Compositions and methods for target RNA delivery

Abstract

Provided herein is a composition, its manufacturing method and method for the targeted delivery of therapeutic agents for modifying target gene expression and function, for example, the protein involved in lipid and cholesterol metabolism, such as PCSK9.Further provided herein is a composition and method for treating coronary artery disease-related conditions.

Description

[Technical Field]

[0001] Cross-reference of related applications

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 078,982 filed September 16, 2020 and U.S. Provisional Application No. 62 / 984,866 filed March 4, 2020, which are incorporated herein by reference in their entirety.

[0002] Sequence List

[0002] This application includes an electronically submitted sequence listing in ASCII format, the entirety of which is incorporated herein by reference. This ASCII copy, created on 3 March 2021, is named 53989706601_SL.txt and has a size of 67,230 bytes.

[0003] Areas of disclosure

[0003] This disclosure relates to compositions and methods for targeted delivery of therapeutic agents such as CRISPR guide RNA and other nucleic acid drugs. [Background technology]

[0004]

[0004] All publications herein are incorporated by reference to the same extent as if each individual publication or patent application were specifically and individually incorporated by reference. The following description contains information that may be useful in understanding this disclosure. It is not acknowledged that any information provided herein is prior art or relating to the claimed invention, or that any publication specifically or implicitly referenced is prior art. [Overview of the project]

[0005]

[0005] In one embodiment, a receptor-targeting conjugate is disclosed herein, wherein formula (V):

[0006] [ka]

[0007] comprising the compound of wherein the plurality of A groups collectively comprise a receptor targeting ligand; each L 1 、L 2 、L 3 、L 4 、L 5 、L 6 、L 7 、L 8 、L 9 、L 10 and L 12 is independently a substituted or unsubstituted C1-C 12 alkylene, a substituted or unsubstituted C1-C 12 heteroalkylene, a substituted or unsubstituted C2-C 12 alkenylene, a substituted or unsubstituted C2-C 12 alkynylene, -(CH2CH2O) m -, -(OCH2CH2) m -, -O-, -S-, -S(=O)-, -S(=O)2-, -S(=O)(=NR 1 )-, -C(=O)-, -C(=N-OR 1 )-, -C(=O)O-, -OC(=O)-, -C(=O)C(=O)-, -C(=O)NR 1 -, -NR 1 C(=O)-, -OC(=O)NR 1 -, -NR 1 C(=O)O-, -NR 1 C(=O)NR 1 -, -C(=O)NR 1 C(=O)-, -S(=O)2NR 1 -, -NR 1 S(=O)2-, -NR 1 -, or -N(OR 1 )-; L 11 is a substituted or unsubstituted -(CH2CH2O) n - or a substituted or unsubstituted -(OCH2CH2) n -; each R 1These are independently H or substituted or unsubstituted C1-C6 alkyl groups; R is a lipid, nucleic acid, amino acid, protein, or lipid nanoparticle; m is an integer selected from 1 to 10; n is a receptor-targeting conjugate, an integer selected from 1 to 200.

[0008]

[0006] Several control devices, each L 1 , L 4 , and L 7 These are independently of substitution or non-substitution of C1-C 12 It is an alkylene. In some embodiments, each L 1 , L 4 , and L 7 These are independently substituted or unsubstituted C2-C6 alkylenes. In some embodiments, each L 1 , L 4 , and L 7 is a C4 alkylene. In some embodiments, each L 2 , L 5 , and L 8 Independently, -C(=O)NR 1 -, -NR 1 C(=O)-, -OC(=O)NR 1 -, -NR 1 C(=O)O-, -NR1C(=O)NR 1 -, or -C(=O)NR 1 C(=O)- is the case in some embodiments, each L 2 , L 5 , and L 8 Independently, -C(=O)NR 1 -or-NR 1 C(=O)- is the case in some embodiments, each L 2 , L 5 , and L 8 is -C(=O)NH-. In some embodiments, each L 3 , L 6 , and L 9 These are independently of substitution or non-substitution of C1-C 12is an alkylene. In some embodiments, each L 3 is a substituted or unsubstituted C2-C6 alkylene. In some embodiments, L 3 is C4 alkylene. In some embodiments, each L 6 and L 9 are independently a substituted or unsubstituted C2-C 10 alkylene. In some embodiments, each L 6 and L 9 are independently a substituted or unsubstituted C2-C6 alkylene. In some embodiments, each L 6 and L 9 is C3 alkylene. In one aspect, provided herein is a receptor targeting conjugate comprising a compound of formula (VI):

[0009]

Chemical formula

[0010] comprising, wherein, a plurality of A groups collectively comprise a receptor targeting ligand; each L 1 , L 2 , L 3 , L 4 , L 5 , L 6 , L 7 , L 8 , L 9 , L 10 and L 12 are independently a substituted or unsubstituted C1-C 12 alkylene, a substituted or unsubstituted C1-C 12 heteroalkylene, a substituted or unsubstituted C2-C 12 alkenylene, a substituted or unsubstituted C2-C 12 alkynylene, -(CH2CH2O) m -, -(OCH2CH2) m -, -O-, -S-, -S(=O)-, -S(=O)2-, -S(=O)(=NR 1)-, -C(=O)-, -C(=N-OR 1 )-, -C(=O)O-, -OC(=O)-, -C(=O)C(=O)-, -C(=O)NR 1 -, -NR 1 C(=O)-, -OC(=O)NR 1 -, -NR 1 C(=O)O-, -NR 1 C(=O)NR 1 -, -C(=O)NR 1 C(=O)-, -S(=O)2NR 1 -, -NR 1 S(=O)2-, -NR 1 -, or -N(OR 1 )-; L 11 is a substituted or unsubstituted -(CH2CH2O) n -, or a substituted or unsubstituted -(OCH2CH2) n -; Each R 1 is independently H or a substituted or unsubstituted C1-C6 alkyl; R is a lipid, nucleic acid, amino acid, protein, or lipid nanoparticle; m is an integer selected from 1 to 10; n is an integer selected from 1 to 200.

[0011]

[0007] In some embodiments, each L 1 , L 4 , and L 7 are independently a substituted or unsubstituted C1-C 12 alkylene or a substituted or unsubstituted C1-C 12 heteroalkylene. In some embodiments, each L 1 , L 4 , and L 7 are independently a substituted or unsubstituted C1-C 12 heteroalkylene. In some embodiments, each L 1 , L 4 , and L 7 are independently a substituted or unsubstituted C1-C 12 heteroalkylene containing 1 to 10 O atomsIt is a heteroalkylene. In some embodiments, each L 1 , L 4 , and L 7 It is independently -(CH2CH2O) p1 -(CH2) q1 - and; in the formula, p1 is 1 to 8; q1 is 1 to 6. In some embodiments, each L 1 , L 4 , and L 7 is -(CH2CH2O)3-(CH2)2-. In some embodiments, each L 1 , L 4 , and L 7 These are independently of substitution or non-substitution of C1-C 12 It is an alkylene. In some embodiments, each L 1 , L 4 , and L 7 These are independently substituted or unsubstituted C2-C6 alkylenes. In some embodiments, each L 1 , L 4 , and L 7 is a C4 alkylene. In some embodiments, each L 2 , L 5 , and L 8 Independently, -C(=O)NR 1 -, -NR 1 C(=O)-, -OC(=O)NR 1 -, -NR 1 C(=O)O-, -NR 1 C(=O)NR 1 -, or -C(=O)NR 1 C(=O)- is the case in some embodiments, each L 2 , L 5 , and L 8 Independently, -C(=O)NR 1 -or-NR 1 C(=O)- is the case in some embodiments, each L 2 , L 5 , and L 8 is -NHC(=O)-. In some embodiments, each L 2 , L 5 , and L 8is -C(=O)NH-. In some embodiments, each L 3 , L 6 , and L 9 These are independently of substitution or non-substitution of C1-C 12 It is a heteroalkylene. In some embodiments, each L 3 , L 6 , and L 9 These are independently substituted or unsubstituted C1-C atoms containing 1-10 oxygen atoms. 12 It is a heteroalkylene. In some embodiments, each L 3 , L 6 , and L 9 It is independently -(CH2CH2O) p2 -(CH2CH2CH2O) q2 - and; in the formula, p2 is 1 to 8; q2 is 1 to 6. In some embodiments, each L 3 , L 6 , and L 9 is -(CH2CH2O)-(CH2CH2CH2O)-. In some embodiments, each L 3 , L 6 , and L 9 It is independently -(CH2CH2CH2O) q3 - and; in the formula, q3 is 1 to 8. In some embodiments, each L 3 , L 6 , and L 9 is -(CH2CH2CH2O)2-. In some embodiments, L 10 C1-C are either substituted or non-substituted. 12 It is an alkylene. 10 L is a substituted or unsubstituted C1-C4 alkylene. In some embodiments, L 10 is a C2 alkylene. In some embodiments, L 11 is -(OCH2CH2) n -. In some embodiments, n is 1 to 100. In some embodiments, n is 2 to 50. In some embodiments, n is 2, 12, 37, or 45. In some embodiments, L 12is -O-, -C(=O)O-, -C(=O)NR 1 -, -NR 1 C(=O)-, or -NR 1 In some embodiments, L 12 is -C(=O)O- or -NR 1 In some embodiments, L 12 In some embodiments, L 12 is -NHC(=O)O-. In some embodiments, L 12 A is -NHC(=O)-. In some embodiments, A binds to a lectin. In some embodiments, this lectin is an asialoglycoprotein receptor (ASGPR). In some embodiments, A is N-acetylgalactosamine (GalNAc) or a derivative.

[0012] [ka]

[0013] or a derivative thereof. A is N-acetylgalactosamine (GalNAc).

[0014] [ka]

[0015] or derivative thereof. 1 R is independently H or -CH3. In some embodiments, each R 1 is H. In some embodiments, R comprises one or more fatty alcohols, fatty acids, glycerolipids, glycerophospholipids, sphingolipids, saccharolipids, polyketides, sterol lipids, and prenolipids. In some embodiments, R comprises one or more fatty alcohols. In some embodiments, each fatty alcohol is independently saturated, monounsaturated, or polyunsaturated fatty alcohol. In some embodiments, the fatty alcohol is one or more C2-C26 Contains fatty alcohols. In some embodiments, the fatty alcohols consist of two or more C2-C2 alcohols. 26 Contains fatty alcohols. In some embodiments, each fatty alcohol is a C12, C14, C16, C18, C20, or C22 fatty alcohol. In some embodiments, each fatty alcohol is independently docosahexaenol, eicosapentaenol, oleyl alcohol, stearyl alcohol, (9Z,12Z)-octadeca-9,12-dien-1-yl alcohol, (Z)-docosa-13-en-1-yl alcohol, docosanyl alcohol, (E)-octadeca-9-en-1-yl alcohol, eicosanyl alcohol, (9Z,12Z,15Z)-octadeca-9,12,15-trien-1-yl alcohol, or palmityl alcohol. In some embodiments, each fatty alcohol is stearyl alcohol. In some embodiments, R contains one or more sterol lipids. In some embodiments, R contains one or more vitamins. In some embodiments, each vitamin is independently vitamin A, vitamin D, vitamin E, or vitamin K. In some embodiments, R comprises nucleic acids. In some embodiments, nucleic acids are, but are not limited to, mRNA, guide RNA, siRNA, antisense oligonucleotides, aptamers, microRNAs, immunostimulatory oligonucleotides, splice-switching oligonucleotides, auto-amplified RNA, circular RNA, or DNA. In some embodiments, R comprises proteins. In some embodiments, proteins are, but are not limited to, gene-editing factor (editor) proteins, antibodies, antigen-binding antibody fragments, or peptides.

[0016]

[0008] In one embodiment, the herein describes a receptor-targeting conjugate comprising a compound derived from Table 4.

[0009] In one embodiment, the nanoparticle composition described herein is a nanoparticle composition comprising: (a) one or more nucleic acid molecular entities; and (b) a receptor targeting conjugate described herein. In some embodiments, the receptor targeting conjugate constitutes about 0.001 mol% to about 20 mol% of the total lipid content present in the nanoparticle composition. In some embodiments, the receptor targeting conjugate constitutes about 0.01 mol% to about 1 mol% of the total lipid content present in the nanoparticle composition. In some embodiments, sterols or derivatives thereof constitute 10 mol% to 70 mol% of the total lipid content present in the nanoparticle composition. In some embodiments, the sterols or derivatives thereof are cholesterol or cholesterol derivatives. In some embodiments, cholesterol or cholesterol derivatives constitute 20 mol% to 50 mol% of the total lipid content present in the nanoparticle composition. In some embodiments, the nanoparticle composition contains phospholipids that constitute 1 mol% to 20 mol% of the total lipid content present in the nanoparticle composition. In some embodiments, phospholipids constitute about 5 mol% to about 15 mol% of the total lipid content present in the nanoparticle composition. In some embodiments, the phospholipid is selected from 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 2-oleoyl-1-palmitoyl-sn-glycero-3-phosphocholine (POPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and sphingomyelin. In some embodiments, the phospholipid is DSPC. In some embodiments, the nanoparticle composition contains stealth lipids constituting 0.1 mol% to 6 mol% of the total lipid content present in the nanoparticle composition. In some embodiments, the stealth lipids constituting about 2.0 mol% to about 2.5 mol% of the total lipid content present in the nanoparticle composition.In some embodiments, the stealth lipid is a PEG lipid having a number-average molecular weight ranging from about 200 Da to about 5000 Da. In some embodiments, the nanoparticle composition contains amino lipids constituting about 10 mol% to about 60 mol% of the total lipid content present in the nanoparticle composition. In some embodiments, the nanoparticle composition contains an antioxidant. In some embodiments, the antioxidant contains ethylenediaminetetraacetic acid (EDTA). In some embodiments, one or more nucleic acid molecular entities include a single guide RNA (sgRNA) or guide RNA (gRNA) that targets a target pathogenic gene generated in hepatocytes. In some embodiments, one or more nucleic acid molecular entities include mRNA encoding a Cas nuclease. In some embodiments, at least one of the one or more nucleic acid molecular entities includes chemical modification. In some embodiments, the chemical modification is 2'-F modification, phosphorothioate internucleotide linkage modification, acyclic nucleotide, LNA, HNA, CeNA, 2'-methoxyethyl, 2'-O-methyl, 2'-O-allyl, 2'-C-allyl, 2'-deoxy, 2'-fluoro, 2'-ON-methylacetamide (2'-O-NMA), 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE), 2'-O-aminopropyl (2'-O-AP), 4'-O-methyl, or 2'-ara-F modification. In some embodiments, the chemical modification is 2'-O-methyl modification.

[0017]

[0010] In one embodiment, a pharmaceutical composition comprising a receptor-targeting conjugate or a nanoparticle composition described herein, and an excipient or carrier is provided herein. In some embodiments, the pharmaceutical composition comprises mRNA encoding a gene-editing factor nuclease. In some embodiments, the pharmaceutical composition comprises one or more guide RNA molecules. In some embodiments, the pharmaceutical composition comprises two or more guide RNA molecules. In some embodiments, the two or more guide RNA molecules target two or more genes of interest. In some embodiments, the mRNA encodes a Cas9 nuclease. In some embodiments, the mRNA encodes a base-editing factor nuclease. In some embodiments, the mRNA and one or more guide RNA molecules are present in the same nanoparticle composition. In some embodiments, the mRNA and one or more guide RNA molecules are present in different nanoparticle compositions. In some embodiments, the ratio of gRNA molecules to mRNA in the pharmaceutical composition is about 0.01 to about 100 by weight or mole. In some embodiments, the ratio of the gRNA molecule to the mRNA in the pharmaceutical composition is about 50:1, about 40:1, about 30:1, about 20:1, about 18:1, about 16:1, about 14:1, about 12:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, or about 1:10 by weight or moles.

[0018]

[0011] In one embodiment, a pharmaceutical composition comprising (a) a first receptor-targeting conjugate or first nanoparticle composition described herein, and (b) a second receptor-targeting conjugate or second nanoparticle composition described herein is provided herein. In some embodiments, the first nanoparticle composition comprises gene editing factor mRNA. In some embodiments, the second nanoparticle composition comprises one or more guide RNA molecules. In some embodiments, the ratio of guide RNA molecules to mRNA in the pharmaceutical composition is about 50:1, about 40:1, about 30:1, about 20:1, about 18:1, about 16:1, about 14:1, about 12:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, or about 1:10 by weight or moles.

[0019]

[0012] In one embodiment, a method for delivering nucleic acids to cells is provided herein, the method comprising the step of contacting cells with a nanoparticle composition or pharmaceutical composition described herein, thereby delivering nucleic acids to the cells. In some embodiments, the cells are contacted in vivo, ex vivo, or intro.

[0020]

[0013] In one embodiment, the Specified herein provides a method for producing a target polypeptide in a cell, comprising the step of contacting the cell with a nanoparticle composition or pharmaceutical composition described herein, thereby enabling the nucleic acid to be translated in the cell to produce a polypeptide.

[0021]

[0014] In one embodiment, a method for treating a disease or condition of interest is provided herein, the method comprising the step of administering a therapeutically effective amount of a pharmaceutical composition described herein to the subject. In some embodiments, the disease or condition is coronary artery disease. In some embodiments, the subject is low-density lipoprotein receptor (LDLR) deficiency. In some embodiments, the subject has heterozygous familial hypercholesterolemia (HeFH), homozygous familial hypercholesterolemia (HoFH), or clinical atherosclerotic cardiovascular disease (ASCVD). In some embodiments, the subject is at high risk of cardiovascular events. In some embodiments, the subject requires further reduction of low-density lipoprotein cholesterol (LDL-C) despite maximum tolerated lipid-lowering therapy.

[0022]

[0015] In one embodiment, a method for delivering a nucleic acid molecular entity to a target liver is provided herein, comprising the step of administering a pharmaceutical composition described herein to the target, thereby delivering a nucleic acid molecular entity.

[0023]

[0016] In one embodiment, the nucleotide conjugate described herein comprises (a) a nucleic acid and (b) a targeting moiety attached to the nucleic acid of (a), having the structure of Table 1. In some embodiments, the targeting moiety further comprises a coupling sequence that hybridizes with the nucleic acid of (a). In one embodiment, the nucleotide conjugate described herein comprises (a) a nucleic acid and (b) a targeting moiety attached to the nucleic acid of (a), comprising a coupling sequence that hybridizes with the nucleic acid of (a). In some embodiments, the nucleic acid comprises a single-stranded, double-stranded, partially double-stranded, or hairpin stem-loop nucleic acid, and the targeting moiety is a receptor targeting moiety. In some embodiments, the targeting moiety binds to a lectin. In some embodiments, the lectin is an asialoglycoprotein receptor (ASGPR). In some embodiments, the targeting moiety comprises one or more N-acetylgalactosamines (GalNAc) or GalNAc derivatives. In some embodiments, the targeting moiety comprises one or more N-acetylgalactosamine (GalNAc) or GalNAc derivatives and a spacer. In some embodiments, the targeting moiety comprises one or more galactose or galactose derivatives. In some embodiments, the targeting moiety comprises one or more galactose or galactose derivatives and a spacer. In some embodiments, the spacer is polyethylene glycol, substituted or unsubstituted C1-C 12The polyethylene glycol comprises alkylene, or both, and has 1 to 5 repeating units. In some embodiments, the targeting moiety is bonded to one or more strands of nucleic acid via one or more linkers. In some embodiments, the targeting moiety includes the structure shown in Table 1. In some embodiments, the coupling sequence hybridizes with the nucleic acid of (a). In some embodiments, the coupling sequence hybridizes with the extension of the nucleic acid of (a). In some embodiments, the targeting moiety is bonded to the 5' end, the 3' end, or the middle of the nucleic acid sequence. In some embodiments, the targeting moiety comprises at least two GalNAc or GalNAc derivatives. In some embodiments, the targeting moiety comprises at least three GalNAc or GalNAc derivatives. In some embodiments, the GalNAc or GalNAc derivatives are bonded to the nucleic acid of (a) via linkers in the targeting moiety, via hybridization of the coupling sequence in the targeting moiety that hybridizes with the nucleic acid of (a), or via a combination thereof. In some embodiments, the targeting moiety comprises at least two galactose or galactose derivatives. In some embodiments, the targeting moiety comprises at least three galactose or galactose derivatives. In some embodiments, the galactose or galactose derivative is linked to the nucleic acid of (a) via a linker in the targeting moiety, via hybridization of coupling sequences of the targeting moiety that hybridize with the nucleic acid of (a), or via a combination thereof. In some embodiments, the targeting moiety comprises at least two coupling sequences that hybridize with the nucleic acid of (a). In some embodiments, at least two coupling sequences are identical. In some embodiments, at least two coupling sequences are different. In some embodiments, the nucleotide conjugate further comprises a second targeting moiety. In some embodiments, the second targeting moiety binds to an asialoglycoprotein receptor (ASGPR).In some embodiments, the second targeting moiety is bonded to one or more strands of nucleic acid via a spacer and / or via one or more linkers. In some embodiments, the second targeting moiety comprises GalNAc or a GalNAc derivative. In some embodiments, the second targeting moiety comprises at least three GalNAc moieties or GalNAc derivatives. In some embodiments, the GalNAc or GalNAc derivative is bonded to nucleic acid (a) via a linker of the targeting moiety, via hybridization of a coupling sequence of the targeting moiety that hybridizes with nucleic acid (a), or via a combination thereof. In some embodiments, the second targeting moiety comprises galactose or a galactose derivative. In some embodiments, the second targeting moiety comprises at least three galactose moieties or galactose derivatives. In some embodiments, the galactose or galactose derivative is linked to the nucleic acid (a) via a linker of the targeting moiety, via hybridization of a coupling sequence of the targeting moiety that hybridizes with the nucleic acid (a), or via a combination thereof. In some embodiments, the second targeting moiety includes the structure shown in Table 1. In some embodiments, the second targeting moiety includes a coupling sequence that hybridizes with the nucleic acid. In some embodiments, the second targeting moiety is linked to the 5' end of the nucleic acid, the 3' end of the nucleic acid, or the middle of the nucleic acid. In some embodiments, the nucleic acid (a) includes RNA or DNA. In some embodiments, the coupling sequence includes RNA, DNA, chemically modified RNA, chemically modified DNA, or a DNA-RNA hybrid.In some embodiments, the coupling sequence comprises one or more of (a), (c), (g), (u), (A)n, (T)n, (U)n, (a)n, or (u)n, where n is an integer greater than or equal to 3, a is 2'-O-methyladenosine (2'-OMe A), c is 2'-O-methylacythidine (2'-OMe-C), g is 2'-O-methylacythidineguanine (2'-OMe-G), and u is 2'-O-methyluridine (2'-OMe-U). In some embodiments, the coupling sequence comprises one or more of (a), (c), (g), (u), (A)n, (T)n, (U)n, (a)n, or (u)n, where n is an integer greater than or equal to 3, a is 2'-O-methyladenosine (2'-OMe A), c is 2'-O-methylacythidine (2'-OMe-C), g is 2'-O-methylacythidineguanine (2'-OMe-G), and u is 2'-O-methyluridine (2'-OMe-U). In some embodiments, (a), (c), (g), or (u) are scattered along the nucleic acid or coupling sequence. In some embodiments, the nucleic acid and coupling sequence contain one or more GC base pairs within the hybridization double helix, the coupling sequence hybridizes with the nucleic acid, and the one or more GC base pairs enhance the stability of the hybridization double helix. In some embodiments, the linker includes a covalent linker. In some embodiments, the linker includes a non-covalent linker. In some embodiments, the linker includes a monovalent linker, a divalent linker, a trivalent linker, or a combination thereof. In some embodiments, the linker includes a biocleavable linker. In some embodiments, the linker includes a non-biodegradable linker. In some embodiments, the linker includes a phosphate, phosphorothioate, amide, ether, oxime, hydrazine, or carbamate. In some embodiments, the linker is a phosphate or phosphorothioate. In some embodiments, the nucleic acid of (a) includes chemical modifications.In some embodiments, the nucleic acid of (a) includes 2'-F modification, phosphorothioate internucleotide linkage modification, acyclic nucleotide, LNA, HNA, CeNA, 2'-methoxyethyl, 2'-O-methyl, 2'-O-allyl, 2'-C-allyl, 2'-deoxy, 2'-fluoro, 2'-ON-methylacetamide (2'-O-NMA), 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE), 2'-O-aminopropyl (2'-O-AP), 4'-O-methyl, or 2'-ara-F modification. In some embodiments, the nucleic acid includes 2'-O-methyl modification. In some embodiments, the nucleic acid includes phosphorothioate internucleotide linkage modification. In some embodiments, the nucleic acid may hybridize with a target sequence within a target gene in the genome. In some embodiments, the nucleic acid includes mRNA, siRNA, shRNA, antisense oligonucleotides, microRNA, anti-microRNA or antimir, supermir, antagomir, ribozymes, triple-stranding oligonucleotides, decoy oligonucleotides, splice-switching oligonucleotides, immunostimulatory oligonucleotides, RNA activators, UI adapters, guide RNA, or any combination thereof. In some embodiments, the nucleic acid encodes a protein. In some embodiments, the nucleic acid is a CRISPR enzyme. In some embodiments, the nucleic acid is a guide RNA that can form a complex with a CRISPR enzyme. In some embodiments, the guide RNA is a single guide RNA or a dual guide RNA. In some embodiments, the CRISPR enzyme is selected from the group consisting of Cas9, Cpf1, CasX, CasY, C2c1, C2c3, and base-editing factor (base editor) fusion proteins. In some embodiments, the nucleic acid further includes mRNA encoding a CRISPR enzyme. In some embodiments, the CRISPR enzyme causes a change in a target sequence. In some embodiments, the target gene is involved in a lipid metabolic pathway.In some embodiments, the target gene is selected from the group consisting of PCSK9, ANGPTL3, APOC3, LPA, APOB, MTP, ANGPTL4, ANGPTL8, APOA5, APOE, LDLR, IDOL, NPC1L1, ASGR1, TM6SF2, GALNT2, GCKR, LPL, MLXIPL, SORT1, TRIB1, MARC1, ABCG5, and ABCG8. In some embodiments, the guide RNA includes a sequence selected from the sequences in Table 3. In some embodiments, the guide RNA includes a sequence selected from the sequences in Table 5.

[0024]

[0017] In one embodiment, what is described herein are particles comprising a described nucleotide conjugate and a described CRISPR enzyme. In some embodiments, the particles are lipid nanoparticles, liposomes, inorganic nanoparticles, or RNPs.

[0025]

[0018] In one embodiment, what is described herein is a cell comprising a nucleotide conjugate as described in any one of the preceding claims. In some embodiments, the cell is a prokaryotic cell, a eukaryotic cell, a vertebrate cell, a mouse cell, a non-human primate cell, or a human cell.

[0026]

[0019] In one embodiment, what is described herein is a pharmaceutical composition comprising a nucleotide conjugate, particle, or cell described herein. In some embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable adjuvant, diluent, carrier, preservative, excipient, buffer, stabilizer, or a combination thereof. In some embodiments, the carrier comprises a solvent, dispersion medium, dispersion or suspension aid, surfactant, isotonic agent, thickener or emulsifier, preservative, lipid, lipidoid, polymer, lipoplex, core-shell nanoparticle, hyaluronidase, nanoparticle mimetic, or a combination thereof.

[0027]

[0020] In one embodiment, a kit comprising the nucleotide conjugate described herein is described.

[0021] In one embodiment, a method for reducing the risk of coronary artery disease in a subject requiring reduction of the risk of coronary artery disease is described herein, comprising the step of administering an effective amount of the nucleotide conjugate described herein to the subject. In one embodiment, a method for reducing the risk of coronary artery disease in a subject requiring reduction of the risk of coronary artery disease is described herein, comprising the step of administering an effective amount of a pharmaceutical composition to the subject, wherein the pharmaceutical composition comprises (a) a nucleic acid and (b) a targeting moiety connected to the nucleic acid of (a), comprising the structure of Table 1. In one embodiment, a method for delivering a nucleic acid to the liver of a subject is described herein, comprising the step of administering to the subject the nucleic acid connected to a targeting moiety comprising the structure of Table 1. In some embodiments, the targeting moiety further comprises a coupling sequence that hybridizes with the nucleic acid of (a). In one embodiment, a method for reducing the risk of coronary artery disease in a subject requiring reduction of the risk of coronary artery disease is described herein, comprising the step of administering an effective amount of a pharmaceutical composition to the subject, wherein the pharmaceutical composition comprises (a) a nucleic acid and (b) a targeting moiety attached to the nucleic acid of (a), the targeting moiety comprising a coupling sequence that hybridizes with the nucleic acid of (a). In one embodiment, a method for delivering a nucleic acid to the liver of a subject is described herein, the method comprising the step of administering the nucleic acid attached to a targeting moiety comprising a coupling sequence that hybridizes with the nucleic acid. In some embodiments, the nucleic acid comprises a single-stranded, double-stranded, partially double-stranded, or hairpin stem-loop nucleic acid, wherein the targeting moiety is a receptor targeting moiety. In some embodiments, the targeting moiety binds to a lectin. In some embodiments, the lectin is an asialoglycoprotein receptor (ASGPR). In some embodiments, the targeting moiety comprises one or more N-acetylgalactosamines (GalNAc) or GalNAc derivatives. In some embodiments, the targeting portion includes at least three GalNAc or GalNAc derivatives.In some embodiments, the targeting moiety comprises one or more galactoses or galactose derivatives. In some embodiments, the targeting moiety comprises at least three galactoses or galactose derivatives. In some embodiments, the nucleic acid comprises (i) a guide RNA and a nuclease mRNA, or (ii) a guide RNA complexed with a nuclease RNP, the guide RNA being capable of guiding the nuclease to a target sequence within the target gene. In some embodiments, the guide RNA comprises a single guide RNA or a dual guide RNA. In some embodiments, the nuclease is a CRISPR enzyme. In some embodiments, the CRISPR enzyme is selected from the group consisting of Cas9, Cpf1, CasX, CasY, C2c1, C2c3, and base-editing factor (base editor) fusion proteins. In some embodiments, the CRISPR enzyme causes an alteration of the target sequence. In some embodiments, administration results in a decrease in the expression of the target gene in the liver of the subject. In some embodiments, the expression of the target gene in the liver of the subject is reduced by at least 1%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more than 99.99% compared to the control tissue of the subject. In some embodiments, the target gene is associated with coronary artery disease. In some embodiments, the target gene is selected from the group consisting of PCSK9, ANGPTL3, APOC3, LPA, APOB, MTP, ANGPTL4, ANGPTL8, APOA5, APOE, LDLR, IDOL, NPC1L1, ASGR1, TM6SF2, GALNT2, GCKR, LPL, MLXIPL, SORT1, TRIB1, MARC1, ABCG5, and ABCG8. In some embodiments, the coupling sequence includes RNA, DNA, chemically modified RNA, chemically modified DNA, or a DNA-RNA hybrid.In some embodiments, the coupling sequence comprises (A)n, (T)n, (U)n, (a)n, or (u)n, where n is an integer greater than or equal to 3, a is 2'-O-methyladenosine (2'-OMe A), and u is 2'-O-methyluridine (2'-OMe U). In some embodiments, the nucleic acid of (a) comprises (A)n, (T)n, (U)n, (a)n, or (u)n, where n is an integer greater than or equal to 3, a is 2'-O-methyladenosine, and u is 2'-O-methyluridine. In some embodiments, the targeting moiety is linked to the nucleic acid of (a) via a linker of the targeting moiety, via hybridization of the coupling sequence of the targeting moiety that hybridizes with the nucleic acid of (a), or via a combination thereof. In some embodiments, the linker comprises a covalent linker. In some embodiments, the linker comprises a phosphate, phosphorothioate, amide, ether, oxime, hydrazine, or carbamate. In some embodiments, the linker is a phosphate or phosphorothioate. In some embodiments, the nucleic acid of (a) comprises chemical modifications. In some embodiments, the nucleic acid of (a) comprises 2'-F modifications, phosphorothioate internucleotide bond modifications, acyclic nucleotides, LNA, HNA, CeNA, 2'-methoxyethyl, 2'-O-methyl, 2'-O-allyl, 2'-C-allyl, 2'-deoxy, 2'-fluoro, 2'-ON-methylacetamide (2'-O-NMA), 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE), 2'-O-aminopropyl (2'-O-AP), or 2'-ara-F modifications. In some embodiments, the nucleic acid comprises 2'-O-methyl modifications. In some embodiments, the nucleic acid comprises phosphorothioate internucleotide bond modifications. In some embodiments, the levels of nucleic acids in the liver of the subject are at least 1.5 times, at least 2 times, at least 2.5 times, at least 3 times, at least 5 times, at least 10 times, at least 15 times, at least 20 times, at least 30 times, at least 40 times, and at least 50 times higher than in other tissues of the subject at least 1 hour, 2 hours, 6 hours, 12 hours, 24 hours, 2 days, 1 week, 2 weeks, 3 weeks, 6 weeks, or 8 weeks after delivery.In some embodiments, the effective dose is approximately 1 mg / kg to approximately 10 mg / kg. In some embodiments, the administration results in a reduction of blood triglycerides and / or low-density lipoprotein cholesterol in subjects requiring a reduction of blood triglycerides and / or low-density lipoprotein cholesterol. In some embodiments, the administration is performed intravenously, intrathecally, intramuscularly, intraventricularly, intracerebrally, intracerebellarly, intraventricularly, intraparenchymal tissue, subcutaneously, or a combination thereof.

[0028]

[0022] In one embodiment, the present invention describes a method for reducing the risk of coronary artery disease in a subject requiring reduction of the risk of coronary artery disease, comprising the step of administering to the subject a pharmaceutical composition comprising (a) (i) a single guide RNA and nuclease mRNA, (ii) a dual guide RNA and nuclease mRNA, (iii) a single guide RNA and RNP, or (iv) a dual guide RNA and RNP, and (b) an asialoglycoprotein receptor (ASGPR) targeting moiety attached to the nucleic acid of (a), wherein the single guide RNA or dual guide RNA comprises four or more 2'-O-methyl modifications and two or more phosphorothioate nucleotide interbondings, the targeting moiety comprises the structure of Table 1, and the guide RNA hybridizes with the PCSK9 gene. In one embodiment, a method is provided for reducing the risk of coronary artery disease in a subject requiring reduction of the risk of coronary artery disease, comprising the step of administering to the subject a pharmaceutical composition comprising (a) (i) a single guide RNA and nuclease mRNA, (ii) a dual guide RNA and nuclease mRNA, (iii) a single guide RNA and RNP, or (iv) a dual guide RNA and RNP, and (b) a targeting moiety attached to the nucleic acid of (a), wherein the single guide RNA or dual guide RNA comprises four or more 2'-O-methyl modifications and two or more phosphorothioate nucleotide interbondings, and the targeting moiety comprises a coupling sequence that hybridizes with the single guide RNA of (a), wherein the guide RNA hybridizes with the PCSK9 gene. In some embodiments, the nuclease mRNA and / or single guide RNA comprises at least one chemical modification.In some embodiments, the chemical modification is selected from the group consisting of 2'-F modification, phosphorothioate internucleotide linkage modification, acyclic nucleotide, LNA, HNA, CeNA, 2'-methoxyethyl, 2'-O-methyl, 2'-O-allyl, 2'-C-allyl, 2'-deoxy, 2'-fluoro, 2'-ON-methylacetamide (2'-O-NMA), 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE), 2'-O-aminopropyl (2'-O-AP), 4'-O-methyl, and 2'-ara-F modification. In some embodiments, administration of the nucleic acid conjugate results in a reduced level of immune response compared to a control nucleic acid conjugate without the aforementioned chemical modification.

[0029]

[0023] In one embodiment, formula (IV) is described herein

[0030] [ka]

[0031] A nucleotide conjugate containing the structure, During the ceremony, Each X is independently either H or a protecting group, and R A is -OX or -NHAc, Y is O or S, W is (a)(i) single guide RNA and nuclease mRNA, (ii) dual guide RNA and nuclease mRNA, (iii) single guide RNA and RNP, or (iv) dual guide RNA and RNP; or (b) Coupling sequence It represents.

[0032]

[0024] In some embodiments, one or more linkers include structures selected from the group consisting of:

[0033] [ka]

[0034] In some embodiments, each linker is independently -(L 1 ) k1 -(L 2 ) k2 -(L 3 ) k3 -(L 4 ) k4 - has the structure, where each of k1, k2, k3, and k4 is independently 0, 1, or 2, L 1 , L 2 , L 3 and L 4 Each of these is independent of -O-, -S-, and S(=O). 1~2 -, -C(=O)-, -C(=S)-, -NR L -, -OC(=O)-, -C(=O)O-, -OC(=O)O-, -C(=O)NR L -, -OC(=O)NR L -, -NR L C(=O)-, -NR L C(=O)NR L -, -P(=O)R L -, -NR L S(=O)(=NR L )-, -NR L S(=O)2-, -S(=O)2NR L -, -N=N-, -(CH2-CH2-O) 1~6 -, linear or branched C 1~6 Alkylene, linear or branched C 2~6 Alkenylene, linear or branched C 2~6 Alkynylene, C3-C8 cycloalkylene, C2-C7 heterocycloalkylene, C6-C 10 Arylenes and C5-C9 heteroarylenes are selected, where the alkylene, alkenylene, alkynylene, cycloalkylene, cycloalkylene, arylene, or heteroarylene may be substituted or unsubstituted, where each R LR is independently H, D, cyano, halogen, substituted or unsubstituted C1-C6 alkyl, -CD3, -OCH3, -OCD3, substituted or unsubstituted C1-C6 haloalkyl, substituted or unsubstituted C1-C6 heteroalkyl, substituted or unsubstituted C3-C8 cycloalkyl, substituted or unsubstituted C2-C7 heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl. In some embodiments, each R L k1, k2, k3, and k4 are independently H, a substituted or unsubstituted C1-C6 alkyl group, -OCH3, a substituted or unsubstituted C1-C6 haloalkyl group, a substituted or unsubstituted C1-C6 heteroalkyl group, a substituted or unsubstituted C3-C8 cycloalkyl group, or a substituted or unsubstituted C2-C7 heterocycloalkyl group. In some embodiments, the sum of k1, k2, k3, and k4 is 1, 2, or 3.

