Compositions and methods for targeted RNA delivery

Abstract

Provided herein are compositions, methods for their production, and methods for targeted delivery of therapeutic agents to alter target gene expression and function, e.g., proteins involved in lipid and cholesterol metabolism, such as PCSK9.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 229,060, filed August 3, 2021, U.S. Provisional Application No. 63 / 246,858, filed September 22, 2021, and U.S. Provisional Application No. 63 / 275,335, filed November 3, 2021, each of which is incorporated by reference in its entirety into this specification.

[0002] Field of Disclosure

[0002] This disclosure relates to methods for forming lipid nanoparticles to improve gene editing capabilities. This disclosure also relates to compositions and methods for targeted delivery of therapeutic agents, such as CRISPR guide RNAs and other nucleic acid drugs. [Background technology]

[0003]

[0003] All publications herein are incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. The following description contains information that may be useful in understanding the present disclosure. No admission is made that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art. Summary of the Invention

[0004] In one aspect, described herein are methods for preparing formulations comprising GalNAc-lipid nanoparticles (GalNAc-LNPs). In some embodiments, the nanoparticles comprise (i) one or more nucleic acid active agents, (ii) one or more lipid excipients selected from a sterol or derivative thereof, a phospholipid, a stealth lipid, and an amino lipid, and / or (iii) a GalNAc-lipid receptor-targeting conjugate. In some embodiments, the method can include providing a first solution comprising one or more nucleic acid active agents in an aqueous buffer. In some embodiments, the method can include providing a second solution comprising (i) at least one of the one or more lipid excipients and (ii) at least a portion of the receptor-targeting conjugate in a water-miscible organic solvent. In some embodiments, the method can include combining an antioxidant with the first solution. In some embodiments, the method can include mixing the first solution with the second solution. In some embodiments, the method may include incubating the mixture of the first and second solutions to form GalNAc-LNPs. In some embodiments, the method may include performing one or more processes selected from dilution, buffer exchange, concentration, filtration, freezing, thawing, incubation, and GalNAc-LNP evaluation.

[0005] In some embodiments, the steps of the method are performed simultaneously. In some embodiments, the steps of the method are performed sequentially.

[0006] In some embodiments, the aqueous buffer comprises polyethylene glycol. In some embodiments, the polyethylene glycol has a number-average molecular weight ranging from about 200 to about 1000 (e.g., about 200, about 400, about 500, about 600, or about 1000). In some embodiments, the method further comprises diluting the GalNAc-lipid in an aqueous solution to produce a diluted GalNAc-LNP solution. In some embodiments, the GalNAc-LNP is configured for direct administration to a subject. In some embodiments, the method further comprises diluting the GalNAc-LNP in solution one or more times. In some embodiments, the method further comprises exchanging the water-miscible organic solvent with a buffer solution one or more times. In some embodiments, the method further comprises concentrating the GalNAc-LNP. In some embodiments, the concentrating step comprises passing the GalNAc-LNP through a membrane. In some embodiments, the method further comprises a second concentration process, wherein the second concentration comprises concentrating said GalNAc-LNPs by passing an exchange buffer through a membrane.

[0006]

[0007] In some embodiments, the method further comprises filtering the GalNAc-LNP through a membrane. In some embodiments, the method further comprises a second incubation after step e, wherein the incubation occurs for about 1 minute to about 120 minutes. In some embodiments, the method further comprises storing the GalNAc-LNP at a temperature of about -80 degrees Celsius (°C) to about 25°C. In some embodiments, the method further comprises storing the GalNAc-LNP at a temperature of about -80 degrees Celsius (°C), or about 2°C to about 8°C.

[0007]

[0008] In some embodiments, the method further comprises (i) thawing stored GalNAc-LNP, (ii) pooling the GalNAc-LNP, (iii) diluting the GalNAc-LNP in a solution, and (iv) filtering the GalNAc-LNP through a membrane prior to administering the dose of GalNAc-LNP to a subject. In some embodiments, the order of performing steps (iii) and (iv) is reversed. In some embodiments, the miscible organic solvent is ethanol. In some embodiments, the antioxidant is ethylenediaminetetraacetic acid (EDTA). In some embodiments, the second solution comprises all of the receptor-targeting conjugate. In some embodiments, at least a portion of the receptor-targeting conjugate is combined with one or more lipids prior to the mixing step.

[0008]

[0009] In some embodiments, the mixing is performed in an in-line mixer, a cross mixer, or a T-mixer device. In some embodiments, the mixing comprises laminar mixing, vortex mixing, turbulent mixing, or a combination thereof. In some embodiments, the method further comprises concentrating the GalNAc-LNPs using a tangential flow filtration (TFF) process. In some embodiments, the method further comprises performing buffer exchange using chromatography, dialysis, or a TFF process.

[0009]

[0010] In some embodiments, the receptor-targeting conjugate comprises one or more N-acetylgalactosamine (GalNAc) or GalNAc derivatives. In some embodiments, the GalNAc-lipid receptor-targeting conjugate is selected from the structures identified in Table 4. In some embodiments, mixing is performed by an in-line mixing device having a first mixing chamber with 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. In some embodiments, the first solution comprises RNA. In some embodiments, the concentration (mol%) of the GalNAc-lipid receptor-targeting conjugate is about 0.01 mol% to about 10 mol%. In some embodiments, the neutral lipid is distearoylphosphatidylcholine (DSPC). In some embodiments, the stealth lipid is polyethylene glycol-dimyristoylglycerol (PEG-DMG). In some embodiments, the stealth lipid concentration in the second solution is between 0 mol% and about 5 mol%. In some embodiments, the nucleic acid drug concentration is between about 0.1 and about 5 mg / mL (e.g., about 0.1, 0.25, 0.5, 0.75, 1, 1.5, 2, 2.5, 3, 4, or 5 mg / mL). In some embodiments, the mixture is incubated for between about 1 minute and about 24 hours. In some embodiments, the mixture is incubated for between about 1 minute and about 120 minutes. In some embodiments, the mixture is incubated for about 1 hour. In some embodiments, the final GalNAc-LNP solution comprises a Tris buffer.

[0010]

[0011] In some embodiments, the final GalNAc-LNP solution further comprises a cryoprotectant. In some embodiments, the cryoprotectant is sucrose. In some embodiments, the concentration of the cryoprotectant in the final solution is about 0.1 mM to about 500 mM. In some embodiments, the concentration of the cryoprotectant in the final solution is about 150 mM to about 500 mM. In some embodiments, the concentration of the cryoprotectant in the final solution is about 300 mM.

[0011]

[0012] In some embodiments, GalNAc-LNP are stored at a temperature of about -80 degrees Celsius (°C). In some embodiments, the final GalNAc-LNP solution does not further contain a cryoprotectant. In some embodiments, the GalNAc-LNP are stored at about 2°C to about 8°C. In some embodiments, the GalNAc-LNP are in a solution having a pH of about 6 to about 9. In some embodiments, the GalNAc-LNP are in a solution having a pH of about 7 to 8 (e.g., 7 to 8, 7.2 to 7.8, 7.3 to 7.7, or 7.4 to 7.6).

[0012]

[0013] In some embodiments, the method further comprises introducing the receptor-targeting conjugate into the second solution at a concentration of at least 0.01 (e.g., at least 0.01, 0.05, 0.1, or 0.5) mol% by total volume. In some embodiments, the method further comprises introducing the receptor-targeting conjugate into the second solution at a concentration of at least 1 mol% by total volume. In some embodiments, the method further comprises introducing the receptor-targeting conjugate into the second solution at a concentration of at least 3 mol% by total volume. In some embodiments, the method further comprises introducing the receptor-targeting conjugate into the second solution at a concentration of at least 5 mol% by total volume.

