Compositions and methods for organ-specific delivery of nucleic acids

Abstract

The present disclosure provides compositions that exhibit preferential targeting or delivery of nucleic acid compositions to specific organs. In some embodiments, the compositions comprise steroids or sterols, ionizable cationic lipids, phospholipids, PEG lipids, and permanent cationic lipids, which can be used to deliver nucleic acids. The invention also provides the use of the composition in the preparation of a medicament for targeted delivery of a therapeutic agent to an organ or cells in an organ.

Description

[0001] This application is a divisional application of the Chinese invention patent application with application number 201980070622.2, application date September 4, 2019, and invention name “Compositions and methods for organ-specific delivery of nucleic acids”.

[0002] This application claims the benefit of priority to U.S. Provisional Application Serial No. 62 / 726,741, filed on September 4, 2018, the entire contents of which are hereby incorporated by reference. Technical Field

[0003] The present disclosure relates generally to the field of molecular biology. More specifically, it relates to tissue-specific delivery of therapeutic agents such as nucleic acids, proteins or small molecule therapeutic agents in lipid nanoparticles. Background Art

[0004] CRISPR / Cas (Clustered Regularly Interspaced Short Palindromic Repeats / CRISPR-associated protein (Cas)) technology can edit the genome in a precise, sequence-dependent manner, resulting in permanent changes. It holds the incredible promise of curing genetic diseases once and for all due to the ability to target disease-causing mutations. To date, successful editing has been primarily mediated by viral vectors, which require laborious customization for each target and present challenges to clinical translation due to immunogenicity, the production of antibodies that prevent repeated administration, and concerns about rare but dangerous integration events. There is a clear need to accomplish CRISPR / Cas editing via synthetic nanoparticles (NPs) to expand the safe and effective application of gene editing.

[0005] CRISPR / Cas achieves sequence-specific DNA editing through RNA-guided CRISPR-associated protein 9 (Cas9) nucleases or its homologs that form 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 a complementary genomic sequence with a 3′ pre-spacer adjacent motif (PAM) sequence. After DNA cleavage, the DSB repair pathway achieves directed mutagenesis, or insertion / deletion (indel) of the target gene. For therapeutic utility, transient Cas9 expression is preferred to limit off-target genomic changes. Since both the Cas9 protein and the sgRNA must be present in the same cell, the co-delivery of Cas9 mRNA and the sgRNA of the targeting sequence in one NP is an attractive approach, especially for in vivo use where tissue penetration and cellular uptake are more challenging. CRISPR / Cas editing using viruses, membrane deformation, ribonucleoprotein complex delivery, and hydrodynamic injection is functional, but has limitations that clinically hinder in vivo therapeutic use, including sustained expression and off-target editing of Cas9. Furthermore, these delivery systems are often not selective for the specific organ in which editing is desired. For example, most lipid nanoparticles accumulate through biological processes in the liver, thereby reducing the efficacy of the composition delivered into the target organ.

[0006] Similarly, other therapeutic agents such as proteins and small molecule therapeutic agents can benefit from organ-specific delivery. Many different types of compounds such as chemotherapeutic agents show significant cytotoxicity. If these compounds can be better directed to the desired organ, fewer off-target effects will be seen.

[0007] Therefore, there remains a need to develop new lipid nanoparticles that show preferential delivery to specific organs. Summary of the invention

[0008] In some aspects, the disclosure provides lipid compositions that demonstrate organ-specific delivery of lipid compositions. These compositions can be used to deliver nucleic acid components to specific organs.

[0009] In some aspects, the present disclosure provides compositions comprising:

[0010] (A) a therapeutic agent; and

[0011] (B) a lipid nanoparticle composition comprising:

[0012] (1) Selective organ-targeting compounds;

[0013] (2) ionizable cationic lipids; and

[0014] (3) phospholipids;

[0015] Wherein the composition preferentially delivers the nucleic acid to a target organ selected from the group consisting of 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, brain stem, cerebellum, spinal cord, eye, ear, tongue, or skin. In some embodiments, the target organ is selected from the group consisting of lung, heart, brain, spleen, lymph node, bone, bone marrow, skeletal muscle, stomach, small intestine, large intestine, kidney, bladder, breast, testis, ovary, uterus, spleen, thymus, brain stem, cerebellum, spinal cord, eye, ear, tongue, or skin.

[0016] In some embodiments, the target organ is a lung, a lymph node, or a 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.

[0017] In some embodiments, the selective organ targeting compound is a permanent cationic lipid. In some embodiments, the permanent cationic lipid is present in a molar percentage of about 5% to about 20% of the lipid nanoparticle composition. In some embodiments, the molar percentage of the permanent cationic lipid is 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 in a molar percentage of about 20% to about 65% of the lipid nanoparticle composition. In some embodiments, the molar percentage of the permanent cationic lipid is about 40% to about 61%. In some embodiments, the molar percentage of the permanent cationic lipid is about 50%.

[0018] In some embodiments, the permanent cationic lipid comprises a quaternary ammonium ion. In some embodiments, the permanent cationic lipid is further defined as:

[0019]

[0020] in:

[0021] R 1 and R 2 are independently alkyl (C8-C24) , alkenyl (C8-C24) or a substituted form of any group;

[0022] R 3 , R 3 ′ and R 3 "are each independently an alkyl (C≤6) or substituted alkyl (C≤6) ;

[0023] X- It is a monovalent anion.

[0024] In some embodiments, R 1 It is alkenyl (C8-C24) or substituted alkenyl (C8-C24) In some embodiments, R 2 It is alkenyl (C8-C24) or substituted alkenyl (C8-C24) In other embodiments, R 1 It is an alkyl (C8-C24) or substituted alkyl (C8-C24) In other embodiments, R 2 It is an alkyl (C8-C24) or substituted alkyl (C8-C24) In some embodiments, R 1 and R 2 In some embodiments, R 3 , R 3 ′ and R 3 ” are the same. In some embodiments, R 3 , R 3 ′ and R 3 " are each methyl. In some embodiments, X - is a halide anion such as bromide or chloride. In some embodiments, the permanent cationic lipid is further defined as:

[0025]

[0026] In other embodiments, the permanent cationic lipid is further defined as:

[0027]

[0028] in:

[0029] R 4 and R 4 ' are each independently alkyl (C6-C24) , alkenyl (C6-C24) or a substituted form of any group;

[0030] R 4 ″ is an alkyl (C≤24) , alkenyl (C≤24) or a substituted form of any group;

[0031] R 4 ″′ is an alkyl (C1-C8) , alkenyl (C2-C8) or a substituted form of either group; and

[0032] X 2It is a monovalent anion.

[0033] In some embodiments, R 4 It is an alkyl (C6-C24) or substituted alkyl (C6-C24) In some embodiments, R 4 ' is an alkyl (C6-C24) or substituted alkyl (C6-C24) In some embodiments, R 4 ″ is an alkyl (C≤24) or substituted alkyl (C≤24) In some embodiments, R 4 ″ is an alkyl (C≤8) or substituted alkyl (C≤8) In some embodiments, R 4 ″′ is an alkyl (C1-C8) or substituted alkyl (C1-C8) In some embodiments, X 2 is a halide such as chloride or bromide. In some embodiments, the permanent cationic lipid is further defined as:

[0034]

[0035] In some embodiments, the permanent cationic lipid is further defined as:

[0036]

[0037] in:

[0038] R 1 and R 2 are independently alkyl (C8-C24) , alkenyl (C8-C24) or a substituted form of any group;

[0039] R 3 , R 3 ′ and R 3 "are each independently an alkyl (C≤6) or substituted alkyl (C≤6) ;

[0040] R 4 It is an alkyl (C≤6) or substituted alkyl (C≤6) ;and

[0041] X - It is a monovalent anion.

[0042] In some embodiments, R 1 It is alkenyl (C8-C24) or substituted alkenyl(C8-C24) In some embodiments, R 2 It is alkenyl (C8-C24) or substituted alkenyl (C8-C24) In other embodiments, R 1 It is an alkyl (C8-C24) or substituted alkyl (C8-C24) In other embodiments, R 2 It is an alkyl (C8-C24) or substituted alkyl (C8-C24) In some embodiments, R 1 and R 2 Both are the same.

[0043] In some embodiments, R 3 , R 3 ′ and R 3 "Each is the same, such as R 3 , R 3 ′ and R 3 " are each methyl. In some embodiments, R 4 It is an alkyl (C≤6) In some embodiments, X - is a halide anion such as bromide or chloride.

[0044] In some embodiments, the permanent cationic lipid is further defined as:

[0045]

[0046] In other embodiments, the selective organ targeting compound is a permanent anionic lipid. In some embodiments, the permanent anionic lipid is present in a molar percentage of about 5% to about 50% of the lipid nanoparticle composition. In some embodiments, the molar percentage of the permanent anionic lipid is about 10% to about 45%. In some embodiments, the molar percentage of the permanent anionic lipid is about 30%. In some embodiments, the permanent anionic lipid comprises a phosphate group.

[0047] In some embodiments, the permanently anionic lipid is further defined as:

[0048]

[0049] in:

[0050] R 1 and R 2 are independently alkyl (C8-C24) , alkenyl (C8-C24) or a substituted form of any group;

[0051] R3 is hydrogen, alkyl (C≤6) or substituted alkyl (C≤6) or -Y 1 -R 4 ,in:

[0052] Y 1 Alkanediyl (C≤6) or substituted alkanediyl (C≤6) ;and

[0053] R 4 It is acyloxy (C≤8-24) or substituted acyloxy (C≤8-24) .

[0054] In some embodiments, R 1 It is alkenyl (C8-C24) or substituted alkenyl (C8-C24) In other embodiments, R 2 It is alkenyl (C8-C24) or substituted alkenyl (C8-C24) In other embodiments, R 1 It is an alkyl (C8-C24) or substituted alkyl (C8-C24) In other embodiments, R 2 It is an alkyl (C8-C24) or substituted alkyl (C8-C24) In some embodiments, R 1 and R 2 Both are the same.

[0055] In some embodiments, R 3 is hydrogen. In other embodiments, R 3 Yes - Y 1 -R 4 ,in:

[0056] Y 1 Alkanediyl (C≤6) or substituted alkanediyl (C≤6) ;and

[0057] R 4 It is acyloxy (C≤8-24) or substituted acyloxy (C≤8-24) .

[0058] In some embodiments, Y 1 is a substituted alkanediyl (C≤6) Such as 2-hydroxypropanediyl. In some embodiments, R 4 It is acyloxy (C≤8-24) In some embodiments, the permanent anionic lipid is further defined as:

[0059]

[0060] In other embodiments, the selective organ targeting compound is C 6 -C 24 In some embodiments, the diacylphosphatidylcholine is present in a molar percentage of about 5% to about 50% of the lipid nanoparticle composition. In some embodiments, the molar percentage of the diacylphosphatidylcholine is about 10% to about 45%, such as about 30%.

[0061] In some embodiments, the selective organ targeting compound comprises at least two fatty acid chains, a quaternary amine and an anionic phosphate group. In some embodiments, the diacylphosphatidylcholine is further defined as:

[0062]

[0063] in:

[0064] R 1 and R 2 are independently alkyl (C8-C24) , alkenyl (C8-C24) or a substituted form of any group;

[0065] R 3 , R 3 ′ and R 3 "are each independently an alkyl (C≤6) or substituted alkyl (C≤6) ;and

[0066] X - It is a monovalent anion.

[0067] In some embodiments, R 1 It is alkenyl (C8-C24) or substituted alkenyl (C8-C24) In some embodiments, R 2 It is alkenyl (C8-C24) or substituted alkenyl (C8-C24) In other embodiments, R 1 It is an alkyl (C8-C24) or substituted alkyl (C8-C24) In other embodiments, R 2 It is an alkyl (C8-C24) or substituted alkyl (C8-C24) In some embodiments, R 1 and R 2 Both are the same.

[0068] In some embodiments, R 3 , R3 ′ and R 3 ” are the same. In some embodiments, R 3 , R 3 ′ and R 3 " are each methyl. In some embodiments, X - is a halide anion such as bromide or chloride. In some embodiments, the diacylphosphatidylcholine is further defined as:

[0069]

[0070] In some embodiments, the ionizable cationic lipid is present in a molar percentage of about 5% to about 30% of the lipid nanoparticle composition. In some embodiments, the molar percentage of the ionizable cationic lipid is about 7.5% to about 20%. In some embodiments, the molar percentage of the ionizable cationic lipid is about 11.9%. In some embodiments, the ionizable cationic lipid is present in a molar percentage of about 15% to about 30% of the lipid nanoparticle composition. In some embodiments, the molar percentage of the ionizable cationic lipid is about 15% to about 25%. In some embodiments, the molar percentage of the ionizable cationic lipid is about 20.3%.

[0071] In some embodiments, the ionizable cationic 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 ionizable cationic lipid is a dendrimer or dendron. In some embodiments, the ionizable cationic lipid comprises at least two C6-C24 alkyl or alkenyl groups. In some embodiments, the ionizable cationic lipid comprises at least two C8-C24 alkyl groups.

[0072] In some embodiments, the phospholipid is present at a molar percentage of about 8% to about 20% of the lipid nanoparticle composition. 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 about 20% to about 23% of the lipid nanoparticle composition. 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.

[0073] In some embodiments, the composition further comprises a steroid. In some embodiments, the steroid is present at a molar percentage of about 39% to about 46% of the lipid nanoparticle composition. In some embodiments, the molar percentage of the steroid is about 40% to about 43%. In some embodiments, the molar percentage of the steroid is about 40.5%. In other embodiments, the steroid is present at a molar percentage of about 15% to about 39% of the lipid nanoparticle composition. In some embodiments, the molar percentage of the steroid is about 20% to about 27.5%. In some embodiments, the molar percentage of the steroid is about 23.8%. In some embodiments, the steroid is cholesterol.

[0074] 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% of the lipid nanoparticle composition. In some embodiments, the molar percentage of the pegylated lipid is about 0.5% to about 5.0%. In other embodiments, the molar percentage of the pegylated lipid is about 2.0% to about 2.8%. In some embodiments, the molar percentage of the pegylated lipid is about 2.4%. In other embodiments, the pegylated lipid is present at a molar percentage of about 3.9% to about 4.6% of the lipid nanoparticle composition. In some embodiments, the molar percentage of the pegylated lipid is about 4.0% to about 4.3%. In some embodiments, the molar percentage of the pegylated lipid is about 4.1%. In some embodiments, the pegylated lipid comprises a PEG component of about 1000 to about 10,000 daltons. In some embodiments, the PEG lipid is a pegylated diacylglycerol. In some embodiments, the PEG lipid is further defined by the formula:

[0075]

[0076] in:

[0077] R 12 and R 13 are independently alkyl (C≤24) , alkenyl (C≤24) or a substituted form of any of these groups;

[0078] R e is hydrogen, alkyl (C≤8) or substituted alkyl (C≤8) ;and

[0079] x is 1-250.

[0080] In some embodiments, the PEG lipid is dimyristoyl-sn-glycerol or a compound of the formula:

[0081]

[0082] in:

[0083] n 1 is 5-250; and

[0084] n 2 and n 3 Each independently is 2-25.

[0085] In some embodiments, the composition comprises cholesterol and DMG-PEG. In some embodiments, the composition comprises DOPE. In other embodiments, the composition comprises DSPC. In some embodiments, the composition further comprises DLin-MC3-DMA. In other embodiments, the composition further comprises C12-200. In some embodiments, the composition further comprises 3A5-SC8, 3A3-SC8, 4A1-SC8, 4A3-SC8, 5A2-SC8 with five tails or 5A2-SC8 with six tails. In some embodiments, the composition further comprises 5A2-SC8. In some embodiments, the composition further comprises DOTAP. In some embodiments, the composition comprises cholesterol, DMG-PEG, DSPC, DLin-MC3-DMA and DOTAP.

[0086] In some embodiments, the therapeutic agent is a small molecule such as a small molecule selected from the group consisting of anticancer agents, antifungal agents, psychiatric agents such as analgesics, consciousness-altering agents such as anesthetics or hypnotics, nonsteroidal anti-inflammatory drugs (NSAIDS), anthelmintics, anti-acne agents, antianginal agents, antiarrhythmic agents, antiasthmatic agents, antibacterial agents, anti-benign prostatic hypertrophy agents, anticoagulants, antidepressants, antidiabetic agents, antiemetics, antiepileptics, antigout agents, antihypertensive agents, anti-inflammatory agents, antimalarials, antimigraine agents, antimuscarinics, antitumor agents, antiobesity agents, anti Osteoporosis agent, anti-Parkinson's syndrome drug, antiproliferative agent, antiprotozoal agent, antithyroid agent, antitussive, anti-incontinence agent, antiviral agent, antianxiety agent, appetite suppressant, beta-blocker, cardiac positive inotropic agent, chemotherapeutic drug, cognitive enhancer, contraceptive, corticosteroid, Cox-2 inhibitor, diuretic, erectile dysfunction improver, expectorant, gastrointestinal agent, histamine receptor antagonist, immunosuppressant, keratolytic agent, lipid regulator, leukotriene inhibitor, macrolide, muscle relaxant, neuroleptic, nutrient, narcotic analgesic, protease inhibitor or sedative. In other embodiments, the therapeutic agent is protein. In other embodiments, the therapeutic agent is nucleic acid such as therapeutic nucleic acid. In some embodiments, the nucleic acid is siRNA, miRNA, primary-miRNA, messenger RNA (mRNA), clustered regularly interspaced short palindromic repeats (CRISPR)-related nucleic acids, single guide RNA (sgRNA), CRISPR-RNA (crRNA), trans-activated crRNA (tracrRNA), plasmid DNA (pDNA), transfer RNA (tRNA), antisense oligonucleotide (ASO), guide RNA, double-stranded DNA (dsDNA), single-stranded DNA (ssDNA), single-stranded RNA (ssRNA) and double-stranded RNA (dsRNA). In some embodiments, the composition comprises a first nucleic acid and a second nucleic acid. In some embodiments, the first nucleic acid is a messenger RNA. In some embodiments, the second nucleic acid is a single guide RNA. In some embodiments, the first nucleic acid is a messenger RNA (mRNA) and a single guide RNA (sgRNA). In some embodiments, the nucleic acid is present in a ratio of about 1:1 to about 1:100 lipid nanoparticle composition: nucleic acid. In some embodiments, the ratio is about 1:10 to about 1:60. In some embodiments, the ratio is about 1:40.

[0087] In some embodiments, the composition further comprises a protein. In some embodiments, the protein is a protein related to translation or transcription. In some embodiments, the protein is related to the CRISPR process. In some embodiments, the protein is a CRISPR-related protein. In some embodiments, the protein is Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, a homologue thereof, or a modified form thereof. In some embodiments, the protein is Cas9. In some embodiments, the protein and the nucleic acid are present in a molar ratio of about 1:1 to about 1:20. In some embodiments, the molar ratio is about 1:1 to about 1:10. In some embodiments, the molar ratio is about 1:3 to about 1:8.

[0088] In some embodiments, the composition has a negative zeta potential. In some embodiments, the zeta potential is -0.25 mV to about -10 mV. In some embodiments, the zeta potential is about -0.5 mV to about -2 mV. In some embodiments, the composition comprises both protein and nucleic acid. In some embodiments, the composition comprises Cas9 protein and a single guide nucleic acid. In some embodiments, the composition comprises Cas9 protein, a single guide nucleic acid, and a donor DNA.

[0089] In another aspect, the present disclosure provides a pharmaceutical composition comprising:

[0090] (A) a composition as described herein; and

[0091] (B) Excipients.

[0092] In some embodiments, the pharmaceutical composition is formulated for administration as follows: orally, intra-adipose, intra-arterial, intra-articular, intra-cranial, intra-dermal, intralesional, intra-muscular, intra-nasal, intra-ocular, intra-pericardial, intra-peritoneal, intra-pleural, intra-prostatic, intra-rectal, intra-thecal, intra-tracheal, intra-tumor, intra-umbilical, intra-vaginal, intra-venous, intra-vesical, intra-vitreal, in the form of liposomes, topically, mucosally, parenterally, rectally, sub-conjunctivally, subcutaneously, sub-lingually, topically, buccally, transdermally, vaginally, in the form of creams, in the form of lipid compositions, via catheters, via lavages, via continuous infusions, via infusions, via inhalation, via injections, via local delivery, or via local perfusions. In some embodiments, the pharmaceutical composition is formulated for intravenous or intra-arterial injection. In some embodiments, the excipient is a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutically acceptable carrier is a solvent or a solution. In some embodiments, the pharmaceutical composition is formulated as a unit dosage.

[0093] In another aspect, the disclosure provides a method of regulating gene expression 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.

[0094] In some embodiments, the cells are contacted in vitro or ex vivo. In some embodiments, the cells are contacted in vivo. In some embodiments, the modulation of gene expression is sufficient to treat a disease or condition such as cancer.

[0095] In yet another aspect, the disclosure provides a method of treating a disease or condition in a patient, the method comprising administering to a patient in need thereof 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 condition.

[0096] In some embodiments, the disease or condition 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 twice or more. In some embodiments, the patient is a mammal such as a human.

[0097] In other aspects, the present disclosure provides a method of preparing lipid nanoparticles, comprising:

[0098] (A) dissolving a selective organ targeting compound, an ionizable cationic lipid and a phospholipid in a first solution to form a lipid solution, wherein the lipid solution is formed in an organic solvent;

[0099] (B) dissolving the therapeutic agent in a buffer, wherein the buffer is a buffer having a pH of about 6.8 to about 7.6 to form a buffered therapeutic agent solution; and

[0100] (C) mixing the lipid solution with a buffered therapeutic agent solution to form lipid nanoparticles. In other aspects, the present disclosure provides a method for preparing lipid nanoparticles, comprising:

[0101] (A) dissolving a permanent cationic lipid, an ionizable cationic lipid, and a phospholipid in a first solution to form a lipid solution, wherein the lipid solution is formed in an organic solvent;

[0102] (B) dissolving the therapeutic agent in a buffer, wherein the buffer is a buffer having a pH of about 6.8 to about 7.6 to form a buffered therapeutic agent solution; and

[0103] (C) The lipid solution is mixed with a buffered therapeutic agent solution to form lipid nanoparticles.

[0104] 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 method has an encapsulation efficiency greater than 80%.

[0105] In another aspect, the present disclosure provides a composition comprising:

[0106] (A) therapeutic agent;

[0107] (B) a lipid nanoparticle composition comprising:

[0108] (1) Ionizable cationic lipids;

[0109] (2) phospholipids; and

[0110] (3) selective organ-targeting compounds;

[0111] wherein the organ targeting ligand causes preferential delivery of the composition to organs other than the liver.

[0112] In yet another aspect, the present disclosure provides a composition comprising:

[0113] (A) therapeutic agent;

[0114] (B) a lipid nanoparticle composition comprising:

[0115] (1) Ionizable cationic lipids;

[0116] (2) phospholipids;

[0117] (3) selective organ-targeting compounds;

[0118] (4) steroids; and

[0119] (5) PEG lipids;

[0120] wherein the organ targeting ligand causes preferential delivery of the composition to organs other than the liver.

[0121] In another aspect, the present disclosure provides a composition comprising a therapeutic agent and a lipid nanoparticle composition comprising:

[0122] (A) ionizable cationic lipids;

[0123] (B) phospholipids; and

[0124] (C) selective organ-targeting compounds;

[0125] wherein the composition has an apparent pK of about 8 to about 13 a And the composition delivers the nucleic acid primarily to the lung.

[0126] In another aspect, the present disclosure provides a composition comprising a therapeutic agent and a lipid nanoparticle composition comprising:

[0127] (A) ionizable cationic lipids;

[0128] (B) phospholipids;

[0129] (C) selective organ-targeting compounds;

[0130] (D) steroids; and

[0131] (E) PEG lipids;

[0132] wherein the composition has an apparent pK of about 8 to about 13 a And the composition delivers the nucleic acid primarily to the lungs.

[0133] In another aspect, the present disclosure provides a composition comprising a therapeutic agent and a lipid nanoparticle composition comprising:

[0134] (A) ionizable cationic lipids;

[0135] (B) phospholipids; and

[0136] (C) selective organ-targeting compounds;

[0137] wherein the composition has an apparent pK of about 3 to about 6 a And the composition delivers the nucleic acid primarily to the spleen.

[0138] In yet another aspect, the present disclosure provides a composition comprising a therapeutic agent and a lipid nanoparticle composition comprising:

[0139] (A) Steroids;

[0140] (B) ionizable cationic lipids;

[0141] (C) phospholipids;

[0142] (D) PEG lipids; and

[0143] (E) Selective organ-targeting compounds;

[0144] wherein the composition has an apparent pK of about 3 to about 6 a And the composition delivers the nucleic acid primarily to the spleen.

[0145] In another aspect, the present disclosure provides a composition comprising a therapeutic agent and a lipid nanoparticle composition comprising:

[0146] (A) ionizable cationic lipids;

[0147] (B) phospholipids; and

[0148] (C)C 6 -C 24 diacylphosphatidylcholine;

[0149] wherein the composition delivers the nucleic acid primarily to the lymph nodes.

[0150] In another aspect, the present disclosure provides a composition comprising a therapeutic agent and a lipid nanoparticle composition comprising:

[0151] (A) Steroids;

[0152] (B) ionizable cationic lipids;

[0153] (C) phospholipids;

[0154] (D) PEG lipids; and

[0155] (E)C 6 -C 24 diacylphosphatidylcholine;

[0156] wherein the composition delivers the nucleic acid primarily to the lymph nodes.

[0157] In yet another aspect, the present disclosure provides a composition comprising a therapeutic agent and a lipid nanoparticle composition comprising:

[0158] (A) ionizable cationic lipids;

[0159] (B) phospholipids; and

[0160] (C) selective organ-targeting compounds;

[0161] wherein the surface of the composition interacts with vitronectin and the composition delivers the nucleic acid primarily to the lung.

[0162] In another aspect, the present disclosure provides a composition comprising a therapeutic agent and a lipid nanoparticle composition comprising:

[0163] (A) ionizable cationic lipids;

[0164] (B) phospholipids;

[0165] (C) selective organ-targeting compounds;

[0166] (D) steroids; and

[0167] (E) PEG lipids;

[0168] wherein the surface of the composition interacts with vitronectin and the composition delivers the nucleic acid primarily to the lung.

[0169] In yet another aspect, the present disclosure provides a composition comprising a therapeutic agent and a lipid nanoparticle composition comprising:

[0170] (A) ionizable cationic lipids;

[0171] (B) phospholipids; and

[0172] (C) selective organ-targeting compounds;

[0173] wherein the surface of the composition interacts with Apo H and the composition delivers the nucleic acid primarily to the spleen.

[0174] In another aspect, the present disclosure provides a composition comprising a therapeutic agent and a lipid nanoparticle composition comprising:

[0175] (A) Steroids;

[0176] (B) ionizable cationic lipids;

[0177] (C) phospholipids;

[0178] (D) PEG lipids; and

[0179] (E) Selective organ-targeting compounds;

[0180] wherein the surface of the composition interacts with Apo H and the composition delivers the nucleic acid primarily to the spleen.

[0181] In yet another aspect, the present disclosure provides a composition comprising a therapeutic agent and a lipid nanoparticle composition, wherein a targeting protein in a protein corona present at the surface of the composition binds to a target protein primarily present in a target organ, wherein the target organ is not the liver.

[0182] In some embodiments, the targeting protein is selected from vitronectin or β2-glycoprotein I (Apo H). In some embodiments, the targeting protein is vitronectin and the target organ is the lung. In other embodiments, the targeting protein is Apo H and the target organ is the spleen.

[0183] In some embodiments, the lipid nanoparticle composition further comprises a selective organ targeting compound that modifies 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 agent, a vitamin, or a protein. In some embodiments, the selective organ targeting compound is a lipid such as a permanent cationic lipid, a permanent anionic lipid, or a phosphatidylcholine. In some embodiments, the lipid nanoparticle composition further comprises an ionizable cationic 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.

[0184] In another aspect, the 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 results in an apparent pK of about 3 to about 6. a Lipid nanoparticle composition.

[0185] In another aspect, the 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 results in an apparent pK of about 8 to about 13. a Lipid nanoparticle composition.

[0186] As used herein, "substantially free" with respect to a particular component is used herein to indicate that the particular component is not intentionally formulated in the composition and / or is present only as a contaminant or in trace amounts. The total amount of the particular component caused by any unintentional contamination of the composition is preferably less than 0.01%. Most preferred are compositions in which the amount of the particular component cannot be detected by standard analytical methods.

[0187] As used herein in the specification and claims, "a" or "an" may refer to one or more. As used herein in the specification and claims, when used in conjunction with the word "comprising", the words "a" or "an" may refer to one or more than one. As used herein, in the specification and claims, "another" or "a further" may refer to at least a second or more.

[0188] As used herein in the specification and claims, the term "about" is used to indicate that a value includes the inherent variation of error for the device, method employed to determine the value, or the variation that exists between study subjects.

[0189] Other objects, features and advantages of the present disclosure will be apparent from the following detailed description. However, it should be understood that although the detailed description and specific examples indicate certain embodiments of the present disclosure, they are given by way of illustration only, because those skilled in the art will appreciate various changes and modifications within the spirit and scope of the present disclosure from the detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0190] The following drawings form part of this 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.

[0191] Fig. 1A-C: DOTAP mDLNP preparation mediates excellent mRNA delivery efficacy and shows tissue-specific delivery characteristics with a given percentage of DOTAP after low-dose intravenous injection. (Fig. 1A) Schematic diagram of DOTAP mDLNP formation and the structure of DOTAP. The molar ratio of 5A2-SC8 / DOPE / Chol / DMG-PEG is fixed to 15 / 15 / 30 / 3 (named mDLNP). Then only the DOTAP ratio is adjusted from 0 to 1200 to produce a series of DOTAP mDLNP preparations and named DOTAPY, wherein Y represents the percentage of DOTAP in total lipids. (Fig. 1B) In vitro images (n=2) of luciferase in major organs 6h after intravenous injection with a dose of 0.1mg / kg Luc mRNA. As the molar percentage of DOTAP increases, luciferase protein expression is transferred from the liver to the spleen and then to the lungs. (Fig. 1C) Quantitative data confirm that DOTAP percentage is a factor in tissue-specific delivery, with mDLNP (0%) being the best for liver, DOTAP10-15 (similar between them) being the best for spleen, and DOTAP50 being the best for lung. Because luciferase expression was detected only in liver, spleen, and lung after intravenous injection, the relative expression in each organ was calculated. Obviously, the higher the percentage of DOTAP (permanent cationic lipid) in the preparation, the less luminescence in the liver, and when>70%, it is close to 0. However, the higher the percentage of DOTAP, the more luminescence in the lung, and when>70%, it is close to 100%. DOTAP5-20 shows a higher percentage in the spleen, and DOTAP10 appears to be the highest.

[0192] Figures 2A1-2F2: The structure of lipids determines the mRNA expression profile after intravenous injection. Generally, as the percentage increases, quaternary lipids change mRNA delivery from the liver to the spleen and then to the lungs, and zwitterionic lipids help deliver mRNA into the spleen at a higher percentage. However, tertiary amine lipids do not change the mRNA expression organ, but improve the delivery efficiency in the liver. In order to further confirm the delivery trend of quaternary lipid mDLNP, two other quaternary lipids DDAB and EPC were selected for in vivo mRNA delivery, and their modification strategy was the same as DOTAP. (Figure 2A1, Figure 2B1) DDAB and EPC show large structural differences between them and DOTAP, including at three comparison levels: the length of the hydrophobic tail, the chemical structure of the saturated and unsaturated bonds and the head group. Formulations containing 5%, 15%, 40% and 50% of quaternary lipids were formed to detect size distribution and in vivo evaluation (0.1mg / kg, 6h, n=2). (Figure 2A2, Figure 2B2) Like DOTAP mDLNP, DDAB and EPC also present similar mRNA delivery profiles. Lower cation percentage (5%) delivers mRNA to liver and spleen, and then more to spleen when increased to 15%. Once increased to 40%, mRNA expression is hardly observed in liver and spleen, but lungs show high luciferase signals, and then decrease at 50%. These results are very similar to DOTAP mDLNP, which suggests that mDLNP functionalized by quaternary lipids is a universal and generalizable strategy for mRNA delivery to targeted tissues. Then, just like the DOTAP strategy, representative zwitterionic lipids DSPC and DOCPe are used to evaluate in vivo mRNA delivery (Figure 2C1, Figure 2D1). The structures of DSPC and DOCPe lipids belong to the general category of zwitterionic lipids. Before intravenous injection, the size distribution of DSPC and DOCPe mDLNP preparations was tested by DLS. In this article, DSPC and DOCPe show two horizontal contrasts in structure: saturation relative to unsaturated hydrophobic tails, and charge position relative to in the head group. (Figure 2C2, Figure 2D2) Interestingly, similar mRNA expression profiles like quaternary lipid preparations were not observed. Instead, DSPC and DOCPe improved mRNA delivery to the spleen within a given range (less than 80% in DSPC and less than 50% in DOCPe), and no signal was seen in the lungs regardless of the percentage (0.1 mg / kg, 6 h, n=2). Inspired by these results, ionizable tertiary amine lipids, DODAP and C12-200 were further tested with the same strategy. DODAP has the same structure as DOTAP except for the head group (quaternary amine relative to tertiary amine), and C12-200 is an effective lipid for siRNA or mRNA delivery, which has a completely different structure from DODAP.(Fig. 2E1, Fig. 2F1) Similarly, the size distribution of the two modified mDLNPs is still good at certain percentages (less than 80%). (Fig. 2E2, Fig. 2F2) Surprisingly, DODAP and C12-200 cannot change the mRNA expression profile (different effects compared to quaternary lipids or zwitterionic lipids). Instead, DODAP and C12-200 increase the delivery of mRNA to the liver. Supporting this, DODAP20 and C12-200 show much better delivery efficacy (0.1 mg / kg, 6h, n=2) than the original mDLNP formulation. As the percentage of DODAP or C12-200 increases (50% or 80%), the luciferase signal decreases a lot, but the liver is still the main organ, not the spleen or lungs.

[0193] Figure 3A-C: To determine why various lipids can induce large differences in mRNA expression in organs, distribution assays and pK a Detection. Biodistribution and pK a All have an effect on the mRNA expression profile in organs. (Fig. 3A) Organ distribution of Cy5.5-LucmRNA preparations delivered by three modified mDLNPs (DOTAP (quaternary lipid), DSPC (zwitterionic lipid) and DODAP (tertiary amine lipid)). C57BL / 6 mice were injected intravenously at a dose of 0.5mg / kg and imaged 6 hours after injection (n=2). Compared with the original mDLNP preparation (without DOTAP), DOTAP changed the organ distribution of mRNA, and both DOTAP10 and DOTAP50 can deliver mRNA to the lungs, and DOTAP50 increases more, which can partially explain why DOTAP preparations mediate mRNA expression in the lungs at a higher percentage. However, even at 80% (DSPC) or 50% (DODAP) percentages, DSPC and DODAP cannot change mRNA distribution more. It is also noted that, as shown in Figures 1 and 2, for DOTAP50 and DSPC80, mRNA is retained in the liver, the former is a lung-targeted NP, and the latter is a spleen-targeted NP. Therefore, distribution is not the only factor explaining this mechanism. (Figure 3B) The pK values ​​of all tested and effective formulations were then measured. a , including original mDLNP, DOTAP, DDAB, EPC, DSCP, DOCPe, DODAP, and C12-200 modified formulations. (Figure 3C) Finally, based on the defined rules, the pK a Here, eight scoring rules were designed as shown in the table. It is obvious that all liver-targeted agents have narrow pK a (~6-7), spleen-targeted formulations have no obvious range, but lung-targeted delivery requires a high pKa (>9.25).