[0035]

[0025] In one embodiment, a method for preparing a formulation comprising nanoparticles is described herein, the nanoparticles comprising (i) one or more nucleic acid molecular entities, (ii) one or more lipids selected from sterols or derivatives thereof, phospholipids, stealth lipids, and aminolipids, and (iii) a receptor targeting conjugate, the method comprising (a) preparing a first solution comprising one or more nucleic acid molecular entities; (b) preparing a second solution comprising at least one of one or more lipids; (c) mixing the first solution and the second solution to produce a mixture comprising nanoparticles comprising one or more nucleic acid molecular entities and one or more lipids; (d) mixing the receptor targeting conjugate with the nanoparticles produced in step (c); (e) incubating the nanoparticles; and (f) optionally performing a buffer exchange process. In some embodiments, the receptor targeting conjugate is combined with one or more lipids after the mixing step in (c). In some embodiments, the receptor-targeting conjugate is added to a dilution buffer, which is then mixed with pre-formed nucleic acid-lipid nanoparticles emerging from an in-line mixing chamber, thereby forming nanoparticles. In some embodiments, the dilution buffer is added to the mixture, and the diluted mixture is held for a certain period of time before the receptor-targeting conjugate is introduced. In some embodiments, the holding time is between 1 and 120 minutes. In some embodiments, the holding time is between 1 and 90 minutes, between 1 and 60 minutes, or between 10 and 40 minutes. In some embodiments, the holding time is approximately 30 minutes. In some embodiments, the receptor-targeting conjugate is introduced into the nanoparticles after buffer exchange. In some embodiments, the receptor-targeting conjugate is introduced into the nanoparticles after buffer exchange and concentration, but before storage. In some embodiments, the receptor-targeting conjugate is introduced into the nanoparticles after storage and thawing, and before administration or evaluation.In one embodiment, a method for preparing a formulation comprising nanoparticles is described herein, the nanoparticles comprising (i) one or more nucleic acid molecular entities, (ii) one or more lipids selected from sterols or derivatives thereof, phospholipids, stealth lipids, and aminolipids, and (iii) a receptor targeting conjugate, the method comprising (a) preparing a first solution comprising one or more nucleic acid molecular entities, (b) preparing a second solution comprising at least one of the one or more lipids, (c) mixing the first solution and the second solution in line to produce a mixture comprising nanoparticles comprising one or more nucleic acid molecular entities and one or more lipids, (d) mixing the receptor targeting conjugate in line with the mixture of step (c) to produce a mixture comprising nanoparticles comprising one or more nucleic acid molecular entities, one or more lipids, and the receptor targeting conjugate, (e) diluting the mixture of step (d) by adding a dilution buffer, and (f) optionally performing a buffer exchange process. In some embodiments, the in-line mixing of step (c) and the in-line mixing of step (d) are carried out sequentially. In one embodiment, a method for preparing a formulation comprising nanoparticles is described herein, the nanoparticles comprising (i) one or more nucleic acid molecular entities, (ii) one or more lipids selected from sterols or derivatives thereof, phospholipids, stealth lipids, and aminolipids, and (iii) receptor targeting conjugates, the method comprising (a) preparing a first solution comprising one or more nucleic acid molecular entities; (b) preparing a second solution comprising at least one of (i) one or more lipids and (ii) at least a portion of the receptor targeting conjugates; (c) mixing the first solution and the second solution to produce a mixture comprising nanoparticles; (d) optionally incubating the nanoparticles; and (e) optionally performing a buffer exchange process. In some embodiments, the second solution comprises all of the receptor targeting conjugates.In one embodiment, a method for preparing a formulation comprising nanoparticles is described herein, the nanoparticles comprising (i) one or more nucleic acid molecular entities, (ii) one or more lipids selected from sterols or derivatives thereof, phospholipids, stealth lipids, and aminolipids, and (iii) a receptor targeting conjugate, the method comprising (a) preparing a first solution comprising one or more nucleic acid molecular entities, (b) preparing a second solution comprising at least one of the one or more lipids, (c) mixing the first solution and the second solution to produce a mixture comprising nanoparticles comprising one or more nucleic acid molecular entities and one or more lipids, (d) combining a receptor targeting conjugate with one or more lipids, wherein at least a portion of the receptor targeting conjugate is combined with one or more lipids before or concurrently with the mixing step, (e) optionally incubating the nanoparticles, and (f) optionally performing a buffer exchange process. In some embodiments, at least a portion of the receptor-targeting conjugate is combined with one or more lipids at the same time as the mixing step. In some embodiments, at least a portion of the receptor-targeting conjugate is combined with one or more lipids before the mixing step. In some embodiments, the receptor-targeting conjugate is combined with one or more lipids in a second solution. In some embodiments, a portion of the receptor-targeting conjugate is combined with one or more lipids in a second solution, and a portion of the receptor-targeting conjugate is combined with one or more lipids after mixing. In some embodiments, a portion of the receptor-targeting conjugate is combined with one or more lipids in a second solution, and a portion of the receptor-targeting conjugate is combined with one or more lipids after the incubation step.In some embodiments, a portion of the receptor-targeting conjugate is combined with one or more lipids in a second solution, and a portion of the receptor-targeting conjugate is combined with one or more lipids after a buffer exchange step. In some embodiments, the method further includes the step of diluting the mixture produced by mixing the first and second solutions by adding a dilution buffer. In some embodiments, the mixture is diluted in line. In some embodiments, the dilution buffer contains at least a portion of the receptor-targeting conjugate. In some embodiments, the dilution buffer contains at least a portion of the stealth lipids. In some embodiments, the first solution contains an aqueous buffer. In some embodiments, the second solution contains ethanol. In some embodiments, the mixing includes laminar mixing, vortex mixing, turbulent mixing, or a combination thereof. In some embodiments, the mixing includes cross-mixing. In some embodiments, the mixing includes in-line mixing. In some embodiments, mixing includes the step of introducing at least a portion of the first solution through a first inlet channel and at least a portion of the second solution through a second inlet channel, wherein the angle between the first and second inlet channels is about 15 to 180 degrees. In some embodiments, mixing includes the step of introducing a portion of the first solution through a third inlet channel. In some embodiments, buffer exchange includes dialysis, chromatography, or tangential flow filtration (TFF). In some embodiments, this method further includes a filtration step. In some embodiments, the receptor targeting conjugate comprises one or more N-acetylgalactosamines (GalNAc) or GalNAc derivatives. In some embodiments, the receptor targeting conjugate comprises one or more galactoses or galactose derivatives. In some embodiments, the receptor targeting conjugate is selected from Table 4. In some embodiments, the receptor targeting conjugate is a targeting conjugate described herein. In some embodiments, the nanoparticles comprise the first nanoparticle composition described herein.In some embodiments, the formulation is a pharmaceutical composition described herein.

[0036]

[0026] In one embodiment, a pharmaceutical composition comprising nanoparticles is described herein, the nanoparticles comprising (i) one or more nucleic acid molecular entities, (ii) one or more lipids selected from sterols or derivatives thereof, phospholipids, stealth lipids, and aminolipids, and (iii) a receptor-targeting conjugate, the formulation being prepared by the method described herein. In one embodiment, a pharmaceutical composition comprising nanoparticles is described herein, the nanoparticles comprising (i) one or more nucleic acid molecular entities, (ii) one or more lipids selected from sterols or derivatives thereof, phospholipids, stealth lipids, and aminolipids, and (iii) a receptor targeting conjugate, the formulation being prepared by a method comprising: (a) preparing a first solution comprising one or more nucleic acid molecular entities; (b) preparing a second solution comprising at least one of the one or more lipids; (c) mixing the first solution and the second solution to produce a mixture comprising nanoparticles comprising one or more nucleic acid molecular entities and one or more lipids; (d) mixing the receptor targeting conjugate with the nanoparticles produced in step (c); (e) incubating the nanoparticles; and (f) optionally performing a buffer exchange process.In one embodiment, a pharmaceutical composition comprising nanoparticles is described herein, the nanoparticles comprising (i) one or more nucleic acid molecular entities, (ii) one or more lipids selected from sterols or derivatives thereof, phospholipids, stealth lipids, and aminolipids, and (iii) a receptor targeting conjugate, the formulation being prepared by a method comprising: (a) preparing a first solution comprising one or more nucleic acid molecular entities; (b) preparing a second solution comprising at least one of the one or more lipids; (c) mixing the first solution and the second solution to produce a mixture comprising nanoparticles comprising one or more nucleic acid molecular entities and one or more lipids; (d) combining the receptor targeting conjugate with one or more lipids, wherein at least a portion of the receptor targeting conjugate is combined with one or more lipids before or concurrently with the mixing step; (e) optionally incubating the nanoparticles; and (f) optionally performing a buffer exchange process.In one embodiment, a pharmaceutical composition comprising nanoparticles is described herein, the nanoparticles comprising (i) one or more nucleic acid molecular entities, (ii) one or more lipids selected from sterols or derivatives thereof, phospholipids, stealth lipids, and aminolipids, and (iii) a receptor targeting conjugate, wherein the formulation is prepared by a method comprising: (a) preparing a first solution comprising one or more nucleic acid molecular entities; (b) preparing a second solution comprising at least one of one or more lipids; (c) mixing the first solution and the second solution in line to produce a mixture comprising nanoparticles comprising one or more nucleic acid molecular entities and one or more lipids; (d) mixing the receptor targeting conjugate in line with the mixture of step (c) to produce a mixture comprising nanoparticles comprising one or more nucleic acid molecular entities, one or more lipids, and the receptor targeting conjugate; (e) diluting the mixture of step (d) by adding a dilution buffer; and (f) optionally performing a buffer exchange process. In one embodiment, a pharmaceutical composition comprising nanoparticles is described herein, wherein the nanoparticles comprise (i) one or more nucleic acid molecular entities, (ii) one or more lipids selected from sterols or derivatives thereof, phospholipids, stealth lipids, and aminolipids, and (iii) a receptor targeting conjugate, wherein the formulation is prepared by a method comprising: (a) preparing a first solution comprising one or more nucleic acid molecular entities; (b) preparing a second solution comprising at least one of (i) one or more lipids and (ii) at least a portion of the receptor targeting conjugate; (c) mixing the first solution and the second solution to produce a mixture comprising nanoparticles; (d) optionally incubating the nanoparticles; and optionally performing a buffer exchange process.

[0037] Embedding by reference

[0027] All publications, references, patents, and patent applications referenced herein are incorporated by reference to the same extent as if each individual publication, patent, or patent application were specifically and individually indicated as being incorporated by reference. In the event of any inconsistency in usage between this document and any document incorporated by reference, the usage in the incorporated reference should be deemed to supplement the usage in this document. In the event of any irreconcilable inconsistency, the usage herein shall prevail.

[0038]

[0028] Novel features of the present invention are described in detail in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by referring to the following detailed description illustrating exemplary embodiments in which the principles of the present invention are utilized, and to the appended drawings. [Brief explanation of the drawing]

[0039] [Figure 1]

[0029] Figures 1A and 1B show HPLC chromatograms of GalNAc lipid incorporation of the compositions of this specification. Figure 1A shows a reference LNP in which GalNAc lipids are absent, and Figure 1B shows an LNP composed of GalNAc lipids. [Figure 2]

[0030] The in vitro PCSK9 gene editing efficiency of the LNP formulations in the compositions of this specification in primary human hepatocytes is shown. [Figure 3]

[0031] This shows PCSK9 gene editing in the livers of wild-type, LDLr- / -, and ApoE- / - mice after post-orbital administration of the LNP composition herein containing SpCas9 mRNA and PCSK9 gRNA in a 1:1 ratio. [Figure 4]

[0032] This shows ANGPTL3 gene editing in LDLr- / - mouse liver after post-orbital administration of the LNP composition herein containing ABE mRNA and ANGPTL3 gRNA in a 1:1 ratio. [Figure 5]

[0033] This shows PCSK9 gene editing in wild-type and LDLr- / - mouse livers after post-orbital administration of an LNP containing ABE mRNA and PCSK9 gRNA in a 1:1 ratio. [Figure 6]

[0034] This shows PCSK9 gene editing in wild-type female mouse hepatocytes after post-orbital administration of the LNP composition of this specification. [Figure 7]

[0035] This shows PCSK9 gene editing in wild-type female mouse hepatocytes after post-orbital administration of the LNP composition described herein. [Figure 8]

[0036] This shows PCSK9 editing in LDLR- / - female mouse hepatocytes after post-orbital administration of the LNP composition herein containing Cas9 mRNA and gRNA. [Figure 9]

[0037] Four common processes for introducing GalNAc-lipids into lipid nanoparticles are shown. [Figure 10]

[0038] Three protocols for preparing lipid nanoparticles, including post-addition of GalNAc-lipids, are shown. [Figure 11]

[0039] Three protocols for preparing lipid nanoparticles, including post-addition of GalNAc-lipids, are shown. [Figure 12]

[0040] Three protocols for preparing lipid nanoparticles are presented, including the addition of GalNAc-lipids to LNP excipients and the division and addition of GalNAc-lipids. [Figure 13]

[0041] Two protocols for preparing lipid nanoparticles are presented, including the addition of GalNAc-lipids to LNP excipients and the division and addition of GalNAc-lipids. [Figure 14]

[0042] Two protocols for preparing lipid nanoparticles, including cross-mixing of GalNAc-lipids, are presented. [Figure 15]

[0043] This document demonstrates PCSK9 editing in LDLR- / - female mouse hepatocytes after post-orbital administration of the LNP composition described herein, which contains PCSK9 ABE mRNA and guide RNA in a 1:1 ratio. [Modes for carrying out the invention]

[0040]

[0044] To provide a complete understanding of the various embodiments, certain details of this description are included. However, those skilled in the art will understand that this disclosure may be carried out without these details. In other examples, well-known structures and / or methods are described in detail, or not, to avoid unnecessarily obscuring the description of embodiments. Unless otherwise required by context, throughout the entire specification and claims, the words and their variations such as “comprise,” “comprises,” and “comprising” should be interpreted in an open and comprehensive sense, i.e., “including, but not limited to.” Furthermore, the headings provided herein are for convenience only and do not construe as defining the scope or meaning of the claimed disclosure. The section headings used herein are for structural purposes only and should not be interpreted as limiting the subject matter described.

[0041]

[0045] Efficient delivery to cells requires specific targeting and substantial protection from the extracellular environment, particularly serum proteins. One way to achieve specific targeting is to conjugate a targeting moiety to a pharmaceutical effector, such as an activator or nucleotide agent, thereby directing the activator or nucleotide effector to a specific cell or tissue, depending on the specificity of the targeting moiety. One way the targeting moiety can improve delivery is through receptor-mediated endocytosis activity. In some cases, this uptake mechanism may involve the movement of a membrane receptor-bound nucleotide agent into the enclosed region via membrane invagination of membrane structures or fusion of the delivery system with the cell membrane. This process is initiated via activation of the cell surface or membrane receptor following binding of a specific ligand to the receptor. Many receptor-mediated endocytosis systems are known and have been studied, including systems that recognize sugars such as galactose, mannose, and mannose-6-phosphate, transferrin, asialoglycoprotein, vitamin B12, insulin, and peptides and proteins such as epidermal growth factor (EGF). Lipophilic moieties, such as cholesterol or fatty acids, can significantly enhance plasma protein binding and consequently prolong the circulating half-life when they bind to highly hydrophilic molecules, such as nucleic acids. Lipophilic conjugates may be used in combination with targeting ligands to improve intracellular transport in targeted delivery approaches.

[0042]

[0046] The asialoglycoprotein receptor (ASGP-R) is a high-capacity receptor that is very abundant on hepatocytes. ASGP-R exhibits 50-fold higher affinity for N-acetyl-D-galactosylamine (GalNAc) than for D-Gal. Previous studies have shown that while the spacing between sugars is important, polyvalence is necessary to achieve high affinity. Herein, we recognize a clear need for novel receptor-specific ligand conjugate RNA or DNA agents and methods for preparing them to address the shortcomings of in vivo delivery of therapeutic agents using the above-mentioned nucleic acids or nucleic acid-related complexes. This disclosure is directed toward this very important objective.

[0043]

[0047] As used herein and in the appended claims, the singular forms “a,” “an,” and “the” refer to multiple objects unless the context explicitly indicates otherwise. It should also be noted that the term “or” is generally used to mean “and / or” unless the context explicitly indicates otherwise.

[0044]

[0048] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art in which this disclosure pertains. Methods and materials similar to or equivalent to those described herein may be used in the implementation or testing of this disclosure, but appropriate methods and materials are described below. All references cited herein are incorporated in their entirety by reference as if they were fully cited. Singleton et al., Dictionary of Microbiology and Molecular Biology, 3rd Edition, J. Wiley & Sons (New York, NY 2001); MarcH, Advanced Organic Chemistry Reactions, Mechanisms and Structure, 5th Edition, J. Wiley & Sons (New York, NY 2001); and Sambrook and Russel, Molecular Cloning: A Laboratory Manual, 3rd Edition, Cold Spring Harbor Laboratory Press (Cold Spring Harbor, NY 2001) provide a general guide to many of the terms used in this application for those skilled in the art.

[0045] Specific definition

[0049] When indicating the number of substituents, the term "one or more" refers to a range from one substituent to the maximum number of substitutions possible, for example, from the substitution of one hydrogen to the substitution of all hydrogens by substituents.

[0046]

[0050] The terms "optional" or "at will" mean that the event or situation described thereafter does not have to occur, but the description includes the cases in which the event or situation may or may not occur.

[0047]

[0051] The term "nucleic acid molecular entity" is used interchangeably with "nucleic acid."

[0052] As used herein, the term “nucleic acid” generally refers to one or more nucleic acid bases, nucleosides, or nucleotides, and this term includes polynucleic acid bases, polynucleosides, and polynucleotides. Nucleic acids may include polynucleotides, mononucleotides, and oligonucleotides. Nucleic acids may comprise DNA, RNA, or mixtures thereof, and may be single-stranded, double-stranded, or partially single-stranded or double-stranded, and may form secondary structures. In some embodiments, nucleic acids have multiple double-stranded and single-stranded segments. For example, nucleic acids may comprise polynucleotides, such as mRNA having multiple double-stranded segments. DNA may be in the form of, for example, antisense molecules, plasmid DNA, pre-condensed DNA, PCR products, vectors, expression cassettes, chimeric sequences, chromosomal DNA, or derivatives and combinations thereof. RNA may be in the form of siRNA, asymmetric interfering RNA (aiRNA), microRNA (miRNA), mRNA, tRNA, rRNA, tRNA, viral RNA (vRNA), CRISPR RNA, base-editing factor RNA, and combinations thereof. Nucleic acids include synthetic, natural, and unnatural nucleic acids that contain known nucleotide analogs or modified backbone residues or bindings that have similar binding properties to the reference nucleic acid. Examples of such analogs include, but are not limited to, phosphorothioates, phosphoramides, methylphosphonates, chiral-methylphosphonates, 2'-O-methylribonucleotides, and peptide-nucleic acids (PNAs). Unless otherwise specified, this term encompasses nucleic acids that contain known analogs of natural nucleotides that have similar binding properties to the reference nucleic acid. Unless otherwise specified, a particular nucleic acid sequence implicitly includes not only the explicitly stated sequence but also its conservatively modified variants (e.g., degenerate codon substitutions), alleles, orthologues, SNPs, and complementary sequences. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with a mixed base and / or deoxyinosine residue.(Batzer et al., Nucleic Acid Res., 19:5081 (1991); Ohtsuka et al., J Biol. Chem., 260:2605-2608 (1985); Rossolini et al., Mal. Cell. Probes, 8:91-98 (1994)). A "nucleotide" is defined as a substituted and / or unsubstituted sugar deoxyribose (DNA), or a substituted and / or unsubstituted sugar ribose (RNA), or a substituted and / or unsubstituted carbon ring, or a substituted and / or unsubstituted acyclic moiety (glycolic nucleic acid, e.g.), a base, and a phosphate group. Nucleotides are linked together via phosphate groups. "Bases" include purines and pyrimidines, which further include, but are not limited to, the natural compounds adenine, thymine, guanine, cytosine, uracil, and inosine, as well as natural analogs and synthetic derivatives of purines and pyrimidines, and modifications that introduce new reactive groups such as amines, alcohols, thiols, carboxylates, and alkyl halides.

[0048]

[0053] The term "gene" refers to a nucleic acid (e.g., DNA or RNA) sequence that contains a partial or full-length coding sequence necessary for the production of a polypeptide or precursor polypeptide.

[0054] As used herein, "gene product" refers to the product of a gene, such as an RNA transcript or polypeptide.

[0049]

[0055] As used herein, the term “polynucleotide” generally refers to a molecule containing two or more bonded nucleic acid subunits, such as nucleotides, and may be used interchangeably with “oligonucleotide.” For example, a polynucleotide may contain one or more nucleotides selected from adenosine (A), cytosine (C), guanine (G), thymine (T), and uracil (U), or their variants. A nucleotide generally contains a nucleoside and at least one, two, three, four, five, six, seven, eight, nine, ten, or more phosphate (PO3) groups. Examples of nucleotides include a nucleic acid base, a pentose (either ribose or deoxyribose), and one or more phosphate groups. Examples of ribonucleotides include nucleotides in which the sugar is ribose. Examples of deoxyribonucleotides include nucleotides in which the sugar is deoxyribose. A nucleotide may be a nucleoside monophosphate, a nucleoside diphosphate, a nucleoside triphosphate, or a nucleoside polyphosphate. For example, the nucleotide may be a deoxyribonucleoside polyphosphate, such as a deoxyribonucleoside triphosphate (dNTP), and exemplary dNTPs include deoxyadenosine triphosphate (dATP), deoxycytidine triphosphate (dCTP), deoxyguanosine triphosphate (dGTP), uridine triphosphate (dUTP), and deoxythymidine triphosphate (dTTP). The dNTP may also include a detectable tag, such as a luminescent tag or marker (e.g., a fluorophore). For example, the nucleotide may be a purine (e.g., A or G, or a variant thereof) or a pyrimidine (e.g., C, T or U, or a variant thereof). In some examples, the polynucleotide is deoxyribonucleic acid (DNA), ribonucleic acid (RNA), or a derivative or variant thereof.Examples of polynucleotides include, but are not limited to, short interfering RNA (siRNA), microRNA (miRNA), plasmid DNA (pDNA), short hairpin RNA (shRNA), small nuclear RNA (snRNA), messenger RNA (mRNA), precursor mRNA (premRNA), antisense RNA (asRNA), and heteronuclear RNA (hnRNA), encompassing both nucleotide sequences and any structural embodiments thereof, such as single-stranded, double-stranded, triple-stranded, helical, hairpin, stem-loop, and bulge. In some cases, polynucleotides are circular. Polynucleotides can have a variety of lengths. For example, polynucleotides can have lengths of at least approximately 7, 8, 9, 10, 20, 30, 40, 50, 100, 200, 300, 400, 500 bases, 1 kilobase (kb), 2kb, 3kb, 4kb, 5kb, 10kb, 50kb, or more. Polynucleotides can be isolated from cells or tissues. For example, polynucleotide sequences may include isolated and purified DNA / RNA molecules, synthetic DNA / RNA molecules, and / or synthetic DNA / RNA analogs.

[0050]

[0056] Polynucleotides may contain one or more nucleotide variants, including non-standard nucleotides, non-natural nucleotides, nucleotide analogs, and / or modified nucleotides. Examples of modified nucleotides, but not limited to, include diaminopurine, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosyl quosine, inosine, N6-isopentenyl adenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, and 7-methyl Examples include tylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylquosin, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid(v), weybutoxosin, pseudouracil, quosin, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methyl ester, 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl)uracil, (acp3)w, and 2,6-diaminopurine. In some cases, nucleotides may include modifications to their phosphate moieties, including modifications to the triphosphate moiety. Non-limiting examples of such modifications include modifications with longer phosphate chains (e.g., phosphate chains with 4, 5, 6, 7, 8, 9, 10 or more phosphate moieties) and modifications with thiol moieties (e.g., α-thiotriphosphate and beta-thiotriphosphate). Nucleic acid molecules may also be modified with base moieties (e.g., one or more atoms that can typically form hydrogen bonds with complementary nucleotides and / or one or more atoms that cannot typically form hydrogen bonds with complementary nucleotides), sugar moieties, or phosphate backbone.Nucleic acid molecules may also contain amine-modifying groups such as aminoallyl 1-dUTP (aa-dUTP) and aminohexylacrylamide-dCTP (aha-dCTP), enabling covalent bonding of amine-reactive moieties such as N-hydroxysuccinimide esters (NHS). Substitutions of standard DNA or RNA base pairs in oligonucleotides of this disclosure may result in higher bit density per cubic mm, greater safety (resistance to accidental or intentional synthesis of natural toxins), easier identification in photoprogrammed polymerases, or lower secondary structures. Such alternative base pairs compatible with native and mutant polymerases for de novo and / or amplification synthesis are described in Betz K, Malyshev Da, Lavergne T, Welte W, Diederichs K, Dwyer TJ, Ordoukhanian P, Romesberg FE, Marx A. Nat. Chem. Biol. 2012 Jul;8(7):612-4, which are incorporated herein by reference for all purposes.

[0051]

[0057] As used herein, the terms “polypeptide,” “protein,” and “peptide” are interchangeable and refer to polymers of amino acid residues linked via peptide bonds, which may consist of two or more polypeptide chains. The terms “polypeptide,” “protein,” and “peptide” refer to polymers of at least two amino acid monomers linked together via amide bonds. The amino acids may be L-optical isomers or D-optical isomers. More specifically, the terms “polypeptide,” “protein,” and “peptide” refer to molecules consisting of two or more amino acids in a specific order, for example, in the order determined by the base sequence of nucleotides of a protein-coding gene or RNA. Proteins are essential for the structure, function, and regulation of the body’s cells, tissues, and organs, and each protein has its own function. Examples include hormones, enzymes, antibodies, and any fragments thereof. In some cases, a protein may be a part of a protein, e.g., a protein domain, subdomain, or motif. In some cases, a protein may be a variant (or mutation) of a protein in which one or more amino acid residues are inserted, deleted, and / or substituted into the amino acid sequence of a naturally occurring (or at least known) protein. The protein or its variant may be natural or recombinant.

[0052]

[0058] As used herein, the terms “intercalate” or “intercalation” refer to the action of a substance (e.g., a small molecule) that enters between consecutive bases of DNA. In some cases, intercalation can interfere with the proper functioning of DNA.

[0053]

[0059] As used herein, “complement” means a sequence complementary to a nucleic acid according to the standard Watson / Crick pairing rules. The complementary sequence may also be a DNA sequence or a sequence of RNA complementary to that complementary sequence, or it may be cDNA. The complement may be fully complementary or partially complementary so that the two sequences hybridize under stringent hybridization conditions. Those skilled in the art will understand that complementary or substantially complementary sequences do not need to hybridize along their entire length. In certain embodiments, the complementary or substantially complementary sequence may include a sequence of bases located at 3' or 5' relative to a sequence of bases that hybridize to the target sequence, and which does not hybridize to the target sequence.

[0054]

[0060] As used herein, “hybridize” refers to the process by which two nucleic acid strands anneal to each other according to the Watson-Crick base pairing rules. Nucleic acid hybridization techniques are well known in the art. See, for example, Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Press, Plainview, NY. A person skilled in the art will understand how to determine the appropriate stringency of hybridization / washing conditions so that sequences with at least a desired level of complementarity will stably hybridize, while sequences with lower complementarity will not. For examples of hybridization conditions and parameters, see, for example, Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Press, Plainview, NY; Ausubel, FM et al., 1994, Current Protocols in Molecular Biology, John Wiley & Sons, Secaucus, NJ. All of these are incorporated herein by reference. In certain embodiments, hybridization may occur between nucleic acid molecules of 20 to 100 nucleotides in length. In some embodiments, hybridization is performed on at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, This can occur between 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or between 100 consecutive nucleotides.In some embodiments, the nucleic acid molecules to be hybridized may contain up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 acceptable mismatches.

[0055]

[0061] As used herein, the term “biological sample” means any biological material from which polynucleotides, polypeptides, biomarkers, and / or metabolites can be prepared and tested. Non-limiting examples include whole blood, plasma, saliva, cheek swabs, fecal specimens, urine specimens, cell clumps, or any other bodily fluids or tissues.

[0056]

[0062] As used herein, terms such as “administer,” “administering,” “administering step,” and “administration” refer to methods that may be used to enable the delivery of a compound or composition to a desired site of biological action. These methods include, but are not limited to, oral (PO), intraduodenal (ID), parenteral injection (e.g., intravenous (IV), subcutaneous (SC), intraperitoneal (IP), intramuscular (IM), intravenous or infusion (INF)), topical (TOP.), and rectal (PR) administration. Those skilled in the art will be familiar with the administration techniques that can be used with the compounds and methods described herein. In some embodiments, the compounds and compositions described herein are administered orally.

[0057]

[0063] As used herein, terms such as “co-administration” mean the administration of a selected therapeutic agent to a single patient and are intended to include therapeutic regimens in which the drugs are administered to the same patient, via different routes of administration, or simultaneously or at different times.

[0058]

[0064] As used herein, the terms “effective dose” or “therapeutic effective dose” refer to a sufficient amount of an agent or compound administered that alleviates to some extent one or more symptoms of the disease or condition being treated (e.g., reduction and / or mitigation of one or more signs, symptoms, or causes of the disease, or any other desired alteration of the biological system). For example, an “effective dose” for therapeutic use may be the amount of an agent that clinically significantly reduces one or more disease symptoms. An appropriate “effective” dose may be determined in individual cases using techniques such as dose escalation studies.

[0059]

[0065] As used herein, the terms “enhance” or “enhance” mean to increase or extend the amount, potency, or duration of a desired effect.

[0066] As used herein, “carbohydrate” refers to a compound that is a carbohydrate itself, consisting of one or more monosaccharide units having at least six carbon atoms (which may be linear, branched, or cyclic) each having an oxygen, nitrogen, or sulfur atom bonded to each carbon atom; or a compound that has as part a carbohydrate portion consisting of one or more monosaccharide units, each having at least six carbon atoms (which may be linear, branched, or cyclic) each having an oxygen, nitrogen, or sulfur atom bonded to each carbon atom. Typical carbohydrates include sugars (monosaccharides, disaccharides, trisaccharides, and oligosaccharides containing about 4 to 9 monosaccharide units), as well as polysaccharides such as starch, glycogen, cellulose, and polysaccharide gum. Specific monosaccharides include sugars with 5 or more carbon atoms (preferably C5 to C8); disaccharides and trisaccharides include sugars having two or three monosaccharide units (preferably C5 to C8).

[0060]

[0067] The term "monosaccharide" encompasses the radicals of allose, altrose, arabinose, cradinose, erythrose, erythrulose, fructose, D-fusitol, L-fusitol, fucosamine, fucose, galactosamine, D-galactosaminitol, N-acetyl-galactosamine, galactose, glucosamine, N-acetyl-glucosamine, glucosaminitol, glucose, glucose-6-phosphate growthglyceraldehyde, L-glycero-D-mannos-heprose, glycerol, glyceron, growth iodose, lyxose, mannosamine, mannose, mannose-6-phosphate, psicose, quinovose, quinovosamine, rhamnitol, rhamnosamine, rhamnose, ribose, ribulose, sedoheptulose, sorbose, tagatose, talose, tartaric acid, trose, xylose, and xylulose. Monosaccharides may be in a D configuration or an L configuration. Monosaccharides may also include deoxy sugars (where the alcoholic hydroxyl group is replaced by hydrogen), amino sugars (where the alcoholic hydroxyl group is replaced by an amino group), thio sugars (where the alcoholic hydroxyl group is replaced by a thiol, or C=O is replaced by C=S, or the ring oxygen in a cyclic form is replaced by sulfur), seleno sugars, tello sugars, aza sugars (where the ring carbon is replaced by nitrogen), imino sugars (where the ring oxygen is replaced by nitrogen), phosphano sugars (where the ring oxygen is replaced by phosphorus), phosphates (where the ring carbon is replaced by phosphorus), C-substituted monosaccharides (where the hydrogen of the non-terminal carbon atom is replaced by carbon), unsaturated monosaccharides, alditols (where the carbonyl group is replaced by a CHOH group), aldonic acids (where the aldehyde group is replaced by a carboxyl group), ketoaldonic acids, uronic acids, aldalic acids, and the like. Examples of amino sugars include amino monosaccharides, preferably galactosamine, glucamine, mannosamine, fucosamine, quinabosamine, neuraminic acid, muramic acid, lactosediamine, acosamine, basilosamine, daunosamine, desosamine, phorosamine, galosamine, canosamine, canosamine, mycaminose, myosamine, perosamine, pneumosamine, purprosamine, and rhodosmin. It is understood that monosaccharides and the like may be further substituted.

[0061]

[0068] As used herein, “N / P ratio” is the molar ratio of ionizable nitrogen atoms (e.g., in the physiological pH range) in lipids (or lipids) to phosphate groups in nucleic acid molecular entities (or nucleic acid molecular entities), for example, in nanoparticle compositions containing lipid components and RNA. Ionizable nitrogen atoms may include, for example, nitrogen atoms that can be protonated at about pH 1, about pH 2, about pH 3, about pH 4, about pH 5, about pH 6, about pH 7, about pH 7.5, or about pH 8 or higher. The physiological pH range may include, for example, the pH ranges of different cellular compartments (organs, tissues, and cells, etc.) and body fluids (blood, CSF, gastric juice, milk, bile, saliva, tears, and urine, etc.). In certain embodiments, the physiological pH range refers to the pH range of blood in mammals, for example, about 7.35 to about 7.45. In some embodiments, ionizable nitrogen atoms refer to nitrogen atoms that can be ionized within a pH range between 5 and 14.

[0062]

[0069] The terms "disaccharides," "trisaccharides," and "polysaccharides" refer to avequoise, acrabose, amicetose, amylopectin, amylose, apiose, alkanose, ascarilose, ascorbic acid, voibinose, cellobiose, cellotriose, cellulose, cacotriose, calcose, chitin, coritose, cyclodextrin, simarose, dextrin, 2-deoxyribose, 2-deoxyglucosediginose, digitalose, digitoxose, evarose, everitose, fructooligosaccharides, galtooligosaccharides, gentianose, genichiobiose, glucan, glucogen, glycogen, witch hazel, heparin, inulin, isolevoglucocenone, isomaltose, isomalttriose, isopanose, cozybiose, lactose, lactosamine, lacto This includes sudiamine, laminarabiose, levoglucosan, levoglucocenone, β-maltose, maltriose, mannan oligosaccharides, amninotriose, melezitose, melibiose, muramic acid, micalose, mycinose, neuraminic acid, migelose, nojirimycon, nobiose, oleandrose, panose, paratose, planteose, primbellose, raffinose, rhodone, rutinose, oleandrose, panose, paratose, planteose, primbellose, raffinose, rosinose, rutinose, sarmentose, sedoheptulose, sedoheptulosan, solatriose, sophorose, stachyose, streptose, sucrose, α,α-trehalose, trahalosamine, turanose, tyberose, xylobiose, umbelliferose, etc. Furthermore, it is understood that disaccharides, trisaccharides, and polysaccharides may be further substituted. Disaccharides include amino sugars and their derivatives, in particular mikaminos derivatized at the C-4' position or 4-deoxy-3-amino-glucose derivatized at the C-6' position.

[0063]

[0070] The terms “subject” or “patient” encompass mammals. Examples of mammals include, but are not limited to, any member of the mammalian class; humans, non-human primates such as chimpanzees, and other ape and monkey species; domesticated animals such as cattle, horses, sheep, goats, and pigs; domesticated animals such as rabbits, dogs, and cats; and laboratory animals, including rodents such as rats, mice, and guinea pigs. In one embodiment, mammal is human. As used herein, the term “animal” includes humans and non-human animals. In one embodiment, “non-human animal” is a mammal, such as a rodent such as a rat or mouse. In one embodiment, non-human animal is a mouse or monkey.

[0064]

[0071] As used herein, the terms “treat,” “treating,” or “treatment” include reducing, relieving, or improving at least one symptom of a disease or condition, preventing additional symptoms, inhibiting a disease or condition, for example, preventing the onset of a disease or condition, alleviating a disease or condition, causing regression of a disease or condition, alleviating a condition caused by a disease or condition, or prophylactic and / or therapeutic cessation of symptoms of a disease or condition. The term “treat” further encompasses the concepts of “prevent,” “preventing,” and “prevention,” as set forth below. It is understood that, though not excluded, a step in treating a disorder or condition does not require the complete elimination of the disorder, condition, or symptoms associated therewith.

[0065]

[0072] The terms "prevent" or "prevent" a disease state mean preventing the development of clinical symptoms of a disease state in individuals who are potentially exposed to or predisposed to a disease state but have not yet experienced or presented symptoms of that state.

[0066]

[0073] The terms “pharmaceutical composition” and “pharmaceutical preparation” (or “preparation”) mean a mixture or solution containing a therapeutically effective amount of an active pharmaceutical ingredient together with one or more pharmaceutically acceptable excipients that are interchangeable and administered to an object, e.g., a human being in need of it.

[0067]

[0074] As used herein, the term “pharmaceutical combination” means a product obtained by mixing or combining multiple active ingredients, and includes both fixed and unfixed combinations of those active ingredients. The term “fixed combination” means that both the active ingredients, e.g., the compounds and adjuvants described herein, are administered to the patient simultaneously in the form of a single entity or dose. The term “unfixed combination” means that the active ingredients, e.g., the compounds and adjuvants described herein, are administered to the patient as separate entities, simultaneously, in combination, or sequentially without specific intervening time limitations, and such administration provides effective levels of the two compounds in the patient’s body. The latter also applies to cocktail therapies, e.g., the administration of three or more active ingredients.

[0068]

[0075] The term “pharmaceutically acceptable” generally refers to the attributes of a material that is safe, non-toxic, not biologically or otherwise undesirable, and useful for preparing a pharmaceutical composition that is acceptable for veterinary and human pharmaceutical use. “pharmaceutically acceptable” may also refer to a material such as a carrier or diluent that does not negate the biological activity or properties of a compound and is relatively non-toxic, and can be administered to an individual without causing an undesirable biological effect or interacting in a harmful manner with any of the components of the composition in which it is contained.

[0069]

[0076] The terms “pharmaceutically acceptable excipient,” “pharmaceutically acceptable carrier,” “pharmaceutically acceptable vehicle,” and “therapeutically inactive excipient” may be used interchangeably and mean any pharmaceutically acceptable component in a pharmaceutical composition that has no therapeutic activity and is nontoxic to the target of administration, such as disintegrants, binders, fillers, solvents, buffers, isotonic agents, stabilizers, antioxidants, surfactants, carriers, diluents, excipients, preservatives, or lubricants used in the formulation of pharmaceuticals.

[0070]

[0077] The terms "base editing" and "base correction" are used interchangeably to describe changes or mutations in bases at a target sequence within a target gene that result in base modification. In certain embodiments, base editing occurs at a single base in the target sequence. In preferred embodiments, base editing does not involve double-strand breaks in the target sequence.

[0071]

[0078] As used herein, the term “siRNA” refers to drugs that mediate targeted cleavage of RNA transcripts. These drugs are associated with a cytoplasmic multiprotein complex known as the RNAi-induced silencing complex (RISC). Drugs effective in inducing RNA interference are also referred to herein as siRNA, RNAi drugs, or iRNA drugs. As used herein, the term siRNA includes microRNA and premicroRNA. As used herein, the terms “siRNA activity” and “RNAi activity” refer to gene silencing by siRNA.

[0072]

[0079] The term "2'-O-methoxyethyl" (also known as 2'-MOE, 2'-O(CH2)2-OCH3, and 2'-O-(2-methoxyethyl)) refers to the O-methoxyethyl modification at the 2' position of the furosyl ring. 2'-O-methoxyethyl modified sugars are modified sugars.

[0073]

[0080] The term "2'-O-methoxyethyl nucleotide" refers to a nucleotide containing a 2'-O-methoxyethyl modified sugar moiety.

[0081] The term "5-methylcytosine" refers to cytosine modified with a methyl group attached to the 5' position. 5-methylcytosine is a modified nucleic acid base.

[0074]

[0082] The term "oxo" refers to an =O substituent.