[0013]

[0014] In some embodiments, the method further comprises introducing the receptor-targeting conjugate into the second solution at a concentration of at least 7 mol% by total volume. In some embodiments, the method further comprises introducing the receptor-targeting conjugate into the second solution at a concentration of at least 9 mol% by total volume. In some embodiments, the method further comprises introducing the receptor-targeting conjugate into the second solution at a concentration of at least 10 mol% by total volume.

[0014]

[0015] In another aspect, described herein are GalNAc-LNPs that can be prepared by the methods described herein. In some embodiments, the distribution of GalNAc-lipids throughout the LNPs is substantially uniform. In some embodiments, the GalNAc-lipids are present in the GalNAc-LNPs at a concentration of about 0.01-0.5 mol%.

[0015]

[0016] In another aspect, described herein are methods of administering GalNAc-LNPs, such as those described herein, to a mammal. In some embodiments, the GalNAc-LNPs comprise one or more gRNAs targeting the LDL receptor (LDLr) gene and Cas9 mRNA. In some embodiments, the method comprises administering to a mammal a dose comprising one or more of said GalNAc-LNPs, thereby increasing blood LDL-C levels by at least 300% compared to a corresponding subject not administered said dose. In some embodiments, the LDL-C level is increased by at least 350%. In some embodiments, the LDL-C level is increased by at least 400%. In some embodiments, the LDL-C level is increased by at least 500%. In some embodiments, the LDL-C level is increased by at least 550%. In some embodiments, the LDL-C level is increased by at least 600%. In some embodiments, the one or more gRNAs comprise GA468 / GA470 and / or GA469 / GA471. In some embodiments, the Cas9 mRNA is MS004. In some embodiments, the mammal is a non-human primate (NHP) (eg, a cynomolgus monkey).

[0016]

[0017] In another aspect, described herein are GalNAc-LNPs comprising an adenine base editor (ABE) mRNA. In some embodiments, the mRNA is MA004. In some embodiments, the ABE mRNA further comprises a 3' untranslated region (UTR) described herein, e.g., a UTR in Table 19. In some embodiments, the GalNAc-LNPs further comprise an ANGPTL3 gRNA described herein. In some embodiments, the GalNAc-LNPs further comprise a PCSK9 gRNA described herein.

[0017]

[0018] In some embodiments, the ABE mRNA further comprises a 5' UTR described herein, e.g., a UTR in Table 19. In some embodiments, the GalNAc-LNP further comprises an ANGPTL3 gRNA described herein. In some embodiments, the GalNAc-LNP further comprises a PCSK9 gRNA described herein.

[0018]

[0019] In some embodiments, the distribution of GalNAc-lipids across the LNP provides a percent (%) of PCSK9 editing in mammalian cells of about 15% to about 60%. In some embodiments, the % PCSK9 editing is about 50% to 60%. In some embodiments, the % PCSK9 editing is about 40% to about 50%. In some embodiments, the % PCSK9 editing is about 30% to about 40%. In some embodiments, the % PCSK9 editing is about 20% to about 30%.

[0019]

[0020] In some embodiments, GalNAc-LNPs provide improved delivery in mammals lacking low-density lipoprotein receptor (LDLr), as determined by a percent editing that is at least 5% higher than the corresponding LNP without the receptor-targeting conjugate. In some embodiments, the percent editing is at least 50% higher than the corresponding LNP without the receptor-targeting conjugate. In some embodiments, the GalNAc-LNPs provide improved delivery in mammals lacking apolipoprotein E (ApoE), as determined by a percent editing that is at least 5% higher than the corresponding LNP without the receptor-targeting conjugate. In some embodiments, the percent editing is at least 50% higher than the corresponding LNP without the receptor-targeting conjugate.

[0020]

[0021] In another aspect, described herein is a GalNAc-LNP comprising a receptor-targeting conjugate, wherein the receptor-targeting conjugate has formula (V):

[0022]

[0021] [ka]

[0022]

[0023] and

[0024] wherein A is a receptor targeting moiety;

[0025] 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 substituted or unsubstituted C1 to C 12 Alkylene, substituted or unsubstituted C1-C 12 Heteroalkylene, substituted or unsubstituted C2-C 12 Alkenylene, 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)N(R 1 )-, -N(R 1 )C(=O)-, -OC(=O)N(R 1 )-, -N(R 1 )C(=O)O-, -N(R 1 )C(=O)N(R 1 )-, -C(=O)N(R 1 )C(=O)-, -S(=O)2N(R 1 )-, -N(R 1 )S(=O)2-, -N(R 1 )-, -N(OR 1 )-, -O[(P=O)O-]O-, -O[(P=O)S-]O-, -(CH2)p -O-, -O-(CH2) p -O-, -O-(CH2) p -, -SS-, or a bond;

[0026] L 11 is -(CH2CH2O) n -, -(OCH2CH2) n - or in conjunction;

[0027] Each R 1 are independently H or substituted or unsubstituted C1-C6 alkyl;

[0028] R is a lipophilic organic residue;

[0029] m is an integer selected from 1 to 10;

[0030] n is an integer selected from 1 to 200;

[0031] p is an integer selected from 1 to 200, GalNAc-LNP.

[0023]

[0032] In another aspect, described herein is a GalNAc-LNP comprising a receptor-targeting conjugate, wherein the receptor-targeting conjugate is a compound of formula (VI):

[0033]

[0024] [ka]

[0025]

[0034] Including,

[0035] wherein A is a receptor targeting moiety;

[0036] Each L 1 , L 2 , L 3 , L 4 , L 5 , L 6 , L 7 , L 8 , L 9 , L 10 and L 12are independently substituted or unsubstituted C1 to C 12 Alkylene, substituted or unsubstituted C1-C 12 Heteroalkylene, substituted or unsubstituted C2-C 12 Alkenylene, 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)N(R 1 )-, -N(R 1 )C(=O)-, -OC(=O)N(R 1 )-, -N(R 1 )C(=O)O-, -N(R 1 )C(=O)N(R 1 )-, -C(=O)N(R 1 )C(=O)-, -S(=O)2N(R 1 )-, -N(R 1 )S(=O)2-, -N(R 1 )-, or -N(OR 1 )-and;

[0037] L 11 is -(CH2CH2O) n -, -(OCH2CH2) n - or in conjunction;

[0038] Each R 1 are independently H or substituted or unsubstituted C1-C6 alkyl;

[0039] R is a lipophilic organic residue;

[0040] m is an integer selected from 1 to 10;

[0041] n is an integer selected from 1 to 200;

[0042] p is an integer selected from 1 to 200, GalNAc-LNP.

[0026]

[0043] In some embodiments, A is N-acetylgalactosamine (GalNAc) or a derivative thereof.

[0044] In another aspect, described herein is a method of gene editing comprising introducing GalNAc-LNPs by administering the GalNAc-LNPs to a non-human primate (NHP) or human subject. In some embodiments, at least one GalNAc-LNP is introduced into the NHP via IV infusion into a peripheral vein (e.g., saphenous or brachial). In some embodiments, each of the at least one GalNAc-LNPs is independently administered at about 1 mg / kg, 2 mg / kg, or 6 mg / kg. In some embodiments, the NHP is treated with a steroid before the introduction of the at least one GalNAc-LNP. In some embodiments, the introduction of the at least one GalNAc-LNP results in at least about 20% (e.g., about 20, 30, 40, 50, 60, 70, 80, 90, or 180 days) of gene editing over a period of at least 15 days (e.g., about 15, 20, 30, 40, 50, 60, 70, 80, 90, or 180 days).

[0027]

[0045] In some embodiments, at least two GalNAc-LNPs are introduced into a non-human primate. In some embodiments, the NHP is treated with a steroid via intramuscular injection. In some embodiments, the steroid comprises dexamethasone. In some embodiments, the steroid is co-administered with famotidine and / or diphenhydramine. In some embodiments, this method is used to generate an LDLR KD / KO NHP. In some embodiments, the NHP has an LDLr knockout.