[0194] Figures 4A-4C : Liver and lung gene editing was achieved in both Td-Tomato and C57BL / 6 mice. (Figure 4A) Schematic diagram showing that co-delivery of Cas9 mRNA and sgTom1 activated Td-Tomato expression in Td-Tomato mice. (Figure 4B) Td-Tomato expression was induced in the liver and lungs when treated with mDLNP and DOTAP50 formulations, respectively. Mice were intravenously injected with mDLNP and DOTAP50 formulations to co-deliver IVT Cas9 mRNA and modified sgTom1 (4 / 1, weight / weight) at a total dose of 2.5 mg / kg (50 μg each), and then the fluorescence of major organs was detected on day 10 after treatment. (Figure 4C) T7E1 assays showed that tissue-specific features were further confirmed by in vivo PTEN editing. C57 BL6 mice were intravenously injected with mDLNP, DODAP20 or DOTAP50 to achieve tissue-specific gene editing, with a total dose of 2.5 mg / kg (50 μg each), a weight ratio of IVT Cas9 mRNA to modified sgPTEN of 4 / 1, and the detection time was 10 days after treatment.

[0195] Figure 5A-C: Characterization of DOTAP mDLNP formulations (n=3). Size, PDI (Figure 5A) and zeta potential (Figure 5B) were examined by dynamic light scattering (DLS). (Figure 5C) Encapsulation efficiency (EE%) was tested by Ribogreen RNA assay.

[0196] Fig. 6A-C: DOTAP preparations show excellent mRNA delivery efficiency, and these goods (such as proteins) that are not well tolerated for ethanol or acidic buffer show delivery potential. (Fig. 6A) DOTAP mDLNPs mediate high Luc mRNA expression in Huh-7 cells and A549 cells, and reveal that 5%-50% of DOTAP percentage is better for mRNA delivery, and 10% is the best. 24h after transfection, Luc mRNA expression and cell viability (n=4) were tested with a dose of 50ng / well mRNA. Here, DOTAP mDLNPs (10mM, pH 4.0) were formed in PBS instead of citric acid buffer. (Fig. 6B and 6C) The reduced ethanol volume percentage does not affect characterization and mRNA delivery efficacy. In order to test the effect of ethanol on mRNA delivery, DOTAP25 was selected as a model and four preparations with different volume ratios (1:3, 1:5, 1:7.5 and 1:10) of ethanol to PBS were formed. All four formulations showed similar EE, size and PDI in FaDu cells (Figure 6B), and exhibited equal mRNA delivery efficacy (Figure 6C) (50ng / well of mRNA, 24h, n=4). Therefore, the formulation was optimized using 1XPBS (pH 7.4) instead of acidic buffer (10mM pH 4.0), and the ethanol percentage was significantly reduced, which provides the possibility that the DOTAP formulation delivers those cargoes that are not well tolerated in high ethanol concentrations or acidic buffers, such as proteins.

[0197] Figure 7 : Quantitative data of biodistribution in major organs. C57BL6 mice were injected intravenously with various Cy5.5-Luc mRNA preparations at a dose of 0.5 mg / kg (n=2). After 6 hours, the heart, lungs, liver, spleen and kidneys were isolated, imaged and quantified.

[0198] Figures 8A-8C:For DOTAP10 preparations formed by PBS or citric acid buffer, there is no big difference in size distribution and Luc mRNA delivery efficacy, but it is not applicable to DSPC50 and DODAP50. In order to test the effect of buffer on mRNA delivery efficacy in vivo, DOTAP10 (quaternary lipid), DSPC50 (zwitterionic lipid) and DODAP50 (tertiary amine lipid) were selected. Each Luc mRNA preparation was intravenously injected into C57 BL6 mice at a dose of 0.1 mg / kg, and after 6 hours, the main organs were separated and imaged (n=2). (Fig. 8A) Between DOTAP10 formed by PBS and citric acid buffer (10mM, pH 4.0), size and delivery efficacy did not change much. (Fig. 8B and 8C) However, DSPC50 and DODAP50 formed by citric acid buffer significantly improved the mRNA delivery effect, although there was no big difference in size distribution. DOTAP (or another permanent cationic lipid) can be added for forming LNP at neutral pH (e.g., 7.4 PBS buffer).

[0199] Fig. 9 : Western blot results of quality test of IVT Cas9 mRNA delivered by mDLNP. In order to achieve tissue-specific gene editing, Cas9 mRNA and sgRNA were designed to be co-delivered. First, Cas9 mRNA was prepared by IVT and quality tested by western blot analysis. In this assay, Cas9 pDNA was delivered by both Lipofectamine 2000 and commercial Cas9 mRNA (TriLink). mDLNP was a positive control, and mCherry mDLNP was a negative control. One day before transfection, 293T cells were seeded in 12-well plates, and the cells were treated for 24 hours per condition before western blotting. The effect of IVT Cas9 mRNA was much better than that of commercial mRNA, so IVT Cas9 mRNA was used for in vivo gene editing.

[0200] Figures 10A and 10B: sgRNA screening and weight ratio (Cas9mRNA / sgRNA) optimization in Td-Tomato mice. In order 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 preparations and td-tomato expression in the liver. Three types of mDLNPs were injected intravenously into mice at a total dose of 3mg / kg (Cas9 / sgRNA, 4 / 1, weight / weight), and organs were imaged on the 7th day. It seems that sgTom1 is the leader among the three candidates. (Figure 10B) SgTom1 was selected and further tested for liver gene editing by different weight ratios (Cas9 / sgRNA) of 2 / 1, 4 / 1 and 6 / 1. mDLNP was used for intravenous injection at a dosage of 3mg / kg, and td-tomato expression was detected on the 7th day. In this example, 4 / 1 performs better than 2 / 1 and 6 / 1.

[0201] Figures 11A-11G: Characterization of Cas9 / sgRNA complexes and DOTNP lipid nanoparticles after encapsulation of Cas9 / sgRNA complexes. Size (FIG. 11A) and zeta potential (FIG. 11B) of Cas9 / sgLUC complexes (mol / mol=1 / 1) in PBS (pH 7.4) and in citrate buffer (pH 4.2). The size of the Cas9 / sgLUC complex prepared in citrate buffer is very large (greater than 100nm) and the zeta potential is positively charged, so it is impossible to be encapsulated by lipid nanoparticles. However, the size of the Cas9 / sgLUC complex prepared in PBS is compact (less than 20nm) and has a negative charge, so it can be encapsulated by lipid nanoparticles. Size (FIG. 11C) and zeta potential (FIG. 11D) of Cas9 / sgLUC complexes prepared with different Cas9 / sgRNA molar ratios (1 / 1, 1 / 3, and 1 / 5). Compared with the Cas9 / sgLUC complex (1 / 1, mol / mol), higher molar ratios (1 / 3 and 1 / 5, mol / mol) showed smaller size and more negative charge, which is beneficial for lipid nanoparticle encapsulation. The size (Figure 11E) and zeta potential (Figure 11F) of DOTNP10 lipid nanoparticles (named DOTNP10-L) encapsulating Cas9 / sgLUC complexes when prepared at different molar ratios (1 / 1, / 3, 1 / 5). (Figure 11G) TEM image of DOTNP10-L (1 / 3, mol / mol). DOTAP lipid nanoparticles are composed of five components (including 5A2-SC8, cholesterol, DOPE, DMG-PEG and DOTAP). The molar ratio of 5A2-SC8, cholesterol, DOPE and DMG-PEG is fixed (15:15:30:5, mol / mol), and DOTNP X Refers to DOTNP with different molar percentages of DOTAP. Here, different sgRNAs were used, including sgLUC, sgGFP, sgTOM, sgPTEN, etc. To distinguish them, the first letter of each gene was added to the end of DOTNP. For example, DOTNP10-L refers to DOTNP10 lipid nanoparticles encapsulating Cas9 / sgLUC complex; DOTNP10-G refers to DOTNP10 lipid nanoparticles encapsulating Cas9 / sgGFP complex.

[0202] Figures 12A-12F:DOTNP lipid nanoparticles can deliver Cas9 / sgRNA complexes into the nucleus and show effective gene editing in vitro. (Figure 12A) Confocal images of Hela-Luc cells after incubation for 1h, 3h, 6h and 24h with DOTNP10 encapsulating Cas9-EGFP / sgLUC complexes (1 / 3, mol / mol) (using 9nM sgRNA). Green: EGFP-fused Cas9 protein; Blue: Nuclei stained with Hoechst 33342. The red arrow indicates the process of DOTNP10 entering the nucleus. (Figure 12B) The percentage of indels at the LUC locus after incubation for 3 days with DOTNP10-L at different molar ratios was analyzed by TIDE sequencing (using 24nM sgRNA). DOTNP10 lipid nanoparticles (DOTNP10-G) encapsulating Cas9 / sgGFP were used as negative controls. Here, two commercial Cas9 proteins (GeneArt Cas9 and Truecut Cas9) were used. (FIG. 12C) T7EI cleavage assay of Hela-Luc cells incubated with different formulations (using 24 nM sgRNA). 1. 100 bp DNA ladder; 2. PBS; 3. DOTNP10-G (1 / 3); 4. DOTNP10-L (1 / 1); 5. DOTNP10-L (1 / 3); 6. DOTNP10-L (1 / 5); 7. DOTNP10-L (1 / 3) prepared in citrate buffer. Two commercial Cas9 proteins (GeneArtCas9 and Truecut Cas9) were used. Among them, a molar ratio of 1 / 3 showed the best gene editing when Truecut Cas9 protein was used. (FIG. 12D) Fluorescence microscopy images of SKOV3-GFP cells incubated with DOTNP10-L and DOTNP10-G (using 24 nM sgRNA). Here, DOTNP10-L was used as a negative control. (FIG. 12E) Flow cytometry analysis of SKOV3-GFP cells incubated with DOTNP10-L and DOTNP10-G. (FIG. 12F) Mean fluorescence intensity of SKOV3-GFP cells incubated with DOTNP10-L and DOTNP10-G measured by flow cytometry.

[0203] Figures 13A and 13B: DOTNP shows tissue-specific gene editing in vivo. (Figure 13A) In vitro images of tdTomato fluorescence in major organs 7 days after intravenous injection of different formulations (1.5mg / kg sgRNA / mouse). DOTNP5-T refers to DOTNP5 lipid nanoparticles encapsulating Cas9 / sgTom complexes; DOTNP10-T refers to DOTNP10 lipid nanoparticles encapsulating Cas9 / sgTom complexes; DOTNP50-T refers to DOTNP50 lipid nanoparticles encapsulating Cas9 / sgTom complexes. tdTomato fluorescence was observed only in the liver in the DOTNP5-T treated group; in the DOTNP10-T group, slight fluorescence was visible in the lungs, and if the DOTAP dose was further increased to 50% (DOTNP50-T), most of the tdTomato fluorescence was observed in the lungs. (FIG. 13B) T7EI cleavage assay (2 mg / kg sgRNA / mouse) in liver and lung organs after incubation with DOTNP5-P (DOTNP5 lipid nanoparticles encapsulating Cas9 / sgPTEN complex), DOTNP10-P (DOTNP10 lipid nanoparticles encapsulating Cas9 / sgPTEN complex), and DOTNP50-P (DOTNP50 lipid nanoparticles encapsulating Cas9 / sgPTEN complex). The results are consistent with those obtained by ex vivo imaging. Gene editing was detected only in the liver after treatment with DOTNP5-P; when incubated with DOTNP10-P, gene editing was obtained in both the liver and lungs; while in the DOTNP50-P treated group, most gene editing was observed in the lungs.

[0204] Figures 14A and 14B show the details of MC3 LNP and DOTAP-modified MC3 formulations, including (Figure 14A) structure of each component, (Figure 14B) molar ratio, total lipid to mRNA weight ratio, size, and PDI.

[0205] Figures 15A and 15B show that DLin-MC3-DMA (Figure 15A) and C12-200 (Figure 15B) were selected and evaluated with the DOTAP strategy at a dosage of 0.1 mg / kg (6h, n=2). As the DOTAP percentage increased from 0 to 50%, the LNPs based on MC3 and C12-200 showed the same mRNA expression profiles, like mDLNPs, in which the luciferase signal was transferred from the liver to the spleen and finally to the lungs.

[0206] Figures 16A and 16B show details of C12-200 LNP and DOTAP-modified C12-200 formulations, including (Figure 16A) structure of each component, (Figure 16B) molar ratio, total lipid to mRNA weight ratio, size, and PDI.

[0207] Figures 17A and 17B show further optimization of (Figure 17A) mDLNPs. As a key lipid in mDLNPs, 5A2-SC8 was used as the "fifth" lipid to modify mDLNPs to form four preparations with an additional percentage of 10% to 30%. (Figure 17B) In vitro luciferase images and quantitative data show that an additional 15% to 25% of 5A2-SC8 significantly improves mRNA delivery efficacy, and 20% has the highest signal (0.05mg / kg, 6h, n=2).

[0208] Fig.18 The structures of 5A2-SC8, DOPE, cholesterol, and DMG-PEG are shown. mDLNPs are an efficient and safe mRNA delivery vehicle for liver-targeted therapeutics developed in previous work and are composed of 5A2-SC8, DOPE, cholesterol, and DMG-PEG in a molar ratio of 15 / 15 / 30 / 3.

[0209] Figures 19A-19GSelective organ targeting (SORT) allows lipid nanoparticles (LNPs) to be systematically and predictably engineered to accurately edit cells in specific organs. (19A) Adding supplementary components (called SORT lipids) to traditional LNPs systematically changes the in vivo delivery profile and mediates tissue-specific delivery based on the percentage and biophysical properties of SORT lipids. This general approach successfully redirects multiple classes of nanoparticles. Bioluminescent images of mice injected intravenously with 0.1 mg / kg luciferase mRNA are shown here, and the luciferase mRNA is in lung and spleen specific DLin-MC3-DMA LNPs (Onpattro SNALP) and liver enhanced 5A2-SC8 degradable dendrimer-based LNPs (DLNPs). SORT lipids are included in 4-component 5A2-SC8, DLin-MC3-DMA and C12-200 LNPs to create 5-component SORT LNPs. (19B) 5A2-SC8 SORT LNPs were formulated at a molar ratio of 5A2-SC8 / DOPE / Chol / DMG-PEG / SORT lipid = 15 / 15 / 30 / 3 / X (mol / mol), where X was adjusted from 0 to 1200 to prepare a series of LNPs with 0% to 100% SORT lipid (fraction of total lipid). Here, the inclusion of a permanent cationic lipid (DOTAP) systematically shifted luciferase protein expression from liver to spleen to lungs (0.1 mg / kg Luc mRNA, 6 h) depending on the percentage of DOTAP. (19C) Quantitative data confirmed that SORT lipid percentage is a factor for tissue-specific delivery; 0% (mDLNP) is optimal for liver; 5-15% is optimal for spleen; and 50% is optimal for lungs. (19D) Relative luciferase expression in each organ confirmed that fractional expression is predictably tunable. (19E) Inclusion of anionic SORT lipids enabled selective delivery of mRNA to the spleen. When 18PA lipids were introduced into mDLNPs to 40%, luciferase expression was observed only in the spleen (0.1 mg / kgLuc mRNA, 6 h). (19F) Ex vivo luminescence images of major organs 6 hours after intravenous injection of DLin-MC3-DMA SORT LNPs at a dose of 0.1 mg / kg Luc mRNA. As the molar percentage of DOTAP increased, luciferase expression shifted from the liver to the lungs. 18PA-mediated sole 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.

[0210] Figures 20A-20C(20A) Details of DOTAP and 18PA SORT LNPs are shown, including molar ratio, molar percentage, weight ratio of total lipid to mRNA, size, PDI, and zeta potential. (20B) LNPs were prepared using a modified ethanol dilution method. SORT lipids were included in the ethanol phase, and sgRNA / mRNA was encapsulated during LNP formation. (20C) The chemical structures of lipids used in standard mDLNP and DOTAP / 18PA SORT preparations are shown. For the development of SORT, ionizable cationic lipids based on degradable dendrimers (called 5A2-SC8) are the focus of LNPs that can deliver siRNA / miRNA to prolong survival in a genetically engineered mouse model of MYC-driven liver cancer and trigger polyploidy in the liver (Zhou et al., 2016; Zhang et al., 2018a; Zhang et al., 2018b). LNP molar compositions optimized for mRNA delivery are focused on mDLNPs named after the liver (Cheng et al., 2018). This liver-targeted basic mRNA formulation of 5A2-SC8 / DOPE / cholesterol / DMG-PEG2000=15 / 15 / 30 / 3 (mol) was prepared and supplemented with SORT lipids to prepare SORT LNPs (details in 20A). For further clarity, the traditional 4-component LNPs consist of ionizable cationic lipids (defined herein as containing a pK a <8 amino groups), zwitterionic phospholipids (defined as lipids with an equal number of positive and negative charges), cholesterol, and poly(ethylene glycol) (PEG) lipids (most commonly PEG2000-DMG). SORT LNPs contain a fifth lipid, such as a permanent cationic lipid (defined as a positively charged lipid with no pK a or pK a >8) or permanently anionic lipids (defined as having a negative charge).

[0211] Figure 21A and 21B show the results of in vitro luciferase (Luc) mRNA delivery of SORT mDLNP modified by DOTAP in (Figure 21A) Huh-7 hepatocytes and (Figure 21B) A549 pneumocytes, which vary with the percentage of DOTAP incorporated. The Luc mRNA delivery results show that the LNP with a percentage of DOTAP of 5%-50% delivers the most mRNA in Huh-7 hepatocytes and A549 pneumocytes. The SORT LNP with 10% DOTAP is much more effective in vitro than the basic mDLNP reported previously. No perceptible cytotoxicity is observed for any preparation, and all are uniform (low PDI), with a diameter in the range of 90nm to 150nm (Figure 20). The measurement of surface charge reveals that DOTAP is encapsulated inside with mRNA, rather than on the LNP surface, because when DOTAP is less than 60%, the zeta potential is close to 0. The surface charge only becomes positive at a percentage above 65% ( FIG. 20 ), which reveals that the PEG lipid-coated SORTLNPs with selective tissue tropism can be found to have a surface charge close to neutral, a property for clinical translation. 4 Cells were seeded into 96-well plates at a density of 10 cells / well. Luc mRNA expression and cell viability were measured 24 hours after treatment at a dose of 50 ng / well of Luc mRNA (n=4).

[0212] Figures 22A-22C 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. 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) liver-targeted basic mRNA formulations were prepared and supplemented with SORT lipids to prepare SORT LNPs later. (22C) Tables and results of other SORT formulations using DLin-MC3-DMA and C12-200. For all DLin-MC3-DMA and C12-200 LNPs, the weight ratio of total lipid / mRNA was 20 / 1 (weight / weight).

[0213] Figures 23A-23CSORT is shown to rely on general biophysical properties rather than exact chemical structure. (23A) SORT lipids can be divided into specific groups with defined biophysical properties. Permanent cationic SORT lipids (DDAB, EPC, and DOTAP) all produce the same mRNA delivery profile (based on SORT lipid percentage, liver to spleen to lung) (0.1 mg / kg LucmRNA, 6h). (23B) Anionic SORT lipids (14PA, 18BMP, 18PA) all produce the same mRNA delivery profile (based on SORT lipid percentage, spleen only). (23C) Ionizable cationic SORT lipids with tertiary amino groups (DODAP, C12-200) enhance liver delivery without any luciferase expression in the lung (0.1 mg / kg Luc mRNA, 6h).

[0214] Figures 24A and 24B show further application of SORT to further enhance mDLNP liver delivery using ionizable cationic lipids as SORT lipids. (24A) Schematic diagram of SORT. (24B) 5A2-SC8 is used as SORT lipid, and the SORT method is used to supplement the basic mRNA mDLNP formulation (5A2-SC8 / DOPE / cholesterol / DMG-PEG2000=15 / 15 / 30 / 3 (mol)) with additional 5A2-SC8. In vitro luciferase images and quantitative data show that when additional 15%-25% SORT lipids are added, the mRNA delivery efficacy is dramatically improved. Maximum expression (0.05mg / kg, 6h, n=2) was produced with 20% incorporation. Therefore, SORT allows the development of second-generation mDLNPs with increased efficacy.

[0215] Figures 25A and 25B show the effect of evaluating zwitterionic SORT lipids. The inclusion of zwitterionic SORT lipids in liver-targeted mDLNPs altered expression from the liver to the spleen as SORT lipid incorporation increased. After intravenous injection, 80% DSPC and 50% DOCPe SORT LNPs delivered mRNA only to the spleen. (25A) Schematic diagram of the SORT method. (25B) Ex vivo luminescent images in major organs 6h after intravenous injection. DSPC and DOCPe (zwitterionic lipids with different structures) improved the delivery of Luc mRNA to the spleen with increasing percentages (0.1 mg / kg, 6h, n=2).

[0216] Figures 26A and 26B SORT was evaluated as a potential strategy to "activate" inactive LNP formulations. (26A) Schematic diagram of supplementing inactive C1 formulations with SORT lipids to test whether SORT can confer activity. (26B) Details of C1 LNP (inactive LNP) and DOTAP (or DODAP) C1 SORT LNP, including lipid molar ratio, molar percentage, total lipid to mRNA weight ratio, size, and PDI. C1 LNPs were prepared in a manner that allowed mRNA encapsulation and favorable biophysical properties (uniform <200nm size). However, there was no protein expression at all after intravenous injection of C1 LNP. Therefore, it was asked whether SORT could "activate" dead LNPs. DODAP and DOTAP SORT lipids were evaluated. DODAP@C1 LNP delivered mRNA to the spleen and liver, and DOTAP@C1LNP delivered mRNA to the lungs and spleen (0.1 mg / kg, 6h, n=2). Therefore, SORT can activate dead LNPs and provide tissue selectivity.

[0217] Figures 27A and 27B show that SORT alters LNP biodistribution and reveals the relative apparent pK a and organ specificity. (27A) Fluorescent Cy5-labeled mRNA was used to track the biodistribution of SORT LNPs. The inclusion of DOTAP as the SORT lipid increased mRNA accumulation in the lungs, which in part explains the ability to deliver RNA to mouse lungs. 18PA increased uptake into the spleen. DODAP slightly increased liver accumulation and reduced spleen accumulation (0.5 mg / kg, 6 h). It should be noted that this data describes the location of SORT LNPs, not the ability to effectively deliver mRNA within cells. (27B) The relative apparent pK values ​​of all 67 effective mRNA preparations were measured by TNS assay. a , and plotted against in vivo delivery efficacy in different organs (functional delivery via mRNA translated into protein). As expected, all liver-targeted formulations had narrow pK a (6-7). Surprisingly, lung-targeted delivery requires a high pK a (>9) and a low pK a (<6) Assisted spleen delivery. It should be noted that all SORT LNPs contain ionizable cationic lipids (for endosomal escape) as well as a mixture of other charged and uncharged lipids (which together mediate tissue tropism).

[0218] Figures 28A and 28B show that Cy5-labeled mRNA was used to track the biodistribution of SORT LNPs. Organ distribution of DSPC mDLNPs after intravenous injection. (28A) Schematic diagram of SORT. (28B) Cy5 fluorescence and quantitative data of major organs treated with DSPC mDLNPs (0.5 mg / kg, 6 h, n=2).

[0219] Fig.29 The modified TNS assay was shown to measure the overall / apparent pK of mRNA preparations. a A total of 67 successful NP formulations (high in vivo potency) were evaluated. The relative pK was estimated when 50% of the normalized signal was generated compared to the basic LNP formulation (without added SORT lipids). a . The TNS assay has historically been used to measure LNPs with a single ionizable cationic lipid and a neutral (non-ionizable) helper lipid, yielding values ​​for the ionization behavior of the cationic lipid trapped within the self-assembled LNP. A modified approach was used here because SORTLNPs (e.g., DOTAP) with a high percentage (>40%) of permanent cationic lipid do not buffer charge well, even though they contain ionizable cationic lipids. Due to the complexity of SORT LNPs containing multiple charged lipids (rather than a single ionizable cationic lipid as in traditional LNPs), the focus was on the relative signal at 50%, which correlates with in vivo tissue specific activity.

[0220] Figures 30A and 30B show that the inclusion of ionizable lipids (e.g., 5A2-SC8) is required for efficacy. LNPs containing SORT lipids but no ionizable cationic lipids are inactive. (30A) Schematic diagram of SORT C2 LNPs. (30B) Details of C2 and SORT lipid C2 LNPs. In vitro luciferase images show that neither DODAP nor DOTAP can achieve significant mRNA delivery with C2 LNPs. These results indicate that successful mRNA delivery requires ionizable amino lipids (0.1 mg / kg, 6 h, n=2).

[0221] Figures 31A-31ETissue-specific gene editing in Td-Tomato mice by Cre mRNA delivery of SORT LNPs is shown. (31A) Schematic showing that delivery of Cre mRNA activates Td-Tom expression in Td-Tom transgenic mice. (31B) mDLNPs and 20% DODAP LNPs specifically induce Td-Tom fluorescence in the liver, and 50% DOTAP LNPs selectively edit the lungs. Td-Tom fluorescence in major organs was detected 2 days after intravenous injection of Cre mRNA-loaded LNPs (0.3 mg / kg). (31C) 30% 18PA SORT LNPs induced gene editing in the spleen (note the high liver background fluorescence in PBS-injected mice). (31D) Confocal microscopy was used to further verify effective tissue editing. Scale bars = 20 μm and 100 μm. (31E) TdTom in defined cell type populations in the liver, lungs, and spleen was quantified using FACS. + Percentage of cells (Day 2, 0.3 mg / kg).

[0222] Fig.32 Display B6.Cg-Gt(ROSA)26Sor tm9(CAG-tdTomato)Hze / J(Ai9) mice exhibited some autofluorescence in the absorption region of TdTom. In addition, there was a large difference in TdTom autofluorescence between different organs (n=2). The liver and kidney showed the highest signal, while the spleen showed the lowest. Although this did not interfere with the detection of editing in most organs (when the excitation settings were appropriately adjusted to eliminate background), it did complicate the detection of spleen TdTom expression because the background spleen was so much lower than other organs.

[0223] Figures 33A-33CIt is shown that CRISPR / Cas gene editing in the spleen was achieved in both Td-Tom transgenic mice 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 activated Td-Tom expression in Td-Tom mice. (33B) Td-Tom expression was induced in the spleen and liver by the spleen-targeted formulation 30% 18PA SORTLNP. Quantitative data showed that editing in the spleen was higher than in the liver. Td-Tom fluorescence was detected in major organs on the second day after intravenous treatment with co-delivery of Cas9 mRNA and modified sgTom1 (2 / 1, weight / weight) at a total dose of 4 mg / kg. (33C) The T7E1 assay indicates that specific PTEN editing of the spleen was obtained by co-delivery of Cas9 mRNA (IVT) and sgPTEN. C57 / BL6 mice were injected intravenously with 30% 18PA SORT LNP (Cas9 mRNA / sgPTEN, 2 / 1, weight / weight) at a total dose of 4 mg / kg, and PTEN editing was detected on day 2. In this case, liver editing was not observed, suggesting that spleen-specific editing can be achieved.

[0224] Fig.34 It was shown that by administering a single 0.3 mg / kg Cre mRNA dose, DODAP-20SORT LNP achieved almost 100% TdTom editing in hepatocytes. As shown in the flow cytometry histogram, there was a complete separation between TdTom-control mice and TdTom+20% DODAP-treated mice. After liver perfusion, the resected livers of mice treated with 20% DODAP SORT LNP were surprisingly bright red compared to the control livers. Even without fluorescence excitation, the liver glowed red due to the complete activation of 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.

[0225] Fig.35 The FACS gating strategy used to analyze TdTom+ expression in lung cells is shown. 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. The gating of Td-Tom+ in cell types was drawn based on PBS-injected control mice. Td-Tom mice were injected with Cre mRNA preparations and Td-Tom+ in a given cell type was detected by flow cytometry 2 days later (n=3).

[0226] Fig.36 The FACS gating strategy for analyzing TdTom+ expression in splenocytes is shown. Ghost Red 780 was used to distinguish live and dead cells. CD44+ was used to distinguish immune cells, then CD3+ and CD11b- for T cells, CD3- and CD11b+ for macrophages, and CD19+ and CD11b- for B cells. The gating of Td-Tom+ in cell types was drawn based on PBS-injected control mice. Td-Tom mice were injected with Cre mRNA preparations, and Td-Tomato+ in a given cell type was detected by flow cytometry 2 days later (n=3).

[0227] Figures 37A-37GSORTLNPs mediate tissue-specific CRISPR / Cas gene editing in Td-Tom transgenic mice and C57 / BL6 wild-type mice by co-delivery of Cas9 mRNA and sgRNA and by delivery of Cas9 RNPs. (37A) Schematic diagram showing that co-delivery of Cas9 mRNA (or Cas9 protein) and sgTom1 activated Td-Tom expression in Td-Tom transgenic mice. (37B) mDLNPs and 20% DODAP LNPs specifically induced Td-Tom fluorescence in the liver, and 50% DOTAP LNPs selectively edited the lungs. Td-Tom fluorescence was detected 10 days after intravenous injection of Cas9 mRNA and modified sgTom1 (4 / 1, weight / weight) at a total dose of 2.5 mg / kg. (37C) tdTom expression was confirmed by confocal imaging of tissue sections. Scale bars = 20 μm and 100 μm. (37D) Cas9 mRNA and sgPTEN were co-delivered in SORT LNPs to selectively edit the liver, lung, and spleen of C57 / BL6 mice (total dose of 2.5 mg / kg (Cas9mRNA / sgPTEN, 4 / 1, wt / wt; measured 10 days after a single injection). T7E1 assay indicated that tissue-specific PTEN editing was achieved. (37E) H&E sections and IHC further confirmed successful PTEN editing. Transparent 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. Td-Tom fluorescence was detected 7 days after intravenous injection of Cas9 / sgTom1 RNP at doses of sgTom1. tdTom expression was confirmed by confocal imaging of tissue sections. 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 lungs of C57 / BL6 mice (1.5 mg / kg sgPTEN; measured 7 days after a single injection). The T7E1 assay indicated that tissue-specific PTEN editing was achieved.

[0228] Fig.38 Evaluation of IVT Cas9 mRNA by western blot is shown. 293T cells were seeded in 12-well plates one day before transfection and cells were treated with each condition for 24 hours before western blot. Cas9 pDNA was delivered by Lipofectamine2000, and mRNA was delivered by mDLNP.

[0229] Figures 39A and 39B show the optimization of the weight ratio of IVT Cas9 mRNA to sgTom1 by the Cas9 mRNA and sgRNA co-delivery strategy. (39A) Schematic diagram showing that co-delivery of Cas9 mRNA and sgTom1 activated Td-Tom expression in transgenic mice. (39B) Td-Tom fluorescence imaging of major organs at day 7 after intravenous injection showed that 2 / 1 Cas9 / sgTom1 (weight / weight) was optimal. The total RNA dose was 1 mg / kg, and IVT Cas9 mRNA and modified sgTom1 were co-encapsulated by mDLNPs.

[0230] Figures 40A-40I A modular approach developed to achieve systemic nanoparticle delivery of CRISPR / Cas9 ribonucleoproteins (RNPs) for tissue-specific genome editing is shown. (40A) The addition of a permanent cationic supplement component (e.g., DOTAP) to a conventional LNP formulation enables encapsulation and protection of the Cas9 / sgRNA complex using a neutral buffer during nanoparticle formation. Precise tuning of 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 size increase may be due to denaturation. (40C) Size distribution of 5A2-DOT-10 encapsulating Cas9 / sgLuc RNPs prepared in PBS and citrate buffer. 5A2-DOT-10 prepared without RNP was used as a control. (40D) Size distribution of Cas9 / sgRNA RNPs with Cas9 / sgLuc molar ratios of 1 / 1, 1 / 3, and 1 / 5. (40E) Size distribution of 5A2-DOT-10 encapsulating Cas9 / sgLuc with molar ratios of 1 / 1, 1 / 3, and 1 / 5. (40F) Zeta potential of Cas9 / sgRNA RNPs showing a decrease in charge. (40G) No significant difference in zeta potential was observed for 5A2-DOT-10 encapsulating Cas9 / sgLuc with different molar ratios. (40H) Time-dependent cellular uptake of 5A2-DOT-10 LNPs encapsulating 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: inhibitor of macropinocytosis; CMZ: inhibitor of clathrin-mediated endocytosis; GEN: inhibitor of caveolae-mediated endocytosis; MβCD: lipid raft-mediated endocytosis; 4 degrees: energy-mediated endocytosis.

[0231] Figures 41A-C show (41A) A table of 5A2-DOT-X LNPs showing the molar ratios and 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. The total lipid / sgRNA ratio for all LNPs was 40:1 (weight). (41B) Gene editing in HeLa-Luc cells treated with different 5A2-DOT-X Cas9 / sgLuc RNP formulations was detected using the T7EI assay. (41C) Gene editing was analyzed using Sanger sequencing and ICE analysis.

[0232] Fig.42 Representative TEM images of 5A2-DOT-10 encapsulating Cas9 / sgLuc RNP complexes at a molar ratio of 1 / 3 are shown. 5A2-DOT-10Cas9 / sgLuc was prepared at a total lipid concentration of 2 mg / mL in PBS buffer. 3 μL of the nanoparticle solution was dropped onto a carbon TEM grid and allowed to settle for 1 minute before being blotted with filter paper. The TEM grid was then imaged using transmission electron microscopy (FEI Tecnai G2 Spirit Biotwin).

[0233] Fig.43 Confocal images are shown showing cellular uptake of PBS (control), free Cas9 / sgLuc complex (control) and 5A2-DOT-10Cas9 / sgLuc in Hela-Luc cells 20 hours after treatment. Cas9-EGFP fusion protein was used to track the subcellular distribution of Cas9 / sgRNA complexes. Cas9 / sgLuc complexes did not show detectable green fluorescence above background (PBS) inside cells, while bright green signals were detected after treatment with 5A2-DOT-10.

[0234] Figures 44A-44HIt was shown that gene editing was performed rapidly and efficiently in vitro. (44A) T7EI cleavage assay of DNA isolated from HeLa-Luc cells treated with various nanoparticles and controls. Efficient gene editing was mediated by 5A2-DOT-10 delivering Cas9 / sgLucRNP (1 / 3 and 1 / 5). Indels (%) at the Luc locus were quantified by ICE analysis. It should be noted that gene editing of LNPs prepared using low pH citrate buffer (currently used and established methods) was 0%. (44B) Fluorescence microscopy images of HeLa-GFP cells after treatment with various preparations. Scale bar = 100 μm. 5A2-DOT-10Cas9 / sgGFP treatment significantly reduced GFP fluorescence. (44C) Flow cytometry analysis of HeLa-GFP cells after treatment with various preparations. Only for the 5A2-DOT-10Cas9 / sgGFP group, the peak of GFP-positive cells shifted completely to the left, indicating that almost all GFP-positive cells turned black. (44D) Time-dependent GFP fluorescence intensity of HeLa-GFP cells after various treatments. Permanent GFP fluorescence loss after day 2 was observed with 5A2-DOT-10Cas9 / sgGFP treatment, while ICE analysis of Sanger sequencing data showed that indels remained above 90% after day 2. (44E and 44F) Mean fluorescence intensity (%) of HeLa-GFP cells after treatment with Cas9 / sgGFP alone, 5A2-SC8 loaded with Cas9 / sgGFP, C12-200, DLin-MC3-DMA LNP formulations containing 10% supplemental DOTAP, traditional C12-200 and DLin-MC3-DMA LNP nanoformulations loaded with Cas9 / sgGFP, and RNAiMAX loaded with Cas9 / sgGFP. GFP fluorescence was significantly reduced after treatment with all three DOTAP-modified formulations. ICE analysis of Sanger sequencing data further confirmed that the highest gene editing efficiency was obtained with 5A2-DOT-10LNP. Mean ± SD mean (n = 3). Statistical significance was determined using a two-sided Student's t-test. t value = 42.69, degrees of freedom (df) = 4 (P < 0.0001); t value = 16.75, degrees of freedom (df) = 4 (P < 0.0001); t value = 37.53, degrees of freedom (df) = 4 (P < 0.0001). P values ​​< 0.05 were considered statistically significant. (44G) 5A2-DOT-10Cas9 / sgGFP LNPs were stored at 4°C for 2 months. Nanoparticle diameter and PDI were monitored over time. (44H) Regular treatment of HeLa-GFP cells with stored LNPs showed no loss of activity, indicating long-term LNP and RNP stability and metastatic potential. All cells in the above experiments were treated with 24 nM sgRNA.