[0083] The term "alkyl" refers to a linear or branched hydrocarbon chain group that has 1 to 20 carbon atoms, which are bonded to the rest of the molecule by single bonds. Alkyl groups containing up to 10 carbon atoms are C1-C 10 Alkyls are called alkyls, and similarly, for example, alkyls containing up to six carbon atoms are C1-C6 alkyls. Alkyls containing other numbers of carbon atoms (and other parts as defined herein) are similarly represented. Alkyls are, but are not limited to, C1-C6 alkyls. 10Examples of alkyl groups include alkyl, C1-C9 alkyl, C1-C8 alkyl, C1-C7 alkyl, C1-C6 alkyl, C1-C5 alkyl, C1-C4 alkyl, C1-C3 alkyl, C1-C2 alkyl, C2-C8 alkyl, C3-C8 alkyl, and C4-C8 alkyl. Typical alkyl groups, though not limited to these, include methyl, ethyl, n-propyl, 1-methylethyl (i-propyl), n-butyl, i-butyl, s-butyl, n-pentyl, 1,1-dimethylethyl (t-butyl), 3-methylhexyl, 2-methylhexyl, and 1-ethyl-propyl. In some embodiments, the alkyl is methyl or ethyl. In some embodiments, the alkyl is -CH(CH3)2 or -C(CH3)3. Unless otherwise specified herein, alkyl groups may be optionally substituted as described below. "Alkylene" or "alkylene chain" refers to a straight or branched divalent hydrocarbon chain that links the rest of the molecule to a radical group. In some embodiments, the alkylene is -CH2-, -CH2CH2-, or -CH2CH2CH2-. In some embodiments, the alkylene is -CH2-. In some embodiments, the alkylene is -CH2CH2-. In some embodiments, the alkylene is -CH2CH2CH2-.

[0075]

[0084] The term "alkoxy" refers to a radical of the formula -OR, where R is a defined alkyl group. Unless otherwise specified herein, the alkoxy group may be optionally substituted as described below. Typical alkoxy groups include, but are not limited to, methoxy, ethoxy, propoxy, butoxy, and pentoxy. In some embodiments, the alkoxy is methoxy. In some embodiments, the alkoxy is ethoxy.

[0076]

[0085] The term "alkylamino" refers to a radical of the formula -NHR or -NRR, where each R is independently an alkyl group as defined above. Unless otherwise specified herein, the alkylamino group may be optionally substituted as described below.

[0077]

[0086] The term "alkenyl" refers to an alkyl group of a type that contains at least one carbon-carbon double bond. In one embodiment, the alkenyl group has the formula -C(R)=CR2, where R refers to the rest of the alkenyl group, which may be the same or different. In some embodiments, R is H or alkyl. In some embodiments, the alkenyl is selected from ethenyl (i.e., vinyl), propenyl (i.e., allyl), butenyl, pentenyl, pentadienyl, etc. Non-limiting examples of alkenyl groups include -CH=CH2, -C(CH3)=CH2, -CH=CHCH3, -C(CH3)=CHCH3, and -CH2CH=CH2. Depending on the structure, the alkenyl group may be monovalent or divalent (i.e., an alkenylene group).

[0078]

[0087] The term "alkynyl" refers to an alkyl group of a type that has at least one carbon-carbon triple bond. Thus, "alkynylene" may refer to a divalent alkynyl group. In one embodiment, the alkenyl group has the formula -C≡CR, where R refers to the rest of the alkynyl group. In some embodiments, R is H or alkyl. In some embodiments, the alkynyl is selected from ethynyl, propynyl, butynyl, pentynyl, hexynyl, etc. Non-limiting examples of alkynyl groups include -C≡CH, -C≡CCH3-C≡CCH2CH3, and -CH2C≡CH.

[0079]

[0088] The term "aryl" refers to an aromatic ring in which each of the ring-forming atoms is a carbon atom. The aryl group may be optionally substituted. Examples of aryl groups include, but are not limited to, phenyl and naphthyl. In some embodiments, the aryl is phenyl. Depending on the structure, the aryl group may be monovalent or divalent (i.e., an "arylene" group). Unless otherwise specified herein, the term "aryl" or the prefix "ar-" (e.g., "aralkyl") means that it includes aryl radicals which may be optionally substituted. In some embodiments, the aryl group is partially reduced to form a cycloalkyl group as defined herein. In some embodiments, the aryl group is completely reduced to form a cycloalkyl group as defined herein. In some embodiments, the aryl group is C6-C 14 It is an aryl group. In some embodiments, the aryl group is C6-C 10 It is Ariel.

[0080]

[0089] The term "cycloalkyl" refers to monocyclic or polycyclic non-aromatic radicals in which each of the ring-forming atoms (i.e., skeletal atoms) is a carbon atom. In some embodiments, cycloalkyls are saturated or partially unsaturated. In some embodiments, cycloalkyls are spirocyclic or crosslinked compounds. In some embodiments, cycloalkyls are condensed with an aromatic ring (in which case the cycloalkyl is bonded via a non-aromatic ring carbon atom). Cycloalkyl groups include those having 3 to 10 ring atoms. Representative cycloalkyls include, but are not limited to, those having 3 to 10 carbon atoms, 3 to 8 carbon atoms, 3 to 6 carbon atoms, or 3 to 5 carbon atoms. Examples of monocyclic cycloalkyl radicals include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. In some embodiments, monocyclic cycloalkyls are cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl. In some embodiments, monocyclic cycloalkyls are cyclopentenyl or cyclohexenyl. In some embodiments, the monocyclic cycloalkyl group is cyclopentenyl. Examples of polycyclic groups include adamantyl, 1,2-dihydronaphthalenyl, 1,4-dihydronaphthalenyl, tetrainyl, dekalinyl, 3,4-dihydronaphthalenyl-1(2H)-one, spiro[2.2]pentyl, norbornyl, and bicyclo[1.1.1]pentyl. Unless otherwise specified herein, the cycloalkyl groups may be optionally substituted. Depending on the structure, the cycloalkyl group may be monovalent or divalent (i.e., a cycloalkylene group).

[0081]

[0090] The term "haloalkyl" refers to an alkyl group in which at least one of the hydrogen atoms of the alkyl group is replaced by the same or different halogen atom, particularly a fluoro atom. Examples of haloalkyls include monofluoro-, difluoro-, or trifluoro-methyl, -ethyl, or -propyl, e.g., 3,3,3-trifluoropropyl, 2-fluoroethyl, 2,2,2-trifluoroethyl, fluoromethyl, or trifluoromethyl. The term "perhaloalkyl" refers to an alkyl group in which all of the hydrogen atoms of the alkyl group are replaced by the same or different halogen atoms.

[0082]

[0091] The term "heteroalkylene" refers to the alkyl group described above, in which one or more carbon atoms of the alkyl group are replaced with O, N, or S atoms. "Heteroalkylene" or "heteroalkylene chain" refers to a straight or branched divalent heteroalkyl chain that links the rest of the molecule to a radical group. Unless otherwise specified herein, heteroalkyl or heteroalkylene groups may be substituted as described below. Representative heteroalkylene groups include, but are not limited to, -OCH2CH2O-, -OCH2CH2OCH2CH2O-, or -OCH2CH2OCH2CH2OCH2CH2O-.

[0083]

[0092] The term "heterocycloalkyl" refers to a cycloalkyl group comprising at least one heteroatom selected from nitrogen, oxygen, and sulfur. Unless otherwise specified herein, heterocycloalkyl groups may be monocyclic or bicyclic ring systems, which may include condensed (when condensed with an aryl or heteroaryl ring, the heterocycloalkyl group is bonded via a non-aromatic ring atom) or bridging ring systems. The nitrogen, carbon, or sulfur atom in the heterocyclyl group may optionally be oxidized. The nitrogen atom may optionally be quaternized. Heterocycloalkyl groups are partially or fully saturated. Examples of heterocycloalkyl groups, though not limited to them, include dioxolanil, thienyl[1,3]dithianil, tetrahydroquinolyl, tetrahydroisoquinolyl, decahydroquinolyl, decahydroisoquinolyl, imidazolinyl, imidazolidinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl, octahydroindolyl, octahydroisoindolyl, 2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolidinyl, oxazolidinyl, piperidinyl, piperazinyl, 4-piperidonyl, pyrrolidinyl, pyrazolidinyl, quinuclidinyl, thiazolidinyl, tetrahydrofuryl, trithianil, tetrahydropyranil, thiomorpholinyl, thiamorpholinyl, 1-oxo-thiomorpholinyl, and 1,1-dioxo-thiomorpholinyl. The term heterocycloalkyl also includes all cyclic forms of carbohydrates, including but not limited to monosaccharides, disaccharides, and oligosaccharides. Unless otherwise specified, heterocycloalkyls have 2 to 12 carbon atoms in the ring. In some embodiments, heterocycloalkyls have 2 to 10 carbon atoms in the ring. In some embodiments, heterocycloalkyls have 2 to 10 carbon atoms and 1 or 2 nitrogen atoms in the ring. In some embodiments, heterocycloalkyls have 2 to 10 carbon atoms and 3 or 4 nitrogen atoms in the ring. In some embodiments, heterocycloalkyls have 2 to 12 carbon atoms, 0 to 2 nitrogen atoms, 0 to 2 oxygen atoms, 0 to 2 phosphorus atoms, and 0 to 1 sulfur atom in the ring.In some embodiments, the heterocycloalkyl group has 2 to 12 carbon atoms, 1 to 3 nitrogen atoms, 0 to 1 oxygen atoms, and 0 to 1 sulfur atoms within the ring. When referring to the number of carbon atoms in a heterocycloalkyl group, it should be understood that the number of carbon atoms in the heterocycloalkyl group is not the same as the total number of atoms constituting the heterocycloalkyl group (i.e., the skeletal atoms of the heterocycloalkyl ring) (including heteroatoms). Unless otherwise specified herein, heterocycloalkyl groups may be optionally substituted. As used herein, the term “heterocycloalkylene” may refer to a divalent heterocycloalkyl group.

[0084]

[0093] The term "heteroaryl" refers to an aryl group containing one or more ring heteroatoms selected from nitrogen, oxygen, and sulfur. Heteroaryls can be monocyclic or bicyclic. Specific examples of monocyclic heteroaryls include pyridinyl, imidazolyl, pyrimidinyl, pyrazolyl, triazolyl, pyrazinyl, tetrazolyl, furyl, thienyl, isoxazolyl, thiazolyl, oxazolyl, isothiazolyl, pyrrolyl, pyridazinyl, triazinyl, oxadiazolyl, thiadiazolyl, flazanyl, indidine, indole, benzofuran, benzothiophene, indazole, benzimidazole, purine, quinoridine, quinoline, isoquinoline, cinnoline, phthalazine, quinazoline, quinoxaline, 1,8-naphthyridine, and pteridine. Specific examples of monocyclic heteroaryls include pyridinyl, imidazolyl, pyrimidinyl, pyrazolyl, triazolyl, pyrazinyl, tetrazolyl, furyl, thienyl, isoxazolyl, thiazolyl, oxazolyl, isothiazolyl, pyrrolyl, pyridazinyl, triazinyl, oxadiazolyl, thiadiazolyl, and flazanil. Specific examples of bicyclic heteroaryls include indidine, indole, benzofuran, benzothiophene, indazole, benzimidazole, purine, quinolidine, quinoline, isoquinoline, cinnoline, phthalazine, quinazoline, quinoxaline, 1,8-naphthyridine, and pteridine. In some embodiments, the heteroaryl is pyridinyl, pyrazinyl, pyrimidinyl, thiazolyl, thienyl, thiadiazolyl, or furyl. In some embodiments, the heteroaryl contains 0 to 6 nitrogen atoms in the ring. In some embodiments, the heteroaryl contains 1 to 4 N atoms in the ring. In some embodiments, the heteroaryl contains 4 to 6 N atoms in the ring. In some embodiments, the heteroaryl contains 0 to 4 N atoms, 0 to 1 O atom, 0 to 1 P atom, and 0 to 1 S atom in the ring. In some embodiments, the heteroaryl contains 1 to 4 N atoms, 0 to 1 O atom, and 0 to 1 S atom in the ring. In some embodiments, the heteroaryl is a C1 to C9 heteroaryl.In some embodiments, the monocyclic heteroaryl is a C1-C5 heteroaryl. In some embodiments, the monocyclic heteroaryl is a 5-membered or 6-membered heteroaryl. In some embodiments, the bicyclic heteroaryl is a C6-C9 heteroaryl. In some embodiments, the heteroaryl group is partially reduced to form a heterocycloalkyl group as defined herein. In some embodiments, the heteroaryl group is completely reduced to form a heterocycloalkyl group as defined herein. Depending on the structure, the heteroaryl group may be monovalent or divalent (i.e., a "heteroarylene" group).

[0085]

[0094] Unless otherwise indicated, terms such as "substituted" and "substituent" may refer to the step of individually and independently replacing one or more hydrogen groups in a given structure with the groups of a particular substituent, and these substituents include, but are not limited to, D, halogens, -CN, -NH2, -NH(alkyl), -N(alkyl)2, -OH, -CO2H, -CO2alkyl, -C(=O)NH2, -C(=O)NH(alkyl), -C(=O)N(alkyl)2, -S(=O)2NH2, -S(=O)2NH(alkyl), -S(=O)2N(alkyl)2, alkyl, cycloalkyl, fluoroalkyl, heteroalkyl, alkoxy, fluoroalkoxy, heterocycloalkyl, aryl, heteroaryl, aryloxy, alkylthio, arylthio, alkylsulfoxide, arylsulfoxide, alkylsulfone, and arylsulfone. In some other embodiments, the optional substituent is independently selected from D, halogen, -CN, -NH2, -NH(CH3), -N(CH3)2, -OH, -CO2H, -CO2(C1~C4 alkyl), -C(=O)NH2, -C(=O)NH(C1-C4 alkyl), -C(=O)N(C1~C4 alkyl)2, -S(=O)2NH2, -S(=O)2NH(C1~C4 alkyl), -S(=O)2N(C1~C4 alkyl)2, C1~C4 alkyl, C3~C6 cycloalkyl, C1~C4 fluoroalkyl, C1~C4 heteroalkyl, C1~C4 alkoxy, C1~C4 fluoroalkoxy, -SC1~C4 alkyl, -S(=O)C1~C4 alkyl, and -S(=O)2(C1~C4 alkyl). In some embodiments, any substituent is independently selected from D, halogen, -CN, -NH2, -OH, -NH(CH3), -N(CH3)2, -NH(cyclopropyl), -CH3, -CH2CH3, -CF3, -OCH3, and -OCF3. In some embodiments, the substituent is substituted with one or two of the aforementioned groups. In some embodiments, any substituent on an aliphatic carbon atom (acyclic or cyclic) is oxo (=O).

[0086]

[0095] The term "unsubstituted" means that a particular group has no substituents. The term "optionally substituted" means that a particular group is either unsubstituted or substituted by one or more substituents independently selected from the group of possible substituents. When indicating the number of substituents, the term "one or more" means from one substituent to as many substitutions as possible, i.e., from the substitution of one hydrogen by a substituent to the substitution of all hydrogens.

[0087]

[0096] "Approximately" means within ±10% of the value. For example, if it says "the marker can be increased by approximately 50%", it means the marker can be increased between 45% and 55%.

[0088]

[0097] "Activator" refers to one or more substances in a pharmaceutical composition that produce a therapeutic effect when administered to an individual.

[0098] “Dosage unit” means the form in which the drug is provided, for example, a pill, a tablet, or other dosage form known in the art. In certain embodiments, the dosage form is a vial containing a lyophilized antisense oligonucleotide. In certain embodiments, the dosage form is a vial containing a reconstituted antisense oligonucleotide.

[0089]

[0099] "Dose" refers to a specific amount of a drug delivered in a single dose or over a specific period. In certain embodiments, a dose may be administered in one, two, or more boluses, tablets, or injections. For example, in certain embodiments where subcutaneous administration is desired, two or more injections may be used to achieve the desired dose, as the desired dose requires an amount that cannot be easily addressed by a single injection. In certain embodiments, the drug is administered by infusion over a long period or continuously. A dose may be expressed as the amount of drug per hour, day, week, or month. Doses may also be expressed in mg / kg or g / kg.

[0090]

[0100] A "modified nucleoside bond" refers to a substitution or modification of a naturally occurring nucleoside bond. For example, a phosphorothioate bond is a modified nucleoside bond.

[0091]

[0101] "Modified nucleic acid bases" refer to any nucleic acid base other than adenine, cytosine, guanine, thymidine, or uracil. For example, 5-methylcytosine is a modified nucleic acid base. "Unmodified nucleic acid bases" refer to the purine bases adenine (A) and guanine (G), as well as the pyrimidine bases thymine (T), cytosine (C), and uracil (U).

[0092]

[0102] A "modified nucleoside" refers to a nucleoside that has at least one modified sugar moiety and / or modified nucleic acid base.

[0103] A "modified nucleotide" refers to a nucleotide having at least one modified sugar moiety, a modified nucleoside bond, and / or a modified nucleic acid base.

[0093]

[0104] A "modified oligonucleotide" refers to an oligonucleotide that contains at least one modified nucleotide.

[0105] "Modified sugars" refer to substitutions or alterations from natural sugars. For example, 2'-O-methoxyethyl modified sugar is a modified sugar.

[0094]

[0106] A "motif" refers to a pattern of chemically distinct regions in an antisense compound.

[0107] "Statins" refer to drugs that inhibit the activity of HMG-CoA reductase.

[0095]

[0108] "Symptoms of cardiovascular disease or disorder" means phenomena that result from or are associated with cardiovascular disease or disorder and that function as indicators of such disease or disorder. For example, angina; chest pain; shortness of breath; palpitations; weakness; dizziness; nausea; sweating; tachycardia; bradycardia; arrhythmia; atrial fibrillation; swelling of the lower extremities; cyanosis; fatigue; syncope; facial numbness; numbness of the hands and feet; claudication or muscle spasms; abdominal distension; or fever are symptoms of cardiovascular disease or disorder.

[0096]

[0109] "Target nucleic acid" and "target sequence" refer to nucleic acids that can be targeted by genome editing compositions. For example, a target DNA sequence within or adjacent to the ANGPTL3 gene can be targeted by a guide nucleotide associated with the Cas9 nuclease.

[0097]

[0110] Methods for detecting and / or measuring polypeptides in biological materials are well known in the art and include, but are not limited to, Western blotting, flow cytometry, ELISA, RIA, and various proteomics techniques. Exemplary methods for measuring or detecting polypeptides are immunoassays such as ELISA. This type of protein quantification may be based on an antibody capable of capturing a specific antigen and a secondary antibody capable of detecting the captured antigen. Exemplary assays for polypeptide detection and / or measurement are described in Harlow, E. and Lane, D., Antibodies: A Laboratory Manual, (1988), Cold Spring Harbor Laboratory Press.

[0098]

[0111] Methods for detecting and / or measuring RNA in biological materials are well known in the art, and include, but are not limited to, Northern blotting, RNA protection assays, and RT-PCR. Appropriate methods are described in Molecular Cloning: A Laboratory Manual (4th edition) by Michael R. Green, Joseph Sambrook, and Peter MacCallum, 2012, 2, 028pp, ISBN 978-1-936113-42-2.

[0099]

[0112] Ribonucleoproteins (RNPs) refer to nuclear proteins that contain RNA. RNPs may also be complexes of ribonucleic acid and RNA-binding proteins. Such combinations are sometimes called protein-RNA complexes. These complexes can function in several biological processes, including, but are not limited to, DNA replication, DNA modification, gene expression, RNA metabolism and modification, and pre-mRNA splicing.

[0100]

[0113] As used herein, the terms “nucleic acid base editing factor (BE)” or “base editing factor (BE)” refer to a fusion protein comprising a composition, for example, a polypeptide capable of performing nucleic acid base modification and a Cas protein. In some embodiments, the fusion protein comprises nuclease-inactive Cas9 (dCas9) fused to a deaminase. In some embodiments, the fusion protein comprises Cas9 nickase fused to a deaminase. In some embodiments, the fusion protein comprises a wild-type Cas9 sequence, for example, a D10X mutation or an H840X mutation of Cas9 numbered with Sequence ID No. 1 (which allows Cas9 to cleave only one strand of a nucleic acid double helix). In some embodiments, the base editing factor comprises a programmable DNA nuclease domain fused to or ligated to a deaminase domain (e.g., an adenosine deaminase domain or a cytidine deaminase domain). Details of the base editing factors (editors) are described in International PCT applications PCT / 2017 / 045381 (WO2018 / 027078) and PCT / US2016 / 058344 (WO2017 / 070632), which are incorporated herein by reference in their entirety.Furthermore, the entire contents of the following are incorporated herein by reference: Komor, AC et al., "Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage," Nature 533, 420-424 (2016); Gaudelli, NM et al., "Programmable base editing of A·T to G·C in genomic DNA without DNA cleavage," Nature 551, 464-471 (2017); Komor, AC et al., "Improved base excision repair inhibition and bacteriophage Mu Gam protein yields C:G-to-T:A base editors with higher efficiency and product purity," Science Advances 3:eaao4774 (2017); Nishida, K. et al., "Targeted nucleotide editing using hybrid prokaryotic and vertebrate adaptive immune systems," Science 353, aaf8729 (2016); Gehrke JM, Cervantes O, Clement MK, Wu See also Y, Zeng J, Bauer DE, Pinello L, and Joung JK. An APOBEC3A-Cas9 base editor with minimized bystander and off-target activities. Nat Biotechnol. 2018 Nov;36(10):977-982.

[0101]

[0114] As used herein, the terms “biomarker” or “marker” are interchangeable to refer to any biochemical marker, serological marker, genetic marker, or other clinical or ultrasound-detectable feature that may be used to classify a patient-derived sample as being associated with a pathological condition, such as cardiovascular disease or disorder.

[0102]

[0115] As used herein, the term “antibody” includes, but is not limited to, a group of immunoglobulin molecules, or fragments of immunoglobulin molecules, which may be polyclonal or monoclonal and may be of any class and isotype. Immunoglobulins have five main classes: IgA, IgD, IgE, IgG, and IgM, some of which may be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1 (human), IgA2 (human), IgAa (canine), IgAb (canine), IgAc (canine), and IgAd (canine). Such fragments generally include portions of antibody molecules that specifically bind to an antigen. For example, fragments of immunoglobulin molecules known in the art as Fab, Fab', or F(ab')2 are included in the meaning of the term antibody.

[0103]

[0116] As used herein, the term “label” refers to a detectable compound, composition, or solid support that can be conjugated directly or indirectly (e.g., by covalent or non-covalent bonds, alone or encapsulated) to a monoclonal antibody or protein. The label may be detectable on its own (e.g., a radioisotope label, a chemiluminescent dye, an electrochemical label, a metal chelate, a latex particle, or a fluorescent label), or, in the case of an enzymatic label, may catalyze a chemical change in a detectable substrate compound or composition (e.g., an enzyme such as horseradish peroxidase or alkaline phosphatase). Labels used in this disclosure may include, but are not limited to, alkaline phosphatases; glucose-6-phosphate dehydrogenase ("G6PDH"); horseradish peroxidase (HRP); chemiluminescent agents such as isoluminol; fluorescent agents such as fluorescein and rhodamine compounds; ribozymes; and dyes. The label may also be a specific binding molecule that is itself detectable (e.g., biotin, avidin, streptavidin, digitoxygenin, maltose, oligohistidine, e.g., hexahistidine (SEQ ID NO: 114), 2,4-dinitrobenzene, phenylarsenate, ssDNA, dsDNA, etc.). The use of the label generates a signal that can be detected by means such as detection or direct visualization of electromagnetic radiation, and optionally measured.

[0104]

[0117] "Substantial binding" or "substantially binding" refers to the amount of specific binding or affinity between molecules in an assay mixture under specific assay conditions. In its broadest embodiment, substantial binding relates to the difference between a first molecule binding to and being unrecognizable by a second molecule, and a difference that enables a meaningful assay and is sufficient to identify specific binding under a specific set of assay conditions, including the relative concentrations of the molecules, as well as incubation time and temperature. In another embodiment, if one molecule exhibits less than 25% of the reactivity shown towards a second molecule, e.g., less than 10%, e.g., less than 5%, compared to the reactivity shown towards a third molecule under a specific set of assay conditions, including the relative concentrations of the molecules and incubation, then it is substantially impossible for one molecule to bind to or recognize another molecule in the sense of cross-reactivity. Specific binding can be tested using several well-known methods, such as immunohistochemical assays, enzyme-linked immunosorbent assays (ELISAs), radioimmunoassays (RIAs), or Western blot assays.

[0105]

[0118] As used herein, the term “substantially identical amino acid sequence” includes amino acid sequences that are similar to, but not identical to, naturally occurring amino acid sequences. For example, an amino acid sequence having substantially the same amino acid sequence as a flagellin protein, such as a polypeptide, may have one or more modifications, such as additions, deletions, or substitutions of amino acids, compared to the amino acid sequence of a naturally occurring flagellin protein, provided that the modified polypeptide substantially retains at least one biological activity of flagellin, such as immunoreactivity. The “similarity percentage” between two sequences is a function of the number of positions containing matching residues or conserved residues shared by the two sequences, divided by the number of positions being compared and multiplied by 100. In this regard, a conserved residue in a sequence is a residue that is physically or functionally similar to its corresponding reference residue, e.g., similar in size, shape, charge, chemical properties, e.g., ability to form covalent or hydrogen bonds.

[0106]

[0119] The term “targeting moiety” refers to any molecule that provides enhanced affinity to a selected target, e.g., a cell, cell type, tissue, organ, body region, or compartment, e.g., a cell, tissue, or organ compartment. Some exemplary targeting moieties include, but are not limited to, antibodies, antigens, carbohydrate base moieties, folates, receptor ligands, carbohydrates, aptamers, integrin receptor ligands, chemokine receptor ligands, transferrin, biotin, serotonin receptor ligands, PSMA, endothelin, GCPII, somatostatin, LDL, and HDL ligands. Carbohydrate-based targeting moieties include, but are not limited to, D-galactose, polyvalent galactose, N-acetyl-D-galactosamine (GalNAc), polyvalent GalNAc, e.g., GalNAc2 and GalNAc3; D-mannose, polyvalent mannose, polyvalent lactose, N-acetyl-glucosamine, polyvalent fucose, glycosylated polyamino acids, and lectins. The term "polyvalent" indicates the presence of multiple monosaccharide units. Such monosaccharide subunits may be linked to one another via glycosidic bonds, or they may be linked to a scaffold molecule.

[0107]

[0120] The term "heterogeneous" refers to any two or more nucleic acid or polypeptide sequences that are not typically found in nature in the same relationship to one another. For example, heterogeneous nucleic acids typically consist of two or more sequences from unrelated genes that are produced by recombination and arranged to create a new functional nucleic acid, such as a promoter from one source and a coding region from another. Similarly, heterogeneous polypeptides often refer to two or more subsequences that are not typically found in nature in the same relationship to one another (e.g., fusion proteins).

[0108]

[0121] As used herein, the term “fragment” includes a peptide, polypeptide, or protein segment of a full-length protein, provided that the fragment retains reactivity with at least one antibody in the serum of a patient with the disease.

[0109]

[0122] An "epitope" is an antigenic determinant on a polypeptide that is recognized for binding by a polypeptide-specific antibody, such as an IBD-related antibody, via a paratope.

[0123] The term “clinical factors” includes symptoms in patients associated with cardiovascular disease. Examples of clinical factors include, but are not limited to, angina; chest pain; shortness of breath; palpitations; weakness; dizziness; nausea; sweating; tachycardia; bradycardia; arrhythmia; atrial fibrillation; swelling of the lower extremities; cyanosis; fatigue; syncope; facial numbness; limb numbness; claudication or muscle spasms; abdominal distension; or fever. In some embodiments, the diagnosis of cardiovascular disease is based on a combination of analyzing the presence or level of one or more markers in a patient using statistical algorithms and determining whether the patient has one or more clinical factors.

[0110]

[0124] The term "prognosis" includes predictions of possible courses and outcomes of a pathological condition, such as cardiovascular disease, or recovery from the disease. In some embodiments, the prognosis of a patient's cardiovascular disease is obtained by using statistical algorithms. For example, the prognosis may include surgery, the occurrence of one or more clinical factors, or recovery from the disease.

[0111]

[0125] This specification provides methods and compositions for the targeted delivery of therapeutic agents, such as nucleic acid agents. The therapeutic agents used herein may be conjugated to or associated with a targeting moiety to assist targeted delivery. For example, the therapeutic agent and the targeting moiety may form a conjugate. The therapeutic agent may include a nucleic acid-induced programmable nuclease system that forms a complex with a nucleic acid, such as a guide RNA. In some embodiments, the guide RNA may be chemically modified. In some embodiments, the modified guide RNA may be used in the preparation of pharmaceuticals for the treatment of any gene-related disease, disorder, or condition in which the gene may be altered, manipulated, edited, and modified by DNA insertion or deletion. According to a further aspect of this disclosure, the modified guide RNA may be used to alter a gene by the step of deleting, substituting, repairing, or inserting DNA. This can be done in microorganisms or animals, particularly mammals, more specifically humans. Human cells or tissues may be genetically modified or corrected in vitro using the guide RNA of this disclosure and a CRISPR / Cas system known in the art, and then returned to a patient in need. In another aspect of the present disclosure, a pharmaceutical composition is provided comprising the modified guide RNA according to the present disclosure, a CRISPR-Cas system, and a pharmaceutically acceptable carrier or excipient. This pharmaceutical composition may comprise a vector or cells having the modified guide RNA according to the present disclosure. In a further aspect of the present disclosure, a composition is provided comprising the modified guide RNA and at least one delivery means selected from GalNAc, polymers, liposomes, peptides, aptamers, antibodies, viral vectors, folates, or transferrin.

[0112] Nuclease system

[0126] This specification provides compositions and methods for targeted delivery of activators or therapeutic agents comprising nucleic acids, polynucleotides, or oligonucleotides. The activator may be a pharmaceutical composition, a drug, a polynucleotide, an oligonucleotide, an RNP, a lipid nanoparticle, or a protein-RNA complex. Targeted delivery as described herein can lead the activator to a specific desired location, e.g., a specific in vivo location, a cell, tissue, or organ, a recognition site within the intracellular matrix, or a specific location within a cell. In some embodiments, the activator includes a guide RNA associated with a nuclease, e.g., a CRISPR nuclease. In some embodiments, the activator includes a nuclease system that can modify the activity and / or function of one or more target genes, e.g., the PCSK9 or ANGPTL3 gene.

[0113]

[0127] In some embodiments, the activator comprises a genome editing composition comprising a nuclease system. In some embodiments, this genome editing composition is a target-specific genome editing composition. In some embodiments, the genome editing composition comprises a nucleic acid-inducible programmable nuclease or a portion thereof. In some embodiments of this disclosure, the nuclease system comprises at least one nuclease. In some embodiments, the nuclease system comprises at least one programmable nuclease. In some embodiments, the nuclease may comprise at least one DNA-binding domain and at least one nuclease domain. In some embodiments, the nuclease domain may be heterogeneous to the DNA-binding domain. In certain embodiments, the nuclease is a DNA endonuclease that can cleave single-stranded or double-stranded DNA. In certain embodiments, the nuclease can cleave RNA.

[0114]

[0128] In some embodiments, the nuclease system may include a Cas protein domain (also called a "Cas nuclease") derived from the CRISPR / Cas system. The Cas protein may include at least one domain that interacts with a guide nucleic acid, such as a guide RNA (gRNA). Furthermore, the Cas protein may be directed to a target sequence by the guide RNA. The guide RNA interacts with the Cas protein and the target sequence so that the Cas protein is directed to the target sequence and can cleave the target sequence. In certain embodiments, such as Cas9, the Cas protein is a single protein effector, an RNA-induced (RNA-guided) nuclease. In some embodiments, this guide RNA provides specificity for target cleavage, and the Cas protein is universal and can be paired with different guide RNAs to cleave different target sequences. The terms Cas protein and Cas nuclease are used interchangeably herein.

[0115]

[0129] In some embodiments, the CRISPR / Cas system may include components of type I, type II, or type III systems, or any orthologues thereof. The updated classification scheme of CRISPR / Cas loci defines class I and class II CRISPR / Cas systems having types I–V or VI. See, for example, Makarova et al., Nat Rev Microbiol, 13(11):722-36 (2015); Shmakov et al., Molecular Cell, 60:385-397 (2015). Class II CRISPR / Cas systems have a single protein effector. Type II, V, and VI Cas proteins may be single-protein RNA-induced endonucleases, referred herein as “class II Cas nucleases.” Examples of class II Cas nucleases include the Cas9, Cpf1, C2c1, C2c2, and C2c3 proteins. The Cpf1 protein, Zetsche et al., Cell, 163:1-13 (2015), is homologous to Cas9 and contains a RuvC-like nuclease domain, S3.

[0116]

[0130] In some embodiments, the Cas protein may be derived from a type II CRISPR / Cas system, i.e., a Cas9 protein derived from the CRISPR / Cas9 system. In some embodiments, the Cas protein may be derived from a class 2 CRISPR / Cas system, i.e., a single-protein Cas nuclease such as the Cas9 protein or the Cpf1 protein. The Cas9 and Cpf1 families of proteins are enzymes with DNA endonuclease activity and can be directed to cleave a desired nucleic acid target by designing an appropriate guide RNA, as further described herein.

[0117]

[0131] The type II CRISPR / Cas system components may be derived from type IIA, type IIB, or type IIC systems. The structure and sequence of the Cas9 nuclease are known to those skilled in the art (incorporated herein by reference, Jinek et al. Science 2012, 337:816-821; Delcheva et al. Nature 2011, 471:602-607). In some embodiments, wild-type Cas9 corresponds to Streptococcus pyogenes Cas9 (NCBI reference number NC_002737.2, SEQ ID NO: 2) and Uniprot reference number Q99ZW2 (SEQ ID NO: 1).

[0118]

[0132] Streptococcus pyogenes Cas9 (wild-type) protein sequence (SEQ ID NO: 1)

[0133]

[0134] Streptococcus pyogenes Cas9 (wild-type) nucleotide sequence (SEQ ID NO: 2)

[0135] The Cas9 strain contains a specific strain of the strain of Streptococcus pyogenes meningitidis、Campylobacter jejuni、Pasteurella multocida、Fibrobacter succinogene、Rhodospirillum rubrum、Nocardiopsis dassonvillei、Streptomyces pristinaespiralis、Streptomyces viridochromogenes、Streptomyces viridochromogenes Streptosporangium roseum Streptosporangium roseum Alicyclobacillus acidocaldarius Bacillus pseudomycoides Bacillus selenitireducens Exiguobacterium sibiricum Lactobacillus delbrueckii Lactobacillus salivarius、Lactobacillus buchneri、Treponema denticola、Microscilla marina、Burkholderiales bacterium、Polaromonas naphthalenivorans、Polaromonas sp.、Crocosphaera watsonii、Cyanothece sp.、Microcystis aeruginosa、Synechococcus sp., Acetohalobium arabaticum, Ammonifex degensii, Caldicellulosiruptor becscii, Candidatus Desulforudis, Clostridium botulinum, Clostridium difficile, Finegoldia magna, Natranaerobius thermophilus, Pelotomaculum thermopropionicum, Acidithiobacillus caldus, Acidithiobacillus ferrooxidans, Allochromatium vinosum, Marinobacter sp., Nitrosococcus halophilus, Nitrosococcus watsoni, Pseudoalteromonas haloplanktis, Ktedonobacter racemifer, Methanohlobium evestigatum, Anabaena variabilis, Nodularia spumigena, Nostoc sp., Arthrospira maxima, Arthrospira platensis, Arthrospira sp., Lyngbya sp., Microcoleus chthonoplastes, Oscillatoria sp.Examples include Petrotoga mobilis, Thermosipho africanus, Streptococcus pasteurianus, Neisseria cinerea, Campylobacter lari, Parvibaculum lavamentivorans, Corynebacterium diphtheria, and Acaryochloris marina. In some embodiments, the Cas9 protein may be derived from Straptococcus pyogenes. In some embodiments, the Cas9 protein may be derived from Straptococcus thermophilus. In some embodiments, the Cas9 protein may be derived from Neisseria meningitidis. In some embodiments, the Cas9 protein may be derived from Staphylococcus aureus.

[0119]

[0136] In some embodiments, the Cas protein may contain two or more nuclease domains. For example, the Cas9 protein may contain at least one RuvC-like nuclease domain (e.g., Cpf1 / Cas12a) and at least one HNH-like nuclease domain (e.g., Cas9). In some embodiments, the Cas9 protein may be capable of introducing a double-strand break (DSB) into a target sequence. In some embodiments, the Cas9 protein may be modified to contain only one functional nuclease domain. For example, the Cas9 protein may be modified so that one of its nuclease domains is mutated or completely or partially deleted, reducing its nucleic acid cleavage activity. In some embodiments, the Cas9 protein may be modified to not contain a functional RuvC-like nuclease domain. In other embodiments, the Cas9 protein may be modified to not contain a functional HNH-like nuclease domain. In some embodiments where only one nuclease domain is functional, the Cas9 protein may be a nickasase capable of introducing a single-strand break ("nick") into a target sequence. In some embodiments, conserved amino acids within the Cas9 protein nuclease domain are substituted to reduce or alter nuclease activity. In some embodiments, the Cas protein nickase may include amino acid substitutions in the RuvC-like nuclease domain. An example amino acid substitution in the RuvC-like nuclease domain is D10A (based on the S. pyogenes Cas9 protein). In some embodiments, the nickase may include amino acid substitutions in the HNH-like nuclease domain. Example amino acid substitutions in the HNH-like nuclease domain are E762A, H840A, N863A, H983A, and D986A (based on the S. pyogenes Cas9 protein). In some embodiments, the nuclease system described herein may include nickases and a pair of guide RNAs complementary to the sense and antisense strands of the target sequence, respectively.Guide RNA can direct the nickase to the target and introduce a double-sided subsequence (DSB) by generating a nick on the opposite strand of the target sequence (i.e., double nicking). Chimeric Cas9 proteins, in which one domain or region of the protein is replaced by a part of a different protein, may also be used. For example, the Cas9 nuclease domain may be replaced by a domain derived from a different nuclease, such as Fok1. The Cas9 protein may also be a modified nuclease.

[0120]

[0137] By aligning wild-type Cas9 and Cas9 sequences from various species, corresponding homologous amino acid residues can be determined, for example, by determining and / or modifying the amino acid residues D10 and H840 of SEQ ID NO: 1, enabling the generation of Cas9 variants with corresponding mutations in homologous amino acid residues. Alignment methods are known to those skilled in the art. For example, alignment may be performed using the NCBI Constraint-based Multiple Alignment Tool (COBALT, accessible at st-va.ncbi.nlm.nih.gov / tools / cobalt).

[0121]

[0138] In alternative embodiments, the Cas protein may be derived from the type I CRISPR / Cas system. In some embodiments, the Cas protein may be a component of the type I CRISPR / Cas system cascade complex. For example, the Cas protein may be the Cas3 protein. In some embodiments, the Cas protein may be derived from the type III CRISPR / Cas system. In some embodiments, the Cas protein may be derived from the type IV CRISPR / Cas system. In some embodiments, the Cas protein may be derived from the type V CRISPR / Cas system. In some embodiments, the Cas protein may be derived from the type VI CRISPR / Cas system. In some embodiments, the Cas protein may have RNA cleavage activity.