[0028]

[0046] In another aspect, described herein are methods for preparing a formulation comprising lipid nanoparticles (LNPs), wherein the nanoparticles comprise (i) one or more nucleic acid active agents and (ii) one or more lipid excipients selected from a sterol or derivative thereof, a phospholipid, a stealth lipid, and an aminolipid. In some embodiments, the method comprises providing a first solution comprising one or more nucleic acid active agents in an aqueous buffer. In some embodiments, the method comprises providing a second solution comprising at least one of one or more lipid excipients in a water-miscible organic solvent. In some embodiments, the method optionally comprises combining an antioxidant with the first solution. In some embodiments, the method comprises mixing the first solution with the second solution. In some embodiments, the method comprises incubating the mixture of the first solution and the second solution to form LNPs. In some embodiments, the method optionally comprises performing one or more processes selected from dilution, buffer exchange, concentration, filtration, freezing, thawing, incubation, and LNP evaluation.

[0029]

[0047] In another aspect, described herein is a compound of formula (VI), or a pharmaceutically acceptable salt thereof:

[0048]

[0030] [ka]

[0031]

[0049] A composition comprising:

[0050] wherein A is a receptor targeting moiety, such as

[0032] [ka]

[0033] ) and;

[0051] L1 , L 4 , and L 7 teeth,

[0034] [ka]

[0035] and;

[0052] L 3 , L 6 , and L 9 teeth,

[0036] [ka]

[0037] and;

[0053] L 2 , L 5 , and L 8 is -N(R 1 )C(=O)- or -C(=O)N(R 1 )-and;

[0054] L 10 is unsubstituted C2 alkylene;

[0055] L 11 is -(OCH2CH2) n+1 - and;

[0056] R 1 is hydrogen;

[0057] L 12 is -N(R 1 )C(=O)O-, and R is

[0038] [ka]

[0039] wherein n is an integer selected from 33, 34, 35, 37, 38, 39, 40, 41, 42, and 43; or

[0058] L 12 is -N(R 1 )C(=O)- or -C(=O)N(R1 )- and R is unsubstituted C 18 ~C 20 alkyl and n is an integer selected from 1, 11, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, and 43.

[0040]

[0059] In some embodiments, the compound is

[0060] 1088(n=33)

[0041] [ka]

[0042]

[0061] 1089(n=34)

[0043] [ka]

[0044]

[0062]

[0063] 1090(n=35)

[0045] [ka]

[0046]

[0064]

[0065] and 1092 (n=37).

[0047] [ka]

[0048]

[0066] is selected from the group:

[0067] In another aspect, described herein is a compound of formula (V), or a pharmaceutically acceptable salt thereof:

[0068]

[0049] [ka]

[0050]

[0069] A composition comprising:

[0070] wherein A is a receptor targeting moiety;

[0071] L 1 , L 3 , L 4 , and L 7 is unsubstituted C4 alkylene;

[0072] L 6 , and L 9 is unsubstituted C3 alkylene;

[0073] L 2 , L 5 , and L 8 is -N(R 1 )C(=O)- or -C(=O)N(R 1 )-and;

[0074] L 10 is unsubstituted C2 alkylene;

[0075] L 11 is -(OCH2CH2) n+1 - and;

[0076] R 1 is hydrogen;

[0077] L 12 is -N(R 1 )C(=O)O-, and R is

[0051] [ka]

[0052] wherein n is an integer selected from 33, 34, 35, 37, 38, 39, 40, 41, 42, and 43; or

[0078] L 12 is -N(R 1 )C(=O)- or -C(=O)N(R 1 )- and R is unsubstituted C 18 ~C20 is alkyl,

[0079] n is an integer selected from 1, 11, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, and 43.

[0053]

[0080]

[0081] In some embodiments, the compound is

[0082] 1101(n=33)

[0054] [ka]

[0055]

[0083] 1102(n=34)

[0056] [ka]

[0057]

[0084] 1103(n=35)

[0058] [ka]

[0059]

[0085] 1105(n=37)

[0060] [ka]

[0061]

[0086] 1106(n=38)

[0062] [ka]

[0063]

[0087] 1107(n=39)

[0064] [ka]

[0065]

[0088] 1108 (n=40)

[0066]

change

[0067]

[0089] 1112(n=1)

[0068]

change

[0069]

[0090] 1113 (n=11)

[0070]

change

[0071]

[0091] 1114 (n=33)

[0072]

change

[0073]

[0092] 1115 (n=34)

[0074]

change

[0075]

[0093] 1116 (n=35)

[0076]

change

[0077]

[0094] 1117 (n=36)

[0078]

change

[0079]

[0095] 1118 (n=37)

[0080]

change

[0081]

[0096] 1119 (n=38)

[0082]

change

[0083]

[0097] 1120 (n=39)

[0084]

change

[0085]

[0098] 1121(n=40)

[0086]

change

[0087]

[0099] 1122 (n=41)

[0088]

change

[0089]

[0100] 1123 (n=42)

[0090]

change

[0091]

[0101] 1124 (n=43)

[0092]

change

[0093]

[0102] 1125(n=1)

[0094]

change

[0095]

[0103]

[0104] 1126 (n=11)

[0096]

change

[0097]

[0105] 1127 (n=33)

[0098]

change

[0099]

[0106] 1128 (n=34)

[0100]

change

[0101]

[0107] 1129 (n=35)

[0102]

change

[0103]

[0108] 1130 (n=36)

[0104]

change

[0105]

[0109] 1131 (n=37)

[0106]

change

[0107]

[0110] 1132 (n=38)

[0108]

change

[0109]

[0111] 1133 (n=39)

[0110]

change

[0111]

[0112] 1134 (n=40)

[0112]

change

[0113]

[0113] 1135(n=41)

[0114]

change

[0115]

[0114] 1136(n=42)

[0116]

change

[0117]

[0115] 1137(n=43)

[0118]

change

[0119]

[0116] 1140(n=33)

[0120]

change

[0121]

[0117] 1141(n=34)

[0122]

change

[0123]

[0118] 1142(n=35)

[0124]

change

[0125]

[0119] 1144(n=37)

[0126]

change

[0127]

[0120] 1145(n=38)

[0128]

change

[0129]

[0121] 1146(n=39)

[0130]

change

[0131]

[0122] 1147(n=40)

[0132]

change

[0133]

[0123] 1148 (n = 41)

[0134] [ka]

[0135]

[0124] 1149 (n = 42)

[0136] [ka]

[0137] and

[0125] 1150 (n = 43)

[0138] [ka]

[0139] is selected from the group: In another aspect, described herein are pharmaceutical formulations comprising GalNAc-LNPs. In some embodiments, the GalNAc-LNPs are

[0127] one or more nucleic acid activators;

[0128] one or more lipid excipients selected from sterols or derivatives thereof, phospholipids, stealth lipids, and aminolipids; and

[0129] The present invention includes a GalNAc-lipid receptor targeting conjugate, wherein the GalNAc-LNP is formulated according to an excipient mol% ratio selected from Table 14, Table 15, Table 16, or Table 17.

[0140] In some embodiments, the GalNAc-LNP comprises an amino lipid, and the amino lipid has the structure VL422.

[0141] [ka]

[0142] It has.

[0131] In some embodiments, the GalNAc-LNP is

[0133] one or more nucleic acid activators;

[0134] One or more lipid excipients selected from sterols or derivatives thereof, phospholipids, stealth lipids, and aminolipids; Structure of VL422

[0143] [ka]

[0144] one or more lipid excipients comprising an amino lipid having

[0136] and (iii) Includes GalNAc-lipid receptor targeting conjugates.

[0145]

[0138] In some embodiments, the GalNAc-LNP comprises an amino lipid, and the amino lipid has the structure 501, 502, 503, 504, 505, 506, or 507.

[0146] In some embodiments, the GalNAc-LNP comprises a stealth lipid, wherein the lipid has the structure of VP158.

[0147] [ka]

[0148]

[0140] It has. In some embodiments, the GalNAc-LNP comprises a stealth lipid, wherein the lipid has the structure of VP159.

