify an atom of a compound or chemical group, means a compound or chemical group that contains a planar, unsaturated ring of atoms stabilized by the interaction of bonds that form the ring.

[0075] The term "alkyl," when used without the "substituted" modifier, refers to a monovalent saturated aliphatic group with a carbon atom as the point of attachment, a straight or branched acyclic structure, and no atoms other than carbon and hydrogen. Examples include the groups -CH3(Me), -CH2CH3(Et), -CH2CH2CH3(n-Pr or propyl), -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 -CHC(CH)(neo-pentyl) are non-limiting examples of alkyl groups. The term "alkanediyl," when used without the "substituted" modifier, refers to a divalent saturated aliphatic group having one or two saturated carbon atoms as points of attachment, a straight or branched acyclic structure, no carbon-carbon double or triple bonds, and no atoms other than carbon and hydrogen. The groups -CH- (methylene), -CHCH-, -CHC(CH)CH-, and -CHCHCH- are non-limiting examples of alkanediyl groups. "Alkane" refers to the class of compounds having the formula HR, where R is alkyl, as that term is defined above. When any of these terms are used in conjunction with the "substituted" modifier, one or more hydrogen atoms are independently replaced with -OH, -F, -Cl, -Br, -I, -NH, -NO, -COH, -COCH, -CN, -SH, -OCH, -OCHCH, -C(O)CH, -NHCH, -NHCHCH, -N(CH), -C(O)NH, -C(O)NHCH, -C(O)N(CH), -OC(O)CH, -NHC(O)CH, -S(O)OH, or -S(O)NH. The following groups are non-limiting examples of substituted alkyl groups: -CHOH, -CHCl, -CF, -CHCN, -CHC(O)OH, -CHC(O)OCH, -CHC(O)NH, -CHC(O)CH, -CHOCH, -CHOC(O)CH, -CHNH, -CHN(CH), and -CHCHCl. ​​The term "haloalkyl" is a subgroup of substituted alkyl limited to the replacement of hydrogen atoms by halo (i.e., -F, -Cl, -Br, or -I) so that no other atoms other than carbon, hydrogen, and halogen are present. The group -CHCl is a non-limiting example of a haloalkyl. The term "fluoroalkyl" is a subgroup of substituted alkyl limited to the replacement of hydrogen atoms by fluoro so that no other atoms other than carbon, hydrogen, and fluorine are present. The groups -CHF, -CF, and -CHCF are non-limiting examples of fluoroalkyl groups.

[0076] The term "cycloalkyl," when used without the "substituted" modifier, refers to a monovalent saturated aliphatic group having a carbon atom as the point of attachment, the carbon atom forming part of one or more non-aromatic ring structures, no carbon-carbon double or triple bonds, and no atoms other than carbon and hydrogen. Non-limiting examples include -CH(CH2)2 (cyclopropyl), cyclobutyl, cyclopentyl, or cyclohexyl (Cy). The term "cycloalkanediyl," when used without the "substituted" modifier, refers to a divalent saturated aliphatic group having two carbon atoms as the point of attachment, no carbon-carbon double or triple bonds, and no atoms other than carbon and hydrogen. Groups: TIFF0007815196000035.tif8128 is a non-limiting example of a cycloalkanediyl group. "Cycloalkane" refers to the class of compounds having the formula H-R, where R is cycloalkyl, as that term is defined above. When any of these terms are used with the "substituted" modifier, one or more hydrogen atoms are independently replaced with -OH, -F, -Cl, -Br, -I, -NH, -NO, -COH, -COCH, -CN, -SH, -OCH, -OCHCH, -C(O)CH, -NHCH, -NHCHCH, -N(CH), -C(O)NH, -C(O)NHCH, -C(O)N(CH), -OC(O)CH, -NHC(O)CH, -S(O)OH, or -S(O)NH.

[0077] The term "alkenyl," when used without the "substituted" modifier, refers to a monovalent unsaturated aliphatic group having a carbon atom as a point of attachment, a straight-chain 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-limiting examples include -CH=CH2 (vinyl), -CH=CHCH3, -CH=CHCH2CH3, -CH2CH=CH2 (allyl), -CH2CH=CHCHCH3, and -CH=CHCH=CH2. The term "alkenediyl," when used without the "substituted" modifier, refers to a divalent unsaturated aliphatic group having two carbon atoms as points of attachment, a straight-chain or branched, straight-chain 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-limiting examples of alkenediyl groups. It is noted that although alkenediyl groups are aliphatic, when joined at both ends, this does not preclude the group from forming part of an aromatic structure. The terms "alkene" and "olefin" are synonymous and refer to the class of compounds having the formula HR, where R is alkenyl as defined above. Similarly, the terms "terminal alkene" and "α-olefin" are synonymous and refer to an alkene that has only one carbon-carbon double bond, which bond is part of a vinyl group at the end of the molecule. When any of these terms are used in conjunction with the "substituted" modifier, one or more hydrogen atoms are independently replaced with -OH, -F, -Cl, -Br, -I, -NH, -NO, -COH, -COCH, -CN, -SH, -OCH, -OCHCH, -C(O)CH, -NHCH, -NHCHCH, -N(CH), -C(O)NH, -C(O)NHCH, -C(O)N(CH), -OC(O)CH, -NHC(O)CH, -S(O)OH, or -S(O)NH. The groups -CH=CHF, -CH=CHCl, and -CH=CHBr are non-limiting examples of substituted alkenyl groups.

[0078] The term "alkynyl," when used without the "substituted" modifier, refers to a monovalent unsaturated aliphatic group having a carbon atom as the point of attachment, a straight 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 preclude the presence of one or more non-aromatic carbon-carbon double bonds. The groups -C≡CH, -C≡CCH, and -CHC≡CCH are non-limiting examples of alkynyl groups. "Alkyne" refers to the class of compounds having the formula HR, where R is alkynyl. When any of these terms are used in conjunction with the "substituted" modifier, one or more hydrogen atoms are independently replaced with -OH, -F, -Cl, -Br, -I, -NH, -NO, -COH, -COCH, -CN, -SH, -OCH, -OCHCH, -C(O)CH, -NHCH, -NHCHCH, -N(CH), -C(O)NH, -C(O)NHCH, -C(O)N(CH), -OC(O)CH, -NHC(O)CH, -S(O)OH, or -S(O)NH.

[0079] The term "aryl," when used without the modifier "substituted," refers to a monovalent unsaturated aromatic group having an aromatic carbon atom as the point of attachment, the carbon atom forming part of one or more six-membered aromatic ring structures, all ring atoms being carbon, and the group consisting of atoms other than carbon and hydrogen. When two or more rings are present, the rings may be fused or unfused. As used herein, this term does not exclude the presence of one or more alkyl or aralkyl groups (carbon number limitations permitting) attached to the first aromatic ring or any additional aromatic rings present. Non-limiting examples of aryl groups include monovalent groups derived from phenyl (Ph), methylphenyl, (dimethyl)phenyl, -CHCHCH(ethylphenyl), naphthyl, and biphenyl. The term "arenediyl," when used without the "substituted" modifier, refers to a divalent aromatic group having two aromatic carbon atoms as attachment points, the carbon atoms forming part of one or more six-membered aromatic ring structures, the ring atoms being all 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 (carbon number limitations permitting) attached to the first aromatic ring or any additional aromatic rings present. When two or more rings are present, the rings may be fused or unfused. Non-fused rings may be linked via one or more of the following: a covalent bond, an alkanediyl, or an alkenediyl group (carbon number limitations permitting). Non-limiting examples of arenediyl groups include: TIFF0007815196000036.tif32140 is an example.

[0080] "Arene" refers to the class of compounds having the formula H-R, where R is aryl, as that term is defined above. Benzene and toluene are non-limiting examples of arenes. When any of these terms are used with the "substituted" modifier, one or more hydrogen atoms are independently replaced with -OH, -F, -Cl, -Br, -I, -NH, -NO, -COH, -COCH, -CN, -SH, -OCH, -OCHCH, -C(O)CH, -NHCH, -NHCHCH, -N(CH), -C(O)NH, -C(O)NHCH, -C(O)N(CH), -OC(O)CH, -NHC(O)CH, -S(O)OH, or -S(O)NH.

[0081] The term "aralkyl," when used without the "substituted" modifier, refers to the monovalent group -alkanediyl-aryl, where the terms alkanediyl and aryl are each used in a manner consistent with the above definitions. Non-limiting examples are phenylmethyl (benzyl, Bn) and 2-phenyl-ethyl. When the term aralkyl is used with the "substituted" modifier, one or more hydrogen atoms from the alkanediyl and / or aryl group are independently replaced with -OH, -F, -Cl, -Br, -I, -NH, -NO, -COH, -COCH, -CN, -SH, -OCH, -OCHCH, -C(O)CH, -NHCH, -NHCHCH, -N(CH), -C(O)NH, -C(O)NHCH, -C(O)N(CH), -OC(O)CH, -NHC(O)CH, -S(O)OH, or -S(O)NH. Non-limiting examples of substituted aralkyls are (3-chlorophenyl)-methyl and 2-chloro-2-phenyl-eth-1-yl.

[0082] The term "heteroaryl," when used without the "substituted" modifier, refers to a monovalent aromatic group having an aromatic carbon or nitrogen atom as the point of attachment, 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. The heteroaryl ring may contain one, two, three, or four ring atoms selected from nitrogen, oxygen, and sulfur. When more than one ring is present, the rings may be fused or unfused. As used herein, the term does not exclude the presence of one or more alkyl, aryl, and / or aralkyl groups (where carbon number limitations permit) attached to the aromatic ring or aromatic ring system. Non-limiting examples of heteroaryl groups include furanyl, imidazolyl, indolyl, indazolyl (Im), isoxazolyl, methylpyridinyl, oxazolyl, phenylpyridinyl, pyridinyl (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 point of attachment. The term "heteroarenediyl", 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 points of attachment, which atoms form one or more aromatic ring structures, at least one ring atom is nitrogen, oxygen, or sulfur, and the divalent group does not consist of atoms other than carbon, hydrogen, aromatic nitrogen, aromatic oxygen, and aromatic sulfur. When more than one ring is present, the rings may be fused or unfused. Non-fused rings may be linked via one or more of the following: a covalent bond, an alkanediyl, or an alkenediyl group (where the carbon number limit permits). As used herein, the term does not exclude the presence of one or more alkyl, aryl, and / or aralkyl groups (where the carbon number limit permits) attached to an aromatic ring or aromatic ring system.Non-limiting examples of heteroarenediyl groups include: TIFF0007815196000037.tif15128 is an example. "Heteroarene" refers to the class of compounds having the formula HR, where R is heteroaryl. Pyridine and quinoline are non-limiting examples of heteroarenes. When these terms are used with the "substituted" modifier, one or more hydrogen atoms are independently replaced with -OH, -F, -Cl, -Br, -I, -NH, -NO, -COH, -COCH, -CN, -SH, -OCH, -OCHCH, -C(O)CH, -NHCH, -NHCHCH, -N(CH), -C(O)NH, -C(O)NHCH, -C(O)N(CH), -OC(O)CH, -NHC(O)CH, -S(O)OH, or -S(O)NH.

[0083] The term "heterocycloalkyl," when used without the "substituted" modifier, refers to a monovalent non-aromatic group having a carbon or nitrogen atom as the point of attachment, 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 can contain one, two, three, or four ring atoms selected from nitrogen, oxygen, or sulfur. When more than one ring is present, the rings can be fused or unfused. As used herein, the term does not exclude the presence of one or more alkyl groups (carbon number limitations permitting) attached 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 aziridinyl, azetidinyl, pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, tetrahydrofuranyl, tetrahydrothiofuranyl, tetrahydropyranyl, pyranyl, oxiranyl, and oxetanyl. The term "N-heterocycloalkyl" refers to a heterocycloalkyl group having a nitrogen atom as the attachment point. 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 attachment points, which form part of one or more ring structures, at least one ring atom being nitrogen, oxygen, or sulfur, and the divalent group consisting of atoms other than carbon, hydrogen, nitrogen, oxygen, and sulfur. When more than one ring is present, the rings may be fused or unfused. Non-fused rings may be linked through one or more of the following: a covalent bond, an alkanediyl, or an alkenediyl group (carbon number limit permitting). As used herein, the term does not preclude the presence of one or more alkyl groups (carbon number limit permitting) attached to the ring or ring system.Likewise, 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 heterocycloalkanediyl groups include: TIFF0007815196000038.tif14128. When these terms are used with the "substituted" modifier, one or more hydrogen atoms are independently replaced with -OH, -F, -Cl, -Br, -I, -NH, -NO, -COH, -COCH, -CN, -SH, -OCH, -OCHCH, -C(O)CH, -NHCH, -NHCHCH, -N(CH), -C(O)NH, -C(O)NHCH, -C(O)N(CH), -OC(O)CH, -NHC(O)CH, -S(O)OH, or -S(O)NH.

[0084] The term "acyl," when used without the "substituted" modifier, 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)CH(acetyl, Ac), -C(O)CHCH, -C(O)CHCHCH, -C(O)CH(CH), -C(O)CH(CH), -C(O)CH(CH), -C(O)CHH, -C(O)CHCHCH, -C(O)CHCH, -C(O)(imidazolyl) are non-limiting examples of acyl groups. A "thioacyl" is defined in a similar manner, except that the oxygen atom of the group -C(O)R is replaced with a sulfur atom, and is -C(S)R. The term "aldehyde" corresponds to an alkane, as defined above, in which at least one of the hydrogen atoms has been replaced with a -CHO group. When any of these terms are used with the "substituted" modifier, one or more hydrogen atoms (including the hydrogen atom directly bonded to the carbon atom of the carbonyl or thiocarbonyl group, if any) are independently replaced with -OH, -F, -Cl, -Br, -I, -NH, -NO, -COH, -COCH, -CN, -SH, -OCH, -OCHCH, -C(O)CH, -NHCH, -NHCHCH, -N(CH), -C(O)NH, -C(O)NHCH, -C(O)N(CH), -OC(O)CH, -NHC(O)CH, -S(O)OH, or -S(O)NH. The groups -C(O)CH2CF3, -CO2H (carboxyl), -CO2CH3 (methylcarboxyl), -CO2CH2CH3, -C(O)NH2 (carbamoyl), and -CON(CH3)2 are non-limiting examples of substituted acyl groups.

[0085] The term "alkoxy" when used without the "substituted" modifier refers to the group -OR, where R is alkyl, as that term is defined above. Non-limiting examples include -OCH(methoxy), -OCHCH(ethoxy), -OCHCHCH, -OCH(CH)(isopropoxy), -OC(CH)(tert-butoxy), -OCH(CH), -O-cyclopentyl, and -O-cyclohexyl. The terms "cycloalkoxy," "alkenyloxy," "alkynyloxy," "aryloxy," "aralkoxy," "heteroaryloxy," "heterocycloalkoxy," and "acyloxy," when used without the "substituted" modifier, refer to the group defined as -OR, where R is cycloalkyl, alkenyl, alkynyl, aryl, aralkyl, heteroaryl, heterocycloalkyl, and acyl, respectively. The term "alkoxydiyl" refers to the divalent group -O-alkanediyl-, -O-alkanediyl-O-, or -alkanediyl-O-alkanediyl-. The terms "alkylthio" and "acylthio," when used without the "substituted" modifier, refer to the group -SR, where R is alkyl and acyl, respectively. The term "alcohol" corresponds to an alkane, as defined above, in which at least one of the hydrogen atoms has been replaced with a hydroxy group. The term "ether" corresponds to an alkane, as defined above, in which at least one of the hydrogen atoms has been replaced with an alkoxy group. When any of these terms are used in conjunction with the "substituted" modifier, one or more hydrogen atoms are independently replaced with -OH, -F, -Cl, -Br, -I, -NH, -NO, -COH, -COCH, -CN, -SH, -OCH, -OCHCH, -C(O)CH, -NHCH, -NHCHCH, -N(CH), -C(O)NH, -C(O)NHCH, -C(O)N(CH), -OC(O)CH, -NHC(O)CH, -S(O)OH, or -S(O)NH.

[0086] The term "alkylamino," when used without the "substituted" modifier, refers to the group -NHR, where R is alkyl, as that term is defined above. Non-limiting examples include -NHCH and -NHCHCH. The term "dialkylamino," when used without the "substituted" modifier, refers to the group -NRR', where R and R' can be the same or different alkyl groups, or R and R' together can represent an alkanediyl. Non-limiting examples of dialkylamino groups include -N(CH) and -N(CH)(CHCH). The terms "cycloalkylamino," "alkenylamino," "alkynylamino," "arylamino," "aralkylamino," "heteroarylamino," "heterocycloalkylamino," "alkoxyamino," and "alkylsulfonylamino," when used without the "substituted" modifier, refer to the group defined as -NHR, where R is cycloalkyl, alkenyl, alkynyl, aryl, aralkyl, heteroaryl, heterocycloalkyl, alkoxy, and alkylsulfonyl, respectively. A non-limiting example of an arylamino group is -NHC6H5. The term "alkylaminodiyl" refers to the divalent group -NH-alkanediyl-, -NH-alkanediyl-NH-, or -alkanediyl-NH-alkanediyl-. The term "amido" (acylamino), when used without the "substituted" modifier, refers to the group -NHR, where R is acyl, as that term is defined above. A non-limiting example of an amido group is -NHC(O)CH3. The term "alkylimino," when used without the "substituted" modifier, refers to the divalent group =NR, where R is alkyl, as that term is defined above.When any of these terms are used in conjunction with the "substituted" modifier, one or more hydrogen atoms bonded to a carbon atom are independently replaced with -OH, -F, -Cl, -Br, -I, -NH, -NO, -COH, -COCH, -CN, -SH, -OCH, -OCHCH, -C(O)CH, -NHCH, -NHCHCH, -N(CH), -C(O)NH, -C(O)NHCH, -C(O)N(CH), -OC(O)CH, -NHC(O)CH, -S(O)OH, or -S(O)NH. The groups NHC(O)OCH and -NHC(O)NHCH are non-limiting examples of substituted amide groups.