[0235] Figures 45A and 45B show gene editing of different nanoformulations in Hela-GFP cells. (45A) Mean fluorescence intensity (%) of Hela-GFP cells after treatment with Cas9 / sgGFP alone, 5A2-DOT-10Cas9 / sgLuc, and 5A2-DOT-10Cas9 / sgGFP (at a total lipid / sgGFP weight ratio of 40:1). (45B) Mean fluorescence intensity (%) of Hela-GFP cells after treatment with 5A2-DOT-10Cas9 / sgGFP prepared at a total lipid / sgGFP weight ratio of 10:1, 20:1, 30:1, and 40:1.

[0236] Figures 46A-46KThe generalizable RNP delivery strategy ( FIG. 40A ) is shown to be universal for ionizable cationic lipid nanoparticles (DLNP, LLNP, SNALP) and for other cationic lipids that are positively charged at pH 7.4 and for other neutral buffers. (46A) Schemes of LNP formulations with different ionizable lipids. (46B) Details of LNP formulations with different ionizable lipids, including determination of the molar ratio and percentage of each component and the weight ratio of total lipid to sgRNA. (46C) Chemical structures of ionizable cationic lipids used in formulations, including 5A2-SC8, C12-200, and Dlin-MC3-DMA. (46D) Mean fluorescence intensity (%) of HeLa-GFP cells after treatment with Cas9 / sgGFP RNPs encapsulated in 5A2-DOT-10, C12-200-DOT-10, and MC3-DOT-10. GFP fluorescence was significantly reduced after treatment with all three formulations. (46E) Preparation schemes of LNP formulations with different permanent cationic lipids. (46F) Details of LNP formulations with different permanent cationic lipids, including determination of the molar ratio and percentage of each component and the weight ratio of total lipid to sgRNA. (46G) Chemical structures of permanent cationic lipids used in formulations, including DOTAP, DDAB, and EPC. (46H) Mean fluorescence intensity (%) of HeLa-GFP cells after treatment with Cas9 / sgGFP RNPs encapsulated in 5A2-DOT-10, 5A2-DDAB-10, and 5A2-EPC-10. Instead of DOTAP, other cationic lipids (DDAB and EPC) can also achieve efficient gene editing. (46I) Schemes of LNP formulations in different buffers. (46J) Mean fluorescence intensity (%) of HeLa-GFP cells after treatment with 5A2-DOT-10 prepared using different buffers, including PBS, Opti-MEM, HEPES, and citrate buffer. Neutral buffers are required for RNP encapsulation and delivery. (46K) Indels (%) at the GFP locus in genomic DNA isolated from HeLa-GFP cells after treatment with 5A2-DOT-10Cas9 / sgGFP LNPs prepared using different buffers were measured using ICE analysis. All neutral buffers showed high gene editing in cells, indicating the importance of neutral buffers in nanoparticle preparation. Please note that Figures 44E and 44F have been reproduced in Figure 45 above to combine related data together for improved clarity.

[0237] Figures 47A-47JEfficient multiplex genome editing was achieved in vivo. (47A) Schematic diagram showing how delivery of Cas9 / sgTOM RNPs activates Td-Tom expression in Td-Tomato transgenic mice. 5A2-DOT-X LNPs were injected into Td-Tom mice locally (by intramuscular or intracerebral injection) and systemically (by intravenous injection through the tail vein). 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-10Cas9 / sgTOM (respectively) showed bright red fluorescence in leg muscles or brain tissue. Successful CRISPR / Cas gene editing was further confirmed by confocal imaging of (47C) muscle and (47E) brain tissue sections. 5A2-DOT-10 has 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 with different molar percentages of DOTAP. Td-Tom fluorescence (as a downstream readout of DNA editing) showed that low DOTAP percentages favored liver editing, while high DOTAP percentages favored 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 tissues after systemic intravenous treatment with 5A2-DOT-5, 5A2-DOT-10, 5A2-DOT-50, and 5A2-DOT-60 encapsulating Cas9 / sgPTEN. Indels (%) were calculated and reported. (47I) 5A2-DOT-50 LNPs (5A2-DOT-50-pool) containing pooled sgRNAs for 6 targets (sgTOM, sgP53, sgPTEN, sgEml4, sgALK, and sgRB1) were intravenously administered to td-Tom mice at a total RNA dose of 2 mg / kg (0.33 mg / kg per sgRNA). Gene editing at the TOM locus was confirmed by in vivo imaging, and (47J) editing of the other 5 loci was confirmed using a T7EI cleavage assay on lung tissue.

[0238] Figures 48A-48H5A2-DOT-X LNPs are shown to simplify the generation of complex mouse models. (48A) To establish an orthotopic liver-specific cancer model, 5A2-DOT-5LNPs encapsulating Cas9 / sgP53 / sgPTEN / sgRB1RNPs were injected into adult C57BL / 6 mice weekly (3 injections, 2.5 mg / kg of total sgRNA, intravenous, n=4). After 12, 15, and 20 weeks, mice were sacrificed and livers were collected to analyze tumor production. (48B) T7EI cleavage results from genomic DNA extracted from the liver confirmed gene editing at all three loci. (48C) Representative photos of mouse livers containing tumors excised 20 weeks after injection. (48D) H&E and Ki67 staining further confirmed progressive tumor formation. Higher expression of the tumor proliferation biomarker Ki67 was detected in tumor lesions. Scale bar = 100 μm. (48E) To create an orthotopic lung-specific cancer model, 5A2-DOT-50LNPs encapsulating Cas9 / sgEml4 / sgAlk RNPs were injected into adult C57BL / 6 mice once (2 mg / kg) or twice (1.5 mg / kg per week for 2 weeks) (iv, n=5). After 10, 16, and 24 weeks, mice were sacrificed and lungs were collected for analysis of tumor production. (48F) T7EI cleavage results from genomic DNA extracted from the lungs confirmed gene editing at the loci of Eml4 and Alk. PCR amplicons of the Eml4-Alk rearrangement were also detected in all lungs treated with 5A2-DOT-50LNP. (48G) 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 progressive tumor formation. Higher expression of tumor proliferation biomarker Ki67 was detected in lung tumor lesions. Scale bar = 100 μm.

[0239] Figures 49A and 49B show the gene editing efficiency of unmodified sgRNA synthesized by in vitro transcription (IVT) compared to chemically modified and synthetic sgRNA (2'-methyl 3'-phosphorothioate modification in the first 3 and last 3 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 to detect the gene editing efficiency of nanoparticles encapsulating Cas9 / IVT sgRNA and Cas9 / chemically modified sgRNA. The cleavage bands at 536 bp and 184 bp were clearly observed in the group treated with modified sgRNA.

[0240] Fig.50 Gene editing of P53, PTEN and RB1 genes in mouse liver after treatment with 5A2-DOT-5LNPs encapsulating Cas9 / sgP53 / sgPTEN / sgRB1 RNPs is shown. Gene editing of liver genomic DNA at the PTEN, P53 and RB1 genomic loci was detected using the T7EI assay after weekly treatment for two weeks. The PBS-treated group was used as a control. Cleavage bands were detected at 261 bp and 215 bp of the PCR amplicon targeting P53; cleavage bands were detected at 345 bp and 293 bp of the PCR amplicon targeting PTEN; and cleavage bands were detected at 395 bp and 207 bp of the PCR amplicon targeting RB1.

[0241] Fig.51 The T7EI assay was used to detect gene editing of P53, PTEN, and RB1 genes in mouse liver after treatment with 5A2-DOT-5LNPs encapsulating Cas9 / sgP53 / sgPTEN / sgRB1 RNPs. PBS-treated and 5A2-DOT-5 alone (no Cas9 / sgRNA)-treated groups were used as controls. The T7EI results of genomic DNA extracted from mouse tumors after 20 weeks of treatment with 5A2-DOT-5LNPs encapsulating Cas9 / sgP53 / sgPTEN / sgRB1 RNPs indicated that knockout of these three genes induced tumorigenesis, as cleavage bands were detected at all three genomic loci.

[0242] Fig.52 Shown are representative photographs of mouse livers and excised tumors excised from mice in the group treated with 5A2-DOT-5LNPs encapsulating Cas9 / sgP53 / sgPTEN / sgRB1 RNPs for 15 weeks.

[0243] Figures 53A-53CH&E and Ki67 staining images of mouse liver after 15 and 20 weeks of treatment with 5A2-DOT-5LNP alone (no Cas9 / sgRNA) (control) are shown (53A), and tumors excised from mice in the group treated with 5A2-DOT-5LNP encapsulating Cas9 / sgP53 / sgPTEN / sgRB1 RNP for 20 weeks (53B). No morphological changes were detected with 5A2-DOT-5LNP treatment alone, indicating that the nanocarrier alone cannot cause tumors. Scale bar: 100 μm. (53C) Large view image of mouse liver tumor generation after 20 weeks of treatment with 5A2-DOT-5LNP encapsulating Cas9 / sgP53 / sgPTEN / sgRB1 RNP. Scale bar: 500 μm.

[0244] Figures 54A-54D Figure 54: Eml4-Alk rearrangement in mouse lungs following 7 days of treatment with 5A2-DOT-50LNPs (2 mg / kg total sgRNA) encapsulating Cas9 / sgEml4 / sgAlk RNPs. Eml4 editing (54A) and Alk editing (54B) were detected in genomic DNA extracted from mouse lungs by T7EI assay. (54C) PCR analysis of genomic DNA extracted from mouse lungs was performed to determine Eml4-Alk inversion. (54D) The PCR amplicons were subcloned, and the sequences of 6 independent clones were listed, and representative chromatograms are presented in the figure above. The chromatograms are exactly the same as predicted for the Eml4-Alk rearrangement. (Prediction = SEQ ID NO: 59; Clone 1 = SEQ ID NO: 59; Clone 2 = SEQ ID NO: 60; Clone 4 = SEQ ID NO: 61; Clone 5 = SEQ ID NO: 61; Clone 6 = SEQ ID NO: 62)

[0245] Fig.55 H&E and Ki67 staining images of mouse livers treated with 5A2-DOT-50 LNPs alone (without Cas9 / sgRNA) for 10 and 16 weeks (LNP dose equal to 1 mg / kg of total sgRNA) are shown. No morphological changes were detected in animals injected with 5A2-DOT-50 LNPs alone. Scale bar: 100 μm.

[0246] Fig.56 Shown are macroscopic images of lung tumors generated in mice after 24 weeks of treatment with 5A2-DOT-50LNPs encapsulating Cas9 / sgEml4 / sgAlk RNPs. Scale bar: 500 μm. Several tumor lesions (highlighted) were observed from H&E and Ki67 staining images.

[0247] Fig.57It was shown that 5A2-DOT-10 LNPs can effectively deliver ovalbumin (OVA) protein into the cytoplasm of HeLa-Luc cells. Cells were treated with free rhodamine-labeled OVA protein and 5A2-DOT-10 LNPs encapsulating rhodamine-labeled OVA for 22 h before imaging by confocal microscopy. DETAILED DESCRIPTION

[0248] Described herein are lipid nanoparticles (LNPs) composed of 1) permanent cationic lipids, 2) ionizable cationic lipids, and 3) phospholipids, and may optionally contain cholesterol and lipid PEG or both. The inclusion of permanent cationic lipids is used to direct LNPs to specific organs such as the lungs, lymph nodes, or spleen. The data presented herein indicate that this effect is universal, and the components are modular, with each category indicating that 5A2-SC8 can be replaced by any ionizable cationic lipid, DOTAP can be replaced by any cationic lipid, and DOPE can be replaced by any phospholipid. In some embodiments, cholesterol and lipid PEG are also included, but formulations without cholesterol or lipid PEG are feasible. These vectors can deliver mRNA, sgRNA, and proteins to specific organs in vivo, thereby solving a major challenge.

[0249] A. Chemical Definition

[0250] When used in the context of chemical groups: "hydrogen" refers to -H; "hydroxy" refers to -OH; "oxo" refers to =O; "carbonyl" refers to -C(=O)-; "carboxyl" refers to -C(=O)OH (also written as -COOH or -CO 2 H); "halo" refers independently to -F, -Cl, -Br or -I; "amino" refers to -NH 2 ; "Hydroxyamino" refers to -NHOH; "Nitro" refers to -NO 2 ; "imino" refers to =NH; "cyano" refers to -CN; "isocyanate" refers to -N=C=O; "azido" refers to -N 3 ; In the monovalent context, "phosphate" refers to -OP(O)(OH) 2 or its deprotonated form; in the divalent context, "phosphate" refers to -OP(O)(OH)O- or its deprotonated form; "thiol" refers to -SH; and "thio" refers to =S; "sulfonyl" refers to -S(O) 2 -; "Hydroxysulfonyl" refers to -S(O) 2 OH; "sulfonamide" means -S(O) 2 NH 2 ; and "sulfinyl" refers to -S(O)-.

[0251] In the context of chemical formulae, the symbol "-" refers to a single bond, "=" refers to a double bond, and "≡" refers to a triple bond. represents an optional bond, which if present is a single bond or a double bond. represents a single bond or a double bond. Thus, for example, include And it should be understood that no such ring atom forms part of more than one double bond. In addition, it should be noted that the covalent bond symbol "-" does not indicate any preferred stereochemistry when connecting one or two stereogenic atoms. Instead, it encompasses all stereoisomers and mixtures thereof. When drawn vertically through a bond (e.g., for a methyl group, ),symbol Indicates the point of attachment for this group. It should be noted that attachment points are usually only identified in this way for larger groups to help the reader identify the point of attachment unambiguously. refers to a single bond where the group attached to the thick end of the wedge "comes out of the page". refers to a single bond where the group attached to the thick end of the wedge "enters the paper". Refers to a single bond, where the geometry (e.g., E or Z) around the double bond is undefined. Therefore, both options and their combinations are contemplated. Any undefined valence on an atom of a structure shown in this application implicitly represents a hydrogen atom bonded to that atom. Bold dots on carbon atoms indicate that the hydrogen attached to that carbon is facing out of the plane of the paper.

[0252] When the group "R" is described as a "floating group" on the ring system, for example, in the following formula:

[0253]

[0254] Then R can replace any hydrogen atom attached to any of the ring atoms, including depicted, implied or explicitly defined hydrogens, as long as a stable structure results. When the group "R" is described as a "floating group" on a fused ring system, for example, in the following formula:

[0255]

[0256] R can replace any hydrogen connected to any ring atom of any one of the fused rings, unless otherwise specified. Replaceable hydrogen includes depicted hydrogen (e.g., hydrogen connected to nitrogen in the above formula), implicit hydrogen (e.g., hydrogen in the above formula that is not shown but understood to be present), clearly defined hydrogen, and optional hydrogen whose presence depends on the identity of the ring atom (e.g., hydrogen connected to the group X when X equals -CH-), as long as a stable structure is formed. In the example shown, R can be located on a 5-membered or 6-membered ring of the fused ring system. In the above formula, the subscript letter "y" immediately following the group "R" included in brackets 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 replaceable hydrogen atoms of the ring or ring system.

[0257] For chemical groups and compound classes, the number of carbon atoms in the group or class is indicated as follows: "Cn" defines the exact number of carbon atoms (n) in the group / class. "C≤n" defines the maximum number of carbon atoms (n) that can be in the group / class, while the minimum number is as small as possible for the group / class in question. For example, it is understood that the group "alkenyl" (C≤8) ” or category “Olefins (C≤8) The minimum number of carbon atoms in an alkoxy group is 2. (C≤10) ” indicates an alkoxy group having 1 to 10 carbon atoms. “Cn-n′” defines the minimum number (n) and maximum number (n′) of carbon atoms in the group. Thus, “alkyl” (C2-10) ” refers to those alkyl groups having 2-10 carbon atoms. These carbon number indicators may precede or follow the chemical group or class they modify, and they may or may not be included in parentheses without indicating any change in meaning. Thus, the terms “C5 olefins,” “C5-olefins,” “olefins (C5) ” and “Olefins C5 " are all synonymous.

[0258] When used to modify a compound or chemical group, the term "saturated" means that the compound or chemical group does not have carbon-carbon double bonds and carbon-carbon triple bonds, unless described below. When the term is used to modify an atom, it means that the atom is not part of any double or triple bond. In the case of substituted forms of saturated groups, one or more carbon-oxygen double bonds or carbon-nitrogen double bonds may be present. And when such bonds exist, carbon-carbon double bonds that may appear as part of keto-enol tautomerism or imine / enamine tautomerism are not excluded. When the term "saturated" is used to modify a solution of a substance, it means that no more of the substance can be dissolved in the solution.

[0259] The term "aliphatic" used without the "substituted" modifier means 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 straight chains, branched chains, or non-aromatic rings (alicyclic). Aliphatic compounds / groups can be saturated, which are linked by a single carbon-carbon bond (alkanes / alkyls), or unsaturated, which have one or more carbon-carbon double bonds (olefins / alkenyls) or have one or more carbon-carbon triple bonds (alkynes / alkynyls).

[0260] The term "aromatic" when used to modify atoms of a compound or chemical group refers to a compound or chemical group that contains a planar unsaturated ring of atoms stabilized by the interactions of the bonds forming the ring.

[0261] The term "alkyl" used without the "substituted" modifier refers to a monovalent saturated aliphatic group having a carbon atom as the point of attachment, having a straight or branched acyclic structure, and having no atoms other than carbon and hydrogen. 3 (Me), -CH 2 CH 3 (Et), -CH 2 CH 2 CH 3 (n-Pr or propyl), -CH(CH 3 ) 2 (i-Pr, i Pr or isopropyl), -CH 2 CH 2 CH 2 CH 3 (n-Bu), -CH(CH 3 )CH 2 CH 3 (sec-butyl), -CH 2 CH(CH 3 ) 2 (isobutyl), -C(CH 3 ) 3 (tert-butyl, tert-butyl, t-Bu or t Bu) and -CH 2 C(CH 3 ) 3 (neopentyl) is a non-limiting example of an alkyl group. The term "alkanediyl" used without the "substituted" modifier means a divalent saturated aliphatic radical having 1 or 2 saturated carbon atoms as points of attachment, having a straight or branched acyclic structure, no carbon-carbon double or triple bonds, and no atoms other than carbon and hydrogen. The group -CH 2 -(Methylene), -CH 2 CH 2-、-CH 2 C(CH 3 ) 2 CH 2 -and-CH 2 CH 2 CH 2 - is a non-limiting example of an alkanediyl group. "Alkanes" refers to the class of compounds having the formula HR in which R is an alkyl group, as that term is defined above. When any of these terms is used with the "substituted" modifier, one or more hydrogen atoms have been independently replaced by -OH, -F, -Cl, -Br, -I, -NH 2 、-NO 2 , -CO 2 H, -CO 2 CH 3 、-CN、-SH、-OCH 3 、-OCH 2 CH 3 、-C(O)CH 3 、-NHCH 3 、-NHCH 2 CH 3 、-N(CH 3 ) 2 、-C(O)NH 2 、-C(O)NHCH 3 、-C(O)N(CH 3 ) 2 、-OC(O)CH 3 、-NHC(O)CH 3 、-S(O) 2 OH or -S(O) 2 NH 2 The following groups are non-limiting examples of substituted alkyl groups: -CH 2 OH, -CH 2 Cl, -CF 3 、-CH 2 CN、-CH 2 C(O)OH, -CH 2 C(O)OCH 3 、-CH 2 C(O)NH 2 、-CH 2 C(O)CH 3 、-CH 2 OCH 3 、-CH 2 OC(O)CH 3 、-CH 2 NH 2 、-CH 2 N(CH3 ) 2 and -CH 2 CH 2 Cl. The term "haloalkyl" is a subset of substituted alkyl groups in which the hydrogen atom replacement is limited to halo (ie, -F, -Cl, -Br or -I) such that there are no atoms other than carbon, hydrogen and halogen. The group -CH 2 Cl is a non-limiting example of a haloalkyl group. The term "fluoroalkyl" is a subset of substituted alkyl groups in which the hydrogen atom replacement is limited to fluorine, such that no other atoms besides carbon, hydrogen and fluorine are present. The group -CH 2 F, -CF 3 and -CH 2 CF 3 are non-limiting examples of fluoroalkyl groups.

[0262] The term "cycloalkyl" used without the "substituted" modifier means 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, having no carbon-carbon double or triple bonds, and having no atoms other than carbon and hydrogen. Non-limiting examples include: -CH(CH 2 ) 2 (cyclopropyl), cyclobutyl, cyclopentyl or cyclohexyl (Cy). The term "cycloalkanediyl" used without the "substituted" modifier means a divalent saturated aliphatic radical having 2 carbon atoms as points of attachment, no carbon-carbon double or triple bonds, and no atoms other than carbon and hydrogen. is a non-limiting example of a cycloalkanediyl group. "Cycloalkanes" refers to the class of compounds having the formula HR in which R is cycloalkyl, as that term is defined above. When any of these terms is used with the "substituted" modifier, one or more hydrogen atoms have been independently replaced by -OH, -F, -Cl, -Br, -I, -NH 2 、-NO 2 , -CO 2 H, -CO 2 CH 3 、-CN、-SH、-OCH 3 、-OCH 2 CH 3 、-C(O)CH 3 、-NHCH 3 、-NHCH 2 CH 3 、-N(CH 3 ) 2 、-C(O)NH 2 、-C(O)NHCH 3 、-C(O)N(CH 3 )2 、-OC(O)CH 3 、-NHC(O)CH 3 、-S(O) 2 OH or -S(O) 2 NH 2 replace.

[0263] The term "alkenyl" used without the "substituted" modifier means a monovalent unsaturated aliphatic group having a carbon atom as the point of attachment, having a straight or branched acyclic structure, at least one non-aromatic carbon-carbon double bond, no carbon-carbon triple bonds, and no atoms other than carbon and hydrogen. Non-limiting examples include: -CH=CH 2 (vinyl), -CH=CHCH 3 、-CH=CHCH 2 CH 3 、-CH 2 CH=CH 2 (allyl), -CH 2 CH=CHCH 3 and -CH=CHCH=CH 2 The term "alkenediyl" used without the "substituted" modifier means a divalent unsaturated aliphatic radical having 2 carbon atoms as points of attachment, having a straight or branched chain, a straight or branched acyclic structure, at least one non-aromatic carbon-carbon double bond, no carbon-carbon triple bonds, and no atoms other than carbon and hydrogen. The radicals -CH=CH-, -CH=C(CH 3 )CH 2 -、-CH=CHCH 2 -and-CH 2 CH=CHCH 2 - is a non-limiting example of an alkene diyl group. It should be noted that although the alkene diyl group is aliphatic, once attached at both ends, it is not excluded that the group forms part of an aromatic structure. The terms "alkene" and "chain alkene" are synonymous and refer to a class of compounds having the formula HR, wherein R is an alkenyl group, as defined above. Similarly, the terms "terminal olefin" and "alpha-olefin" are synonymous and refer to an olefin having exactly one carbon-carbon double bond, wherein the bond is part of the vinyl group at the end of the molecule. When any of these terms is used with the "substituted" modifier, one or more hydrogen atoms have been independently replaced by -OH, -F, -Cl, -Br, -I, -NH 2 、-NO 2 , -CO 2 H, -CO 2 CH 3 、-CN、-SH、-OCH 3 、-OCH 2 CH3 、-C(O)CH 3 、-NHCH 3 、-NHCH 2 CH 3 、-N(CH 3 ) 2 、-C(O)NH 2 、-C(O)NHCH 3 、-C(O)N(CH 3 ) 2 、-OC(O)CH 3 、-NHC(O)CH 3 、-S(O) 2 OH or -S(O) 2 NH 2 The groups -CH=CHF, -CH=CHCl, and -CH=CHBr are non-limiting examples of substituted alkenyl groups.

[0264] The term "alkynyl" used without the "substituted" modifier means a monovalent unsaturated aliphatic group having a carbon atom as the point of attachment, having a straight or branched acyclic structure, at least one carbon-carbon triple bond, and no atoms other than carbon and hydrogen. The term alkynyl as used herein does not exclude the presence of one or more non-aromatic carbon-carbon double bonds. The groups -C≡CH, -C≡CCH 3 and -CH 2 C≡CCH 3 are non-limiting examples of alkynyl groups. "Alkyne" refers to the class of compounds having the formula HR, in which R is an alkynyl group. When any of these terms is used with the "substituted" modifier, one or more hydrogen atoms have been independently replaced by -OH, -F, -Cl, -Br, -I, -NH 2 、-NO 2 , -CO 2 H, -CO 2 CH 3 、-CN、-SH、-OCH 3 、-OCH 2 CH 3 、-C(O)CH 3 、-NHCH 3 、-NHCH 2 CH 3 、-N(CH 3 ) 2 、-C(O)NH 2 、-C(O)NHCH 3 、-C(O)N(CH 3 ) 2 、-OC(O)CH 3 、-NHC(O)CH3 、-S(O) 2 OH or -S(O) 2 NH 2 replace.

[0265] The term "aryl" used in the absence of a "substituted" modifier refers to a monovalent unsaturated aromatic group having an aromatic carbon atom as a point of attachment, the carbon atom forming a portion of one or more 6-membered aromatic ring structures, wherein the ring atoms are all carbon, and wherein the group is not composed of atoms other than carbon and hydrogen. If there is more than one ring, the ring may be fused or unfused. The term used herein does not exclude the presence of one or more alkyl or aralkyl groups (carbon number restrictions permitting) that are attached to the first aromatic ring or any additional aromatic rings present. Non-limiting examples of aryl groups include phenyl (Ph), methylphenyl, (dimethyl)phenyl, -C 6 H 4 CH 2 CH 3 The term "arene diyl" used in the absence of "substituted" modifiers refers to a divalent aromatic group having 2 aromatic carbon atoms as attachment points, wherein the carbon atoms form a part for one or more 6-membered aromatic ring structures, wherein the ring atoms are all carbons, and wherein the monovalent group is not composed of atoms other than carbon and hydrogen. The term used herein does not exclude the presence of one or more alkyl, aryl or aralkyl (carbon number restrictions allow) that are connected to the first aromatic ring or any additional aromatic rings present. If there is more than one ring, the ring can be fused or unfused. Unfused rings can be connected by one or more of the following: covalent bonds, alkane diyl or alkene diyl groups (carbon number restrictions allow). The non-limiting examples of arene diyl groups include:

[0266]

[0267] "Aromatic hydrocarbons" refers to the class of compounds having the formula HR, in which R is an aryl group, as that term is defined above. Benzene and toluene are non-limiting examples of aromatic hydrocarbons. When any of these terms is used with the "substituted" modifier, one or more hydrogen atoms have been independently replaced by -OH, -F, -Cl, -Br, -I, -NH 2 、-NO 2 , -CO 2 H, -CO 2 CH 3 、-CN、-SH、-OCH 3 、-OCH 2 CH 3 、-C(O)CH 3 、-NHCH 3、-NHCH 2 CH 3 、-N(CH 3 ) 2 、-C(O)NH 2 、-C(O)NHCH 3 、-C(O)N(CH 3 ) 2 、-OC(O)CH 3 、-NHC(O)CH 3 、-S(O) 2 OH or -S(O) 2 NH 2 replace.

[0268] The term "aralkyl" used without the "substituted" modifier refers to the monovalent group -alkanediyl-aryl, wherein the terms alkanediyl and aryl are each used in a manner consistent with the definitions provided above. 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 groups have been independently replaced by -OH, -F, -Cl, -Br, -I, -NH 2 、-NO 2 , -CO 2 H, -CO 2 CH 3 、-CN、-SH、-OCH 3 、-OCH 2 CH 3 、-C(O)CH 3 、-NHCH 3 、-NHCH 2 CH 3 、-N(CH 3 ) 2 、-C(O)NH 2 、-C(O)NHCH 3 、-C(O)N(CH 3 ) 2 、-OC(O)CH 3 、-NHC(O)CH 3 、-S(O) 2 OH or -S(O) 2 NH 2 Non-limiting examples of substituted aralkyl groups are: (3-chlorophenyl)-methyl and 2-chloro-2-phenyl-eth-1-yl.

[0269] The term "heteroaryl" used in the absence of a "substituted" modifier refers to a monovalent aromatic group having an aromatic carbon atom or nitrogen atom as a point of attachment, wherein the carbon atom or nitrogen atom forms a part of one or more aromatic ring structures, wherein at least one ring atom is nitrogen, oxygen or sulfur, and wherein the heteroaryl is not composed of atoms other than carbon, hydrogen, aromatic nitrogen, aromatic oxygen and aromatic sulfur. The heteroaryl ring can contain 1,2,3 or 4 ring atoms selected from nitrogen, oxygen and sulfur. If there is more than one ring, the ring can be fused or unfused. The term used herein does not exclude the presence of one or more alkyl, aryl and / or aralkyl (carbon number restrictions allow) that are connected to an aromatic ring or aromatic ring system. The non-limiting example of heteroaryl includes furanyl, imidazolyl, indolyl, indazolyl (Im), isoxazolyl, methylpyridyl, oxazolyl, phenylpyridyl, pyridyl (pyridyl), pyrrolyl, pyrimidyl, pyrazinyl, quinolyl, quinazolinyl, quinoxalinyl, triazinyl, tetrazolyl, thiazolyl, thienyl and triazolyl.Term " N-heteroaryl " represents the heteroaryl with nitrogen atom as attachment point.Term " heteroarene diyl " used in the case of not having " substituted " modifier represents divalent aromatic group, it has 2 aromatic carbon atoms, 2 aromatic nitrogen atoms or 1 aromatic carbon atom and 1 aromatic nitrogen atom as 2 attachment points, the atom forms a part of one or more aromatic ring structures, wherein at least one ring atom is nitrogen, oxygen or sulfur, and wherein the divalent group is not composed of atoms other than carbon, hydrogen, aromatic nitrogen, aromatic oxygen and aromatic sulfur. If there is more than one ring, the rings may be fused or unfused. Unfused rings may be connected by one or more of: covalent bonds, alkanediyl or alkenediyl groups (carbon number restrictions permitting). The term as used herein does not exclude the presence of one or more alkyl, aryl and / or aralkyl groups (carbon number restrictions permitting) connected to an aromatic ring or aromatic ring system. Non-limiting examples of heteroarenediyls include:

[0270]

[0271] "Heteroarene" refers to the class of compounds having the formula HR, where R is heteroaryl. Pyridine and quinoline are non-limiting examples of heteroarene. When these terms are used with the "substituted" modifier, one or more hydrogen atoms have been independently replaced by -OH, -F, -Cl, -Br, -I, -NH 2 、-NO 2 , -CO 2 H, -CO 2 CH 3 、-CN、-SH、-OCH 3 、-OCH 2 CH 3 、-C(O)CH 3 、-NHCH 3、-NHCH 2 CH 3 、-N(CH 3 ) 2 、-C(O)NH 2 、-C(O)NHCH 3 、-C(O)N(CH 3 ) 2 、-OC(O)CH 3 、-NHC(O)CH 3 、-S(O) 2 OH or -S(O) 2 NH 2 replace.

[0272] The term "heterocycloalkyl" used without the "substituted" modifier means a monovalent non-aromatic group having a carbon atom or a nitrogen atom as a point of attachment, wherein the carbon atom or the nitrogen atom forms a part of one or more non-aromatic ring structures, wherein at least one ring atom is nitrogen, oxygen or sulfur, and wherein the heterocycloalkyl is not composed of atoms other than carbon, hydrogen, nitrogen, oxygen and sulfur. The heterocycloalkyl ring may contain 1, 2, 3 or 4 ring atoms selected from nitrogen, oxygen or sulfur. If there is more than one ring, the ring may be fused or unfused. The term used herein does not exclude the presence of one or more alkyl groups (carbon number restrictions allow) connected to a ring or ring system. Also, the term does not exclude the presence of one or more double bonds in a ring or ring system, provided that the resulting group is still non-aromatic. The non-limiting example of heterocycloalkyl includes aziridine, azetidinyl, pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, tetrahydrofuranyl, tetrahydrothienyl, tetrahydropyranyl, pyranyl, oxacyclopropyl and oxetanyl.Term " N-heterocycloalkyl " represents the heterocycloalkyl with nitrogen atom as the attachment point.N-pyrrolidinyl is an example of such a group.Term " heterocycloalkanediyl " used in the case of not having " substituted " modifier represents the divalent cyclic group with 2 carbon atoms, 2 nitrogen atoms or 1 carbon atom and 1 nitrogen atom as 2 attachment points, the atom forms the part of one or more ring structures, wherein at least one ring atom is nitrogen, oxygen or sulfur, and wherein the divalent group is not composed of the atom except carbon, hydrogen, nitrogen, oxygen and sulfur.If there is more than one ring, the ring can be fused or not fused. The unfused rings may be connected by one or more of the following: covalent bonds, alkanediyl or alkenediyl groups (carbon number restrictions permitting). The term as used herein does not exclude the presence of one or more alkyl groups (carbon number restrictions permitting) attached to a ring or ring system. Also, the term does not exclude the presence of one or more double bonds in a ring or ring system, provided that the resulting group is still non-aromatic. Non-limiting examples of heterocycloalkanediyl include:

[0273]

[0274] When these terms are used with the "substituted" modifier, one or more hydrogen atoms have been independently replaced by -OH, -F, -Cl, -Br, -I, -NH 2 、-NO 2 , -CO 2 H, -CO 2 CH 3 、-CN、-SH、-OCH 3 、-OCH 2 CH 3 、-C(O)CH 3 、-NHCH 3 、-NHCH 2 CH 3 、-N(CH 3 ) 2 、-C(O)NH 2 、-C(O)NHCH 3 、-C(O)N(CH 3 ) 2 、-OC(O)CH 3 、-NHC(O)CH 3 、-S(O) 2 OH or -S(O) 2 NH 2 replace.

[0275] The term "acyl" used without the "substituted" modifier refers to the group -C(O)R, in which R is hydrogen, alkyl, cycloalkyl, alkenyl, aryl, aralkyl or heteroaryl, and those terms are as defined above. The groups -CHO, -C(O)CH 3 (Acetyl, Ac), -C(O)CH 2 CH 3 、-C(O)CH 2 CH 2 CH 3 、-C(O)CH(CH 3 ) 2 、-C(O)CH(CH 2 ) 2 、-C(O)C 6 H 5 、-C(O)C 6 H 4 CH 3 、-C(O)CH 2 C 6 H 5, -C(O)(imidazolyl) are non-limiting examples of acyl groups. "Thioacyl" is defined in a similar manner, except that the oxygen atom of the group -C(O)R has been replaced with a sulfur atom, -C(S)R. The term "aldehyde" corresponds to an alkane as defined above, in which at least one hydrogen atom has been replaced by a -CHO group. When any of these terms is used with the "substituted" modifier, one or more hydrogen atoms (including hydrogen atoms directly attached to the carbon atom of the carbonyl or thiocarbonyl group, if any) have been independently replaced by -OH, -F, -Cl, -Br, -I, -NH 2 、-NO 2 , -CO 2 H, -CO 2 CH 3 、-CN、-SH、-OCH 3 、-OCH 2 CH 3 、-C(O)CH 3 、-NHCH 3 、-NHCH 2 CH 3 、-N(CH 3 ) 2 、-C(O)NH 2 、-C(O)NHCH 3 、-C(O)N(CH 3 ) 2 、-OC(O)CH 3 、-NHC(O)CH 3 、-S(O) 2 OH or -S(O) 2 NH 2 Replacement. Group -C(O)CH 2 CF 3 , -CO 2 H (carboxyl), -CO 2 CH 3 (methylcarboxyl), -CO 2 CH 2 CH 3 、-C(O)NH 2 (carbamoyl) and -CON(CH 3 ) 2 are non-limiting examples of substituted acyl groups.