[0149] [ka]

[0150]

[0142] It has.

[0143] In some embodiments, GalNAc-LNPs comprise at least two GalNAc-lipid acceptor targeting conjugates. In some embodiments, GalNAc-LNPs comprise about 0-1 mol% total GalNAc-lipid acceptor targeting conjugates. In some embodiments, GalNAc-LNPs comprise about 0-0.5 mol% total GalNAc-lipid acceptor targeting conjugates. In some embodiments, GalNAc-LNPs comprise about 0-0.25 mol% total GalNAc-lipid acceptor targeting conjugates. In some embodiments, GalNAc-LNPs comprise about 0-0.1 mol% total GalNAc-lipid acceptor targeting conjugates. In some embodiments, GalNAc-LNPs comprise about 0-0.05 mol% total GalNAc-lipid acceptor targeting conjugates. In some embodiments, the GalNAc-LNPs comprise about 0-0.01 mol% total GalNAc-lipid acceptor targeting conjugates. In some embodiments, the GalNAc-LNPs comprise GalNAc-lipid 1079. In some embodiments, the GalNAc-LNPs comprise GalNAc-lipid 1004.

[0151] In some embodiments, GalNAc-LNPs comprise about 40-60 mol% amino lipid. In some embodiments, GalNAc-LNPs comprise about 45 mol% amino lipid. In some embodiments, GalNAc-LNPs comprise about 50 mol% amino lipid. In some embodiments, GalNAc-LNPs comprise about 55 mol% amino lipid. In some embodiments, GalNAc-LNPs comprise about 34-35 (e.g., 34.1, 34.6, or 34.9) mol% cholesterol or cholesterol derivative. In some embodiments, GalNAc-LNPs comprise about 37.1-37.3 (e.g., 37.2) mol% cholesterol or cholesterol derivative. In some embodiments, GalNAc-LNPs comprise about 37.6-37.8 (e.g., 37.7) mol% cholesterol or cholesterol derivative. In some embodiments, GalNAc-LNPs comprise about 37.9-38.0 (e.g., 37.95) mol% cholesterol or cholesterol derivative. In some embodiments, GalNAc-LNPs comprise about 38.1-38.3 (e.g., 38.2) mol% cholesterol or cholesterol derivative. In some embodiments, GalNAc-LNPs comprise about 38.3-38.5 (e.g., 38.4) mol% cholesterol or cholesterol derivative. In some embodiments, GalNAc-LNPs comprise about 4-10 (e.g., 4.7, 9, or 10) mol% neutral lipid. In some embodiments, GalNAc-LNPs comprise about 1-3 (e.g., 1.3, 1.6, 2.1, or 3) mol% stealth lipid.

[0152] In another aspect, described herein are methods for assaying the amount of GalNAc lipids on the surface of GalNAc-LNPs. In some embodiments, the methods include contacting the GalNAc-LNPs with an ASPGR protein, where the ASPGR protein is labeled with a detectable marker; and measuring a signal shift of the detectable marker in the presence of the GalNAc-LNPs.

[0153] In some embodiments, the signal shift is an optical shift. In some embodiments, the signal shift is measured using biolayer interferometry. In some embodiments, the detectable marker is a His tag. In some embodiments, the ASPGR protein is recombinant human ASPGR protein.

[0154] In one aspect, described herein is a method for preparing a formulation comprising GalNAc-lipid nanoparticles (GalNAc-LNPs), the nanoparticles comprising (i) one or more nucleic acid active agents, (ii) one or more lipid excipients selected from a sterol or derivative thereof, a phospholipid, a stealth lipid, and an amino lipid, and (iii) a GalNAc-lipid receptor targeting conjugate, the method comprising the steps of: (a) providing a first solution comprising the one or more nucleic acid active agents in an aqueous buffer; and (b) dissolving (i) at least one of the one or more lipid excipients, and and (ii) providing a second solution comprising at least a portion of a receptor-targeting conjugate in a water-miscible organic solvent; (c) combining an antioxidant with the first solution; (d) mixing the first solution with the second solution; (e) incubating the mixture of the first and second solutions to form GalNAc-LNPs; and (f) optionally performing one or more processes selected from dilution, buffer exchange, concentration, filtration, freezing, thawing, incubation, and GalNAc-LNP evaluation. In one aspect, the GalNAc-LNPs are further diluted in an aqueous solution to produce a diluted GalNAc-LNP solution. In some embodiments, the GalNAc solution is configured for direct administration. In some embodiments, the GalNAc-LNP solution is further diluted one or more times. In one aspect, the water-miscible organic solvent is exchanged with a buffer solution one or more times. In one aspect, the GalNAc-LNPs are further concentrated. In some embodiments, GalNAc-LNP is concentrated by passing the GalNAc-LNP through a membrane. In some embodiments, GalNAc-LNP is concentrated twice by passing the GalNAc-LNP through a membrane. In one aspect, GalNAc-LNP is filtered through a membrane. In one aspect, GalNAc-LNP is incubated twice for a period ranging from about 1 minute to about 120 minutes. In one aspect, GalNAc-LNP is stored at a temperature ranging from about -80°C to about 25°C. In some embodiments, GalNAc-LNP is stored at about -80°C.In some embodiments, the GalNAc-LNPs are stored at about 2°C to about 8°C. In one aspect, the method may further include (i) thawing the stored GalNAc-LNPs, (ii) pooling the GalNAc-LNPs, (iii) diluting the GalNAc-LNPs in the solution, and (iv) filtering the GalNAc-LNPs through a membrane prior to administering the dose of GalNAc-LNPs to the subject or mammal. In some embodiments, the order of performing steps (iii) and (iv) is reversed. In some embodiments, the miscible organic solvent is ethanol. In some embodiments, the antioxidant is ethylenediaminetetraacetic acid (EDTA). In one aspect, the second solution contains all of the receptor-targeting conjugates. In one aspect, at least a portion of the receptor-targeting conjugates are combined with one or more lipids prior to the mixing step. In some embodiments, the mixing is performed in an in-line mixer, cross mixer, or T-mixer device. In some embodiments, the mixing comprises laminar mixing, vortex mixing, turbulent mixing, or a combination thereof. In one aspect, the method further comprises concentrating the GalNAc-LNPs using a tangential flow filtration (TFF) process. In one aspect, the method further comprises performing buffer exchange using chromatography, dialysis, or a TFF process. In one aspect, the receptor-targeting conjugate comprises one or more N-acetylgalactosamine (GalNAc) or GalNAc derivatives. In some embodiments, the GalNAc-lipid receptor-targeting conjugate is selected from the structures identified in Table 4. In one aspect, the mixing is performed by an in-line mixing device having a first mixing chamber comprising a first port for separately introducing a first solution into the first mixing chamber and a second port for separately and simultaneously introducing a second solution into the first mixing chamber. In some embodiments, the first solution comprises RNA. In one embodiment, the concentration (mol %) of the GalNAc-lipid acceptor targeting conjugate is about 0.01 mol % to about 10 mol %. In one embodiment, the neutral lipid is distearoylphosphatidylcholine (DSPC).In one aspect, the stealth lipid is polyethylene glycol-dimyristoylglycerol (PEG-DMG). In some embodiments, the stealth lipid concentration in the second solution is between 0 mol% and about 5 mol%. In one aspect, the nucleic acid drug concentration is about 1 mg / mL. In one aspect, the mixture is incubated for between about 1 minute and about 24 hours. In some embodiments, the mixture is incubated for between about 1 minute and about 120 minutes. In some embodiments, the mixture is incubated for about 1 hour. In one aspect, the final GalNAc-LNP solution comprises a Tris buffer. In one aspect, the final GalNAc-LNP solution further comprises a cryoprotectant. In some embodiments, the cryoprotectant is sucrose. In some embodiments, the cryoprotectant in the final solution is between about 0.1 mM and about 500 mM. In some embodiments, the cryoprotectant concentration in the final solution is between about 150 mM and about 500 mM. In some embodiments, the cryoprotectant in the final solution is about 300 mM. In one aspect, the GalNAc-LNP is stored at a temperature of about -80 degrees Celsius (°C). In one aspect, the final GalNAc-LNP solution does not further comprise a cryoprotectant. In some embodiments, the GalNAc-LNP is stored at about 2°C to about 8°C. In one aspect, the GalNAc-LNP is in a solution having a pH of about 7 to about 8. In some embodiments, the GalNAc-LNP is in a solution having a pH of about 7.4. In one aspect, the method further comprises introducing a receptor-targeting conjugate into the second solution at a concentration of at least 0.1 mol% by total volume. In some embodiments, the receptor-targeting conjugate is introduced into the second solution at a concentration of at least 1 mol% by total volume. In some embodiments, the receptor-targeting conjugate is introduced into the second solution at a concentration of at least 3 mol% by total volume. In some embodiments, the receptor-targeting conjugate is introduced into the second solution at a concentration of at least 5 mol% by total volume, hi some embodiments, the receptor-targeting conjugate is introduced into the second solution at a concentration of at least 7 mol% by total volume.In some embodiments, the receptor-targeting conjugate is introduced into the second solution at a concentration of at least 9 mol% by total volume, hi some embodiments, the receptor-targeting conjugate is introduced into the second solution at a concentration of at least 10 mol% by total volume.