[0087] The use of the words "a" or "an," when used in conjunction with the word "comprising" in the claims and / or this specification, can mean "one," but is also consistent with the meanings of "one or more," "at least one," and "one or more."

[0088] Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value, or the variation that exists among test subjects.

[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 a different level of polymerization and therefore a different molar mass. Average molecular weight can be used to describe the molecular weight of multiple polymer molecules. Average molecular weight is typically synonymous with average molar mass. In particular, there are three main types of average molecular weight: number-average molar mass, weight-average (mass) molar mass, and Z-average molar mass. In the context of this application, unless otherwise specified, 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, average molecular weight can be used to describe the PEG component present in the lipid.

[0090] The terms "comprise," "have," and "include" are open-ended linking verbs. Any form or tense of one or more of these verbs, such as "comprises," "comprising," "has," "having," "includes," and "including," is also open-ended. For example, any method that "comprises," "has," or "includes" one or more steps is not limited to having only those one or more steps, but also covers other unlisted steps.

[0091] The term "effective," as that term is used in the specification and / or claims, means sufficient to achieve a desired, expected, or intended result. An "effective amount," "therapeutically effective amount," or "pharmaceutically effective amount," when used in the context of treating a patient or subject with a compound, means the amount of a compound that, when administered to a subject or patient for treating a disease, is sufficient to effect such treatment for the disease.

[0092] As used herein, "IC 50 The term "inhibitory dose" refers to an inhibitory dose that is 50% of the maximal 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 a process, i.e., an enzyme, cell, cell receptor, or microorganism) by half.

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

[0094] As used herein, the term "patient" or "subject" refers to a living mammalian organism, such as a human, monkey, cow, sheep, goat, dog, cat, mouse, rat, guinea pig, or transgenic species thereof. In certain embodiments, the patient or subject is a primate. Non-limiting examples of human subjects are adults, juveniles, infants, and fetuses.

[0095] As generally used herein, "pharmaceutically acceptable" refers to compounds, materials, compositions, and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues, organs, and / or body fluids of human beings and animals without undue toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0096] "Pharmaceutically acceptable salts" refers to salts of the compounds of the present disclosure that are pharmaceutically acceptable as defined above and possess the desired pharmacological activity. Such salts include salts with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, etc.; or salts with 1,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, 2-naphthalenesulfonic acid, 3-phenylpropionic acid, 4,4'-methylenebis(3-hydroxy-2-ene-1-carboxylic acid), 4-methylbicyclo[2.2.2]oct-2-ene-1-carboxylic acid, acetic acid, aliphatic mono- and dicarboxylic acids, aliphatic sulfuric acids, aromatic sulfuric acids, benzenesulfonic acid, benzoic acid, camphorsulfonic acid, carbonic acid, cinnamic acid, citric acid, cyclopentanepropionic acid, ethanesulfonic acid, fumaric acid, glucoheptonic acid, etc. Pharmaceutically acceptable salts include acid addition salts formed with organic acids such as gluconic acid, glutamic acid, glycolic acid, heptanoic acid, hexanoic acid, hydroxynaphthoic acid, lactic acid, lauryl sulfuric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, muconic acid, o-(4-hydroxybenzoyl)benzoic acid, oxalic acid, p-chlorobenzenesulfonic acid, phenyl-substituted alkanoic acids, propionic acid, p-toluenesulfonic acid, pyruvic acid, salicylic acid, stearic acid, succinic acid, tartaric acid, tert-butylacetic acid, trimethylacetic acid, and the like. Pharmaceutically acceptable salts also include base addition salts that may be formed when acidic protons present are capable of reacting with inorganic or organic bases. Acceptable inorganic bases include sodium hydroxide, sodium carbonate, potassium hydroxide, aluminum hydroxide, and calcium hydroxide. Acceptable organic bases include ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine, and the like. It should be understood that the particular anion or cation forming a part of any salt of the present disclosure is not critical, so long as the salt, as a whole, is pharmacologically acceptable. Further examples of pharmaceutically acceptable salts and their methods of preparation and use are provided 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 vehicle, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, that is involved in carrying or transporting a chemical substance.

[0098] "Prevention" or "preventing" includes (1) inhibiting the onset of disease in a subject or patient who may be at risk and / or predisposed to the disease, but who has not yet experienced or displayed any or all of the symptoms or symptomatology of the disease, and / or (2) delaying the onset of symptoms or symptomatology of the disease in a subject or patient who may be at risk and / or predisposed to the disease, but who has not yet experienced or displayed any or all of the symptoms or symptomatology of the disease.

[0099] A "repeating unit" is the simplest structural entity of the backbone and / or polymer of a particular material, e.g., either organic, inorganic, or organometallic. In the case of a polymer chain, the repeating units are linked sequentially along the chain, like beads on a necklace. For example, polyethylene -[-CH2CH2-] n In -, the repeat unit is -CH2CH2-. The subscript "n" indicates the degree of polymerization, i.e., the number of repeat units linked together. If the value of "n" is left undefined or is missing, it specifies the repeat of the formula within the brackets, not just the polymeric nature of the material. The concept of a repeat unit applies equally where the linkages between repeat units extend three-dimensionally, such as in metal-organic frameworks, modified polymers, thermosetting polymers, etc. Within the context of dendrimers, repeat units may also be described as branching units, internal layers, or generations. Similarly, end groups may also be described as surface groups.

[0100] "Stereoisomers" or "optical isomers" are isomers of a given compound that have the same atoms bonded to the same other atoms but differ in the three-dimensional arrangement of those atoms. "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. Chiral molecules contain chiral centers, also called stereogenic centers or stereogenic centers, which are any points, but not necessarily atoms, in a molecule that bear groups such that the interchange of any two groups leads to a stereoisomer. In organic compounds, chiral centers are typically carbon, phosphorus, or sulfur atoms, although other atoms can be stereocenters in organic and inorganic compounds. Molecules can have multiple stereocenters, resulting in many stereoisomers. In compounds whose stereoisomerism is due to tetrahedral stereogenic centers (e.g., tetrahedral carbon), the total number of hypothetical possible stereoisomers is 2. n where n is the number of tetrahedral stereocenters. Molecules with symmetry often have fewer than the maximum possible number of stereoisomers. A 50:50 mixture of enantiomers is called a racemic mixture. Alternatively, a mixture of enantiomers may be enantiomerically enriched, such that one enantiomer is present in an amount greater than 50%. Typically, enantiomers and / or diastereomers can be resolved or separated using techniques known in the art. For any stereocenter or axis of chirality where stereochemistry is not defined, it is contemplated that that stereocenter or axis of chirality may exist as its R-, S-, or mixtures of R- and S-forms, including racemic and non-racemic mixtures. 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 "treating" includes (1) inhibiting a disease (e.g., halting further development of the pathology and / or symptomatology) in a subject or patient experiencing or exhibiting the pathology or symptomatology of the disease, (2) ameliorating a disease (e.g., reversing the pathology and / or symptomatology) in a subject or patient experiencing or exhibiting the pathology or symptomatology of the disease, and / or (3) making any measurable reduction in a disease in a subject or patient experiencing or exhibiting the pathology or symptomatology of the disease.

[0102] The foregoing definitions supersede any conflicting definitions in any reference incorporated herein by reference. However, the fact that a particular term is defined should not be considered to indicate that any term not defined is unclear. Rather, all terms used are considered to describe the present disclosure in terms such that one skilled in the art can understand the scope and practice the present disclosure.

[0103] B. Cationic Ionizable Lipids In some aspects of the present disclosure, a composition is provided that contains a compound containing 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 a pH greater than 8, 9, 10, 11, or 12.The ionizable cationic group may contain one or more protonatable amines that can form cationic groups at physiological pH.The cationic ionizable lipid compound also contains one or more lipid components, for example, C6-C 24 They may also contain two or more fatty acids with alkyl or alkenyl carbon groups. These lipid groups may be attached via ester bonds or may be further attached 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 sequential or generational addition of branched layers to or from a core, characterized by a core, at least one internal branched layer, and surface branched layers. (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, internal layers (or "generations") of repeating units regularly attached to the internal core, and an external surface of terminal groups attached to the outermost generation. A "dendron" is a dendrimer species having branches emanating from a focal point that are or can be attached to the core directly or through a linking moiety to form larger dendrimers. In some embodiments, the dendrimer structure has repeating groups radiating from a central core, each serving as a repeating unit for each branch. 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 emanating from a single focal point).

[0105] Although dendrimers are polymers, they are preferred over conventional polymers because they have a controllable architecture, a single molecular weight, a large number of controllable surface functional groups, and traditionally assume a spherical structure after reaching a certain generation. Dendrimers can be prepared by sequential reactions of individual repeat units to produce monodisperse, dendritic, and / or generationally structured polymer structures. Individual dendrimers consist of a central core molecule with dendritic wedges attached to one or more functional moieties on the central core. Depending on the assembly monomers used during preparation, the dendrimer surface layer can have a variety of functional groups disposed thereon, including anionic, cationic, hydrophilic, or lipophilic groups.

[0106] Their physical properties can be adjusted by modifying the core, the functional groups and / or chemical nature of the repeating units, and the surface or terminal groups. Some properties that can be varied include, but are not limited to, solubility, toxicity, immunogenicity, and bioadhesion ability. Dendrimers are often described by the number of repeating units in their generations or branches. A dendrimer consisting only of the core molecule is said to be generation 0, while each successive repeating unit along all branches is generation 1, generation 2, etc., up to the terminal or surface groups. In some embodiments, half-generations are possible resulting from only the first condensation reaction with an amine, but not the second condensation reaction with a thiol.

[0107] The preparation of dendrimers requires a level of synthetic control, achieved through a series of stepwise reactions involving the construction of the dendrimer by each successive group. Dendrimer synthesis can be convergent or divergent. During diverse dendrimer synthesis, the molecule is assembled from the core to the periphery by a stepwise process of attaching one generation to the previous generation and subsequently altering the functional groups for the next step of the reaction. Functional group conversion is necessary to prevent uncontrolled polymerization. Such polymerization results in highly branched molecules that are not monodisperse, otherwise known as hyperbranched polymers. As dendrimer repeat units continue to react, steric effects result in spherical or globular molecules until steric overcrowding prevents complete reaction at a particular generation, destroying the monodispersity of the molecule. Thus, in some embodiments, dendrimers of generations G1 to G10 are specifically contemplated. In some embodiments, dendrimers contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 repeat units, or any range derivable therein. In some embodiments, the dendrimers used herein are G0, G1, G2, or G3, however, the number of possible generations (such as 11, 12, 13, 14, 15, 20, or 25) can be increased by reducing the spacing units in the branched polymer.

[0108] Furthermore, dendrimers possess two major chemical environments: an environment created by specific surface groups at the end of their formation, and an interior of the dendritic structure that may be shielded from the bulk medium and surface groups by higher-order structures. Because of these different chemical environments, dendrimers have found many different potential applications, including therapeutic applications.

[0109] In some aspects, dendrimers that can be used in the compositions of the present invention are constructed using the differential reactivity of acrylate and methacrylate groups with amines and thiols. 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 can contain groups that are degradable under physiological conditions. In some embodiments, these repeating units can contain one or more germinal diether, ester, amide, or disulfide groups. In some embodiments, the core molecule is a monoamine, allowing for dendritic polymerization in only one direction. In other embodiments, the core molecule is a polyamine with multiple different dendritic branches, each of which can contain one or more repeating units. Dendrimers can 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 (-NH) or a carboxylic acid (-COH). In still other embodiments, 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 lipid of the present disclosure comprises one or more asymmetrically substituted carbon or nitrogen atoms, and can be isolated in optically active form or racemic form.Therefore, unless specific stereochemistry or isomeric form is specifically indicated, all chiral, diastereomeric, racemic, epimeric and all geometric isomeric forms of chemical formula are intended.The cationic ionizable lipid can occur as racemic compounds and racemic mixtures, single enantiomers, diastereomeric mixtures and individual diastereomers.In some embodiments, a single diastereomer is obtained.The chiral center of the cationic ionizable lipid of the present disclosure can have S or R configuration.Furthermore, it is contemplated that one or more of the cationic ionizable lipids can exist as structural isomers.In some embodiments, the compounds have the same formula but have different bonding properties with the core nitrogen atom. Without wishing to be bound by any theory, it is believed that such cationic ionizable lipids exist because starting monomer first reacts with primary amine, and then statistically reacts with any secondary amine present.Therefore, structural isomers can present a mixture of fully reacted primary amine and then reacted secondary amine.

[0111] The chemical formula used to represent the cationic ionizable lipid of the present disclosure will typically only show one of several different tautomers.For example, it is known that many types of ketone groups exist in equilibrium with corresponding enol groups.Similarly, many types of imine groups exist in equilibrium with enamine groups.No matter which tautomer is depicted for a given formula, all tautomers of a given chemical formula are intended, no matter which one is the most common.

[0112] The cationic ionizable lipids of the present disclosure, whether used in the indications described herein or otherwise, may also be advantageous in that they may be more effective, less toxic, longer acting, more potent, produce fewer side effects, be more readily absorbed, and / or have a better pharmacokinetic profile (e.g., higher oral bioavailability and / or lower clearance) than compounds known in the prior art, and / or have other useful pharmacological, physical, or chemical properties compared to compounds known in the prior art.

[0113] Furthermore, the atoms that make up the cationic ionizable lipids of the present disclosure are intended to include all isotopic forms of such atoms.Isotopes as used herein include atoms with the same atomic number but different mass numbers.By way of general example, and without limitation, hydrogen isotopes include tritium and deuterium, and carbon isotopes include tetrahydrogen and tetrahydrofuran. 13 C and 14 Contains C.

[0114] It should be recognized that the specific anion or cation that forms part of any salt form of cationic ionizable lipid provided herein is not important, as long as the salt as a whole is pharmacologically acceptable.Further examples of pharmaceutically acceptable salts and their preparation and use are provided in Handbook of Pharmaceutical Salts: Properties, and Use (2002), which is incorporated herein by reference.

[0115] In some embodiments, the cationic ionizable lipid is present in an amount 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 derivable 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 derivable therein.

[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 selective delivery of the composition to a particular organ, which may be a lipid, small molecule therapeutic, sugar, vitamin, or protein.

[0117] In some embodiments, a 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 derivable therein. 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. A lipid is a compound having a C6 to C 24 A small molecule therapeutic agent is a compound containing less than 100 non-hydrogen atoms and weighing less than 2,000 daltons. A sugar is a compound with the molecular formula C n H 2n O nA molecule or combination of 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 without a regular tertiary structure are sometimes called peptides. Proteins may also include intact proteins with tertiary structure. Vitamins are macronutrients and include vitamin A, vitamin B1, vitamin B2, vitamin B3, vitamin B5, vitamin B6, vitamin B7, vitamin B9, and vitamin B 12 , vitamin C, vitamin D, vitamin E, and vitamin K.

[0119] 1. Permanent cationic lipids In some aspects, the present disclosure provides one or more lipids, which have one or more hydrophobic components and permanent cationic groups.Permanent cationic lipids can contain a group that has a positive charge regardless of pH.One of the permanent cationic groups that can be used in permanent cationic lipids is a quaternary ammonium group.These permanent cationic lipids have the following formula: comprising a structure as described in TIFF0007815196000039.tif13128, During the ceremony, At least one of Y1, Y2, and Y3 is X2N + with the proviso that R3R4R5, Y1, Y2, or Y3 are each independently X1C(O)R1 or X2N + R3R4R5; R1 is C1~C 24 Alkyl, C1-C 24 Substituted alkyl, C1-C 24 Alkenyl, C1-C 24 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 C1-C6 alkanediyl or C1-C6 substituted alkanediyl; R3, R4, and R5 are each independently C1 to C 24 Alkyl, C1-C 24 Substituted alkyl, C1-C 24 Alkenyl, C1-C 24 is a substituted alkenyl; A1 is the 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 has the formula: Further defined by TIFF0007815196000040.tif13128, During the ceremony, At least one of R6 to R9 is C8 to C 24 R6 to R9 are each independently a group selected from C1 to C 24 Alkyl, C1-C 24 Substituted alkyl, C1-C 24 Alkenyl, C1-C 24 is a substituted alkenyl; and A2 is a monovalent anion.

[0121] In another embodiment, the permanent cationic lipid has the formula: Further defined by TIFF0007815196000041.tif24128, During the ceremony, R1 and R2 are each independently alkyl (C8~C24) , alkenyl (C8~C24) or a substituted form 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 - is a monovalent anion.

[0122] In some embodiments, the permanent cationic lipid is present in an amount of about 4 to about 16 molar percent of the total lipid composition. The composition may contain about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 molar percent, or any range derivable therein. In other embodiments, the composition may comprise about 18 to about 66 molar percent of 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 molar percent, or any range derivable therein.

[0123] 2. Permanent anionic lipids In some aspects, the present disclosure provides one or more lipids having one or more hydrophobic moieties and a permanent anionic group. One anionic group that can be used in a permanent anionic lipid is a phosphate group. The phosphate group is deprotonated at a pH below 8, 9, 10, 11, 12, 13, or 14, and can be a compound with a negative charge. The hydrophobic moiety can be one or more C6-C 24 It may be an alkyl or alkenyl group. The compound may have one hydrophobic group, two hydrophobic groups, or three hydrophobic groups.