[0276] The term "alkoxy" used without the "substituted" modifier refers to the group -OR, in which R is alkyl, as that term is defined above. Non-limiting examples include: -OCH 3 (methoxy), -OCH 2 CH 3 (ethoxy), -OCH2 CH 2 CH 3 、-OCH(CH 3 ) 2 (isopropyloxy), -OC(CH 3 ) 3 (tert-butyloxy), -OCH(CH 2 ) 2 , -O-cyclopentyl and -O-cyclohexyl. The terms "cycloalkoxy", "alkenyloxy", "alkynyloxy", "aryloxy", "aralkyloxy", "heteroaryloxy", "heterocycloalkyloxy" and "acyloxy" used without the "substituted" modifier refer to a group defined as -OR, wherein 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" used without the "substituted" modifier refer to the group -SR, wherein R is an alkyl and an acyl, respectively. The term "alcohol" corresponds to an alkane as defined above, wherein at least one hydrogen atom has been replaced with a hydroxyl group. The term "ether" corresponds to an alkane as defined above, wherein at least one hydrogen atom has been replaced with an alkoxy group. When any of these terms is used with the "substituted" modifier, one or more hydrogen atoms have been independently replaced by -OH, -F, -Cl, -Br, -I, -NH 2 、-NO 2 , -CO 2 H, -CO 2 CH 3 、-CN、-SH、-OCH 3 、-OCH 2 CH 3 、-C(O)CH 3 、-NHCH 3 、-NHCH 2 CH 3 、-N(CH 3 ) 2 、-C(O)NH 2 、-C(O)NHCH 3 、-C(O)N(CH 3 ) 2 、-OC(O)CH 3 、-NHC(O)CH 3 、-S(O) 2 OH or -S(O) 2 NH 2 replace.

[0277] The term "alkylamino" used without the "substituted" modifier refers to the group -NHR, in which R is alkyl, as that term is defined above. Non-limiting examples include: -NHCH 3 and -NHCH 2 CH 3 The term "dialkylamino" used without the "substituted" modifier refers to the group -NRR', wherein R and R' may be the same or different alkyl groups, or R and R' may be taken together to represent an alkanediyl group. Non-limiting examples of dialkylamino groups include: -N(CH 3 ) 2 and -N(CH 3 )(CH 2 CH 3 ). The terms "cycloalkylamino," "alkenylamino," "alkynylamino," "arylamino," "aralkylamino," "heteroarylamino," "heterocycloalkylamino," "alkoxyamino," and "alkylsulfonylamino" used without the "substituted" modifier refer to a radical defined as -NHR, wherein R is cycloalkyl, alkenyl, alkynyl, aryl, aralkyl, heteroaryl, heterocycloalkyl, alkoxy, and alkylsulfonyl, respectively. A non-limiting example of an arylamino radical is -NHC 6 H 5 The term "alkylaminodiyl" refers to the divalent radical -NH-alkanediyl-, -NH-alkanediyl-NH-, or -alkanediyl-NH-alkanediyl-. The term "acylamino" (acylamino) used without the "substituted" modifier refers to the radical -NHR in which R is acyl, as that term is defined above. A non-limiting example of an acylamino radical is -NHC(O)CH 3 The term "alkylimino" used without the "substituted" modifier refers to a divalent radical =NR in which R is an alkyl group, as that term is defined above. When any of these terms is used with the "substituted" modifier, one or more hydrogen atoms attached to the carbon atom have been independently replaced by -OH, -F, -Cl, -Br, -I, -NH 2 、-NO 2 , -CO 2 H, -CO 2 CH 3 、-CN、-SH、-OCH 3 、-OCH 2 CH 3 、-C(O)CH 3 、-NHCH 3 、-NHCH 2 CH 3 、-N(CH 3 ) 2 、-C(O)NH2 、-C(O)NHCH 3 、-C(O)N(CH 3 ) 2 、-OC(O)CH 3 、-NHC(O)CH 3 、-S(O) 2 OH or -S(O) 2 NH 2 Replacement. Group -NHC(O)OCH 3 and -NHC(O)NHCH 3 are non-limiting examples of substituted amido groups.

[0278] When used in conjunction with the term "comprising" in the claims and / or the specification, the use of the word "a" or "an" can mean "one", but it is also consistent with the meaning of "one or more", "at least one", and "one or more than one".

[0279] Throughout this application, the term "about" is used to indicate that a value includes the inherent variation for the device or method employed to determine the value or the variation that exists between study subjects.

[0280] As used in the present application, the term "mean molecular weight" represents the relationship between the number of moles of each polymer substance and the molar mass of the substance. Specifically, each polymer molecule can have different polymerization levels and therefore different molar masses. Mean molecular weight can be used to represent the molecular weight of multiple polymer molecules. Mean molecular weight is usually synonymous with average molar mass. Specifically, there are three main types of mean molecular weights: number average molar mass, weight (mass) average molar mass and Z-average molar mass. In the context of the present application, unless otherwise noted, mean molecular weight represents the number average molar mass or weight average molar mass of the formula. In some embodiments, mean molecular weight is number average molar mass. In some embodiments, mean molecular weight can be used to describe the PEG components present in lipids.

[0281] The terms "comprises," "having," and "includes" 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," are also open-ended. For example, any method that "comprises," "has," or "includes" one or more steps is not limited to having only those one or more steps, and also encompasses other unlisted steps.

[0282] The term "effective," as the term is used in this specification and / or claims, means sufficient to achieve a desired, expected, or intended result. When used in the context of treating a patient or subject with a compound, "effective amount," "therapeutically effective amount," or "pharmaceutically effective amount" means an amount of the compound that, when administered to a subject or patient for treating a disease, is sufficient to achieve such treatment for the disease.

[0283] The term “IC 50 ” represents the inhibitory dose that obtains 50% of the maximal response. This quantitative measure indicates the amount of a particular drug or other substance (inhibitor) required 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.

[0284] "Isomers" of a first compound are individual compounds wherein each molecule contains the same constituent atoms as the first compound, but wherein the three-dimensional configuration of those atoms is different.

[0285] As used herein, the term "patient" or "subject" refers to a living mammalian organism, such as a human, monkey, cow, sheep, goat, dog, cat, mouse, rat, guinea pig, or a transgenic species thereof. In certain embodiments, the patient or subject is a primate. Non-limiting examples of human subjects are adults, adolescents, infants, and fetuses.

[0286] As used generally herein, "pharmaceutically acceptable" refers to compounds, materials, compositions and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues, organs and / or body fluids of human beings and animals without excessive toxicity, irritation, allergic response, or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[0287] "Pharmaceutically acceptable salt" refers to a salt of a compound of the present disclosure that is pharmaceutically acceptable as defined above and has the desired pharmacological activity. Such salts include acid addition salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like; or organic acids such as 1,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, 2-naphthalenesulfonic acid, 3-phenylpropionic acid, 4,4'-methylenebis(3-hydroxy-2-ene-1-carboxylic acid), 4-methylbicyclo[2.2.2]oct-2-ene-1-carboxylic acid, acetic acid, aliphatic mono- and dicarboxylic acids, aliphatic sulfuric acid, aromatic sulfuric acid, benzenesulfonic acid, benzoic acid, camphorsulfonic acid, carbonic acid, Pharmaceutically acceptable salts include cinnamic acid, citric acid, cyclopentanepropionic acid, ethanesulfonic acid, fumaric acid, glucoheptonic acid, gluconic acid, glutamic acid, glycolic acid, enanthic acid, caproic 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 acid, propionic acid, p-toluenesulfonic acid, pyruvic acid, salicylic acid, stearic acid, succinic acid, tartaric acid, tert-butylacetic acid, trimethylacetic acid, etc. Pharmaceutically acceptable salts also include base addition salts that can be formed when the acidic protons present can react with inorganic bases 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, etc. It should be appreciated that the specific anion or cation forming part of any salt of the present disclosure is not critical, so long as the salt as a whole is pharmacologically acceptable. Other examples of pharmaceutically acceptable salts and their methods of preparation and use are presented in Handbook of Pharmaceutical Salts: Properties, and Use (PH Stahl & C. G. Wermuth, ed., Verlag Helvetica Chimica Acta, 2002).

[0288] As used herein, the term "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition or vehicle involved in carrying or transporting a chemical agent, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material.

[0289] "Prevention" or "preventing" includes: (1) inhibiting the onset of a disease in a subject or patient who may be at risk for and / or susceptible to the disease but does not yet experience or display any or all symptoms or signs of the disease; and / or (2) slowing the onset of symptoms or signs of a disease in a subject or patient who may be at risk for and / or susceptible to the disease but does not yet experience or display any or all symptoms or signs of the disease.

[0290] A "repeat unit" is the simplest structural entity of some material, e.g., a framework and / or a polymer, whether organic, inorganic, or metallo-organic. In the case of a polymer chain, the repeat units are linked together one after another along the chain, like beads of a necklace. For example, in polyethylene-[-CH 2 CH 2 -] n -, the repeating unit is -CH 2 CH 2 -. The subscript "n" indicates the degree of polymerization, that is, the number of repeating units linked together. When the value of "n" is undefined or in the absence of "n", it simply indicates the repetition of the formula within the brackets and the polymeric nature of the material. The concept of repeating units also applies to situations where the connectivity between repeating units extends three-dimensionally, such as in metal organic frameworks, modified polymers, thermosetting polymers, etc. In the context of dendrimers, repeating units can also be described as branching units, inner layers or generations. Similarly, end-capping groups can also be described as surface groups.

[0291] "Stereoisomers" or "optical isomers" are isomers of a given compound in which the same atoms are bonded to the same other atoms, but in which the three-dimensional configuration of those atoms is different. "Enantiomers" are stereoisomers of a given compound that are mirror images of each other, like the left and right hands. "Diastereomers" are stereoisomers of a given compound that are not enantiomers. Chiral molecules contain a chiral center (also called a stereocenter or stereocenter), which is any point (although not necessarily an atom) in a molecule that carries multiple groups, such that the interchange of any two groups produces a stereoisomer. In organic compounds, the chiral center is usually a carbon, phosphorus, or sulfur atom, although other atoms may also serve as stereocenters in organic and inorganic compounds. A molecule can have multiple stereocenters, giving rise to its many stereoisomers. In compounds whose stereoisomerism is due to tetrahedral stereocenters (e.g., tetrahedral carbon), it is assumed that the total number of possible stereoisomers will not exceed 2. n, where n is the number of tetrahedral stereocenters. Molecules with symmetry often have a smaller number than the maximum possible number of stereoisomers. A 50:50 mixture of enantiomers is called a racemic mixture. Alternatively, a mixture of enantiomers can be enantiomerically enriched so that one enantiomer is present in an amount greater than 50%. Typically, enantiomers and / or diastereomers can be split or separated using techniques known in the art. It is expected that for any stereocenter or chiral axis whose stereochemistry has not yet been defined, the stereocenter or chiral axis can exist in its R form, S form, or as a mixture of the R form and S form (including racemic mixtures and non-racemic mixtures). The phrase "substantially free of other stereoisomers" used herein means that the composition contains ≤15%, more preferably ≤10%, even more preferably ≤5%, or most preferably ≤1% of one or more other stereoisomers.

[0292] "Treatment" or "treating" includes (1) inhibiting a disease in a subject or patient who is experiencing or exhibiting symptoms or signs of the disease (e.g., arresting further development of the symptoms and / or signs), (2) ameliorating a disease in a subject or patient who is experiencing or exhibiting symptoms or signs of the disease (e.g., reversing the symptoms and / or signs), and / or (3) achieving any measurable reduction in a disease in a subject or patient who is experiencing or exhibiting symptoms or signs of the disease.

[0293] The above definitions supersede any conflicting definitions in any references incorporated herein by reference. However, the fact that certain terms are defined should not be taken to mean that any term not defined is undefined. Instead, all terms used are considered to describe the present disclosure in a manner that allows a person of ordinary skill to understand the scope and practice the present disclosure.

[0294] B. Cationic ionizable lipids

[0295] In some aspects of the present disclosure, compositions are provided comprising compounds comprising lipophilic and cationic components, 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 higher than 8, 9, 10, 11, or 12. The ionizable cationic group can contain one or more protonatable amines that can form cationic groups at physiological pH. The cationic ionizable lipid compound can also further comprise one or more lipid components, such as two or more having C 6 -C 24Fatty acids with alkyl or alkenyl carbon groups. These lipid groups can be linked by ester bonds, or can be further added to the sulfur atom by Michael addition. In some embodiments, these compounds can be dendrimers, dendrons, polymers, or combinations thereof.

[0296] In some embodiments, these cationic ionizable lipids are dendrimers, which are polymers that exhibit regular dendritic branches, formed by adding branching layers to or from a core in sequence or by generation, and characterized by a core, at least one internal branching layer, and a surface branching layer (see Petar R. Dvornic and Donald A. Tomalia in Chem. in Britain, 641-645, August 1994). In other embodiments, the term "dendrimer" as used herein is intended to include, but is not limited to, a molecular architecture having an inner core, an inner layer (or "generation") of repeating units regularly connected to the starting core, and an outer surface connected to the end-capping groups of the outermost generation. A "dendron" is a dendritic polymer substance with branches emanating from a focal point, which is a core, or can be connected to a core directly or through a linking moiety to form a larger dendrimer. In some embodiments, the dendritic polymer structure has repeating groups radiating from a central core, which double with each repeating unit for each branch. In some embodiments, the dendritic polymers described herein can be described as small molecules, medium-sized molecules, lipids, or lipid-like substances. These terms can be used to describe compounds described herein that have a dendron-like appearance (e.g., molecules radiating from a single focus).

[0297] Although dendrimers are polymers, they may be superior to traditional polymers because they have a controlled structure, a single molecular weight, numerous and controllable surface functional groups, and traditionally adopt a globular conformation after reaching a certain number of generations. Dendrimers can be prepared by sequential reaction of each repeating unit to produce a monodisperse, tree-like and / or generation-structured polymer structure. A single dendrimer consists of a central core molecule with dendritic wedges attached to one or more functional sites on the central core. Depending on the assembly monomers used in the preparation process, the dendrimer surface layer can have a variety of functional groups disposed thereon, including anionic, cationic, hydrophilic, or lipophilic groups.

[0298] The functional groups and / or chemical properties of the core, repeating units and surfaces or end-capping groups can be changed to adjust their physical properties. Some characteristics that can be changed include, but are not limited to solubility, toxicity, immunogenicity and biological adhesion ability. Dendritic polymers are often described by the number of repeating units in their generations or branches. The dendritic polymers that are only composed of the core molecule are referred to as the 0th generation, and each continuous repeating unit along all branches is the 1st generation, the 2nd generation and so on, until the end-capping or surface groups. In some embodiments, a half generation may only be produced by the first condensation reaction with amine rather than the second condensation reaction with thiol.

[0299] The preparation of dendritic polymers requires a level of synthetic control achieved through a series of stepwise reactions, which includes building up the dendritic polymer through each successive group. Dendritic polymer synthesis can be convergent or divergent. During divergent dendritic polymer synthesis, the molecules are assembled from the core to the periphery in a stepwise process, which includes connecting one generation to the previous generation and then changing the functional group for the next reaction stage. Functional group conversion is necessary to prevent uncontrolled polymerization. Such polymerization will result in highly branched molecules that are not monodisperse, and are otherwise known as hyperbranched polymers. Due to steric effects, dendritic polymer repeat units continue to react to produce spherical or globular molecules until steric overcrowding prevents complete reaction at a particular generation and destroys the monodispersity of the molecule. Therefore, in some embodiments, dendritic polymers of generations G1-G10 are specifically contemplated. In some embodiments, the dendritic polymer comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 repeating units, or any range derivable therein. In some embodiments, the dendritic polymer used herein is G0, G1, G2 or G3. However, the possible number of generations can be increased (such as 11, 12, 13, 14, 15, 20 or 25) by reducing the spacer unit in the branched polymer.

[0300] Furthermore, dendrimers have two main chemical environments: the environment created by the specific surface groups on the end capping groups, and the interior of the dendrimer that may be shielded from the bulk medium and surface groups due to the higher order structure. Due to these different chemical environments, dendrimers have found many different potential uses, including in therapeutic applications.

[0301] In some aspects, the differential reactivity of acrylate and methacrylate groups with amines and thiols is used to assemble dendritic polymers that can be used in the present composition. Dendritic polymers can include secondary or tertiary amines and thioethers formed by the reaction of acrylate groups with primary or secondary amines, and methacrylates with thiol groups. In addition, the repeating units of the dendritic polymers can contain groups that are degradable under physiological conditions. In some embodiments, these repeating units can contain one or more germinated diether, ester, amide or disulfide groups. In some embodiments, the core molecule is a monoamine that allows dendritic polymerization in only one direction. In other embodiments, the core molecule is a polyamine with multiple different dendritic branches, each branch can contain one or more repeating units. Dendritic polymers can be formed by removing one or more hydrogen atoms from the core. In some embodiments, these hydrogen atoms are on heteroatoms such as nitrogen atoms. In some embodiments, the end-capping group is a lipophilic group such as a long-chain alkyl or alkenyl group. In other embodiments, the end-capping group is a long-chain haloalkyl or haloalkenyl group. In other embodiments, the end-capping group is a lipophilic group containing an ionizable group such as an amine (-NH 2 ) or carboxylic acid (-CO 2 In other embodiments, the capping group is an aliphatic or aromatic group containing one or more hydrogen bond donors such as a hydroxide group, an amide group, or an ester.

[0302] The cationic ionizable lipids of the present disclosure may contain one or more asymmetrically substituted carbon or nitrogen atoms and may be separated in optically active or racemic forms. Thus, unless a specific stereochemistry or isomeric form is specifically indicated, all chiral, diastereoisomer, racemic form, epimeric form and all geometric isomeric forms of the chemical formula are meant. Cationic ionizable lipids may exist as racemates and racemic mixtures, single enantiomers, diastereomeric mixtures and each diastereomer. In some embodiments, a single diastereomer is obtained. The chiral center of the cationic ionizable lipids of the present disclosure may have an S or R configuration. In addition, it is expected that one or more of the cationic ionizable lipids may exist as structural isomers. In some embodiments, the compound has the same chemical formula, but the connectivity with the nitrogen atom of the core is different. Without wishing to be bound by any theory, it is believed that such cationic ionizable lipids exist because the starting monomer reacts first with the primary amine and then statistically reacts with any secondary amine present. Thus, the structural isomers may present a fully reacted primary amine and then a mixture of reacted secondary amines.

[0303] Chemical formulae for representing cationic ionizable lipids of the present disclosure will typically show only one of several different tautomers that are possible. For example, many types of keto groups are known to exist in equilibrium with the corresponding enol groups. Similarly, many types of imino groups exist in equilibrium with enamine groups. Regardless of which tautomer is depicted for a given formula, and regardless of which tautomer is the most common, all tautomers of a given chemical formula are intended.

[0304] The cationic ionizable lipids of the present disclosure may also have the following advantages: 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), and / or have other useful pharmacological, physical or chemical properties compared to compounds known in the prior art (whether used for the indications described herein or otherwise).

[0305] In addition, the atoms that make up the cationic ionizable lipids of the present disclosure are intended to include all isotopic forms of such atoms. As used herein, isotopes include those atoms having the same atomic number but different mass numbers. As a general example and not limitation, isotopes of hydrogen include tritium and deuterium, and isotopes of carbon include 13 C and 14 C.

[0306] It should be appreciated that the specific anion or cation forming part of any salt form of the cationic ionizable lipids provided herein is not critical, so long as the salt as a whole is pharmacologically acceptable. Other examples of pharmaceutically acceptable salts and methods of their preparation and use are presented in Handbook of Pharmaceutical Salts: Properties, and Use (2002), which is incorporated herein by reference.

[0307] In some embodiments, the ionizable cationic lipid is present in an amount of about 20 to about 23. In some embodiments, the mole percentage is about 20, 20.5, 21, 21.5, 22, 22.5, to about 23, or any range derivable therein. In other embodiments, the mole percentage is about 7.5 to about 20. In some embodiments, the mole 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.

[0308] C. Selective Organ Targeting (SORT) Compounds

[0309] In some aspects, the disclosure comprises one or more selective organ targeting (SORT) compounds that result in the selective delivery of a composition to a specific organ. The compound can be a lipid, a small molecule therapeutic, a sugar, a vitamin, or a protein.

[0310] In some embodiments, the selective organ targeting (SORT) compound is present in the composition in a molar ratio of about 2%, 4%, 5%, 10%, 15%, 20%, 22%, 24%, 26%, 28%, 30%, 32%, 34%, 36%, 38%, 40%, 45%, 50%, 55%, 60%, 65%, to about 70%, or any range 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%.

[0311] In some embodiments, the SORT compound can be a lipid. A lipid is a lipid having two or more C 6 -C 24 Small molecule therapeutics are compounds containing fewer than 100 non-hydrogen atoms and weighing less than 2,000 Daltons. n H 2n O n (where n is 3 to 7), or a combination of multiple molecules of this formula. A protein is an amino acid sequence containing at least 3 amino acid residues. A protein without a formal tertiary structure may also be called a peptide. A protein may also include a complete protein with a tertiary structure. Vitamins are macronutrients and consist of one or more selected from vitamin A, vitamin B 1 , Vitamin B 2 , Vitamin B 3 , Vitamin B 5 , Vitamin B 6 , Vitamin B 7 , Vitamin B 9 , Vitamin B 12 , vitamin C, vitamin D, vitamin E and vitamin K.

[0312] 1. Permanent cationic lipids

[0313] In some aspects, the disclosure provides one or more lipids having one or more hydrophobic components and permanent cationic groups. The permanent cationic lipid can contain a group with a positive charge (regardless of pH). A permanent cationic group that can be used in the permanent cationic lipid is a quaternary ammonium group. These permanent cationic lipids include structures such as those described in the following formula:

[0314]

[0315] in:

[0316] Y 1 , Y 2 or Y 3 Each independently is X 1 C(O)R 1 or X 2 N + R 3 R 4 R 5 ; As long as Y 1 , Y 2 and Y 3 At least one of them is X 2 N + R 3 R 4 R 5 ;

[0317] R 1 It is C 1 -C 24 Alkyl, C 1 -C 24 Substituted alkyl, C 1 -C 24 Alkenyl, C 1 -C 24 substituted alkenyl;

[0318] X 1 Is O or NR a , where R a It is hydrogen, C 1 -C 4 Alkyl or C 1 -C 4 Substituted alkyl;

[0319] X 2 It is C 1 -C 6 Alkanediyl or C 1 -C 6 Substituted alkanediyl;

[0320] R 3 , R 4 and R 5 Each is independently C 1 -C 24 Alkyl, C 1 -C 24 Substituted alkyl, C 1 -C 24 Alkenyl, C 1 -C 24 substituted alkenyl;

[0321] A 1 is an anion, which has a charge equal to that of the X 2 N + R 3 R 4 R 5 The number of groups.

[0322] In another embodiment, the permanent cationic lipid is further defined by the formula:

[0323]

[0324] in:

[0325] R 6 -R 9 Each is independently C 1 -C 24 Alkyl, C 1 -C 24 Substituted alkyl, C 1 -C 24 Alkenyl, C 1 -C 24 Substituted alkenyl; as long as R 6 -R 9 At least one of them is C 8 -C 24 and

[0326] A 2 It is a monovalent anion.

[0327] In another embodiment, the permanent cationic lipid is further defined by the formula:

[0328]

[0329] in:

[0330] R 1 and R 2 are independently alkyl (C8-C24) , alkenyl (C8-C24) or a substituted form of any group;

[0331] R 3 , R 3 ′ and R 3 "are each independently an alkyl (C≤6) or substituted alkyl (C≤6) ;

[0332] R 4 It is an alkyl (C≤6) or substituted alkyl (C≤6) ;and

[0333] X- It is a monovalent anion.

[0334] In some embodiments, the permanent cationic lipid is present in an amount of about 4 to about 16 molar percentages of a total lipid composition. The composition can contain about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 molar percentages or any range that can be derived therefrom. In other embodiments, the composition can include about 18 to about 66 molar percentages of a total lipid composition. In some embodiments, the composition can 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 percentages or any range that can be derived therefrom.

[0335] 2. Permanent anionic lipids

[0336] In some aspects, the present disclosure provides one or more lipids having one or more hydrophobic components and permanent anionic groups. One anionic group that can be used in permanent anionic lipids is a phosphate group. The phosphate group can be a compound that is deprotonated and has a negative charge at a pH below 8, 9, 10, 11, 12, 13, or 14. The hydrophobic component can be one or more C 6 -C 24 The compound may have one hydrophobic group, two hydrophobic groups, or three hydrophobic groups.

[0337] In some embodiments, the permanent anionic lipid has the structure of the following formula:

[0338]

[0339] in:

[0340] R 1 and R 2 are independently alkyl (C8-C24) , alkenyl (C8-C24) or a substituted form of any group;

[0341] R 3 is hydrogen, alkyl (C≤6) or substituted alkyl (C≤6) or -Y 1 -R 4 ,in:

[0342] Y 1 Alkanediyl (C≤6) or substituted alkanediyl (C≤6) ;and

[0343] R4 It is acyloxy (C≤8-24) or substituted acyloxy (C≤8-24) .

[0344] 3. Phosphatidylcholine

[0345] In some aspects, the present disclosure provides one or more lipids having one or more hydrophobic components, a cationic amine group, and a negatively charged phosphate group. The cationic amine group can be a quaternary amine having three methyl groups attached to a nitrogen atom. The hydrophobic component can be one or more C 6 -C 24 Alkyl or alkenyl. The compound may have one hydrophobic group, two hydrophobic groups or three hydrophobic groups. In some embodiments, the phosphatidylcholine compound is further defined as:

[0346]

[0347] in:

[0348] R 1 and R 2 are independently alkyl (C8-C24) , alkenyl (C8-C24) or a substituted form of any group;

[0349] R 3 , R 3 ′ and R 3 "are each independently an alkyl (C≤6) or substituted alkyl (C≤6) ;and

[0350] X - It is a monovalent anion.

[0351] D. Additional lipids in lipid nanoparticles

[0352] In some aspects of the present disclosure, a composition containing one or more lipids is mixed with a cationic ionizable lipid to produce a composition. In some embodiments, the cationic ionizable lipid is mixed with 1, 2, 3, 4 or 5 different types of lipids. It is contemplated that a cationic ionizable lipid can be mixed with a single type of multiple different lipids. In some embodiments, the cationic ionizable lipid composition comprises at least a steroid or steroid derivative, a PEG lipid and a phospholipid.

[0353] In some embodiments, the lipid nanoparticle is preferentially delivered to a target organ. In some embodiments, the target organ is selected from lung, heart, brain, spleen, bone marrow, bone, skeletal muscle, stomach, small intestine, large intestine, kidney, bladder, breast, liver, testis, ovary, uterus, spleen, thymus, brain stem, cerebellum, spinal cord, eye, ear, tongue or skin. Alternatively, the composition can be preferentially delivered to a target organ system, such as a part of the nervous system, cardiovascular system or respiratory system or one of these organ systems. The term "preferential delivery" used herein is used to represent a composition delivered to a target organ or organ system at least 25% of the amount applied. The term is used to represent a composition delivered to a target organ or organ system at least 25%, 50% or at least 75% of the amount applied.

[0354] 1. Steroids and steroid derivatives

[0355] In some aspects of the present disclosure, cationic ionizable lipids are mixed with one or more steroids or steroid derivatives to produce a composition. In some embodiments, the steroid or steroid derivative comprises any steroid or steroid derivative. As used herein, in some embodiments, the term "steroid" is a class of compounds with a tetracyclic 17-carbon ring structure, which may further comprise one or more substitutions, including alkyl, alkoxy, hydroxyl, oxo, acyl, or a double bond between two or more carbon atoms. In one aspect, the ring structure of a steroid comprises three fused cyclohexyl rings and a fused cyclopentyl ring, as shown in the following formula:

[0356]

[0357] In some embodiments, the steroid derivative comprises the above ring structure with one or more non-alkyl substitutions. In some embodiments, the steroid or steroid derivative is a sterol, wherein the formula is further defined as:

[0358]

[0359] In some embodiments of the present disclosure, the steroid or steroid derivative is cholestane or a cholestane derivative. In cholestane, the ring structure is further defined by the formula:

[0360]

[0361] As described above, cholestane derivatives include non-alkyl substitutions of one or more of the above ring systems. In some embodiments, the cholestane or cholestane derivative is cholestene or a cholestene derivative or a sterol or a sterol derivative. In other embodiments, the cholestane or cholestane derivative is cholestene and a sterol or a derivative thereof.

[0362] In some embodiments, the composition may further comprise a molar percentage of steroid 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.

[0363] 2. PEG or PEGylated lipids

[0364] In some aspects of the present disclosure, the polymer is mixed with one or more PEGylated lipids (or PEG lipids) to produce a lipid composition. In some embodiments, the present disclosure includes the use of any lipid that has been connected to a PEG group. In some embodiments, the PEG lipid is a diglyceride, which also includes a PEG chain connected to a glycerol group. In other embodiments, the PEG lipid is a compound containing one or more C6-C24 long-chain alkyl or alkenyl or C6-C24 fatty acid groups connected to a linker group with a PEG chain. Some non-limiting examples of PEG lipids include PEG-modified phosphatidylethanolamine and phosphatidic acid, PEG-conjugated ceramides, PEG-modified dialkylamines and PEG-modified 1,2-diacyloxypropane-3-amines, PEG-modified diacylglycerols and dialkylglycerols. In some embodiments, PEG-modified distearoyl phosphatidylethanolamine or PEG-modified dimyristoyl-sn-glycerol. In some embodiments, PEG is measured 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, 12,500 to about 15,000. Some non-limiting examples of lipids that may be used in the present disclosure are taught in US Pat. No. 5,820,873, WO 2010 / 141069, or US Pat. No. 8,450,298, which are incorporated herein by reference.

[0365] In another aspect, the PEG lipid has the formula:

[0366]

[0367] Where: R 12 and R 13 are independently alkyl (C≤24) , alkenyl (C≤24) or a substituted form of any of these groups; R e is hydrogen, alkyl (C≤8) or substituted alkyl (C≤8) ; and x is 1-250. In some embodiments, R e It is an alkyl (C≤8) Such as methyl. 12 and R 13 are independently alkyl (C≤4-20) In some embodiments, x is 5-250. In one embodiment, x is 5-125 or x is 100-250. In some embodiments, the PEG lipid is 1,2-dimyristoyl-sn-glycerol, methoxypolyethylene glycol.

[0368] In another aspect, the PEG lipid has the formula:

[0369]

[0370] Where: n 1 is an integer between 1 and 100, and n 2 and n 3 Each is independently selected from an integer between 1 and 29. In some embodiments, n 1 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, n 1 is about 30 to about 50. In some embodiments, n 2 is 5-23. In some embodiments, n 2 is from 11 to about 17. In some embodiments, n 3 is 5-23. In some embodiments, n 3 It is 11 to about 17.

[0371] In some embodiments, the composition can further include a PEG lipid of about 4.0 to about 4.6 and a molar percentage of the total lipid composition. In some embodiments, the molar percentage is about 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, to about 4.6 or any range that can be derived therefrom. In other embodiments, the molar percentage is about 1.5 to about 4.0. In some embodiments, the molar percentage is about 1.5, 1.75, 2, 2.25, 2.5, 2.75, 3, 3.25, 3.5, 3.75, to about 4.0 or any range that can be derived therefrom.

[0372] 3. Phospholipids

[0373] In some aspects of the present disclosure, the polymer is mixed with one or more phospholipids to produce a composition. In some embodiments, any lipid also comprising a phosphate group. In some embodiments, the phospholipid is a structure containing one or two long-chain C6-C24 alkyl or alkenyl, glycerol or sphingosine, one or two phosphate groups and optional small organic molecules. In some embodiments, the small organic molecule is an alkoxy group substituted with an amino acid, sugar or amino group, such as choline or ethanolamine. In some embodiments, the phospholipid is phosphatidylcholine. In some embodiments, the phospholipid is distearoylphosphatidylcholine or dioleoylphosphatidylethanolamine.

[0374] In some embodiments, the composition may further comprise a mole percentage of phospholipids to total lipid composition of about 20 to about 23. In some embodiments, the mole percentage is about 20, 20.5, 21, 21.5, 22, 22.5, to about 23, or any range derivable therein. In other embodiments, the mole percentage is about 7.5 to about 20. In some embodiments, the mole 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.

[0375] E. Therapeutic agents

[0376] 1. Nucleic Acids

[0377] 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 existing with the lipid composition in a weight ratio of about 5:1 to about 1:100. In some embodiments, the weight ratio of the nucleic acid to the lipid composition is about 5:1, 2.5:1, 1:1, 1:5, 1:10, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, 1:50, 1:60, 1:70, 1:80, 1:90 or 1:100 or any range that can be derived therefrom. In some embodiments, the weight ratio is about 1:40. In addition, it should be clear that the present disclosure is not limited to the specific nucleic acids disclosed herein. The present disclosure is not limited in scope to any particular source, sequence or type of nucleic acid, however, those of ordinary skill in the art can readily identify related homologs in various other nucleic acid sources, including nucleic acids from non-human species (e.g., mice, rats, rabbits, dogs, monkeys, gibbons, chimpanzees, apes, baboons, cattle, pigs, horses, sheep, cats and other species). It is contemplated that the nucleic acids used in the present disclosure may comprise sequences based on naturally occurring sequences. Considering the degeneracy of the genetic code, a sequence having at least about 50%, usually at least about 60%, more usually about 70%, most usually about 80%, preferably at least about 90% and most preferably about 95% of the nucleotides is identical to the nucleotide sequence of the naturally occurring sequence. In another embodiment, the nucleic acid is a sequence complementary to a naturally occurring sequence, or is complementary at 75%, 80%, 85%, 90%, 95% and 100%. Longer polynucleotides encoding 250, 500, 1000, 1212, 1500, 2000, 2500, 3000 or more are encompassed herein.

[0378] 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). Also contemplated are cDNA plus natural introns or introns from another gene; such engineered molecules are sometimes referred to as "mini-genes". At a minimum, these and other nucleic acids of the present disclosure may be used as molecular weight standards in, for example, gel electrophoresis.

[0379] The term "cDNA" is intended to represent a DNA prepared using messenger RNA (mRNA) as a template. Compared with genomic DNA or DNA polymerized from a genomic, non-processed or partially processed RNA template, the advantage of using cDNA is that cDNA primarily contains the coding sequence of the corresponding protein. All or part of the genomic sequence may sometimes be preferred, such as when the non-coding region is required for optimal expression, or when the non-coding region, such as an intron, will be targeted in an antisense strategy.

[0380] In some embodiments, the nucleic acid comprises one or more antisense segments that inhibit the expression of a gene or gene product. Antisense methods utilize the fact that nucleic acids tend to pair with "complementary" sequences. Complementary means that polynucleotides are polynucleotides that can perform base pairing according to standard Watson-Crick complementarity rules. That is, larger purines will perform base pairing with smaller pyrimidines, to form a combination of guanine (G:C) paired with cytosine and adenine (A:T) paired with thymine in the case of DNA, or to form adenine (A:U) paired with uracil in the case of RNA. In the hybridization sequence, less common bases such as inosine, 5-methylcytosine, 6-methyladenine, hypoxanthine, etc. will not interfere with pairing.