[0155]

[0148] In one aspect, described herein are GalNAc-LNPs prepared according to the methods described herein, wherein the distribution of GalNAc-lipids throughout the LNP is substantially uniform. In one aspect, the GalNAc-LNPs have GalNAc-lipids present in the GalNAc-LNPs at a concentration of 5 mol%. In one aspect, GalNAc-LNPs prepared according to the methods described herein, wherein administration of a dose comprising one or more GalNAc-LNPs to a mammal increases blood LDL levels by at least 300% relative to a corresponding subject not administered the dose. In some embodiments, LDL levels are increased by at least 350%. In some embodiments, LDL levels are increased by at least 400%. In some embodiments, LDL levels are increased by at least 500%. In some embodiments, LDL levels are increased by at least 550%. In some embodiments, LDL levels are increased by at least 600%.

[0156] In one aspect, described herein are GalNAc-LNPs prepared according to the methods described herein, further comprising an adenine base editor (ABE) mRNA. In some embodiments, the mRNA is MA004. In some embodiments, the ABE mRNA further comprises a 3' untranslated region (UTR) described herein. In some embodiments, the GalNAc-LNPs further comprise an ANGPTL3 gRNA described herein. In some embodiments, the GalNAc-LNPs further comprise a PCSK9 gRNA. In some embodiments, the ABE mRNA further comprises a 5' UTR described herein. In some embodiments, the GalNAc-LNPs further comprise an ANGPTL3 gRNA. In some embodiments, the GalNAc-LNPs further comprise a PCSK9 gRNA.

[0157] In one aspect, described herein are GalNAc-LNPs comprising a PCSK9 gRNA, wherein the distribution of GalNAc-lipids across the LNP results in a percent (%) of PCSK9 editing in mammalian cells of about 15% to about 60%. In some embodiments, the % PCSK9 editing is about 50% to 60%. In some embodiments, the % PCSK9 editing is about 40% to about 50%. In some embodiments, the % PCSK9 editing is about 30% to about 40%. In some embodiments, the % PCSK9 editing is about 20% to about 30%.

[0158] In one aspect, described herein are GalNAc-LNPs comprising an ANGPTL3 gRNA, wherein the distribution of GalNAc-lipids across the LNP results in a percent (%) of ANGPTL3 editing in mammalian cells of about 15% to about 60%. In some embodiments, the % ANGPTL3 editing is about 50% to 60%. In some embodiments, the % ANGPTL3 editing is about 40% to about 50%. In some embodiments, the % ANGPTL3 editing is about 30% to about 40%. In some embodiments, the % ANGPTL3 editing is about 20% to about 30%.

[0159] In one aspect, GalNAc-LNPs provide improved delivery in mammals lacking the low-density lipoprotein receptor (LDLr), as determined by a percent editing that is at least 5% higher than the corresponding LNP without the receptor-targeting conjugate. In some embodiments, the percent editing is at least 50% higher than the corresponding LNP without the receptor-targeting conjugate. In one aspect, GalNAc-LNPs provide improved delivery in mammals lacking apolipoprotein E (ApoE), as determined by a percent editing that is at least 5% higher than the corresponding LNP without the receptor-targeting conjugate. In some embodiments, the percent editing is at least 50% higher than the corresponding LNP without the receptor-targeting conjugate.

[0160] In one aspect, described herein is a GalNAc-LNP prepared according to the methods described herein, wherein the receptor-targeting conjugate has the formula (V):

[0161] [ka]

[0162] and wherein A is a receptor targeting moiety; 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 substituted or unsubstituted C1 to C 12 Alkylene, substituted or unsubstituted C1-C 12 Heteroalkylene, substituted or unsubstituted C2-C 12 Alkenylene, substituted or unsubstituted C2-C 12Alkynylene, -(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)N(R 1 )-, -N(R 1 )C(=O)-, -OC(=O)N(R 1 )-, -N(R 1 )C(=O)O-, -N(R 1 )C(=O)N(R 1 )-, -C(=O)N(R 1 )C(=O)-, -S(=O)2N(R 1 )-, -N(R 1 )S(=O)2-, -N(R 1 )-, -N(OR 1 )-, -O[(P=O)O-]O-, -O[(P=O)S-]O-, -(CH2) p -O-, -O-(CH2) p -O-, -O-(CH2) p -, -SS-, or a bond; L 11 is -(CH2CH2O) n -, -(OCH2CH2) n - or in conjunction; Each R 1 are independently H or substituted or unsubstituted C1-C6 alkyl; R is a lipophilic organic residue; m is an integer selected from 1 to 10; n is an integer selected from 1 to 200; p is an integer selected from 1 to 200, GalNAc-LNP.

[0163] In one aspect, described herein is a GalNAc-LNP prepared according to the methods described herein, wherein the receptor-targeting conjugate has the formula (VI):

[0164] [ka]

[0165] and wherein A is a receptor targeting moiety; 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 substituted or unsubstituted C1 to C 12 Alkylene, substituted or unsubstituted C1-C 12 Heteroalkylene, substituted or unsubstituted C2-C 12 Alkenylene, 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)N(R 1 )-, -N(R 1 )C(=O)-, -OC(=O)N(R 1 )-, -N(R 1 )C(=O)O-, -N(R 1 )C(=O)N(R 1 )-, -C(=O)N(R 1 )C(=O)-, -S(=O)2N(R 1 )-, -N(R 1 )S(=O)2-, -N(R 1 )-, or -N(OR 1 )-and; L 11 is -(CH2CH2O) n -, -(OCH2CH2) n - or in conjunction; Each R 1 are independently H or substituted or unsubstituted C1-C6 alkyl; R is a lipophilic organic residue; m is an integer selected from 1 to 10; n is an integer selected from 1 to 200; p is an integer selected from 1 to 200, GalNAc-LNP.

[0166]

[0155] In one embodiment, the A moiety described in formula (V) and formula (VI) is N-acetylgalactosamine (GalNAc) or a derivative thereof.

[0156] In one aspect, described herein is a method for editing a gene, comprising introducing GalNAc-LNP into a non-human primate (NHP), wherein the NHP has an LDLr knockout.

[0167]

[0157] In one aspect, described herein is a method for preparing a formulation comprising lipid nanoparticles (LNPs), wherein the nanoparticles comprise (i) one or more nucleic acid active agents and (ii) one or more lipid excipients selected from a sterol or derivative thereof, a phospholipid, a stealth lipid, and an amino lipid, the method comprising the steps of: (a) providing a first solution comprising the one or more nucleic acid active agents in an aqueous buffer; (b) providing a second solution comprising at least one of the one or more lipid excipients in a water-miscible organic solvent; (c) combining an antioxidant with the first solution; (d) mixing the first solution and the second solution; (e) incubating the mixture of the first solution and the second solution to form LNPs; and (f) optionally performing one or more processes selected from dilution, buffer exchange, concentration, filtration, freezing, thawing, incubation, and LNP evaluation.