[0124] In some embodiments, the permanent anionic lipid has the formula: It has the structure TIFF0007815196000042.tif24128, During the ceremony, R1 and R2 are each independently alkyl (C8~C24) , alkenyl (C8~C24) or a substituted form of either group; R3 is hydrogen, alkyl (C≦6) , or substituted alkyl (C≦6) or -Y1-R4, During the ceremony, Y1 is alkanediyl (C≦6) or substituted alkanediyl (C≦6) and R4 is acyloxy(C≦8~24) or substituted acyloxy (C≦8~24) is.

[0125] 3. Hos Fa Phosphidinecholine a tidylcholine) In some aspects, the present disclosure provides one or more lipids having one or more hydrophobic moieties, a cationic amine group, and a negatively charged phosphate group. The cationic amine group may be a quaternary amine with three methyl groups attached to the nitrogen atom. The hydrophobic moiety may be one or more C6-C 24 The hydrophobic group may be an alkyl or alkenyl group. The compound may have one hydrophobic group, two hydrophobic groups, or three hydrophobic groups. In some embodiments, Fa The tidylcholine compound is further defined as TIFF0007815196000043.tif24128, During the ceremony, R1 and R2 are each independently alkyl (C8~C24) , alkenyl (C8~C24) or a substituted form of either group; R3, R3', and R3'' are each independently alkyl (C≦6) or substituted alkyl (C≦6) and X - is a monovalent anion.

[0126] D. Additional Lipids in Lipid Nanoparticles In some aspects of the present disclosure, the composition containing one or more kinds of lipids is mixed with cationic ionizable lipid to produce composition.In some embodiments, cationic ionizable lipid is mixed with 1, 2, 3, 4 or 5 different types of lipid.It is intended that cationic ionizable lipid can be mixed with multiple different lipids of one type.In some embodiments, cationic ionizable lipid composition comprises at least steroid or steroid derivative, PEG lipid and phospholipid.

[0127] In some embodiments, lipid nanoparticles are preferentially delivered to a target organ. In some embodiments, the target organ is selected from the lung, heart, brain, spleen, bone marrow, bone, skeletal muscle, stomach, small intestine, large intestine, kidney, bladder, breast, liver, testis, ovary, uterus, spleen, thymus, brainstem, cerebellum, spinal cord, eye, ear, tongue, or skin. Alternatively, the composition can be preferentially delivered to a target organ system such as the nervous system, cardiovascular system, or respiratory system, or a part of one of these organ systems. As used herein, the term "preferentially delivered" refers to a composition in which at least 25% of the administered amount is delivered to a target organ or organ system. This 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 the present disclosure, cationic ionizable lipids are mixed with one or more steroids or steroid derivatives to form compositions.In some embodiments, the steroid or steroid derivative comprises any steroid or steroid derivative.As used herein, in some embodiments, the term "steroid" refers to a class of compounds having a tetracyclic 17-carbon ring structure, which may further comprise one or more substitutions, including alkyl groups, alkoxy groups, hydroxy groups, oxo groups, acyl groups, or double bonds between two or more carbon atoms.In one aspect, the ring structure of the steroid comprises three fused cyclohexyl rings and a fused cyclopentyl ring, as shown in the following formula: TIFF0007815196000044.tif17128. In some embodiments, the steroid derivative comprises the above ring structure with one or more non-alkyl substitutions. In some embodiments, the steroid or steroid derivative has the formula: It is a sterol, further defined as in TIFF0007815196000045.tif18128.

[0129] In some embodiments of the present disclosure, the steroid or steroid derivative is cholestane or a cholestane derivative. In cholestane, the ring structure has the formula: As further defined by TIFF0007815196000046.tif35128. As described above, the cholestane derivative contains one or more non-alkyl substitutions of the ring system. In some embodiments, the cholestane or cholestane derivative is cholestene or a cholestene derivative or a sterol or a sterol derivative. In other embodiments, the cholestane or cholestane derivative is both cholestene and a sterol or a derivative thereof.

[0130] In some embodiments, the composition may further comprise a molar percentage of steroid relative 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, to about 46, or any range derivable therein. In other embodiments, the molar percentage of steroid relative 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 derivable therein.

[0131] 2. PEG or PEGylated lipids In some aspects of the present disclosure, the polymer is mixed with one or more PEGylated lipids (or PEG-lipids) to create a lipid composition. In some embodiments, the present disclosure includes using any lipid to which a PEG group is attached. In some embodiments, the PEG-lipid is a diglyceride that also contains a PEG chain attached to a glycerol group. In other embodiments, the PEG-lipid is a compound containing one or more C6-C24 long-chain alkyl or alkenyl groups or C6-C24 fatty acid groups attached to a linker group via a PEG chain. Some non-limiting examples of PEG-lipids include PEG-modified phosphatidylethanolamine and phosphatidic acid, PEG-ceramide-conjugated PEG-modified dialkylamine and PEG-modified 1,2-diacyloxypropan-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, the PEG modification has a molecular weight of about 100 to about 15,000. In some embodiments, the molecular weight is about 200 to about 500, about 400 to about 5,000, about 500 to about 3,000, or about 1,200 to about 3,000. The molecular weight of the PEG modification is about 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, or 12,500 to about 15,000. Some non-limiting examples of lipids that can be used in the present disclosure are taught by U.S. Pat. No. 5,820,873, WO 2010 / 141069, or U.S. Pat. No. 8,450,298, which are incorporated herein by reference.

[0132] In another aspect, the PEG lipid has the formula: TIFF0007815196000047.tif18128In formula, R12 and R 13 are each independently an alkyl (C≦24) , alkenyl (C≦24) or a substituted version of any of these groups; R e is hydrogen, alkyl (C≦8) or substituted alkyl (C≦8) and x is 1 to 250. In some embodiments, R e is an alkyl group such as methyl. (C≦8) R 12 and R 13 are each independently an 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 aspect, the PEG lipid has the formula: TIFF0007815196000048.tif38128 wherein 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 derivable therein. In some embodiments, n1 is about 30 to about 50. In some embodiments, n2 is 5 to 23. In some embodiments, n2 is 11 to about 17. In some embodiments, n3 is 5 to 23. In some embodiments, n3 is 11 to about 17.

[0134] In some embodiments, the composition may further comprise a molar percentage of PEG lipid relative 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 derivable 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 derivable therein.

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

[0136] In some embodiments, the composition may further comprise a molar percentage of phospholipids relative 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 derivable 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 derivable therein.

[0137] E. Therapeutic Agents 1. Nucleic acids In some aspects of the present disclosure, the lipid composition comprises one or more nucleic acids. In some embodiments, the lipid composition comprises one or more nucleic acids present at a weight ratio to lipid composition of about 5:1 to about 1:100. In some embodiments, the weight ratio of nucleic acid to 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 derivable therein. In some embodiments, the weight ratio is about 1:40. Furthermore, it should be apparent that the present disclosure is not limited to the specific nucleic acids disclosed herein. However, the present disclosure is not limited in scope to any particular source, sequence, or type of nucleic acid, as those skilled in the art can readily identify related homologs in a variety of other sources of nucleic acid, including nucleic acids from species other than humans (e.g., mouse, rat, rabbit, dog, monkey, gibbon, chimpanzee, ape, baboon, cow, pig, horse, sheep, cat, and other species). It is contemplated that the nucleic acids used in the present disclosure may include sequences based on naturally occurring sequences. Given the degeneracy of the genetic code, sequences having at least about 50%, typically at least about 60%, more typically about 70%, most typically about 80%, preferably at least about 90%, and most preferably about 95% of the nucleotides are identical to the nucleotide sequence of a native sequence. In another embodiment, the nucleic acid is a complementary sequence to a native sequence, or is 75%, 80%, 85%, 90%, 95%, or 100% complementary. Longer polynucleotides encoding 250, 500, 1000, 1212, 1500, 2000, 2500, 3000 or more are contemplated in the present invention.

[0138] The nucleic acids used herein may be derived from genomic DNA, i.e., cloned directly from the genome of a particular organism. However, in preferred embodiments, the nucleic acids will comprise complementary DNA (cDNA). cDNAs containing natural introns or introns derived from other genes are also contemplated; such engineered molecules are sometimes referred to as "minigenes." At a minimum, these and other nucleic acids of the present 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. The advantage of using cDNA, as opposed to genomic DNA or DNA polymerized from an unprocessed or partially processed RNA template, is that the cDNA contains primarily the coding sequence of the corresponding protein. There may be cases where a complete or partial genomic sequence is preferred, 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, the nucleic acid comprises one or more antisense segments that inhibit the expression of a gene or gene product. Antisense methodology takes advantage of the fact that nucleic acids tend to pair with "complementary" sequences. Complementary means that the polynucleotides are capable of base pairing 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 (A:T) in the case of DNA, or adenine paired with uracil (A:U) in the case of RNA. The inclusion of 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 antisense polynucleotides are introduced into target cells, they specifically bind to their target polynucleotides and interfere with transcription, RNA processing, transport, translation, and / or stability. Antisense RNA constructs, or DNA encoding such antisense RNAs, can be used to inhibit gene transcription or translation, or both, in host cells, either in vitro or in vivo, for example, in host animals, including human subjects.

[0142] Antisense constructs can be designed to bind to promoters and other control regions, exons, introns, or even exon-intron boundaries of genes. It is contemplated that the most effective antisense constructs will contain regions complementary to intron / exon splice junctions. Therefore, preferred embodiments are proposed to include antisense constructs with complementarity to regions within 50-200 bases of intron-exon splice junctions. It has been observed that some 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. It is easy to test whether too much exon DNA has been included by simply testing the construct in vitro to determine whether normal cellular function or expression of the associated gene bearing the complementary sequence is affected.

[0143] As stated above, "complementary" or "antisense" refers to polynucleotide sequences that are substantially complementary over their entire length and have few base mismatches. For example, a sequence 15 bases in length can be referred to as complementary if it has a complementary nucleotide at position 13 or 14. Of course, a fully complementary sequence would be one that is completely complementary over its entire length and has no base mismatches. Other sequences with lower degrees of homology are also contemplated. For example, antisense constructs (e.g., ribozymes; see below) can be designed that have limited regions of high homology and also contain non-homologous regions. These molecules will have less than 50% homology but will bind to the target sequence under appropriate conditions.

[0144] 2. Modified Nucleobases In some embodiments, the nucleic acids of the present disclosure contain one or more modified nucleosides containing a modified sugar moiety. Such compounds containing one or more sugar-modified nucleosides may have desirable properties, such as increased nuclease stability or increased binding affinity with target nucleic acids, compared to oligonucleotides containing only nucleosides containing natural sugar moieties. In some embodiments, the modified sugar moiety is a substituted sugar moiety. In some embodiments, the modified sugar moiety is a sugar surrogate. Such sugar surrogates may contain 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-bridging 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 is allyl, amino, azido, thio, O-allyl, O-C1-C1-C2-C3 ...3-C1-C3-C3-C1-C3-C3-C3-C3-C3-C3-C3-C3-C3-C3-C3-C3-C3-C3-C3-C 10 Alkyl, O--C1~C 10substituted alkyl; 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 a substituted or unsubstituted C1-C 10 and alkyl. Examples of 5'-position sugar substituents 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-bridging sugar substituents, such as TF-5'-methyl sugar moieties (see, e.g., PCT International Application WO 2008 / 101157 for additional 5',2'-bis-substituted sugar moieties and nucleosides).

[0146] Nucleosides that include a 2'-substituted sugar moiety are referred to as 2'-substituted nucleosides. In some embodiments, 2'-substituted nucleosides include halo, allyl, amino, azido, SH, CN, OCN, CF, OCF, O, S, or N(R m )-alkyl; O, S, or N(R m )-alkenyl; O, S or N(R m )-alkynyl; O-Alkylenyl-O-alkyl, alkynyl, alkaryl, aralkyl, O-alkaryl, O-aralkyl, O(CH2)2SCH3, O(CH2)2--O--N(R m )(R n ) or O--CH2--C(=O)--N(R m )(R n ), wherein each R m and R n are independently H, an amino protecting group, or a substituted or unsubstituted C1-C 10 These 2'-substituents can be further substituted with one or more substituents independently selected from hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro (NO), thiol, thioalkoxy (S-alkyl), halogen, alkyl, aryl, alkenyl, and alkynyl.

[0147] In some embodiments, the 2'-substituted nucleoside is selected from the group consisting of F, NH, N, OCF, O--CH, O(CH)NH, CH-CH=CH, O--CH-CH=CH, OCHCHOCH, O(CH)SCH, O--(CH)--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 ), wherein each R m and R n are independently H, an amino protecting group, or a substituted or unsubstituted C1-C 10 It is alkyl.

[0148] In some embodiments, 2'-substituted nucleosides comprise a sugar moiety that includes 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 that includes a 2'-substituent selected from F, O--CH3, and OCH2CH2OCH3.

[0150] Certain modified sugar moieties include a bridging sugar substituent that forms a second ring resulting in a bicyclic sugar moiety. In some such embodiments, the bicyclic sugar moiety includes a bridge between the 4' and 2' furanose ring atoms. Examples of such 4'-2' sugar substituents include -[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(CHOCH3)--O-2', and analogs thereof (see, e.g., U.S. Pat. No. 7,399,845); 4'-C(CH3)(CH3)--O-2' and analogs thereof (see, e.g., WO 2009 / 006478); 4'-CH2--N(OCH3)-2' and analogs thereof (see, e.g., WO2008 / 150729); 4'-CH2--O--N(CH3)-2' (See, e.g., US 2004 / 0171570 published September 2, 2004); 4'-CH2--O--N(R)-2', and 4'-CH2--N(R)--O-2'-, where each R is independently H, a protecting group, or a C1-C 12 alkyl; 4'-CH2--N(R)--O-2', where R is H, C1-C 12 alkyl, or protecting group (see U.S. Pat. No. 7,427,672); 4'-CH2--C(H)(CH3)-2' (see, e.g., Chattopadhyaya et al., J. Org. Chem., 2009, 74, 118-134); and 4'-CH2--C(=CH2)-2' and analogs thereof (see PCT International Application WO 2008 / 154401).

[0151] In some embodiments, such 4'-2' bridges are independently —[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)—; wherein x is 0, 1, or 2; n is 1, 2, 3, or 4; Each R a and R b are independently H, a protecting group, hydroxyl, C1-C 12 Alkyl, substituted C1-C 12 Alkyl, C2-C 12 Alkenyl, substituted C2-C 12 Alkenyl, C2-C 12 Alkynyl, substituted C2-C 12 Alkynyl, C5-C 20 Aryl, substituted C5-C 20 aryl, heterocyclic group, substituted heterocyclic group, heteroaryl, substituted heteroaryl, C5-C7 cycloaliphatic group, substituted C5-C7 cycloaliphatic 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 to C 12 Alkyl, substituted C1-C 12 Alkyl, C2-C 12 Alkenyl, substituted C2-C 12 Alkenyl, C2-C 12 Alkynyl, substituted C2-C 12 Alkynyl, 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 aminoalkyl, 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') BNAs, (B) β-D-methyleneoxy (4'-CH2--O-2') BNAs (also known as locked nucleic acids or LNAs), (C) ethyleneoxy (4'-(CH2)2--O-2') BNAs, (D) aminooxy (4'-CH2--O--N(R)-2') BNAs, (E) oxyamino (4'-CH2--N(R)--O-2') BNAs, (F) methyl(methyleneoxy) (4'-CH(CH3)--O-2') BNAs (also known as constrained ethyl or cEt), (G) methylene-thio (4'-CH2--S-2') BNAs, (H) These include, but are not limited to, methylene-amino (4'-CH2-N(R)-2') BNAs, (I) methyl carbocyclic (4'-CH2--CH(CH3)-2') BNAs, (J) propylene carbocyclic (4'-(CH2)3-2') BNAs, and (K) methoxy(ethyleneoxy) (4'-CH(CH2OMe)-O-2') BNAs (also known as constrained MOEs or cMOEs).

[0153] Further bicyclic sugar moieties are known in the art, for example: 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 Publication Nos. 2004 / 0171570, 2007 / 0287831, and 2008 / 0039618; U.S. Patent Application Nos. 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; and PCT International Application Nos. 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 isomeric configuration.For example, nucleosides comprising a 4'-2' methylene-oxy bridge can be in the α-L configuration or the β-D configuration.Previously, α-L-methyleneoxy (4'-CH2-O-2') bicyclic nucleosides have been incorporated into antisense oligonucleotides that exhibit 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-bridging sugar substituents and one or more bridging sugar substituents (e.g., 5'-substituted and 4'-2'-bridging sugars; PCT International Application WO 2007 / 134181, where LNA is substituted with, e.g., 5'-methyl or 5'-vinyl groups).

[0156] In some embodiments, the modified sugar moiety is a sugar surrogate. In some such embodiments, the oxygen atom of a natural sugar is replaced with, for example, a sulfur, carbon, or nitrogen atom. In some such embodiments, such modified sugar moieties also include bridging and / or non-bridging substituents as described above. For example, certain sugar surrogates include a 4'-sulfur atom and substitutions at the 2'-position (see, e.g., Published U.S. Patent Application No. 2005 / 0130923) and / or the 5'-position. As a further example, carbocyclic bicyclic nucleosides with a 4'-2' bridge have been described (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 surrogate contains a ring other than five atoms. For example, in some embodiments, the sugar surrogate contains a six-membered tetrahydropyran. Such tetrahydropyrans can be further modified or substituted. Nucleosides containing such modified tetrahydropyrans include, but are not limited to, hexitol nucleic acid (HNA), anitol nucleic acid (ANA), mannitol nucleic acid (MNA) (see Leumann, C J. Bioorg. & Med. Chem. (2002) 10:841-854), and fluoro-HNA (F-HNA).