[0381] Targeting double-stranded (ds) DNA with polynucleotides results in triple helix formation; targeting RNA will result in double helix formation. Antisense polynucleotides specifically bind to their target polynucleotides when introduced into target cells 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 in vitro or in vivo (such as in host animals including human subjects).

[0382] Antisense constructs can be designed to bind the promoter and other control regions, exons, introns or even exon-intron boundaries of genes. It is expected that the most effective antisense constructs will include a region complementary to the intron / exon splicing junction. Therefore, a preferred embodiment is proposed to include an antisense construct with a region within the 50-200 base of the intron-exon splicing junction that has complementarity. It has been observed that some exon sequences can be included in the construct without seriously affecting its target selectivity. The amount of the exon material included will vary according to the specific exon and intron sequence used. Can simply include too much exon DNA by easily testing as follows: test the construct in vitro to determine whether normal cell function is affected or whether the expression of the associated gene with the complementary sequence is affected.

[0383] As mentioned above, "complementary" or "antisense" refers to polynucleotide sequences that are substantially complementary over their entire length and have very few base mismatches. For example, when a sequence of 15 bases in length has complementary nucleotides at 13 or 14 positions, they can be called complementary. Naturally, a fully complementary sequence would be a sequence that is fully complementary over its entire length and has no base mismatches. Other sequences with a lower degree of homology are also contemplated. For example, antisense constructs can be designed that have limited regions of high homology but also contain non-homologous regions (e.g., ribozymes; see below). These molecules, although having less than 50% homology, will bind to the target sequence under appropriate conditions.

[0384] 2. Modified Nucleobases

[0385] In some embodiments, the nucleic acid of the present disclosure comprises one or more modified nucleosides, and the nucleosides comprise modified sugar moieties. Such compounds comprising one or more sugar-modified nucleosides may have desired characteristics, such as enhanced nuclease stability or increased binding affinity to target nucleic acids relative to oligonucleotides comprising only nucleosides containing naturally occurring sugar moieties. In some embodiments, the modified sugar moieties are substituted sugar moieties. In some embodiments, the modified sugar moieties are sugar surrogates. Such sugar surrogates may comprise one or more substitutions corresponding to the substitutions of the substituted sugar moieties.

[0386] In some embodiments, the modified sugar moiety is a substituted sugar moiety comprising one or more non-bridging sugar substituents, including, but not limited to, substituents at the 2' and / or 5' positions. Examples of sugar substituents suitable for the 2'-position include, but are not limited to: 2'-F, 2'-OCH 3 ("OMe" or "O-methyl") and 2'-O(CH 2 ) 2 OCH 3 ("MOE"). In certain embodiments, the sugar substituent at the 2' position is selected from the group consisting of allyl, amino, azido, thio, O-allyl, O--C 1 -C 10 Alkyl, O--C 1 -C 10 Substituted alkyl; OCF 3 , O(CH 2 ) 2 SCH 3 , O(CH 2 ) 2 --O--N(Rm)(Rn) and O--CH 2 --C(=O)--N(Rm)(Rn), wherein each Rm and Rn is independently H or substituted or unsubstituted C 1 -C 10 Alkyl. Examples of sugar substituents at the 5'-position include, but are not limited to: 5'-methyl (R or S); 5'-vinyl and 5'-methoxy. In some embodiments, the substituted sugar comprises more than one non-bridging sugar substituent, for example, TF-5'-methyl sugar moiety (for additional 5',2'-disubstituted sugar moieties and nucleosides, see, e.g., PCT International Application WO 2008 / 101157).

[0387] Nucleosides containing 2'-substituted sugar moieties are referred to as 2'-substituted nucleosides. In some embodiments, the 2'-substituted nucleosides contain 2'-substituents selected from the group consisting of halogen, allyl, amino, azido, SH, CN, OCN, CF 3 、OCF 3 , O, S or N (R m )-alkyl; O, S or N(R m )-alkenyl; O, S or N(R m )-alkynyl; O-alkylalkenyl-O-alkyl, alkynyl, alkaryl, aralkyl, O-alkaryl, O-aralkyl, O(CH 2 ) 2 SCH 3 , O(CH 2 ) 2 --O--N(R m )(R n ) or O--CH 2 --C(=O)--N(R m )(R n ), where each R m and R n are independently H, an amino protecting group or a substituted or unsubstituted C 1 -C 10 These 2'-substituents may be further substituted by one or more substituents independently selected from hydroxyl, amino, alkoxy, carboxyl, benzyl, phenyl, nitro (NO 2 ), mercapto, thioalkoxy (S-alkyl), halogen, alkyl, aryl, alkenyl, and alkynyl.

[0388] In some embodiments, the 2'-substituted nucleoside comprises a 2'-substituent selected from the group consisting of: F, NH 2 、N 3 、OCF 3 、O--CH 3 , O(CH 2 ) 3 NH 2 , CH 2 —CH=CH 2 、O--CH 2 —CH=CH 2 、OCH 2 CH 2 OCH 3 , O(CH 2 ) 2 SCH 3 、O--(CH 2 ) 2 --O--N(R m )(Rn )、O(CH 2 ) 2 O(CH 2 ) 2 N(CH 3 ) 2 and N-substituted acetamides (O--CH 2 --C(=O)--N(R m )(R n ), where each R m and R n are independently H, an amino protecting group or a substituted or unsubstituted C 1 -C 10 alkyl.

[0389] In some embodiments, the 2'-substituted nucleoside comprises a sugar moiety comprising a 2'-substituent selected from the group consisting of: F, OCF 3 、O--CH 3 、OCH 2 CH 2 OCH 3 , O(CH 2 ) 2 SCH 3 , O(CH 2 ) 2 --O--N(CH 3 ) 2 、--O(CH 2 ) 2 O(CH 2 ) 2 N(CH 3 ) 2 and O--CH 2 --C(=O)--N(H)CH 3 .

[0390] In some embodiments, the 2'-substituted nucleoside comprises a moiety selected from the group consisting of F, O--CH 3 and OCH 2 CH 2 OCH 3 The 2'-substituent is the sugar portion.

[0391] Certain modified sugar moieties include a bridging sugar substituent that forms a second ring, thereby generating 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' to 2' sugar substituents include, but are not limited to: - [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'-CH 2 -2', 4'-(CH 2 ) 2 -2', 4'-(CH 2 )--O-2'(LNA);4'-(CH 2 )--S-2';4'-(CH 2 ) 2 --O-2'(ENA); 4'-CH(CH 3 )--O-2'(cEt) and 4'-CH(CH 2 OCH 3 )--O-2' and its analogs (see, e.g., U.S. Pat. No. 7,399,845); 4'-C(CH 3 )(CH 3 )--O-2' and its analogs, (see, for example, WO 2009 / 006478); 4'-CH 2 --N(OCH 3 )-2' and its analogs (see, for example, WO2008 / 150729); 4'-CH 2 --O--N(CH 3 )-2' (see, for example, US 2004 / 0171570, published on September 2, 2004); 4'-CH 2 --O--N(R)-2', and 4'-CH 2 --N(R)--O-2'-, wherein each R is independently H, a protecting group or C 1 -C 12 Alkyl; 4'-CH 2 --N(R)--O-2', where R is H, C 1 -C 12 Alkyl or protecting group (see, U.S. Patent 7,427,672); 4'-CH 2 --C(H)(CH 3 )-2' (see, e.g., Chattopadhyaya et al., J. Org. Chem., 2009, 74, 118-134); and 4'-CH 2 --C(=CH 2 )-2' and its analogs (see, PCT International Application WO 2008 / 154401).

[0392] In some embodiments, such a 4'-to-2' bridge independently comprises from 1 to 4 linking groups independently selected from the following: --[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:

[0393] x is 0, 1 or 2;

[0394] n is 1, 2, 3 or 4;

[0395] Each R a and R b is independently H, a protecting group, a hydroxyl group, C 1 -C 12 alkyl, substituted C 1 -C 12 alkyl, C 2 -C 12 alkenyl, substituted C 2 -C 12 alkenyl, C 2 -C 12 alkynyl, substituted C 2 -C 12 alkynyl, C 5 -C 20 aryl, substituted C 5 -C 20 aryl, a heterocyclic residue, a substituted heterocyclic residue, a heteroaryl, a substituted heteroaryl, C 5 -C 7 alicyclic residue, substituted C 5 -C 7 alicyclic residue, a halogen, OJ 1 、NJ 1 J 2 、SJ 1 、N 3 、COOJ 1 、 an acyl group (C(=O)--H), a substituted acyl group, CN, a sulfonyl group (S(=O) 2 -J 1 ) or a sulfinyl group (S(=O)-J 1);and

[0396] Each J 1 and J 2 Independently H, C 1 -C 12 Alkyl, substituted C 1 -C 12 Alkyl, C 2 -C 12 Alkenyl, substituted C 2 -C 12 Alkenyl, C 2 -C 12 Alkynyl, substituted C 2 -C 12 Alkynyl, C 5 -C 20 Aryl, substituted C 5 -C 20 Aryl, acyl (C(=O)--H), substituted acyl, heterocyclic residue, substituted heterocyclic residue, C 1 -C 12 Aminoalkyl, substituted C 1 -C 12 Aminoalkyl or a protecting group.

[0397] Nucleosides containing a bicyclic sugar moiety are referred to as bicyclic nucleosides or BNAs. Bicyclic nucleosides include, but are not limited to: (A) α-L-methyleneoxy (4'-CH 2 --O-2')BNA, (B)β-D-methyleneoxy (4'-CH 2 --O-2')BNA (also called locked nucleic acid or LNA), (C)ethyleneoxy (4'-(CH 2 ) 2 --O-2')BNA, (D)aminooxy (4'-CH 2 --O--N(R)-2')BNA, (E)oxyamino(4'-CH 2 --N(R)--O-2')BNA, (F)methyl(methyleneoxy)(4'-CH(CH 3 )--O-2')BNA (also known as constrained ethyl or cEt), (G)methylene-sulfide (4'-CH 2 --S-2')BNA, (H)methylene-amino (4'-CH2-N(R)-2')BNA, (I)methylcarbocyclyl (4'-CH 2 --CH(CH 3 )-2')BNA, (J)propylene carbocyclyl (4'-(CH 2 ) 3 -2')BNA and (K)methoxy(ethyleneoxy)(4'-CH(CH2 OMe)-O-2')BNA (also known as restricted MOE or cMOE).

[0398] Other 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 (July 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. Patents 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 Publication Nos. US2004 / 0171570, US 2007 / 0287831, and US2008 / 0039618; U.S. Serial 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.

[0399] In some embodiments, bicyclic sugar moieties and nucleosides incorporating such bicyclic sugar moieties are further defined by isomeric configurations. For example, nucleosides comprising a 4'-2' methylene-oxy bridge may be in an α-L configuration or in a β-D configuration. Previously, α-L-methyleneoxy (4'-CH 2--O-2') bicyclic nucleosides have been incorporated into antisense oligonucleotides that exhibit antisense activity (Frieden et al., Nucleic Acids Research, 2003, 21, 6365-6372).

[0400] 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' bridged sugars; PCT International Application WO 2007 / 134181, wherein LNA is substituted with, e.g., 5'-methyl or 5'-vinyl).

[0401] In some embodiments, the modified sugar moiety is a sugar substitute. In some such embodiments, the oxygen atom of a naturally occurring sugar is replaced by, for example, sulfur, carbon or nitrogen atom. In some such embodiments, such a modified sugar moiety also includes bridging and / or non-bridging substituents as described above. For example, some sugar substitutes include 4'-sulfur atoms and substitutions at 2'-positions (see, for example, disclosed U.S. Patent Application US2005 / 0130923) and / or 5' positions. As another example, carbocyclic bicyclic nucleosides with 4'-2' bridges have been described (see, for example, Freier et al., Nucleic Acids Research, 1997, 25 (22), 4429-4443 and Albaek et al., J. Org. Chem., 2006, 71, 7731-7740).

[0402] In some embodiments, the sugar substitute comprises a ring with a non-5-atom. For example, in some embodiments, the sugar substitute comprises a 6-membered tetrahydropyran. Such tetrahydropyran can be further modified or substituted. Nucleosides comprising such modified tetrahydropyrans include, but are not limited to, hexitol nucleic acids (HNA), anitol nucleic acids (ANA), mannitol nucleic acids (MNA) (see Leumann, C J. Bioorg. and Med. Chem. (2002) 10: 841-854) and fluoro-HNA (F-HNA).

[0403] In some embodiments, a modified THP nucleoside of Formula VII is provided, wherein q 1 ,q 2 ,q 3 ,q 4 ,q 5 ,q 6 and q 7 Each is H. In certain embodiments, q 1 ,q 2 ,q 3 ,q 4 ,q5 ,q 6 and q 7 At least one of is not H. In some embodiments, q 1 ,q 2 ,q 3 ,q 4 ,q 5 ,q 6 and q 7 In some embodiments, a THP nucleoside of Formula VII is provided, wherein R 1 and R 2 In certain embodiments, R 1 is fluorine and R 2 Yes H, R 1 is methoxy and R 2 is H, and R 1 is methoxyethoxy and R 2 It's H.

[0404] Many other bicyclic and tricyclic sugar surrogate ring systems are also known in the art and can be used to modify nucleosides for incorporation into antisense compounds (see, for example, review article: Leumann, JC, Bioorganic and Medicinal Chemistry, 2002, 10, 841-854).

[0405] Combinations of modifications are also provided, including but not limited to, such as 2'-F-5'-methyl substituted nucleosides (see PCT International Application WO 2008 / 101157 for other disclosed 5',2'-disubstituted nucleosides) and replacement of the ribosyl ring oxygen atom with S and further substitution at the 2'-position (see U.S. Patent Publication No. US 2005 / 0130923), or alternatively 5'-substitution of bicyclic nucleic acids (see PCT International Application WO 2007 / 134181, wherein 4'-CH 2 --O-2' bicyclic nucleosides are further substituted at the 5' position with 5'-methyl or 5'-vinyl). The synthesis and preparation of carbocyclic bicyclic nucleosides as well as their oligomerization and biochemical studies have also been described (see, for example, Srivastava et al., 2007).

[0406] In some embodiments, the disclosure provides oligonucleotides comprising modified nucleosides. Those modified nucleotides can include modified sugars, modified nucleoside bases and / or modified bonds. Select specific modifications so that the oligonucleotides obtained have desired characteristics. In some embodiments, the oligonucleotides comprise one or more RNA sample nucleosides. In some embodiments, the oligonucleotides comprise one or more DNA sample nucleotides.

[0407] In some embodiments, the nucleosides of the present disclosure comprise one or more unmodified nucleobases. In certain embodiments, the nucleosides of the present disclosure comprise one or more modified nucleobases.

[0408] In some embodiments, the modified nucleoside base is selected from: universal bases, hydrophobic bases, promiscuous bases, bases with expanded size, and fluorinated bases as defined herein. 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6, and O-6 substituted purines as defined herein, 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-propynylCH 3 ) uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azouracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxy and other 8-substituted adenines and guanines, 5-halo, especially 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, hybrid bases, size-expanded bases and fluorinated bases. Other 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), G-clamps such as substituted phenoxazine cytidines (e.g., 9-(2-aminoethoxy)-H-pyrimido[5,4-13][1,4]benzoxazin-2(3H)-one), carbazole cytidine ( 2H-pyrimido[4,5-b]indol-2-one), pyridoindolecytidine (H-pyrido[3',2':4,5]pyrrolo[2,3-d]pyrimidin-2-one). Modified nucleoside bases may also include those in which the purine or pyrimidine base is replaced by other heterocycles, such as 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone. Other nucleoside bases include those disclosed in U.S. Pat. No. 3,687,808; those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, Kroschwitz, JI, ed., John Wiley and Sons, 1990, 858-859; those disclosed by Englisch et al., 1991; and those disclosed by Sanghvi, YS, 1993.

[0409] Representative U.S. patents that teach the preparation of some of the above-mentioned modified nucleobases, as well as other modified nucleobases, include, but are not limited to, U.S. Patents 3,687,808; 4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121; 5,596,091; 5,614,617; 5,645,985; 5,681,941; 5,750,692; 5,763,588; 5,830,653 and 6,005,096, each of which is incorporated herein by reference in its entirety.

[0410] In some embodiments, the present disclosure provides oligonucleotides comprising linked nucleosides. In such embodiments, the nucleosides can be linked together using any internucleoside bond. The two main categories of internucleoside linking groups are defined by the presence or absence of a phosphorus atom. Representative phosphorus-containing internucleoside bonds include, but are not limited to, phosphodiester (P=O), phosphotriester, methylphosphonate, phosphoramidate, and phosphorothioate (P=S). Representative non-phosphorus-containing internucleoside linking groups include, but are not limited to, methylenemethylimino (--CH 2 --N(CH 3 )--O--CH 2 --), thiodiesters (--O--C(O)--S--), thiocarbamates (--O--C(O)(NH)--S--); siloxanes (--O--Si(H) 2 --O--); and N,N'-dimethylhydrazine (--CH2 --N(CH 3 )--N(CH 3 )--). Compared to the natural phosphodiester bond, the modified bond can be used to change (usually increase) the nuclease resistance of the oligonucleotide. In some embodiments, the internucleoside bond with a chiral atom can be prepared as a racemic mixture or a single enantiomer. Representative chiral bonds include, but are not limited to, alkyl phosphonates and thiophosphates. The preparation of phosphorus-containing and non-phosphorus-containing internucleoside bonds is well known to those skilled in the art.

[0411] The oligonucleotides described herein contain one or more asymmetric centers and thus give rise to enantiomers, diastereomers, and other stereoisomeric configurations, which can be defined in terms of absolute stereochemistry as (R) or (S), α or β (e.g., for sugar anomers), or (D) or (L) (e.g., for amino acids, etc.). All such possible isomers are included in the antisense compounds provided herein, as well as their racemic and optically pure forms.

[0412] Neutral internucleoside bonds include, but are not limited to, phosphotriester, methylphosphonate, MMI (3'-CH 2 --N(CH 3 )--O-5'), amide-3(3'-CH 2 --C(=O)--N(H)-5'), amide-4 (3'-CH 2 --N(H)--C(=O)-5'), methylal (3'-O--CH 2 --O-5') and thioformal (3'-S--CH 2 --O-5'). Other neutral internucleoside bonds include nonionic bonds including siloxanes (dialkylsiloxanes), carboxylates, carboxamides, sulfides, sulfonates, and amides (see, e.g., Carbohydrate Modifications in Antisense Research; YS Shanghvi and PD Cook, eds., ACS Symposium Series 580; Chapters 3 and 4, 40-65). Other neutral internucleoside bonds include N, O, S, and CH 2 Non-ionic bonds of components.

[0413] Additional modifications may also be made at other positions on the oligonucleotide, particularly at the 3' position of the sugar on the 3' terminal nucleotide and at the 5' position of the 5' terminal nucleotide. For example, one additional modification of the ligand-conjugated oligonucleotides of the present disclosure involves chemically attaching to the oligonucleotide one or more additional non-ligand moieties or conjugates that enhance the activity, cellular distribution or cellular uptake of the oligonucleotide. Such moieties include, but are not limited to, 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; Svinar et al., 1993), and oligonucleotides such as oligonucleotides. Chuk 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), polyamines or polyethylene glycol chains (Manoharan et al., 1995), or adamantaneacetic acid (Manoharan et al., 1995), a palmityl moiety (Mishra et al., 1995), or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., 1996).

[0414] Representative U.S. patents that teach the preparation of such oligonucleotide conjugates include, but are not limited to, U.S. Patents 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717; 5,580,731; 5,580,731; 5,591,584; 5,109,124; 5,118, 802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958, 013; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241,5,391,723; 5,416, 5,599,923; 5,599,928 and 5,688,941, each of which is incorporated herein by reference.

[0415] 3. Egg white

[0416] In some embodiments, the composition may further include one or more proteins. Some proteins may include enzymes such as nucleases. The compositions described herein may include one or more CRISPR-related proteins (e.g., CRISPR enzymes), including Cas proteins. 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, Csf1, Csf2, Csf3, Csf4, homologs thereof, or modified forms 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.

[0417] The protein in the compositions described herein can be Cas9 (e.g., from Streptococcus pyogenes or Streptococcus pneumoniae (S.pneumonia)). The CRISPR enzyme can guide the cutting of one or both chains at the target sequence position, such as within the target sequence and / or within the complementary sequence of the target sequence. The CRISPR enzyme can be mutated relative to the corresponding wild-type enzyme so that the mutated CRISPR enzyme lacks the ability to cut one or both chains of the target polynucleotide containing the target sequence. For example, an aspartate-to-alanine substitution (D10A) in the RuvC I catalytic domain of Cas9 from Streptococcus pyogenes converts Cas9 from a nuclease that cuts two chains to a nickase (cutting a single strand). In some embodiments, the Cas9 nickase can be used in combination with a guide sequence (e.g., two guide sequences) that target the sense and antisense strands of a DNA target, respectively. This combination allows both chains to be nicked and used to induce NHEJ or HDR.

[0418] In some embodiments, the present disclosure provides compounds containing one or more therapeutic proteins. Therapeutic proteins that can be included in the composition include a wide range of molecules such as cytokines, chemokines, interleukins, interferons, growth factors, coagulation factors, anticoagulants, blood factors, bone morphogenic proteins, immunoglobulins, and enzymes. Some non-limiting examples of specific therapeutic proteins include erythropoietin (EPO), granulocyte colony stimulating factor (G-CSF), α-galactosidase A, α-L-iduronidase, thyrotropin α, N-acetylgalactosamine-4-sulfatase (rhASB), dornase alfa, tissue-type plasminogen activator (TPA) Activase, glucocerebrosidase, interferon (IF) β-1a, interferon β-1b, interferon γ, interferon α, TNF-α, IL-1 to IL-36, human growth hormone (rHGH), human insulin (BHI), human chorionic gonadotropin α, darbepoetin α, follicle stimulating hormone (FSH), and factor VIII.

[0419] 4. Small molecule therapeutics

[0420] In some aspects, the present disclosure provides a composition comprising a therapeutic agent. The therapeutic agent can be a small molecule such as 7-methoxypteridine, 7-methylpteridine, abacavir, abafungin, abarelix, acebutolol, acenaphthene, acetaminophen, acetanilide, acetazolamide, acetohexamide, acitretin, acrivastine, adenine, adenosine, alafloxacin, albendazole, salbutamol, alclofenac, aldesleukin, alemtuzumab, alfuzosin, alitretinoin, alobarbital, allopurinol, all-trans retinoic acid (ATRA), aloprin, alprazolam, alprenolol, hexamethylmelamine, amifostine, amiloride, aminoglutethimide, aminopyrine, amiodarone hydrochloride, amitriptyline, amlodipine, amobarbital, amodiaquine, amoxapine, amphetamine, amphetamine, amphotericin, amphotericin B, ampicillin, amprenavir, amsacrine, amyl nitrate, amobarbital, anastrozole, anrinone, anthracene, anthracycline antibiotics, aprobital, arsenic trioxide, asparaginase, aspirin, astemizole, atenolol, atorvastatin, atovaquone, atrazine, atropine, atropine azathioprine, auranofin, azacitidine, azapropazone, azathioprine, azintamide, azithromycin, aztreonam, baclofen, barbiturates, live bacillus Calmette-Guérin, beclomethasone, bendrofluazide, benezepril, benidipine, benolate, benperidol, benzazepam, benzodiazepine amide, benzanthracene, benzathine penicillin, benzhexol hydrochloride, benznidazole, benzodiazepines, benzoic acid, hydroxynaphthofenine, betamethasone, bevacizumab (atorvastatin), bexarotene, bezafibrate, bicalutamide, bifonazole, biperiden, bisacodyl, bisantrene, bleomycin, bleomycin, bortezomib, brinzolamide, bromazepam, bromocriptine mesylate, bromperidol, brotizolam, budesonide, bumetanide, bupropion, busulfan, butalbital, butamben, butenafine hydrochloride, butalbital, butalbital (n-butalbital), butoconazole, butoconazole nitrate, butylparaben, caffeine, calcifediol, calcipotriene (calcip rotriene), calcitriol, captestosterone, canbendazole, camphor, camptothecin, camptothecin analogs, candesartan, capecitabine, capsaicin, captopril, carbamazepine, carbimazole, cypermethrin, carboplatin, carbromide, carimazole, carmustine, cefmandole, cefazolin, cefixime, ceftazidime, cefuroxime axetil, celecoxib, cephradine, cerivastatin, cetrizine, cetuximab, chlorambucil, chloramphenicol, chlordiazepoxide, clomethiazole, chloroquine, chlorothiazide, chlorpheniramine, chlorproguanil hydrochloride, chlorpromazine, chlorsulfuronamide, chlorprothixene, chlorpyrifos, chlortetracycline, chlorthalidone, chlorzoxazone, cholecalciferol, , cilostazol, cimetidine, cinnarizine, cinoxacin, ciprofibrate, ciprofloxacin hydrochloride, cisapride, cisplatin, citalopram, cladribine, clarithromycin, clemastine fumarate, clioquinol, clobazam, clofarabine, clofazimine, clofibrate, clomiphene citrate, clomipramine, clonazepam, clopidogrel, clotiazepam, clotrimazole, clotrimazole, cloxacillin, clozapine, cocaine, codeine, colchicine, colistin, conjugated estrogens, corticosterone, cortisone, cortisone acetate, cyclizine, cyclohexylbarbital, cyclobenzaprine, cyclobutane-spirobarbiturate, cyclohexane-spirobarbiturate, cycloheptane-spirobarbiturate, cyclohexane-spirobarbiturate, cyclopentane-spirobarbiturate, cyclophosphamide, cyclopro Alkane-spirobarbiturate, cycloserine, cyclosporine, cyproheptadine, cyproheptadine hydrochloride, cytarabine, cytosine, dacarbazine, dactinomycin, danazol, dananthrone, dantrolene sodium, dapsone, erythropoietin alfa, darodipine, daunorubicin, decoquinate, dehydroepiandrosterone, delavirdine, demethylchlorocycline, denileukin, deoxycorticosterone, deoxymethasone, dexamethasone, dextroamphetamine, dexchlorpheniramine, dexfenfluramine, dexrazoxane, dextropropoxyphene, diatrizoate, diazepam, diazoxide, dichlorophen, 2,4-dichloropropionic acid, diclofenac, dicoumarol, didanosine, diflunisal, digitoxin, digoxin, dihydrocodeine, dihydroequilin, dihydroergotamine mesylate, diiodohydroxyquinoline, diltiazem hydrochloride, diloxanide furoate, Dimenhydrinate, Demopramine, Dinitolamide, Diosgenin, Diphenoxylate Hydrochloride, Biphenyl, Dipyridamole, Dirithromycin, Disopyramide, Disulfiram, Diuron, Docetaxel, Domperidone, Donepezil, Doxazosin, Doxazosin Hydrochloride, Doxorubicin (Neutral), Doxorubicin Hydrochloride, Doxycycline, Drostanolone Propionate, Droperidol, Diprophylline, Echinocandin, Econazole, Econazole Nitrate, Efavirenz, Eli Lilly, Enalapril, Enmoumab, Enoximone, Epinephrine, Epipodophyllotoxin Derivatives, Epirubicin, Epoetin Alfa, Iprosartan (eposartan), Dehydroequilin, Equilin, Ergocalciferol, Ergotamine Tartrate, Erlotinib, Erythromycin, Estradiol, Estramustine, Estriol, Estrone, ethacrynic acid, ethambutol, ethinyl acetamide, ethionamide, profenamide hydrochloride, ethyl 4-aminobenzoate (benzocaine), ethyl parahydroxybenzoate, ethinyl estradiol, etodolac, etomidate, etoposide, acitretin, exemestane, felbamate, felodipine, fenbendazole, fenbuconazole, fenbufen, fenfos, fenclorac, fenfluramine, fenofibrate, fenoldepam, fenoprofen calcium, fenoxycarb, fenpicrol, fentanyl, fenticonazole, fexofenadine, filgrastim, finasteride, flecainide acetate, floxuridine, fludarabine, fluconazole, fluconazole, flucytosine, fludioxonil, fludrocortisone, fludrocortisone acetate, flufenamic acid,Fluanisone, flunarizine hydrochloride, flunisolide, flunitrazepam, flucortolone, flumethuron, fluorene, fluorouracil, fluoxetine hydrochloride, fluoxymesterone, flupentixol decanoate, triflupentixol decanoate (fluphenthixol decanoate), flurazepam, 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, glibenclamide, glyceryl trinitrate (nitroglycerin), goserelin acetate, grepafloxacin, griseofulvin, guaifenesin, guanabenzyl acetate, guanine, halofantrine hydrochloride, haloperidol, hydrochlorothiazide, heptylbarbital, hesperidin, hexachlorobenzene, hexylbarbital, histrelin acetate, hydrocodone pine, hydroflumethiazide, hydroxyurea, scopolamine, hypoxanthine, ibritumomab tiuxetan, ibuprofen, idarubicin, allyl butalbital, ifosfamide, ihydroequilenin, imatinib mesylate, imipenem, indapamide, indinavir, indomethacin, indoprofen, 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, kaline, labetalol, lamivudine, lamotrigine, lanosprazole, L-DOPA, leflunomide, lenalidomide, letrozole, folinic acid, leuprolide acetate, levamisole, levofloxacin, lidocaine, lindole, lisinopril, lomefloxacin, lomustine, loperamide, loratadine, lorazepam, lorefloxacin, clometazepam, losartan mesylate, lovastatin, lisuride maleate, maprotiline hydrochloride, mazindol, mebendazole, meclizine hydrochloride, meclofenamic acid, medazepam, medroxyprogesterone acetate, mefenamic acid, mefluoxetine hydrochloride Quinone, megestrol acetate, melphalan, mepenzolate bromide, meprobamate, meptazolol, mercaptopurine, mesalamine, mesna, mesoridazine, mestranol, methadone, methaqualone, methocarbamol, mephenytoin, methotrexate, methoxsalen, methsuximide, methylchlorothiazide, methylphenidate, methylphenobarbital, methylparaben, methylprednisolone, methyltestosterone, methysergide maleate, metoclopramide, metolazone, metoprolol, metronidazole, mianserin hydrochloride, miconazole, midazolam, mifepristone, miglitol, minocycline, minoxidil, mitomycin C, mitotane, mitoxantrone, mycophenolate mofetil, molindone, montelukast, morphine, moxifloxacin hydrochloride,Nabumetone, nadolol, nalbuphine, nalidixic acid, nandrolone, tetracene, naphthalene, naproxen, naratriptan hydrochloride, natamycin, nelarabine, nelfinavir, nevirapine, nicardipine hydrochloride, nicotinamide, nicotinic acid, acenocoumarol, nifedipine, nilutamide, nimodipine, nimozole, nisoldipine, nitrazepam, nitrofurantoin, nitrofurazone, nizatidine, romostat , norethindrone, norfloxacin, norgestrel, nortriptyline hydrochloride, nystatin, estradiol, ofloxacin, olanzapine, omeprazole, omoconazole, ondansetron hydrochloride, opreleukin, ornidazole, oxaliplatin, oxaniquine, octadalafil pamoate, oxaprozin, oxamit, oxazepam, oxcarbazepine, oxfendazole, oxiconazole, oxprenolol, hydroxybutazone , hydroxybenzylamine hydrochloride, paclitaxel, palifermin, pamidronate, para-aminosalicylic acid, pantoprazole, methylethyldione, paroxetine hydrochloride, pegaspargase, pegfilgrastim, pemetrexed disodium, penicillamine, pentaerythritol tetranitrate, pentazocin, pentazocine, pentobarbital, pentobarbitone, pentostatin, pentoxifylline, perphenazine, perphenazine pimozide, perylene, phenylacetylurea, phenacetin, phenanthren, phenindione, phenobarbital, phenolbarbital, phenolphthalein, phenoxybenzamine, phenoxybenzamine hydrochloride, phenoxymethylpenicillin, phensuximide, phenylbutazone, phenytoin, pindolol, pioglitazone, pipobroman, piroxicam , pizotifen maleate, platinum compounds, plicamycin, polyene, polymyxin B, porfibril sodium, posaconazole (Posa), pramipexole, prasterone, pravastatin, praziquantel, prazosin, prazosin hydrochloride, prednisolone, prednisone, primidone, probarbital, probenecid, probucol, procarbazine, prochlorperazine, progesterone, proguanil hydrochloride, promethazine, propofol, propoxur, propranolol, propylparaben, propylthiouracil, prostaglandins, pseudoephedrine, pteridine-2-methyl-thiol, pteridine-2-thiol, pteridine-4-methyl-thiol, pteridine-4-thiol, pteridine-7-methyl-thiol, pteridine-7-thiol, pyrantel pamoate, pyrazinamide, pyrene, pyridostigmine, pyrimethamine, quetiapine, imipazone Lin, quinapril, quinidine, quinidine sulfate, quinine, quinine sulfate, rabeprazole sodium, ranitidine hydrochloride, rasburicase, ravuconazole, repaglinide, dicyclopental, reserpine, retinoic acid, rifabutin, rifampicin, rifapentine, rimexolone, risperidone, ritonavir, rituximab, rizatriptan benzoate, rofecoxib, ropinirole hydrochloride, rosiglitazone, saccharin, salbutamol, salicylamide, salicylic acid, saquinavir, sargramostim, butabarbital, secobarbital, sertaconazole, sertindole, sertraline hydrochloride, simvastatin, sirolimus, sorafenib, sparfloxacin, spiramycin, spironolactone, dihydrotestosterone, stanozolol, stavudine, diethylstilbestrol, streptozocin, strychnine, sulconazole, sulconazole nitrate,Sulfacetamide, sulfadiazine, sulfamethazine, sulfadimethoxine, sulfamethoxazole, sulfa, sulfathiazole, sulindac, sulphabenzamide, sulphacetamide, sulphadiazine, sulphadoxine, sulfisoxazole, sulphamerazine, sulpha-methoxazole, sulfamethoxazole Sulphapyridine, sulfasalazine, sulfinpyridine, sulpiride, thiothiazide, sumatriptan succinate, sunitinib maleate, tacrine, tacrolimus, talbutal, tamoxifen citrate, tamulosin, targretin, taxane, tazarotene, telmisartan, temazepam, temozolomide, teniposide, tenoxicam, terazosin, terazosin hydrochloride, terbinafine hydrochloride, terbutaline sulfate, terconazole, terfenadine, testosterone , testosterone, tetracycline, tetrahydrocannabinol, tetraoxoprine, thalidomide, thebaine, theobromine, theophylline, thiabendazole, thiamphenicol, thioguanine, thioridazine, thiotepa, ethotoin, thymine, tiagabine hydrochloride, tibolone, ticlopidine, tinidazole, tioconazole, tirofiban, tizanidine hydrochloride, tolazamide, tolbutamide, tolcapone, topiramate, topotecan, toremifene, tositumomab, tramadol, trastuzumab, trazodone hydrochloride, tretinoin, triamcinolone, triamterene, triazolam , triazoles, triflupromazine, trimethoprim, trimipramine maleate, benzylphenidate, troglitazone, tromethamine, tropicamide, trovafloxacin, tadalafil, ubidecarenone (coenzyme Q10), undecylenic acid, uracil, uracil nitrogen mustard, uric acid, valproic acid, valrubicin, valsartan, vancomycin, venlafaxine hydrochloride, vigabatrin, pentobarbital, vinblastine, vincristine, vinorelbine, voriconazole, xanthine, zafirlukast, zidovudine, zileuton, zoledronate, zoledronic acid, zolmitriptan, zolpidem and zopiclone. ,

[0421] F. Kit

[0422] The present disclosure also provides kits. Any components disclosed herein can be combined in the form of a kit. In some embodiments, the kit comprises a composition as described above or in the claims.