[0122] Fusion protein

[0139] Compositions and methods for targeted modification of genes, such as PCSK9, ANGPTL3, APOC3, LPA, APOB, MTP, ANGPTL4, ANGPTL8, APOA5, APOE, LDLR, IDOL, NPC1L1, ASGR1, TM6SF2, GALNT2, GCKR, LPL, MLXIPL, SORT1, TRIB1, MARC1, ABCG5, or ABCG8, are provided herein. In certain examples, the modification may be ex vivo or in vivo. In preferred embodiments, the targeted modification may be directed to a specific type of organ, tissue, or cell, such as hepatocytes of the liver. In some embodiments, the target gene is genetically modified with a genome editing composition comprising a fusion protein. Thus, in some embodiments, fusion proteins for targeted modification of genes are provided herein. In some embodiments, the fusion protein comprises a target-specific nuclease domain. In some embodiments, the fusion protein comprises a nucleic acid-inducible programmable nuclease domain. In some embodiments, the nucleic acid-inducible programmable nuclease may comprise at least one DNA-binding domain and at least one nuclease domain. In some embodiments, the nuclease domain may be heterogeneous to the DNA-binding domain. In some embodiments, the nuclease domain may be modified such that it mutates and its nuclease cleavage activity is reduced. In some embodiments, nuclease activity is completely lost. In some embodiments, nuclease activity is partially reduced. In some embodiments, the modified nuclease domain may comprise a modified Cas protein domain. In certain embodiments, the modified Cas protein domain is modified Cas9. In some embodiments, the modified Cas9 domain is a nuclease-inactive Cas9 (dCas9) domain. In some embodiments, the modified dCas9 domain is a nickasase domain.In some embodiments, the modified Cas9 domain includes at least one substitution selected from D10A, N497A, R661A, Q695A, E762A, H840A, N863A, Q926A, H983A, and D986A, based on the S.pyogenes Cas9 protein. In some embodiments, the modified nuclease domain is a catalytically inactive Cpf1 domain, a catalytically inactive Cas13a domain, a catalytically inactive Cas13b domain, or a catalytically inactive Cas13c domain. In some embodiments, the modified nuclease domain is a catalytically inactive CasX, CasY, Cpf1, C2c1, C2c2, C2c3, and Argonaute protein domain.

[0123]

[0140] In some embodiments, the fusion proteins described herein include one or more functional domains in addition to the nuclease domain. At least one protein domain may be located at the N-terminus, C-terminus, or internal position of the fusion protein. In some embodiments, two or more heterologous protein domains are located at one or more positions on the fusion protein. Non-limiting examples of functional domains include repressor domains, activator domains, methyltransferase domains, and demethylase domains. In some embodiments, the functional domain includes a base editing enzyme domain. In some embodiments, the functional domain is a cytidine deaminase domain. For example, cytidine deaminase deaminates a specific cytidine to uracil, resulting in a UG mismatch, which is then separated via a cell repair mechanism to form a UA base pair, followed by a TA base pair, thereby creating a C→T substitution. The cytidine deaminase domain and cytidine-deaminase fusion protein sequences are known to those skilled in the art, as described in Komor et al., Science Advances 2017, 3(8):eaao4774; Komor et al., Nature 2016, 533:420-424. In some embodiments, the functional domain is the adenine deaminase domain. For example, the adenine deaminase domain can deaminate adenosine to produce inosine, which then base-pairs with cytidine and is subsequently modified to guanine by cell repair mechanisms, thereby converting A to G. An exemplary adenosine deaminase fusion protein is described in its entirety by reference in Gaudelli et al., Nature 2017 551(7681):464-471.

[0124]

[0141] In some embodiments, the fusion protein described herein includes a nuclear localization signal (NLS). In some embodiments, the fusion protein may include two, three, four, or five NLSs. In some embodiments, the fusion protein may include one to ten NLSs. The NLS sequences may be fused at the N-terminus and / or C-terminus of the fusion protein. In some embodiments, the NLS may be a single subsequence, such as SV40 NLS, PKKKRKV (SEQ ID NO: 3), or PKKKRRV (SEQ ID NO: 4). In some embodiments, the NLS may be a two-subsequence, such as the NLS of nucleoplasmin, KRPAATKKAGQAKKKK (SEQ ID NO: 5). In some embodiments, the NLS may be genetically modified from its wild-type counterpart. In a preferred embodiment, the fusion protein includes the sequence ABE7.10 (SEQ ID NO: 6).

[0125]

[0142] In some embodiments, the fusion protein may further include a tag domain. In some embodiments, the tag domain may include a fluorescent tag, a purified tag, an epitope tag, or a reporter gene tag. In some embodiments, the tag domain may include a fluorescent protein domain. Non-limiting examples of suitable fluorescent proteins include green fluorescent proteins (e.g., GFP, GFP-2, tagGFP, turboGFP, sfGFP, EGFP, Emerald, Azami Green, Monomeric Azami). Green fluorescent proteins (e.g., CopGFP, AceGFP, ZsGreenl), yellow fluorescent proteins (e.g., YFP, EYFP, Citrine, Venus, YPet, PhiYFP, ZsYellowl), blue fluorescent proteins (e.g., EBFP, EBFP2, Azurite, mKalamal, GFPuv, Sapphire, T-sapphire), cyan fluorescent proteins (e.g., ECFP, Cerulean, CyPet, AmCyanl, Midoriishi-Cyan), red fluorescent proteins (e.g., mKate, mKate2, mPlum, DsRed monomer, mCherry, mRFP1, DsRed-Express, DsRed2, DsRed-Monomer, HcRed-Tandem, HcRedl, AsRed2, eqFP611, mRasberry, mStrawberry, Jred), and orange fluorescent proteins (mOrange, mKO, Kusabira-Orange, Monomeric). Examples include Kusabira-Orange, mTangerine, tdTomato, or any other suitable fluorescent protein. In some embodiments, the tag domain may include a purified tag and / or an epitope tag.Examples of non-limiting tags include glutathione-S-transferase (GST), chitin-binding protein (CBP), maltose-binding protein (MBP), thioredoxin (TRX), poly(NANP), tandem affinity purification (TAP) tag, myc, AcV5, AU1, AU5, E, ECS, E2, FLAG, HA, nus, Softag1, Softag3, Strep, SBP, Glu-Glu, HSV, KT3, S, S1, T7, V5, VSV-G, 6×His (SEQ ID NO: 114), biotin carboxyl carrier protein (BCCP), and calmodulin. In some embodiments, the tag domain may include a reporter gene domain. Non-exclusive exemplary reporter genes include glutathione-S-transferase (GST), horseradish peroxidase (HRP), chloramphenicol acetyltransferase (CAT), beta-galactosidase, beta-glucuronidase, luciferase, or fluorescent proteins.

[0126]

[0143] In further embodiments, the nuclease in the nuclease system may include one or more programmable nucleases other than Cas proteins. For example, the nuclease may be selected from meganucleases (e.g., homing endonucleases), ZFNs, TALENs, and megaTALs.

[0127]

[0144] Naturally occurring meganucleases can recognize and cleave double-stranded DNA sequences of approximately 12–40 base pairs and are generally classified into five families. In some embodiments, meganucleases may be selected from the LAGLIDADG family, GIY-YIG family, HNH family, His-Cys box family, and PD-(D / E)XK family. In some embodiments, the DNA-binding domain of a meganuclease may be engineered to recognize and bind to sequences other than its congener target sequences. In some embodiments, the DNA-binding domain of a meganuclease may be fused to a heterologous nuclease domain. In some embodiments, a meganuclease, such as a homing endonuclease, may be fused to a TAL module to create a hybrid protein, such as a "megaTAL" protein. MegaTAL proteins may have improved DNA target specificity by recognizing target sequences in both the DNA-binding domain of the meganuclease and the TAL module.

[0128]

[0145] ZFNs are fusion proteins containing a zinc finger DNA-binding domain ("zinc finger" or "ZF") and a nuclease domain. Each naturally occurring ZF can bind to three consecutive base pairs (DNA triplets), and ZF repeats combine to recognize a target DNA sequence and provide sufficient affinity. Therefore, engineered ZF repeats may be combined to recognize longer DNA sequences, such as 9 bp, 12 bp, 15 bp, or 18 bp. In some embodiments, ZFNs may contain ZF fused to a nuclease domain derived from a restriction endonuclease. For example, the restriction endonuclease may be FokI. In some embodiments, the nuclease domain may include a dimerization domain, for example, when the nuclease dimerizes to become active. A pair of ZFNs, each containing a ZF repeat and a nuclease domain, may be designed to target a target sequence containing two semi-targets recognized by each ZF repeat on the opposite strand of the DNA molecule, with an interconnection sequence (sometimes called a spacer in the literature) between them. For example, the interconnection sequence may be 5–7 bp in length. When both ZFNs of the pair bind, the nuclease domain may dimerize, introducing a DSB within the interconnection sequence. In some embodiments, the dimerization domain of the nuclease domain may include a knob-into-hole motif that facilitates dimerization. For example, the ZFN FokI may include a knob-into-hole motif in its dimerization domain.

[0129]

[0146] The DNA-binding domain of a TALEN typically contains a variable number of 34 or 35 amino acid repeats ("modules" or "TAL modules"), each module binding to a single DNA base pair, A, T, G, or C. Adjacent residues at positions 12 and 13 of each module ("variable repeat duodecimal residues" or RVD) specify the single DNA base pair to which the module binds. While a module used to recognize G may also have affinity for A, TALENs benefit from a simple recognition code (one module for each of the four bases), which greatly simplifies the customization of DNA-binding domains to recognize specific target sequences. In some embodiments, a TALEN may also contain a nuclease domain derived from a restriction endonuclease. For example, the restriction endonuclease may be FokI. In some embodiments, the nuclease domains may be dimerized to become active, and pairs of TALENS may be designed to target a target sequence containing two halves of the target sequence recognized by each DNA-binding domain on the opposite strand of a DNA molecule with interconnecting sequences between them. For example, each half-target sequence may be in the range of 10–20 bp, and the interconnection sequence may be 12–19 bp in length. When both TALENs of a pair bind, the nuclease domain may dimerize, introducing a double-sided split (DSB) within the interconnection sequence. In some embodiments, the dimerization domain of the nuclease domain may contain a knob-into-hole motif that facilitates dimerization. For example, the dimerization domain of FokI may contain a knob-into-hole motif.

[0130]

[0147] Certain embodiments of this disclosure also provide nucleic acids encoding the nuclease systems described herein, provided on a vector. In some embodiments, the nucleic acid may be a DNA molecule. In other embodiments, the nucleic acid may be an RNA molecule. In some embodiments, the nucleic acid encoding the nuclease may be an mRNA molecule.

[0131]

[0148] In some embodiments, the nucleic acid encoding the nuclease may be a codon optimized for efficient expression in one or more eukaryotic cell types. In some embodiments, the nucleic acid encoding the nuclease may be a codon optimized for efficient expression in one or more mammalian cells. In some embodiments, the nucleic acid encoding the nuclease may be a codon optimized for efficient expression in human cells. Codon optimization methods, including codon usage tables and codon optimization algorithms, are available in the art.

[0132] Guide polynucleotides

[0149] In some embodiments of this disclosure, the CRISPR / Cas nuclease system comprises at least one guide polynucleotide, such as guide RNA. In some embodiments, the guide RNA and Cas protein may form a ribonucleoprotein (RNP), such as a CRISPR / Cas complex. The guide RNA may guide the Cas protein to a target sequence on a target nucleic acid molecule, where the guide RNA hybridizes and the Cas protein cleaves the target sequence. In some embodiments, the CRISPR / Cas complex may be a Cpf1 / guide RNA complex. In some embodiments, the CRISPR complex may be a type II CRISPR / Cas9 complex. In some embodiments, the Cas protein may be a Cas9 protein. In some embodiments, the CRISPR / Cas9 complex may be a Cas9 / guide RNA complex.

[0133]

[0150] A guide nucleic acid (e.g., guide RNA) can bind to a Cas protein and target the Cas protein to a specific position within a target polynucleotide. The guide nucleic acid may include a nucleic acid targeting segment and a Cas protein binding segment.

[0134]

[0151] The guide nucleic acid may refer to another nucleic acid, for example, a nucleic acid that can hybridize to a target polynucleotide in the cell's genome. The guide nucleic acid may be RNA, for example, guide RNA. The guide nucleic acid may be DNA. The guide nucleic acid may contain both DNA and RNA. The guide nucleic acid may be single-stranded. The guide nucleic acid may be double-stranded. The guide nucleic acid may contain nucleotide analogs. The guide nucleic acid may contain modified nucleotides. The guide nucleic acid may be programmed or designed to bind to the nucleic acid sequence in a site-specific manner.

[0135]

[0152] The guide nucleic acid may contain one or more modifications to provide novel or improved characteristics to the nucleic acid. The guide nucleic acid may contain nucleic acid affinity tags. The guide nucleic acid may contain synthetic nucleotides, synthetic nucleotide analogs, nucleotide derivatives, and / or modified nucleotides.

[0136]

[0153] The guide nucleic acid may contain a nucleic acid target region (e.g., a spacer region) complementary to the protospacer sequence in the target polynucleotide, for example, at or near the 5' or 3' end. The spacer of the guide nucleic acid may interact with the protospacer in a sequence-specific manner via hybridization (base pairing). The protospacer sequence may be located at the 5' or 3' end of the protospacer adjacent motif (PAM) of the target polynucleotide. The nucleotide sequence of the spacer region may vary to determine the location within the target nucleic acid in which the guide nucleic acid can interact. The spacer region of the guide nucleic acid may be designed or modified to hybridize to any desired sequence within the target nucleic acid.

[0137]

[0154] The guide nucleic acid may comprise two separate nucleic acid molecules, which can be called dual guide nucleic acids. The guide nucleic acid may comprise a single nucleic acid molecule, which can be called a single guide nucleic acid (e.g., sgRNA). In some embodiments, the guide nucleic acid is a single guide nucleic acid comprising a fused CRISPR RNA (crRNA) and a transactivated crRNA (tracrRNA). In some embodiments, the guide nucleic acid is a single guide nucleic acid comprising crRNA. In some embodiments, the guide nucleic acid is a single guide nucleic acid comprising crRNA but lacking tracRNA. In some embodiments, the guide nucleic acid is a dual guide nucleic acid comprising an unfused crRNA and a tracrRNA. An exemplary dual guide nucleic acid may comprise a crRNA-like molecule and a tracrRNA-like molecule. An exemplary single guide nucleic acid may comprise a crRNA-like molecule. An exemplary single guide nucleic acid may comprise a fused crRNA-like molecule and a tracrRNA-like molecule.

[0138]

[0155] The crRNA may include a nucleic acid target segment of the guide nucleic acid (e.g., a spacer region) and a sequence of nucleotides that can form half of the double-stranded Cas protein-binding segment of the guide nucleic acid.

[0139]

[0156] The tracrRNA may contain a sequence of nucleotides that form the remaining half of the double helix of the Cas protein-binding segment of the gRNA. The sequence of nucleotides of the crRNA may be complementary to the sequence of nucleotides of the tracrRNA and may hybridize with it to form the double helix of the Cas protein-binding domain of the guide nucleic acid.

[0140]

[0157] crRNA and tracrRNA can hybridize to form guide nucleic acids. crRNA can also provide a single-stranded nucleic acid targeting segment (e.g., a spacer region) that hybridizes to a target nucleic acid recognition sequence (e.g., a protospacer). The sequence of the crRNA or tracrRNA molecule containing the spacer region may be designed to be specific to the species in which the guide nucleic acid is used.

[0141]

[0158] Guide RNAs for the CRISPR / Cas9 system typically include CRISPR RNA (crRNA) and tracr RNA (tracr). Guide RNAs for the CRISPR / Cpf1 system usually include crRNA. In some embodiments, the crRNA may include a targeting sequence that is complementary to and hybridizes with a target sequence on a target nucleic acid molecule. The crRNA may also include a sequence that is complementary to and hybridizes with a portion of the tracrRNA. In some embodiments, the crRNA may be structured to resemble a naturally occurring crRNA transcribed from a bacterial CRISPR locus, where the targeting sequence acts as a spacer for the CRISPR / Cas9 system.

[0142]

[0159] The guide RNA can target any desired sequence via the targeting sequence of the crRNA. In some embodiments, the degree of complementarity between the targeting sequence of the guide RNA and the target sequence on the target nucleic acid molecule may be about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100%. In some embodiments, the targeting sequence of the guide RNA and the target sequence on the target nucleic acid molecule may be 100% complementary. In other embodiments, the targeting sequence of the guide RNA and the target sequence on the target nucleic acid molecule may contain at least one mismatch. For example, the targeting sequence of the guide RNA and the target sequence on the target nucleic acid molecule may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mismatches. In some embodiments, the targeting sequence of the guide RNA and the target sequence on the target nucleic acid molecule may contain 1 to 6 mismatches. In some embodiments, the targeting sequence of the guide RNA and the target sequence on the target nucleic acid molecule may contain five or six mismatches.

[0143]

[0160] The length of the targeting sequence may depend on the CRISPR / Cas9 system and the components used. For example, different Cas9 proteins from different bacterial species have varying optimal targeting sequence lengths. Therefore, the targeting sequence may include nucleotide lengths of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, or more than 50. In some embodiments, the targeting sequence may include lengths of 18 to 30 nucleotides. In some embodiments, the targeting sequence may include lengths of 19 to 24 nucleotides. In some embodiments, the targeting sequence may include lengths of 20 nucleotides.

[0144]

[0161] crRNA and tracr may contain any sequence that has sufficient complementarity to facilitate the formation of a functional CRISPR / Cas9 complex. In some embodiments, the complementary sequence between crRNA and tracr may contain all or part of a naturally occurring crRNA sequence (also called a “tag” or “handle”) that is complementary to tracr RNA in the same CRISPR / Cas9 system. In some embodiments, the complementary sequence may contain all or part of a naturally occurring repetitive sequence derived from the CRISPR / Cas9 system. In some embodiments, the complementary sequence may contain a shortened or modified tag or handle sequence. In some embodiments, the degree of complementarity between tracr RNA and the portion of the complementary region that hybridizes with tracr RNA along the shorter of the two sequences may be about 40%, 50%, 60%, 70%, 80% or more, but less than 100%. In some embodiments, one or more bulge structures are present on the tracr and / or wobble base pairing, so the tracr RNA and the portion hybridizing with the tracr RNA are not 100% complementary along the shorter of the two sequences. The length of the tracr RNA portion complementary to tracr may depend on the CRISPR / Cas9 system or tracr RNA used. For example, the complementary portion may be 10 to 50 nucleotides long, or longer than 50 nucleotides. In some embodiments, the complementary portion may be 15 to 40 nucleotides long. In other embodiments, the complementary portion may be 20 to 30 nucleotides long. In yet another embodiment, the complementary portion may be 22 nucleotides long. For example, when using dual guide RNA, there may be no upper limit to the length of the complementary portion.

[0145]

[0162] In some embodiments, the tracr RNA may comprise all or part of a wild-type tracr RNA sequence derived from a naturally occurring CRISPR / Cas9 system. In some embodiments, the tracr RNA may comprise a truncated or modified variant of the wild-type tracr RNA. The length of the tracr RNA may depend on the CRISPR / Cas9 system used. In some embodiments, the tracr RNA may comprise nucleotide lengths of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, or more than 100. In certain embodiments, the tracr RNA is at least 26 nucleotides long. In additional embodiments, the tracr RNA is at least 40 nucleotides long. In some embodiments, the tracr RNA may comprise certain secondary structures, such as one or more hairpin or stem-loop structures, or one or more bulge structures.

[0146]

[0163] In some embodiments, the guide RNA may comprise two RNA molecules, referred herein as “dual guide RNA” or “dgRNA”. In some embodiments, the dgRNA may comprise a first RNA molecule comprising crRNA and a second RNA molecule comprising tracr RNA. The first and second RNA molecules may form an RNA double helix via base pairing between the flagpole on the crRNA and the tracr RNA.

[0147]

[0164] In some embodiments, the guide RNA may comprise a single RNA molecule, referred to herein as “single guide RNA” or “sgRNA”. In some embodiments, the sgRNA may comprise a crRNA covalently bound to the tracr RNA. In some embodiments, the crRNA and tracr RNA may be covalently bound via a linker. In some embodiments, the single guide RNA may comprise a stem-loop structure via base pairing between a flagpole on the crRNA and the tracr RNA.

[0148]

[0165] Certain embodiments of this disclosure also provide nucleic acids, such as vectors, that encode the guide RNA described herein. In some embodiments, the nucleic acid may be a DNA molecule. In other embodiments, the nucleic acid may be an RNA molecule. In some embodiments, the nucleic acid may include a nucleotide sequence encoding crRNA. In some embodiments, the nucleotide sequence encoding crRNA includes a targeting sequence adjacent to all or part of a naturally occurring CRISPR / Cas system-derived repetitive sequence. In some embodiments, the nucleic acid may include a nucleotide sequence encoding tracr RNA. In some embodiments, crRNA and tracr RNA may be encoded by two distinct nucleic acids. In some embodiments, crRNA and tracr RNA may be encoded by a single nucleic acid. In some embodiments, crRNA and tracr RNA may be encoded by opposite strands of a single nucleic acid. In other embodiments, crRNA and tracr RNA may be encoded by the same strand of a single nucleic acid.

[0149]

[0166] In certain embodiments, two or more guide RNAs may be used with the CRISPR / Cas nuclease system. Each guide RNA may contain different targeting sequences so that the CRISPR / Cas system cleaves two or more target sequences. In some embodiments, one or more guide RNAs may have the same or different properties, such as activity or stability within the Cas9 RNP complex. When two or more guide RNAs are used, each guide RNA may be encoded on the same vector or on different vectors. The promoters used to drive the expression of the two or more guide RNAs may be the same or different.

[0150]

[0167] Methods for selecting guide RNAs for efficient targeting with high specificity and low off-target effects are known to those skilled in the art. For programmable base editing, the selection of genomic sequences containing the target sequence may be as described in Komor et al., Nature, 533, 420-424 (2016), incorporated herein by reference. The priority of guide RNA sequences and PAMs defines the genomic target sequences of programmable nuclease domains (e.g., Cas9, dCas9, Cas9n, Cpfl, NgAgo domains). Hsu et al. (Nature biotechnology, 2013, 31(9):827-832), Fusi et al. (bioRxiv021568;doi:http: / / dx.doi.org / 10.1101 / 021568), Chari et al. (Nature Methods, 2015, 12(9):823-6), Doench et al. (Nature Biotechnology, 2014, 32(12):1262-7), Wang et al. (Science, 2014, 343)(6166):80-4), Moreno-Mateos et al. (Nature Methods, 2015, 12(10):982-8), Housden et al. (Science Signaling, 2015, 8(393):rs9), Haeussler et al., (Genome Methods for reducing off-target binding, as described in Biol. 2016, 17:148), are incorporated herein by reference. The possibility of bulge formation between guide RNA and target DNA, and other parameters that may affect target sequence binding, may also be considered, as described in Bae et al. (Bioinformatics, 2014, 30, 1473-5), and also incorporated herein by reference in Houston et al. (Science Signaling, 2015, 8(393):rs9) and Farboud et al. (Genetics, 2015, 199(4):959-71).

[0151] RNA modification

[0168] This specification provides modified RNA molecules suitable for targeted ex vivo and in vivo delivery systems. Modified RNA molecules may comprise two or more linked ribonucleic acid subunits. Examples of non-restrictive modified RNAs include CRISPR guide RNA, short interfering RNA (siRNA), microRNA (miRNA), short hairpin RNA (shRNA), small nuclear RNA (snRNA), messenger RNA (mRNA), precursor mRNA (premRNA), antisense RNA (asRNA), and heteronuclear RNA (hnRNA). The modified RNAs described herein encompass both RNA sequences and any structural embodiments thereof, such as single-stranded, double-stranded, triple-stranded, circular, helical, hairpin, stem-loop, bulge, etc. Modified RNAs may have lengths of at least approximately 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100, 200, 300, 400, or 500 bases. Modified RNA can be at least about 1 kilobase (kb), 2kb, 3kb, 4kb, 5kb, 10kb, 20kb, 50kb, or longer in length. In some embodiments, the modified RNA is CRISPR guide RNA (gRNA). The gRNA may be a single guide RNA or a dual guide RNA. In some embodiments, the modified RNA is mRNA. In some embodiments, the mRNA may be isolated from cells or from tissue. In some embodiments, the mRNA may be transcribed from DNA. In some embodiments, the mRNA may be chemically synthesized.

[0152]

[0169] In certain embodiments, the modified RNA molecules provided herein are resistant to degradation by RNases or other exonucleases. In certain embodiments, the modified RNA molecules provided herein are stabilized to prevent degradation by endonucleases. In some embodiments, the modified RNA molecules provided herein are suitable for in vivo delivery and induce less cellular immune receptor activation (e.g., TLRs, RIG-I) compared with unmodified RNA. RNA modifications are as described in whole in Diebold (2008) Adv Drug Deliv Rev. Apr 29;60(7):813-23) and Sorrentino (1998) Cellmol Life Sci. Aug;54(8):785-94, both of which are incorporated herein by reference.

[0153]

[0170] In addition to “unmodified” or “natural” nucleic acid bases such as the purine nucleic acid bases adenine (A) and guanine (G), and the pyrimidine nucleic acid bases thymine (T), cytosine (C), and uracil (U), many modified nucleic acid bases or nucleic acid base mimetics known to those skilled in the art are suitable for the compounds described herein. Modified or replaced unmodified or natural nucleic acid bases may provide oligonucleotides with improved properties. For example, nuclease-resistant oligonucleotides may be prepared using these bases, or using any one of synthetic and natural nucleic acid bases (e.g., inosine, xanthine, hypoxanthine, nubularin, isoguanisine, or tubercidine) and the oligomeric modifications described herein. Alternatively, substituted or modified analogs of any of the above bases and “universal bases” may be used. When a natural base is replaced by a non-natural and / or universal base, the nucleotide is said herein to contain a modified nucleic acid base and / or nucleic acid base modification. Modified nucleic acid bases and / or nucleic acid base modifications also include natural, non-natural, and universal bases, including conjugate moieties, e.g., ligands described herein. Preferred conjugate moieties for conjugation with nucleic acid bases include cationic amino groups that can be conjugated to nucleic acid bases via linkers having suitable alkyl, alkenyl, or amide bonds.

[0154]

[0171] As used herein, “unmodified” or “natural” nucleic acid bases include the purine bases adenine (A) and guanine (G), as well as the pyrimidine bases thymine (T), cytosine (C), and uracil (U). Exemplary modified nucleic acid bases include, but are not limited to, other synthetic and natural nucleic acid bases such as inosine, xanthine, hypoxanthine, nuvalarin, isoguanisine, tubercidine, 2-(halo)adenine, 2-(alkyl)adenine, 2-(propyl)adenine, 2-(amino)adenine, 2-(aminoalkyll)adenine, 2-(aminopropyl)adenine, 2-(methylthio)-N6-(isopentenyl)adenine, 6-(alkyl)adenine, 6-(methyl)adenine, 7-(deaza)adenine, 8-(alkenyl)adenine, 8-(alkyl)adenine, 8-(alkynyl)adenine, 8-(amino)adenine, 8-(halo)adenine, 8-(hydroxyl)adenine, 8-(thioalkyl)adenine, 8-(thiol)adenine, N6-(isopentyl)adenine, N6-(methyl)adenine, N6, N6-(dimethyl)adenine, 2-(alkyl)guanine, 2-(propyl)guanine, 6-(alkyl)guanine, 6-(methyl)guanine, 7-(a Lukyl)guanine, 7-(methyl)guanine, 7-(deaza)guanine, 8-(alkyl)guanine, 8-(alkenyl)guanine, 8-(alkynyl)guanine, 8-(amino)guanine, 8-(halo)guanine, 8-(hydroxyl)guanine, 8-(thioalkyl)guanine, 8-(thiol)guanine, N-(methyl)guanine, 2-(thio)cytosine, 3-(deaza)-5-(aza)cytosine, 3-(alkyl)cytosine, 3-(methyl)cytosine, 5-(alkyl)cytosine , 5-(alkynyl)cytosine, 5-(halo)cytosine, 5-(methyl)cytosine, 5-(propynyl)cytosine, 5-(propynyl)cytosine, 5-(trifluoromethyl)cytosine, 6-(azo)cytosine, N4-(acetyl)cytosine, 3-(3-amino-3-carboxypropyl)uracil, 2-(thio)uracil, 5-(methyl)-2-(thio)uracil, 5-(methylaminomethyl)-2-(thio)uracil, 4-(thio)uracil, 5-(methyl)-4-(thio)uracil,5-(methylaminomethyl)-4-(thio)uracil, 5-(methyl)-2,4-(dithio)uracil, 5-(methylaminomethyl)-2,4-(dithio)uracil, 5-(2-aminopropyl)uracil, 5-(alkyl)uracil, 5-(alkynyl)uracil, 5-(allylamino)uracil, 5-(aminoallyl)uracil, 5-(aminoalkyl)uracil, 5-(guanidiniumalcyl)uracil, 5-(1,3-diazole-1-alkyl)uracil, 5-(cyanoalkyl)uracil, 5-(dialkylaminoalkyl)uracil, 5 -(dimethylaminoalkyl)uracil, 5-(halo)uracil, 5-(methoxy)uracil, uracil-5-oxyacetic acid, 5-(methoxycarbonylmethyl)-2-(thio)uracil, 5-(methoxycarbonyl-methyl)uracil, 5-(propynyl)uracil, 5-(propynyl)uracil, 5-(trifluoromethyl)uracil, 6-(azo)uracil, dihydrouracil, N-(methyl)uracil, 5-uracil (i.e., pseudouracil), 2-(thio)pseudracil, 4-(thio)pseudracil, 2,4-(dithio)pseudra Uracil, 5-(alkyl)pseudracil, 5-(methyl)pseudracil, 5-(alkyl)-2-(thio)pseudracil, 5-(methyl)-2-(thio)pseudracil, 5-(alkyl)-4-(thio)pseudracil, 5-(methyl)-4-(thio)pseudracil, 5-(alkyl)-2,4-(dithio)pseudracil, 5-(methyl)-2,4-(dithio)pseudracil, 1-methylpseudracil (N1-methylpseudracil), 1-substituted pseudouracil, 1-substituted 2(thio)-pseudracil, 1- Substituting 4-(thio)pseudracil, 1-substituting 2,4-(dithio)pseudracil, 1-(aminocarbonylethylenyl)-pseudracil, 1-(aminocarbonylethylenyl)-2(thio)-pseudracil, 1-(aminocarbonylethylenyl)-4-(thio)pseudracil, 1-(aminocarbonylethylenyl)-2,4-(dithio)pseudracil, 1-(aminoalkylaminocarbonylethylenyl)-pseudracil, 1-(aminoalkylamino-carbonylethylenyl)-2(thio)-pseudracil,1-(aminoalkylaminocarbonylethylenyl)-4-(thio)pseudracil, 1-(aminoalkylaminocarbonylethylenyl)-2,4-(dithio)pseudracil, 1,3-(diaza)-2-(oxo)-phenoxazine-1-yl, 1-(aza)-2-(thio)-3-(aza)-phenoxazine-1-yl, 1,3-(diaza)-2-(oxo)-phenthiadin-1-yl, 1-(aza)-2-(thio)-3-(aza)-phenthiadin-1-yl, 7-substituted 1,3-(diaza)-2-(oxo)-phenoxazine-1-yl 7-substituted-1-(aza)-2-(thio)-3-(aza)-phenoxazine-1-yl, 7-substituted-1,3-(diaza)-2-(oxo)-phenthiadin-1-yl, 7-substituted-1-(aza)-2-(thio)-3-(aza)-phenthiadin-1-yl, 7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazine-1-yl, 7-(aminoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazine-1-yl, 7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)- Phenthiadin-1-yl, 7-(aminoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenthiadin-1-yl, 7-(guanidiniumalcylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxadin-1-yl, 7-(guanidiniumalcylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxadin-1-yl, 7-(guanidiniumalcylhydroxy)-1-(aza)-2- (Thio)-3-(aza)-phenthiadin-1-yl, 1,3,5-(triaza)-2,6-(dioxa)-naphthalene, inosine, xanthine, hypoxanthine, nubralin, tubercidine, isoguanisine, inosinyl, 2-aza-inosinyl, 7-deaza-inosinyl, nitroimidazolyl, nitropyrazolyl, nitrobenzimidazolyl, nitroindazolyl, aminoindolyl, pyrrolopyrimidinyl, 3-(methyl)isocarbostyrillyl, 5-(methyl)isocarbostyrillyl, 3-(methyl)-7-(propynyl)isocarbostyrillyl,7-(aza)indolyl, 6-(methyl)-7-(aza)indolyl, imidizopyridinyl, 9-(methyl)-imidizopyridinyl, pyrrolopyridinyl, isocarbostyrillyl, 7-(propynyl)isocarbostyrillyl, propynyl-7-(aza)indolyl, 2,4,5-(trimethyl)phenyl, 4-(methyl)indolyl, 4,6-(dimethyl)indolyl, phenyl, naphthalenyl, anthracenyl, phenanthracenyl, pyrenyl, s Tilbenyl, tetracenyl, pentacenyl, difluorotolyl, 4-(fluoro)-6-(methyl)benzimidazole, 4-(methyl)benzimidazole, 6-(azo)thymine, 2-pyridinone, 5-nitroindole, 3-nitropyrrole, 6-(aza)pyrimidine, 2-(amino)purine, 2,6-(diamino)purine, 5-substituted pyrimidine, N2-substituted purine, N6-substituted purine, O6-substituted purine, substituted 1,2,4-triazole, py llo-pyrimidine-2-on-3-yl, 6-phenyl-pyrrolo-pyrimidine-2-on-3-yl, para-substituted-6-phenyl-pyrrolo-pyrimidine-2-on-3-yl, ori / zo-substituted-6-phenyl-pyrrolo-pyrimidine-2-on-3-yl, bis-ori / zo-substituted-6-phenyl-pyrrolo-pyrimidine-2-on-3-yl, para-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidine-2-on-3-yl Examples include ori / zo-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidine-2-on-3-yl, bis-ori / zo-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidine-2-on-3-yl, pyridopyrimidine-3-yl, 2-oxo-7-amino-pyridopyrimidine-3-yl, 2-oxo-pyridopyrimidine-3-yl, or any O-alkylated or N-alkylated derivative thereof. Alternatively, substitutions or modified analogs of any of the above bases and “universal bases” may be used. A universal nucleic acid base is any nucleic acid base that can form base pairs with all four naturally occurring nucleic acid bases without substantially affecting melting behavior, recognition by intracellular enzymes, or the activity of oligonucleotide double helixes. Some exemplary universal nucleic acid bases are, but are not limited to:2,4-Difluorotoluene, Nitropyrrolyl, Nitroindolyl, 8-Aza-7-Deazadenine, 4-Fluoro-6-Methylbenzimidazul, 4-Methylbenzimidazul, 3-Methylisocarbostyrillyl, 5-Methylisocarbostyrillyl, 3-Methyl-7-Propynylisocarbostyrillyl, 7-Azaindolyl, 6-Methyl-7-Azaindolyl, Imidizopyridinyl, 9-Methylimidizopyridinyl, Pyrrolopyridinyl, I Examples include socarbostyrillyl, 7-propynylisocarbostyrillyl, propynyl-7-azaindryl, 2,4,5-trimethylphenyl, 4-methyllinolyl, 4,6-dimethylindolyl, phenyl, naptarenyl, anthracenyl, phenantracenyl, pyrenyl, stilbenyl, tetracerenyl, pentacerenyl, and their structural derivatives (see, for example, Loakes, 2001, Nucleic Acids Research, 29, 2437-2447, whose wholes are incorporated herein by reference). Further examples of nucleic acid bases include those disclosed in U.S. Patent No. 3,687,808; those disclosed in International Application No. PCT US09 / 038425, filed March 26, 2009; those disclosed in the Concise Encyclopedia Of Polymer Science And Engineering, pp. 858-859, edited by Kroschwitz, JI, John Wiley & Sons, 1990; those disclosed in English et al., Angewandte Chemie, International Edition, 1991, 30, 613; those disclosed in Modified Nucleosides in Biochemistry, Biotechnology and Medicine, edited by Herdewijin, P., Wiley-VCH, 2008; and those disclosed in Sanghvi, YS, Chapter 15, dsRNA Research and Applications, pp. 289-302, edited by Crooke, ST and Lebleu, B., CRC Press, 1993. All of the above is incorporated herein by reference.

[0155]

[0172] In some embodiments, the modified RNA described herein is modified to bind a delivery and / or targeting moiety, such as GalNAc. Preferably, GalNAc may bind to the 3' end, the 5' end, or both of the RNA. In some embodiments, GalNAc is bound to the 3' end. In some embodiments, the modified RNA exhibits improvements compared to its unmodified counterpart. Such improvements may relate to improved specificity (e.g., reduced off-target effects or the need for lower concentrations of gRNA), improved stability (e.g., resistance to enzymes such as nucleases), improved functionality, or reduced immunogenicity or immunostimulatory properties. In some embodiments, the modified RNA exhibits improved properties that enable efficient transfection into cells and / or delivery and maintenance of RNA into organisms, tissues, fluids, or cells, resulting in the function of the RNA, e.g., guide RNA. Methods for measuring these improved properties compared to their unmodified counterparts are known to those skilled in the art and are described herein. Thus, in some embodiments, modified RNA with increased stability compared to its unmodified counterpart is provided herein. An unmodified equivalent means an RNA, such as a guide RNA, that targets the same specific gene sequence, interacts with the same Cas9 or CRISPR nuclease, and contains native nucleotides. Improved stability includes improved stability or resistance to enzymes, such as nucleases, that may be present in cells, tissues, or bodily fluids and could contribute to the degradation of RNA that would otherwise be impaired. In certain embodiments, increased stability includes increased serum stability. In some embodiments, modified guide RNAs having increased CRISPR activity compared to an unmodified equivalent are provided herein. Methods for measuring CRISPR activity are described herein. In some embodiments, modified guide RNAs having reduced immunostimulatory activity compared to an unmodified equivalent are provided herein. Methods for measuring immunostimulation are described herein.

[0156]

[0173] This specification provides modified mRNA molecules for targeted delivery. For example, mRNA encoding a CRISPR enzyme, such as Cas9, Cas12b, or a base-editing factor (BE), may be modified to target a specific tissue. The mRNA may have at least one nucleotide modified at the 2' position and / or by skeletal modification. In some embodiments, the nucleotides in the mRNA may include thioate modifications. In some embodiments, the mRNA may include one or more modifications from 2'-OMe, 2'-F, N-1-methyl-pseuduridine, 5-methyluridine, 5-methoxyuridine, and 5-ethoxyuridine.

[0157]

[0174] In certain embodiments, the mRNA sequences provided herein include fully modified or partially modified mRNA. In some embodiments, the mRNA includes chemical modifications to fragments or multiple full-length fragments. Non-limiting exemplary modifications and modification patterns of mRNA nucleotides or segments thereof are shown in Tables 2 and 3.

[0158]

[0175] Provided herein are modified guide RNAs for use in CRISPR / Cas systems, which may be modified by chemical modification and / or skeletal modification of at least one nucleotide at the 2' position. Skeletal modifications may include thioate modifications. In certain embodiments, the nucleotide to be modified is selected from a group of nucleotides that interact with Cas amino acids in the Cas protein to result in the binding of the guide RNA to Cas. In certain embodiments, this modification may include modifications in which the 2'-OH on the nucleotide is replaced by at least one of H, -OR, -R, -O-C1~C6-alkylene-OR, -O-C1~C6-alkylene-OH, halo, -SH, -SR, -NH2, -NHR, -N(R)2, -C1~C6-alkylene-NH2, -C1~C6-alkylene-NHR, -C1~C6-alkylene-N(R)2, or CN, where each R is independently a C1~C6 alkyl, a C2~C6 alkenyl, or a C2~C6 alkynyl, and halo is F, CI, Br, or I. In some cases, this modification is 2'-O-methyl and / or 2'-F. In some embodiments, this modification includes one or more of the following modifications: 2'-F, phosphorothioate internucleotide linkage modification, acyclic nucleotide, LNA, HNA, CeNA, 2'-methoxyethyl, 2'-O-methyl, 2'-O-allyl, 2'-C-allyl, 2'-deoxy, 2'-ON-methylacetamide (2'-O-NMA), 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE), 2'-O-aminopropyl (2'-O-AP), and 2'-ara-F modification. In some embodiments, the modification includes 2'-MOE. In some embodiments, the modification includes phosphorothioate internucleotide linkage modification. In some embodiments, the modification includes 4-O-alkyllibosaccharides such as 4'-methoxy and 4'-ethoxy modifications.