[0158] In some embodiments, modified THP nucleosides of Formula VII are provided wherein 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 other than 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 wherein 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 bicyclic and tricyclic sugar surrogate ring systems are also known in the art that 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] Combinations of modifications are also provided, including, but not limited to, 2'-F-5'-methyl substituted nucleosides (see PCT International Application WO 2008 / 101157 for other disclosed 5',2'-bissubstituted nucleosides), as well as substitution of the ribosyl ring oxygen atom with 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, in which 4'-CH2--O-2' bicyclic nucleosides are further substituted at the 5' position with a 5'-methyl or 5'-vinyl group). The synthesis and preparation of carbocyclic bicyclic nucleosides have also been described, along with their oligomerization and biochemical studies (see, e.g., Srivastava et al., 2007).

[0161] In some embodiments, the present disclosure provides oligonucleotides containing modified nucleosides. These modified nucleotides may contain modified sugars, modified nucleobases, and / or modified linkages. The specific modifications are selected so that the resulting oligonucleotide possesses desired characteristics. In some embodiments, the oligonucleotide contains one or more RNA-like nucleosides. In some embodiments, the oligonucleotide contains one or more DNA-like nucleotides.

[0162] In some embodiments, nucleosides of the present disclosure comprise one or more unmodified nucleobases. In certain embodiments, nucleosides of the present disclosure comprise one or more modified nucleobases.

[0163] In some embodiments, modified nucleobases include universal bases, hydrophobic bases, promiscuous bases, size-extended bases, and fluorinated bases, as defined herein, 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6, and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil; 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-propynyl (CH3)uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-aza (azo)uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8- Selected from thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo, particularly 5-bromo, 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, promiscuous bases, size-extended bases, and fluorinated bases as defined herein. Further modified nucleobases include tricyclic pyrimidines such as phenoxazine cytidine ([5,4-b][1,4]benzoxazin-2(3H)-one), phenothiazine cytidine (1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), substituted phenoxazine cytidines (e.g., 9-(2-aminoethoxy)-H-pyrimido[5,4-13][1,4]benzoxazin-2(3H)-one), carbazole cytidine ( 2G-clamps include pyridoindole cytidine (H-pyrimido[4,5-b]indol-2-one), pyridoindole cytidine (H-pyrido[3',2':4,5]pyrrolo[2,3-d]pyrimidin-2-one). Modified nucleobases can also include those in which the purine or pyrimidine base is replaced with other heterocycles, such as 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine, and 2-pyridone. Additional nucleobases include those disclosed in U.S. Pat. No. 3,687,808, The Concise Encyclopedia of Polymer Science and Engineering, Kroschwitz, JI, Ed., John Wiley & Sons, 1990, pp. 858-859; Englisch et al., 1991; and Sanghvi, YS, 1993.

[0164] Representative United States patents that teach the preparation of some of the above and other modified nucleobases 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; 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 present disclosure provides oligonucleotides comprising linked nucleosides. In such embodiments, the nucleosides can be linked using any internucleoside linkage. Two main classes of internucleoside linking groups are defined by the presence or absence of a phosphorus atom. Representative phosphorus-containing internucleoside linkages include, but are not limited to, phosphodiester (P=O), phosphotriester, methylphosphonate, phosphoramidate, and phosphorothioate (P=S). Representative non-phosphorus-containing internucleoside linkages include, but are not limited to, methylenemethylimino (--CH2--N(CH3)--O--CH2--), thiodiester (--O--C(O)--S--), thionocarbamate (--O--C(O)(NH)--S--); siloxane (--O--Si(H)2--O--); and N,N'-dimethylhydrazine (--CH2--N(CH3)--N(CH3)--). Compared to native phosphodiester linkages, modified linkages can be used to alter, typically increase, the nuclease resistance of oligonucleotides. In some embodiments, internucleoside linkages with chiral atoms can be prepared as racemic mixtures or as separate enantiomers. Representative chiral linkages include, but are not limited to, alkylphosphonates and phosphorothioates. Methods for the preparation of phosphorus-containing and non-phosphorus-containing internucleoside linkages are well known to those skilled in the art.

[0166] The oligonucleotides described herein contain one or more asymmetric centers and thus give rise to enantiomers, diastereomers, and other stereoisomeric configurations that can be defined in terms of absolute stereochemistry as (R) or (S), α or β, such as in the case of sugar anomers, or (D) or (L), such as in the case of amino acids. Included in the antisense compounds provided herein are all such possible isomers, as well as their racemic and optically pure forms.

[0167] Neutral internucleoside linkages include, but are not limited to, phosphotriester, methylphosphonate, 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'). Additional neutral internucleoside linkages include nonionic linkages, including siloxanes (dialkylsiloxanes), carboxylate esters, carboxamides, sulfides, sulfonate esters, and amides (see, for example, "Carbohydrate Modifications in Antisense Research" by Y.S. Sanghvi and P.D. Cook, Eds., ACS Symposium Series 580; Chapters 3 and 4, 40-65). Additional neutral internucleoside linkages include nonionic linkages containing mixed N, O, S, and CH moieties.

[0168] Further modifications can also be made at other positions on the oligonucleotide, particularly the 3' position of the sugar on the 3'-terminal nucleotide and the 5' position of the 5'-terminal nucleotide. For example, one further modification of the ligand-conjugated oligonucleotides of the present disclosure involves chemically linking one or more additional non-ligand moieties or conjugates to the oligonucleotide that enhance the activity, cellular distribution, or cellular uptake of the oligonucleotide. Such moieties include lipid moieties such as cholesterol moieties (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., 1990), polyamine or polyethylene glycol chains (Manoharan et al., 1995), or adamantane acetic acid (Manoharan et al., 1995), palmityl moieties (Mishra et al., 1995), or octadecylamine or hexylamino-carbonyl-oxycholesterol moieties (Crooke et al., 1996).

[0169] Representative United States patents that teach 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; 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; 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 comprise one or more proteins.Some proteins may include enzymes such as nuclease enzymes.The compositions described herein may include one or more CRISPR-related proteins (e.g., CRISPR enzymes), including Cas proteins. Non-limiting 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, homologs thereof, or modified versions thereof. These enzymes are known. For example, the amino acid sequence of the S. pyogenes Cas9 protein can be found in the SwissProt database under accession number Q99ZW2.

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

[0172] In some embodiments, the present disclosure provides compounds containing one or more therapeutic proteins. The therapeutic proteins that can be included in the compositions include a wide range of molecules, such as cytokines, chemokines, interleukins, interferons, growth factors, clotting factors, anticoagulants, blood factors, bone morphogenetic proteins, immunoglobulins, and enzymes. Some non-limiting examples of specific therapeutic proteins include erythropoietin (EPO), granulocyte colony-stimulating factor (G-CSF), α-galactosidase A, α-L-iduronidase, thyrotropin α, N-acetylgalactosamine-4-sulfatase (rhASB), dornase alfa, tissue plasminogen activator (TPA), activase, glucocerebrosidase, interferon (IF) beta-1a, interferon beta-1b, interferon gamma, interferon α, TNF-α, IL-1 through 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 therapeutics In some aspects, the disclosure provides compositions comprising a therapeutic agent, the therapeutic agent being a small molecule, such as 7-methoxypteridine, 7-methylpteridine, abacavir, abafungin, abarelix, acebutolol, acenaphthene, acetaminophen, acetanilide, acetazolamide, acetohexamide, acitretin, acrivastine, adenine, adenosine, alatrofloxacin, albendazole, albuterol, alclofenac, aldesleukin, alemtuzumab, alfuzosin, alitretinoin, allobarbital, allopurinol, all-trans retinoic acid (ATRA), aloxiprine, alprazolam, alprenolol, altretamine, amifostine, amiloride, aminoglutethimide, aminopyrine, or amiodarone HCl. l, amitriptyline, amlodipine, amobarbital, amodiaquine, amoxapine, amphetamine, amphotericin, amphotericin B, ampicillin, amprenavir, amsacrine, amyl nitrate, amylobarbitone, anastrozole, anrinone, anthracene, anthracycline, aprobarbital, arsenic trioxide, asparaginase, aspirin, astemizole, atenolol, atorvastatin, atovaquone, atrazine, atropine, azathioprine, auranofin, azacitidine, azapropazone, azathioprine, azintamide, azithromycin, aztreonam, baclofen, barbitone, BCG Live, beclamide, beclomethasone, bendroflumethiazide, benezepril, benidipine, benorylate, benperidol, bentazepam, benzamide, benzanthracene, benzathine penicillin, benzhexol HCl, benznidazole, benzodiazepine, benzoic acid, bephenium hydroxynaphthoate, betamethasone, bevacizumab (Avastin), bexarotene, bezafibrate, bicalutamide, bifonazole, biperiden, bisacodyl, bisantrene, bleomycin, bortezomib, brinzolamide, bromazepam, bromocriptine mesylate, bromperidol, brotizolam, budesonide, bumetanide, bupropion,Busulfan, butalbital, butamben, butenafine HCl, butobarbitone, butobarbitone (butetal), butoconazole, butoconazole nitrate, butylparaben, caffeine, calcifediol, calciprotriene, calcitriol, calsterone, campbendazole, camptothecin, camptothecin analogs, candesartan, capecitabine, capsaicin, captopril, carbamazepine, carbimazole, carbofuran, carboplatin, carbromal, calimazole imazole), carmustine, cefamandole, cefazolin, cefixime, ceftazidime, cefuroxime axetil, celecoxib, cephradine, cerivastatin, cetrizine, cetuximab, chlorambucil, chloramphenicol, chlordiazepoxide, chlormethiazole, chloroquine, chlorothiazide, chlorpheniramine, chlorproguanil HCl, chlorpromazine, chlorpropamide, chlorprothixene, chlorpyrifos, chlortetracycline, chlorthalidone, chlorzoxazone, cholecalciferol Ferrol, chrysene, cilostazol, cimetidine, cinnarizine, cinoxacin, ciprofibrate, ciprofloxacin HCl, cisapride, cisplatin, citalopram, cladribine, clarithromycin, clemastine fumarate, clioquinol, clobazam, clofarabine, clofazimine, clofibrate, clomiphene citrate, clomipramine, clonazepam, clopidogrel, clotiazepam, clotrimazole, cloxacillin, clozapine, cocaine, codeine, colchicine, colistin, conjugated esophageal squamous cell carcinoma (ESC) trogen, corticosterone, cortisone, cortisone acetate, cyclizine, cyclobarbital, cyclobenzaprine, cyclobutane-spirobarbiturate, cycloethane-spirobarbiturate, cycloheptane-spirobarbiturate, cyclohexane-spirobarbiturate, cyclopentane-spirobarbiturate, cyclophosphamide, cyclopropane-spirobarbiturate, cycloserine, cyclosporine, cyproheptadine, cyproheptadine HCl, cytarabine, cytosine, dacarbazine, dactinomycin, danazol, danthrone,Dantrolene sodium, dapsone, darbepoetin alfa, dalodipine, daunorubicin, decoquinate, dehydroepiandrosterone, delavirdine, demeclocycline, denileukin, deoxycorticosterone, deoxymetasone, dexamethasone, dexamphetamine, dexchlorpheniramine, dexfenfluramine, dexrazoxane, dextropropoxyphene, diamorphine, diatrizoate, diazepam, diazoxide, dichlorophen, dichlorprop, diclofenac, dicumarol, didanosine, diflunisal, digitoxin Dimethicone, digoxin, dihydrocodeine, dihydroequilin, dihydroergotamine mesylate, diiodohydroxyquinoline, diltiazem HCl, diloxanide furoate, dimenhydrinate, dimorpholamine, dinitrumide, diosgenin, diphenoxylate HCl, diphenyl, dipyridamole, dirithromycin, disopyramide, disulfiram, diuron, docetaxel, domperidone, donepezil, doxazosin, doxazosin HCl, doxorubicin (neutral), doxorubicin HCl, doxycycline, dromostanolone propionate, docetaxel Loperidol, dyphylline, echinocandins, econazole, econazole nitrate, efavirenz, ellipticine, enalapril, enlimomab, enoximone, epinephrine, epipodophyllotoxin derivatives, epirubicin, epoetin alfa, eposartan, equilenin, equilin, ergocalciferol, ergotamine tartrate, erlotinib, erythromycin, estradiol, estramustine, estriol, estrone, ethacrynic acid, ethambutol, echinamate, ethiazolin namid, ethopropazine HCl, ethyl-4-aminobenzoate (benzocaine), ethylparaben, ethinylestradiol, etodolac, etomidate, etoposide, etretinate, exemestane, felbamate, felodipine, fenbendazole, fenbuconazole, fenbufen, fenchlorphos, fenclofenac, fenfluramine, fenofibrate, fenoldepam, fenoprofen calcium, fenoxycarb, fenpiclonil, fentanyl,Fenticonazole, fexofenadine, filgrastim, finasteride, flecamide acetate, floxuridine, fludarabine, fluconazole, flucytosine, fludioxonil, fludrocortisone, fludrocortisone acetate, flufenamic acid, flunanisone, flunarizine HCl, flunisolide, flunitrazepam, fluocortolone, fluometuron, fluorene, fluorouracil, fluoxetine HCl, fluoxymesterone, flupentixol decanoate, flupentixol decanoate, flu Lurazepam, flurbiprofen, fluticasone propionate, fluvastatin, folic acid, fosenopril, fosphenytoin sodium, frovatriptan, furosemide, fulvestrant, furazolidone, gabapentin, G-BHC (lindane), gefitinib, gemcitabine, gemfibrozil, gemtuzumab, glafenine, glibenclamide, gliclazide, glimepiride, glipizide, glutethimide, glyburide, glycerin trinitrate (nitroglycerin), goserelin acetate, grepafloxacin, griseofulvin, guaifenesin, guanabolic acid Benz, guanine, halofantrine HCl, haloperidol, hydrochlorothiazide, heptabarbital, heroin, hesperetin, hexachlorobenzene, hexetal, histrelin acetate, hydrocortisone, hydroflumethiazide, hydroxyurea, hyoscyamine, hypoxanthine, ibritumomab, ibuprofen, idarubicin, idobutal, ifosfamide, ihydroequilenin, imatinib mesylate, imipenem, indapamide, indinavir, indomethacin, indoprofen, interf Interferon alpha-2a, interferon alpha-2b, iodamide, iopanoic acid, iprodione, irbesartan, irinotecan, isavuconazole, isocarboxazid, isoconazole, isoguanine, isoniazid, isopropyl barbiturate, isoproturon, isosorbide dinitrate, isosorbide mononitrate, isradipine, itraconazole, itraconazole, itraconazole (Itra), ivermectin, ketoconazole, ketoprofen, ketorolac, khellin, labetalol, lamivudine, lamotrigine, lanatoside C, lansoprazole,L-dopa, leflunomide, lenalidomide, letrozole, leucovorin, leuprolide acetate, levamisole, levofloxacin, lidocaine, linuron, lisinopril, lomefloxacin, lomustine, loperamide, loratadine, lorazepam, lomefloxacin, lormetazepam, losartan mesylate, lovastatin, lisuride maleate, maprotiline HCl, mazindol, mebendazole, meclizine HCl, meclofenamic acid, medazepam, medigoxin, medroxyprogesterone acetate, mefenamic acid, mefloquine HCl, acetic acid Megestrol acetate, melphalan, mepenzolate bromide, meprobamate, meptazinol, mercaptopurine, mesalazine, mesna, mesoridazine, mestranol, methadone, methaqualone, methocarbamol, methoine, methotrexate, methoxsalen, methsuximide, methyclothiazide, methylphenidate, methylphenobarbitone, methyl-p-hydroxybenzoate, methylprednisolone, methyltestosterone, methyprylon, methysergide maleate, metoclopramide, metolazone, metoprolol, metronidazole , mianserin HCl, miconazole, midazolam, mifepristone, miglitol, minocycline, minoxidil, mitomycin C, mitotane, mitoxantrone, mofetil mycophenolate, molindone, montelukast, morphine, moxifloxacin HCl, nabumetone, nadolol, nalbuphine, nalidixic acid, nandrolone, naphthacene, naphthalene, naproxen, naratriptan HCl, natamycin, nelarabine, nelfinavir, nevirapine, nicardipine HCl, nicotinamide, nicotinic acid, nicoumarin, Nifedipine, nilutamide, nimodipine, nimorazole, nisoldipine, nitrazepam, nitrofurantoin, nitrofurazone, nizatidine, nofetumomab, norethisterone, norfloxacin, norgestrel, nortriptyline HCl, nystatin, estradiol, ofloxacin, olanzapine, omeprazole, omoconazole, ondansetron HCl, oprelvequin, ornidazole, oxaliplatin, oxamniquine, oxantelembonate, oxaprozin, oxatomide, Oxazepam, oxcarbazepine, oxfendazole, oxiconazole, oxprenolol, oxyphenbutazone, oxyphencyclimine HCl, paclitaxel, palifermin, pamidronate, p-aminosalicylic acid, pantoprazole, paramethadione, paroxetine HCl, pegademase, pegaspargase, pegfilgrastim, pemetrexed disodium, penicillamine, pentaerythritol trinitrate, pentazocine, pentobarbital, pentobarbitone, pentostatin, pentoxifylline, Perphenazine, perphenazine pimozide, perylene, phenacemide, phenacetin, phenanthrene, phenindione, phenobarbital, phenolbarbitone, phenolphthalein, phenoxybenzamine, phenoxybenzamine HCl, phenoxymethylpenicillin, phensuccinimide, phenylbutazone, phenytoin, pindolol, pioglitazone, pipoproman, piroxicam, pizotifen maleate, platinum compounds, plicamycin, polyenes, polymyxin B, porfimer sodium, porosa Conazole (Posa), Pramipexole, Prasterone, Pravastatin, Praziquantel, Prazosin, Prazosin HCl, Prednisolone, Prednisone, Primidone, Probarbital, Probenecid, Probucol, Procarbazine, Prochlorperazine, Progesterone, Proguanil HCl, Promethazine, Propofol, Propoxur, Propranolol, Propylparaben, Propylthiouracil, Prostaglandins, Pseudoephedrine, Pteridine-2-methyl-thiol, Pteridine-2-thiol, Pteridine-4-methyl- Thil-thiol, pteridine-4-thiol, pteridine-7-methyl-thiol, pteridine-7-thiol, pyrantelembonate, pyrazinamide, pyrene, pyridostigmine, pyrimethamine, quetiapine, quinacrine, quinapril, quinidine, quinidine sulfate, quinine, quinine sulfate, rabeprazole sodium, ranitidine HCl, rasburicase, ravuconazole, repaglinide, reposal, reserpine, retinoids, rifabutin, rifampicin, rifapentine, rimexolone, risperidone,Ritonavir, rituximab, rizatriptan benzoate, rofecoxib, ropinirole HCl, rosiglitazone, saccharin, salbutamol, salicylamide, salicylic acid, saquinavir, sargramostim, secbutabarbital, secobarbital, sertaconazole, sertindole, sertraline HCl, simvastatin, sirolimus, sorafenib, sparfloxacin, spiramycin, spironolactone, stanolone, stanozolol, stavudine, stilbestrol, streptozocin, strychnine, sulconazole, sulconitrate benzodiazepine, sulfacetamide, sulfadiazine, sulfamerazine, sulfamethazine, sulfamethoxazole, sulfanilamide, sulfathiazole, sulindac, sulfabenzamide, sulfacetamide, sulfadiazine, sulfadoxine, sulfafurazole, sulfamerazine, sulfamethoxazole, sulfapyridine, sulfasalazine, flufinpyrazone, sulpiride, sulthiame, sumatriptan succinate, sunitinib maleate, tacrine, tacrolimus, talbutal, tamoxifen citrate, tamurosin (t amulosin), targretin, taxanes, 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, thotoin, thymine, tiagabine H Cl, tibolone, ticlopidine, tinidazole, tioconazole, tirofiban, tizanidine HCl, tolazamide, tolbutamide, tolcapone, topiramate, topotecan, toremifene, tositumomab, tramadol, trastuzumab, trazodone HCl, tretinoin, triamcinolone, triamterene, triazolam, triazole, triflupromazine, trimethoprim, trimipramine maleate, triphenylene, troglitazone, tromethamine, tropicamide, trovafloxacin, tibamate, ubidecarenone (coenzyme Q10),may be undecenoic acid, uracil, uracil mustard, uric acid, valproic acid, valrubicin, valsartan, vancomycin, venlafaxine HCl, vigabatrin, vinbarbital, vinblastine, vincristine, vinorelbine, voriconazole, xanthine, zafirlukast, zidovudine, zileuton, zoledronate, zoledronic acid, zolmitriptan, zolpidem, and zopiclone.