[0423] The kit will typically include at least one vial, tube, flask, bottle, syringe or other container in which the components can be placed, and preferably appropriately aliquoted. Where there is more than one component in the kit, the kit will typically also contain a second, third or other additional container in which the additional components can be separately placed. However, various combinations of components can be included in the container. In some embodiments, all 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.

[0424] The kit of the present disclosure will also typically include packaging for accommodating various containers, which are tightly sealed for commercial sale. Such packaging may include cardboard or injection or blow molded plastic packaging to hold the desired container therein. The kit may also include instructions for using the kit components. The instructions may include achievable variants.

[0425] F. Examples

[0426] The following examples are included to demonstrate the preferred embodiments of the present disclosure. It will be appreciated by those skilled in the art that the techniques disclosed in the following examples represent techniques that the inventors have found to work well in the practice of the present disclosure, and thus can be considered to constitute the preferred modes of its practice. However, it will be appreciated by those skilled in the art that many changes can be made in the disclosed specific embodiments and similar or similar results can still be obtained without departing from the spirit and scope of the present disclosure.

[0427] Example 1 - Preparation of DOTAP-modified lipid nanoparticles

[0428] Lipid nanoparticles (LNPs) are the most effective carrier classes for in vivo nucleic acid delivery. Historically, effective LNPs are composed of 4 components: ionizable cationic lipids, zwitterionic phospholipids, cholesterol, and lipid poly(ethylene glycol) (PEG). However, these LNPs only result in the general delivery of nucleic acids, rather than the delivery of targeted organs or tissues. LNPs are usually only delivered to the liver with RNA. Therefore, new LNP preparations are sought to attempt to provide targeted nucleic acid delivery.

[0429] Four standard types of lipids were mixed in a 15:15:30:3 molar ratio with or without the addition of permanent cationic lipids. Briefly, LNPs were prepared by mixing 5A2-SC8 (ionizable cationic), DOPE (zwitterionic), cholesterol, DMG-PEG, and DOTAP (permanent cationic) in the ratios shown in Table 0.1.

[0430] Table 0.1: Molar ratios and percentages of lipids in modified LNPs.

[0431]

[0432] To prepare mDLNP formulations, 5A2-SC8, DOPE, cholesterol, and DMG-PEG were dissolved in ethanol at a given molar ratio (15:15:30:3). mRNA was dissolved in citrate buffer (10 mM, pH 4.0). The mRNA was then diluted into the lipid solution to achieve a weight ratio of 40:1 (total lipid: mRNA) by quickly mixing the mRNA into the lipid solution at a volume ratio of 3:1 (mRNA: lipid, v / v). The solution was then incubated at room temperature for 10 min. To form DOTAP-modified mDLNP formulations, mRNA was dissolved in 1× PBS or citrate buffer (10 mM, pH 4.0) and quickly mixed into ethanol containing 5A2-SC8, DOPE, cholesterol, DMG-PEG, and DOTAP to fix a weight ratio of 40:1 (total lipid: mRNA) and a volume ratio of 3:1 (mRNA: lipid). As shown in Table 1, each formulation is referred to as DOTAPX, where X represents the molar percentage of DOTAP in the total lipids.

[0433] Example 2 - Characterization of DOTAP-modified mDLNP preparations.

[0434] In order to characterize different mDLNP preparations, size, polydispersity index and zeta potential were checked by dynamic light scattering, and each preparation was carried out 3 times respectively. Size and polydispersity index are shown in Fig. 5 A, showing that no matter how DOTAP concentration, all preparations fall within the size range of about 90nm to about 160nm, and polydispersity index changes from about 0.1 to about 0.3, showing the relative uniformity of size. In Fig. 5 B, the zeta potential of each preparation is shown, and it is shown that the zeta potential usually increases with the concentration of DOTAP.

[0435] Next, the encapsulation efficiency was tested using Ribogreen RNA assay (Zhao et al., 2016). In short, when mRNA was dissolved in an acidic buffer (10mM citrate, pH 4), mRNA was encapsulated with about 85% efficiency by mDLNPs without DOTAP (Fig. 5C). Low pH is required to protonate ionizable amines in ionizable cationic lipids (e.g., 5A2-SC8, C12-200, DLin-MC3-DMA) to allow electrostatic complexation of negatively charged mRNA. For all other preparations in the figure, mRNA dissolved in PBS was mixed at pH 7.4. Obviously, for low concentrations of DOTAP, the encapsulation efficiency is low, but when the molar percentage of DOTAP is higher than 25%, the encapsulation efficiency increases to>80% (Fig. 5C). For all preparations with a molar percentage greater than 25% DOTAP, the encapsulation efficiency is between about 80% and about 95%. Therefore, the potential for mixing with neutral pH PBS is a feature of the permanent cationic lipid strategy. This strategy allows tissue-specific delivery and high Cas9 protein encapsulation. The addition of permanent cationic lipids allows the formation of LNPs at neutral pH. These encapsulation results are the results when PBS is used as a buffer. When an acidic buffer (e.g., citrate buffer (10mM, pH 4.0)) is used, the encapsulation efficiency is high (>90%) for all preparations of 0-100% DOTAP.

[0436] Finally, the pK was determined using the 2-(p-toluidine)-6-naphthalenesulfonic acid (TNS) assay. a (Figure 3B) (Zhao et al., 2016). Plotting pK based on the defined rules a The relationship between liver-targeted agents and tissue-specific mRNA delivery. As shown in the table, 8 rules were designed for scoring. a (~6-7), spleen-targeted formulations have no obvious range, but lung-targeted delivery requires a high pK a (>9.25). Comprehensive consideration of distribution and pK a It was concluded that the internal charge of the NP is a factor affecting mRNA distribution and that the overall / apparent LNP pK a It is another factor that determines the profile of mRNA-mediated protein expression in an organ.

[0437] Example 3 - Efficacy of Permanently Cationic Lipid-Modified mDLNPs for mRNA Delivery

[0438] In order to check that the LNP including permanent cationic lipid can deliver the delivery efficacy of active goods in vitro, the mDLNP modified by DOTAP is loaded with the mRNA encoding luciferase, and the mRNA transfection Huh-7 hepatocytes and A549 adenocarcinoma human alveolar basal epithelial cells are transfected with 50ng / hole. These cells are cultured for 24 hours, and then luciferase expression and cell viability are checked. As shown in Fig. 6 A, for mRNA delivery and expression in Huh-7 hepatocytes in vitro, the DOTAP percentage of 5%-50% is better, and 10% DOTAP seems to have maximum luciferase delivery and expression (Fig. 6 A). It should be noted that the delivery characteristics may be different in vivo. Generally, due to other internal barriers, organ distribution and cell characteristics of SORT LNP, these studies may not be used to predict activity in vivo, nor can they be used to predict tissue tropism. In addition, cell viability was checked, and it was found that 10% DOTAP produced high viability (Fig. 6 A) in the mDLNP showing robust luciferase expression. The same transfection inspection with A549 lung cancer cell line showed similar results, with cells transfected with DOTAP10 formulation showing almost twice the fluorescence of any other formulation, and maintaining high cell viability (Fig. 6A). DOTAP SORT LNPs are formed in PBS (pH 7.4), rather than in citrate buffer (10mM, pH 4.0), but can be formed in either buffer system. This is a property, because it allows encapsulation and delivery of unstable goods, such as proteins, in ethanol or acidic buffers.

[0439] To determine the effect of ethanol concentration in the formulation, DOTAP25 was selected and prepared with various ratios of ethanol to PBS (1:3, 1:5, 1:7.5 and 1:10) (Figure 6B). All four formulations showed similar encapsulation efficiency, size and PDI (Figure 2B1 and Figure 2B2). The mRNA delivery efficiency was also measured by transfecting FaDu hypopharyngeal cancer cells with 50ng / well of mRNA in each formulation. The delivery efficiency of each formulation was similar, and the effect on cell viability was also very small (Figure 6C). Therefore, these formulations appear to be suitable for many cell types, including liver, lung and larynx. Further, the formulation has also been successfully modified to use 1×PBS (pH7.4) instead of acidic buffer (pH 4.0), and these data show that the ethanol percentage can be significantly reduced, which provides the possibility that DOTAP formulations can deliver cargo (such as proteins) that are much more sensitive to high ethanol concentrations and acidic buffers.

[0440] Then, in order to test the ability of these mDLNPs to deliver mRNA in vivo, mice were injected with the Luc mRNA dosage of 0.1mg / kg in each preparation. Fig. 1B shows the in vitro images of luciferase in major organs 6 hours after the intravenous injection of each preparation. Interestingly, along with the increase of the molar percentage of DOTAP, luciferase expression is transferred from liver to spleen, and then to lung, which proves that organ-specific delivery. These data are quantitatively disclosed, revealing that the DOTAP percentage is a factor of tissue-targeted delivery, and mDLNP (0% DOTAP) is best for liver delivery, and 5-15% DOTAP is best for spleen delivery, and DOTAP50 (50%) is best for lung delivery (Fig. 1B). Assuming that luciferase expression is only detected in liver, spleen and lung after intravenous injection, the percentage (Fig. 1C) of luciferase expressed in each organ can be calculated. These data clearly indicate that as the DOTAP molar percentage in the formulation increases, delivery to the liver and expression therein decrease, and when the DOTAP percentage is greater than 70%, expression close to zero is seen in the liver (Figure 1B, 1C). However, the greater the DOTAP percentage, the more luminescence is seen in lung tissue, and when the DOTAP percentage is greater than 80%, luminescence close to 100% is seen in the lung (Figure 1C). Concentrations of DOTAP 5 and 30 molar percentages show higher luminescence percentages in spleen tissue, while DOTAP10 shows the highest relative luminescence compared to other tissues in the spleen (Figure 1C). These results indicate that, after injection, lipid concentrations can be adjusted for specific tissue delivery.

[0441] In order to test the organ biodistribution of specific DOTAP formulations in more depth, PBS or liver-targeted NPs (mDLNPs), spleen-targeted NPs (DOTAP10), and lung-targeted NPs (DOTAP50) were injected into C57BL / 6 mice (n=2) at a dose of 0.5 mg / kg Cy5-Luc mRNA (dye-labeled mRNA to track RNA LNPs). Six hours after injection, major organs were collected and imaged (Fig. 3A). The organ distribution of the formulations changed with the amount of DOTAP, and with the increase in the percentage of DOTAP, the accumulation in the liver gradually moved to the lungs, but regardless of the percentage of DOTAP, there was still NP in the liver ( Figure 7 , Figure 8). Considering this data together with the data in Figure 1, it is clear that organ distribution is not sufficient to analyze the delivery efficacy of targeted tissues (mRNA translation into protein). Further considering the similar size distribution and EE, zeta potential and pK between these formulations a May play a role in mRNA expression in target tissues.

[0442] In order to understand whether the effect of DOTAP addition to mDLNP is limited or whether the distribution shown above is universal for mDLNPs formulated with permanent cationic lipids, mDLNPs (Figure 2A1) containing another popular cationic lipid, didodecyl dimethyl ammonium bromide (DDAB) were produced. DDAB has two hydrophobic tails with 18 carbon atoms and no unsaturated bonds, and has a head group completely different from DOTAP (Figure 1C). DDAB5, DDAB15, DDAB40 and DDAB50 formulations were selected for in vivo delivery (0.1 mg / kg, 6h, n=2). Similar to the DOTAP formulation above, even if the DDAB percentage in NPs changed 10 times (5% to 50%), there was almost no difference in size distribution (Figure 2A1). Similar to DOTAP NPs, in vivo luciferase expression showed the following trend: as the DDAB percentage increased, luminescence was transferred from the liver to the spleen and then to the lungs (Figure 2A2).

[0443] mDLNPs were formed with a third permanent cationic lipid with a head group 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine chloride, which has a similar structure to DOTAP but with a shorter 14-carbon hydrophobic tail ((14:0)EPC) (Figure 2B1). Similar to the DDAB strategy, (14:0)EPC5, (14:0)EPC15, (14:0)EPC40, and (14:0)EPC50 formulations were prepared and examined for 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 EPC molar percentage increased (14:0) (Figure 2B2). Taking all these data together, including the different hydrophobic tails, saturated and unsaturated bonds, and different head groups, it appears that cationic lipid-formulated mDLNPs are versatile for tissue-targeted mRNA delivery.

[0444] In order to understand whether the effect of DOTAP addition to LNP is specific to permanent cationic lipids, the effect of adding zwitterionic lipids instead of permanent cationic lipids in mDLNP preparations was examined. Two representative zwitterionic lipids were tested, phospholipids with different chemical structures: DSPC and DOCPe. In addition, tests were also carried out to determine whether the addition of zwitterionic lipids (rather than permanent cationic lipids) would affect tissue-specific delivery efficacy. Figures 2C1 and 2D1 show the chemical structures of DSPC and DOCPe lipids (zwitterionic lipids). There are differences in the position of the functional head groups with positive and negative charges and the position of the hydrophobic domains (saturated relative to unsaturated), thereby suggesting that any observed effect of zwitterionic lipids is universal / universal. mDLNPs prepared with DSPC or DOCPe are similar (Figures 2C1, 2D1). Interestingly, the inclusion of zwitterionic lipids in 5-component modified DLNPs does not change the protein expression profile from liver to lung like DOTAP and other permanent cationic lipids. In contrast, both DSPC and DOCPe improved mRNA delivery to the spleen within a given range (less than 80% in DSPC and less than 50% in DOCPe). There was no protein expression in the lungs for any percentage (0.1 mg / kg, 6 h, n=2) (Figure 2C2, 2D2). Therefore, the inclusion of additional zwitterionic lipids can help spleen delivery, but cannot modulate the delivery efficacy from liver to spleen to lungs as the inclusion of permanent cationic lipids does.

[0445] In order to understand whether the effect of the addition of DOTAP to LNP is specific to permanent cationic lipids, the effect of adding ionizable cationic lipids instead of permanent cationic lipids in mDLNP preparations was examined. Two representative ionizable cationic lipids with different chemical structures were tested: C12-200 and DODAP. Except for the head group (quaternary amine relative to tertiary amine), DODAP has the same structure as DOTAP. C12-200 is an effective lipidoid for siRNA and mRNA delivery containing ionizable tertiary amines (also without quaternary amines), which has a completely different structure from DODAP. (Figure 2E1, 2F1) Similarly, the size distribution of the two modified mDLNPs is still uniform in some percentages (less than 80%). (Figure 2E1, 2F1) Surprisingly, the inclusion of ionizable cationic lipids in 5-component modified DLNPs did not change the protein expression profile from liver to spleen and then to lungs like DOTAP and other permanent cationic lipids. On the contrary, the inclusion of ionizable cationic lipids into DLNPs increases the mRNA delivery efficacy of mRNA to the liver. Compared with the original mDLNP (5A2-SC8 only) (0.1 mg / kg, 6h, n=2) without additional ionizable cationic lipids, these drugs show much better delivery efficacy. As the percentage of DODAP or C12-200 increases (50% or 80%), the luciferase signal decreases a lot, but the liver is still the main organ, not the spleen or lung. Therefore, it is concluded that the organ-specific effect can be attributed to the inclusion of permanent cationic lipids at a specific ratio. In addition, the data show that permanent cationic lipids produce different effects from ionizable cationic lipids. These data further show that these trends are universal in terms of lipid classes.

[0446] Example 4 - CRISPR / Cas9 gene editing using modified mDLNPs that co-deliver Cas9 mRNA and sgRNA

[0447] First, three sgRNAs targeting Td-Tomato mice were compared to determine which sgRNA was the 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 and were more successful than sgTom2 in inducing TdTomato (Figure 10A). Considering the weak PAM of sgLoxP (NAG), sgTom1 was ultimately selected for further experiments.

[0448] In view of the tissue-specific mRNA (luc mRNA) delivery shown with DOTAP NP, and both DDAB and EPC-modified NPs showed similar delivery trends, DOTAP-modified mDLNPs were then used for Cas9 mRNA / sgRNA co-delivery, aiming to achieve tissue-specific gene editing. In order to examine co-delivery in vivo, genetically engineered mice containing a homozygous ROSA26 promoter Lox-Stop-Lox tdTomato (tdTO) box present in all cells were used (Figure 4A). Co-delivery of DOTAP-modified mDLNPs containing Cas9-mRNA and sgRNA for LoxP or for Tom enables the deletion of the stop box and the induction of tdTO expression (Figure 4B). Mice were intravenously injected with mDLNPs and DOTAP50 formulations at a total dose of 2.5 mg / kg (50 ug each) to co-deliver IVT Cas9 mRNA and modified sgTom1 (4 / 1, weight / weight), and then the fluorescence of major organs was detected on the 10th day after treatment (Figure 4B). Liver- and lung-specific CRISPR / Cas gene editing was achieved. Spleen-specific editing was also achieved. However, due to very high background red autofluorescence, quantification of spleen editing was not possible using this TdTomato reporter mouse.

[0449] To further examine tissue-specific editing, PTEN was selected as an endogenous target. C57BL / 6 mice were intravenously injected with mDLNP, DODAP20, or DOTAP50 to achieve tissue-specific gene editing. The total dose was 2.5 mg / kg (50 ug each), the weight ratio of IVT Cas9 mRNA to modified sgPTEN was 4 / 1, and the detection time was 10 days after treatment. sgRNA targeting PTEN was used. T7E1 assay showed that tissue-specific characteristics were further confirmed with in vivo PTEN editing. (Figure 4C).

[0450] Example 5 - CRISPR / Cas9 gene editing using modified mDLNPs delivering Cas9 protein / sgRNA ribonucleoprotein (RNP)

[0451] Based on the following findings: the inclusion of permanent cationic lipids (e.g., DOTAP) in traditional LNP formulations containing ionizable cationic lipids, zwitterionic lipids, cholesterol, and PEGylated lipids, it was studied whether this formulation method can also deliver other cargoes that are sensitive to ethanol and / or low pH acidic aqueous buffers. The key element of the DOTAP strategy is that PBS at neutral pH can be used to prepare the formulation. Therefore, it was checked whether the method can also encapsulate and deliver large proteins (such as Cas9) for gene editing applications. Therefore, DOTNP lipid nanoparticles are composed of five components: modular inclusion of ionizable cationic lipids (e.g., 5A2-SC8), zwitterionic lipids (e.g., DOPE), cholesterol, DMG-PEG, and permanent cationic lipids (e.g., DOTAP). The molar ratio of 5A2-SC8, cholesterol, DOPE, and DMG-PEG is fixed (15:15:30:5, mol / mol), and DOTNPX refers to DOTNP with different molar percentages of DOTAP.

[0452] Check whether the Cas9 / sgRNA complex characterized first is sensitive to acidic pH. The size (diameter) (Figure 11A) and zeta potential (Figure 11B) of the Cas9 / sgLUC complex (mol / mol=1 / 1) were measured in PBS (pH 7.4) and in citrate buffer (pH 4.2). The size of the Cas9 / sgLUC complex prepared in citrate buffer is large (greater than 100nm), and the zeta potential is positively charged. These two properties (size larger than typical effective LNP) and positive charge (charge is incompatible with complexes with positively charged lipids) make it impossible to be effectively encapsulated by lipid nanoparticles. However, the size of the Cas9 / sgLUC complex prepared in PBS is compact (less than 20nm) and has a negative charge. Therefore, when formulated at neutral pH, it can be encapsulated by lipid nanoparticles. Next, Cas9 / sgRNA complexes with different Cas9 protein to sgRNA molar ratios were prepared and characterized. The size (FIG. 11C) and zeta potential (FIG. 11D) of the Cas9 / sgLUC complex prepared with different Cas9 / sgRNA molar ratios (1 / 1, 1 / 3, and 1 / 5). Compared with the Cas9 / sgLUC complex (1 / 1, mol / mol), the higher molar ratio (1 / 3 and 1 / 5, mol / mol) showed a smaller size and more negative charge, which may be beneficial for lipid nanoparticle encapsulation. Prepare the composition and characterize the DOTNP lipid nanoparticles after encapsulating the Cas9 / sgRNA complex. When prepared in different molar ratios (1 / 1, / 3, 1 / 5), the size (FIG. 11E) and zeta potential (FIG. 11F) of the DOTNP10 lipid nanoparticles (named DOTNP10-L) encapsulating the Cas9 / sgLUC complex. The data indicate that Cas9 / sgRNARNP is encapsulated into monodisperse LNP. Figure 11G shows a TEM image of DOTNP10-L (1 / 3, mol / mol) LNP with encapsulated RNP. After this initial study, different sgRNAs were used, including sgLUC, sgGFP, sgTOM, and sgPTEN. To distinguish them, the first letter of each gene was added to the end of DOTNP. DOTNP10-L refers to DOTNP10 lipid nanoparticles encapsulating Cas9 / sgLUC complexes; DOTNP10-G refers to DOTNP10 lipid nanoparticles encapsulating Cas9 / sgGFP complexes.

[0453]

[0454] Table 7. Characterization of DOTAP10, DSPC50, and DODAP50 formulations formed with PBS and citric acid buffers, including size, PDI, and encapsulation efficacy.

[0455]

[0456] Table 8. List of all primers used in this study, including the length of the PCR product and its purpose (Cas9 = SEQ ID NO: 3-4; Ca9 Seq-1 = SEQ ID NO: 5; Ca9 Seq-2 = SEQ ID NO: 6; Ca9 Seq-3 = SEQ ID NO: 7; Ca9 Seq-4 = SEQ ID NO: 8; Ca9 Seq-5 = SEQ ID NO: 9; Ca9 Seq-6 = SEQ ID NO: 10; Ca9 Seq-7 = SEQ ID NO: 11; PTEN = SEQ ID NO: 14-15; IVT sgTom1 = SEQ ID NO: 44-45; IVT sgTom2 = SEQ ID NO: 46-47; IVT sgLoxP = SEQ ID NO: 48-49)

[0457] In order to examine whether DOTNP lipid nanoparticles can deliver Cas9 / sgRNA RNP complexes into the cell nucleus and mediate efficient gene editing in vitro, a series of experiments were performed. First, DOTNPs containing Cas9 / sgRNA RNPs labeled with green fluorescent EGFP were tracked by confocal microscopy (Figure 12A). After incubation with DOTNP10 (using 9nM sgRNA) encapsulating Cas9-EGFP / sgLUC complexes (1 / 3, mol / mol) for 1h, 3h, 6h, and 24h, images of Hela-Luc cells showed that DOTNP was internalized into the cells and Cas9 RNP was transported into the nucleus. Green: EGFP-fused Cas9 protein; Blue: cell nucleus stained with Hoechst 33342. The red arrow indicates the process of DOTNP10 entering the cell nucleus. (Figure 12B)

[0458] Next, it was checked whether the DOTNP lipid nanoparticles can deliver Cas9 / sgRNA RNP complexes and whether the targeted luciferase DNA can be cut. After incubation with DOTNP10-L of different molar ratios (using 24nM sgRNA) for 3 days, the percentage of DNA insertion and deletion (insertion and deletion) at the LUC locus was quantified using TIDE determination. The DOTNP10 lipid nanoparticles (DOTNP10-G) encapsulating Cas9 / sgGFP were used as negative controls. Here, two commercial Cas9 proteins (GeneArt Cas9 and Truecut Cas9) (Figure 12B) were used. Then, the T7EI cutting assay of Hela-Luc cells incubated with different preparations (24nM sgRNA) was carried out to demonstrate DNA editing (Figure 12C). In the conditions tested, when using Truecut Cas9 protein, a molar ratio of 1 / 3 showed optimal gene editing. Then, the editing of GFP was tested using fluorescence microscopy (Figure 12D). Images of SKOV3-GFP cells incubated with DOTNP10-L (control, not targeting GFP) and DOTNP10-G (targeting GFP) (24 nM sgRNA) show on-target editing confirmed by disappearance of GFP protein expression. Finally, SKOV3-GFP cells incubated with DOTNP10-L and DOTNP10-G were analyzed using flow cytometry ( FIG. 12E ). ( FIG. 12F ) Mean fluorescence intensity of SKOV3-GFP cells incubated with DOTNP10-L and DOTNP10-G obtained by flow cytometry shows editing of GFP by CRSPR / Cas.

[0459] Next, we examined whether DOTNP lipid nanoparticles could deliver Cas9 / sgRNA RNP complexes in vivo to achieve CRISPR / Cas-mediated gene editing. As before, a genetically engineered TdTomato mouse model was used. 1.5 mg / kg of sgRNA was delivered to each mouse using the following formulations: DOTNP5-T refers to DOTNP5 LNP encapsulating the Cas9 / sgTom complex; DOTNP10-T refers to DOTNP10 LNP encapsulating the Cas9 / sgTom complex; DOTNP50-T refers to DOTNP50 LNP encapsulating the Cas9 / sgTom complex. After intravenous injection of these formulations, tdTomato fluorescence was quantified in vitro in major organs 7 days after injection (Figure 13A). TdTomato fluorescence was observed only in the liver of the DOTNP5-T treated group; in the DOTNP10-T group, slight fluorescence was seen in the lungs, and if the dose of DOTAP was further increased to 50% (DOTNP50-T), most of the tdTomato fluorescence was observed in the lungs. Therefore, similar to the mRNA delivery experiments outlined above, the DOTAP method also allows tissue-specific gene editing of Cas9 / sgRNA ribonucleoprotein (RNP) complexes. To further examine delivery, LNPs containing sgRNA for PTEN were delivered. Using T7EI cleavage assays of liver and lung organs, it was determined that intravenous injection (2 mg / kg sgRNA / mouse) of DOTNP5-P (DOTNP5 LNP encapsulating Cas9 / sgPTEN complex), DOTNP10-P (DOTNP10 LNP encapsulating Cas9 / sgPTEN complex), and DOTNP50-P (DOTNP50 LNP encapsulating Cas9 / sgPTEN complex) mediated gene editing (Figure 13B). The results were consistent with those obtained by ex vivo imaging. Gene editing was detected only in the liver after treatment with DOTNP5-P; when incubated with DOTNP10-P, gene editing was obtained in both the liver and lungs; while in the DOTNP50-P treated group, most gene editing was observed in the lungs.

[0460] The data provided herein show that lipid nanoparticles can be prepared to have different compositions, so that payload is specifically targeted to specific tissues. Specifically, lipid nanoparticles with low concentrations of permanent cationic lipids (≤10%) effectively deliver nucleic acids to the liver, and LNPs with less than 30% permanent cationic lipids effectively deliver nucleic acids to the spleen, and LNPs with greater than 30% permanent cationic lipids effectively deliver nucleic acids to the lungs. These findings appear to be universal, with almost no effect on headgroup, saturation and tail length.

[0461] Example 6 - Addition of another lipid to the known four lipid compositions results in a change in delivery target

[0462] The generality of the scheme (method) for inclusion of a "fifth" lipid into established 4-component LNPs was then explored.

[0463] In order to check whether the inclusion of permanent cationic lipid (such as DOTAP) can change the tissue specificity of other ionizable cationic lipids, two well-known and fully established ionizable cationic lipid LNP systems were selected.Select DLin-MC3-DMA, and prepare it with DSPC, cholesterol and PEG-DMG.Produce the molar composition identical with Patisiran / Onpattro (Alnylam Pharmaceuticals), and supplement 15% or 50% DOTAP (the extra 5th lipid in Onpattro 4 lipid formulations) (Figure 14).DLin-MC3-DMA LNP is considered to " the gold standard " that siRNA and mRNA are delivered.So far, only show that they are delivered to liver after intravenous administration.As shown in Figure 15 A, DOTAP changes the mRNA expression overview (0.1mg / kg luciferase mRNA, 6h) of LNP in organs based on DLin-MC3-DMA. As the percentage of DOTAP increases, luciferase signal is transferred from liver to spleen, and finally to lung, which is exactly the same as the phenomenon about 5A2-SC8 mDLNP. In order to further study the universality of this scheme, we include DOTAP in C12-200 LNP (Figure 16). Although DLin-MC3-DMA is a two-tail lipid with a single dimethylamine head group, it is considered to be a stable nucleic acid lipid nanoparticle (SNALP), but C12-200 is a representative "lipidoid" that can be formulated into lipid-like LNP. All three are ionizable cationic lipids. Identical to the results using 5A2-SC8 and DLin-MC3-DMA, 15% or 50% DOTAP included in C12-200 LNP changes the luciferase protein expression after mRNA delivery from liver to spleen to lung (Figure 15 B). Thus, the fifth lipid method (such as adding permanent cationic lipids) can be extended to other ionizable cationic lipid LNPs.

[0464] Example 7 - Addition of another lipid to the known four lipid compositions results in improved delivery

[0465] Furthermore, the generalizability of this approach was explored by investigating whether additional ionizable cationic lipids would improve hepatic delivery.

[0466] To examine whether ionizable cationic lipids generally promote liver delivery, additional 5A2-SC8 ionizable cationic lipids were included as a "fifth" lipid into LNPs containing appropriate ratios of 5A2-SC8, DOPE, cholesterol, and PEG DMG. Additional 5A2-SC8 was included at 10-30% (Figures 17A and Fig.18 ). To avoid saturated luminescence, a low dose of 0.05 mg / kg of mRNA was tested (intravenously, 6 h). As shown in Figure 17B, in vitro images and quantitative data confirmed that the additional 15% to 25% of 5A2-SC8 added did help improve the mRNA delivery efficacy in the liver. 5A2-SC8^20 (5A2-SC8 LNP + 20% additional 5A2-SC8) increased luciferase to 2-3 times that of the original mDLNP formulation.

[0467] Example 8 - Studies Related to Selective Organ Targeting Compositions

[0468] The present disclosure describes a strategy called selective organ targeting (SORT), which allows nanoparticles to be systematically engineered to accurately deliver a variety of 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 ionizable cationic lipids, zwitterionic phospholipids, cholesterol and poly (ethylene glycol) (PEG) lipids. The present disclosure shows that the addition of supplementary components (called SORT compounds or selective organ targeting compounds) accurately changes the RNA delivery profile in vivo and mediates tissue-specific gene delivery and editing, which varies with the percentage and biophysical properties of the added SORT lipids. The present disclosure shows evidence for the theory of tissue-specific delivery, establishes that the method can be used for various nanoparticle systems, and provides methods for LNP design to edit treatment-related cells.

[0469] Effective intracellular delivery materials often rely on an optimal balance of ionizable amines for binding and release of RNA (pK a6.0-6.5) and stabilized hydrophobic nanoparticles (Kanasty et al., 2013; Jayaraman et al., 2012; Nelson et al., 2013; Hao et al., 2015). Without wishing to be bound by any theory, it is believed that internal and / or external charge may be a factor regulating tissue tropism. Intravenous administration of the developed SORT LNPs achieves high levels of tissue-specific gene editing. SORT is compatible with various methods that employ gene editing mechanisms, including mRNA, Cas9 mRNA / sgRNA, and Cas9 RNP (systemic RNP delivery). SORT LNPs targeting the lungs edited 40% of epithelial cells and 65% of endothelial cells; SORTLNPs targeting the spleen edited 13% of B cells and 10% of T cells; and after a single low-dose injection, the enhanced SORT LNPs targeting the liver edited 93% of hepatocytes.

[0470] A. Discovery and development of SORT

[0471] To examine the hypothesis that internal charge modulation can mediate tissue-specific delivery, a strategy was conceived to add a fifth lipid to an already established LNP composition that has demonstrated efficacy in hepatocytes. The rationale is to tailor effective LNP formulations without disrupting the core 4-component ratios that are typically used to mediate RNA encapsulation and endosomal escape (Wittrup et al., 2015; Cheng et al., 2018).

[0472] The permanent cationic lipids (defined as positively charged lipids with no pK a or pK a >8) added to the degradable dendrimer named 5A2-SC8 used in mDLNPs, which is ionizable (pK a<8) cationic lipids (Zhou et al., 2016; Zhang et al., 2018a; Zhang et al., 2018b), the 5A2-SC8 effectively delivers fumaryl acetoacetate hydrolase (FAH) mRNA to hepatocytes and prolongs survival in FAH knockout mice (Cheng et al., 2018). The initial basic mDLNP formulation consists 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, weight / weight) (Figure 20). A series of LNPs (Figures 19B and 20) were then formed by systematically increasing the percentage of additional permanent cationic lipids from 5% to 100% of total lipids. 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), a well-known quaternary amino lipid, was initially selected as the SORT lipid to be added to the LNP formulation. Therefore, the DOTAP-modified SORT formulation contained 5 lipid components, in which 5A2-SC8 / DOPE / Chol / DMG-PEG was fixed at 15 / 15 / 30 / 3 (mol / mol), and DOTAP was added at a molar ratio of 0 to 1200 to prepare a titration series of formulations (Figure 20).

[0473] The effect of SORT modification was then evaluated by intravenous (IV) delivery of luciferase (Luc) mRNA at a dose of 0.1 mg / kg. As the molar percentage of DOTAP increases, the resulting luciferase protein expression gradually shifts from the liver to the spleen, and then to the lungs, thereby demonstrating a clear and precise organ-specific delivery trend with a threshold value that allows unique lung delivery (Figure 19B). The DOTAP percentage is a key factor in adjusting tissue specificity. Basic LNP (0% DOTAP) is optimal for liver delivery, which is expected because they have been optimized for hepatocyte delivery (Cheng et al., 2018). By adding 10-15% DOTAP, the resulting SORT LNP can now deliver mRNA to cells in the spleen. Further increasing permanent cationic SORT lipids, it was found that 50% DOTAP was optimal for lung delivery (Figure 19C). It is worth noting that although 50% DOTAP SORT LNP effectively delivers mRNA to the lungs in vivo, they are not so effective for in vitro delivery (Figure 21). In addition, 50% DOTAP SORT LNP has a neutral zeta potential surface charge (-0.52mV) (Figure 20), indicating that tissue tropism is not due to MPS uptake associated with positive charge. Calculating the relative expression in each organ, the application of DOTAP as a SORT lipid completely changed the delivery from liver to lung (Figure 19D). It is estimated that more than 99% of current intravenous nanomedicines are sequestered by MPS (Wilhelm et al., 2016; Gustafson et al., 2015), so these new SORT nanoparticles overcome a long-standing challenge in nanomedicine.

[0474] The functional role of the permanently cationic SORT lipids was elucidated. Without wishing to be bound by any theory, it is believed that the inclusion of other lipids may also alter tissue tropism. To explore this potential, negatively charged 1,2-dioleoyl-sn-glycero-3-phosphate (18PA) was incorporated as a SORT lipid in a similar manner to DOTAP (Figure 20). At 10-40% 18PA incorporation, SORTLNP now mediated complete selective delivery to the spleen, with no luciferase expression in any other organ (Figure 19E). Therefore, negatively charged SORT lipids allow for precise delivery to the spleen. These results suggest that the SORT lipid percentage can be adjusted for specific tissue mRNA delivery by intravenous injection.