[0159]

[0176] Preferably, the modified guide RNA can be matched to the CRISPR / Cas system of S. pyogenes, or any other CRISPR / Cas system, such as RNA found in Staphylococcus aureus or Staphylococcus haemolyticus. This modification or a similar modification pattern may also be used to guide RNA from Cpf1 derived from Lachnospiraceae bacterium ND2006 or Cpf1 derived from Acidominococcus species BV3L6.

[0160]

[0177] In certain embodiments, the guide RNA sequence includes a fully modified single guide RNA. In some embodiments, the guide RNA includes chemical modifications to the tracr RNA portion. Non-limiting exemplary nucleotide modifications and modification patterns of guide RNA according to this disclosure are shown in Tables 2 and 3.

[0161]

[0178] The modified guide RNAs described herein can be used in complex with a CRISPR / Cas system or a CRISPR / Cas enzyme to induce a change in a target gene or DNA sequence. The CRISPR / Cas enzyme may include CRISPR nucleases such as Cas9, Cpf1, C2c1, C2c2, or C2c3. In some embodiments, the CRISPR / Cas enzyme may include a nuclease-inactive Cas9 or a CRISPR nuclease with modified or reduced nuclease activity, such as Cpf1. For example, mutations can be introduced into one or both nuclease subdomains of the Cas9 enzyme to produce Cas9 nickas or nuclease-inactive Cas9. Exemplary inactivation mutations in Cas9 include changes at positions D10, E762, H840, N854, N863, or D986 in SEQ ID NO: 1. For example, the D10A mutation in the RuvC subdomain and the H840A mutation in the HNH subdomain of Cas9 result in Cas9 nuclease inactivation. The D10A mutation in the RuvC subdomain or the H840A mutation in the HNH subdomain of Cas9 produce Cas9 nickase. Additional amino acid substitutions in Cas9 are discussed in WO15 / 89354, which is incorporated herein by reference in its entirety.

[0162]

[0179] The modified guide RNA may share sequence identity with or hybridize with a target nucleotide, such as a target gene or target DNA sequence. In some embodiments, the modified guide RNA has at least 100%, 99%, 98%, 96%, 95%, 90%, 85%, 80%, 75%, or 70% correspondence or identity with the target nucleotide of the gene or target DNA.

[0163]

[0180] The nucleotides described herein may be synthetic or chemically modified. For example, the guide RNA provided herein may be synthetic or chemically modified guide RNA. The nucleotides in the modified guide RNA may be one or more nucleotides in the binding region between the guide RNA and Cas9, and / or nucleotides in the binding region between the guide RNA and target DNA. The remaining unmodified nucleotides in the guide RNA may be nucleotides that need to be identified to minimize the binding of Cas9 to the 2'-OH position of the base. In some embodiments, the nucleotides may be modified at the 2' position of the sugar moiety of the nucleotide. In some embodiments, the 2'-OH group of the sugar moiety is replaced by a group selected from H, OR, R, halo, SH, SR, H2, NHR, N(R)2, or CN, where R is a C1-C6 alkyl, alkenyl, or alkynyl, and halo is F, Cl, Br, or I. Other modifications include inverted (deoxy) base-free, amino, fluoro, chloro, bromo, CN, CF, methoxy, imidazole, carboxylate, thioate, C1-C10 (CI to CIO) lower alkyl, substituted lower alkyl, alkaryl or aralkyl, heterocycloalkyl (heterozycloalkyl); heterocycloalkaryl; aminoalkylamino; polyalkylamino or substituted silyl. Methods for producing RNA with specific sequences and modifications are known to those skilled in the art, for example, in Dellinger et al. (2011), J.Am.Chem.Soc, 133, 11540; U.S. Patent No. 8,202,983; Kumar et al. (2007), J.Am.Chem.Soc, 129, 6859-64; WO2013176844, which are incorporated herein by reference in their entirety.

[0164]

[0181] In some embodiments, the polynucleotides or oligonucleotides provided herein may be synthetic. For example, the guide RNA may be a chemically synthesized guide RNA. The yield of synthetic RNA is based on sequence and modification. 2'-O-methyl modification has been shown to enhance the effectiveness or efficiency of coupling during RNA synthesis and thus increase the yield of chemically synthesized RNA. Furthermore, nucleotides may be modified by phosphorothioates. Phosphothioate (phosphorothioate) (PS) bonds replace uncrosslinked oxygen atoms in the phosphate backbone of oligonucleotides with sulfur atoms. Accordingly, exemplary nucleotides of this disclosure include, but are not limited to, ribonucleic acid (RNA), deoxyribonucleic acid (DNA), threose nucleic acid (TNA), glycol nucleic acid (GNA), peptide nucleic acid (PNA), locked nucleic acid (LNAs, for example, LNA having a β-D-ribo configuration, α-LNA having an α-L-ribo configuration (a diastereomer of LNA), 2'-amino-LNA having 2'-amino functionalization, and 2'-amino-α-LNA having 2'-amino functionalization) or hybrids thereof.

[0165] Conjugate for targeted delivery

[0182] This specification provides conjugates suitable for targeted delivery of drugs such as mRNA, guide RNA, miRNA, siRNA, DNA, peptides, or other micromolecules or macromolecules. The conjugate may comprise one or more aptamers, ligands, or moieties for targeted delivery ex vivo or in vivo. In some embodiments, the conjugate comprises a targeting moiety (or ligand), a linker, and an activator (or payload) attached to the targeting moiety. The activator may be a therapeutic, prophylactic, or diagnostic / prognostic agent. The activator may have the ability to manipulate physiological functions in a target (e.g., gene expression). The activator may be a guide RNA, mRNA, miRNA, siRNA, DNA, or peptide. The activator may be bound to the targeting moiety via a linker, via non-covalent bonding, via nucleic acid base pairing, or by any combination thereof. In some embodiments, the conjugate may be a conjugate between a single activator having formula (I):XYZ and a single targeting moiety, where X is the targeting moiety; Y is the linker; and Z is the guide RNA. In certain embodiments, one targeting ligand may be conjugated to two or more activators, and this conjugate has the formula:X-(YZ)n. For example, this conjugate may include guide RNA and mRNA. In certain embodiments, one activator may be linked to two or more targeting ligands, and this conjugate has the formula:(XY)nZ. In other embodiments, one or more targeting moieties may be bound to one or more active payloads, the conjugate formula of which may be (XYZ)n. In various combinations, the formula of the conjugate may be, for example, XYZYX, (XYZ)nYZ, or XY-(XYZ)n, where X is the targeting moiety; Y is the linker; and Z is the activator, e.g., guide RNA. The number of components in the conjugate may vary depending on the type of drug, the size of the conjugate, the delivery target, the particles used to package the conjugate, other activators (e.g., immunological adjuvants), and the route of administration.The presences of X, Y, and Z may be the same or different, and for example, the conjugate may include multiple types of targeting parts, multiple types of linkers, and / or multiple types of activators, where n is an integer of 1 or more. In some embodiments, n is an integer between 1 and 50, or between 2 and 20, or between 5 and 40. In some embodiments, n may be an integer of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

[0166]

[0183] In some embodiments, activators, such as guide RNA, can be delivered to cells and tissues using viruses, polymers and liposome formulations, cell-permeable peptides, aptamers, ligands, or conjugate and antibody approaches. A portion or ligand may guide the guide RNA to a specific organ, tissue, or cell, such as hepatocytes in the liver, and may be referred to as a targeting portion. In some embodiments, the targeting portion modifies one or more properties of the adherent molecule (e.g., mRNA or guide RNA), including but not limited to pharmacodynamics, pharmacokinetics, binding, absorption, cell distribution, cell uptake, charge, and clearance.

[0167]

[0184] Exemplary moieties that may bind to the activators described herein include, but are not limited to, intercalators, reporter molecules, polyamines, polyamides, polyethylene glycol, thioethers, polyethers, cholesterol, thiocholesterol, cholic acid moieties, folates, lipids, phospholipids, biotin, phenazine, phenanthridine, anthraquinone, adamantane, acridine, fluorescein, rhodamine, coumarin, pigments, and lipid moieties such as cholesterol moieties (Letsinger et al., Proc). .Natl.Acad.Sci.USA, 1989, 86, 6553); Cholic acid (Manoharan et al., Bioorg.Med.Chem.Lett., 1994, 4, 1053); Thioethers, e.g., hexyl-S-tritylthiole (Manoharan et al., Ann.NY.Acad.Sci., 1992, 660, 306; Manoharan et al., Bioorg.Med.Chem.Lett., 1993, 3, 2765); Thiocholesterol (Oberhauser et al., Nucl.Acids Res., 1992, 20, 533); aliphatic chains, e.g., dodecanediol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10, 111; Kabanov et al., FEBS Lett., 1990, 259, 327; Svinarchuk et al., Biochimie, 1993, 75, 49); phospholipids, e.g., di-hexadecyl-rac-glycerol or triethylammonium-l,2-di-0-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651; Shea et al., Nucl. Acids Res., 1990, 18, 3777); polyamine or polyethylene glycol chains (Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969); adamantane acetate (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651); palmityl group (Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229); or octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al.,Examples include J. Pharmacol. Exp. Ther., 1996, 277, 923) (all cited references are incorporated herein by reference in their entirety). The targeting portion includes, but is not limited to, naturally occurring molecules or recombinant or synthetic molecules, including, GalNAc or its derivatives (e.g., dimers, trimers, or tetramers of GalNAc or its derivatives), polylysine (PLL), poly-L-aspartic acid, poly-L-glutamic acid, styrene-maleic anhydride copolymer, poly(L-lactide-co-glycated) copolymer, divinyl ether-maleic anhydride copolymer, and N-(2-hydroxypropyl)methacrylate. Docopolymer (HMPA), polyethylene glycol (PEG, e.g., PEG-2K, PEG-5K, PEG-10K, PEG-12K, PEG-15K, PEG-20K, PEG-40K), MPEG, [MPEG]2, polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacrylic acid), N-isopropylacrylamide polymer, polyphosphatidine, polyethyleneimine, cationic group, spermine, spermidine, polyamine, pseudopeptide-polyamine, peptide-mimicking polyamine, Endormer polyamines, arginine, amidine, protamine, cationic lipids, cationic porphyrins, quaternary salts of polyamines, tyrotropin, melanotropin, lectins, glycoproteins, surfactant protein A, mucin, glycosylated polyamino acids, transferrin, bisphosphonates, polyglutamic acid, polyaspartic acid, aptamers, asialofetin, hyaluronic acid, procollagen, immunoglobulins (e.g., antibodies), insulin, transferrin, albumin, glucalbumin conjugate Intercalating agents (e.g., acridine), crosslinking agents (e.g., psoralen, mitomycin C), porphyrins (e.g., TPPC4, texaphylline, saffrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g., EDTA), lipophilic molecules (e.g., steroids, bile acids, cholesterol, cholic acid, adamantane acetate, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, geranyloxyhexyl group,Hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, 03-(oleoyl)lithocholic acid, 03-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine), peptides (e.g., α-helical peptides, amphiphilic peptides, RGD peptides, cell-permeable peptides, endosomal degradation / fusion peptides), alkylating agents, phosphates, amino acids, mercaptosaccharides, polyamino acids, alkyl groups, substituted alkyl groups, radiolabeled markers, enzymes, haptens (e.g., biotin), transport / absorption enhancers Drugs (e.g., naproxen, aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, bisimidazole, histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+ complexes of tetraazamacro rings), dinitrophenyl, HRP, AP, antibodies, hormones and hormone receptors, lectins, carbohydrates, polyunsaturated carbohydrates, vitamins (e.g., vitamin A, vitamin E, vitamin K, vitamin B, e.g., folic acid, B12, riboflavin, biotin, and pyridoxal), vitamin cofactors, lipopolysaccharides, p38 MAP kinase activators, NF-κB activators, taxons, vincristine, vinblastine, cytochalasin, nocodazole, japraquinolide, latranculine A, phalloidin, swinholide A, indanosine, myoserbine, tumor necrosis factor alpha (TNFalpha), interleukin-1 beta, gamma interferon, natural or recombinant low-density lipoprotein (LDL), natural or recombinant high-density lipoprotein (HDL), and cell permeabilizers (e.g., helical cell permeabilizers), peptides and peptide mimetic molecules. Gands, for example, those having naturally occurring or modified peptides such as D or L peptides; α, β, or γ peptides; N-methyl peptides; azapeptides; peptides having one or more amides, i.e., peptides in which peptide bonds are replaced by one or more urea, thiourea, carbamate, or sulfonylurea bonds; or cyclic peptides; amphiphilic peptides, examples including, but not limited to, secropine, lycotoxin, paradaxin, buforin, CPF, bombinin-like peptide (BLP), cathelicidine,Examples include ceratotoxin, S. clava peptide, rhinoceros blenny intestinal antimicrobial peptide (HFIAP), magainin, brevinin-2, dermaceptin, melittin, pleurocidine, H2A peptide, African clawed frog peptide, esculentinis-1, and kaerin. Peptide mimes (also referred to herein as oligopeptide mimes) are molecules that can be folded into a defined three-dimensional structure similar to that of natural peptides. Peptides or peptide mimetic ligands or moieties may be about 5 to 50 amino acid lengths, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acid lengths. In some embodiments, the targeting moiety may be other peptides, such as somatostatin, octeotide, LHRH (luteinizing hormone-releasing hormone), epidermal growth factor receptor (EGFR) binding peptides, aptides or bidentate peptides, RGD-containing peptides, protein scaffolds, such as fibronectin domains, single-domain antibodies, stable scFv, or other homing peptides. As a non-limiting example, the protein or peptide-based targeting moiety may be a protein, such as thrombospongin, tumor necrosis factor (TNF), annexin V, interferon, angiostatin, endostatin, cytokines, transferrin, GM-CSF (granulocyte-macrophage colony-stimulating factor), or growth factors such as vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), platelet-derived growth factor (PDGF), basic fibroblast growth factor (bFGF), and epidermal growth factor (EGF). In some embodiments, the targeting moiety may be an antibody, The protein scaffold may be an antibody fragment, RGD peptide, folic acid, or prostate-specific membrane antigen (PSMA). In some embodiments, the protein scaffold may be an antibody-derived protein scaffold. Non-limiting examples include single-domain antibodies (dAb), nanobodies, single-chain variable fragments (scFv), antigen-binding fragments (Fab), avibodies, minibodies, CH2D domains, Fcab, and bispecific T-cell engager (BiTE) molecules. In some embodiments, the scFv is a stable scFv, where the scFv has ultrastable properties. In some embodiments,Nanobodies may originate from the single variable domain (VHH) of camelid antibodies.

[0168]

[0185] In some embodiments, the targeting moiety recognizes or binds to target cells, markers, or molecules exclusively or predominantly present on the surface of specific cells. For example, the targeting moiety may bind to tumor antigens and induce activators, such as guide RNA-Cas complexes, to malignant cells. In some embodiments, the targeting moiety recognizes intracellular proteins. In some embodiments, the targeting moiety induces the conjugate to specific tissues, cells, or intracellular locations. The targeting moiety may induce the conjugate in culture, throughout the organism, or both. In any case, the targeting moiety may bind to receptors present on or inside the target cell, and this targeting moiety binds to the receptor with effective specificity, affinity, and binding strength. In other embodiments, the targeting moiety targets the conjugate to specific tissues such as the liver, kidney, lung, or pancreas. In other cases, the targeting moiety may induce the conjugate to reticular endothelial or lymphoid cells, or to specialized phagocytic cells such as macrophages or eosinophils. In some embodiments, the targeting moiety may recognize RTK receptors, EGF receptors, serine or threonine kinases, G protein-coupled receptors, methyl CpG-binding proteins, cell surface glycoproteins, cancer stem cell antigens or markers, carbonic anhydrase, cytolytic T lymphocyte antigens, DNA methyltransferases, extracellular enzymes, glycosylphosphatidylinositol-anchored coreceptors, glypican-associated endometrial proteoglycans, heat shock proteins, hypoxia-inducible proteins, multidrug resistance transporters, tumor-associated macrophage markers, tumor-associated carbohydrate antigens, TNF receptor family members, transmembrane proteins, tumor necrosis factor receptor superfamily members, tumor differentiation antigens, zinc-dependent metalloexopeptidases, zinc transporters, sodium-dependent transmembrane transport proteins, members of the SIGLEC family of lectins, or matrix metalloproteinases.

[0169]

[0186] In some embodiments, the conjugate described herein, for example, the guide RNA conjugate, comprises at least one N-acetyl-galactosamine (GalNAc), N-Ac-glucosamine (GluNAc), or mannose (e.g., mannose-6-phosphate). In some embodiments, the targeting portion comprises at least one N-acetyl-galactosamine (GalNAc), N-Ac-glucosamine (GluNAc), or mannose (e.g., mannose-6-phosphate).

[0170]

[0187] In some embodiments, the conjugates described herein comprise one or more targeting moieties comprising N-acetylgalactosamine (GalNAc) or a GalNAc derivative. Such conjugates are also referred to herein as GalNAc conjugates. In some embodiments, the conjugate targets RNA to specific cells, such as liver cells. In some embodiments, the GalNAc derivative may be conjugated via a linker, such as a divalent or trivalent branched linker.

[0171]

[0188] In some embodiments, the conjugate described herein is a carbohydrate conjugate. In some embodiments, the carbohydrate conjugate comprises a monosaccharide. In some embodiments, the monosaccharide is N-acetylgalactosamine (GalNAc). GalNAc and GalNAc derivatives can bind to the asialoglycoprotein receptor (ASGPR), also known as the Ashwell-Morell receptor, which is a lectin primarily expressed in hepatocytes of the liver.

[0172]

[0189] GalNAc conjugates are described, for example, in U.S. Patent No. 8,106,022, the entire contents of which are incorporated herein by reference. In some embodiments, the GalNAc conjugate functions as a ligand that targets guide RNA to specific cells. In some embodiments, the GalNAc conjugate targets guide RNA to hepatocytes by acting, for example, as a ligand to the asialoclycoprotein receptor of hepatocytes (e.g., hepatocytes). In some embodiments, the carbohydrate conjugate comprises one or more GalNAc derivatives. The GalNAc derivatives may be conjugated via a linker, for example, a divalent or trivalent branched linker. In some embodiments, the GalNAc conjugate is conjugated to the 3' end of the sense strand. In some embodiments, the GalNAc ligand is conjugated to an activator (e.g., the 3' end of the guide RNA) via a linker, for example, a linker described herein. In some other embodiments, the GalNAc ligand is conjugated to the activator (e.g., to the 5' end of the guide RNA) via a linker, for example, the linker described herein.

[0173]

[0190] In some embodiments, the GalNAc ligand may be conjugated to a shorter oligonucleotide via a linker and spacer, where the shorter oligonucleotide conjugate is complementary to a segment of RNA. The RNA encompasses all lengths, structures, and forms of RNA molecules, including, for example, the mRNA of interest and the guide RNA of interest. In some embodiments, the short-mer-GalNAc conjugate and RNA constitute a pharmaceutical composition. For example, shorter GalNAc-binding oligonucleotides and RNA, e.g., coupling sequences, may together constitute a pharmaceutical composition via WHC bonds of complementary nucleotides. The shorter oligonucleotide conjugate may include nucleotide lengths of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, or greater than 50. In some embodiments, the coupling sequence may have a length of 15 to 40 nucleotides. In some embodiments, the coupling sequence may have a length of 19 to 30 nucleotides. In some embodiments, the coupling sequence may have a length of 20 to 24 nucleotides.

[0174]

[0191] In some embodiments, pharmaceutical compositions comprising one or more GalNAc conjugate shorter oligonucleotides and one or more RNAs are provided herein. In some embodiments, a single GalNAc conjugate shorter oligonucleotide, for example, a GalNAc conjugate RNA, may be complementary to multiple oligonucleotide segments within the RNA. For example, a single GalNAc conjugate shorter may contain coupling sequences complementary to multiple segments within the RNA. In some embodiments, multiple GalNAc ligand conjugate shorter oligonucleotides complementary to multiple oligonucleotide segments within the RNA may constitute a pharmaceutical composition.

[0175]

[0192] In certain embodiments, the targeting portion of the conjugate described herein includes a ligand having the structure shown in Table 1 below.

[0176] [Table 1-1]

[0177] [Table 1-2]

[0178] [Table 1-3]

[0179]

[0193] As shown in Table 1, each of t, n, p, q, and m is independently 0 or an integer from 1 to 30. In some embodiments, each of t, n, p, q, and m in Table 1 is independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 13, 15, 16, 17, 18, 19, or 20. In some embodiments, each of t, n, p, q, and m in Table 1 is independently 0, 1, 2, 3, 4, or 5. In some embodiments, each of t, n, p, q, and m in Table 1 is independently 0, 1, 2, or 3. In some embodiments, each of t, n, p, q, and m in Table 1 is independently 1 or 2. Therefore, it should be understood that in some embodiments of the compounds in Table 1, t is intended to be between 0 and 10. In some embodiments, t is between 1 and 5. In some embodiments, t is 10 to 20. In some embodiments, t is 1 or 2. In some embodiments, t is 1. In some embodiments, t is 2. In some embodiments of the compounds in Table 1, m is 0 to 10. In some embodiments, m is 1 to 5. In some embodiments, m is 10 to 20. In some embodiments, m is 1 or 2. In some embodiments, m is 1. In some embodiments, m is 2. In some embodiments of the compounds in Table 1, n is 0 to 10. In some embodiments, n is 1 to 5. In some embodiments, n is 10 to 20. In some embodiments, n is 1 or 2. In some embodiments, n is 1. In some embodiments, n is 2. In some embodiments of the compounds in Table 1, p is 0 to 10. In some embodiments, p is 1 to 5. In some embodiments, p is 10 to 20. In some embodiments, p is 1 or 2. In some embodiments, p is 1. In some embodiments, p is 2. In some embodiments of the compounds in Table 1, q is 0 to 10. In some embodiments, q is 1 to 5. In some embodiments, q is 10 to 20. In some embodiments, q is 1 or 2. In some embodiments, q is 1. In some embodiments, q is 2.In some embodiments, each R is OH, NHC(O)CH3, or a combination thereof. In some embodiments, in compounds (1-1a), (1-2a), (1-3a), (1-4a), (1-5a), (1-6a), (1-7a), (1-8a), (1-9a), (1-10a), (1-11a), (1-12a), (1-16a), (1-21a), (1-22a), (1-23a), (1-24a), (1-25a), and (1-26a) of Table 1, x is 0 or an integer from 1 to 5. In some embodiments, in compounds (1-1b), (1-2b), (1-3b), (1-4b), (1-5b), (1-6b), (1-7b), (1-8b), (1-9b), (1-10b), (1-11b), (1-12b), (1-16b), (1-21b), (1-22b), (1-23b), (1-24b), (1-25b), and (1-26b) of Table 1, x is 0 or an integer from 1 to 5. In some embodiments, x is 1. In some embodiments, x is 2. In some embodiments, x is 0. In some embodiments, x is 3. In some embodiments, x is 4. In some embodiments, x is 5.

[0180]

[0194] The targeting moiety can be conjugated to a nucleic acid base, a sugar moiety, or a nucleic acid, such as a guide RNA or mRNA nucleoside bond. Conjugation to purine nucleic acid bases or their derivatives can occur at any position, including intraring and extraring atoms. In some embodiments, the 2, 6, 7, or 8 positions of the purine nucleic acid base are conjugated to a moiety. Conjugation to pyrimidine nucleic acid bases or their derivatives can also occur at any position. In some embodiments, the 2, 5, and 6 positions of the pyrimidine nucleic acid base may be substituted by a moiety. When a moiety conjugates to a nucleic acid base, a preferred position is one that does not interfere with hybridization, i.e., does not interfere with the hydrogen bonding interactions necessary for base pairing.

[0181]

[0195] Conjugation of the sugar moiety of a nucleoside can occur at any carbon atom. Examples of carbon atoms in the sugar moiety that can bond to the conjugate moiety include the 2', 3', and 5' carbon atoms. The gamma position can also bond to the conjugate moiety, such as a debasic residue. Nucleoside bonds can also have a conjugate moiety. In the case of phosphorus-containing bonds (e.g., phosphodiesters, phosphorothioates, phosphorodithioates, phosphoramidates, etc.), the conjugate moiety can bond directly to the phosphorus atom or to an O, N, or S atom bonded to the phosphorus atom. In the case of amine or amide-containing nucleoside bonds (e.g., PNA), the conjugate moiety can bond to the nitrogen atom of the amine or amide or to an adjacent carbon atom.

[0182]

[0196] Numerous methods exist for preparing oligonucleotide conjugates. Generally, oligonucleotides are bonded to a conjugate moiety by contacting a reactive group on the oligonucleotide (e.g., OH, SH, amine, carboxyl, aldehyde, etc.) with a reactive group on the conjugate moiety. In some embodiments, one reactive group is electrophilic and the other is nucleophilic. For example, the electrophile may be a carbonyl-containing functional group, and the nucleophile may be an amine or a thiol. Methods for conjugating nucleic acids and related oligomeric compounds with and without linking groups are well described in the literature, e.g., Manoharan in Antisense Research and Application, edited by Crooke and LeBleu, CRC Press, Boca Raton, Fls., 1993, Chapter 17, which are incorporated herein by reference in their entirety.

[0183]

[0197] The targeting moiety may bind to the activators or therapeutic nucleic acids described herein, such as guide RNA, via RNA-RNA or RNA-DNA base pairing and hybridization. While not intended to be bound by any theory, the targeting moiety may include a coupling sequence that recognizes or binds to the activator, e.g., guide RNA or mRNA. In some embodiments, the targeting moiety includes a coupling sequence that can hybridize to the 5', 3', or intermediate portion of the guide RNA. The guide RNA hybridizing with the coupling sequence may include an elongation segment. For example, the coupling sequence may hybridize to the elongation segment of the guide RNA, thereby directing the guide RNA to a desired in vivo, exovivo, intercellular, or intracellular location, while the guide RNA function, such as interaction with CRISPR enzymes or binding to target sequences, remains unaffected. In some embodiments, the guide RNA includes an elongation segment containing a polynucleotide tail. In some embodiments, the guide nucleic acid includes a poly(A) tail, a poly(U) tail, or a poly(T) tail, each capable of hybridizing with a poly(A) tail of the coupling sequence. In some embodiments, the guide nucleic acid may be a guide RNA containing a (A)n or (U)n sequence. In some embodiments, the guide nucleic acid may contain DNA and may contain a (A)n or (T)n sequence. In some embodiments, the coupling sequence may contain a (A)n (SEQ ID NO: 115), (U)n (SEQ ID NO: 116), or (T)n (SEQ ID NO: 117) sequence. As readily available, n can be any integer from 1 to 200.

[0184]

[0198] The coupling sequence may share sequence identity or complementarity with the nucleic acid activator or a portion thereof. In some embodiments, the coupling sequence may share at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity with the guide RNA or a portion thereof described herein. In some embodiments, the coupling sequence may share at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity with the complementary sequence of the guide RNA described herein, or partial complementarity of such guide RNA.In some mechanisms, the coupling sequence is at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, at least 40, at least 41, at least 42, at least 43, at least 44, at least 45, at least 46, at least 47, at least 48, at least 49, at least 50, at least 51 of the guide RNA. This may include identity or complementarity with 52, at least 53, at least 54, at least 55, at least 56, at least 57, at least 58, at least 59, at least 60, at least 61, at least 62, at least 63, at least 64, at least 65, at least 66, at least 67, at least 68, at least 69, at least 70, at least 71, at least 72, at least 73, at least 74, at least 75, at least 76, at least 77, at least 78, at least 79, at least 80, at least 81, at least 82, at least 83, at least 84, at least 85, at least 86, at least 87, at least 88, at least 89, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, at least 99, or at least 100 consecutive nucleic acid bases.

[0185]

[0199] In some embodiments, the targeting portion may include or be associated with a chemically modified coupling sequence. In some embodiments, the coupling sequence includes an elongation portion that hybridizes with a therapeutic nucleic acid, e.g., guide RNA, or a portion thereof. In some embodiments, the elongation portion of the coupling sequence may be chemically modified. In some embodiments, the therapeutic nucleic acid, e.g., guide RNA, may include an elongation portion. In some embodiments, the elongation portion of the guide RNA may be chemically modified. Non-limiting examples of elongation portions of guide RNA and complementary or substantially complementary coupling sequence elongations are shown in Table 2 below.

[0186] [Table 2-1]

[0187] [Table 2-2]

[0188]

[0200] As used in Table 2, uppercase A, C, G, and U refer to ribonucleotides containing the nucleic acid bases adenine, cytosine, guanidine, and uracil, respectively. Lowercase a, c, g, and u refer to modified (e.g., 2'-OMe or 2'-MOE) ribonucleotides containing the nucleic acid bases adenine, cytosine, guanidine, and uracil, respectively. The letter "T" refers to thymidine or deoxythymidine. The letter "s" refers to phosphorus-containing bonds (such as phosphorothioate (PS) bonds, phosphodiester bonds, or phosphorodithioate bonds). As used in Table 2, "(GalNAc)" refers to a targeting moiety, such as a targeting moiety containing GalNAc or a derivative thereof. As used in Table 2, “(GalNAc)” also encompasses a targeting moiety including multiple GalNAc structures or derivatives thereof, e.g., dimers, trimers, tetramers or derivatives of GalNAc (including the GalNAc structures listed in Table 1). In some embodiments, “s” represents a phosphorothioate (PS) bond. As disclosed herein, nucleotide sequences and modification patterns include all lengths, structures, and RNA types or fragments thereof, CRISPR guide RNAs, e.g., sgRNA, dual guide RNA, or mRNA. For example, the nucleotide sequences and modification patterns listed in Table 2 above may represent RNA sequences and modification patterns in single guide RNA, dual guide RNA, nuclease mRNA, or any fragment or segment thereof.

[0189]

[0201] Table 3 below shows non-limiting examples of receptor targeting moieties and guide RNAs conjugated to coupling sequences containing targeting moieties. The (GalNAc) conjugate moiety is covalently bound to the 3' and / or 5' ends of the guide RNA, and / or covalently bound to the 3' and / or 5' ends of the guide RNA, with an additional nucleotide spacer between this ligand and the guide RNA. Guide RNA conjugates 3-1 and 3-2 (Table 3) are representative examples of direct conjugation of GalNAc ligand to guide RNA. The guide RNA is conjugated from 3-10 to 3-21, and this GalNAc ligand is conjugated to the 3' / 5' ends of the additional 3' and / or 5' nucleotide spacer. The guide RNA strand is extended to the desired number of nucleotides to the 3' end, the 5' end, or both ends. (GalNAc) conjugates to the 3', 5', or both ends of an oligonucleotide complementary to the elongating nucleotide of the guide RNA chain to form a complementary double helix linked to a single chemical. 3-3 to 3-8 of this conjugate design are constructed from an elongating nucleotide spacer and a spacer complementary strand containing the GalNAc ligand. As used in Table 3, uppercase A, C, G, and U refer to ribonucleotides containing the nucleic acid bases adenine, cytosine, guanidine, and uracil, respectively. Lowercase a, c, g, and u refer to modified (e.g., 2'-OMe or 2'-MOE) ribonucleotides containing the nucleic acid bases adenine, cytosine, guanidine, and uracil, respectively. The letter "T" refers to thymidine or deoxythymidine. The letter "s" refers to a phosphate bond (such as a phosphorothioate (PS) bond, phosphodiester bond, or phosphorodithioate bond). In some embodiments, "s" represents a PS bond. As used in Table 3, "(GalNAc)" refers to a targeting moiety, such as one containing GalNAc or a derivative thereof. As used in Table 3, "(GalNAc)" also encompasses a targeting moiety containing multiple GalNAc structures or derivatives thereof, such as dimers, trimers, tetramers, or derivatives thereof of GalNAc.

[0190] [Table 3-1]

[0191] [Table 3-2]

[0192] [Table 3-3]

[0193]

[0202] As disclosed herein, nucleotide sequences and modification patterns include RNA or its fragments of all lengths, structures, and types, CRISPR guide RNAs, such as sgRNA, dual guide RNA, or mRNA. For example, the nucleotide sequences and modification patterns listed in Table 3 above may represent RNA sequences and modification patterns in single guide RNA, dual guide RNA, nuclease mRNA, or any fragment or segment thereof.

[0194]

[0203] A targeting moiety may be bound to the nucleic acid described herein via a carrier. The carrier may include (i) at least one “skeletal binding point,” preferably two “skeletal binding points,” and (ii) at least one “tethering binding point.” As used herein, “skeletal binding point” refers to a binding point that is available for and suitable for the incorporation of a carrier monomer into a functional group, e.g., a hydroxyl group, or generally into the backbone of an oligonucleotide, e.g., a phosphate or modified phosphate, e.g., a sulfur-containing backbone. A “tethering binding point” (TAP) refers to an atom of the carrier monomer to which the selected moiety is bound, e.g., a carbon atom or a heteroatom (different from the atom providing the skeletal binding point). The selected moiety may be, for example, a carbohydrate, e.g., a monosaccharide, disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, or polysaccharide. Optionally, the selected moiety is bound to the carrier monomer by an intervening tether. Thus, the carrier often includes a functional group such as an amino group, or generally provides a binding point suitable for the incorporation or tethering of another chemical substance, such as a ligand, into a constituent atom. Representative U.S. patents teaching the preparation of nucleic acid conjugates include, but are not limited to, U.S. Patents No. 4,828,979; No. 4,948,882; No. 5,218,105; No. 5,525,465; No. 5,541,313; No. 5,545,730; No. 5,552,538; No. 5,578,717; No. 5,580,731; No. 5,580,731; No. 5,591,584; No. 5,109,124; No. 5,118,802; No. 5,138,045; and others, the contents of which are incorporated herein by reference in their entirety. 5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; No. 4,587,044; No. 4,605,735; No. 4,667,025; No. 4,762,779; No. 4,789,737; No. 4,824,941; No. 4,835,263; No. 4,876,335; No. 4,904,582; No. 4,958,013; Same No. 5,082,830; Same No. 5,112,963; Same No. 5,214,136; Same No. 5,082,830; Same No. 5,112,963;Same No. 5,149,782; Same No. 5,214,136; Same No. 5,245,022; Same No. 5,254,469; Same No. 5,258,506 ; Same No. 5,262,536; Same No. 5,272,250; Same No. 5,292,873; Same No. 5,317,098; Same No. 5,371,241 No. 5,391,723; No. 5,416,203, No. 5,451,463; No. 5,510,475; No. 5,512,66 No. 7; No. 5,514,785; No. 5,565,552; No. 5,567,810; No. 5,574,142; No. 5,585,4 No. 81; No. 5,587,371; No. 5,595,726; No. 5,597,696; No. 5,599,923; No. 5,599, No. 928; No. 5,672,662; No. 5,688,941; No. 5,714,166; No. 6,153,737; No. 6,172 Examples include No. 208; Nos. 6,300,319; Nos. 6,335,434; Nos. 6,335,437; Nos. 6,395,437; Nos. 6,444,806; Nos. 6,486,308; Nos. 6,525,031; Nos. 6,528,631; and Nos. 6,559,279.

[0195]

[0204] The targeting moiety may be bound to an activator, such as guide RNA, via a linker. The linker may bind to one or more activators and targeting moiety ligands to form a conjugate, which releases at least one activator, such as guide RNA or a guide RNA-Cas complex, upon delivery to target cells. The linker may be bound to the targeting moiety and activator by a functional group independently selected from ester bonds, disulfides, amides, acylhydrazones, ethers, carbamates, carbonates, and ureas. Alternatively, the linker may be bound to either the targeting moiety or the activator by an inseparable group, such as one provided by a conjugation between a thiol and a maleimide, or between an azide and an alkyne. In some embodiments, the targeting moiety comprises one or more linkers. In some embodiments, one or more linkers described herein connect a portion of the targeting moiety to a different portion of the targeting moiety. For example, the targeting moiety may comprise two, three, four, five or more GalNAc structures or derivatives thereof connected by one or more linkers. In some embodiments, two or more GalNAc structures or derivatives thereof in the targeting moiety are linked by one or more non-cleavable linkers. In some embodiments, the conjugate described herein includes an activator directly bound to the sugar moiety of the targeting moiety.

[0196]

[0205] Each linker may independently contain one or more functional groups selected from the group consisting of ethylene glycol, propylene glycol, amide, ester, ether, alkyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl, where each of the alkyl, alkenyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl groups is independently selected from halogen, cyano, nitro, hydroxyl, carboxyl, carbamoyl, ether, alkoxy, aryloxy, amino, amide, carbamate, alkyl, alkenyl, alkynyl, aryl, arylalkyl, cycloalkyl, heteroaryl, and heterocyclyl. The linker may be substituted with one or more selected groups, where each of the carboxyl, carbamoyl, ether, alkoxy, aryloxy, amino, amide, carbamate, alkyl, alkenyl, alkynyl, aryl, arylalkyl, cycloalkyl, heteroaryl, or heterocyclyl groups is optionally substituted with one or more groups independently selected from halogen, cyano, nitro, hydroxyl, carboxyl, carbamoyl, ether, alkoxy, aryloxy, amino, amide, carbamate, alkyl, alkenyl, alkynyl, aryl, arylalkyl, cycloalkyl, heteroaryl, or heterocyclyl groups. In some embodiments, the linker independently comprises a phosphate, phosphorothioate, amide, ether, oxime, hydrazine, or carbamate. As intended herein, in some embodiments, the targeting conjugate of formula (V), (VI), (VIa), or (VIb) should be understood to include the linker described herein. For example, groups R and L 1 ~L 12 Each of these may include one or more linkers.

[0197]

[0206] In some embodiments, the linker is C1~C 10 Linear alkyl, C1-C 10 Linear O-alkyl, C1~C 10 Linear substituted alkyl, C1~C 10 Linear substituted O-alkyl, C4~C13 Branched alkyl, C4~C 13 Branched chain O-alkyl, C2~C 12 Linear alkenyls, C2~C 12 Linear O-alkenyl, aralkyl, C3~C 12 Linear substituted alkenyls, C3-C 12 The following may be independently included: linearly substituted O-alkenyls, polyethylene glycol, polylactic acid, polyglycolic acid, poly(lactide-co-glycolide), polycarprolactone, polycyanoacrylate, ketone, aryl, heterocyclic, succinate ester, amino acid, aromatic group, ether, crown ether, urea, thiourea, amide, purine, pyrimidine, bipyridine, indole derivatives acting as crosslinking agents, chelating agents, aldehydes, ketones, bisamines, bisalcohols, heterocyclic structures, aziline, disulfide, thioether, hydrazone, and combinations thereof. For example, the linker may be a C3 linear alkyl or a ketone. The alkyl chain of the linker may be substituted with one or more substituents or heteroatoms. In some embodiments, the alkyl chain of the linker may be optionally interrupted by one or more atoms or groups selected from -O-, -C(=O)-, -NR, -OC(=O)-NR-, -S-, and -SS-.