[0174] F. Kit The present disclosure also provides kits. Any of the components disclosed herein can be combined in the form of a kit. In some embodiments, the kit includes the above or claimed composition.

[0175] A kit generally comprises at least one vial, test tube, flask, bottle, syringe or other container, in which components can be placed, and preferably dispensed appropriately.If there are two or more components in the kit, the kit also generally comprises a second, third or other additional container, in which additional components can be placed separately.However, various combinations of components can be contained 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] Kits of the present disclosure also typically include packaging to contain the various containers in a sealed container for commercial sale. Such packaging may include cardboard or injection- or blow-molded plastic packaging in which the desired containers are retained. The kit may also include instructions for utilizing the kit components. The instructions may include executable variations. [Example]

[0177] F. Working Example The following examples are included to demonstrate preferred embodiments of the present disclosure. It should be understood by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventors to work well in the practice of the present disclosure, and can therefore be considered to constitute preferred modes for its practice. However, those skilled in the art should, in light of the present disclosure, recognize that many changes can be made in the specific embodiments disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

[0178] Example 1: Preparation of DOTAP-modified lipid nanoparticles Lipid nanoparticles (LNPs) are the most effective class of carriers for in vivo nucleic acid delivery. Historically, effective LNPs have been composed of four components: cationic ionizable lipids, zwitterionic phospholipids, cholesterol, and the lipid poly(ethylene glycol) (PEG). However, these LNPs deliver nucleic acids only systemically, without organ- or tissue-targeted delivery. LNPs typically deliver RNA only to the liver. Therefore, new LNP formulations are being explored in an attempt 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 a permanent cationic lipid. Briefly, LNPs were prepared by mixing 5A2-SC8 (cationic ionizable), DOPE (zwitterionic), cholesterol, DMG-PEG, and DOTAP (permanent cationic) in the ratios shown in Table 1.

[0180] Table 1. Molar ratios and mole percentages of lipids in modified LNPs TIFF0007815196000049.tif96170

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

[0182] Example 2: Characterization of DOTAP-modified mDLNP formulations To characterize the various mDLNP formulations, the size, polydispersity index, and zeta potential were measured by dynamic light scattering in triplicate for each formulation. The size and polydispersity index are shown in Figure 5A. Regardless of DOTAP concentration, all formulations fall within a size range of approximately 90 nm to approximately 160 nm, whereas the polydispersity index, which indicates relative size uniformity, varies from approximately 0.1 to approximately 0.3. The zeta potential of each formulation is shown in Figure 5B. This indicates 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 an acidic buffer (10 mM citrate, pH 4), mRNA was encapsulated in mDLNPs without DOTAP at 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 complexation with the negatively charged mRNA. For all other formulations in this table, mixing was performed with mRNA dissolved in PBS at pH 7.4. Obviously, encapsulation efficiency was low when low concentrations of DOTAP were used, but increased to >80% when the molar percentage of DOTAP was greater than 25% (Figure 5C). Encapsulation efficiency ranged from approximately 80% to approximately 95% for all formulations with DOTAP molar percentages greater than 25%. Thus, the potential for using neutral pH PBS mixtures is a hallmark of the permanent cationic lipid strategy. This strategy allows for tissue-specific delivery and high-quantity Cas9 protein encapsulation. The addition of permanent cationic lipids allows LNP formation at neutral pH. These encapsulation results are based on the use of PBS as the buffer. When an acidic buffer is used (e.g., citrate buffer (10 mM, pH 4.0)), encapsulation efficiency is high (>90%) for all formulations from 0 to 100% DOTAP.

[0184] Finally, the pKa was determined using the 2-(p-toluidino)-6-naphthalenesulfonic acid (TNS) assay (Figure 3B) (Zhao et al., 2016). Based on the 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, the pKa of liver-targeted formulations was narrow (approximately 6-7), while that of spleen-targeted formulations lacked a significant range, whereas 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 affecting 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 mDLNPs modified with permanent cationic lipids for mRNA delivery To investigate the in vitro delivery efficacy of LNPs containing permanent cationic lipids, we loaded DOTAP-modified mDLNPs with luciferase-encoding mRNA and transfected Huh-7 liver cells and A549 adenocarcinoma human alveolar basal epithelial cells with 50 ng / well of mRNA. After 24 hours of culture, luciferase expression and cell viability were examined. As shown in Figure 6A, 5% to 50% DOTAP percentages were better for mRNA delivery and expression in Huh-7 liver cells in vitro, with 10% DOTAP appearing to exhibit the greatest luciferase delivery and expression (Figure 6A). Note that in vivo delivery characteristics may differ. In general, SORT LNPs have additional in vivo barriers, organ distribution, and cellular specificity, so these studies may not be useful for predicting in vivo activity or tissue tropism. Furthermore, cell viability was examined and 10% DOTAP was found to result in high viability in mDLNPs, which showed robust luciferase expression (Figure 6A). Similar results were obtained when the same transfection was performed using the A549 lung cancer cell line. Cells transfected with the DOTAP10 formulation exhibited nearly twice the fluorescence of the other formulations and maintained high cell viability (Figure 6A). Although DOTAP SORT LNPs were formed in PBS (pH 7.4) rather than citrate buffer (10 mM, pH 4.0), they can be formed in either buffer system. This is a unique feature, as it allows for the encapsulation and delivery of cargoes, such as proteins, that are not stable in either ethanol or acidic buffers.

[0186] To confirm 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 carcinoma cells with 50 ng / well of mRNA 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 multiple cell types, including liver, lung, and pharynx. Furthermore, this formulation was also successfully modified to use 1x PBS (pH 7.4) instead of acidic buffer (pH 4.0). These data indicate that the percentage of ethanol can be drastically reduced. This suggests that DOTAP formulations may be capable of delivering cargoes, such as proteins, that are highly 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 a 0.1 mg / kg dose of Luc mRNA 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 mDLNPs (0% DOTAP) being best for liver delivery, whereas 5-15% DOTAP was best for the spleen, and DOTAP 50 (50%) was best for lung delivery (Figure 1B). Assuming luciferase expression was only detected in the liver, spleen, and lungs after IV injection, the percent of luciferase expressed in each organ can be calculated (Figure 1C). These data clearly show that as the DOTAP mole percentage in the formulation increases, liver delivery and expression decrease, with near-zero expression observed in the liver at DOTAP percentages above 70% (Figure 1B, 1C). However, the greater the DOTAP percentage, the more luminescence observed in lung tissue, with near-100% luminescence observed in the lung at DOTAP percentages above 80% (Figure 1C). DOTAP concentrations of 5 and 30 mole percent showed a high percentage of luminescence in spleen tissue, while DOTAP 10 showed the highest relative luminescence in the spleen compared to other tissues (Figure 1C). These results demonstrate that lipid concentrations can be tailored for specific tissue delivery after injection.

[0188] To further examine the organ 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 a dose of 0.5 mg / kg Cy5-Luc mRNA (a dye-labeled mRNA that tracks RNA LNPs). Six hours after injection, major organs were collected and imaged (Figure 3A). The organ distribution of the formulations varied with the DOTAP dose, with liver accumulation gradually shifting to the lung as the DOTAP percentage increased. However, NPs remained present in the liver regardless of the DOTAP percentage (Figures 7 and 8). Considering this data together with that in Figure 1, it is clear that organ distribution is not sufficient to analyze tissue-targeted delivery efficacy (mRNA translation into protein). Furthermore, given the similar size distribution and EE between 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 was limited or whether the distribution demonstrated above was universal for mDLNPs formulated with permanent cationic lipids, we created mDLNPs containing another popular cationic lipid, didodecyldimethylammonium bromide (DDAB) (Figure 2A1). DDAB has two hydrophobic tails with 18 carbon atoms and no unsaturated bonds, creating a completely different head group from DOTAP (Figure 1C). DDAB5, DDAB15, DDAB40, and DDAB50 formulations were selected for in vivo delivery (0.1 mg / kg, 6 h, n = 2). Similar to the DOTAP formulations above, there was little difference in size distribution (Figure 2A1), but the percentage of DDAB in the NPs varied 10-fold (5% to 50%). In vivo luciferase expression showed a trend toward luminescence migration from the liver to the spleen and then to the lungs as the percentage of DDAB increased, similar to DOTAP NPs (Figure 2A2).

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

[0191] In an attempt to understand whether the effects of adding DOTAP to LNPs are specific to permanent cationic lipids, we investigated the effect of adding zwitterionic lipids to mDLNP formulations instead of permanent cationic lipids. Two representative zwitterionic lipids, phosplolipids with different chemical structures: DSPC and DOCPe, were tested. This was also tested to determine whether the addition of zwitterionic lipids (instead of permanent cationic lipids) would affect tissue-specific delivery efficacy. Figures 2C1 and 2D1 show the chemical structures of DSPC and DOCPe lipids (zwitterionic lipids). Differences exist in the location of the positively and negatively charged functional head groups and the hydrophobic domains (saturated vs. unsaturated). This suggests that the observed effects are general / universal across zwitterionic lipids. mDLNPs formulated with DSPC or DOCPe were similar (Figures 2C1 and 2D1). Interestingly, the inclusion of zwitterionic lipids in the five-component modified DLNPs did not alter the protein expression profile from the liver to the lung, as was the case with DOTAP and other permanent cationic lipids. Instead, both DSPC and DOCPe improved mRNA delivery to the spleen within the specified range (less than 80% for DSPC and less than 50% for DOCPe). There was no protein expression in the lung at any percentage (0.1 mg / kg, 6 h, n = 2) (Figures 2C2, 2D2). Thus, although the inclusion of additional zwitterionic lipids can aid spleen delivery, it cannot adjust the efficacy of liver-to-spleen-to-lung delivery, as can the inclusion of permanent cationic lipids.

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

[0193] Example 4: CRISPR / Cas9 gene editing using modified mDLNPs co-delivering Cas9 mRNA and sgRNA First, we compared three sgRNAs targeting Td-Tomato mice to determine which sgRNA would be most effective in subsequent experiments. These sgRNAs were sgTom1, sgTom2, and sgLoxP. As shown in Figure 10A, sgTom1 and sgLoxP were delivered and expressed with similar results. They were more successful than sgTom2 in inducing TdTomato (Figure 10A). Given 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 with DDAB and EPC showed similar delivery trends, we next used DOTAP-modified mDLNPs to co-deliver Cas9 mRNA / sgRNA with the goal of achieving tissue-specific gene editing. To investigate co-delivery in vivo, we used genetically engineered mice containing a homozygous Rosa26 promoter Lox-Stop-Lox tdTomato (tdTO) cassette present in all cells (Figure 4A). Co-delivery of Cas9 mRNA with DOTAP-modified mDLNPs harboring sgRNAs against LoxP or Tomato deleted the Stop cassette and induced tdTO expression (Figure 4B). Mice were IV-injected with mDLNPs 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). Fluorescence in major organs was then detected 10 days after treatment (Figure 4B). Liver- and lung-specific CRISPR / Cas gene editing was achieved. Spleen-specific editing was also achieved. However, spleen editing could not be quantified using this TdTomato reporter mouse due to very strong background red autofluorescence.

[0195] To further investigate tissue-specific editing, PTEN was selected as an endogenous target. To achieve tissue-specific gene editing, C57BL / 6 mice were intravenously injected 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 10 days after treatment. PTEN-targeting sgRNA was used. T7E1 assays further confirmed the tissue-specific characteristics of in vivo PTEN editing (Figure 4C).

[0196] Example 5: CRISPR / Cas9 gene editing using modified mDLNPs delivering Cas9 protein / sgRNA ribonucleoproteins (RNPs) Building on the discovery of including a permanent cationic lipid (e.g., DOTAP) in a conventional LNP formulation containing a cationic ionizable lipid, a zwitterionic lipid, cholesterol, and a PEGylated lipid, we investigated whether this formulation methodology could also deliver other cargoes that are sensitive to ethanol and / or low pH acidic aqueous buffers. A key element of the DOTAP strategy is the ability to prepare the formulation using neutral PBS. Therefore, we investigated whether this methodology could also encapsulate and deliver large proteins, such as Cas9, for gene editing applications. Thus, DOTNP lipid nanoparticles consist of five components: a cationic ionizable lipid (e.g., 5A2-SC8), a zwitterionic lipid (e.g., DOPE), cholesterol, DMG-PEG, and a modular 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 DOTNPs containing different molar percentages of DOTAP.

[0197] We first investigated whether the characterized Cas9 / sgRNA complexes are sensitive to acidic pH. We measured the size (diameter) (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). Cas9 / sgLUC complexes prepared in citrate buffer are large (>100 nm) and have a positive zeta potential. These two characteristics (larger than typical effective LNPs) and a positive charge (incompatible with complexing with positively charged lipids) preclude effective encapsulation by lipid nanoparticles. However, Cas9 / sgLUC complexes prepared in PBS are small (<20 nm) and negatively charged. Therefore, they can be encapsulated by lipid nanoparticles when formulated at neutral pH. Next, we prepared and characterized Cas9 / sgRNA complexes with different Cas9 protein:sgRNA molar ratios. Figure 11C) and zeta potential (Figure 11D) of Cas9 / sgLUC complexes prepared at different Cas9 / sgRNA molar ratios (1 / 1, 1 / 3, and 1 / 5). Compared with the Cas9 / sgLUC complex (1 / 1, mol / mol), the larger molar ratios (1 / 3 and 1 / 5, mol / mol) resulted in smaller size and a more 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. Figure 11E) and zeta potential (Figure 11F) of DOTNP10 lipid nanoparticle-encapsulated Cas9 / sgLUC complexes (designated DOTNP10-L) prepared at different molar ratios (1 / 1, 1 / 3, and 1 / 5) demonstrate the encapsulation of Cas9 / sgRNA RNPs into monodisperse LNPs. Figure 11G shows a TEM image of DOTNP10-L (1 / 3, mol / mol) LNPs containing encapsulated RNPs. Following this initial study, we used different sgRNAs, including sgLUC, sgGFP, sgTOM, and sgPTEN. To distinguish between these, we added the first letter of each gene to the end of DOTNP.For example, DOTNP10-L refers to a DOTNP10 lipid nanoparticle-encapsulated Cas9 / sgLUC complex, and DOTNP10-G refers to a 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. TIFF0007815196000050.tif45151

[0199] Table 8 lists all primers used in this study, including the length of the PCR product and its purpose (Cas9 = SEQ ID NOs: 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 NOs: 14-15; IVT sgTom1 = SEQ ID NOs: 44-45; IVT sgTom2 = SEQ ID NOs: 46-47; IVT sgLoxP = SEQ ID NOs: 48-49). TIFF0007815196000051.tif124170

[0200] To investigate whether DOTNP lipid nanoparticles can deliver Cas9 / sgRNA RNP complexes to the nucleus and mediate efficient gene editing in vitro, we performed a series of experiments. First, we tracked DOTNPs containing Cas9 / sgRNA RNPs tagged with green fluorescent EGFP by confocal microscopy (Figure 12A). Images of Hela-Luc cells (using 9 nM sgRNA) after incubation with DOTNP10-encapsulated Cas9-EGFP / sgLUC complexes (1 / 3, mol / mol) for 1, 3, 6, and 24 hours showed that DOTNPs were internalized into the cells and Cas9 RNPs were transported to the nucleus. Green: EGFP-fused Cas9 protein; Blue: nuclei stained with Hoechst 33342. Red arrows indicate the process of DOTNP10 entering the nucleus (Figure 12B).