[0475] B. SORT can be extended to other LNP types and lipid classes

[0476] Then it was explored whether the SORT method can be applied to other categories of established 4-component LNPs to test whether SORT is universal. First, DLin-MC3-DMA was prepared with DSPC, cholesterol and PEG-DMG, which has the same molar composition (30) (Figure 22) as Onpattro (Patisiran) approved by FDA-, and is considered to be the "gold standard" for siRNA and mRNA delivery. So far, they have only been shown to be delivered to the liver after intravenous administration, which has also been confirmed here (Figure 19F). As expected, supplementing DLin-MC3-DMA LNP with DOTAP changes the protein expression profile of Onpattro preparations. As the SORT lipid percentage increases, luciferase signal is transferred from the liver to the spleen and then to the lungs, which is exactly the same as the phenomenon of the 5A2-SC8 DLNP tested initially. In order to further study the universality of the scheme, DOTAP is included in C12-200 LNP (Figures 19G and 22), which also well verifies the delivery of RNA to the liver (Kove et al., 2010; Kauffman et al., 2015). The results of 5A2-SC8 and DLin-MC3-DMA LNP are the same, and the inclusion of 15% or 50% DOTAP in C12-200 LNP changes the luciferase protein expression after mRNA delivery from liver to spleen to lung (Figures 19G and 22). In addition, the inclusion of 18PA as a SORT lipid reflects the results of 5A2-SC8, and mediates the unique delivery of DLin-MC3-DMA SNALP and C12-200 LLNP to the spleen for Luc mRNA (Figures 19F-G). DLin-MC3-DMA is a two-tail lipid with a single dimethylamine head group, which forms a stable nucleic acid lipid nanoparticle (SNALP), and C12-200 is a representative lipidoid that forms a lipid-like LNP (LLNP). Therefore, it is shown that the SORT method can be extended to other types of ionizable cationic lipid LNPs, which will make the existing liver-targeted LNPs easily change to deliver mRNA to the spleen or lungs. Specifically, SORT technology could enable the rapid redevelopment of FDA-approved Onpattro for the treatment of diseases of the lung and spleen.

[0477] To understand whether the observed tissue tropism profiles are specific to exact chemical structures or generalizable to limited chemical classes, a variety of permanent cationic, anionic, zwitterionic, and ionizable cationic SORT lipids were evaluated (Figure 23). First, 5A2-SC8 LNPs were generated with two other permanent cationic lipids: didodecyldimethylammonium bromide (DDAB) and 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine chloride (EPC). These lipids all contain quaternary amino groups, but there are major chemical differences in the polar head group, linker region, and hydrophobic domains (e.g., saturation). LNPs containing 5%, 15%, 40%, 50%, and 100% DDAB or EPC were formulated and characterized. The in vivo luciferase expression profile matched that of DOTAP LNPs, with luminescence activity systemically shifting from the liver to the spleen and then to the lungs as the percentage of DDAB or EPC increased (0.1 mg / kg, 6h). Once the percentage increased to 40%, high luciferase signals were observed only in the lungs (Figure 23A). 1,2-dimyristoyl-sn-glycerol-3-phosphate (14PA) and sn-(3-oleoyl-2-hydroxy)-glycerol-1-phosphate-sn-3'-(1', 2'-dioleoyl)-glycerol (18BMP) were generated as representative anionic lipids, which have very different structures compared to 18PA. All anionic SORT lipids promote exclusive delivery to the spleen (Figure 23B). This flexibility provides a method for balancing multiple factors (including efficacy, selectivity and tolerability) by optimizing SORT compounds.

[0478] Inspired by these findings, other ionizable cationic lipids were added to the established formulations. As expected, the addition of DODAP or C12-200 to 5A2-SC8 LNPs did not significantly alter tissue tropism, but surprisingly did enhance liver delivery by >10-fold at 20% incorporation ( FIG. 23C ). Supplementation of the established 5A2-SC8 LNPs with additional 5A2-SC8 as a SORT lipid significantly improved liver mRNA delivery, producing 10-fold increases in liver mRNA delivery at a very low dose of 0.05 mg / kg. 7 Photons / sec / cm 2 . Therefore, SORT provides a new strategy to further improve the liver-targeted LNP system (Figure 24). The effect of using zwitterionic lipids (DOCPe and DSPC) was also evaluated as SORT lipids. Although the tissue tropism was found to shift from the liver to the spleen, it was not as selective as the application of cationic or anionic SORT lipids (Figure 25).

[0479] To test the limitations of the SORT method, SORT was examined to see if it could "activate" an originally inactive preparation. In fact, supplementing a completely inactive preparation with DODAP or DOTAP resulted in tissue-specific delivery to the spleen and lungs (Figure 26). Combining these results, SORT is a modular and universal strategy for achieving tissue-targeted delivery.

[0480] C. SORT changes protein corona, LNP biodistribution, and apparent pK a To mediate organ-specific delivery

[0481] Mechanism experiments were carried out to explore how and why additional lipids are included in the defined categories to control the delivery of mRNA to different organs. Logically, LNPs delivered to cells in the lung should be biodistributed (accumulated) in the lungs. Cy5-labeled mRNA was delivered to track the in vivo distribution (Figures 27 A and 28) of 5A2-SC8 LNPs containing SORT lipids with lung (DOTAP quaternary amino lipids), spleen (18PA anionic lipids and DSPC zwitterionic lipids) and liver (DODAP ionizable tertiary amino lipids) tropism. All LNPs were injected intravenously with a dosage of 0.5mg / kg Cy5-labeled mRNA, and imaging was performed after 6 hours. As shown in Figure 27 A, DOTAP has changed biodistribution, and lung accumulation gradually increases as a function of DOTAP percentage. The incorporation of 18PA has increased the uptake into the spleen. DODAP has slightly increased liver accumulation, and has reduced spleen accumulation. Interestingly, even if the SORT LNPs specific to the lungs and spleen are still accumulated in the liver, there is no protein expression at all in the liver. This suggests that organ biodistribution is required for organ-specific efficacy but is not the only factor explaining the mechanism of tissue-targeted delivery.

[0482] Without wishing to be bound by any theory, it is believed that changes in biodistribution and activity in defined cell populations may be due to changes in the protein corona, where the binding of specific proteins creates a functionally active biological identity. Using quantitative mass spectrometry analysis, it was found that the addition of SORT molecules greatly altered the specific proteins that were most tightly bound and the overall protein corona composition. Without wishing to be bound by any theory, it is believed that the lung-specific SORT LNPs selectively and most abundantly bind vitronectin, which can interact with positively charged lipids and bind to the αvβ3 integrin that is highly expressed on endothelial and epithelial cells of the lung. This mechanism of endogenous targeting is in direct contrast to adenoviruses that utilize the αvβ5 integrin to target the bronchial epithelium. The spleen-specific SORT LNPs most tightly bind to β2-glycoprotein I, which has been shown to interact with negatively charged lipids and may play a role in splenic localization with immune cell populations in the spleen. It is worth noting that a complex mixture of bound proteins may also play a role. This collective effect is also seen as a potential mechanism for targeting multiple cell types and a way to further enrich specificity for specific cell types in one organ using alternative SORT molecules. It has been previously shown that apolipoprotein E binds to DLin-MC3-DMAOnpattro LNPs and loses efficacy in Apo E knockout animals. Therefore, there is strong evidence that Apo E is required for receptor-mediated targeting and hepatocyte uptake (presumably through the LDL receptor) and that the protein corona mechanism described can control cell specificity and efficacy. It is therefore encouraging to see that mDLNPs also closely associate with Apo E, providing further evidence of their hepatocyte efficacy and enhancing the validity of the protein corona assay. Liver-enhanced SORT LNPs retain Apo E binding but are also enriched in albumin, suggesting possible proliferation of cell types within the liver. These data cumulatively show that the chemical structure of SORT molecules can target specific protein coronas that alter organ tropism and cell specificity. Without wishing to be bound by any theory, it is believed that the identity of the SORT molecule can control the identity of the protein corona, suggesting that the SORT molecule can include sugars, lipids, small molecule therapeutics, vitamins, small molecules, hydrophilic molecules, hydrophobic molecules, amphiphilic molecules, peptides, proteins, etc.

[0483] For apparent / total pK a was examined because it has been identified as a parameter that correlates LNPs with functional activity. For example, it has been shown that at pK around 6.4 a Optimal for delivery to hepatocytes (Jayaraman et al., 2012). For all effective in vivo formulations (67 LNPs), the apparent pK was analyzed using the TNS assay. a(Figures 27B and 29, Table 1). Because SORT involves the inclusion of additional charged lipids, the resulting TNS titration curve captures the ionization behavior of the more complex mixed material LNP. Thus, the relative pK is estimated alternatively when 50% of the normalized signal is generated. a When the relative pK is plotted against tissue tropism a When SORT LNPs are grouped into defined apparent pK a As expected, all effective liver-targeted agents had a very narrow pK in the well-defined range of 6-7. a (Jayaraman et al., 2012). All lung-targeted formulations were located in the high pK a In contrast, spleen-tropic SORT LNPs were grouped into low pK a These results confirm that 6-7 are optimal for delivery to the liver but reveal a high pK a Mediates lung delivery and has a low pK a mediated spleen delivery. It should be noted that all SORT LNPs still contain ionizable cationic lipids, which are thought to be useful for endosomal escape due to their ability to acquire charge (Wittrup et al., 2015). Control experiments were performed and confirmed that the inclusion of ionizable cationic lipids is required for efficacy (Figure 30). Therefore, SORT allows the retention of specific microparticles with desired molar ratios with the pK required for efficacy. a molecules, while the inclusion of SORT lipids changes the apparent pK a Without wishing to be bound by any theory, it is believed that a two-part mechanism may be at work. SORT LNPs selectively bind to specific proteins in serum that enable receptor-mediated efficacy in lung or spleen cells, much like how lipoprotein particles (e.g., LDL) naturally transport cholesterol. This controlled and predictable endogenous targeting mechanism enables SORT LNPs to reach non-liver targets. The second part involves how the SORT molecule alters the properties of non-liver-targeted SORT LNPs so that they no longer have the physiochemical properties of liver efficacy (e.g., overall / apparent pK a 6.4), which provides precision. It should also be noted that other and more complex factors, such as cell-specific endocytic trafficking differences, may also play a role. Considering the results, it is suggested that the internal charge of the LNP nanostructure mediates biodistribution and that the apparent pK a Correlated with the protein expression profile in a specific organ. This specific value can be used to continue developing other organ-specific nanoparticles.

[0484] Table 1: Details of DDAB, EPC, 14PA, 18BMP, DODAP, C12-200, 5A2-SC8, DSPC, and DOCPe modified mDLNP formulations (SORT LNPs), including the molar ratio and percentage of each component, total lipid to mRNA weight ratio, size, and PDI.

[0485]

[0486] a X stands for DDAB, EPC, 14PA, 18BMP, DODAP, C12-200, 5A2-SC8, DSPC, and DOCPe.

[0487] D.SORT allows lung-, liver-, and spleen-specific gene editing after intravenous administration

[0488] Given the ability of SORT LNP to target specific organs, these findings are then applied to tissue-specific gene editing by intravenous injection. CRISPR / Cas (clustered regularly interspaced short palindromic repeats / CRISPR-associated proteins (Cas)) technology can edit the genome in a precise and sequence-dependent manner and has been rapidly developed for a variety of applications, including potential correction for pathogenic 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 administration of injections (Zuris et al., 2015; Sun et al., 2015; Chew et al., 2016; Staahl et al., 2017). However, many serious genetic disorders are caused by mutations in cells deep in organs, in which case specific cells need to be corrected to cure the disease. Such corrections can be best achieved by systemic administration. Recently, intravenous co-delivery of Cas9 mRNA and sgRNA has been reported to be a safe and effective strategy to achieve gene editing (Miller et al., 2017; Yin et al., 2017; Finn et al., 2018). However, to date, there have been no reports on the rational engineering of LNPs to edit cells in organs other than the liver.

[0489] In order to examine and quantify the ability of SORT LNP to mediate organ-specific gene editing, genetically engineered tdTomato (tdTom) reporter mice were used, which contain a stop box flanked by LoxP (Tabebordbar et al., 2016), which prevents the expression of tdTom protein (Staahl et al., 2017). Once the stop box is deleted, tdTom fluorescence is turned on, allowing detection of gene-edited cells (Figure 31A). Initially, Cre recombinase mRNA (Cre mRNA) was delivered to activate tdTom in edited cells. In selected organs treated with SORT LNP selective for liver, lung and spleen, fluorescent tissue is easily visualized (Figure 31B). It should be noted that a separate control must be used for each experiment because these mice have some background organ fluorescence, with the weakest fluorescence in the spleen compared to other organs (Figures 31C and 32). This makes spleen-specific detection more challenging to distinguish in the tdTom mouse model. When endogenous PTEN was subsequently edited, spleen-specific SORT LNPs showed clean DNA cleavage by T7E1 assay only in spleen ( FIG. 33C ), without any DNA cleavage in liver or lung. Nevertheless, tdTom-positive cells were easily seen by confocal imaging of tissue sections ( FIG. 31D ).

[0490] E.SORT achieves high levels of editing in specific and therapeutically relevant cell populations

[0491] Using flow cytometry of single cells extracted from edited organs, gene editing of specific cell types in the liver, lungs, and spleen was quantified (Figure 31E). After a single injection of 0.3mg / kg Cre mRNA, liver-specific SORT (20% DODAP) 5A2-SC8 LNP edited about 93% of all hepatocytes in the liver (Figures 31E and 34). This is the highest level of hepatocyte gene editing reported to date. At the same dose, lung-specific SORT (50% DOTAP) 5A2-SC8 LNP edited about 40% of all epithelial cells, about 65% of all endothelial cells, and about 20% of immune cells in the lungs (Figures 31E and 35). Given that epithelial cells are the main target for correcting mutations in CFTR that cause cystic fibrosis, this result establishes lung-specific SORT LNP as a compelling delivery system and is immediately applied to correct CFTR mutations. Finally, spleen-specific SORT (30% 18PA) 5A2-SC8 LNPs edited approximately 13% of all B cells, approximately 10% of all T cells, and approximately 20% of all macrophages (Figures 31E and 36). Due to the improved selectivity relative to previous studies, spleen-specific SORT LNPs may be used to treat non-Hodgkin's B-cell lymphoma and other immune disorders. Although the initial focus was on quantification in a single low-dose injection, higher levels of editing can be achieved by administering higher doses or multiple injections.

[0492] F.SORT allows tissue-specific gene editing via intravenous co-delivery of Cas9 mRNA / sgRNA and via delivery of Cas9 RNP

[0493] Then the ability of SORT LNP to achieve tissue-specific CRISPR / Cas gene editing by intravenous co-delivery of Cas9 mRNA and sgRNA in a single nanoparticle was examined (Figures 37A, 38 and 39, Table 2). The SORTLNP targeting liver and lungs was injected with a dose of 2.5 mg / kg of total RNA (4: 1mRNA: sgRNA, wt: wt), and quantitative gene editing was performed 10 days after a single intravenous injection. As shown in Figure 37B, for mice treated with basic LNP and 20% DODAP SORT LNP, strong tdTom fluorescence in the liver was observed, and strong fluorescence was observed in the lungs of mice treated with 50% DOTAP SORT LNP. All results are consistent with the Luc mRNA delivery results. Due to the rapid renewal of spleen immune cells in mice (Kamath et al., 2000), the weight ratio of Cas9 / sgRNA was optimized to 2 / 1 (Figure 39), and spleen editing was tested two days after injection. Taking into account background autofluorescence, bright tdTom fluorescence was observed in the spleen of 30% 18PA-treated mice, and a clear T7E1 cleavage band was detected only in DNA isolated from the spleen (no editing in liver or lung) (Figure 33). Fluorescence was then confirmed by imaging tissue sections with confocal microscopy (Figure 37C).

[0494] Next, direct delivery of Cas9 RNP was explored, which is the most challenging strategy for synthetic vectors. The application of permanent cationic SORT lipids enables the Cas9 protein / sgtdTom complex to be encapsulated and control tissue tropism. Intravenous injection of 7% DOTAPSORT LNP achieved liver editing, while 55% DOTAP SORT LNP achieved exclusive lung editing (Figure 37F). These data indicate that the described method can achieve liver, lung and spleen specific CRISPR / Cas gene editing.

[0495] In order to go beyond reporter mice, the ability of editing endogenous targets of tissue-specific LNPs was tested. PTEN was selected because it is a very determined tumor suppressor expressed in most cells. Wild-type C57BL / 6 mice were injected with SORT LNPs (total RNA of 2.5 mg / kg) loaded with Cas9 mRNA and sgPTEN. The generation of insertion and deletion (indel) was quantitatively measured 10 days after a single intravenous injection. As shown in Figure 37D, clear DNA cleavage bands were observed in specific tissues by T7E1 determination, indicating that basic LNP and 20% DODAP SORT LNP mediate effective PTEN editing in the liver, while in the lung or spleen, it is not mediated at all. It is worth noting that 50% DOTAP SORT LNP only shows PTEN editing in the lung. In order to further confirm PTEN editing, H&E staining and immunohistochemistry (IHC) of tissue sections were performed. As shown in Figure 37E, the cells in the tissue sections clearly show clear cytoplasm, which is a known phenotype (Xue et al., 2014) of PTEN loss caused by lipid accumulation. In addition, negative staining of PTEN was observed in IHC sections of both liver and lung tissues, providing clear evidence for PTEN editing. Although it is more challenging to distinguish spleen-specific 18PA SORT LNP editing in the tdTom mouse model, clear spleen PTEN editing can be observed in wild-type mice by optimizing the Cas9 / sgPTEN weight ratio (2 / 1) and detection time (2 days). By T7E1 assay performed on mice injected with 18PA SORT LNP, no editing of DNA in the liver or lung was observed (Figure 33). Finally, SORT was applied to Cas9 RNP and endogenous editing of PTEN was examined. As before, 7% and 55% of DOTAP SORT LNPs containing Cas9 protein / sgPTEN were able to perform liver- and lung-specific editing, respectively (Figure 37G). These results targeting endogenous genes demonstrate rationally guided tissue-selective gene editing achieved by synthetic vectors.

[0496] G. Materials and Methods

[0497] I. Materials

[0498] 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), dimethyldioctadecyl ammonium (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)-glycero-1-phospho-sn-3'-(1',2'-dioleoyl)-glycerol (ammonium salt) (18:1Hemi BMP, 18BMP), 1,2-dioleoyl-3-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-glycero-methoxy(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 Kit (WMCO, 3.5 kDa) was purchased from Sigma-Aldrich. 4',6-diamidino-2-phenylindole dihydrochloride (DAPI) was purchased from ThermoFisher 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 The modified sgTom1 and sgPTEN (Table 2) were purchased from Synthego.

[0499] Table 2: Relative apparent pKa values ​​of SORT LNPs measured by TNS assay

[0500]

[0501] II. Nanoparticle Formation

[0502] LNP preparations loaded with RNA were formed using an ethanol dilution method (Zhou et al., 2016). Targeted liver mRNA preparations (mDLNP) were developed and reported in previous papers (Cheng et al., 2018), and basic preparations were prepared as previously described (Jayaraman et al., 2012; Love et al., 2010). Unless otherwise stated, all lipids with a specified molar ratio were dissolved in ethanol, and RNA was dissolved in 10mM citrate buffer (pH 4.0). The two solutions were quickly mixed with a 3:1 water to ethanol volume ratio (3:1, water: ethanol, vol: vol) to reach a final weight ratio of 40: 1 (total lipid: mRNA), and then incubated at room temperature for 10min. To prepare SORT LNP formulations containing anionic SORT lipids (such as 18PA, 14PA, and 18BMP), the anionic lipids were first dissolved in tetrahydrofuran (THF), then mixed with other lipid components in ethanol, and finally obtained as described above The formulation containing mRNA buffer (10mM, pH 3.0). All formulations are named based on the additional lipids. Taking DOTAP mDLNP as an example, the internal molar ratio of mDLNP is fixed, as reported in the published paper, with 5A2-SC8 / DOPE / cholesterol / DMG-PEG of 15 / 15 / 30 / 3 (Cheng et al., 2018). DOTAP was dissolved as an additional lipid in the above ethanol lipid mixture in the specified amount to make the molar ratio of 5A2-SC8 / DOPE / cholesterol / DMG-PEG / DOTAP equal to 15 / 15 / 30 / 3 / X, and then rapidly mixed with the mRNA aqueous solution following the above standard protocol to finally produce SORT LNPs named Y% DOTAP, where Y refers to the molar percentage of DOTAP in the total lipid. Formulations with other additional lipids were similarly formed using the above method (Figure 20 and Table 3). For Cas9 / sgRNA ribonucleoprotein (RNP) encapsulation, 1×PBS was used for formulation, and the molar ratio of Cas9 and sgRNA was fixed at 1:3. After SORT LNP formation, the fresh LNP preparation was diluted to 0.5ng / μL mRNA with 1×PBS (final ethanol concentration <5%) for in vitro assays and size detection. For in vivo experiments, preparations were dialyzed (Pur-A-Lyzer Midi Dialysis Kit, WMCO 3.5 kDa, Sigma-Aldrich) against 1× PBS for 2 h and diluted to 15 μL / g with PBS for intravenous (IV) injection.

[0503] Table 3: sgRNA sequences

[0504]

[0505] III. Characterization of mRNA Preparations

[0506] Size distribution and polydispersity index (PDI) were measured using dynamic light scattering (DLS, Malvern MicroV; He-Ne laser, λ = 632 nm) and zeta potential was measured after dilution with 1× PBS. a The 2-(p-toluidine)-6-naphthalenesulfonic acid (TNS) assay (Cheng et al., 2018; McLaughlin and Harary, 1976; Bailey and Cullis, 1994; Heyes et al., 2005) was used with some modifications. mRNA preparations (60 μM total lipids) and TNS probes (2 μM) were incubated 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) for 5 min. Ex =321nm and λ Em =445 nm The mean fluorescence intensity of each well (black bottom 96-well plate) was measured using a Tecan plate reader and the data were normalized to the value of pH 2.5. a It is defined by the pH at half-maximal fluorescence. Although this approach can be used to estimate the overall / apparent pK of the LNP for most all LNPs a , but it cannot be used for SORT LNPs containing >40% permanent cationic lipids because these LNPs are always charged. Therefore, when 50% of the normalized signal is generated, the relative pK is estimated compared to the base LNP formulation (without added SORT lipids). a This alternative calculation does not change the pK of most LNPs. a , but did allow estimates of permanently cationic SORT LNPs that are consistent with experimental results for tissue-selective RNA delivery. It can therefore be recommended to use the standard TNS assay when the LNP contains a single ionizable cationic lipid, and to use the alternative 50% normalized signal approach for systems such as SORT that contain a complex mixture of multiple lipids carrying multiple charge states.

[0507] IV. In vitro luciferase expression and cell viability assay

[0508] One day before transfection, Huh-7 or A549 cells were plated at 1 × 10 4The density of cells was inoculated into a white 96-well plate. The culture medium was replaced with 150 μL fresh DMEM culture medium (5% FBS), and then 50 μL Luc mRNA preparation was added, and 25 ng mRNA was fixed in each well. After incubation for another 24 hours, mRNA expression and cytotoxicity were detected using the ONE-Glo+Tox kit based on Promega's standard protocol.

[0509] V. Animal Experiments

[0510] All animal experiments were approved by the Institutional Animal Care and Use Committee of The University of Texas Southwestern Medical Center and were in accordance with applicable local, state, and federal regulations. C57BL / 6 mice were obtained from the UTSW Mouse Breeding Core Facility. B6.Cg-Gt(ROSA)26Sor tm9(CAG-tdTomato)Hze / J mice (also referred to as Ai9 or Ai9 (RCL-tdT) mice) were obtained from The Jackson Laboratory (007909) and bred to maintain homozygous expression of the Cre reporter allele, which has a stop box flanked by loxP, which prevents transcription of the red fluorescent tdTomato protein driven by the CAG promoter. After Cre-mediated recombination, Ai9 mice will express tdTomato fluorescence. Ai9 mice are isogenic on a C57BL / 6J genetic background.

[0511] VI. In vivo Luc mRNA delivery and biodistribution

[0512] Various Luc mRNA preparations were injected intravenously into C57BL / 6 mice weighing 18-20 g at a dose of 0.1 or 0.05 mg / kg. n = 2-4 per group. After 6 hours, mice were injected intraperitoneally (IP) with D-luciferin (150 mg / kg) and imaged by the IVISLumina system (Perkin Elmer). For biodistribution, Cy5-Luc mRNA preparations were injected intravenously into C57BL / 6 mice at a dose of 0.5 mg / kg. Ex vivo imaging (Cy5 channel) was performed 6 hours after injection.

[0513] VII. mRNA Synthesis

[0514] Optimized Cre recombinase mRNA and Cas9 mRNA were generated by in vitro transcription (IVT). In short, NLS-Cre fragments and Cas9 fragments were prepared by PCR procedures using pCAG-CreERT2 and pSpCas9(BB)-2A-GFP (PX458) as PCR templates, respectively. These fragments were then cloned into pCS2+MT vectors with optimized 5'(3')-untranslated regions (UTRs) and polyadenylic acid sequences. The IVT reaction was performed following the standard protocol, but N1-methylpseudouridine-5'-triphosphate was used instead of the typical UTP. Finally, the mRNA was capped (Cap-1) by Vaccinia Capping Enzyme and 2'-O-methyltransferase (NEB). Table 4 shows the primers used herein.

[0515] Table 4: Primers, including the length of the PCR product and its purpose

[0516]

[0517] The coding sequences of NLS-Cre and Cas9 are as follows:

[0518] NLS-Cre:

[0519]

[0520] SV40 NLS-Cas9-nucleoplasmin NLS:

[0521]

[0522] VIII. Western Blot

[0523] The quality of IVT Cas9 mRNA was analyzed by Western blotting. One day before transfection, 293T cells were plated at 1 × 10 5 Cells were seeded into 12-well plates at a density of 10 cells / well. The cells were treated with various preparations in a total volume of 600 μL for another 24 h, 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 Lipofectamine2000 / Cas9pDNA (0.5 μgpDNA / well). After washing 3 times with 1×PBS, 100 μL of lysis buffer (50 mM Tris HCl, pH 7.4, containing 150 mM NaCl, 1 mM EDTA and 1% TRITON X-100) and 1 μL of protein inhibitor cocktail (100×, ThermoFisher) were added to each well and shaken at room temperature for 20 min. Cell lysates were collected in 1.6 mL tubes and centrifuged at 4 ° C for 10 min (13,000 g). If not used immediately, the supernatant was collected in a new tube and stored at -80 ° C. Before Western blotting, protein concentration was measured using a BCA assay kit (ThermoFisher). 15 μg of total protein was loaded and separated by 4-20% polyacrylamide gel (ThermoFisher). The separated protein was then transferred into a polyvinylidene membrane (BioRad) and blocked with 5% BSA (dissolved in PBST) for 1 hour at room temperature. The first antibody was applied overnight at 4 ° C. After washing four times with PBST, the membrane was incubated with the second antibody at room temperature for 1 hour, and then imaged with ECL substrate after washing four times with PBST (ThermoFisher).

[0524] IX. Gene Editing (Cre mRNA) in the Td-Tomato Mouse Model

[0525] Cre mRNA preparations were prepared as described above and injected intravenously (0.3 mg / kg Cre mRNA). Two days later, mice (n=4 per group) were sacrificed and major organs were imaged by IVIS Lumina system (Perkin Elmer).

[0526] X. Cell Isolation and Staining for Flow Cytometry

[0527] To test Td-Tomato in each organ cell type + Cells were isolated and stained after being treated with Cre mRNA preparation (0.3 mg / kg) for 2 days and then analyzed by flow cytometry.

[0528] For hepatocyte separation, two-step collagenase perfusion was performed as described above (Cheng et al., 2018). In short, mice were anesthetized and fixed with isoflurane. Perfusion was started with liver perfusion medium (Thermo Fisher Scientific, 17701038) for 7-10 min, and then replaced with liver digestion medium (Thermo Fisher Scientific, 17703034) for another 7-10 min. The liver was collected into a plate containing 10 mL of liver digestion medium and cut to release hepatocytes. The released hepatocytes were then collected and washed twice with hepatocyte washing medium (Thermo Fisher Scientific, 17704024) and once with 1 × PBS. After further separation by coarse filtration and low-speed (50 × g) centrifugation, hepatocytes were analyzed by FACS Aria IISORP machine (BD Biosciences).

[0529] For the separation and staining of spleen cell types, the removed spleen was minced with a sterile blade and homogenized in 250 μL of 1× digestion medium (45 units / μL collagenase I, 25 units / μL DNase I and 30 units / μL hyaluronidase). The spleen solution was transferred into a 15mL tube containing 5-10mL 1× digestion medium. Then, the spleen solution was filtered using a 70μm filter and washed once with 1×PBS. By centrifugation at a speed of 300×g for 5min, a cell pellet was obtained. The supernatant was removed, and the cell pellet was resuspended in 2mL 1×RBC lysis buffer (BioLegend, 420301) and incubated on ice for 5min. After incubation, 4mL cell staining buffer (BioLegend) was added to stop RBC lysis. The solution was centrifuged again at 300×g for 5min to obtain a cell pellet. Single cells were resuspended in cell staining buffer and added to a flow tube containing antibodies (100μL total volume). The cells were incubated with antibodies in the dark at 4 ° C for 20 min. The stained cells were washed twice with 1 mL 1 × PBS and then resuspended in 500 μL 1 × PBS for flow cytometry analysis. The antibodies used were Pacific Blue anti-mouse CD45 (BioLegend, 103126), Alexa Fluor 488 anti-mouse / human CD11b (BioLegend, 101217), Alexa Fluor 647 anti-mouse CD19 (BioLegend, 115522) and PerCP-Cyanine5.5 anti-mouse CD3e (145-2C11) (Tonbo Biosciences, 65-0031). Ghost Dye Red780 (Tonbo Biosciences, 13-0865-T500) was used to distinguish live cells.

[0530] For the separation and staining of lung cell types, the separated lungs were minced with sterile blades and then transferred into 15 mL tubes containing 10 mL 2 × digestion medium (90 units / μL collagenase I, 50 units / μL DNA enzyme I and 60 units / μL hyaluronidase) and incubated at 37 ° C for 1 h under shaking. After incubation, any remaining lung tissue was homogenized. The following steps are similar to the above spleen protocol. The antibodies used here are Pacific Blue anti-mouse CD45 (BioLegend, 103126), AlexaFluor 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) is used to distinguish living cells.

[0531] XI. Gene Editing in the Td-Tomato Mouse Model (Cas9 mRNA / sgRNA and Cas9 / sgRNA RNP)

[0532] To evaluate in vivo gene editing, Td-Tom mice were selected, with comparable body weight and the same sex. Cas9mRNA and sgRNA were co-delivered to tdTomato (td-Tom) mice. Cas9mRNA / sgTom1 (4 / 1, weight / weight) were co-delivered through various formulations, with a total RNA dose equal to 2.5 mg / kg. Ten days after intravenous injection, the major organs were removed and imaged on the IVIS Lumina system. For the formulation targeting the spleen, the total RNA dose was 4 mg / kg, and 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 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) on a Cryostat instrument (Leica Biosystems). Mounted tissue sections were stained with 4,6-diamidino-2-phenylindole (DAPI, Vector Laboratories) and then imaged by confocal microscopy on a Zeiss LSM 700.

[0533] XII. Gene Editing in C57BL / 6 Mice (Cas9 mRNA / sgPTEN and Cas9 / sgRNARNP)

[0534] To examine endogenous gene editing in vivo, PTEN was selected. Wild-type C57BL / 6 mice were injected intravenously with various vectors (n=2-4 per group) by co-delivering Cas9 mRNA and modified sgPTEN (4 / 1, mRNA / sgRNA, weight / weight) at a total dose of 2.5 mg / kg. Ten days later, tissues were collected and genomic DNA was extracted using the PureLink Genomic DNA Mini Kit (ThermoFisher). For preparations targeting the spleen, the total RNA dose was 4 mg / kg, Cas9 mRNA / sgTom1 was 2 / 1 (weight / weight), 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 per group). After obtaining the PTEN PCR product, the T7E1 assay (NEB) was performed to confirm the gene editing efficacy by standard protocols. In addition, tissue sections were evaluated for PTEN editing by H&E staining and immunohistochemistry (IHC). Briefly, paraformaldehyde (PFA)-fixed tissues were embedded in paraffin, sectioned, and H&E stained by the Molecular Pathology Core at UTSW. 4 μm sections were performed in a standard manner and IHC was performed using the Elite ABC Kit and DAB Substrate (Vector Laboratories).

[0535] Example 9: Formulations using neutral buffer

[0536] Cas9 RNPs were observed to denature in acidic buffers, resulting in an increase in hydrodynamic size from 10 nm to 150 nm (Figure 40B). This makes it difficult, if not impossible, to encapsulate the RNPs into monodisperse nanoparticles. These studies have focused on lipid nanoparticles (LNPs) because they are the most effective class of RNA delivery vehicles in preclinical models and in humans (Wood, 2018) (Wang et al., 2017; Doudna and Charpentier, 2014; Hajj and Whitehead, 2017; Sander and Joung, 2014). Of the four components of LNPs [ionizable cationic lipids, zwitterionic phospholipids, cholesterol, and poly(ethylene glycol) (PEG) lipids)], pK aIonizable cationic lipids of about 6.4 are useful for activity because they bind negatively charged RNA at mixed pH (e.g., pH 4 when amines are protonated), lose charge at neutral pH before cellular uptake, and then obtain charge again as the pH in endosomes decreases to fuse with endosomal membranes and be able to release goods to cytoplasm. However, this feature prevents effective encapsulation of goods at neutral pH because ionizable cationic lipids are uncharged at neutral pH. In order to overcome this challenge, a fifth component is added, particularly a positively charged cationic lipid at neutral pH, which allows the use of neutral buffers (rather than acidic buffers) to encapsulate RNA and protein, thereby retaining the tertiary structure and stability of RNP (Figure 40 A).

[0537] To evaluate this strategy, 5A2-SC8 was selected as an ionizable cationic lipid because 5A2-SC8 LNPs safely delivered short siRNA / miRNA and long mRNA to mice with impaired liver function, including MYC-driven liver cancer (Zhou et al., 2016; Zhang et al., 2018a; Zhang et al., 2018b) or genetic knockout of fumaryl acetoacetate hydrolase (FAH) (Cheng et al., 2018). The introduction of permanent cationic lipids (e.g., DOTAP) into the traditional 4-component 5A2-SC8 LNP formulation actually allows controlled self-assembly by mixing an ethanol solution of lipids with a PBS solution of RNPs (1 / 3, v / v). The incorporation of DOTAP was evaluated from 5-60 mol% of total lipids (Figure 41), which revealed higher gene editing levels of 10-20% in vitro and the formation of stable RNP-loaded nanoparticles with a size of <200 nm ( Fig.42). Initially, using sgRNA targeting reporter luciferase (sgLuc), LNP size with 10 mol% DOTAP incorporation was observed (5A2-DOT-10:5A2-SC8 / DOPE / Chol / DMG-PEG / DOTAP=15 / 15 / 30 / 3 / 7 (mol / mol)), and particles prepared using PBS buffer were slightly larger than nanoparticles without RNP loading. The same LNP prepared using low pH buffer did not change size, suggesting that RNP was not encapsulated (Figure 40C). In order to determine the optimal molar ratio between Cas9 protein and sgRNA, 1 / 1, 1 / 3, and 1 / 5 (mol / mol) Cas9 / sgRNA complexes were prepared, which reduced RNP size (Figure 40D) and increased negative charge (Figure 40F). After encapsulation, the RNP ratio did not change the size or zeta potential of the resulting LNP (Figures 40E, 40G). The surface charge of all LNPs was neutral, which not only indicates successful encapsulation, but also can be used to minimize in vivo uptake by the immune mononuclear phagocyte system (MPS). To further examine whether 5A2-DOT-10 can successfully mediate the delivery of RNPs into the nucleus, LNPs with encapsulated fluorescent EGFP-fused Cas9 protein were tracked. Free RNPs alone cannot enter cells because no green fluorescence above background was detected ( Fig.43 ). After 3 hours of 5A2-DOT-10 treatment, bright green fluorescence was observed in the cytoplasm of the cells. It was then observed that the EGFP-fused Cas9 protein gradually entered the nucleus within 6 hours due to the presence of a nuclear localization signal on Cas9 (Figure 40H). Endocytosis is energy-dependent and mainly depends on lipid rafts, because treatment with MβCD (an inhibitor of lipid raft-based endocytosis) significantly inhibited the cellular uptake of nanoparticles (Figure 40I).