[0168] Incorporation by Reference

[0158] All publications, references, patents, and patent applications mentioned in this specification are incorporated herein by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. In the event of a conflict in usage between this document and a document incorporated by reference, the usage in the incorporated reference should be considered supplemental to the usage in this document. In the event of an irreconcilable conflict, the usage in this specification shall control.

[0169]

[0159] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings. [Brief explanation of the drawings]

[0170] [Figure 1A]

[0160] Figures 1A-1B show HPLC chromatograms of GalNAc lipid incorporation in compositions herein. Figure 1A shows a reference LNP lacking GalNAc lipid, and Figure 1B shows an LNP comprised of GalNAc lipid. [Figure 1B]

[0160] Figures 1A-1B show HPLC chromatograms of GalNAc lipid incorporation in compositions herein. Figure 1A shows a reference LNP lacking GalNAc lipid, and Figure 1B shows an LNP comprised of GalNAc lipid. [Figure 2]

[0161] Shows the in vitro PCSK9 gene editing efficiency in primary human hepatocytes of the LNP formulation in the composition of the present specification. [Figure 3]

[0162] PCSK9 gene editing in wild-type, LDLr- / -, and ApoE- / - mouse livers after retroorbital administration of the LNP composition herein carrying SpCas9 mRNA and PCSK9 gRNA at a 1:1 ratio. [Figure 4]

[0163] Shows ANGPTL3 gene editing in the liver of LDLr- / - mice after retro-orbital administration of the LNP composition herein carrying ABE mRNA and ANGPTL3 gRNA at a 1:1 ratio. [Figure 5]

[0164] Shows PCSK9 gene editing in wild-type and LDLr- / - mouse livers after retroorbital administration of LNPs carrying ABE mRNA and PCSK9 gRNA at a 1:1 ratio. [Figure 6]

[0165] Shows PCSK9 gene editing in wild-type female mouse hepatocytes after retroorbital administration of the LNP composition of the present specification. [Figure 7]

[0166] Shows PCSK9 gene editing in wild-type female mouse hepatocytes after retroorbital administration of the LNP composition of the present specification. [Figure 8]

[0167] Shows PCSK9 editing in LDLR- / - female mouse hepatocytes after retroorbital administration of the LNP composition herein carrying Cas9 mRNA and gRNA. [Figure 9]

[0168] Four general processes for incorporating GalNAc-lipids into lipid nanoparticles are presented. [Figure 10]

[0169] Three protocols for preparing lipid nanoparticles involving post-addition of GalNAc-lipids are presented. [Figure 11]

[0170] Three protocols for preparing lipid nanoparticles involving post-addition of GalNAc-lipids are presented. [Figure 12]

[0171] Three protocols for preparing lipid nanoparticles are presented, including the addition of GalNAc-lipids to the LNP excipient and the split addition of GalNAc-lipids. [Figure 13]

[0172] Two protocols for preparing lipid nanoparticles are presented, including the addition of GalNAc-lipids to the LNP excipient and the split addition of GalNAc-lipids. [Figure 14]

[0173] Two protocols for preparing lipid nanoparticles containing GalNAc-lipid cross-mixing are presented. [Figure 15]

[0174] 1 shows PCSK9 editing in LDLR− / − female mouse hepatocytes following retroorbital administration of the LNP composition herein carrying PCSK9 ABE mRNA and guide RNA at a 1:1 ratio. [Figure 16]

[0175] 1 shows PCSK9 gene editing in liver tissue isolated from female LDLR− / − mice (n=5), also referred to herein as homozygous LDLR knockout (“KO”), following retroorbital injection of LNPs carrying mRNA MA004 and gRNA GA256 at a dose of 0.25 mg / kg. [Figure 17]

[0176] 1 shows PCSK9 gene editing in liver tissue isolated from female LDLR KO mice (n=5) after retroorbital injection of LNPs carrying mRNA MA004 and gRNA GA256 at a dose of 0.125 mg / kg. [Figure 18]

[0177] Figure 1 shows PCSK9 gene editing in liver tissue isolated from female LDLR KO mice (n = 4–5) and WT mice (n = 4–5) after retroorbital injection of LNPs carrying mRNA MA004 and gRNA GA256 at a dose of 0.125 mg / kg in LDLR KO and 0.05 mg / kg in WT mice. [Figure 19]

[0178] 1 shows PCSK9 gene editing in liver tissue isolated from female LDLR KO mice (n=5) after retroorbital injection of LNPs carrying mRNA MA004 and gRNA GA256 at a dose of 0.125 mg / kg in LDLR KO. [Figure 20]

[0179] Figure 1 shows ANGPTL3 gene editing in liver tissue isolated from female homozygous LDLR KO mice (n=5), female WT mice (n=5), and female heterozygous LDLR knockout mice (n=5) after retroorbital injection of LNPs bearing no GalNAc and bearing mRNA MA004 and gRNA GA260 at doses of 1, 0.25, and 0.05 mg / kg in all three mouse types. [Figure 21]

[0180] Figure 1 shows PCSK9 gene editing in liver tissue isolated from female LDLR KO mice (n=5) and female WT mice (n=5) after retroorbital injection of LNPs carrying mRNA MA004 and gRNA GA256 at a dose of 0.05 mg / kg in LDLR KO and WT mice. [Figure 22]

[0181] Figure 1 shows PCSK9 gene editing in liver tissue isolated from female LDLR KO mice (n=5) and female WT mice (n=5) after retroorbital injection of LNPs carrying mRNA MA004 and gRNA GA256 at doses of 0.125 mg / kg in LDLR KO and 0.125 mg / kg and 0.05 mg / kg in WT mice. [Figure 23]

[0182] Figure 1 shows PCSK9 gene editing in liver tissue isolated from female LDLR KO mice (n=5) and female WT mice (n=5) after retroorbital injection of LNPs carrying mRNA MA004 and gRNA GA256 at a dose of 0.05 mg / kg in LDLR KO and 0.05 mg / kg in WT mice. [Figure 24]

[0183] Figure 1 shows PCSK9 gene editing in liver tissue isolated from female LDLR KO mice (n=5) and female WT mice (n=5) after retroorbital injection of LNPs carrying mRNA MA004 and gRNA GA256 at a dose of 0.05 mg / kg in LDLR KO and 0.05 mg / kg in WT mice. [Figure 25]

[0184] Figure 1 shows PCSK9 gene editing in liver tissue isolated from female LDLR KO mice (n=5) and female WT mice (n=5) after retroorbital injection of LNPs carrying mRNA MA004 and gRNA GA257 at a dose of 0.05 mg / kg in LDLR KO and 0.05 mg / kg in WT mice. [Figure 26]

[0185] Graph showing the percentage of ANGPTL3 gene editing in liver tissue isolated from female homozygous LDLR KO mice (n=5), female WT mice (n=5), and female ApoE knockout mice following retro-orbital injection of LNPs carrying mRNA MA004 and ANGPTL3 gRNA GA260 at doses of 0.025, 0.05, 0.1, and 0.25 mg / kg in all three mouse types to demonstrate dose response. [Figure 27]

[0186] 1 is a graph showing the percentage of ANGPTL3 gene editing in liver tissue isolated from WT NHPs after administration of 1 mg / kg of LNPs made with GA097 and mRNA MA004. [Figure 28]

[0187] 1 is a graph showing LDL levels in the blood of NHPs after administration of LNP A or LNP B at 1 or 2 mg / kg doses. Both LNP A and LNP B were loaded with mRNA MS004 and one of two different guide pairs targeting LDLR: GA468 / GA470 or GA469 / GA471. This treatment then converted the NHPs from WT to LDLR KO / KD NHPs. [Figure 29]