[0201] Next, we investigated whether DOTNP lipid nanoparticles could deliver the Cas9 / sgRNA RNP complex and cleave the target luciferase DNA. The percentage of DNA indels (insertions and deletions) at the LUC locus after 3 days of incubation with different molar ratios of DOTNP10-L 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 verify DNA editing, we performed a T7EI cleavage assay (24 nM sgRNA) of Hela-Luc cells incubated with different formulations (Figure 12C). Of the conditions tested, a 1 / 3 molar ratio showed the best gene editing when using Truecut Cas9 protein. GFP editing was then examined using a fluorescence microscope (Figure 12D). Images of SKOV3-GFP cells (24 nM sgRNA) incubated with DOTNP10-L (control, not targeting GFP) and DOTNP10-G (targeting GFP). On-target editing was demonstrated by the 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) The mean fluorescence intensity of SKOV3-GFP cells incubated with DOTNP10-L and DOTNP10-G by flow cytometry demonstrated 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 engineered TdTomato mouse model. 1.5 mg / kg of sgRNA was delivered per mouse using the following formulations: DOTNP5-T refers to DOTNP5 LNP-encapsulated Cas9 / sgTom complex; DOTNP10-T refers to DOTNP10 LNP-encapsulated Cas9 / sgTom complex; and DOTNP50-T refers to 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. When the DOTAP dose was further increased to 50% (DOTNP50-T), most 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 with the Cas9 / sgRNA ribonucleoprotein (RNP) complex. To further investigate delivery, LNPs containing an 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) confirmed 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, gene editing was mostly 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 specific 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 are effective in delivering nucleic acids to the lung. These findings appear to be universal, with head, saturation, and tail length having little effect.

[0204] Example 6: Adding additional lipids to four known lipid compositions alters delivery targets We then explored the generalizability of the approach (methodology) of including a "fifth" lipid in established four-component LNPs.

[0205] To investigate whether the inclusion of a permanent cationic lipid (e.g., DOTAP) can alter the tissue specificity of other cationic ionizable lipids, we selected two well-known and well-established cationic ionizable lipid LNP systems. DLin-MC3-DMA was selected and formulated with DSPC, cholesterol, and PEG-DMG. The same molar composition as Patisiran / Onpattro (Alnylam Pharmaceuticals) was produced, with the addition of 15% or 50% DOTAP (an extra fifth lipid in the Onpattro 4 lipid formulation) (Figure 14). DLin-MC3-DMA LNPs are considered the "gold standard" for siRNA and mRNA delivery. To date, these have only been shown to deliver to the liver after IV administration. As shown in Figure 15A, DOTAP altered the mRNA expression profile in organs of DLin-MC3-DMA-based LNPs (0.1 mg / kg luciferase mRNA, 6 h). As the percentage of DOTAP increased, the luciferase signal migrated from the liver to the spleen and finally to the lung. This was exactly the same phenomenon as observed with 5A2-SC8 mDLNPs. To further explore the generality of this approach, we included DOTAP in C12-200 LNPs (Figure 16). DLin-MC3-DMA is a lipid with two tails and one dimethylamine head group, which is considered a stable nucleic acid lipid nanoparticle (SNALP), whereas C12-200 is a typical "lipidoid" that can be formulated into lipid-like LNPs. All three are cationic ionizable lipids. Consistent with the results using 5A2-SC8 and DLin-MC3-DMA, the inclusion of 15 or 50% DOTAP in C12-200 LNPs shifted luciferase protein expression after mRNA delivery from the liver to the spleen to the lung (Figure 15B). Therefore, the fifth lipid methodology (e.g., adding a permanent cationic lipid) is generalizable to other cationic ionizable lipid LNPs.

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

[0207] To investigate whether cationic ionizable lipids generally enhance liver delivery, we included an additional 5A2-SC8 cationic ionizable lipid as a "fifth" lipid in LNPs containing appropriate ratios of 5A2-SC8, DOPE, cholesterol, and PEG-DMG. The extra 5A2-SC8 was included at 10-30% (Figures 17A and 18). To avoid luminescence saturation, a low mRNA dose of 0.05 mg / kg was tested (IV, 6 hours). As shown in Figure 17B, both ex vivo imaging and quantitative data demonstrated that increasing the extra 5A2-SC8 by 15-25% helped improve mRNA delivery efficacy in the liver. 5A2-SC8^20 (5A2-SC8 LNPs + 20% extra 5A2-SC8) increased luciferase activity by 2-3-fold compared to the original mDLNP formulation.

[0208] Example 8: Studies related to selective organ-targeting compositions This disclosure describes a strategy termed selective organ targeting (SORT), which allows nanoparticles to be systematically engineered to precisely deliver diverse cargoes, including mRNA, Cas9 mRNA / sgRNA, and Cas9 ribonucleoprotein (RNP) complexes, to the lungs, spleen, and liver of mice after intravenous (IV) administration (Figure 19A). Traditional LNPs are composed of cationic ionizable lipids, zwitterionic phospholipids, cholesterol, and poly(ethylene glycol) (PEG) lipids. This disclosure demonstrates that the addition of auxiliary components (termed SORT compounds or selective organ targeting compounds) precisely alters in vivo RNA delivery profiles and mediates tissue-specific gene delivery and editing as a function of the percentage and biophysical properties of added SORT lipids. This disclosure provides proof of theory for tissue-specific delivery, establishes the utility of this methodology for a variety of nanoparticle systems, and provides a method for designing LNPs for therapeutically relevant cell editing.

[0209] Effective intracellular delivery materials have traditionally relied on an optimal balance of ionizable amines (pKa 6.0–6.5) for RNA binding and release and nanoparticles for stabilizing hydrophobicity (Kanasty et al., 2013; Jayaraman et al., 2012; Nelson et al., 2013; Hao et al., 2015). Without being bound by any theory, it is believed that internal and / or external charges may be factors that modulate tissue tropism. The developed SORT LNPs enabled high-level tissue-specific gene editing upon intravenous administration. SORT is compatible with various methods of delivering gene editing machinery, including mRNA, Cas9 mRNA / sgRNA, and Cas9 RNP (systemic RNP delivery). Lung-targeted SORT LNPs edited 40% of epithelial cells and 65% of endothelial cells. Spleen-targeted SORT LNPs edited 13% of B cells and 10% of T cells. Enhanced liver-targeted SORT LNPs edited 93% of hepatocytes after a single low-dose injection.

[0210] A. Discovery and Development of SORT To explore the hypothesis that tissue-specific delivery can be mediated by modulating internal charge, a strategy was devised to add a fifth lipid to an established LNP composition and validated for efficacy in hepatocytes. The rationale was to tailor an effective LNP formulation without disrupting the core quaternary component ratio typically used to mediate RNA encapsulation and endosomal escape (Wittrup et al., 2015; Cheng et al., 2018).

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

[0212] The effects of SORT modifications were then evaluated by intravenously (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 shifted sequentially from the liver to the spleen and then to the lungs. This demonstrated a clear and precise organ-specific delivery trend, with a threshold that enabled exclusive pulmonary delivery (Figure 19B). The DOTAP percentage was a key factor regulating tissue specificity. Base LNP (0% DOTAP) was optimal for liver delivery, which was expected since it had previously been optimized for hepatocyte delivery (Cheng et al., 2018). With the addition of 10–15% DOTAP, the resulting SORT LNP was able to deliver mRNA to cells within the spleen. Further increasing the permanent cationic SORT lipid, 50% DOTAP was found to be optimal for pulmonary delivery (Figure 19C). It is noteworthy that 50% DOTAP SORT LNPs were effective in delivering mRNA to the lung 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 positive charge. When calculating relative expression in each organ, delivery was completely shifted from liver to lung when DOTAP was used as the SORT lipid (Figure 19D). Thus, given that >99% of current IV nanomedicines are estimated to be sequestered by MPS (Wilhelm et al., 2016; Gustafson et al., 2015), these new SORT nanoparticles overcome a long-standing challenge in nanomedicine.

[0213] Without being bound by any theory, now that the functional role of the permanent cationic SORT lipid has been elucidated, it is conceivable that the inclusion of other lipids may also alter tissue tropism. To explore this potential, we incorporated negatively charged 1,2-dioleoyl-sn-glycero-3-phosphate (18PA) as a SORT lipid, similar to DOTAP (Figure 20). At 10–40% 18PA incorporation, SORT LNPs mediated completely selective delivery to the spleen, with no luciferase expression in other organs (Figure 19E). Thus, the negatively charged SORT lipid allows for specific delivery to the spleen. These results demonstrate that the SORT lipid percentage can be tailored for specific tissue mRNA delivery via IV injection.

[0214] B. SORT is generalizable to other LNP types and lipid classes To test the generality of SORT, we next explored the applicability of the SORT methodology 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 deliver to the liver after IV administration, and this was confirmed here (Figure 19F). As expected, adding DOTAP to DLin-MC3-DMA LNPs altered the protein expression profile of the Onpattro formulation. As the SORT lipid percentage increased, the luciferase signal shifted from the liver to the spleen to the lungs. This was exactly the same phenomenon as with the 5A2-SC8 DLNPs we initially tested. To further explore the generality of this approach, we included DOTAP in C12-200 LNPs (Figures 19G and 22). C12-200 LNPs have also been well-documented for RNA delivery to the liver (Kove et al., 2010; Kauffman et al., 2015). Identical to the results using 5A2-SC8 and DLin-MC3-DMA LNPs, including 15% or 50% DOTAP in C12-200 LNPs shifted luciferase protein expression after mRNA delivery from the liver to the spleen to the lung (Figures 19G and 22). Furthermore, the inclusion of 18PA as a sorting lipid mediated exclusive Luc mRNA delivery to the spleen for both DLin-MC3-DMA SNALP and C12-200 LLNPs, recapitulating the results using 5A2-SC8 (Figures 19F-G). DLin-MC3-DMA is a lipid with two tails and one dimethylamine head group 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 was shown to be generalizable to other classes of cationic ionizable lipid LNPs. This allows existing liver-targeting LNPs to be easily modified to deliver mRNA to the spleen or lung.Specifically, SORT technology may enable the rapid redevelopment of the FDA-approved Onpattro to treat diseases of the lungs and spleen.

[0215] To understand whether the observed tissue tropism profiles were specific to a precise chemical structure or generalizable to defined chemical classes, multiple permanent cationic, anionic, zwitterionic, and cationic ionizable SORT lipids were evaluated (Figure 23). Initially, 5A2-SC8 LNPs were prepared with two additional permanent cationic lipids: didodecyldimethylammonium bromide (DDAB) and 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine chloride (EPC). While all of these lipids contain quaternary amino groups, there are significant chemical differences (e.g., degree of saturation) in the polar headgroup, 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 LNPs. As the percentage of DDAB or EPC increased, luminescence activity systematically shifted from the liver to the spleen and then to the lungs (0.1 mg / kg, 6 h). When the percentage increased to 40%, high luciferase signals were observed exclusively in the lungs (Figure 23A). Representative anionic lipids with different structures compared to 18PA were engineered: 1,2-dimyristoyl-sn-glycero-3-phosphate (14PA) and sn-(3-oleoyl-2-hydroxy)-glycerol-1-phospho-sn-3'-(1',2'-dioleoyl)-glycerol (18BMP). All anionic SORT lipids promoted delivery exclusively to the spleen (Figure 23B). This versatility paves the way for optimizing SORT compounds to balance multiple factors, including potency, selectivity, and tolerability.

[0216] These findings inspired us to add other cationic ionizable lipids to established formulations. As expected, adding DODAP or C12-200 to 5A2-SC8 LNPs 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 LNPs dramatically improved liver mRNA delivery, achieving a 10-fold increase at the extremely low dose of 0.05 mg / kg. 7 photon / sec / cm 2 Thus, SORT offers a new strategy to further improve liver-targeted LNP systems (Figure 24). The effect of using zwitterionic lipids (DOCPe and DSPC) as SORT lipids was also evaluated. Tissue tropism was found to be from the liver to the spleen, but 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, the addition of DODAP or DOTAP to a completely inactive formulation resulted in tissue-specific delivery to the spleen and lung (Figure 26). Taken together, these results demonstrate that SORT is a modular and universal strategy for achieving tissue-targeted delivery.

[0218] C.SORT Mediates Organ-Specific Delivery of Protein Corona, LNP Biodistribution, and Apparent pK a Change Mechanistic experiments were performed to explore how and why the inclusion of extra lipids from defined categories controls mRNA delivery to different organs. It stands to reason that LNPs intended for delivery to cells in the lung must biodistribute (accumulate) in the lung. Cy5-labeled mRNA was delivered to track the in vivo distribution of 5A2-SC8 LNPs containing SORT lipids with tropism for the lung (DOTAP quaternary amino lipid), spleen (18PA anionic lipid and DSPC zwitterionic lipid), and liver (DODAP ionizable tertiary amino lipid) (Figures 27A and 28). All LNPs were injected IV at a dose of 0.5 mg / kg Cy5-labeled mRNA and imaged 6 hours later. As shown in Figure 27A, DOTAP altered biodistribution, with lung accumulation progressively increasing as a function of DOTAP percentage. Incorporation of 18PA increased spleen uptake. DODAP slightly increased liver accumulation and decreased spleen accumulation. Interestingly, there was no protein expression in the liver for lung-specific and spleen-specific SORT LNPs, yet these LNPs still accumulated in the liver, suggesting that organ biodistribution is necessary for organ-specific efficacy but is not the only factor explaining the mechanism of tissue-targeted delivery.

[0219] Without being bound by any theory, we believe that changes in biodistribution and activity in defined cell populations may be due to changes in the protein corona. Binding of specific proteins in the protein corona creates functionally active biological identities. Using quantitative mass spectrometry, we found that the addition of SORT molecules dramatically altered both the specific proteins most closely bound and the overall protein corona composition. Without being bound by any theory, we believe that lung-specific SORT LNPs selectively and most abundantly bound 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 with adenoviruses, which utilize αvβ5 integrin to target bronchial epithelia. Spleen-specific SORT LNPs bound most closely to β2-glycoprotein I, which has been shown to interact with negatively charged lipids and may play a role in splenic localization of immune cell populations in the spleen. It is worth noting that the complex mixture of bound proteins may also play a role. This ensemble effect is also a potential mechanism for targeting multiple cell types and is considered one way to further enhance specificity for specific cell types within a given organ using alternative SORT molecules. It has previously been shown that apolipoprotein E binds to DLin-MC3-DMA Onpattro LNPs and that efficacy is lost in ApoE knockout animals. Thus, there is strong evidence that ApoE is required for receptor-mediated targeting and uptake in hepatocytes, presumably via the LDL receptor, and that the described protein corona mechanism can control cell specificity and efficacy. Therefore, it is encouraging to see that mDLNPs also bind strongly to ApoE, providing further evidence of their hepatocyte efficacy and strengthening the validity of the protein corona assay. Liver-enhanced SORT LNPs retain ApoE binding but are also enriched in albumin.This suggests that cell types may have proliferated within the liver. These data collectively demonstrate that the chemical structure of the SORT molecule can direct a specific protein corona, altering organ tropism and cell specificity. Without being bound by any theory, it is believed that the identity of the SORT molecule can control the identity of the protein corona. This suggests that SORT molecules can include sugars, lipids, small molecule therapeutics, vitamins, small molecules, hydrophilic molecules, hydrophobic molecules, amphipathic molecules, peptides, proteins, and more.

[0220] Apparent / overall pK as a parameter correlating LNP and functional activity a has been established, the apparent / overall pK a For example, delivery to hepatocytes requires a pK of approximately 6.4. a has been shown to be optimal (Jayaraman et al., 2012). Using the TNS assay, the apparent pK a were analyzed (Figures 27B and 29, Table 1). Because SORT involves the inclusion of additional charged lipids, the resulting TNS titration curve captures the more complex ionization behavior of mixed-species LNPs. Therefore, instead, the relative pK at which 50% of the normalized signal occurs is used. a The relative pK for tissue tropism was estimated. a When plotting the apparent pK a As expected, the pK of all effective liver-targeting formulations was a pK was very narrow and within the well-established range of 6–7 (Jayaraman et al., 2012). All lung-targeted formulations had high pK a In contrast, spleen-tropic SORT LNPs had low pK a These results support that 6–7 is optimal for hepatic delivery, but the high pK a mediates pulmonary delivery and has a low pK aThis study highlights the finding that SORT mediates splenic delivery. It is important to note that all SORT LNPs still contain cationic ionizable lipids, which are considered useful for endosomal escape due to their ability to acquire a charge (Wittrup et al., 2015). Control experiments were performed, confirming that the inclusion of cationic ionizable lipids is necessary for efficacy (Figure 30). Thus, SORT is able to achieve the specific microspecies pK required for efficacy at the desired molar ratio. a While the inclusion of SORT lipids allows for the retention of molecules with an apparent pK a Without being bound by any theory, it is believed that a two-part mechanism may play a role. SORT LNPs selectively bind to specific proteins in serum, enabling receptor-mediated efficacy in cells located in the lung or spleen. This is very similar to how lipoprotein particles (e.g., LDL) naturally transport cholesterol. This controlled and predictable endogenous targeting mechanism allows SORT LNPs to reach non-hepatic targets. The second part involves altering the physiochemical properties (e.g., overall / apparent pK) of the SORT molecule for hepatic efficacy. a This involves altering the properties of non-liver-targeted SORT LNPs so that they no longer have the apparent pK (6.4). This provides precision. We also note that other, more complex factors, such as differences in cell-specific endocytic trafficking, may also play a role. Considering the results, we believe that the internal charge of the LNP nanostructure mediates biodistribution and the apparent pK a It is suggested that the expression of nanoparticles correlates with the protein expression profile in specific organs. This special value can be used to continue developing other organ-specific nanoparticles.