[0538] In order to quantify the gene editing efficacy, HeLa-Luc and HeLa-GFP reporter cells were used. Examining different Cas9 / sgLuc ratios, gene editing was higher at 1 / 3 and 1 / 5 (Figure 44A). The results of the T7 endonuclease I (T7EI) assay confirmed that most of all target DNA bands (720bp) were cut into two cleavage bands (536bp and 184bp). No cleavage bands were observed in the control treatment group. In order to test the hypothesis that a neutral pH buffer is required to encapsulate RNP and maintain Cas9 function, the gene editing efficiency of 5A2-DOT-10 prepared using pH 4 citrate buffer was also evaluated. No cleavage bands were observed at all (Figure 44A). The negative results were further confirmed by Sanger sequencing, providing additional evidence that conventional acid-based formulation methods cannot produce effective NPs. Switching to cells expressing GFP, 5A2-DOT-10 encapsulating Cas9 / sgGFP induced insertions and deletions in GFP DNA and knocked out almost all GFP expression. The control group showed fluorescence intensity similar to that of PBS-treated cells (Figure 44B), which was confirmed by flow cytometry (Figures 44C and 45). Permanent gene editing was manifested by the infinite loss of GFP in growing cells and confirmed by Sanger sequencing, where the inference (ICE) analysis of CRISPR editing showed that the insertion and deletion reached 95% (Figure 44D). With an eye on clinical transformation, the stability of 5A2-DOT-10 loaded with RNP was monitored at 4 ° C for 2 months. LNP did not change size and remained uniform (PDI <0.2) (Figure 44G). Continuous testing of 5A2-DOT-10 nanoparticles revealed constant gene editing activity, even after 60 days of storage (Figure 44H).

[0539] The strategy of adding permanent cationic lipids to the classic 4-component LNPs to achieve efficient RNP delivery is not limited to dendrimer-based ionizable lipids, 5A2-SC8. To demonstrate this, supplementary DOTAP was included in nanoformulations prepared using other types of ionizable materials: the well-known DLin-MC3-DMA lipid used in the FDA-approved Onpattro (Wood, 2018) and C12-200 lipidoids (Figures 46A-B). Even though they have very different chemical structures compared to 5A2-SC8 (Figure 46C), all DOTAP-modified nanoparticles can effectively edit cells, while previously established C12-200 or MC3 formulations without DOTAP showed low editing efficiency (Figure 44E). 5A2-DOT-10 also achieved higher editing efficiency than the positive control RNAiMAX. Because 5A2-DOT-10LNP is more effective than MC3-DOT-10 and C12-200-DOT-10, all subsequent experiments are carried out using 5A2-SC8. Except DOTAP, other cationic lipids (including DDAB and EPC) are also introduced into LNP preparations (Figure 46 E-G). The results of all three kinds of cationic lipids with different chemical structures are similar (Figure 46 H). These results indicate that this strategy is general for ionizable cationic lipid nanoparticles (DLNP, LLNP, SNALP) and other cationic lipids with positive charge at pH7.4. Because this method allows adjustment of the Onpattro preparation of FDA-approval to realize the delivery of RNP, this scheme provides different directions for the treatment of clinically convertible human diseases.

[0540] The key to successful RNP delivery is to replace the standard acidic buffer with PBS buffer to maintain protein stability. In order to test whether this method is compatible with other neutral buffers, LNPs in PBS, Opti-MEM culture medium and HEPES were prepared. The preparation prepared in citrate buffer (pH 4) was used as a control (Figure 46I). Using LNPs prepared under all three neutral buffer conditions rather than in acidic buffer, significant and equivalent gene editing (>90%) (Figure 44F) was achieved. The ICE analysis of the sequencing results is consistent with the results shown by flow cytometry (Figure 46K).

[0541] To check in vivo gene editing, 5A2-DOT-10 encapsulating Cas9 / sgTOM complex was delivered to Td-Tomato mouse model (Figure 47A). In these mice, the deletion of CRISPR-mediated Lox-Stop-Lox box opened the downstream tdTom expression in the cells successfully edited. The 5A2-DOT-10LNP loaded with Cas9 / sgTOM RNP was injected into the left leg of the mouse by intramuscular injection at a dose of 1mg / kg sgTOM. Due to the previous use for direct injection gene editing (Zuris et al., 2015), RNAiMAX, which forms a complex with Cas9 / sgTOM RNP, was used for comparison. Higher Td-Tom fluorescence (Figure 47B) was observed in the muscles treated with 5A2-DOT-10 than in the mice treated with RNAiMAX. Imaging of tissue sections further confirmed that gene editing produced brighter red fluorescence (Figure 47C) in the 5A2-DOT-10 treatment group. 5A2-DOT-10 was injected into the brain of Td-Tom mice (0.15 mg / kg sgTOM). Again, bright red signals were observed near the injection site, confirming editing of the mouse brain (Figure 47D-E).

[0542] It was evaluated that the improved stability and efficacy of 5A2-DOT-10 can mediate successful systemic gene editing in tissues. In order to examine this RNP delivery strategy, LNPs with different molar percentages of DOTAP (5-60%) were prepared, and RNPs were delivered to Td-Tom mice IV (1.5mg / kg sgTOM). Seven days after injection of 5A2-DOT-5, Td-Tom fluorescence was observed only in the liver. Increasing the percentage of incorporated DOTAP from 5% to 60% resulted in gradual fluorescence from the liver to the lungs (CRISPR-guided gene editing). 5A2-DOT-60 mainly achieved lung editing (Figure 47F). These results indicate that deep tissue editing can be achieved in a tissue-specific manner by adjusting the internal lipid component chemistry and molar ratio. Tissue-specific editing (Figure 47G) was further confirmed by confocal imaging of tissue sections. Editing of the endogenous target Pten was then evaluated by systemic injection of LNPs encapsulating Cas9 / sgPTEN RNPs into wild-type C57BL / 6 mice. Clear 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).

[0543] In order to evaluate whether it is possible to edit multiple genes simultaneously in vivo, Cas9 protein and six different sgRNAs were loaded into 5A2-DOT-50. sgTOM, sgP53, sgPTEN, sgEml4, sgALK and sgRB1 were loaded into Cas9 protein for encapsulation. Then, Td-Tom mice were treated with 5A2-DOT-50 (combination) by tail vein injection (0.33mg / kg of each sgRNA). One week later, bright Td-Tom fluorescence was detected in the lungs, indicating the gene editing of TOM (Figure 47I). Clear T7EI cleavage bands were observed at all other 5 genomic loci, indicating that 5A2-DOT-50 can simultaneously and effectively edit multiple genes at low doses (Figure 47J). Quantitative analysis revealed a target editing efficiency of up to 22% in the lungs (Figures 47 and 48). This article uses sgRNAs with terminal modifications of the first and last 3 nucleotides to enhance sgRNA stability and reproducibility (Figure 49) (Finn et al., 2018; Hendel et al., 2015). Reports have shown that precise modification of other nucleotides can increase in vivo gene editing by 2-4 times compared to terminally modified sgRNAs (Finn et al., 2018; Yin et al., 2017), indicating that the editing efficiency reported herein may be higher with further sgRNA optimization. Nevertheless, the high efficacy and tissue specificity of 5A2-DOT-50 allow simultaneous editing of 6 targets in the lung with a single injection.

[0544] Traditionally, animal models are generated by transgenic or genetic engineering in embryonic stem cells, which is time-consuming and expensive. Direct mutation of tumor and other disease-related genes in adult mice using CRISPR / Cas provides a feasible solution for the rapid generation of models. This can only be achieved using expensive lentiviruses that must be engineered for each target and injected into the liver by hydrodynamic injection (Xue et al., 2014; Maddalo et al., 2014). Since mutation of multiple genes is usually required to generate functional cancer models, it is highly desirable to develop an inexpensive and effective multiplexing solution based on non-viral nanoparticles. Because 5A2-DOT-X LNPs are efficient, can edit multiple targets simultaneously, can be administered repeatedly, and provide tissue specificity, they provide a way to generate a variety of animal models.

[0545] 5A2-DOT-5 was used to selectively knock out three tumor suppressor genes (P53, PTEN and RB1) in the liver at the same time. These genes have been identified in many human cancers (including liver cancer). C57BL / 6 mice were treated by intravenous injection of 2.5mg / kg of total sgRNA for 3 weeks each week, and the gene editing efficiency in mouse liver was detected (Figure 48A). Clear cutting bands (Figures 48B, 50 and 51) were observed in all three gene loci after treatment of 2, 12, 15 and 20 weeks by T7EI determination. Over time, the cutting bands were much brighter, indicating tumor growth. When mice were killed at 15 weeks and 20 weeks, visible tumors were found on the liver, and several metastatic tumors (Figures 48C and 52) were found in the abdominal cavity. Tumor generation (Figures 48D and 53) was also detected by H&E staining and IHC staining of targeted tumor proliferation biomarker Ki67 at different time points.

[0546] 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 (especially non-small cell lung cancer) (Maddalo et al., 2014; Blasco et al., 2014). The Eml4-Alk fusion protein produced after rearrangement between Eml4 and Alk promotes cancer development. Using the high efficacy and lung targeting specificity of 5A2-DOT-50, we injected once (total sgRNA at a dose of 2 mg / kg) or twice (total sgRNA at a dose of 1.5 mg / kg, once a week) intravenous dose and evaluated the tumor generation process (Figure 48E). At all time points examined, insertion and deletion generation can be detected in the extracted lung DNA from both groups of mice (Figures 48F and 54). Clear gene rearrangement bands were detected in the lungs of 5A2-DOT-50-treated mice, confirming the successful generation of chromosomal rearrangements (Figures 48F and 54). Over time, the Eml4-Alk rearrangement bands were much brighter, indicating the proliferation of edited cells. The sequencing results after subcloning these PCR amplicons further confirmed the Eml4-Alk rearrangement (Figures 48G and 54). After 16 and 24 weeks of H&E staining and Ki67 staining, several tumor lesions were observed in the lung (Figures 48H, 55 and 56). These results show that a single injection of 5A2-DOT-50LNP can successfully produce chromosome rearrangements and lead to lung tumors in adult mice. Therefore, these LNPs are positioned to accelerate the in situ creation of multiple disease models.

[0547] B. Materials and Methods

[0548] I. Materials.

[0549] 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-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), dimethyldioctadecyl ammonium (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-glycero-methoxy(poly(ethylene glycol) MW 2000) (DMG-PEG2000) was purchased from NOF America Corporation. ONE-Glo+Tox luciferase reporter assay kit was purchased from Promega Corporation. Pur-A-Lyzer Midi Dialysis Kit (WMCO, 3.5 kDa) was purchased from Sigma-Aldrich. 4',6-diamidino-2-phenylindole dihydrochloride (DAPI), Hoechst33342, DLS Ultramicro cuvettes, Lipofectamine RNAiMAX transfection reagent, and Lab-Tek chambered coverslip 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.

[0550] II. Preparation of Cas9 / sgRNA complex.

[0551] Separate solutions of Cas9 protein and sgRNA were mixed together in equal volumes in labeled buffer. After mixing, they were incubated at room temperature for 5 minutes to allow the formation of RNPs for complete Cas9 / sgRNA complex self-assembly. The molar ratios of Cas9 protein to sgRNA used were 1 / 1, 1 / 3, and 1 / 5.

[0552] III. Optimized Nanoparticle Formulation and Characterization.

[0553] Ionizable cationic 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, and permanent cationic lipids (DOTAP, DDAB, or EPC) were dissolved in ethanol at given molar ratios. Cas9 / sgRNA complexes were dissolved in 1× PBS buffer. The Cas9 / sgRNARNP complex solution in PBS buffer was quickly mixed with a pipette into the ethanol solution of lipids at a volume ratio of 3:1 (Cas9 / sgRNARNP:lipid, v / v) to make a total lipid to sgRNA weight ratio of 40:1 (weight), and then incubated at room temperature for 15 min. Afterwards, the fresh preparations were directly characterized and used for in vitro assays. For animal experiments, the preparations were dialyzed (Pur-A-Lyzer Midi Dialysis Kit, WMCO 3.5 kDa) against 1× PBS for 1 h to remove ethanol before local injection (intramuscular or intracerebral injection) or systemic injection (intravenous injection). The size distribution and zeta potential of the nanoformulations were measured using dynamic light scattering (DLS, Malvern; He-Ne laser, λ = 632 nm; detection angle = 173°).

[0554] IV. RNAiMAX formulation.

[0555] To prepare RNAiMAX complexed RNPs, Cas9 / sgRNA complexes were prepared in Opti-MEM and gently mixed with Lipofectamine RNAiMAX transfection reagent diluted in Opti-MEM (at a dose of 1 μL RNAiMAX / μg sgRNA). The mixture solution was incubated at room temperature for 30 minutes to complete the complexation.

[0556] V. Standard LNP preparation.

[0557] To prepare C12-200 and MC3 LNPs encapsulating RNPs, the Cas9 / sgRNARNP complex solution in citrate buffer (pH 4.0) was quickly mixed into the lipid ethanol solution with a pipette at a volume ratio of 3:1 (Cas9 / sgRNARNP: total lipid, v / v), so that the weight ratio of total lipid to sgRNA was 40:1 (weight / weight), and then incubated at room temperature for 15 minutes. For C12-200 LNP, the molar ratio of C12-200 / DOPE / Chol / DMG-PEG was 35 / 16 / 46.5 / 2.5; for MC3 LNP, the molar ratio of DLin-MC3-DMA / DSPC / Chol / DMG-PEG was 50 / 10 / 38.5 / 1.5.

[0558] VI. Cellular uptake and uptake mechanism of 5A2-DOT-10Cas9 / sgLuc treatment

[0559] To examine cellular uptake, HeLa-Luc cells were plated at 2 × 10 4 The cells were seeded into Lab-Tek Chambered Coverglass (8-well) at a density of 10 cells / well and incubated overnight at 37°C. The old medium was then replaced with 150 μL of fresh DMEM containing 10% FBS and treated with 50 μL of 5A2-DOT-10 encapsulating Cas9-EGFP / sgLuc RNP (9 nM sgRNA / well). At 1 h, 3 h, 6 h, and 24 h after treatment, the cells were washed 3 times with PBS and stained with Hoechst (0.1 mg / mL) for 15 min at 37°C, and then imaged by confocal microscopy (Zeiss LSM 700).

[0560] To examine the uptake mechanism, an assay for specific inhibition of the endocytosis pathway was evaluated using Hela-Luc cells. 5A2-DOT10 treatment alone was used as a control. HeLa-Luc cells were plated at 5 × 10 5 The cells were seeded in 12-well plates at a density of 10 cells / well and incubated in DMEM complete medium for 24 hours. The cells were then washed with PBS and subsequently pre-incubated at 37°C for 1 h with one of the following endocytosis inhibitors dissolved in Opti-MEM: 20 μM chlorpromazine (CMZ, an inhibitor of clathrin-mediated endocytosis), 2 mM amiloride (AMI, an inhibitor of macropinocytosis), 200 μM genistein (GEN, an inhibitor of caveolae-mediated endocytosis), 5 mM methyl-β-cyclodextrin (MβCD, an inhibitor of lipid raft-mediated endocytosis). Next, the culture medium was removed and replaced with complete DMEM culture medium containing 5A2-DOT-10Cas9 / sgLuc (24 nM sgLuc) for another 30 min. Thereafter, the culture medium was removed and the cells were washed 3 times with PBS. The cells were then collected and analyzed by flow cytometry. All experiments were performed in triplicate. Here, Cas9-EGFP protein was used to formulate Cas9 / sgLuc complex. To evaluate whether it was energy-dependent endocytosis, cells were also pre-incubated at 4°C for 1 h and then treated with complete DMEM medium containing 24 nM 5A2-DOT-10Cas9 / sgLuc (24 nM sgLuc) for another 30 min before analysis by flow cytometry.

[0561] VII. T7EI assay for detecting genome editing.

[0562] For in vitro genomic DNA editing analysis, HeLa-Luc cells were cultured at 1.5 × 10 5 The cell density of cells / well was inoculated into 12-well plates and incubated overnight. Then, different nanoformulations containing 24nM sgRNA were added to the cells. After 3 days, the cells were collected, washed and resuspended in 50μL of 1× passive lysis buffer (Promega) with 2μL proteinase K (Thermofisher). Afterwards, the cleavage PCR program (65°C for 15 minutes, 95°C for 10 minutes) was run to obtain cell lysates. The targeted genomic loci were then amplified using the following PCR amplification program (95°C for 5 minutes; (95°C for 30 seconds; 60-64°C for 30 seconds; 72°C for 1 minute) for a total of 40 cycles; 72°C for 7 minutes, and then maintained at 4°C). Cell lysates were used as DNA templates. The amplicon was then purified using a PCR purification kit (Qiagen), and 200ng of purified DNA was added to 19μL of annealing reactions containing 1×NEBuffer 2. The PCR product was then annealed in a thermal cycler using the following conditions (95°C for 5 minutes, then the mixture was cooled from 95°C to 85°C at a ramp rate of -2°C / second, then cooled from 85°C to 25°C at a ramp rate of -0.1°C / second, and then maintained at 4°C) to form heteroduplex DNA. Then, 1 μL of T7EI (NEB) was added and incubated at 37°C for 15 minutes. The cleavage reaction was then stopped by adding 1.5uL of 0.25M EDTA. Next, the digested DNA was analyzed using 2.5% agarose gel electrophoresis. All primers used for T7EI assay are listed in Table 5.

[0563] Table 5: sgRNA sequences

[0564]

[0565]

[0566] For in vivo genomic DNA editing analysis, genomic DNA was extracted from tissues using PureLink Genomic DNA MiniKit (Invitrogen) according to the manufacturer's instructions. Subsequently, the aforementioned procedure was as described above for T7EI detection.

[0567] The fragmented PCR products were analyzed and the indel percentage was calculated based on the following formula:

[0568] %Gene modification = 100 × (1-(1-cleavage score) 1 / 2 )

[0569] where the cleavage fraction is the sum of the cleavage product peaks divided by the sum of the cleavage product peaks and the parent peak.5

[0570] VIII. Sanger sequencing for detection of genome editing.

[0571] The purified PCR amplicon of the T7EI assay was sequenced along with its forward primer by the McDermott Center Sequencing core facility at the University of Texas Southwestern Medical Center. Finally, the sequencing data were analyzed using the online analysis software ICE Analysis, which is a web tool provided by Synthego.

[0572] IX. In vitro gene editing in Hela-GFP cells.

[0573] Incubate the cells in DMEM containing 10% FBS and 1% penicillin / streptomycin at 37 °C / 5% CO. 2 For the experiment, HeLa-GFP cells were cultured at 1.5 × 10 5 The cell density of individual cells / well was inoculated into 12-well plates and incubated overnight. Then, the culture medium was replaced with 0.5mL fresh complete DMEM, and then 100uL nanoparticle dispersion was added (the final concentration of sgRNA was fixed to 24nM). Three days after treatment, cells were analyzed using a fluorescence microscope (Keyence). For flow cytometry analysis, cells were collected, washed with PBS, resuspended in PBS, and analyzed using BD analyzer LSRFortessaSORP (BD Biosciences).

[0574] Stability of X.5A2-DOT-10Cas9 / sgGFP.

[0575] To measure stability, 5A2-DOT-10LNPs encapsulating Cas9 / sgGFP RNP complexes were prepared and then stored at 4°C for 2 months. After storage for different times, the size and PDI of these nanoparticles were tested, and their gene editing efficiency was also evaluated in HeLa-GFP cells by adding nanoparticles (24nM sgRNA dose) and quantifying gene editing after 3 days. For each time point, aliquots of the stored 5A2-DOT-10LNPs encapsulating Cas9 / sgGFP RNPs were extracted and analyzed (size, PDI, potency).

[0576] XI. Animal experiments.

[0577] All animal experiments were approved by the Institutional Animal Care and Use Committee of The University of Texas Southwestern Medical Center and were in accordance with applicable local, state, and federal regulations. C57BL / 6 mice were obtained from the UTSW Mouse Breeding Core Facility. B6.Cg-Gt(ROSA)26Sor tm9(CAG-tdTomato)Hze / J mice (also referred to as Ai9 or Ai9 (RCL-tdT) mice) were obtained from The Jackson Laboratory (007909) and bred to maintain homozygous expression of the Cre reporter allele, which has a stop box flanked by loxP, which prevents transcription of the red fluorescent tdTomato protein driven by the CAG promoter. After Cas9 / sgRNARNP-mediated gene editing, Ai9 mice will express tdTomato fluorescence. Ai9 mice are isogenic on a C57BL / 6J genetic background.

[0578] XII. In vivo gene editing.

[0579] For gene editing in muscle, 5A2-DOT-10LNP encapsulating the Cas9 / sgTOM RNP complex was injected into the left leg of Td-Tomato mice at a dose of 1 mg / kg sgTOM by intramuscular injection. RNAiMAX encapsulating the Cas9 / sgTOM RNP complex was used as a positive control. After 7 days of treatment, muscle tissues from all treatment groups were collected and imaged using the IVISLumina system (Perkin Elmer). Afterwards, the muscle tissues were embedded in optimal cutting temperature (OCT) compound and cut into 10 μm slices. The slices were fixed with 4% paraformaldehyde (Thermo Fisher Scientific) for 20 minutes and washed 3 times with PBS buffer. Then, a drop of ProLong GoldMountant containing DAPI (Thermo Fisher Scientific) was applied to each slide. A coverslip was placed and the slides were imaged by confocal microscopy (Zeiss LSM 700). For gene editing in the brain, Td-Tomato mice were injected with 5A2-DOT-10LNP encapsulating the Cas9 / sgTOM RNP complex at a dose of 0.15 mg / kg sgTOM by intracerebral injection. After 6 days of treatment, the brain was excised and imaged using the IVIS Lumina system. Cryosections of the brain were prepared according to the above protocol and imaged by confocal microscopy.

[0580] For gene editing by intravenous injection, Td-Tomato mice were treated with 5A2-DOT-X LNPs containing different percentages of DOTAP at a dose of 1.5 mg / kg sgTOM by tail vein injection. After 7 days of treatment, all organs were collected and imaged using the IVIS Lumina system. Cryosections of these tissues were prepared according to the above protocol and imaged by confocal microscopy.

[0581] XIII. PCR for Eml4-Alk rearrangement.

[0582] In vivo Eml4-Alk rearrangement was tested by nested PCR (Blasco et al., 2014). For the first round of PCR, 40ng of genomic DNA was used as a template, and the PCR program was 95°C for 5 minutes; (95°C for 30 seconds; 64°C for 30 seconds; 72°C for 30 seconds) for a total of 18 cycles; 72°C for 7 minutes, and then kept at 4°C. For the second round of PCR, 1 μl of the first round of PCR products (100 dilution) was used for PCR reactions (95°C for 5 minutes; (95°C for 30 seconds; 68°C for 30 seconds; 72°C for 30 seconds) for a total of 30 cycles; 72°C for 7 minutes, and then kept at 4°C. The primers used in the PCR reactions are listed in Table 6.

[0583] Table 6: List of primers

[0584]

[0585]

[0586] XIV. H&E staining and immunohistochemistry (IHC).

[0587] Briefly, tissues fixed with 10% formalin solution were embedded in paraffin, sectioned, and stained with H&E by the Molecular Pathology Core at UTSW. 4 μm sections were prepared in a standard manner and used for IHC using the Elite ABC Kit and DAB Substrate (Vector Laboratories).

[0588] XV. Statistical analysis.

[0589] Statistical analysis was performed using two-sided Student's t-test by GraphPad Prism software version 7.04 (GraphPad Software, USA). No adjustments were made in any statistical test. P values ​​< 0.05 were considered statistically significant.

[0590] ***

[0591] According to the present disclosure, all methods disclosed and claimed in this article can be realized and executed without too much experimentation. Although the compositions and methods of the present disclosure have been described in terms of preferred embodiments, it is obvious to those skilled in the art that changes can be applied to the steps or step sequences of the methods and methods described herein without departing from the concept, spirit and scope of the present disclosure. More specifically, it is obvious that certain medicaments that are chemically and physiologically related can be used to replace the medicaments described herein while achieving the same or similar results. All such similar substitutions and modifications that are obvious to those skilled in the art are considered to be within the spirit, scope and concept of the present disclosure defined by the appended claims.

[0592] References

[0593] The following references are specifically incorporated herein by reference, to the extent that they provide exemplary operating details or other details supplementary to those described herein.

[0594] Amoasii et al., Science, 362:86-91, 2018.

[0595] Bailey and Cullis, Biochemistry, 33:12573-12580, 1994.

[0596] Blasco et al., Cell Rep., 9:1219-1227, 2014.

[0597] Cheng et al., Adv. Mater., 30:e1805308, 2018.

[0598] Chew et al., Nature methods, 13:868, 2016. [0599...

Claims

1. A composition comprising a lipid nanoparticle composition assembled with a therapeutic agent, the lipid nanoparticle composition comprising: (1) a cationic selective organ targeting (SORT) lipid that is different from (2) an ionizable cationic lipid, wherein the cationic SORT lipid is an ionizable cationic SORT lipid or a permanent cationic SORT lipid; wherein the cationic SORT lipid is present in the lipid nanoparticle composition at a molar percentage of 5% or more, wherein the molar percentage is determined based on the total moles of lipid present in the lipid nanoparticle composition; (2) the ionizable cationic lipid; (3) phospholipids; (4) steroids; and (5) polyethylene glycol-conjugated (PEGylated) lipids; wherein the ionizable cationic SORT lipid is 1,2-dioleoyl-3-dimethylammonium-propane (DODAP); wherein the permanent cationic SORT lipid has a structure of formula (I), formula (II) or formula (III), or a pharmaceutically acceptable salt thereof; in, In formula (I): R 1 and R 2 are independently alkyl C8-C24 , substituted alkyl C8-C24 , alkenyl C8-C24 or substituted alkenyl C8-C24 ; R 3 , R 3 ′ and R 3 "are each independently an alkyl C≤6 or substituted alkyl C≤6 ;and X - It is a monovalent anion; Wherein, in formula (II): R 4 and R 4 ' are each independently alkyl C6-C24 , substituted alkyl C6-C24 , alkenyl C6-C24 or substituted alkenyl C6-C24 ; R 4 ″ is an alkyl C≤24 , substituted alkyl C≤24 , alkenyl C≤24 or substituted alkenyl C≤24 ; R 4 ″′ is an alkyl C1-C8 , substituted alkyl C1-C8 , alkenyl C2-C8 or substituted alkenyl C2-C8 ;and X 2 is a monovalent anion; and Wherein, in formula (III): R 1 and R 2 are independently alkyl C8-C24 , substituted alkyl C8-C24 , alkenyl C8-C24 or substituted alkenyl C8-C24 ; R 3 , R 3 ′ and R 3 "are each independently an alkyl C≤6 or substituted alkyl C≤6 ; R 4 It is an alkyl C≤6 or substituted alkyl C≤6 ;and X - It is a monovalent anion; wherein the lipid nanoparticle composition is characterized by an apparent ionization constant (pK) of 3 to 6 or from 8 to 13 a ), determined by 2-(p-toluidine)-6-naphthalenesulfonic acid titration method.

2. The composition of claim 1, wherein the composition is characterized by a zeta (ζ) potential of -0.25 to -10 mV as measured by dynamic light scattering.

3. The composition of claim 1, wherein the cationic SORT lipid is present in the lipid nanoparticle composition at a molar percentage of 5% to 20%.

4. The composition of claim 1, wherein the cationic SORT lipid is present in the lipid nanoparticle composition at a molar percentage of 20% to 65%.

5. The composition of claim 1, wherein the permanent cationic SORT lipid has the following structural formula:

6. The composition of claim 1, wherein the permanent cationic SORT lipid has the following structural formula:

7. The composition of claim 1, wherein the permanent cationic SORT lipid has the following structural formula:

8. The composition of claim 1, wherein the ionizable cationic lipid is present in the lipid nanoparticle composition at a molar percentage of 5% to 30%.

9. The composition of claim 8, wherein the ionizable cationic lipid is present in the lipid nanoparticle composition at a molar percentage of 15% to 30%.

10. The composition of claim 1, wherein the ionizable cationic lipid comprises an ammonium group that is positively charged at physiological pH and contains at least two hydrophobic groups.

11. The composition of claim 10, wherein the ionizable cationic lipid comprises at least two C 6 -C 24 Alkyl or alkenyl.

12. The composition of claim 1, wherein the phospholipid is present in the lipid nanoparticle composition at a molar percentage of 8% to 20%.

13. The composition of claim 1, wherein the phospholipid is present in the lipid nanoparticle composition at a molar percentage of 20% to 23%.

14. The composition of claim 1, wherein the phospholipid is 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine or 1,2-distearoyl-sn-glycero-3-phosphocholine.

15. The composition of claim 1, wherein the steroid is present in the lipid nanoparticle composition at a molar percentage of 39% to 46%.

16. The composition of claim 1, wherein the steroid is present in the lipid nanoparticle composition at a molar percentage of 15% to 39%.

17. The composition of claim 1, wherein the steroid is cholesterol.

18. The composition of claim 1, wherein the PEGylated lipid is present in the lipid nanoparticle composition at a molar percentage of 0.5% to 10.0%.

19. The composition of claim 18, wherein the PEGylated lipid is present in the lipid nanoparticle composition at a molar percentage of 3.9% to 4.6%.

20. The composition of claim 1, wherein the PEGylated lipid comprises a polyethylene glycol (PEG) component of 1000 to 10,000 Daltons.

21. The composition of claim 1, wherein the PEGylated lipid is a PEGylated diacylglycerol.

22. The composition of claim 1, wherein the PEGylated lipid has the following structural formula: in: R 12 and R 13 are independently alkyl C≤24 , substituted alkyl C≤24 , alkenyl C≤24 or substituted alkenyl C≤24 ; R e Is hydrogen, alkyl C≤8 or substituted alkyl C≤8 ;and x is 1-250.

23. The composition of claim 1, wherein the PEGylated lipid is dimyristoyl-sn-glycerol or a compound having the following structural formula: in: n 1 is 5-250; and n 2 and n 3 Each independently is 2-25.

24. The composition of claim 1, wherein the therapeutic agent is a small molecule.

25. The composition of claim 1, wherein the therapeutic agent is a protein.

26. The composition of claim 1, wherein the therapeutic agent is a nucleic acid.

27. The composition of claim 26, wherein the nucleic acid is a therapeutic nucleic acid.

28. The composition of claim 26, wherein the nucleic acid is selected from the group consisting of short interfering ribonucleic acid (siRNA), micro-ribonucleic acid (miRNA), pri-miRNA, messenger ribonucleic acid (mRNA), clustered regularly interspaced short palindromic repeats (CRISPR)-related nucleic acid, single guide ribonucleic acid (sgRNA), CRISPR-RNA (crRNA), trans-activating crRNA (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) and double-stranded ribonucleic acid (dsRNA).

29. The composition of claim 26, wherein the nucleic acid is present in the lipid nanoparticle composition at a ratio of 1:1 to 1:100 of the nucleic acid to the lipid nanoparticle composition.

30. The composition of claim 1, wherein the therapeutic agent comprises a protein.

31. The composition of claim 30, wherein the therapeutic agent comprises both a protein and a nucleic acid.

32. The composition according to any one of claims 1-31, wherein the composition is a pharmaceutical composition further comprising an excipient.

33. The composition of claim 32, wherein the excipient is a pharmaceutically acceptable excipient.

34. Use of a composition comprising a gene or transcript editing agent and a lipid composition in the preparation of a medicament for targeted delivery of a gene or transcript editing agent to an organ or a cell in an organ, Wherein the lipid composition comprises: (1) ionizable cationic lipids; and (2) a SORT lipid which is a permanently cationic SORT lipid; wherein the composition is configured to provide a modified expression profile of a target gene or transcript in the organ or the cell compared to that achieved in the absence of the SORT compound, wherein the organ is the lung or spleen, and wherein the cell is selected from cells from the lung or the spleen.

35. The use of claim 34, wherein the gene or transcript editing agent comprises an enzyme.

36. The use according to claim 35, wherein the enzyme comprises CRISPR-associated protein 9 (Cas9).

37. Use according to claim 35, wherein the enzyme is a stand-alone enzyme.

38. The use according to claim 35, wherein the enzyme is a fusion enzyme.

39. The use according to claim 34, wherein the surface of the lipid composition is bound to apolipoprotein E (ApoE), vitronectin or apolipoprotein H (Apo H) as determined by quantitative mass spectrometry.

40. The use according to claim 34, wherein the organ or cells in the organ is the lung or spleen, or cells in the lung or spleen.

41. Use of a composition comprising a therapeutic agent and a lipid composition in the preparation of a medicament for non-hepatic targeted delivery of the therapeutic agent, the lipid composition comprising: (1) ionizable cationic lipids; and (2) a SORT lipid different from the ionizable cationic lipid, wherein the SORT lipid is selected from the group consisting of a cationic SORT lipid, a zwitterionic SORT lipid, and an anionic SORT lipid; in, When administered to a subject, the surface of the lipid composition interacts with apolipoprotein E (Apo E) to a lesser extent than with endogenous proteins other than Apo E, thereby providing a lower amount or activity of the therapeutic agent in the liver or cells in the liver than would be achieved in the absence of the SORT lipid.

42. The use of claim 41, wherein after said administration, in said subject, said endogenous protein is bound to said surface of said SORT lipid composition in an amount greater than Apo E.

43. The use according to claim 41, wherein the endogenous protein is vitronectin, β2-glycoprotein 1 or apolipoprotein H (Apo H).

44. The use according to claim 41, wherein the endogenous protein is β2-glycoprotein 1 or apolipoprotein H (ApoH).

45. The use of claim 41, wherein the endogenous protein is not β2-glycoprotein 1 or apolipoprotein H (ApoH); and wherein in the subject, the surface of the SORT lipid composition interacts with Apo H to a lesser extent than with the endogenous protein.

46. ​​The use according to claim 45, wherein the endogenous protein is vitronectin.

47. The use according to claim 41, in, The use provides a greater amount or higher activity of the therapeutic agent in a non-hepatic organ in the subject or in cells in the non-hepatic organ than is achieved in the absence of the SORT lipid.

48. The use of claim 47, wherein the non-hepatic organ or cells in the non-hepatic organ comprises cells in the lung, spleen, bone marrow, lymph nodes, or any of the foregoing organs.

49. The use according to claim 48, wherein the non-hepatic organ or cells in the non-hepatic organ comprises the lung or cells in the lung.

50. The use according to claim 48, wherein the non-hepatic organ or cells in the non-hepatic organ comprises spleen, bone marrow, lymph node, or cells in any of the foregoing organs.

51. A composition comprising: (a) chemotherapeutic agents; and (b) a lipid nanoparticle composition comprising: (1) a SORT compound comprising a cationic SORT lipid, an anionic SORT lipid or a zwitterionic SORT lipid; and (2) Ionizable cationic lipids.

52. A composition comprising a messenger RNA (mRNA) and a lipid composition, wherein the mRNA encodes a cystic fibrosis transmembrane regulator (CFTR) protein, wherein the lipid composition comprises: (i) about 5 to about 50 mole percent zwitterionic SORT lipid; (ii) at least about 5% by mole of an ionizable cationic lipid, wherein the ionizable cationic lipid comprises an ammonium group that is positively charged at a pH of about 6 to about 8 and at least two C 6 -C 24 Alkyl or alkenyl; (iii) from about 15 to about 46 mole percent of a steroid or steroid derivative; and (iv) about 0.5% to about 10% by mole of polymer-conjugated lipid; wherein the molar percentage is determined based on the total lipid present in the lipid composition; wherein the composition is formulated for inhalation delivery to the lung.

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