[0198]

[0207] In some embodiments, the linker may be cleavable and release an activator upon cleavage. The cleavable functional group may be hydrolyzed in vivo or may be designed to be enzymatically hydrolyzed by, for example, cathepsin B. As used herein, “cleavable” linker refers to any linker that can be cleaved physically or chemically. Examples of physical cleavage may be cleavage by light, radioactive emission, or heat, while examples of chemical cleavage include cleavage by redox reactions, hydrolysis, or pH-dependent cleavage.

[0199]

[0208] A linker is a direct bond, or an atom such as oxygen or sulfur, NR 1Units such as C(O), C(O)NH, SO, SO2, SO2NH, or chains of atoms, for example, but not limited to substituted or unsubstituted alkyls, substituted or unsubstituted alkenyls, substituted or unsubstituted alkynyls, arylalkyls, arylalkenyls, arylalkynyls, heteroarylalkyls, heteroarylalkenyls, heteroarylalkynyls, heterocyclylalkyls, heterocyclylalkenyls, heterocyclylalkynyls, heterocyclylalkynyls, aryls, heteroaryls, heterocyclyls, cycloalkyls, cycloalkenyls, alkylarylalkyls, alkylarylalkenyls, alkylarylalkynyls, alkenylarylalkyls, alkenylarylalkenyls, alkenylarylalkynyls, alkenylarylalkynyls, alkynylarylalkyls, alkynylarylalkenyls, alkynylarylalkynyls, alkylheteroarylalkyls, alkylheteroarylalkenyls, alkylheteroarylalkynyls, alkenylheteroarylalkyls, alkenylheteroarylalkyls The following may be included: alkenyl, alkenyl heteroarylalkynyl, alkynyl heteroarylalkyl, alkynyl heteroarylalkenyl, alkynyl heteroarylalkynyl, alkylheterocyclylalkyl, alkylheterocyclylalkenyl, alkylheterocyclylalkynyl, alkenyl heterocyclylalkyl, alkenyl heterocyclylalkenyl, alkenyl heterocyclylalkynyl, alkynyl heterocyclylalkyl, alkynyl heterocyclylalkenyl, alkynyl heterocyclylalkynyl, alkylaryl, alkenylaryl, alkynylaryl, alkylheteroaryl, alkenyl heteroaryl, alkynyl heteroaryl, and alkynyl heteroaryl, where one or more methylene groups may be interrupted or terminated by O, S, S(O), SO2, N(R'), C(O), substituted or unsubstituted aryl groups, substituted or unsubstituted heteroaryl groups, or substituted or unsubstituted heterocycles; where R' is hydrogen, acyl, aliphatic, or substituted aliphatic. In one embodiment, the linker is 1 to 24 atoms, preferably 4 to 24 atoms, preferably 6 to 18 atoms, more preferably 8 to 18 atoms, and most preferably 8 to 16 atoms.

[0200]

[0209] In one embodiment, the linker is -[(PQ”-R)qX-(P'Q'”-R')q']q”-T-, where P, R, T, P', R' and T are, for each presence, independently non-existent or non-existent CO, NH, O, S, OC(O), NHC(O), CH2, CH2NH, CH2O; NHCH(Ra)C(O), -C(O)-CH(Ra)-NH-, CH-NO,

[0201] [ka]

[0202] or heterocyclyl; Q'' and Q''' are, for each presence, independently absent or -(CH2)n-, -C(R1)(R2)(CH2)n-, -(CH2)nC(R1)(R2)-, -(CH2CH2O)mCH2CH2-, or -(CH2CH2O)mCH2CH2NH-; X is absent or a cleavable linking group; Ra is H or an amino acid side chain; R1 and R2 are, for each presence, independently H, CH3, OH, SH, or N(RN)2; RN is, for each presence, independently H, methyl, ethyl, propyl, isopropyl, butyl, or benzyl; q, q', and q'' are, for each presence, independently 0 to 20, where this repeating unit may be the same or different; n is, for each presence, independently 1 to 20; and m is, for each presence, independently 0 to 50.

[0203]

[0210] In one embodiment, the linker comprises at least one cleavable linking group. In a particular embodiment, the linker is a branched linker. The branching point of the branched linker may be at least trivalent, but may be a tetravalent, pentavalent or hexavalent atom, or a group exhibiting multiple such valencies. In a particular embodiment, the branching point is -N, -N(O)-C, -OC, -SC, -SS-C, -C(O)N(O)-C, -OC(O)N(O)-C, -N(O)C(O)-C, or -N(O)C(O)OC; where Q is independently, for each presence, H or optionally substituted alkyl. In other embodiments, the branching point is glycerol or a glycerol derivative.

[0204]

[0211] In one embodiment, the linker may be cleaved by an enzyme. In a non-limiting example, the linker may be a polypeptide moiety cleaved by an intracellular peptidase (e.g., AA in WO2010093395 to Govindan, the contents of which are incorporated herein by reference in their entirety). Govindan teaches that AA in the linker may be a dipeptide, tripeptide, or tetrapeptide such as Ala-Leu, Leu-Ala-Leu, and Ala-Leu-Ala-Leu. In another example, the cleavable linker may be a branched peptide. A branched peptide linker may contain two or more amino acid moieties that provide an enzymatic cleavage site. Any branched peptide linker disclosed in Dubowchik's WO1998019705 (the contents of which are incorporated herein by reference in their entirety) may be used as a linker in the conjugate of this disclosure. As an alternative, the linker may include a lysosome-cleavable polypeptide disclosed in U.S. Patent No. 8,877,901 to Govindan et al., the contents of which are incorporated in their entirety by reference herein. As an alternative, the linker may include a protein peptide sequence that is selectively enzymatically cleavable by a tumor-associated protease, such as any Y and Z structures disclosed in U.S. Patent No. 6,214,345 to Firestone et al., the contents of which are incorporated in their entirety by reference herein.

[0205]

[0212] In some embodiments, the linker may include a cleavable linking group. A cleavable linking group is a group that is sufficiently stable extracellularly but is cleaved upon entering a target cell, releasing the two parts held together by the linker. In preferred embodiments, the cleavable linking group is cleaved at least 10 times, preferably at least 100 times faster, inside the target cell or under first reference conditions (e.g., which may be selected to mimic or represent intracellular conditions) or second reference conditions (e.g., which may be selected to mimic or represent conditions found in blood or serum) than in the blood of the subject. The cleavable linking group may be susceptible to the presence of cleavage agents, such as pH, redox potential, or degradation molecules. Generally, cleavage agents are more preferentially found inside cells or at higher levels or activity than in serum or blood. Examples of such degrading agents include redox agents that are selected for specific substrates or that do not have substrate specificity, such as oxidases or reductases or reducing agents such as mercaptans present in cells (which can degrade redox-cleavable linking groups by reduction); esterases; endosomes or drugs that can create an acidic environment, such as those that lower the pH to 5 or below; and enzymes that can hydrolyze or degrade acid-cleavable linking groups by acting as common acids, peptidases (which may be substrate-specific), and phosphatases.

[0206]

[0213] Cleavable linking groups, such as disulfide bonds, can be susceptible to pH fluctuations. While human serum has a pH of 7.4, the average intracellular pH is slightly lower, ranging from approximately 7.1 to 7.3. Endosomes have a more acidic pH, ranging from 5.5 to 6.0, and lysosomes have an even more acidic pH, around 5.0. Some linkers possess cleavable linking groups that are cleaved at a favorable pH, thereby releasing cationic lipids from intracellular ligands or into desired compartments within the cell.

[0207]

[0214] Linkers may contain cleavable linking groups that can be cleaved by specific enzymes. The type of cleavable linking group incorporated into a linker may depend on the target cell. For example, liver targeting ligands may bind to cationic lipids via linkers containing ester groups. Because liver cells are rich in esterases, linkers are cleaved more efficiently in liver cells than in cell types that are not rich in esterases. Other cell types rich in esterases include lung, renal cortex, and testicular cells.

[0208]

[0215] One class of cleavable linking groups is the redox cleavable linking group, which is cleaved by reduction or oxidation. An example of a reductively cleavable linking group is the disulfide linking group (-SS-). To determine whether a candidate cleavable linking group is a suitable “reductively cleavable linking group,” or whether it is suitable for use with, for example, a particular RNA moiety and a particular targeting agent, the methods described herein may be examined. For example, a candidate may be evaluated by incubation with dithiothreitol (DTT) or other reducing agents using reagents known in the art that mimic the cleavage rate observed in cells, e.g., target cells. Candidates may also be evaluated under conditions selected to mimic blood or serum conditions. In a preferred embodiment, the candidate compound is cleaved up to 10% in blood. In a preferred embodiment, a useful candidate compound is degraded at least 2, 4, 10, or 100 times faster intracellularly (or under in vitro conditions selected to mimic intracellular conditions) compared to in blood (or under in vitro conditions selected to mimic extracellular conditions). The cleavage rate of candidate compounds can be determined using standard enzyme kinetic assays under conditions selected to mimic intracellular media and compared to conditions selected to mimic extracellular media.

[0209]

[0216] In some embodiments, the linker may include a phosphate-based cleavable linking group. The phosphate-based cleavable linking group is cleaved by agents that decompose or hydrolyze the phosphate group. Examples of agents that cleave phosphate groups in cells include enzymes such as intracellular phosphatases. Examples of phosphate-based linking groups (i.e., phosphorus-containing linkers) are -P(O)(ORk)-, O-, -OP(S)(ORk)-O-, -OP(S)(SRk)-O-, -SP(O)(ORk)-O-, -OP(O)(ORk)-S-, -SP(O)(ORk)-S-, -OP(S)(ORk)-S-, -SP(S)(ORk)-O-, -OP(O)(Rk)-O-, -OP(S)(Rk)-O-, -SP(O)(Rk)-O-, -SP(S)(Rk)-O-, -SP(O)(Rk)-S-, -OP(S)(Rk)-S-. In some embodiments, the phosphate-based linking groups are -OP(O)(OH)-O-, -OP(S)(OH)-O-, -OP(S)(SH)-O-, -SP(O)(OH)-O-, -OP(O)(OH)-S-, -SP(O)(OH)-S-, -OP(S)(OH)-O-, -SP(S)(OH)-O-, -OP(O)(H)-O-, -OP(S)(H)-O-, -SP(O)(H)-O-, -SP(S)(H)-O-, -SP(O)(H)-S-, -OP(S)(H)-SS-. In some embodiments, the phosphate-based linker is -OP(O)(OH)-O-.

[0210]

[0217] In some embodiments, the linker may include an acid-cleavable linking group. An acid-cleavable linking group is a linking group that is cleaved under acidic conditions. In preferred embodiments, the acid-cleavable linking group is cleaved in an acidic environment with a pH of about 6.5 or less (e.g., about 6.0, 5.5, 5.0 or less), or by a drug such as an enzyme that can act as a general acid. Within cells, certain low-pH organelles such as endosomes and lysosomes may provide an environment for cleaving acid-cleavable linking groups. Examples of acid-cleavable linking groups, but not limited to, include hydrazones, esters, and amino acid esters. The acid-cleavable group may have the general formula -C=NN-, C(O)O, or -OC(O). In preferred embodiments, the carbon bonded to the oxygen of the ester (alkoxy group) is an aryl group, a substituted alkyl group, or a tertiary alkyl group such as dimethylpentyl or t-butyl. These candidates may be evaluated using methods similar to those described above.

[0211]

[0218] In some embodiments, the linker may include an ester-based linking group. The ester-based cleavable linking group is cleaved by enzymes such as esterases and amidases in cells. Examples of ester-based cleavable linking groups include, but are not limited to, esters of alkylene, alkenylene, and alkynylene groups. The ester-cleavable linking group has the general formula -C(O)O- or -OC(O)-. These candidates can be evaluated using methods similar to those described above.

[0212]

[0219] In some embodiments, the linker may include a peptide-based linking group. Peptide-based cleavable linking groups are cleaved by enzymes such as peptidases and proteases within the cell. Peptide-based cleavable linking groups are peptide bonds formed between amino acids to produce oligopeptides (e.g., dipeptides, tripeptides, etc.) and polypeptides. Peptide-based cleavable linking groups do not include amide groups (-C(O)NH-). Amide groups can be formed between any alkylene, alkenylene, or alkynelene. Peptide bonds are a special type of amide bond formed between amino acids to produce peptides and proteins. Peptide-based cleavable linking groups are generally limited to peptide bonds (i.e., amide bonds) formed between amino acids that produce peptides and proteins, and do not include entire amide functional groups. Peptide-based cleavable linking groups have the general formula -NHCHRAC(O)NHCHRBC(O)-, where RA and RB are the R groups of two adjacent amino acids.

[0213]

[0220] Linkers containing peptide bonds may be used when targeting peptidase-rich cell types such as liver cells and synovial cells.

[0221] Generally, the suitability of a candidate cleavable linking group can be evaluated by testing the ability of a degrading agent (or condition) to cleave the candidate linking group. It would also be desirable to test the candidate cleavable linking group for its ability to resist cleavage in blood or other non-target tissues. Thus, if a first state is selected to demonstrate cleavage in target cells and a second state is selected to demonstrate cleavage in other tissues or body fluids, e.g., blood or serum, the relative sensitivity to cleavage between the first and second states can be determined. Evaluations can be performed in cell-free systems, intracellular, cell cultures, organ or tissue cultures, or whole animals. It may be helpful to perform initial evaluations in cell-free or culture conditions and confirm them with further evaluations in whole animals. In preferred embodiments, a useful candidate compound is cleaved at least 2, 4, 10, or 100 times faster intracellularly (or under in vitro conditions selected to mimic intracellular conditions) compared with blood or serum (or under in vitro conditions selected to mimic extracellular conditions).

[0214]

[0222] In some embodiments, the conjugate described herein is of formula (I),

[0215] [ka]

[0216] It includes the structure, In the formula, each X is independently H or a protecting group, and W represents an activator or coupling sequence. One or more linkers of formula (I) each independently comprise the linkers described herein. In some embodiments, each protecting group of formula (I) is independently 4-acetoxy-2,2-dimethylbutanoyl (ADMB), 3-(2-hydroxyphenyl)-3,3-dimethylpropanoate (DMBPP), 3-(2-hydroxy-4,6-dimethylphenyl)-3,3-dimethylpropanoate group (TMBPP), methylsulfonylethoxycarbonyl (Msc), 2,2-dimethyltrimethylene (DMTM) phosphate, 2-pyridylmethyl, ethyl mandelate, (phenylthiomethyl)benzyl, pen The protecting groups are selected from tafluoropropionyl (PFP), benzoyl (Bz), acetyl (Ac), basilosamine (Bac), benzyl (Bn), 1-benzenesulfinylpiperidine (BSP), tert-butoxycarbonyl (Boc), benzylidene acetal, propargyl, naphthylpropargyl, carbonate, dichloroacetyl, tert-butylsilylene, tetraisopropyldisiloxanylidene (TIPDS), methoxybenzyl (PMB), xylylene, and p-methoxyphenyl (MP). Exemplary protecting groups are further disclosed in Guo et al., Molecules 2010, 15, 7235-7265, the whole of which is incorporated herein by reference. In some embodiments, X is H. In some embodiments, each X is independently selected from H and Bz. In some embodiments of formula (I), W is an activator. In some embodiments, W is a nucleic acid. In some embodiments, W is gRNA. In some embodiments, W is a single-stranded, double-stranded, partially double-stranded, or hairpin stem-loop nucleic acid. In some embodiments of formula (I), W is a coupling sequence. In some embodiments, W comprises an RNA or DNA sequence. W may comprise one or more modified DNA or RNA bases. The nucleic acid bases may comprise any chemical modifications described herein. In some embodiments, the nucleic acid bases comprise 2'-OH or 2'-OMe modifications.For example, W may include one or more 2'-OMe modified adenine, cytosine, guanidine, and uracil, referred to as (a), (c), (g), or (u). In some embodiments, the modified RNA, e.g., gRNA or mRNA, contains at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 50 or more modified nucleic acid bases. In some embodiments, the modified RNA contains one or more modified nucleic acid bases near the 5' end, near the 3' end, or in the middle of the sequence. The modified nucleic acid bases in the modified RNA may or may not be consecutive. In some embodiments, the modified RNA contains one or more 2'-OMe modifications scattered along the length of the sequence. In some embodiments, the modified RNA contains one or more 2'OH modifications scattered along the length of the sequence. In some embodiments, the modified RNA contains alternating 2'-OH and 2'OMe modifications. In some embodiments, W comprises (A)n, (T)n, (U)n, (a)n, or (u)n, where n is an integer greater than or equal to 3, a is 2'-O-methyladenosine (2'-OMe A), and u is 2'-O-methyluridine (2'-OMe U). In some embodiments, W comprises (u)n, where n is an integer from 3 to 50 (SEQ ID NO: 118). In some embodiments, W comprises (u)n, where n is an integer from 3 to 20 (SEQ ID NO: 119) or from 3 to 15 (SEQ ID NO: 120). In some embodiments, W comprises one or more nucleotide sequences complementary to the coupling sequence. In some embodiments, W comprises one or more guanines or cytidines. In some embodiments, W comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 50 or more guanines or cytidines. In some embodiments, one or more guanines or cytidines are complementary to one or more cytinidines or guanines in the coupling sequence. In some embodiments, the guanines or cytidines are located at the end of W or the coupling sequence.While not intended to be bound by any theory, guanine-cytidine pairings are thought to form a “GC lock” or “CG lock” that increases binding affinity. The guanine and / or cytidine in the W or coupling sequence may be consecutive or not, and may include one of the chemical modifications described herein, e.g., either the 2'-OMe or 2'-OH modification.

[0217]

[0223] In some embodiments, the conjugate of formula (I) is formula (Ia)

[0218] [ka]

[0219] It includes the structure.

[0224] In some embodiments, the conjugate of formula (I) is formula (Ib)

[0220] [ka]

[0221] It includes the structure.

[0225] In some embodiments, the conjugate described herein is of formula (II)

[0222] [ka]

[0223] It includes the structure, In the formula, each X is independently H or a protecting group, Z is a modified or unmodified C5 or C6 monosaccharide, and W represents an activator or coupling sequence. One or more linkers of formula (II) may each independently comprise the linkers described herein. In some embodiments, each protecting group of formula (II) is independently 4-acetoxy-2,2-dimethylbutanoyl (ADMB), 3-(2-hydroxyphenyl)-3,3-dimethylpropanoate (DMBPP), 3-(2-hydroxy-4,6-dimethylphenyl)-3,3-dimethylpropanoate group (TMBPP), methylsulfonylethoxycarbonyl (Msc), 2,2-dimethyltrimethylene (DMTM) phosphate, 2-pyridylmethyl, ethyl mandelate, (phenylthiomethyl)benzyl, phenylthiomethyl The following are selected from ionofluoropropionyl (PFP), benzoyl (Bz), acetyl (Ac), basilosamine (Bac), benzyl (Bn), 1-benzenesulfinylpiperidine (BSP), tert-butoxycarbonyl (Boc), benzylidene acetal, propargyl, naphthylpropargyl, carbonate, dichloroacetyl, tert-butylsilylene, tetraisopropyldisiloxanylidene (TIPDS), methoxybenzyl (PMB), xylylene, and p-methoxyphenyl (MP). In some embodiments, X is H. In some embodiments, each X is independently selected from H and Bz. In some embodiments of formula (II), Z is galactose or mannose. In some embodiments of formula (II), Z is GalNAc. In some embodiments of formula (II), W is an activator. In some embodiments, W is a nucleic acid. In some embodiments, W is gRNA. In some embodiments, W is a single-stranded, double-stranded, partially double-stranded, or hairpin stem-loop nucleic acid. In some embodiments of formula (II), W is a coupling sequence. In some embodiments, W comprises an RNA or DNA sequence. In some embodiments, W comprises (A)n, (T)n, (U)n, (a)n, or (u)n, where n is an integer greater than or equal to 3, a is 2'-O-methyladenosine (2'-OMe A), and u is 2'-O-methyluridine (2'-OMe U).In some embodiments, W includes (u)n, where n is an integer between 3 and 50 (sequence number 118). In some embodiments, W includes (u)n, where n is an integer between 3 and 20 (sequence number 119) or between 3 and 15 (sequence number 120).

[0224]

[0226] In some embodiments, the conjugate of formula (II) is formula (IIa)

[0225] [ka]

[0226] It includes the structure.

[0227] In some embodiments, the conjugate of formula (II) is formula (IIb)

[0227] [ka]

[0228] It includes the structure.

[0228] In some embodiments, the conjugate of formula (II) is formula (IIc)

[0229] [ka]

[0230] It includes the structure.

[0229] In some embodiments, the conjugate described herein is of formula (III),

[0231] [ka]

[0232] It includes the structure, In the formula, each X is independently H or a protecting group, Z is a modified or unmodified C5 or C6 monosaccharide, and W represents an activator or coupling sequence. One or more linkers of formula (III) each independently comprises the linkers described herein. In some embodiments, each of the protecting groups of formula (III) is independently 4-acetoxy-2,2-dimethylbutanoyl (ADMB), 3-(2-hydroxyphenyl)-3,3-dimethylpropanoate (DMBPP), 3-(2-hydroxy-4,6-dimethylphenyl)-3,3-dimethylpropanoate group (TMBPP), methylsulfonylethoxycarbonyl (Msc), 2,2-dimethyltrimethylene (DMTM) phosphate, 2-pyridylmethyl, ethyl mandelate, (phenylthiomethyl)benzyl, phenylthiomethyl The following are selected from ionofluoropropionyl (PFP), benzoyl (Bz), acetyl (Ac), basilosamine (Bac), benzyl (Bn), 1-benzenesulfinylpiperidine (BSP), tert-butoxycarbonyl (Boc), benzylidene acetal, propargyl, naphthylpropargyl, carbonate, dichloroacetyl, tert-butylsilylene, tetraisopropyldisiloxanylidene (TIPDS), methoxybenzyl (PMB), xylylene, and p-methoxyphenyl (MP). In some embodiments, X is H. In some embodiments, each X is selected from H and Bz. In some embodiments of formula (III), Z is galactose or mannose. In some embodiments of formula (III), Z is GalNAc. In some embodiments of formula (III), W is an activator. In some embodiments, W is a nucleic acid. In some embodiments, W is gRNA. In some embodiments, W is a single-stranded, double-stranded, partially double-stranded, or hairpin stem-loop nucleic acid. In some embodiments of formula (III), W is a coupling sequence. In some embodiments, W comprises an RNA or DNA sequence. In some embodiments, W comprises (A)n, (T)n, (U)n, (a)n, or (u)n, where n is an integer greater than or equal to 3, a is 2'-O-methyladenosine (2'-OMe A), and u is 2'-O-methyluridine (2'-OMe U).In some embodiments, W includes (u)n, where n is an integer between 3 and 50 (sequence number 118). In some embodiments, W includes (u)n, where n is an integer between 3 and 20 (sequence number 119) or between 3 and 15 (sequence number 120).

[0233]

[0230] In some embodiments, the conjugate of formula (III) is formula (IIIa),

[0234] [ka]

[0235] It includes the structure.

[0231] In some embodiments, the conjugate of formula (III) is formula (IIIb),

[0236] [ka]

[0237] It includes the structure.

[0232] In some embodiments, the conjugate of formula (III) is formula (IIIc),

[0238] [ka]

[0239] It includes the structure, In the formula, Y is either O or S.

[0233] In some embodiments, the conjugate of formula (III) is formula (IIId),

[0240] [ka]

[0241] It includes the structure, In the formula, Y is either O or S.

[0234] In some embodiments, the conjugate of formula (III) is formula (IIIe),

[0242] [ka]

[0243] It includes the structure, In the formula, Y is either O or S.

[0235] In some embodiments, the conjugate described herein is of formula (IV),

[0244] [ka]

[0245] It includes the structure, In the formula, each X is independently either H or a protecting group, and R Ais -OX or -NHAc, Y is O or S, and W represents an activator or coupling sequence. One or more linkers of formula (IV) may each independently comprise the linkers described herein. In some embodiments, each protecting group of formula (IV) may independently be 4-acetoxy-2,2-dimethylbutanoyl (ADMB), 3-(2-hydroxyphenyl)-3,3-dimethylpropanoate (DMBPP), 3-(2-hydroxy-4,6-dimethylphenyl)-3,3-dimethylpropanoate group (TMBPP), methylsulfonylethoxycarbonyl (Msc), 2,2-dimethyltrimethylene (DMTM) phosphate, 2-pyridylmethyl, ethyl mandelate, (phenylthiomethyl)benzyl, phenylthiomethyl X is selected from fluoropropionyl (PFP), benzoyl (Bz), acetyl (Ac), basilosamine (Bac), benzyl (Bn), 1-benzenesulfinylpiperidine (BSP), tert-butoxycarbonyl (Boc), benzylidene acetal, propargyl, naphthylpropargyl, carbonate, dichloroacetyl, tert-butylsilylene, tetraisopropyldisiloxanylidene (TIPDS), methoxybenzyl (PMB), xylylene, and p-methoxyphenyl (MP). In some embodiments, X is H. In some embodiments, each X is independently selected from H and Bz. In some embodiments, R A is -OX. In some embodiments, R A is -OH. In some embodiments, R Ais -NHAc. In some embodiments of formula (IV), W is an activator. In some embodiments, W is a nucleic acid. In some embodiments, W is gRNA. In some embodiments, W is a single-stranded, double-stranded, partially double-stranded, or hairpin stem-loop nucleic acid. In some embodiments of formula (IV), W is a coupling sequence. In some embodiments, W comprises an RNA or DNA sequence. In some embodiments, W comprises (A)n, (T)n, (U)n, (a)n, or (u)n, where n is an integer greater than or equal to 3, a is 2'-O-methyladenosine (2'-OMe A), and u is 2'-O-methyluridine (2'-OMe-U). In some embodiments, W comprises (u)n, where n is an integer from 3 to 50 (SEQ ID NO: 118). In some embodiments, W comprises (u)n, where n is an integer from 3 to 20 (SEQ ID NO: 119) or from 3 to 15 (SEQ ID NO: 120).

[0246]

[0236] In some embodiments, the conjugate of formula (IV) includes structures 1-1, 1-2, 1-5, 1-6, 1-9, 1-10, 1-11, or 1-12, as shown in Table 1.

[0247]

[0237] In some embodiments of formula (I), formula (Ia), formula (II), formula (IIa), formula (IIc), formula (III), formula (IIIa), formula (IIIb), formula (IIIc), formula (IIId), formula (IIIe), or formula (IV), “one or more linkers” referred to in the boxes of the formulas above include a structure selected from the group consisting of:

[0248] [ka]

[0249] And each linker is independent. In some embodiments of formula (I), formula (Ia), formula (Ib), formula (II), formula (IIa), formula (IIb), formula (IIc), formula (III), formula (IIIa), formula (IIIb), formula (IIIc), formula (IIId), formula (IIIe), or formula (IV), where each linker is independent, -(L 1 ) k1 -(L 2 ) k2 -(L 3 ) k3 -(L 4 ) k4 - has the structure, where each of k1, k2, k3, and k4 is independently 0, 1, or 2, and L 1 , L 2 , L 3 and L 4 Each of these can independently be an oxo, ester, amide, amino, C1-C3 alkylene, and -(CH2-CH2-O) 1~3 -Selected from. In some embodiments, the sum of k1, k2, k3, and k4 is an integer of 1 or more. In some embodiments, the sum of k1, k2, k3, and k4 is an integer of 2 or more. As those skilled in the art will recognize, "N" refers to nitrogen, "

[0250] [ka]

[0251] " represents a connection point.

[0238] In some embodiments of formula (I), formula (Ia), formula (Ib), formula (II), formula (IIa), formula (IIb), formula (IIc), formula (III), formula (IIIa), formula (IIIb), formula (IIIc), formula (IIId), formula (IIIe), or formula (IV), each of the linkers in the formula is independently -(L 1 ) k1 -(L 2 ) k2 -(L 3 ) k3 -(L 4 ) k4- has the structure, where each of k1, k2, k3, and k4 is independently 0, 1, or 2, and L 1 , L 2 , L 3 and L 4 Each of these is independent of -O-, -S-,S(=O) 1~2 -, -C(=O)-, -C(=S)-, -NR L -, -OC(=O)-, -C(=O)O-, -OC(=O)O-, -C(=O)NR L -, -OC(=O)NR L -, -NR L C(=O)-, -NR L C(=O)NR L -, -P(=O)R L -, -NR L S(=O)(=NR L )-, -NR L S(=O)2-, -S(=O)2NR L -, -N=N-, -(CH2-CH2-O) 1~6 -, linear or branched C 1~6 Alkylene, linear or branched C 2~6 Alkenylene, linear or branched C 2~6 Alkynylene, C3-C8 cycloalkylene, C2-C7 heterocycloalkylene, C6-C 10 Arylenes and C5-C9 heteroarylenes are selected, where alkylenes, alkenylenes, alkynylenes, cycloalkylenes, cycloalkylenes, arylenes, or heteroarylenes are substituted or unsubstituted, where each R L R is independently H, D, cyano, halogen, substituted or unsubstituted C1-C6 alkyl, -CD3, -OCH3, -OCD3, substituted or unsubstituted C1-C6 haloalkyl, substituted or unsubstituted C1-C6 heteroalkyl, substituted or unsubstituted C3-C8 cycloalkyl, substituted or unsubstituted C2-C7 heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl. In some embodiments, each R LThese are independently H, a substituted or unsubstituted C1-C6 alkyl group, -OCH3, a substituted or unsubstituted C1-C6 haloalkyl group, a substituted or unsubstituted C1-C6 heteroalkyl group, a substituted or unsubstituted C3-C8 cycloalkyl group, or a substituted or unsubstituted C2-C7 heterocycloalkyl group.

[0252]

[0239] In some embodiments of formula (I), formula (Ia), formula (Ib), formula (II), formula (IIa), formula (IIb), formula (IIc), formula (III), formula (IIIa), formula (IIIb), formula (IIIc), formula (IIId), formula (IIIe), or formula (IV), each linker independently is as follows:

[0253] [ka]

[0254] The formula includes a structure selected from, where each of p, q, m, and n is independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 13, 15, 16, 17, 18, 19, or 20. In some embodiments, each of p, q, m, and n is independently 0, 1, 2, 3, 4, or 5.

[0255]

[0240] In some embodiments of formula (I), formula (Ia), formula (Ib), formula (II), formula (IIa), formula (IIb), formula (IIc), formula (III), formula (IIIa), formula (IIIb), formula (IIIc), formula (IIId), formula (IIIe), or formula (IV), the “one or more linkers” referred to in the boxes of the formulas above are,

[0256] [ka]

[0257] [ka]

[0258] [ka]

[0259] [ka]

[0260] The structure includes the following, where each of p, q, m, and n is independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 13, 15, 16, 17, 18, 19, or 20. In some embodiments, each of p, q, m, and n is independently 0, 1, 2, 3, 4, or 5.

[0261]

[0241] In some embodiments of formula (I), formula (Ia), formula (Ib), formula (II), formula (IIa), formula (IIb), formula (IIc), formula (III), formula (IIIa), (IIIb), formula (IIIc), formula (IIId), formula (IIIe), or formula (IV), W comprises one or more modified DNA or RNA bases. The nucleic acid bases may include any chemical modifications described herein. In some embodiments, the nucleic acid bases include 2'-OH or 2'-OMe modifications. For example, W may comprise one or more 2'-OMe modified adenine, cytosine, guanidine, and uracil, referred to as (a), (c), (g), or (u). In some embodiments, the modified RNA, e.g., gRNA or mRNA, contains at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 50 or more modified nucleic acid bases. In some embodiments, the modified RNA contains one or more modified nucleic acid bases near the 5' end, near the 3' end, or in the middle of the sequence. The modified nucleic acid bases in the modified RNA may be consecutive or not. In some embodiments, the modified RNA contains one or more 2'-OMe modifications scattered along the length of the sequence. In some embodiments, the modified RNA contains one or more 2'OH modifications scattered along the length of the sequence. In some embodiments, the modified RNA contains alternating 2'-OH and 2'OMe modifications. In some embodiments, W comprises (A)n, (T)n, (U)n, (a)n, or (u)n, where n is an integer greater than or equal to 3, a is 2'-O-methyladenosine (2'-OMe A), and u is 2'-O-methyluridine (2'-OMe-U). In some embodiments, W comprises (u)n, where n is an integer from 3 to 50 (SEQ ID NO: 118). In some embodiments, W comprises (u)n, where n is an integer from 3 to 20 (SEQ ID NO: 119) or from 3 to 15 (SEQ ID NO: 120). In some embodiments, W comprises one or more nucleotide sequences complementary to the coupling sequence. In some embodiments, W comprises one or more guanines or cytidines.In some embodiments, W contains at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 50 or more guanines or cytidines. In some embodiments, one or more guanines or cytidines are complementary to one or more cytinidines or guanines in the coupling sequence. In some embodiments, the guanines or cytidines are at the end of W or the coupling sequence. While not intended to be bound by any theory, guanine-cytidine pairings are thought to form a “GC lock” or “CG lock” that increases binding affinity. The guanines and / or cytidines in W or the coupling sequence may be consecutive or not, and may include any one of the chemical modifications described herein, e.g., 2'-OMe or 2'-OH modifications.

[0262] Receptor-targeting conjugate

[0242] The key to achieving nucleic acid-based therapies is the safe and effective delivery of payloads to specific cell types and tissues. Lipid nanoparticles (LNPs) represent the most advanced nonviral drug delivery technology platform to date. LNPs can physically pass through blood vessels to reach hepatocytes [Am.J.Patel.2010, 176, 14-21]. It has also been shown that apolipoprotein E (ApoE) protein, after PEG lipids diffuse from the LNP surface, binds to LNPs in the bloodstream in a near-neutral charge state and functions as an endogenous ligand for hepatocytes, representing a low-density lipoprotein receptor (LDLr) [Mol.Ther., 2010, 18, 1357-1364]. Therefore, two key factors controlling the efficient hepatic delivery of LNPs are hypothesized to be 1) effective PEG lipid desorption from the LNP surface in serum, and 2) ApoE binding to LNPs. The endogenous ApoE-mediated LDLr-dependent LNP delivery pathway described above is not likely to be an effective pathway for achieving LNP-based liver gene delivery in the LDLr-deficient patient population.

[0263]

[0243] In one embodiment, an LNP comprising a receptor-targeting conjugate is described herein. In some embodiments, what is described herein is a receptor-targeting conjugate. An LNP having a target conjugate is configured to have a receptor-targeting moiety on the surface or periphery of a particle. In one embodiment, a low mol% of the receptor-targeting conjugate is used while constituting the targeted LNP to achieve a low surface density of the targeting moiety on the surface / periphery of the particle. In another embodiment, a high mol% of the receptor-targeting conjugate is used while constituting the targeted LNP to achieve a high surface density of the targeting moiety on the surface / periphery of the particle. In another embodiment, a desired mol% of the receptor-targeting conjugate is used to achieve a range of surface densities of the targeting moiety on the surface / periphery of the particle. In some embodiments, the receptor-targeting conjugate comprises a targeting moiety (or ligand), a linker, and a lipophilic moiety linked to the targeting moiety. In some embodiments, the receptor-targeting moiety (or ligand) targets a lectin receptor. In some embodiments, the lectin receptor is an asialoglycoprotein receptor (ASGPR). In some embodiments, the receptor targeting moiety is a GalNAc or a GalNAc derivative that targets the ASGPR. In one embodiment, the receptor targeting conjugate comprises one GalNAc moiety or a derivative thereof. In another embodiment, the receptor targeting conjugate comprises two GalNAc moieties or derivatives thereof. In yet another embodiment, the receptor targeting conjugate comprises three GalNAc moieties or derivatives thereof. In yet another embodiment, the receptor targeting conjugate is lipophilic. In some embodiments, the receptor targeting conjugate comprises one or more GalNAc moieties and one or more lipid moieties, i.e., GalNAc-lipids. In some embodiments, the receptor targeting conjugate is a GalNAc-lipid.

[0264]

[0244] This disclosure provides tissue-specific and efficient LNP delivery to hepatocytes in an LDLr-independent manner. The trivalent GalNAc moiety developed by this disclosure is conjugated to hydrophobic glycerol-based dialkyl lipid chains, sterols (e.g., cholesterol), and hydrophobic α-tocopherol via different PEG spacers. These GalNAc conjugate lipids are then formulated with various excipients to generate LNPs that hold GalNAc ligands ranging from low to high surface densities, custom-designed to target asialoglycoprotein receptors (ASGPRs) highly expressed on the surface of hepatocytes.

[0265]

[0245] Ligands on the surface of the manipulated LNP promote ASGPR-mediated tissue-specific uptake into hepatocytes. Different GalNAc-LNPs are configured to avoid ApoE binding and enable GalNAc-ASGPR interaction to promote clathrin-mediated uptake into hepatocytes. By using the PEG-lipids described herein in combination with GalNAc-lipids having various PEG-tethers, the PEG shedding kinetics are modulated and the net surface charge density of the GalNAc-LNP particles is adjusted to obtain GalNAc-LNPs lacking endogenous ApoE binding properties, which deliver particles carrying a hepatocyte-specific RNA payload at a safe and effective dose in preclinical animal models lacking LDLR. Further dose optimization in preclinical animal models will enable GalNAc-LNP (or LNP) to proceed to clinical development to treat LDLR-deficient patient populations and induce genome editing at a therapeutically viable, safe, and effective dose.

[0266]

[0246] Accordingly, in one embodiment, the compound of formula (V) is disclosed herein:

[0267] [ka]

[0268] It is a receptor-targeting conjugate that includes During the ceremony, Multiple A groups collectively contain receptor-targeting ligands; L 1 , L 2 , L 3 , L 4 , L 5 , L 6 , L 7 , L 8 , L 9 , L 10 and L 12 Each of these independently determines whether C1-C is a substitution or non-substitution. 12 Alkylene, substituted or unsubstituted C1-C 12 Heteroalkylenes, substituted or unsubstituted C2-C 12 Alkenylenes, substituted or unsubstituted C2-C2 12 Alkynylene, -(CH2CH2O) m -,-(OCH2CH2) m -, -O-, -S-, -S(=O)-, -S(=O)2-, -S(=O)(=NR 1 )-, -C(=O)-, -C(=N-OR 1 )-, -C(=O)O-, -OC(=O)-, -C(=O)C(=O)-, -C(=O)NR 1 -, -NR 1 C(=O)-, -OC(=O)NR 1 -, -NR 1 C(=O)O-, -NR 1 C(=O)NR 1 -, -C(=O)NR 1 C(=O)-, -S(=O)2NR 1 -, -NR 1 S(=O)²⁻, -NR 1 -, -N(OR 1 )-,-O[(P=O)O - ]O- or -O[(P=O)S - ] O- or bonded; L 11 This is a substituted or unsubstituted -(CH2CH2O) n -, substitution or non-substitution of -(OCH2CH2) n -, or substituted or unsubstituted -(CH2) n -, or combination; Each R1 These are independently H or substituted or unsubstituted C1-C6 alkyl groups; R is a lipid, nucleic acid, amino acid, protein, or lipid nanoparticle; m is an integer selected from 1 to 10; n is an integer selected from 1 to 200.