[0221] Table 1. Details of mDLNP formulations (SORT LNPs) modified with 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. TIFF0007815196000052.tif177170 a X represents DDAB, EPC, 14PA, 18BMP, DODAP, C12-200, 5A2-SC8, DSPC, and DOCPe.

[0222] D.SORT enables lung-, liver-, and spleen-specific gene editing after IV administration Given the ability of SORT LNPs to target specific organs, these findings were then applied to tissue-specific gene editing via IV injection. CRISPR / Cas (clustered regularly interspaced short palindromic repeats / CRISPR-associated protein (Cas)) technology allows for precise, sequence-dependent genome editing and has rapidly evolved for use in a wide variety of applications, 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 severe genetic disorders result from mutations in cells deep within organs, and correcting these specific cells is necessary to cure the disease. Such correction may be best achieved by systemic administration. Recently, IV co-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 rationally engineered LNPs to edit cells in organs other than the liver.

[0223] To examine and quantify the ability of SORT LNPs to mediate organ-specific gene editing, we utilized genetically engineered tdTomato (tdTom) reporter mice containing a LoxP-flanked stop cassette (Tabebordbar et al., 2016) that blocks expression of the tdTom protein (Staahl et al., 2017). Upon deletion of the stop cassette, tdTom fluorescence was turned on, allowing detection of gene-edited cells (Figure 31A). To activate tdTom in edited cells, we first delivered Cre recombinase mRNA (Cre mRNA). Fluorescent tissue was readily visible in selected organs treated with liver-selective SORT LNPs, lung-selective SORT LNPs, 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. The background organ fluorescence in the spleen was the weakest compared to the other organs (Figures 31C and 32). This makes spleen-specific detection difficult to discern in the tdTom mouse model. When endogenous PTEN was subsequently edited, spleen-specific SORT LNPs showed clear DNA breaks only in the spleen by T7E1 assay (Figure 33C), but not in the liver or lung. Nevertheless, tdTom-positive cells were readily detected by confocal imaging of tissue sections (Figure 31D).

[0224] E.SORT enables high-level editing in specific and therapeutically relevant cell populations We quantified gene editing in specific cell types within the liver, lung, and spleen using flow cytometry of single cells extracted from the edited organs (Figure 31E). Liver-specific SORT(20% DODAP)5A2-SC8 LNPs 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 LNPs edited approximately 40% of all epithelial cells, 65% of all endothelial cells, and 20% of immune cells in the lung 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 LNPs as a compelling delivery system with immediate application for correcting CFTR mutations. Finally, spleen-specific SORT(30%18PA)5A2-SC8 LNPs edited approximately 13% of total B cells, 10% of total T cells, and 20% of total macrophages (Figures 31E and 36). Due to improved selectivity over previous studies, spleen-specific SORT LNPs may be applicable to treating non-Hodgkin's B-cell lymphoma and other immune disorders. While the initial focus was on quantification with a single low-dose injection, higher levels of editing may be achievable by administering higher doses or multiple injections.

[0225] F.SORT enables tissue-specific gene editing by IV co-delivery of Cas9 mRNA / sgRNA and delivery of Cas9 RNP We next investigated the ability of SORT LNPs to achieve tissue-specific CRISPR / Cas gene editing by co-delivering Cas9 mRNA and sgRNA in a single nanoparticle via IV co-delivery (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, strong tdTom fluorescence was observed in the liver of both basal LNP- and 20% DODAP SORT LNP-treated mice, and in the lungs of 50% DODAP SORT LNP-treated mice. All results were consistent with Luc mRNA delivery. Due to the rapid turnover of splenic immune cells in mice (Kamath et al., 2000), the Cas9 / sgRNA weight ratio was optimized to 2 / 1 (Figure 39), and spleen editing was tested 2 days after injection. Taking background autofluorescence into account, bright tdTom fluorescence was observed in the spleens of 30% 18PA-treated mice. A clear T7E1 cleavage band was detected exclusively in DNA isolated from spleens (no liver or lung editing) (Figure 33). Fluorescence was then confirmed by imaging tissue sections with confocal microscopy (Figure 37C).

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

[0227] We tested the ability of tissue-specific LNPs to edit endogenous targets in go beyond reporter mice. PTEN was chosen 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. Ten days after a single IV injection, the occurrence of insertions and deletions (indels) was quantified. As shown in Figure 37D, the T7E1 assay revealed distinct DNA break bands in specific tissues. This demonstrated that both basic LNPs and 20% DODAP SORT LNPs mediated effective PTEN editing in the liver, but not in the lung or spleen. Notably, 50% DODAP SORT LNPs showed PTEN editing exclusively in the lung. 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. While spleen-specific 18PA SORT LNP editing was difficult to identify in the tdTom mouse model, clear spleen PTEN editing could be observed in wild-type mice using an optimized weight ratio of Cas9 / sgPTEN (2 / 1) and a detection time of 2 days. T7E1 assays performed on 18PA SORT LNP-injected mice did not detect DNA editing in either the liver or lung (Figure 33). Finally, SORT was applied to Cas9 RNPs to examine endogenous PTEN editing. As before, 7% and 55% DOTAP SORT LNPs containing Cas9 protein / sgPTEN enabled liver- and lung-specific editing, respectively (Figure 37G). These results demonstrated that rationally guided tissue-selective gene editing targeting endogenous genes was achieved using 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 by 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-sn-dimethylammonium-propane (DODAP), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 2-((2,3-bis(oleoyloxy)propyl)dimethylammonio)ethyl ethyl 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-Dimyristoyl-sn-glycerol-methoxyl (poly((ethylene glycol) MW 2000) (DMG-PEG2000) was purchased from NOF America Corporation. Cas9 protein was purchased from Thermo Fisher. ONE-Glo + Tox Luciferase Reporter Assay Kit was purchased from Promega Corporation. Pur-A-Lyzer Midi Dialysis Kits (WMCO, 3.5 kDa) 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 TIFF0007815196000053.tif114170

[0230] II. Nanoparticle formation RNA-loaded LNP formulations were formed using the ethanol dilution method ( Zhou et al., 2016 ). Liver-targeted mRNA formulations (mDLNPs) were developed and reported in a previous paper ( Cheng et al., 2018 ). Base formulations were prepared as previously described (Jayaraman et al., 2012; Love et al., 2010). Unless otherwise noted, all lipids with the specified molar ratios were dissolved in ethanol, and RNA was dissolved in 10 mM citrate buffer (pH 4.0). The two solutions were quickly mixed at a 3:1 aqueous:ethanol ratio by volume (3:1, aqueous:ethanol, vol:vol) to achieve a final weight ratio (total lipids:mRNA) of 40:1 and then incubated at room temperature for 10 min. 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, formulations were obtained using mRNA buffer (10 mM, pH 3.0) as described above. All formulations were named based on the additional lipid. Taking mDLNPs as an example, the internal molar ratio of mDLNPs was fixed at 15 / 15 / 30 / 3 (5A2-SC8 / DOPE / cholesterol / DMG-PEG) as reported in a published paper (Cheng et al., 2018). DOTAP was dissolved in the specified amount as an additional lipid in the above ethanolic lipid mixture to achieve a molar ratio of 15 / 15 / 30 / 3 / X (5A2-SC8 / DOPE / cholesterol / DMG-PEG / DOTAP). This was quickly mixed with the aqueous mRNA solution according to the standard protocol described above to finally obtain SORT LNPs. This was designated Y%DOTAP, where Y represents the molar percentage of DOTAP in the total lipids. Similarly, formulations containing other additional lipids were formed using the above method (Figure 20 and Table 3). For Cas9 / sgRNA ribonucleoprotein (RNP) encapsulation, 1x PBS was used in the formulation, and the molar ratio of Cas9 to sgRNA was fixed at 1:3.After SORT LNP formation, fresh LNP formulations were diluted with 1x PBS to 0.5 ng / μL mRNA (final ethanol concentration <5%) for in vitro assays and size detection. For in vivo experiments, formulations were dialyzed (Pur-A-Lyzer Midi Dialysis Kits, WMCO 3.5 kDa, Sigma-Aldrich) against 1x PBS for 2 hours and diluted to 15 μL / g with PBS for intravenous (IV) injection.

[0231] Table 3: sgRNA sequences TIFF0007815196000054.tif34170

[0232] III. Characterization of mRNA formulations Size distribution and polydispersity index (PDI) were measured using dynamic light scattering (DLS, Malvern MicroV model; He-Ne laser, λ = 632 nm), and zeta potential was measured after dilution with 1x PBS. a To measure λ, 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). The mRNA formulation (60 μM total lipid) and TNS probe (2 μM) were incubated for 5 min with a series of buffers containing 10 mM HEPES, 10 mM MES (4-morpholineethanesulfonic acid), 10 mM ammonium acetate, and 130 mM NaCl (pH range 2.5–11). The mean fluorescence intensity of each well (black-bottom 96-well plate) was measured using a Tecan plate reader at λ. Ex = 321 nm and λ Em = 445 nm and the data were normalized to the value at pH 2.5. Typically, the apparent pK a was defined as the pH at which half of the fluorescence maximum occurs. This method allows us to estimate the overall / apparent pK of most LNPs. aWhile this was useful for estimating the 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 was used to estimate the pK at which 50% of the normalized signal occurred compared to the base LNP formulation (no added SORT lipids). a This alternative calculation yielded pK a The TNS assay was estimated for permanently cationic SORT LNPs without changing the charge state, and was consistent with experimental results for tissue-selective RNA delivery. Therefore, it may be suggested that the standard TNS assay be used when LNPs contain a single cationic ionizable lipid, while the alternative 50% normalized signal method be used for systems such as SORT, which contain complex mixtures of multiple lipids with various charge states.

[0233] IV. In vitro luciferase expression and cell viability assay Huh-7 or A549 cells were plated in white 96-well plates at 1 × 10 cells per well the day before transfection. 4 Cells were seeded at a density of 1000 cells / well. The medium was replaced with 150 μL of fresh DMEM medium (5% FBS), and then 50 μL of Luc mRNA preparation was added, containing 25 ng of mRNA per well. After a further 24 h of incubation, 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 Institutional Animal Care and Use Committees of The University of Texas Southwestern Medical Center and were consistent 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)HzeAi9 / J mice (also known as Ai9 or Ai9(RCL-tdT) mice) were obtained from the Jackson Laboratory (007909) and bred to maintain homozygous expression of a Cre reporter allele containing a loxP-flanked STOP cassette that blocks 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 a C57BL / 6J genetic background.

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

[0236] VII.mRNA synthesis Optimized Cre recombinase mRNA and Cas9 mRNA were generated by in vitro transcription (IVT). Briefly, the NLS-Cre and Cas9 fragments were prepared by PCR using pCAG-CreERT2 and pSpCas9(BB)-2A-GFP (PX458) as PCR templates, respectively. These fragments were then cloned into the pCS2+MT vector, which contains an optimized 5' (3') untranslated region (UTR) and poly(A) sequence. The IVT reaction was performed according to standard protocols, except that the typical UTP was replaced with N1-methylpseudouridine-5'-triphosphate. Finally, the mRNA was capped (Cap-1) using vaccinia capping enzyme and 2'-O-methyltransferase (NEB). Table 4 lists the primers used herein.

[0237] Table 4. PCR product lengths and primers for their purposes TIFF0007815196000055.tif116170

[0238] The coding sequences of NLS-Cre and Cas9 are as follows: TIFF0007815196000056.tif17145TIFF0007815196000057.tif221146TIFF0007815196000058.tif209146

[0239] VIII. Western Blot The quality of IVT Cas9 mRNA was analyzed by Western blot. The day before transfection, 293T cells were plated in 12-well plates at 1 × 10 5Cells were seeded at a density of 100 cells / well in a total volume of 600 μL. Cells were treated 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), for an additional 24 hours. After washing three times with 1x 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 (100x, Thermo Fisher Scientific) were added to each well and incubated at RT for 20 minutes. The cell lysates were collected and placed in 1.6 mL tubes and centrifuged at 13,000 g for 10 minutes at 4°C. The supernatants were collected and placed in new tubes. If not used immediately, they were stored at -80°C. Prior to Western blotting, protein concentrations were measured using a BCA assay kit (ThermoFisher). Fifteen 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) for 1 hour at room temperature. Primary antibodies were applied overnight at 4°C. After washing four times with PBST, the membranes were incubated with secondary antibodies for 1 hour at room temperature and then imaged using ECL substrate, followed by four washes with PBST (ThermoFisher).

[0240] IX. Gene editing (Cre mRNA) in the Td-Tomato mouse model Cre mRNA was prepared as described above and injected intravenously (0.3 mg / kg Cre mRNA). Two days later, 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 in each organ cell type + To test the cells, they were treated with a Cre mRNA preparation (0.3 mg / kg) for 2 days before isolation and staining, 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 using liver perfusion medium (Thermo Fisher Scientific, 17701038) for 7–10 min, then switched to liver digestion medium (Thermo Fisher Scientific, 17703034) for an additional 7–10 min. The liver was collected in a plate containing 10 mL of liver digestion medium and cut to dissociate hepatocytes. Dissociated hepatocytes were then collected and washed twice with hepatocyte wash medium (Thermo Fisher Scientific, 17704024) and once with 1x PBS. After further isolation by filtration and low-speed (50 × g ) centrifugation, hepatocytes were analyzed on a FACS Aria II SORP machine (BD Biosciences).

[0243] For isolation and staining of splenic cell types, the removed spleens were minced with a sterile blade and homogenized in 250 μL of 1x 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 1x digestion medium. The spleen solution was then filtered through a 70 μm filter and washed once with 1x 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 1x 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 a cell pellet. Single cells were resuspended in cell staining buffer and added to the flow tube containing the antibody (100 μL total volume). The cells were incubated with the antibody for 20 minutes at 4°C in the dark. The stained cells were washed twice with 1 mL of 1x PBS and then resuspended in 500 μL of 1x 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-Cyanine5.5 anti-mouse CD3e (145-2C11) (Tonbo Biosciences, 65-0031). Ghost Dye Red 780 (Tonbo Biosciences, 13-0865-T500) was used to identify live cells.

[0244] For isolation and staining of lung cell types, the isolated lungs were minced with a sterile blade and then transferred to a 15 mL tube containing 10 mL of 2x 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 here 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). Ghost Dye Red 780 (Tonbo Biosciences, 13-0865-T500) was used to identify live cells.

[0245] XI. Gene editing in the Td-Tomato mouse model (Cas9 mRNA / sgRNA and Cas9 / sgRNA RNP) To evaluate in vivo gene editing, Td-Tom mice of comparable weight and gender were selected. Cas9 mRNA and sgRNA were co-delivered into tdTomato (td-Tom) mice. Cas9 mRNA / sgTom1 (4 / 1, wt / wt) were co-delivered in various formulations at a total RNA dose equal to 2.5 mg / kg. Ten days after IV injection, major organs were removed and imaged using the IVIS Lumina system. For the spleen-targeting 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 per group). To confirm Td-Tom expression, tissue sections were further prepared and imaged by confocal microscopy. Briefly, tissue blocks were embedded in optimal cutting temperature compound (OCT) (Sakura Finetek) and cryosectioned (8 μm) using a cryostat (Leica Biosystems). Mounted tissue slices were stained with 4,6-diamidino-2-phenylindole (DAPI, Vector Laboratories) and then imaged by confocal microscopy on a Zeiss LSM 700.

[0246] XII. Gene editing in C57BL / 6 mice (Cas9 mRNA / sgPTEN and Cas9 / sgRNA RNP) PTEN was selected to investigate endogenous gene editing in vivo. Wild-type C57BL / 6 mice were intravenously injected with various carriers to co-deliver 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). After 10 days, tissues were collected, 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, the Cas9 mRNA / sgTom1 ratio was 2 / 1 (wt / wt), and the 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 the detection time was 7 days after injection (n = 2–4 mice / group). After obtaining the PTEN PCR product, a T7E1 assay (NEB) was performed to confirm gene editing efficacy using standard protocols. Furthermore, PTEN editing was assessed on tissue sections by H&E staining and immunohistochemistry (IHC). Briefly, paraformaldehyde (PFA)-fixed tissues were embedded in paraffin, sectioned, and stained with H&E by the Molecular Pathology Core at UTSW. Four-micron sections were prepared using standard methods and detected for IHC using the Elite ABC Kit and DAB Substrate (Vector Laboratories).

[0247] Example 9: Formulation with Neutral Buffer Cas9 RNPs were observed to denature in acidic buffers, thereby increasing their hydrodynamic size from 10 nm to 150 nm (Figure 40B). This makes RNP encapsulation into monodisperse nanoparticles difficult, if not impossible. These studies have focused on lipid nanoparticles (LNPs) because they are the most effective class of RNA delivery carriers (Wang et al., 2017; Doudna & Charpentier, 2014; Hajj & Whitehead, 2017; Sander & Joung, 2014) in preclinical models and humans (Wood, 2018). Of the four LNP components [cationic ionizable lipids, zwitterionic phospholipids, cholesterol, and poly(ethylene glycol) (PEG) lipids)], the pK a Cationic ionizable lipids with a pH of approximately 6.4 are useful for RNA encapsulation because they bind to negatively charged RNA at mixed pHs (e.g., pH 4, where amines are protonated), lose their charge at neutral pH, are taken up by cells, and then regain their charge when the intraendosomal pH drops to fuse with the endosomal membrane and release the cargo into the cytoplasm. However, this feature hinders 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, allows RNA and proteins to be encapsulated using neutral buffers (instead of acidic buffers), thus preserving the tertiary structure and stability of RNPs (Figure 40A).