[0188] 1 is a graph showing the percentage of LDLR gene editing in liver tissue isolated from original WT NHPs administered LNP A or LNP B at a dose of 1 or 2 mg / kg. Both LNP A and LNP B are loaded with mRNA MS004 and one of two different guide pairs targeting LDLR: GA468 / GA470 or GA469 / GA471. This treatment then converted the NHPs from WT to LDLR KO / KD NHPs. [Figure 30]

[0189] FIG. 10 is a graph showing ANGPTL3 protein levels from LDLR KO / KD NHP treated with LNPs loaded with MA004 mRNA and GA347 guide RNA targeting ANGPTL3 at a dose of 2 mg / kg, as described in Example 49. [Figure 31]

[0190] FIG. 10 is a graph showing the percentage of ANGPTL3 editing in the liver of LDLR KO / KD NHPs treated with LNPs loaded with MA004 mRNA and GA347 guide RNA targeting ANGPTL3 at a dose of 2 mg / kg, as described in Example 49. [Figure 32]

[0191] Graph showing the percentage of ANGPTL3 gene editing in liver tissue isolated from female LDLR KO mice (n=5) and female WT mice (n=5) after retroorbital injection of LNPs carrying mRNA MA004 and gRNA GA260 at a dose of 0.1 mg / kg in homozygous LDLR KO and 0.1 mg / kg in WT mice. [Figure 33]

[0192] 1 is a graph showing the percentage of LDLR gene editing in liver tissue isolated from original WT NHPs administered LNP C at a dose of 2 mg / kg, loaded with mRNA MS004 and the guide pair GA468 / GA470 targeting LDLR. [Figure 34]

[0193] 1 is a graph showing LDL levels in the blood of original WT NHPs after administration of LNP C at a dose of 2 mg / kg, loaded with mRNA MS004 and the guide pair GA468 / GA470 targeting LDLR. [Figure 35]

[0194] 9-14 are flow charts illustrating the LNP manufacturing process, as described and shown in connection with FIGS. 9-14, which achieves large-scale manufacturing while being scalable to larger quantities and capable of stable storage for long periods prior to use. [Figure 36]

[0195] 1 is a graph showing ANGPTL3 levels after 2 weeks of treatment from LDLR / KD NHP treated with LNPs loaded with MA004 mRNA and GA347 guide RNA targeting ANGPTL3 at a dose of 2 mg / kg. [Figure 37]

[0196] 1 is a graph showing triglyceride levels after 2 weeks of treatment from LDLR / KD NHP treated with LNPs loaded with MA004 mRNA and GA347 guide RNA targeting ANGPTL3 at a dose of 2 mg / kg. [Figure 38A]

[0197] Figure 38A shows base editing and ANGPTL3 protein expression in blood after administration of LNPs made with different GalNAc-ligands, such as 1004, 1002, 1078, and 1014. Figure 38B shows the corresponding ANGPTL3 blood protein expression normalized to pre-treatment levels in WT and LDLR KO mice. [Figure 38B]

[0197] Figure 38 shows base editing and ANGPTL3 protein expression in blood after administration of LNPs made with different GalNAc-ligands, such as 1004, 1002, 1078, and 1014. Figure 38A shows ANGPTL3 adenine base editing. Figure 38B shows the corresponding ANGPTL3 blood protein expression normalized to pre-treatment levels in WT and LDLR KO mice. [Figure 39]

[0198] Lectin column affinity data for LNPs made with and without GalNAc-lipids are shown. [Figure 40A]

[0199]

[0041] Figure 40 shows the results of a biolayer interferometry (BLI) assay. Figure 40A shows a schematic of the assay. [Figure 40B]

[0199] Figure 40B shows the results of a biolayer interferometry (BLI) assay. Figure 40B shows data from LNPs without GalNAc-lipids. [Figure 40C]

[0199] Figure 40C shows the results of a biolayer interferometry (BLI) assay. Figure 40C shows data from LNPs with GalNAc-lipids. [Figure 41]

[0200] Graph showing the percentage of ANGPTL3 gene editing in liver tissue isolated from female LDLR KO (LDLR- / -) mice (n=5), female LDLR+ / - heterozygous mice (n=5), and female WT mice (n=5) following retroorbital injection of LNPs carrying mRNA MA004 and gRNA GA260 at a dose of 0.25 mg / kg in all three types of mice. [Figure 42]

[0201] Graph showing the percentage of ANGPTL3 gene editing in liver tissue isolated from female LDLR KO (LDLR- / -) mice (n=5), female LDLR+ / - heterozygous mice (n=5), and female WT mice (n=5) following retro-orbital injection of LNPs carrying mRNA MA004 and gRNA GA260 at doses of 0.1 mg / kg, 0.25 mg / kg, and 0.5 mg / kg in all three types of mice. [Figure 43]

[0202] 42 shows the corresponding ANGPTL3 protein expression in the blood of the LDLR− / −, LDLR+ / −, and WT mice shown in FIG. 41 9 days after injection of LNPs carrying mRNA MA004 and gRNA GA260 at a dose of 0.25 mg / kg. [Figure 44]

[0203] The corresponding ANGPTL3 protein expression in the blood of LDLR- / -, LDLR+ / -, and WT mice shown in Figure 42 is shown 9 days after injection of LNPs carrying mRNA MA004 and gRNA GA260 at doses of 0.1, 0.25, and 0.5 mg / kg. [Figure 45]

[0204] 1 is a graph showing the percentage of LDLR gene editing in liver tissue isolated from original WT NHPs administered LNP C at a dose of 2 mg / kg. This treatment then converted the NHPs from WT to LDLR KD NHPs. [Figure 46]

[0205] 1 is a graph showing LDLR pg / mg liver protein levels in liver tissue isolated from original WT NHPs administered LNP C at a dose of 2 mg / kg. This treatment then converted the NHPs from WT to LDLR KD NHPs. [Figure 47]

[0206] 1 is a graph showing LDLR pg / mg liver protein levels in liver tissue isolated from original WT NHPs administered LNP A or LNP B at doses of 1 or 2 mg / kg, which treatment then converted the NHPs from WT to LDLR KD NHPs. [Figure 48]

[0207] 1 is a graph showing the extended time course of LDL levels (mg / dL) in the blood of original WT NHPs after administration of LNP C at a dose of 2 mg / kg, which then converted the NHPs from WT to LDLR KD NHPs. [Figure 49]

[0208] 10 is a graph showing the percentage of ANGPTL3 editing in the liver of LDLR KD NHPs treated with LNPs loaded with MA004 mRNA and GA347 guide RNA targeting ANGPTL3 at a dose of 2 mg / kg. [Figure 50]

[0209] 36 is a graph showing ANGPTL3 blood protein levels over time after treatment isolated from WT NHPs treated with LNPs loaded with MA004 mRNA and GA347 guide RNA targeting ANGPTL3 at a dose of 2 mg / kg. This is the time course of FIG. [Figure 51]

[0210] 1 is a graph showing ANGPTL3 blood protein levels over time after treatment isolated from LDLR KD NHPs treated with LNPs loaded with MA004 mRNA and GA347 guide RNA targeting ANGPTL3 at a dose of 2 mg / kg. [Figure 52]

[0211] 1 is a graph showing LDL blood levels over time after treatment isolated from LDLR KD NHPs treated with LNPs loaded with MA004 mRNA and GA347 guide RNA targeting ANGPTL3 at a dose of 2 mg / kg. [Figure 53]

[0212] 28 is a graph showing LDL levels as a percent of baseline in the blood of NHPs after administration of LNP A or LNP B at doses of 1 or 2 mg / kg. This treatment then converted the NHPs from WT to LDLR KD NHPs. This is the time course of FIG. 28. [Figure 54]

[0213] 1 is a graph showing LDL blood levels over time after treatment isolated from LDLR KD NHPs treated with LNPs loaded with MA004 mRNA and GA347 guide RNA targeting ANGPTL3 at a dose of 2 mg / kg. [Figure 55]

[0214] 31 is a graph showing the percentage of ANGPTL3 editing in the liver of LDLR KD NHPs treated with LNPs loaded with MA004 mRNA and GA347 guide RNA targeting ANGPTL3 at a dose of 2 mg / kg. This figure is Figure 31 showing replication of NHPs. DETAILED DESCRIPTION OF THE INVENTION

[0171]

[0215] Certain details are set forth in this description to provide a thorough understanding of various embodiments. However, those skilled in the art will understand that the present disclosure may be practiced without these details. In other instances, well-known structures and / or methods have not been shown or described in detail to avoid unnecessarily obscuring the description of the embodiments. Unless the context otherwise requires, throughout the following specification and claims, words such as "comprise" and variations thereof, such as "comprises" and "comprising," should be interpreted in an open and inclusive sense, i.e., as "including, but not limited to." Furthermore, the headings provided herein are for convenience only and do not interpret the scope or meaning of the claimed disclosure. The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described.