[0269]

[0247] In some embodiments, the receptor targeting conjugate is a compound of formula (V):

[0270] [ka]

[0271] Including, in the formula, Multiple A groups collectively contain receptor-targeting ligands; L 1 , L 2 , L 3 , L 4 , L 5 , L 6 , L 7 , L 8 , L 9 , L 10 and L 12 Each of these independently determines whether C1-C is a substitution or non-substitution. 12 Alkylene, substituted or unsubstituted C1-C 12 Heteroalkylenes, substituted or unsubstituted C2-C 12 Alkenylenes, substituted or unsubstituted C2-C2 12 Alkynylene, -(CH2CH2O) m -,-(OCH2CH2) m -, -O-, -S-, -S(=O)-, -S(=O)2-, -S(=O)(=NR 1 )-, -C(=O)-, -C(=N-OR 1 )-, -C(=O)O-, -OC(=O)-, -C(=O)C(=O)-, -C(=O)NR 1 -, -NR 1 C(=O)-, -OC(=O)NR 1 -, -NR 1C(=O)O-, -NR 1 C(=O)NR 1 -, -C(=O)NR 1 C(=O)-, -S(=O)2NR 1 -, -NR 1 S(=O)²⁻, -NR 1 -, or -N (OR 1 )-and; L 11 This is a substituted or unsubstituted -(CH2CH2O) n -, or the substituted or unsubstituted -(OCH2CH2) n -and; Each R 1 These are independently H or substituted or unsubstituted C1-C6 alkyl groups; R is a lipid, nucleic acid, amino acid, protein, or lipid nanoparticle; m is an integer selected from 1 to 10; n is an integer selected from 1 to 200.

[0272]

[0248] In some embodiments, L 11 is -(CH2CH2O) n -or-(OCH2CH2) n - is

[0249] In some embodiments of the compound of formula (V), A binds to a lectin. In some embodiments, the lectin is an asialoglycoprotein receptor (ASGPR). In some embodiments, A comprises one or more N-acetylgalactosamines (GalNAc) or GalNAc derivatives.

[0273]

[0250] In some embodiments of the compound of formula (V), A is N-acetylgalactosamine (GalNAc) or a derivative thereof. In some embodiments, A is GalNAc. In some embodiments, A is galactose or contains galactose.

[0274]

[0251] In some embodiments of the compound of formula (V), L 1 , L 4 , and L7 Each of these is independently a substitute or non-substitute C1-C 12 It is an alkylene. In some embodiments of the compound of formula (V), each L 1 , L 4 , and L 7 These are independently substituted or unsubstituted C2-C6 alkylenes. In some embodiments of the compound of formula (V), each L 1 , L 4 , and L 7 It is a C4 alkylene.

[0275]

[0252] In some embodiments of the compound of formula (V), each L 2 , L 5 , and L 8 Independently, -C(=O)NR 1 -, -NR 1 C(=O)-, -OC(=O)NR 1 -, -NR 1 C(=O)O-, -NR 1 C(=O)NR 1 -, or -C(=O)NR 1 It is C(=O)-. In some embodiments of the compound of formula (V), each L 2 , L 5 , and L 8 Independently, -C(=O)NR 1 -or-NR 1 It is C(=O)-. In some embodiments of the compound of formula (V), each L 2 , L 5 , and L 8 This is -C(=O)NH-.

[0276]

[0253] In some embodiments of the compound of formula (V), each L 3 , L 6 , and L 9 These are independently of substitution or non-substitution of C1-C 12 It is an alkylene. In some embodiments of the compound of formula (V), each L 3 is a substituted or unsubstituted C2-C6 alkylene. In some embodiments of the compound of formula (V), L 3is a C4 alkylene. In some embodiments of the compound of formula (V), L 6 and L 9 Each of these can be independently substituted or non-substituted C2-C 10 It is an alkylene. In some embodiments of the compound of formula (V), each L 6 and L 9 These are independently substituted or unsubstituted C2-C6 alkylenes. In some embodiments of the compound of formula (V), each L 6 and L 9 It is C3 alkylene.

[0277]

[0254] In some embodiments of the compound of formula (V), R 1 H is H. In some embodiments, R 1 is a substituted or unsubstituted C1-C6 alkyl group. In some embodiments, R 1 It is methyl.

[0278]

[0255] In another embodiment, disclosed herein is a receptor-targeting conjugate which is of formula (VI):

[0279] [ka]

[0280] Contains the compound, During the ceremony, Multiple A groups collectively contain receptor-targeting ligands. L 1 , L 2 , L 3 , L 4 , L 5 , L 6 , L 7 , L 8 , L 9 , L 10 and L 12 Each of these independently determines whether C1-C is a substitution or non-substitution. 12 Alkylene, substituted or unsubstituted C1-C 12 Heteroalkylenes, substituted or unsubstituted C2-C12 Alkenylenes, substituted or unsubstituted C2-C2 12 Alkynylene, -(CH2CH2O) m -,-(OCH2CH2) m -, -O-, -S-, -S(=O)-, -S(=O)2-, -S(=O)(=NR 1 )-, -C(=O)-, -C(=N-OR 1 )-, -C(=O)O-, -OC(=O)-, -C(=O)C(=O)-, -C(=O)NR 1 -, -NR 1 C(=O)-, -OC(=O)NR 1 -, -NR 1 C(=O)O-, -NR 1 C(=O)NR 1 -, -C(=O)NR 1 C(=O)-, -S(=O)2NR 1 -, -NR 1 S(=O)²⁻, -NR 1 -, -N(OR 1 )-,-O[(P=O)O - ]O-, -O[(P=O)S - ]O-, or bonded; L 11 This is a substituted or unsubstituted -(CH2CH2O) n -, substitution or non-substitution of -(OCH2CH2) n -, substituted or unsubstituted -(CH2) n -, or combination; Each R 1 These are independently H or substituted or unsubstituted C1-C6 alkyl groups; R is a lipid, nucleic acid, amino acid, protein, or lipid nanoparticle; m is an integer selected from 1 to 10; n is an integer selected from 1 to 200.

[0281]

[0256] In some embodiments, disclosed herein is a receptor-targeting conjugate, which has formula (VI):

[0282] [ka]

[0283] Contains the compound, During the ceremony, Multiple A groups collectively contain receptor-targeting ligands; L 1 , L 2 , L 3 , L 4 , L 5 , L 6 , L 7 , L 8 , L 9 , L 10 and L 12 Each of these independently determines whether C1-C is a substitution or non-substitution. 12 Alkylene, substituted or unsubstituted C1-C 12 Heteroalkylenes, substituted or unsubstituted C2-C 12 Alkenylenes, substituted or unsubstituted C2-C2 12 Alkynylene, -(CH2CH2O) m -,-(OCH2CH2) m -, -O-, -S-, -S(=O)-, -S(=O)2-, -S(=O)(=NR 1 )-, -C(=O)-, -C(=N-OR 1 )-, -C(=O)O-, -OC(=O)-, -C(=O)C(=O)-, -C(=O)NR 1 -, -NR 1 C(=O)-, -OC(=O)NR 1 -, -NR 1 C(=O)O-, -NR 1 C(=O)NR 1 -, -C(=O)NR 1 C(=O)-, -S(=O)2NR 1 -, -NR 1 S(=O)²⁻, -NR 1 -, or -N (OR 1 )-and; L 11 This is a substituted or unsubstituted -(CH2CH2O) n -, or the substituted or unsubstituted -(OCH2CH2) n -and; Each R1 These are independently H or substituted or unsubstituted C1-C6 alkyl groups; R is a lipid, nucleic acid, amino acid, protein, or lipid nanoparticle; m is an integer selected from 1 to 10; n is an integer selected from 1 to 200.

[0284]

[0257] In some embodiments, L 11 This is -(CH2CH2O)n- or -(OCH2CH2)n-.

[0258] In some embodiments of the compound of formula (VI), A binds to a lectin. In some embodiments, the lectin is an asialoglycoprotein receptor (ASGPR). In some embodiments, A comprises one or more N-acetylgalactosamines (GalNAc) or GalNAc derivatives.

[0285]

[0259] In some embodiments of the compound of formula (VI), A is N-acetylgalactosamine (GalNAc) or a derivative thereof. In some embodiments, A is GalNAc.

[0286]

[0260] In some embodiments of the compound of formula (VI), each L 1 , L 4 , and L 7 These are independently of substitution or non-substitution of C1-C 12 Alkylene or substituted or unsubstituted C1-C 12 It is a heteroalkylene.

[0287]

[0261] In some embodiments of the compound of formula (VI), each L 1 , L 4 , and L 7 These are independently of substitution or non-substitution of C1-C 12 It is a heteroalkylene.

[0262] In some embodiments of the compound of formula (VI), each L 1 , L 4 , and L 7These are independently substituted or unsubstituted C1-C atoms containing 1-10 oxygen atoms. 12 It is a heteroalkylene.

[0288]

[0263] In some embodiments of the compound of formula (VI), each L 1 , L 4 , and L 7 It is independently -(CH2CH2O) p1 -(CH2) q1 - and in the formula, p1 is between 1 and 8; q1 is between 1 and 6.

[0289]

[0264] In some embodiments of the compound of formula (VI), each L 1 , L 4 , and L 7 It is -(CH2CH2O)3-(CH2)2-.

[0265] In some embodiments of the compound of formula (VI), each L 1 , L 4 , and L 7 These are independently of substitution or non-substitution of C1-C 12 It is alkylene.

[0290]

[0266] In some embodiments of the compound of formula (VI), each L 1 , L 4 , and L 7 These are independently substituted or unsubstituted C2-C6 alkylenes.

[0267] In some embodiments of the compound of formula (VI), each L 1 , L 4 , and L 7 It is a C4 alkylene.

[0291]

[0268] In some embodiments of the compound of formula (VI), each L 2 , L 5 , and L 8 Independently, -C(=O)NR 1 -, -NR 1 C(=O)-, -OC(=O)NR 1 -, -NR 1 C(=O)O-, -NR 1C(=O)NR 1 -, or -C(=O)NR 1 C(=O)-

[0292]

[0269] In some embodiments of the compound of formula (VI), each L 2 , L 5 , and L 8 Independently, -C(=O)NR 1 -or-NR 1 C(=O)-

[0270] In some embodiments of the compound of formula (VI), each L 2 , L 5 , and L 8 This is -NHC(=O)-.

[0293]

[0271] In some embodiments of the compound of formula (VI), each L 2 , L 5 , and L 8 This is -C(=O)NH-.

[0272] In some embodiments of the compound of formula (VI), each L 3 , L 6 , and L 9 These are independently of substitution or non-substitution of C1-C 12 It is a heteroalkylene.

[0294]

[0273] In some embodiments of the compound of formula (VI), each L 3 , L 6 , and L 9 These are independently substituted or unsubstituted C1-C atoms containing 1-10 oxygen atoms. 12 Heteroalkylenes are present.

[0295]

[0274] In some embodiments of the compound of formula (VI), each L 3 , L 6 , and L 9 It is independently -(CH2CH2O) p2 -(CH2CH2CH2O) q2- and; where p2 is 1 to 8; q2 is 1 to 6. In some embodiments, p2 is 1. In some embodiments, p2 is 2. In some embodiments, p2 is 3. In some embodiments, p2 is 4. In some embodiments, p2 is 5. In some embodiments, p2 is 6. In some embodiments, p2 is 7. In some embodiments, p2 is 8. In some embodiments, q2 is 1. In some embodiments, q2 is 2. In some embodiments, q2 is 3. In some embodiments, q2 is 4. In some embodiments, q2 is 5. In some embodiments, q2 is 6.

[0296]

[0275] In some embodiments of the compound of formula (VI), each L 3 , L 6 , and L 9 It is -(CH2CH2O)-(CH2CH2CH2O)-.

[0276] In some embodiments of the compound of formula (VI), each L 3 , L 6 , and L 9 It is independently -(CH2CH2CH2O) q3 - and; where q3 is 1 to 8. In some embodiments, q3 is 1. In some embodiments, q3 is 2. In some embodiments, q3 is 3. In some embodiments, q3 is 4. In some embodiments, q3 is 5. In some embodiments, q3 is 6. In some embodiments, q3 is 7. In some embodiments, q3 is 8.

[0297]

[0277] In some embodiments of the compound of formula (VI), each L 3 , L 6 , and L 9 This is -(CH2CH2CH2O)2-.

[0278] In some embodiments, the compound of formula (VI) is formula (VIa):

[0298] [ka]

[0299] It has a structure, During the ceremony, Each q4 is between 1 and 10.

[0300]

[0279] In some embodiments of the compound of formula (VIb), q4 is 1 to 8. In some embodiments, q4 is 1 to 4. In some embodiments, q4 is 1 to 3. In some embodiments, q4 is 1. In some embodiments, q4 is 2. In some embodiments, q4 is 3. In some embodiments, q4 is 4. In some embodiments, q4 is 5.

[0301]

[0280] In some embodiments of the compound of formula (V) or formula (VI), L 10 C1-C are either substituted or non-substituted. 12 It is an alkylene. 10 L is a substituted or unsubstituted C1-C4 alkylene. In some embodiments, L 10 It is a C2 alkylene.

[0302]

[0281] In some embodiments, the compound of formula (VI) is formula (VIb):

[0303] [ka]

[0304] It has a structure, During the ceremony, r is between 1 and 4.

[0305]

[0282] In some embodiments of the compound of formula (VIb), r is 1, 2, or 3. In some embodiments, r is 1 or 2. In some embodiments, r is 2 or 3. In some embodiments, r is 1. In some embodiments, r is 2. In some embodiments, r is 3. In some embodiments, r is 4.

[0306]

[0283] In some embodiments of the compounds of formula (V), formula (VI), formula (VIa), or formula (VIb), L 11 is -(OCH2CH2) n-. In some embodiments, n is 1 to 100. In some embodiments, n is 2 to 50. In some embodiments, n is 10 to 50. In some embodiments, n is 20 to 50. In some embodiments, n is 30 to 50. In some embodiments, n is 40 to 50. In some embodiments, n is 2, 12, 37, or 45. In some embodiments, n is 1. In some embodiments, n is 2. In some embodiments, n is 3. In some embodiments, n is 4. In some embodiments, n is 5. In some embodiments, n is 6. In some embodiments, n is 7. In some embodiments, n is 8. In some embodiments, n is 9. In some embodiments, n is 10. In some embodiments, n is 11. In some embodiments, n is 12. In some embodiments, n is 13. In some embodiments, n is 14. In some embodiments, n is 15. In some embodiments, n is 16. In some embodiments, n is 17. In some embodiments, n is 18. In some embodiments, n is 19. In some embodiments, n is 20. In some embodiments, n is 21. In some embodiments, n is 22. In some embodiments, n is 23. In some embodiments, n is 24. In some embodiments, n is 25. In some embodiments, n is 26. In some embodiments, n is 27. In some embodiments, n is 28. In some embodiments, n is 29. In some embodiments, n is 30. In some embodiments, n is 31. In some embodiments, n is 32. In some embodiments, n is 33. In some embodiments, n is 34. In some embodiments, n is 35. In some embodiments, n is 36. In some embodiments, n is 37. In some embodiments, n is 38. In some embodiments, n is 39.In some embodiments, n is 40. In some embodiments, n is 41. In some embodiments, n is 42. In some embodiments, n is 43. In some embodiments, n is 44. In some embodiments, n is 45. In some embodiments, n is 46. In some embodiments, n is 47. In some embodiments, n is 48. In some embodiments, n is 49. In some embodiments, n is 50. In some embodiments, n is at least 1. In some embodiments, n is at least 2. In some embodiments, n is at least 3. In some embodiments, n is at least 4. In some embodiments, n is at least 5. In some embodiments, n is at least 6. In some embodiments, n is at least 7. In some embodiments, n is at least 8. In some embodiments, n is at least 9. In some embodiments, n is at least 10. In some embodiments, n is at least 11. In some embodiments, n is at least 12. In some embodiments, n is at least 13. In some embodiments, n is at least 14. In some embodiments, n is at least 15. In some embodiments, n is at least 16. In some embodiments, n is at least 17. In some embodiments, n is at least 18. In some embodiments, n is at least 19. In some embodiments, n is at least 20. In some embodiments, n is at least 21. In some embodiments, n is at least 22. In some embodiments, n is at least 23. In some embodiments, n is at least 24. In some embodiments, n is at least 25. In some embodiments, n is at least 26. In some embodiments, n is at least 27. In some embodiments, n is at least 28. In some embodiments, n is at least 29.In some embodiments, n is at least 30. In some embodiments, n is at least 31. In some embodiments, n is at least 32. In some embodiments, n is at least 33. In some embodiments, n is at least 34. In some embodiments, n is at least 35. In some embodiments, n is at least 36. In some embodiments, n is at least 37. In some embodiments, n is at least 38. In some embodiments, n is at least 39. In some embodiments, n is at least 40. In some embodiments, n is at least 41. In some embodiments, n is at least 42. In some embodiments, n is at least 43. In some embodiments, n is at least 44. In some embodiments, n is at least 45. In some embodiments, n is at least 46. In some embodiments, n is at least 47. In some embodiments, n is at least 48. In some embodiments, n is at least 49. In some embodiments, n is at most 2. In some embodiments, n is at most 3. In some embodiments, n is at most 4. In some embodiments, n is at most 5. In some embodiments, n is at most 6. In some embodiments, n is at most 7. In some embodiments, n is at most 8. In some embodiments, n is at most 9. In some embodiments, n is at most 10. In some embodiments, n is at most 11. In some embodiments, n is at most 12. In some embodiments, n is at most 13. In some embodiments, n is at most 14. In some embodiments, n is at most 15. In some embodiments, n is at most 16. In some embodiments, n is at most 17. In some embodiments, n is at most 18. In some embodiments, n is at most 19. In some embodiments, n is at most 20.In some embodiments, n is at most 21. In some embodiments, n is at most 22. In some embodiments, n is at most 23. In some embodiments, n is at most 24. In some embodiments, n is at most 25. In some embodiments, n is at most 26. In some embodiments, n is at most 27. In some embodiments, n is at most 28. In some embodiments, n is at most 29. In some embodiments, n is at most 30. In some embodiments, n is at most 31. In some embodiments, n is at most 32. In some embodiments, n is at most 33. In some embodiments, n is at most 34. In some embodiments, n is at most 35. In some embodiments, n is at most 36. In some embodiments, n is at most 37. In some embodiments, n is at most 38. In some embodiments, n is at most 39. In some embodiments, n is at most 40. In some embodiments, n is at most 41. In some embodiments, n is at most 42. In some embodiments, n is at most 43. In some embodiments, n is at most 44. In some embodiments, n is at most 45. In some embodiments, n is at most 46. In some embodiments, n is at most 47. In some embodiments, n is at most 48. In some embodiments, n is at most 49. In some embodiments, n is at most 50.

[0307]

[0284] In some embodiments of the compounds of formula (V), formula (VI), formula (VIa), or formula (VIb), L 12 is -O-, -C(=O)O-, -C(=O)NR 1 -, -NR 1 C(=O)-, or -NR 1 In some embodiments, L 12 is -C(=O)O- or -NR 1In some embodiments, L 12 In some embodiments, L 12 is -NHC(=O)O-. In some embodiments, L 12 This is -NHC(=O)-.

[0308]

[0285] In some embodiments of the compounds of formula (VI), formula (VIa), or formula (VIb), R 1 H is H. In some embodiments, R 1 is a substituted or unsubstituted C1-C6 alkyl group. In some embodiments, R 1 It is methyl.

[0309]

[0286] According to the formulas mentioned above, in some embodiments of the compound of formula (V) or (VI), L 1 C1-C are either substituted or non-substituted. 12 Alkylene, substituted or unsubstituted C1-C 12 Heteroalkylenes, substituted or unsubstituted C2-C 12 Alkenylenes, substituted or unsubstituted C2-C2 12 Alkynylene, -(CH2CH2O) m -,-(OCH2CH2) m -, -O-, -S-, -S(=O)-, -S(=O)2-, -S(=O)(=NR1)-, -C(=O)-, -C(=N-OR1)-, -C(=O)O-, -OC(=O)-, -C(=O)C(=O)-, -C(=O)NR1-, -NR1C(=O)-, -OC(=O)NR1-, -NR1C(=O)O-, -NR1C(=O)NR1-, -C(=O)NR1C(=O)-, -S(=O)2NR1-, -NR1S(=O)2-, -NR1-, -N(OR1)-, -O[(P=O)O-]O-, -O[(P=O)S-]O-, or bond. In some embodiments, L 1 C1-C are either substituted or non-substituted. 12 It is an alkylene. 1 C1-C are either substituted or non-substituted. 12 It is a heteroalkylene. In some embodiments, L1 C2~C is either substituted or non-substituted. 12 It is an alkenylene. 1 C2~C is either substituted or non-substituted. 12 In some embodiments, L 1 is -(CH2CH2O) m -or-(OCH2CH2) m - is. In some embodiments, L 1 is -O-. In some embodiments, L 1 is -S-. In some embodiments, L 1 In some embodiments, L 1 is -S(=O)2-. In some embodiments, L 1 In some embodiments, L 1 In some embodiments, L 1 is -C(=N-OR1)-. In some embodiments, L 1 In some embodiments, L 1 In some embodiments, L 1 In some embodiments, L 1 is -C(=O)NR 1 - is. In some embodiments, L 1 -NR 1 In some embodiments, L 1 is -OC(=O)NR 1 - is. In some embodiments, L 1 -NR 1 In some embodiments, L 1 -NR1C(=O)NR 1 - is. In some embodiments, L 1 is -C(=O)NR 1 In some embodiments, L 1 is -S(=O)2NR 1 - is. In some embodiments, L1 -NR 1 In some embodiments, L 1 -NR 1 - is. In some embodiments, L 1 is -N(OR 1 )-. In some embodiments, L 1 is -O[(P=O)O-]O-. In some embodiments, L 1 is -O[(P=O)S-]O-. In some embodiments, L 1 It is a combination.

[0310]

[0287] According to the formulas mentioned above, in some embodiments of the compound of formula (V) or (VI), L 2 C1-C are either substituted or non-substituted. 12 Alkylene, substituted or unsubstituted C1-C 12 Heteroalkylenes, substituted or unsubstituted C2-C 12 Alkenylenes, substituted or unsubstituted C2-C2 12 Alkynylene, -(CH2CH2O) m -,-(OCH2CH2) m -, -O-, -S-, -S(=O)-, -S(=O)2-, -S(=O)(=NR 1 )-, -C(=O)-, -C(=N-OR 1 )-, -C(=O)O-, -OC(=O)-, -C(=O)C(=O)-, -C(=O)NR 1 -, -NR 1 C(=O)-, -OC(=O)NR 1 -, -NR 1 C(=O)O-, -NR 1 C(=O)NR 1 -, -C(=O)NR 1 C(=O)-, -S(=O)2NR 1 -, -NR 1 S(=O)²⁻, -NR 1 -, -N(OR 1 )-,-O[(P=O)O - ]O-, -O[(P=O)S - ]O-, or bond. In some embodiments, L 2C1-C are either substituted or non-substituted. 12 It is an alkylene. 2 C1-C are either substituted or non-substituted. 12 It is a heteroalkylene. In some embodiments, L 2 C2~C is either substituted or non-substituted. 12 It is an alkenylene. 2 C2~C is either substituted or non-substituted. 12 In some embodiments, L 2 ( is -CCH2CH2O) m -or-(OCH2CH2) m - is. In some embodiments, L 2 is -O-. In some embodiments, L 2 is -S-. In some embodiments, L 2 In some embodiments, L 2 is -S(=O)2-. In some embodiments, L 2 -S(=O)(=NR 1 )-. In some embodiments, L 2 In some embodiments, L 2 -C(=N-OR 1 )-. In some embodiments, L 2 In some embodiments, L 2 In some embodiments, L 2 In some embodiments, L 2 is -C(=O)NR 1 - is. In some embodiments, L 2 -NR 1 In some embodiments, L 2 In some embodiments, L 2 is -OC(=O)NR 1 - is. In some embodiments, L 2 -NR 1In some embodiments, L 2 -NR 1 C(=O)NR 1 - is. In some embodiments, L 2 is -C(=O)NR 1 In some embodiments, L 2 is -S(=O)2NR 1 - is. In some embodiments, L 2 -NR 1 In some embodiments, L 2 -NR 1 - is. In some embodiments, L 2 is -N(OR 1 )-. In some embodiments, L 2 is -O[(P=O)O - ]O-. In some embodiments, L 2 is -O[(P=O)S - ]O-. In some embodiments, L 2 It is a combination.

[0311]

[0288] According to the formulas mentioned above, in some embodiments of the compound of formula (V) or (VI), L 3 C1-C are either substituted or non-substituted. 12 Alkylene, substituted or unsubstituted C1-C 12 Heteroalkylenes, substituted or unsubstituted C2-C 12 Alkenylenes, substituted or unsubstituted C2-C2 12 Alkynylene, -(CH2CH2O) m -,-(OCH2CH2) m -, -O-, -S-, -S(=O)-, -S(=O)2-, -S(=O)(=NR 1 )-, -C(=O)-, -C(=N-OR 1 )-, -C(=O)O-, -OC(=O)-, -C(=O)C(=O)-, -C(=O)NR 1 -, -NR 1 C(=O)-, -OC(=O)NR 1 -, -NR 1 C(=O)O-, -NR1 C(=O)NR 1 -, -C(=O)NR 1 C(=O)-, -S(=O)2NR 1 -, -NR 1 S(=O)²⁻, -NR 1 -, -N(OR 1 )-,-O[(P=O)O - ]O-, -O[(P=O)S - ]O-, or bond. In some embodiments, L 3 C1-C are either substituted or non-substituted. 12 It is an alkylene. 3 is unsubstituted C 3~4 It is an alkylene. 3 is unsubstituted C 1~4 It is an alkylene. 3 C1-C are either substituted or non-substituted. 12 It is a heteroalkylene. In some embodiments, L 3 C2~C is either substituted or non-substituted. 12 It is an alkenylene. 3 C2~C is either substituted or non-substituted. 12 In some embodiments, L 3 is -(CH2CH2O) m -or-(OCH2CH2) m - is. In some embodiments, L 3 is -O-. In some embodiments, L 3 is -S-. In some embodiments, L 3 In some embodiments, L 3 is -S(=O)2-. In some embodiments, L 3 -S(=O)(=NR 1 )-. In some embodiments, L 3 In some embodiments, L 3 -C(=N-OR 1 )-. In some embodiments, L 3In some embodiments, L 3 In some embodiments, L 3 In some embodiments, L 3 is -C(=O)NR 1 - is. In some embodiments, L 3 -NR 1 In some embodiments, L 3 is -OC(=O)NR 1 - is. In some embodiments, L 3 -NR 1 In some embodiments, L 3 -NR 1 C(=O)NR 1 - is. In some embodiments, L 3 is -C(=O)NR 1 In some embodiments, L 3 is -S(=O)2NR 1 - is. In some embodiments, L 3 -NR 1 In some embodiments, L 3 -NR 1 - is. In some embodiments, L 3 is -N(OR 1 )-. In some embodiments, L 3 is -O[(P=O)O - ]O-. In some embodiments, L 3 is -O[(P=O)S - ]O-. In some embodiments, L 3 It is a combination.

[0312]

[0289] According to the formulas mentioned above, in some embodiments of the compound of formula (V) or (VI), L 4 C1-C are either substituted or non-substituted. 12 Alkylene, substituted or unsubstituted C1-C 12 Heteroalkylenes, substituted or unsubstituted C2-C 12Alkenylenes, substituted or unsubstituted C2-C2 12 Alkynylene, -(CH2CH2O) m -,-(OCH2CH2) m -, -O-, -S-, -S(=O)-, -S(=O)2-, -S(=O)(=NR 1 )-, -C(=O)-, -C(=N-OR 1 )-, -C(=O)O-, -OC(=O)-, -C(=O)C(=O)-, -C(=O)NR 1 -, -NR 1 C(=O)-, -OC(=O)NR 1 -, -NR 1 C(=O)O-, -NR 1 C(=O)NR 1 -, -C(=O)NR 1 C(=O)-, -S(=O)2NR 1 -, -NR 1 S(=O)²⁻, -NR 1 -, -N(OR 1 )-,-O[(P=O)O - ]O-, -O[(P=O)S - ]O-, or bond. In some embodiments, L 4 C1-C are either substituted or non-substituted. 12 It is an alkylene. 4 is an unsubstituted C4 alkylene. In some embodiments, L 4 C1-C are either substituted or non-substituted. 12 It is a heteroalkylene. In some embodiments, L 4 C2~C is either substituted or non-substituted. 12 It is an alkenylene. 4 C2~C is either substituted or non-substituted. 12 In some embodiments, L 4 is -(CH2CH2O) m -or-(OCH2CH2) m - is. In some embodiments, L 4 is -O-. In some embodiments, L 4 is -S-. In some embodiments, L 4In some embodiments, L 4 is -S(=O)2-. In some embodiments, L 4 -S(=O)(=NR 1 )-. In some embodiments, L 4 In some embodiments, L 4 -C(=N-OR 1 )-. In some embodiments, L 4 In some embodiments, L 4 In some embodiments, L 4 In some embodiments, L 4 is -C(=O)NR 1 - is. In some embodiments, L 4 -NR 1 In some embodiments, L 4 is -OC(=O)NR 1 - is. In some embodiments, L 4 -NR 1 In some embodiments, L 4 -NR 1 C(=O)NR 1 - is. In some embodiments, L 4 is -C(=O)NR 1 In some embodiments, L 4 is -S(=O)2NR 1 - is. In some embodiments, L 4 -NR 1 In some embodiments, L 4 -NR 1 - is. In some embodiments, L 4 is -N(OR 1 )-. In some embodiments, L 4 is -O[(P=O)O - ]O-. In some embodiments, L 4 is -O[(P=O)S -]O-. In some embodiments, L 4 It is a combination.

[0313]

[0290] According to the formulas mentioned above, in some embodiments of the compound of formula (V) or (VI), L 5 C1-C are either substituted or non-substituted. 12 Alkylene, substituted or unsubstituted C1-C 12 Heteroalkylenes, substituted or unsubstituted C2-C 12 Alkenylenes, substituted or unsubstituted C2-C2 12 Alkynylene, -(CH2CH2O) m -,-(OCH2CH2) m -, -O-, -S-, -S(=O)-, -S(=O)2-, -S(=O)(=NR 1 )-, -C(=O)-, -C(=N-OR 1 )-, -C(=O)O-, -OC(=O)-, -C(=O)C(=O)-, -C(=O)NR 1 -, -NR 1 C(=O)-, -OC(=O)NR 1 -, -NR 1 C(=O)O-, -NR 1 C(=O)NR 1 -, -C(=O)NR 1 C(=O)-, -S(=O)2NR 1 -, -NR 1 S(=O)²⁻, -NR 1 -, -N(OR 1 )-,-O[(P=O)O - ]O-, -O[(P=O)S - ]O-, or bond. In some embodiments, L 5 C1-C are either substituted or non-substituted. 12 It is an alkylene. 5 C1-C are either substituted or non-substituted. 12 It is a heteroalkylene. In some embodiments, L 5 C2~C is either substituted or non-substituted. 12 It is an alkenylene. 5 C2~C is either substituted or non-substituted. 12is an alkynylene. In some embodiments, L 5 is -(CH2CH2O) m - or -(OCH2CH2) m -. In some embodiments, L 5 is -O-. In some embodiments, L 5 is -S-. In some embodiments, L 5 is -S(=O)-. In some embodiments, L 5 is -S(=O)2-. In some embodiments, L 5 is -S(=O)(=NR 1 ). In some embodiments, L 5 is -C(=O)-. In some embodiments, L 5 is -C(=N-OR 1 ). In some embodiments, L 5 is -C(=O)O-. In some embodiments, L 5 is OC(=O)-. In some embodiments, L 5 is -C(=O)C(=O)-. In some embodiments, L 5 is -C(=O)NR 1 . In some embodiments, L 5 is -NR 1 C(=O)-. In some embodiments, L 2 is -NRHC(=O)-. In some embodiments, L 5 is -OC(=O)NR 1 . In some embodiments, L 5 is -NR 1 C(=O)O-. In some embodiments, L 5 is -NR 1 C(=O)NR 1 . In some embodiments, L 5 is -C(=O)NR 1 C(=O)-. In some embodiments, L 5 is -S(=O)2NR 1 . In some embodiments, L 5 is -NR 1In some embodiments, L 5 -NR 1 - is. In some embodiments, L 5 is -N(OR 1 )-. In some embodiments, L 5 is -O[(P=O)O - ]O-. In some embodiments, L 5 is -O[(P=O)S - ]O-. In some embodiments, L 5 It is a combination.

[0314]

[0291] According to the formulas mentioned above, in some embodiments of the compound of formula (V) or (VI), L 6 C1-C are either substituted or non-substituted. 12 Alkylene, substituted or unsubstituted C1-C 12 Heteroalkylenes, substituted or unsubstituted C2-C 12 Alkenylenes, substituted or unsubstituted C2-C2 12 Alkynylene, -(CH2CH2O) m -,-(OCH2CH2) m -, -O-, -S-, -S(=O)-, -S(=O)2-, -S(=O)(=NR 1 )-, -C(=O)-, -C(=N-OR 1 )-, -C(=O)O-, -OC(=O)-, -C(=O)C(=O)-, -C(=O)NR 1 -, -NR 1 C(=O)-, -OC(=O)NR 1 -, -NR 1 C(=O)O-, -NR 1 C(=O)NR 1 -, -C(=O)NR 1 C(=O)-, -S(=O)2NR 1 -, -NR 1 S(=O)²⁻, -NR 1 -, -N(OR 1 )-,-O[(P=O)O - ]O-, -O[(P=O)S - ]O-, or bond. In some embodiments, L 6is a substituted or unsubstituted C1-C 12 alkylene. In some embodiments, L 6 is an unsubstituted C 3-4 alkylene. In some embodiments, L 6 is an unsubstituted C 1~4 alkylene. In some embodiments, L 6 is a substituted or unsubstituted C1-C 12 heteroalkylene. In some embodiments, L 6 is a substituted or unsubstituted C2-C 12 alkenylene. In some embodiments, L 6 is a substituted or unsubstituted C2-C 12 alkynylene. In some embodiments, L 6 is -(CH2CH2O) m - or -(OCH2CH2) m -. In some embodiments, L 6 is -O-. In some embodiments, L 6 is -S-. In some embodiments, L 6 is -S(=O)-. In some embodiments, L 6 is -S(=O)2-. In some embodiments, L 6 is -S(=O)(=NR 1 )-. In some embodiments, L 6 is -C(=O)-. In some embodiments, L 6 is -C(=N-OR 1 )-. In some embodiments, L 6 is -C(=O)O-. In some embodiments, L 6 is OC(=O)-. In some embodiments, L 6 is -C(=O)C(=O)-. In some embodiments, L 6 is -C(=O)NR 1 -. In some embodiments, L 6 is -NR 1 C(=O)-. In some embodiments, L 6 is -OC(=O)NR 1- is. In some embodiments, L 6 -NR 1 In some embodiments, L 6 -NR 1 C(=O)NR 1 - is. In some embodiments, L 6 is -C(=O)NR 1 In some embodiments, L 6 is -S(=O)2NR 1 - is. In some embodiments, L 6 -NR 1 In some embodiments, L 6 -NR 1 - is. In some embodiments, L 6 is -N(OR 1 )-. In some embodiments, L 6 is -O[(P=O)O - ]O-. In some embodiments, L 6 is -O[(P=O)S - ]O-. In some embodiments, L 6 It is a combination.

[0315]

[0292] According to the formulas mentioned above, in some embodiments of the compound of formula (V) or (VI), L 7 C1-C are either substituted or non-substituted. 12 Alkylene, substituted or unsubstituted C1-C 12 Heteroalkylenes, substituted or unsubstituted C2-C 12 Alkenylenes, substituted or unsubstituted C2-C2 12 Alkynylene, -(CH2CH2O) m -,-(OCH2CH2) m -, -O-, -S-, -S(=O)-, -S(=O)2-, -S(=O)(=NR 1 )-, -C(=O)-, -C(=N-OR 1 )-, -C(=O)O-, -OC(=O)-, -C(=O)C(=O)-, -C(=O)NR 1 -, -NR 1C(=O)-, -OC(=O)NR 1 -, -NR 1 C(=O)O-, -NR 1 C(=O)NR 1 -, -C(=O)NR 1 C(=O)-, -S(=O)2NR 1 -, -NR 1 S(=O)²⁻, -NR 1 -, -N(OR 1 )-,-O[(P=O)O - ]O-, -O[(P=O)S - ]O-, or bond. In some embodiments, L 7 C1-C are either substituted or non-substituted. 12 It is an alkylene. 7 is an unsubstituted C4 alkylene. In some embodiments, L 7 C1-C are either substituted or non-substituted. 12 It is a heteroalkylene. In some embodiments, L 7 C2~C is either substituted or non-substituted. 12 It is an alkenylene. 7 C2~C is either substituted or non-substituted. 12 In some embodiments, L 7 is -(CH2CH2O) m -or-(OCH2CH2) m - is. In some embodiments, L 7 is -O-. In some embodiments, L 7 is -S-. In some embodiments, L 7 In some embodiments, L 7 is -S(=O)2-. In some embodiments, L 7 -S(=O)(=NR 1 )-. In some embodiments, L 7 In some embodiments, L 7 -C(=N-OR 1 )-. In some embodiments, L 7In some embodiments, L 7 In some embodiments, L 7 In some embodiments, L 7 is -C(=O)NR 1 - is. In some embodiments, L 7 -NR 1 In some embodiments, L 7 is -OC(=O)NR 1 - is. In some embodiments, L 7 -NR 1 In some embodiments, L 7 -NR 1 C(=O)NR 1 - is. In some embodiments, L 7 is -C(=O)NR 1 In some embodiments, L 7 is -S(=O)2NR 1 - is. In some embodiments, L 7 -NR 1 In some embodiments, L 7 -NR 1 - is. In some embodiments, L 7 is -N(OR 1 )-. In some embodiments, L 7 is -O[(P=O)O - ]O-. In some embodiments, L 7 is -O[(P=O)S - ]O-. In some embodiments, L 7 It is a combination.

[0316]

[0293] According to the formulas mentioned above, in some embodiments of the compound of formula (V) or (VI), L 8 C1-C are either substituted or non-substituted. 12 Alkylene, substituted or unsubstituted C1-C 12 Heteroalkylenes, substituted or unsubstituted C2-C 12Alkenylenes, substituted or unsubstituted C2-C2 12 Alkynylene, -(CH2CH2O) m -,-(OCH2CH2) m -, -O-, -S-, -S(=O)-, -S(=O)2-, -S(=O)(=NR 1 )-, -C(=O)-, -C(=N-OR 1 )-, -C(=O)O-, -OC(=O)-, -C(=O)C(=O)-, -C(=O)NR 1 -, -NR 1 C(=O)-, -OC(=O)NR 1 -, -NR 1 C(=O)O-, -NR 1 C(=O)NR 1 -, -C(=O)NR 1 C(=O)-, -S(=O)2NR 1 -, -NR 1 S(=O)²⁻, -NR 1 -, -N(OR 1 )-,-O[(P=O)O - ]O-, -O[(P=O)S - ]O- or bonded. In some embodiments, L 8 C1-C are either substituted or non-substituted. 12 It is an alkylene. 8 C1-C are either substituted or non-substituted. 12 It is a heteroalkylene. In some embodiments, L 8 C2~C is either substituted or non-substituted. 12 It is an alkenylene. 8 C2~C is either substituted or non-substituted. 12 In some embodiments, L 8 is -(CH2CH2O) m -or-(OCH2CH2) m - is. In some embodiments, L 8 is -O-. In some embodiments, L 8 is -S-. In some embodiments, L 8 In some embodiments, L 8is -S(=O)2-. In some embodiments, L 8 -S(=O)(=NR 1 )-. In some embodiments, L 8 In some embodiments, L 8 -C(=N-OR 1 )-. In some embodiments, L 8 In some embodiments, L 8 In some embodiments, L 8 In some embodiments, L 8 is -C(=O)NR 1 - is. In some embodiments, L 8 -NR 1 In some embodiments, L 8 is -OC(=O)NR 1 - is. In some embodiments, L 8 -NR 1 In some embodiments, L 2 In some embodiments, L 8 -NR 1 C(=O)NR 1 - is. In some embodiments, L 8 is -C(=O)NR 1 In some embodiments, L 8 is -S(=O)2NR 1 - is. In some embodiments, L 8 -NR 1 In some embodiments, L 8 -NR 1 - is. In some embodiments, L 8 is -N(OR 1 )-. In some embodiments, L 8 is -O[(P=O)O - ]O-. In some embodiments, L 8 is -O[(P=O)S -]O-. In some embodiments, L 8 It is a combination.