[0248] To evaluate this strategy, we selected 5A2-SC8 as a cationic ionizable lipid because 5A2-SC8 LNPs safely deliver short siRNA / miRNA and long mRNA to liver-compromised mice with MYC-driven liver cancer (Zhou et al., 2016; Zhang et al., 2018a; Zhang et al., 2018b) or a fumarylacetoacetate hydrolase (FAH) gene knockout (Cheng et al., 2018). Indeed, when a permanent cationic lipid (e.g., DOTAP) was introduced into a conventional four-component 5A2-SC8 LNP formulation, controlled self-assembly occurred by mixing an ethanol solution of the lipid with a PBS solution of the RNP (1 / 3, v / v). Incorporation of DOTAP at 5–60 mol% relative to the total lipid was evaluated (Figure 4). These results demonstrated that 10–20% RINPs induced high levels of gene editing in vitro and the formation of stable RNP-loaded nanoparticles with sizes <200 nm (Figure 42). First, we observed 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 with a reporter luciferase-targeting sgRNA (sgLuc). This was slightly larger than nanoparticles without RNP loading. Identical LNPs prepared in a low pH buffer did not change size, indicating that RNPs were not encapsulated (Figure 40C). To determine the optimal molar ratio of Cas9 protein to sgRNA, we prepared Cas9 / sgRNA complexes at 1 / 1, 1 / 3, and 1 / 5 (mol / mol). 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, indicating not only successful encapsulation but also useful for minimizing in vivo uptake by the immune mononuclear phagocyte system (MPS).To further investigate whether 5A2-DOT-10 can successfully mediate RNP delivery to the nucleus, we tracked LNPs with encapsulated fluorescent EGFP-fused Cas9 protein. Free RNP alone failed to enter cells, as no green fluorescence was detectable above background (Figure 43). After 3 hours of treatment with 5A2-DOT-10, bright green fluorescence was observed in the cytoplasm of cells. Then, due to the presence of a nuclear localization signal in Cas9, the EGFP-fused Cas9 protein was observed to gradually enter the nucleus within 6 hours (Figure 40H). Endocytosis is energy-dependent and primarily lipid raft-dependent, as treatment with MβCD, an inhibitor of lipid raft-based endocytosis, significantly inhibited cellular uptake of nanoparticles (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 highest at 1 / 3 and 1 / 5 (Figure 44A). Results of a T7 endonuclease I (T7EI) assay demonstrated that most of the target DNA band (720 bp) was cleaved into two cleavage bands (536 bp and 184 bp). No cleavage bands were observed in the control treatment group. To test the hypothesis that a neutral pH buffer is required to encapsulate RNPs while protecting Cas9, we also evaluated the gene editing efficiency of 5A2-DOT-10 prepared with a pH 4 citrate buffer. No cleavage bands were observed (Figure 44A). Furthermore, negative results were confirmed by Sanger sequencing. This provided further evidence that traditional acid-based formulation methods do not produce effective NPs. Upon switching to GFP-expressing cells, 5A2-DOT-10-encapsulated Cas9 / sgGFP induced indels in the GFP DNA, knocking out nearly 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 the inconspicuous loss of GFP in growing cells and confirmed by Sanger sequencing. Inference of CRISPR Edits (ICE) analysis revealed that indels reached 95% (Figure 44D). With an eye toward clinical translation, the stability of RNP-loaded 5A2-DOT-10 was monitored for 2 months at 4°C. LNPs did not change size and remained uniform (PDI < 0.2) (Figure 44G). Continuous testing of 5A2-DOT-10 nanoparticles revealed consistent gene editing activity even after 60 days of storage (Figure 44H).

[0250] The strategy of adding permanent cationic lipids to classical four-component LNPs for efficient RNP delivery is not limited to the dendrimer-based ionizable lipid 5A2-SC8. To demonstrate this, we included supplemental DOTAP in nanoformulations prepared with other classes of ionizable materials: the well-known DLin-MC3-DMA lipid used in the FDA-approved Onpattro (Wood, 2018) and C12-200 lipidoid (Figure 46A-B). Although these have significantly different chemical structures compared to 5A2-SC8 (Figure 46C), all DOTAP-modified nanoparticles were able to efficiently edit cells, whereas previously established C12-200 or MC3 formulations without DOTAP showed lower editing efficiencies (Figure 44E). 5A2-DOT-10 also achieved higher editing efficiency than the positive control RNAiMAX. Because 5A2-DOT-10 LNPs were more effective than MC3-DOT-10 and C12-200-DOT-10, all subsequent experiments were performed with 5A2-SC8. In addition to DOTAP, other cationic lipids, including DDAB and EPC, were also incorporated into the LNP formulation (Figure 46E-G). These results were similar for all three cationic lipids with different chemical structures (Figure 46H). These results demonstrate 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 for RNP delivery, making this approach potentially clinically translatable for the treatment of various human diseases.

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

[0252] To investigate in vivo gene editing, we delivered 5A2-DOT-10-encapsulated Cas9 / sgTOM complexes to the Td-Tomato mouse model (Figure 47A). In successfully edited cells of these mice, CRISPR-mediated deletion of the Lox-Stop-Lox cassette turns on 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 has previously been 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). Again, bright red signals were observed near the injection site, confirming editing in the mouse brain (Figures 47D-E).

[0253] The improved stability and potency of 5A2-DOT-10 enabled successful systematic gene editing in the evaluated tissues. To investigate this RNP delivery strategy, we prepared LNPs with various DOTAP molar percentages (5–60%) and delivered the RNPs IV into Td-Tom mice (1.5 mg / kg sgTOM). Seven days after injection of 5A2-DOT-5, Td-Tom fluorescence was observed exclusively 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 demonstrate that tissue-specific editing of deep tissues can be achieved by adjusting the internal lipid component chemistry and molar ratio. Tissue-specific editing was further confirmed by confocal imaging of tissue sections (Figure 47G). We then assessed editing of the endogenous target, Pten, by systemically injecting LNP-encapsulated Cas9 / sgPTEN RNP into wild-type C57BL / 6 mice. Distinct T7EI cleavage bands were detected only in the liver 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 the potential for simultaneous editing of multiple genes in vivo, we loaded Cas9 protein and six different sgRNAs into 5A2-DOT-50. sgTOM, sgP53, sgPTEN, sgEml4, sgALK, and sgRB1 were loaded and encapsulated into Cas9 protein. Td-Tom mice were then treated with pooled 5A2-DOT-50 via tail vein injection (0.33 mg / kg of each sgRNA). Bright Td-Tom fluorescence was detected in the lungs one week later, indicating gene editing of TOM (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 was up to 22% in the lungs (Figures 47 and 48). To enhance sgRNA stability and reproducibility, we used sgRNAs with terminal modifications at the first and last three nucleotides (Figure 49) (Finn et al., 2018; Hendel et al., 2015). Reports have shown that precise modifications at additional nucleotides can increase in vivo gene editing by 2-4 fold compared to end-modified sgRNAs (Finn et al., 2018; Yin et al., 2017). This suggests that further optimization of sgRNAs could potentially increase the editing efficiency reported here. Nevertheless, the high potency and tissue specificity of 5A2-DOT-50 allowed simultaneous editing of six targets in the lung with a single injection.

[0255] Animal models are traditionally generated through transgenesis or genetic engineering in embryonic stem cells, which is time-consuming and expensive. Direct mutation of tumor-associated and other disease-related genes in adult mice using CRISPR / Cas is a feasible approach for rapid model generation. This can only be achieved using costly lentiviruses, which must be engineered target-by-target and hydrodynamically injected into the liver (Xue et al., 2014; Maddalo et al., 2014). Because multiple gene mutations are typically required to generate functional cancer models, 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 administered repeatedly, and confer tissue specificity, paving the way for the generation of a wide variety of animal models.

[0256] Using 5A2-DOT-5, we simultaneously knocked out three tumor suppressor genes (P53, PTEN, and RB1) selectively in the liver. These genes have been identified in many human cancers, including those of the liver. 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, clear cleavage bands were observed at all three gene loci by T7EI assay (Figures 48B, 50, and 51). As time progressed, these cleavage bands became significantly brighter, indicating tumor growth. When the mice were sacrificed at 15 and 20 weeks, visible tumors were found in the liver, along with several metastatic tumors in the abdominal cavity (Figures 48C and 52). Tumor development by H&E staining and IHC staining targeting the tumor proliferation biomarker Ki67 (Figs. 48D and 53) was also detected at various time points.

[0257] To generate a challenging lung cancer mouse model, we focused on the Eml4-Alk chromosomal rearrangement, a complex mutation found in many solid human tumors, particularly non-small cell lung cancer (NSCLC) (Maddalo et al., 2014; Blasco et al., 2014). The Eml4-Alk fusion protein, generated after rearrangement between Eml4 and Alk, promotes cancer development. Taking advantage of the high potency and lung-targeting specificity of 5A2-DOT-50, we injected one (2 mg / kg total sgRNA dose) or two (weekly, 1.5 mg / kg total sgRNA dose) IV doses and evaluated the tumorigenesis process (Figure 48E). At all time points examined, indels were detectable in lung DNA extracted from both groups of mice (Figures 48F and 54). Distinct gene rearrangement bands were detected in the lungs of 5A2-DOT-50-treated mice. This confirmed that chromosomal rearrangement had been successfully induced (Figures 48F and 54). As time progressed, the Eml4-Alk rearrangement band became significantly brighter, suggesting the proliferation of edited cells. Sequencing of these PCR amplicons after subcloning further confirmed the Eml4-Alk rearrangement (Figures 48G and 54). Several tumor lesions were observed in the lungs by H&E and Ki67 staining at 16 and 24 weeks (Figures 48H, 55, and 56). These results demonstrate that a single injection of 5A2-DOT-50 LNPs can successfully induce chromosomal rearrangement and lead to the development of lung tumors in adult mice. Therefore, these LNPs are well-positioned to accelerate the in situ generation of various disease models.

[0258] B. Materials and Methods I. Material 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 published protocols. 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-Dioleoyl-3-trimethylammonium-propane (DOTAP), dimethyldioctadecylammonium (DDAB), 1,2-Dimyristoyl-sn-glycero-3-ethylphosphocholine (EPC), and 1,2-Distearoyl-sn-glycero-3-phosphocholine (DSPC) were purchased from Avanti Polar Lipids. Cholesterol was purchased from Sigma-Aldrich. 1,2-Dimyristoyl-sn-glycerol-methoxyl (poly((ethylene glycol) MW2000) (DMG-PEG2000) was purchased from NOF America Corporation. The ONE-Glo + Tox Luciferase Reporter Assay Kit was purchased from Promega Corporation. Pur-A-Lyzer Midi Dialysis Kits (WMCO, 3.5 kDa) were purchased from Sigma-Aldrich. 4',6-Diamidino-2-phenylindole dihydrochloride (DAPI), Hoechst 33342, DLS Ultramicro cuvettes, Lipofectamine RNAiMAX Transfection Reagent, and Lab-Tek chambered cover glass units were purchased from Thermo Fisher Scientific. Cas9 protein and Ki-67 monoclonal antibody were purchased from Thermofisher. GenCrispr NLS Cas9-EGFP nuclease was purchased from GenScript. Modified sgRNA was purchased from Synthego.

[0259] II. Preparation of Cas9 / sgRNA complex Separate solutions of Cas9 protein and sgRNA in the noted buffer were mixed together in equal volumes. After mixing, the mixture was incubated at room temperature for 5 minutes to allow the Cas9 / sgRNA complex to completely self-assemble into RNPs. The molar ratios of Cas9 protein to sgRNA used were 1 / 1, 1 / 3, and 1 / 5.

[0260] III. Optimized Nanoparticle Formulation and Characterization Cationic ionizable lipids (5A2-SC8, C12-200, or DLin-MC3-DMA) (Zhou et al., 2016; Jayaraman et al., 2012; Love et al., 2010), zwitterionic lipids (DOPE or DSPC), cholesterol, DMG-PEG,...

Claims

1. 1. A selective organ targeting (SORT) lipid composition for use in a method for targeted delivery of a therapeutic agent to the lung or spleen, the SORT lipid composition comprising: (i) a cationic ionizable lipid; and (ii) a cationic SORT lipid that is separate from the cationic ionizable lipid. Including, the cationic SORT lipid is a cationic ionizable SORT lipid or a permanent cationic SORT lipid; the cationic ionizable SORT lipid is 1,2-dioleoyl-3-dimethylammonium-propane (DODAP); The permanent cationic SORT lipid has the structure of Formula (I), Formula (II), or Formula (III), or a pharmaceutically acceptable salt, stereoisomer, or tautomer thereof: (In formula (I), R 1 and R 2 are each independently an alkyl (C8～C24) , alkenyl (C8～C24) or a substituted form of either group; R 3 , R 3 ', and R 3 '' are each independently an alkyl (C≦6) or substituted alkyl (C≦6) and; X - is a monovalent anion); (In formula (II), R 4 and R 4 ' are each independently alkyl (C6～C24) , alkenyl (C6～C24) or a substituted form of either group; R 4 '' is an alkyl (C≦24) , alkenyl (C≦24) or a substituted form of either group; R 4 ''' is an alkyl (C1～C8) , alkenyl (C2～C8) or a substituted form of either group; and X 2 is a monovalent anion); (In formula (III), R 1 and R 2 are each independently alkyl (C8～C24) , alkenyl (C8～C24) or a substituted form of either group; R 3 , R 3 ', and R 3 '' are each independently alkyl (C≦6) or substituted alkyl (C≦6) and; R 4 But alkyl (C≦6) or substituted alkyl (C≦6) and X - is a monovalent anion); The method includes administering the therapeutic agent in combination with the SORT lipid composition to a subject in need thereof; wherein upon administration, the surface of the SORT lipid composition interacts with apolipoprotein E (Apo E) to a lesser extent compared to endogenous proteins in the subject that are not Apo E, thereby providing a lesser amount or activity of the therapeutic agent in the liver or cells therein in the subject compared to that achieved in the absence of the SORT lipid.

2. 10. The composition of claim 1, wherein upon administration, an endogenous protein in the subject that is not Apo E binds to the surface of the SORT lipid composition in the subject in an amount greater than Apo E.

3. 2. The composition of claim 1, wherein the endogenous protein in the subject that is not Apo E is vitronectin, β2 glycoprotein 1, or apolipoprotein H (Apo H).

4. 2. The composition of claim 1, wherein the endogenous protein in the subject that is not Apo E is β2 glycoprotein 1 or apolipoprotein H (Apo H).

5. 2. The composition of claim 1, wherein the endogenous non-Apo E protein in the subject is not β2 glycoprotein 1 or apolipoprotein H (Apo H), and the surface of the SORT lipid composition interacts with Apo H to a lesser extent than the endogenous protein in the subject.

6. The composition of claim 5, wherein the endogenous protein in the subject that is not Apo E, not β2-glycoprotein I, and not Apo H is vitronectin.

7. The composition of claim 1 , wherein the administering step comprises systemic administration.

8. 10. The composition of claim 1, wherein the administering step comprises intravenous administration.

9. 10. The composition of claim 1, wherein the method provides a greater amount or activity of the therapeutic agent in the lung or spleen or cells therein in the subject compared to that achieved in the absence of the SORT lipid.

10. 10. The composition of claim 1, wherein the SORT lipid is present in the SORT lipid composition at a molar percentage of about 5% to about 65%.

11. 10. The composition of claim 1, wherein the cationic SORT lipid is a permanent cationic SORT lipid.

12. 10. The composition of claim 1, wherein said cationic ionizable lipid is present in said SORT lipid composition at a molar percentage of about 5% to about 30%.

13. 10. The composition of claim 1, wherein the cationic ionizable lipid is a dendrimer or dendron, or a pharmaceutically acceptable salt thereof.

14. 10. The composition of claim 1, further comprising a phospholipid, a polymer-conjugated lipid, a steroid or steroid derivative, or a combination thereof, separate from the SORT lipid.

15. 10. The composition of claim 1, further comprising a polymer-bound lipid separate from the SORT lipid.

16. 16. The composition of claim 15, wherein the polymer-bound lipid comprises one or more hydrophobic moieties, each of which comprises at least 6 carbon atoms.

17. 16. The composition of claim 15, wherein the polymer-bound lipid comprises one or more hydrophobic moieties, each containing from 6 to 24 carbon atoms.

18. 16. The composition of claim 15, wherein the polymer-conjugated lipid comprises a polymer component having an average molecular weight of about 100 Daltons (Da) to about 15,000 Daltons.

19. 16. The composition of claim 15, wherein the polymer-conjugated lipid is a polyethylene glycol (PEG)-conjugated lipid.

20. 2. The composition of claim 1, wherein the therapeutic agent comprises a small-interfering ribonucleic acid (siRNA), microribonucleic acid (miRNA), primary microribonucleic acid (pri-miRNA), messenger ribonucleic acid (mRNA), clustered regularly interspaced short palindromic repeats (CRISPR)-associated nucleic acid, CRISPR-RNA (crRNA), single guide ribonucleic acid (sgRNA), trans-activating CRISPR ribonucleic acid (tracrRNA), plasmid deoxyribonucleic acid (pDNA), transfer ribonucleic acid (tRNA), antisense oligonucleotide (ASO), guide ribonucleic acid, double-stranded deoxyribonucleic acid (dsDNA), single-stranded deoxyribonucleic acid (ssDNA), single-stranded ribonucleic acid (ssRNA), double-stranded ribonucleic acid (dsRNA), protein, CRSIPR-associated (Cas) protein, or a combination thereof.

21. 10. The composition of claim 1, wherein said therapeutic agent is present in a ratio of about 1:1 to about 1:100 relative to said SORT lipid composition.

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