[0317]

[0294] According to the formulas mentioned above, in some embodiments of the compound of formula (V) or (VI), L 9 C1-C are either substituted or non-substituted. 12 Alkylene, substituted or unsubstituted C1-C 12 Heteroalkylenes, substituted or unsubstituted C2-C 12 Alkenylenes, substituted or unsubstituted C2-C2 12 Alkynylene, -(CH2CH2O) m -,-(OCH2CH2) m -, -O-, -S-, -S(=O)-, -S(=O)2-, -S(=O)(=NR 1 )-, -C(=O)-, -C(=N-OR 1 )-, -C(=O)O-, -OC(=O)-, -C(=O)C(=O)-, -C(=O)NR 1 -, -NR 1 C(=O)-, -OC(=O)NR 1 -, -NR 1 C(=O)O-, -NR 1 C(=O)NR 1 -, -C(=O)NR 1 C(=O)-, -S(=O)2NR 1 -, -NR 1 S(=O)²⁻, -NR 1 -, -N(OR 1 )-,-O[(P=O)O - ]O-, -O[(P=O)S - ]O-, or bond. In some embodiments, L 9 C1-C are either substituted or non-substituted. 12 It is an alkylene. 9 is unsubstituted C 3~4 It is an alkylene. 9 is unsubstituted C 1~4 It is an alkylene. 9 C1-C are either substituted or non-substituted. 12 It is a heteroalkylene. In some embodiments, L9 C2~C is either substituted or non-substituted. 12 It is an alkenylene. 9 C2~C is either substituted or non-substituted. 12 In some embodiments, L 9 is -(CH2CH2O) m -or-(OCH2CH2) m - is. In some embodiments, L 9 is -O-. In some embodiments, L 9 is -S-. In some embodiments, L 9 In some embodiments, L 9 is -S(=O)2-. In some embodiments, L 9 -S(=O)(=NR 1 )-. In some embodiments, L 9 In some embodiments, L 9 -C(=N-OR 1 )-. In some embodiments, L 9 In some embodiments, L 9 In some embodiments, L 9 In some embodiments, L 9 is -C(=O)NR 1 - is. In some embodiments, L 9 -NR 1 In some embodiments, L 9 is -OC(=O)NR 1 - is. In some embodiments, L 9 -NR 1 In some embodiments, L 9 -NR 1 C(=O)NR 1 - is. In some embodiments, L 9 is -C(=O)NR 1 In some embodiments, L 9is -S(=O)2NR 1 - is. In some embodiments, L 9 -NR 1 In some embodiments, L 9 -NR 1 - is. In some embodiments, L 9 is -N(OR 1 )-. In some embodiments, L 9 is -O[(P=O)O - ]O-. In some embodiments, L 9 is -O[(P=O)S - ]O-. In some embodiments, L 9 It is a combination.

[0318]

[0295] According to the formulas mentioned above, in some embodiments of the compound of formula (V) or (VI), L 10 C1-C are either substituted or non-substituted. 12 Alkylene, substituted or unsubstituted C1-C 12 Heteroalkylenes, substituted or unsubstituted C2-C 12 Alkenylenes, substituted or unsubstituted C2-C2 12 Alkynylene, -(CH2CH2O) m -,-(OCH2CH2) m -, -O-, -S-, -S(=O)-, -S(=O)2-, -S(=O)(=NR 1 )-, -C(=O)-, -C(=N-OR 1 )-, -C(=O)O-, -OC(=O)-, -C(=O)C(=O)-, -C(=O)NR 1 -, -NR 1 C(=O)-, -OC(=O)NR 1 -, -NR 1 C(=O)O-, -NR 1 C(=O)NR 1 -, -C(=O)NR 1 C(=O)-, -S(=O)2NR 1 -, -NR 1 S(=O)²⁻, -NR 1 -, -N(OR 1 )-,-O[(P=O)O- ]O-, -O[(P=O)S - ]O-, or bond. In some embodiments, L 10 C1-C are either substituted or non-substituted. 12 It is an alkylene. 10 C1-C are either substituted or non-substituted. 12 It is a heteroalkylene. In some embodiments, L 10 C2~C is either substituted or non-substituted. 12 It is an alkenylene. 10 C2~C is either substituted or non-substituted. 12 In some embodiments, L 10 is -(CH2CH2O) m -or-(OCH2CH2) m - is. In some embodiments, L 10 is -O-. In some embodiments, L 10 is -S-. In some embodiments, L 10 In some embodiments, L 10 is -S(=O)2-. In some embodiments, L 10 -S(=O)(=NR 1 )-. In some embodiments, L 10 In some embodiments, L 10 -C(=N-OR 1 )-. In some embodiments, L 10 In some embodiments, L 10 In some embodiments, L 10 In some embodiments, L 10 is -C(=O)NR 1 - is. In some embodiments, L 10 -NR 1 In some embodiments, L 10 is -OC(=O)NR 1 - is. In some embodiments, L 10-NR 1 In some embodiments, L 10 -NR 1 C(=O)NR 1 - is. In some embodiments, L 10 is -C(=O)NR 1 In some embodiments, L 10 is -S(=O)2NR 1 - is. In some embodiments, L 10 -NR 1 In some embodiments, L 10 -NR 1 - is. In some embodiments, L 10 is -N(OR 1 )-. In some embodiments, L 10 is -O[(P=O)O - ]O-. In some embodiments, L 10 is -O[(P=O)S - ]O-. In some embodiments, L 10 L is a substituted or unsubstituted C1-C6 alkylene. In some embodiments, L 10 L is a substituted or unsubstituted C1-C3 alkylene. In some embodiments, L 10 L is a substituted or unsubstituted C2-C3 alkylene. In some embodiments, L 10 is -CH2CH2-. In some embodiments, L 10 It is a combination.

[0319]

[0296] According to the formulas mentioned above, in some embodiments of the compound of formula (V) or (VI), L 11 This is a substituted or unsubstituted -(CH2CH2O) n -, substitution or non-substitution of -(OCH2CH2) n -, substituted or unsubstituted -(CH2) n -, or combination. In some embodiments, L 11 -(CH2CH2O) is either substituted or unsubstituted. n - is. In some embodiments, L11 -(OCH2CH2) is either substituted or unsubstituted. n - is. In some embodiments, L 11 -(CH2) is either substituted or unsubstituted. n - is. In some embodiments, L 11 This is a combination. In some embodiments, n is 30 to 50. In some embodiments, n is 30 to 40. In some embodiments, n is 40 to 50.

[0320]

[0297] According to the formulas mentioned above, in some embodiments of the compound of formula (V) or (VI), L 12 C1-C are either substituted or non-substituted. 12 Alkylene, substituted or unsubstituted C1-C 12 Heteroalkylenes, substituted or unsubstituted C2-C 12 Alkenylenes, substituted or unsubstituted C2-C2 12 Alkynylene, -(CH2CH2O) m -,-(OCH2CH2) m -, -O-, -S-, -S(=O)-, -S(=O)2-, -S(=O)(=NR 1 )-, -C(=O)-, -C(=N-OR 1 )-, -C(=O)O-, -OC(=O)-, -C(=O)C(=O)-, -C(=O)NR 1 -, -NR 1 C(=O)-, -OC(=O)NR 1 -, -NR 1 C(=O)O-, -NR 1 C(=O)NR 1 -, -C(=O)NR 1 C(=O)-, -S(=O)2NR 1 -, -NR 1 S(=O)²⁻, -NR 1 -, -N(OR 1 )-,-O[(P=O)O - ]O-, -O[(P=O)S - ]O-, or bond. In some embodiments, L 12 C1-C are either substituted or non-substituted. 12It is an alkylene. 12 C1-C are either substituted or non-substituted. 12 It is a heteroalkylene. In some embodiments, L 12 C2~C is either substituted or non-substituted. 12 It is an alkenylene. 12 C2~C is either substituted or non-substituted. 12 In some embodiments, L 12 is -(CH2CH2O) m -or-(OCH2CH2) m - is. In some embodiments, L 12 is -O-. In some embodiments, L 12 is -S-. In some embodiments, L 12 In some embodiments, L 12 is -S(=O)2-. In some embodiments, L 12 -S(=O)(=NR 1 )-. In some embodiments, L 12 In some embodiments, L 12 -C(=N-OR 1 )-. In some embodiments, L 12 In some embodiments, L 12 In some embodiments, L 12 In some embodiments, L 12 is -C(=O)NR 1 - is. In some embodiments, L 12 -NR 1 In some embodiments, L 12 is -OC(=O)NR 1 - is. In some embodiments, L 12 -NR 1 In some embodiments, L 12 -NR 1 C(=O)NR 1- is. In some embodiments, L 12 is -C(=O)NR 1 In some embodiments, L 12 is -S(=O)2NR 1 - is. In some embodiments, L 12 -NR 1 In some embodiments, L 12 -NR 1 - is. In some embodiments, L 12 is -N(OR 1 )-. In some embodiments, L 12 is -O[(P=O)O - ]O-. In some embodiments, L 12 is -O[(P=O)S - ]O-. In some embodiments, L 12 L is a substituted or unsubstituted C1-C6 alkylene. In some embodiments, L 12 L is a substituted or unsubstituted C1-C3 alkylene. In some embodiments, L 12 L is a substituted or unsubstituted C2-C3 alkylene. In some embodiments, L 12 L is -CH2CH2-. In some embodiments of the compounds of formula (V) or (VI), L 12 -NR 1 In some embodiments, L 12 is a bond. In some embodiments, L 12 is an organic molecular residue that intercalates with group R. In some embodiments, L 12These can interact with base pairs ionically / electrostatically or covalently. Some non-limiting examples of organic molecular residues that intercalate with the group R include berberine, ethidium bromide, daunomycin, thalidomide, doxorubicin (adriamycin), aflatoxin B1, amsacrine, acridines (e.g., proflavin, quinacrine, acridine orange, pyrazoloacrine), acriflavin, amonafide, 1,10-phenanthroline, metal cations with polycyclic aromatic ligands (e.g., metals such as Rh(III), ligands such as Ir(III), dipyridine, terpyridine), bleomycin, actinomycin D, and ellipticin.

[0321]

[0298] In some embodiments of the compound of formula (V) or (VI), m is an integer selected from 1 to 10. In some embodiments, m is selected from 1 to 3. In some embodiments, m is selected from 1 to 5. In some embodiments, m is selected from 3 to 8. In some embodiments, m is selected from 2 to 5. In some embodiments, m is selected from 5 to 10. In some embodiments, m is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, m is 1. In some embodiments, m is 2. In some embodiments, m is 3.

[0322]

[0299] In some embodiments of the compound of formula (V) or (VI), n is an integer selected from 1 to 200. In some embodiments, n is selected from 1 to 20. In some embodiments, n is selected from 1 to 50. In some embodiments, n is selected from 1 to 100. In some embodiments, n is selected from 50 to 100. In some embodiments, n is selected from 25 to 50. In some embodiments, n is selected from 30 to 40. In some embodiments, n is selected from 25 to 75. In some embodiments, n is selected from 100 to 200. In some embodiments, n is selected from 50 to 150. In some embodiments, n is selected from 150 to 200.

[0323]

[0300] In some embodiments of the compounds of formula (VI), formula (VIa), or formula (VIb), m is an integer selected from 1 to 10. In some embodiments, m is selected from 1 to 3. In some embodiments, m is selected from 1 to 5. In some embodiments, m is selected from 3 to 8. In some embodiments, m is selected from 2 to 5. In some embodiments, m is selected from 5 to 10. In some embodiments, m is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, m is 1. In some embodiments, m is 2. In some embodiments, m is 3.

[0324]

[0301] In some embodiments of the compound of formula (VI), formula (VIa), or formula (VIb), n is an integer selected from 1 to 200. In some embodiments, n is selected from 1 to 20. In some embodiments, n is selected from 1 to 50. In some embodiments, n is selected from 1 to 100. In some embodiments, n is selected from 50 to 100. In some embodiments, n is selected from 25 to 50. In some embodiments, n is selected from 30 to 40. In some embodiments, n is selected from 25 to 75. In some embodiments, n is selected from 100 to 200. In some embodiments, n is selected from 50 to 150. In some embodiments, n is selected from 150 to 200.

[0325]

[0302] In some embodiments of the compounds of formula (V), formula (VI), formula (VIa), or formula (VIb), each R 1 In some embodiments, R is independently H or -CH3. 1 H is H.

[0326]

[0303] In some embodiments of the compounds of formula (V), formula (VI), formula (VIa), or formula (VIb), R comprises one or more fatty alcohols, fatty acids, glycerolipids, glycerophospholipids, sphingolipids, saccharolids, polyketides, sterol lipids, and prenolipids. In some embodiments, R comprises one or more fatty alcohols. In some embodiments, each fatty alcohol is independently saturated, monounsaturated, or polyunsaturated fatty alcohol. In some embodiments, the fatty alcohol comprises one or more C2-C 26 Contains fatty alcohols. In some embodiments, the fatty alcohols consist of two or more C2-C2 alcohols. 26Contains fatty alcohols. In some embodiments, each fatty alcohol is a C12, C14, C16, C18, C20, or C22 fatty alcohol. In some embodiments, each fatty alcohol is independently docosahexaenol, eicosapentaenol, oleyl alcohol, stearyl alcohol, (9Z,12Z)-octadeca-9,12-dien-1-yl alcohol, (Z)-docosa-13-en-1-yl alcohol, docosanyl alcohol, (E)-octadeca-9-en-1-yl alcohol, eicosanyl alcohol, (9Z,12Z,15Z)-octadeca-9,12,15-trien-1-yl alcohol, or palmityl alcohol. In some embodiments, each fatty alcohol is stearyl alcohol. In some embodiments, R contains one or more sterol lipids. In some embodiments, R contains one or more vitamins. In some embodiments, each vitamin is independently vitamin A, vitamin D, vitamin E, or vitamin K.

[0327]

[0304] In some embodiments, the R group represented by formula (V), formula (VI), formula (VIa), or formula (VIb) comprises the payload described herein. In some embodiments, the R group represented by formula (V), formula (VI), formula (VIa), or formula (VIb) comprises lipids.

[0328]

[0305] In some embodiments, the R group obtained by formula (V), formula (VI), formula (VIa), or formula (VIb) comprises a nucleic acid. In some embodiments, this nucleic acid is a single-stranded nucleic acid. In some embodiments, the single-stranded nucleic acid is DNA. In some embodiments, the single-stranded nucleic acid is RNA. In some embodiments, this nucleic acid is a double-stranded nucleic acid. In some embodiments, this double-stranded nucleic acid is DNA. In some embodiments, this double-stranded nucleic acid is RNA. In some embodiments, this double-stranded nucleic acid is a DNA-RNA hybrid. In some embodiments, the nucleic acid is messenger RNA (mRNA), microRNA, asymmetric interfering RNA (aiRNA), small hairpin RNA (shRNA), or dicer substrate dsRNA. In some embodiments, the nucleic acid is mRNA. In some embodiments, R comprises an mRNA molecule encoding a Cas nuclease, i.e., Cas nuclease mRNA. In some embodiments, R comprises one or more guide RNAs or nucleic acids encoding guide RNAs. In some embodiments, R comprises a template nucleic acid for repair or recombination. In some embodiments, R comprises mRNA encoding a gene editing factor nuclease. In some embodiments, R comprises mRNA encoding a base editing factor nucleas...

Claims

1. Formula (V): 【Chemistry 1】 Formula (V) A compound of, During the ceremony, Each A group contains an N-acetylgalactosamine (GalNAc) moiety. L １ , ２ , １ , L ２ , L ３ , L ４ , L ５ , L ６ , L ７ , L ８ , L ９ , L １０ , and L １２ each of which is independently a substituted or unsubstituted C １ to C １２ alkylene, a substituted or unsubstituted C １ to C １２ heteroalkylene, a substituted or unsubstituted C ２ to C １２ alkenylene, a substituted or unsubstituted C ２ to C １２ alkynylene, -(CH ２ CH ２ O) ｍ -, -(OCH ２ CH ２ ) ｍ -, -O-, -S-, -S(=O)-, -S(=O)[[ID= / / ]] ２ -, -S(=O)(=NR １ ) -, -C(=O)-, -C(=N-OR １ )-, -C(=O)O-, -OC(=O)-, -C(=O)C(=O)-, -C(=O)NR １ -, -NR １ C(=O)-, -OC(=O)NR １ -, -NR １ C(=O)O-, -NR １ C(=O)NR １ -, -C(=O)NR １ C(=O)-, -S(=O) ２ NR １ -, -NR １ S(=O) ２ -, -NR １ -, or -N(OR １ )-; L １１ is a substituted or unsubstituted - (CH ２ CH ２ O) ｎ - or substituted or unsubstituted - (OCH ２ CH ２ ) ｎ - and; Each R １ Independently, H or substituted or unsubstituted C １ ~C ６ It is alkyl; R comprises lipids or lipid nanoparticles; m is an integer selected from 1 to 10; A compound where n is an integer selected from 1 to 200.

2. Each of L1, L4, and L7 is independently a C2-C6 alkylene, Each of L2, L5, and L8 is independently -C(=O)NR1- or -NR1C(=O)-, L3, L6, and L9 are each independently C2-C12 alkylenes. L10 is C1-C6 alkylene, L 12 is -NR 1 C(=O)- or -NR 1 C(=O)O-, L 11 is -(CH 2 CH 2 O) n- or -(OCH 2 CH 2) n-, Each R1 is independently either H or CH3; n is an integer selected from 1 to 50. The compound according to claim 1.

3. L 1, L ３ , L ４ , and L ７ Each of them is - (CH ２ ) ４ - including; L 2, L ５ , and L ８ Each of them contains -C(=O)NH-; L 6 and L ９ Each of them is - (CH ２ ) ３ - including; L 10 is - (CH ２ ) １～３ -ien-CH ２ CH ２ O- or -CH ２ It is O-; L 11 is - (CH ２ CH ２ O) ｎ - or - (OCH ２ CH ２ ) ｎ - and in the formula n is an integer selected from 1 to 50; L 12 is -NH(CO)O- or -NH(CO)-; R is dialkylglycerol, diacylglycerol, sterol, C １０ ~C ３０ n-alkyl groups containing carbon atoms, C １０ ~C ３０ The compound according to claim 1, selected from the group consisting of branched alkyl groups containing carbon atoms and tocopherols.

4. The compound according to claim 1, wherein n is an integer from 2 to 50.

5. The compound according to claim 1, wherein n is 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, or 49.

6. The compound according to claim 1, wherein n is 33, 34, 35, 36, 37, 38, or 39.

7. Each of the above A units 【Chemistry 2】 The compound according to claim 1.

8. The compound according to claim 1, selected from 1001-1019, 1060, 1065, 1066, and 1075-1085 in Table 4. 【Request Item 9】 【Chemistry 3】 1004 is, or 【Chemistry 3-1】 (Here, p and q are each 2, and n is one of 33-35 or 37-39) The compound according to claim 1.

10. The compound according to claim 1, wherein R comprises one or more of fatty alcohols, fatty acids, glycerolipids, glycerophospholipids, sphingolipids, glycolipids, polyketides, or sterols.

11. The compound according to claim 1, wherein R is a lipid nanoparticle comprising one or more mRNAs encoding one or more gene editing nucleases or base editing factors, and one or more guide RNAs.

12. Formula (V): 【Chemistry 4】 Formula (V) A compound of, During the ceremony, Each A group contains an N-acetylgalactosamine (GalNAc) moiety. L １ 、L ２ 、L ３ 、L ４ 、L ５ 、L ６ 、L ７ 、L ８ 、L ９ 、L １０ 、and L １２ each is, independently, a substituted or unsubstituted C １ ~C １２ alkylene, a substituted or unsubstituted C １ ~C １２ heteroalkylene, a substituted or unsubstituted C ２ ~C １２ alkenylene, a substituted or unsubstituted C ２ ~C １２ alkynylene, -(CH ２ CH ２ O) ｍ -, -(OCH ２ CH ２ ) ｍ -, -O-, -S-, -S(=O)-, -S(=O) ２ -, -S(=O)(=NR １ )-, -C(=O)-, -C(=N-OR １ )-, -C(=O)O-, -OC(=O)-, -C(=O)C(=O)-, -C(=O)NR １ -, -NR １ C(=O)-, -OC(=O)NR １ -, -NR １ C(=O)O-, -NR １ C(=O)NR １ -, -C(=O)NR １ C(=O)-, -S(=O)​​​​​​​​​​​​ L １１ is a substituted or unsubstituted - (CH ２ CH ２ O) ｎ - or substituted or unsubstituted - (OCH ２ CH ２ ) ｎ - and; Each R １ Independently, H or substituted or unsubstituted C １ ~C ６ It is alkyl; R contains lipids; m is an integer selected from 1 to 10; A compound where n is an integer selected from 1 to 200.

13. The compound according to claim 12, wherein R comprises one or more fatty alcohols, fatty acids, glycerolipids, glycerophospholipids, sphingolipids, glycolipids, polyketides, or sterols.

14. L 1, L ３ , L ４ , and L ７ Each of them is - (CH ２ ) ４ - including; L 2, L ５ , and L ８ Each of them contains -C(=O)NH-; L 6 and L ９ Each of them is - (CH ２ ) ３ - including; L 10 is - (CH ２ ) １～３ -ien-CH ２ CH ２ O- or -CH ２ It is O-; L 11 is - (CH ２ CH ２ O) ｎ - or - (OCH ２ CH ２ ) ｎ - and in the formula n is an integer selected from 1 to 50; L 12 is -NH(CO)O- or -NH(CO)-; R is dialkylglycerol, diacylglycerol, sterol, C １０ ~C ３０ n-alkyl groups containing carbon atoms, C １０ ~C ３０ The compound according to claim 12, selected from the group consisting of branched alkyl groups containing carbon atoms and tocopherols.

15. The compound according to any one of claims 12 to 14, wherein n is an integer from 2 to 50.

16. The compound according to any one of claims 12 to 14, wherein n is 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, or 49.

17. The compound according to any one of claims 12 to 14, wherein n is 33, 34, 35, 36, 37, 38, or 39.

18. Each of the above A units 【Transformation 5】 The compound according to any one of claims 12 to 14.

19. Lipid nanoparticles comprising the compound according to any one of claims 1 to 18.

20. The lipid nanoparticles according to claim 19, further comprising mRNA encoding a guide polynucleotide and a gene editing factor nuclease.

21. The lipid nanoparticle according to claim 20, wherein the ratio of guide polynucleotide to mRNA is 10:1 to 1:10 by weight.

22. The lipid nanoparticle according to claim 20, wherein the ratio of guide polynucleotides to mRNA is 2:1 to 1:2 by weight.

23. The lipid nanoparticle according to claim 20, wherein the ratio of guide polynucleotides to mRNA is 1:1 by weight.

24. The lipid nanoparticle according to claim 20, wherein the guide polynucleotide is a single guide RNA comprising a tracr sequence and a spacer sequence.

25. The tracr sequence is a chemically modified nucleic acid sequence, gUUUUAGagcuaGaaauagcaaGUUaAaAuAaggcuaGUccGUUAucAAcuuGaaaaagugGcaccgagucggugcusususu (Nucleic acid 21-100 of sequence number 126) Including, here, The lipid nanoparticles according to claim 24, wherein 1) uppercase A, U, G, and C represent adenosine, uridine, guanosine, and cytidine, respectively; 2) lowercase a, u, g, and c represent adenosine, uridine, guanosine, and cytidine modified with 2'-O-methyl, respectively; and 3) lowercase s represents a phosphorothioate (PS) bond.

26. The lipid nanoparticle according to claim 20, wherein the gene editing factor nuclease comprises a Cas protein.

27. ​​The lipid nanoparticle according to claim 26, wherein the Cas protein is the Cas9 protein.

28. The lipid nanoparticle according to claim 20, wherein the gene editing factor nuclease comprises a base editing factor including Cas9 niccasase and deaminase.

29. The lipid nanoparticle according to claim 28, wherein the deaminase is adenosine deaminase.

30. Lipid nanoparticles according to any one of claims 19 to 29, further comprising an excipient containing a lipid selected from sterols, phospholipids, stealth lipids, aminolipids, or a combination thereof.

31. The compound constitutes 0.001 mol% to 2.0 mol% of the total lipid content of the lipid nanoparticles, The amino lipids constitute 1 mol% to 65 mol% of the total lipid content of the lipid nanoparticles. The stealth lipids constitute 0.1 mol% to 10 mol% of the total lipid content of the lipid nanoparticles. The sterols constitute 20 mol% to 50 mol% of the total lipid content of the lipid nanoparticles. The lipid nanoparticles according to claim 30, wherein the phospholipids constitute 1 mol% to 20 mol% of the total lipid content of the lipid nanoparticles.

32. The compound constitutes about 0.01 mol%, about 0.05 mol%, or about 0.1 mol% of the total lipid content of the lipid nanoparticles. The amino lipids constitute approximately 45 mol%, 46 mol%, 47 mol%, 48 mol%, 49 mol%, 50 mol%, 51 mol%, 52 mol%, 53 mol%, 54 mol%, or 55 mol% of the total lipid content of the lipid nanoparticles. The stealth lipids constitute 0.1 mol% to 6.0 mol% of the total lipid content of the lipid nanoparticles. The sterols constitute approximately 35 mol%, 36 mol%, 37 mol%, 38 mol%, 39 mol%, 40 mol%, 41 mol%, 42 mol%, 43 mol%, 44 mol%, 45 mol%, 46 mol%, 47 mol%, 48 mol%, or 50 mol% of the total lipid content of the lipid nanoparticles. The lipid nanoparticles according to claim 30, wherein phospholipids constitute about 9 mol%, about 10 mol%, or about 11 mol% of the total lipid content of the lipid nanoparticles.

33. a) The compound is 【Chemistry 5-1】 This includes, where p and q are 2, and n is 33 to 39. b) The aminolipid comprises any of the following: 【Chemistry 5-2】 ,or That combination, c) The stealth lipid comprises any of the following: 【Chemistry 5-3】 or a combination thereof, d) The phospholipid comprises any of the following: 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), dimyristoylphosphatidylcholine (DMPC), dioleoylphosphatidylcholine (DOPC), or a combination thereof. Lipid nanoparticles according to claim 30.

34. a) The compound is 【Chemistry 5-4】 This includes, where p and q are 2, and n is 33 to 39. b) The amino lipids are 【Transformation 5-5】 Includes, c) The stealth lipid is [Chemistry 5-6] including and d) The phospholipid contains 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), Lipid nanoparticles according to claim 30.

35. a) The compound described in Claims 1 to 14 constitutes about 0.05 mol% of the total lipid content of the lipid nanoparticles, b) The aminolipids [Transformation 5-7] These constitute approximately 48 mol%, 49 mol%, 50 mol%, 51 mol%, or 52 mol% of the total lipid content of the lipid nanoparticles. c) The stealth lipid is [Transformation 5-8] These constitute approximately 2.0 mol%, 2.1 mol%, 2.2 mol%, 2.3 mol%, 2.4 mol%, 2.5 mol%, 2.6 mol%, 2.7 mol%, 2.8 mol%, 2.9 mol%, or 3.0 mol% of the total lipids present in the lipid nanoparticles. d) The sterols constitute approximately 35 mol%, approximately 36 mol%, approximately 37 mol%, approximately 38 mol%, approximately 39 mol%, approximately 40 mol%, or approximately 41 mol% of the total lipids present in the lipid nanoparticles. e) The phospholipid is 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), which constitutes approximately 9.0 mol% of the total lipids present in the lipid nanoparticles. Lipid nanoparticles according to claim 30.

36. Use of lipid nanoparticles according to any one of claims 19 to 35 in the manufacture of a pharmaceutical product for editing genes in a target area.

37. A composition for use in a method for reducing the risk of coronary artery disease in subjects requiring reduction of the risk of coronary artery disease, comprising lipid nanoparticles encapsulating a payload containing one or more pharmaceutically active agents, wherein the lipid nanoparticles are of formula (V): 【Transformation 6】 Formula (V) Contains the compound, During the ceremony, Each A group contains an N-acetylgalactosamine (GalNAc) moiety; L １ , L ２ , L ３ , L ４ , L ５ , L ６ , L ７ , L ８ , L ９ , L １０ and L １２ Each of these is an independent substitution or non-substitution of C. １ ~C １２ Alkylene, substituted or unsubstituted C １ ~C １２ Heteroalkylenes, substituted or unsubstituted C ２ ~C １２ Alkenylenes, substituted or unsubstituted carbon atoms ２ ~C １２ Alkinylene, -(CH ２ CH ２ O) ｍ -, - (OCH ２ CH ２ ) ｍ -, -O-, -S-, -S(=O)-, -S(=O) ２ -, -S(=O)(=NR １ )-, -C(=O)-, -C(=N-OR １ )-, -C(=O)O-, -OC(=O)-, -C(=O)C(=O)-, -C(=O)NR １ -, -NR １ C(=O)-, -OC(=O)NR １ -, -NR １ C(=O)O-, -NR １ C(=O)NR １ -, -C(=O)NR １ C(=O)-, -S(=O) ２ NR １ -, -NR １ S (=O) ２ -, -NR １ -, or -N (OR １ ) - and; L １１ is a substituted or unsubstituted - (CH ２ CH ２ O) ｎ - or substituted or unsubstituted - (OCH ２ CH ２ ) ｎ - and; Each R １ Independently, H or substituted or unsubstituted C １ ~C ６ It is alkyl; R contains lipids; m is an integer selected from 1 to 10; A composition in which n is an integer selected from 1 to 200.

38. L 1, L ３ , L ４ , and L ７ Each of them is - (CH ２ ) ４ - including; L 2, L ５ , and L ８ Each of them contains -C(=O)NH-; L 6 and L ９ Each of them is - (CH ２ ) ３ - including; L 10 is - (CH ２ ) １～３ -ien-CH ２ CH ２ O- or -CH ２ It is O-; L 11 is - (CH ２ CH ２ O) ｎ - or - (OCH ２ CH ２ ) ｎ - and in the formula n is an integer selected from 1 to 50; L 12 is -NH(CO)O- or -NH(CO)-; R is dialkylglycerol, diacylglycerol, sterol, C １０ ~C ３０ n-alkyl groups containing carbon atoms, C １０ ~C ３０ The composition according to claim 37, selected from the group consisting of branched alkyl groups containing carbon atoms and tocopherols.

39. Each of the above A units 【Transformation 7】 The composition according to claim 37 or 38.

40. The composition according to any one of claims 37 to 39, wherein the compound is a compound selected from 1001 to 1019, 1060, 1065, 1066, and 1075 to 1085 of Table 4.

41. The composition according to any one of claims 37 to 40, wherein the compound is compound 1004 of Table 4.

42. The composition according to claim 41, wherein the lipid nanoparticles containing the compound provide improved delivery in LDLr-deficient mammals, determined by an edit percentage at least 50% higher than that of the corresponding lipid nanoparticles without the compound.

43. The composition according to claim 41, wherein the lipid nanoparticles containing the compound provide improved delivery in mammals lacking ApoE, as determined by an edit percentage at least 50% higher than that of the corresponding lipid nanoparticles without the compound.

44. The composition according to claim 37, wherein one or more activators comprise a plurality of nucleic acids.

45. The composition according to claim 37, wherein the one or more activators comprises mRNA encoding a gene editing factor nuclease or a base editing factor, and one or more guide RNAs.

46. The composition according to claim 37, wherein the compound constitutes about 0.001 mol% to about 0.5 mol% of the total excipients in the lipid nanoparticles.

47. A method for preparing a formulation containing GalNAc-lipid nanoparticles, wherein the nanoparticles comprise (i) one or more nucleic acid activators, (ii) a lipid excipient comprising sterols, phospholipids, stealth lipids, and aminolipids, and (iii) a GalNAc-lipid conjugate, and the method is The steps include: preparing a first solution containing one or more nucleic acid activators in an aqueous buffer; (i) preparing a second solution containing at least a portion of the lipid excipient and (ii) the GalNAc lipid conjugate in a water-miscible organic solvent such as ethanol; The steps include: mixing the first solution and the second solution; The steps include: incubating a mixture of the first and second solutions to form GalNAc lipid nanoparticles; Optionally, the steps include performing one or more dilution, buffer exchange, concentration, filtration, and GalNAc-lipid nanoparticle evaluation processes. Includes, The GalNAc lipid conjugate is given by formula (V): 【Transformation 8】 Formula (V) A compound of, During the ceremony, Each A group contains an N-acetylgalactosamine (GalNAc) moiety. L １ , L ２ , L ３ , L ４ , L ５ , L ６ , L ７ , L ８ , L ９ , L １０ , and L １２ Each of these independently represents a substitution or non-substitution of C. １ ~C １２ Alkylene, substituted or unsubstituted C １ ~C １２ Heteroalkylenes, substituted or unsubstituted C ２ ~C １２ Alkenylenes, substituted or unsubstituted carbon atoms ２ ~C １２ Alkinylene, -(CH ２ CH ２ O) ｍ -, - (OCH ２ CH ２ ) ｍ -, -O-, -S-, -S(=O)-, -S(=O) ２ -, -S(=O)(=NR １ )-, -C(=O)-, -C(=N-OR １ )-, -C(=O)O-, -OC(=O)-, -C(=O)C(=O)-, -C(=O)NR １ -, -NR １ C(=O)-, -OC(=O)NR １ -, -NR １ C(=O)O-, -NR １ C(=O)NR １ -, -C(=O)NR １ C(=O)-, -S(=O) ２ NR １ -, -NR １ S (=O) ２ -, -NR １ -, or -N (OR １ ) - and; L １１ is a substituted or unsubstituted - (CH ２ CH ２ O) ｎ - or substituted or unsubstituted - (OCH ２ CH ２ ) ｎ - and; Each R １ Independently, H or substituted or unsubstituted C １ ~C ６ It is alkyl; R contains lipids; m is an integer selected from 1 to 10; n is an integer selected from 1 to 200, in this method.

48. The method according to claim 47, wherein the GalNAc-lipid conjugate is selected from the structures specified in Table 4.

49. The method according to claim 47, wherein the mixing is performed by an inline mixing apparatus having a first mixing chamber comprising a first port for separately introducing the first solution into the first mixing chamber and a second port for separately and simultaneously introducing the second solution into the first mixing chamber.

50. The method according to claim 49, further comprising the step of adding a second portion of a GalNAc lipid conjugate after mixing the first solution and the second solution, wherein the addition of the second portion of the GalNAc lipid conjugate is pre-dissolved in a water-miscible organic solvent, combined with an aqueous solution to form an aqueous dilution buffer, which is connected to the first mixing chamber of the inline mixer before incubation and mixed with the pre-mixed first and second solutions in a second mixing chamber located downstream thereof.

51. The method according to claim 50, further comprising a buffer exchange process after the addition of the second portion of the GalNAc lipid conjugate.

52. A method for preparing a formulation containing GalNAc-lipid nanoparticles, wherein the nanoparticles comprise (i) one or more nucleic acid molecular entities, (ii) one or more lipid excipients comprising sterols, phospholipids, stealth lipids, or aminolipids, and (iii) one or more GalNAc-lipid conjugates, and the method is The steps include: preparing a first solution containing one or more nucleic acid molecular entities in an aqueous buffer; The steps include: preparing a second solution containing at least one of the one or more lipid excipients in a water-miscible organic solvent; The steps include providing a third solution comprising at least a portion of a GalNAc lipid conjugate; A step of mixing the first solution, the second solution, and the third solution in one or more mixing chambers, wherein each solution is introduced separately into the one or more mixing chambers via an inlet port; The steps include: incubating a mixture of the first, second, and third solutions to form GalNAc lipid nanoparticles; Optionally, the steps include performing one or more dilution, buffer exchange, concentration, filtration, and GalNAc-lipid nanoparticle evaluation processes. Includes, The GalNAc lipid conjugate is given by formula (V): 【Chemistry 9】 Formula (V) A compound of, During the ceremony, Each A group contains an N-acetylgalactosamine (GalNAc) moiety. L １ , L ２ , L ３ , L ４ , L ５ , L ６ , L ７ , L ８ , L ９ , L １０ , and L １２ Each of these independently represents a substitution or non-substitution of C. １ ~C １２ Alkylene, substituted or unsubstituted C １ ~C １２ Heteroalkylenes, substituted or unsubstituted C ２ ~C １２ Alkenylenes, substituted or unsubstituted carbon atoms ２ ~C １２ Alkinylene, -(CH ２ CH ２ O) ｍ -, - (OCH ２ CH ２ ) ｍ -, -O-, -S-, -S(=O)-, -S(=O) ２ -, -S(=O)(=NR １ )-, -C(=O)-, -C(=N-OR １ )-, -C(=O)O-, -OC(=O)-, -C(=O)C(=O)-, -C(=O)NR １ -, -NR １ C(=O)-, -OC(=O)NR １ -, -NR １ C(=O)O-, -NR １ C(=O)NR １ -, -C(=O)NR １ C(=O)-, -S(=O) ２ NR １ -, -NR １ S (=O) ２ -, -NR １ -, or -N (OR １ ) - and; L １１ is a substituted or unsubstituted - (CH ２ CH ２ O) ｎ - or substituted or unsubstituted - (OCH ２ CH ２ ) ｎ - and; Each R １ Independently, H or substituted or unsubstituted C １ ~C ６ It is alkyl; R contains lipids; m is an integer selected from 1 to 10; n is an integer selected from 1 to 200, in this method.

53. The method according to claim 52, wherein the first solution comprises an aqueous buffer, and the second solution and the third solution are prepared independently from a water-miscible alcohol.

54. The method according to claim 52, wherein the first solution, the second solution, and the third solution are introduced into a mixer simultaneously.

55. The method according to claim 52, wherein the first solution, the second solution, and the third solution are sequentially introduced into a mixer before incubation.

56. The method according to claim 52, wherein the first solution, the second solution, and the third solution are combined in an inline mixer device having a first mixing chamber connected to a second downstream mixing chamber, the first and second solutions are premixed in the first mixing chamber and immediately flow into the second mixing chamber, and the third solution is mixed with the first and second solutions in the second mixing chamber, wherein the third solution optionally comprises a lipid excipient pre-dissolved in an aqueous dilution buffer or a water-miscible organic solvent diluted therein.

57. The method according to claim 52, wherein the water-miscible organic solvent is ethanol.

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