[0172]

[0216] Efficient delivery to cells requires specific targeting and substantial protection from the extracellular environment, especially serum proteins. One way to achieve specific targeting is to conjugate a targeting moiety with a pharmaceutical effector, such as an active agent or a nucleic acid agent, thereby directing the active agent or pharmaceutical effector to specific cells or tissues according to the specificity of the targeting moiety. One way that a targeting moiety can improve delivery is through receptor-mediated endocytosis activity. In some cases, this uptake mechanism may involve the transfer of a nucleic acid agent bound to a membrane receptor into the enclosed area by the membrane through the invagination of a membrane structure or by the fusion of the delivery system with the cell membrane. This process is initiated through the activation of cell surface or membrane receptors following the binding of specific ligands to the receptor. Many receptor-mediated endocytosis systems are known and have been studied, including systems that recognize sugars such as galactose, mannose, mannose-6-phosphate, peptides and proteins such as transferrin, asialoglycoprotein, vitamin B12, insulin, and epidermal growth factor (EGF). Lipophilic moieties such as cholesterol or fatty acids, when conjugated to highly hydrophilic molecules such as nucleic acids, can significantly enhance plasma protein binding and, consequently, extend circulatory half-life. Lipophilic conjugates may be used in combination with targeting ligands to improve intracellular trafficking in targeted delivery approaches.

[0173]

[0217] The asialoglycoprotein receptor (ASGP-R) is a high-capacity receptor highly abundant on hepatocytes. ASGP-R exhibits a 50-fold higher affinity for N-acetyl-D-galactosylamine (GalNAc) than for D-Gal. Previous studies have shown that, while intersaccharide spacing is also important, achieving high affinity requires multivalency. The present inventors have now recognized a clear need for novel receptor-specific ligand-conjugated RNA or DNA agents and methods for their preparation that address the shortcomings of in vivo delivery of therapeutic agents using nucleic acids or nucleic acid-associated complexes described above. The present disclosure is directed toward this very important goal.

[0174]

[0218] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. It should also be noted that the term "or" is generally used in its sense to include "and / or" unless the content clearly dictates otherwise.

[0175]

[0219] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs. In practicing or testing this disclosure, methods and materials similar or equivalent to those described herein can be used, and suitable methods and materials are described below. All references cited herein are incorporated by reference in their entirety as if fully set forth. Singleton et al., Dictionary of Microbiology and Molecular Biology, 3rd Edition, J. Wiley & Sons (New York, NY 2001); Marc H, Advanced Organic Chemistry Reactions, Mechanisms and Structure, 5th Edition, J. Wiley & Sons (New York, NY 2001); and Sambrook and Russell, Molecular Cloning: A Laboratory Manual, 3rd Edition, Cold Spring Harbor Laboratory Press (Cold Spring Harbor, NY 2001) provide those skilled in the art with a general guide to many of the terms used in this application.

[0176] Specific Definitions

[0220] When referring to the number of substituents, the term "one or more" refers to a range from one substituent to the maximum number of substitutions possible, e.g., replacement of one hydrogen to replacement of all hydrogens by substituents.

[0177]

[0221] The term "optional" or "optionally" means that the subsequently described event or circumstance need not occur, but that the description includes cases where the event or circumstance occurs and cases where it does not occur.

[0178]

[0222] The term "nucleic acid molecular entity" is used interchangeably with "nucleic acid."

[0223] As used herein, the term "nucleic acid" generally refers to one or more nucleobases, nucleosides, or nucleotides, and includes polynucleobases, polynucleosides, and polynucleotides. Nucleic acids can include polynucleotides, mononucleotides, and oligonucleotides. Nucleic acids can include DNA, RNA, or mixtures thereof, and can be single-stranded, double-stranded, or partially single- or double-stranded, and can form secondary structures. In some embodiments, nucleic acids have multiple double-stranded and single-stranded segments. For example, nucleic acids can include polynucleotides, such as mRNA, with multiple double-stranded segments therein. DNA can 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 of these groups. RNA can be in the form of siRNA, asymmetric interfering RNA (aiRNA), microRNA (miRNA), mRNA, tRNA, rRNA, tRNA, viral RNA (vRNA), CRISPR RNA, base editor RNA, and combinations thereof. Nucleic acids include synthetic, natural, and non-natural nucleic acids containing known nucleotide analogs or modified backbone residues or linkages that have similar binding properties to the reference nucleic acid. Examples of such analogs include, but are not limited to, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2'-O-methyl ribonucleotides, and peptide-nucleic acids (PNAs). Unless otherwise specified, the term encompasses nucleic acids containing known analogs of natural nucleotides that have similar binding properties to the reference nucleic acid. Unless otherwise specified, a particular nucleic acid sequence implicitly encompasses not only the sequence explicitly indicated, but also its conservatively modified variants (e.g., degenerate codon substitutions), alleles, orthologs, 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 mixed-base and / or deoxyinosine residues.(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" comprises a substituted and / or unsubstituted sugar deoxyribose (DNA), or a substituted and / or unsubstituted sugar ribose (RNA), or a substituted and / or unsubstituted carbocyclic moiety, or a substituted and / or unsubstituted acyclic moiety (glycol nucleic acids, for example), a base, and a phosphate group. Nucleotides are linked together via the phosphate group. "Base" includes purines and pyrimidines, which further include the natural compounds adenine, thymine, guanine, cytosine, uracil, inosine, as well as natural analogs and synthetic derivatives of purines and pyrimidines, including modifications that place new reactive groups such as, but not limited to, amines, alcohols, thiols, carboxylates, and alkyl halides.

[0179]

[0224] The term "gene" refers to a nucleic acid (eg, DNA or RNA) sequence that comprises partial or full-length coding sequences necessary for the production of a polypeptide or precursor polypeptide.

[0225] As used herein, "gene product" refers to the product of a gene, such as an RNA transcript or a polypeptide.

[0180]

[0226] As used herein, the term "polynucleotide" generally refers to a molecule containing two or more linked nucleic acid subunits, e.g., 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 variants thereof. A nucleotide generally contains a nucleoside and at least one, two, three, four, five, six, seven, eight, nine, ten, or more phosphate (PO3) groups. A nucleotide includes a nucleobase, a five-carbon sugar (either ribose or deoxyribose), and one or more phosphate groups. Ribonucleotides include nucleotides in which the sugar is ribose. Deoxyribonucleotides include nucleotides in which the sugar is deoxyribose. A nucleotide may be a nucleoside monophosphate, nucleoside diphosphate, nucleoside triphosphate, or 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 a 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.Exemplary 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 (pre-mRNA), antisense RNA (asRNA), and heteronuclear RNA (hnRNA), and include both nucleotide sequences and any structural embodiments thereof, such as single-stranded, double-stranded, triple-stranded, helical, hairpin, stem loop, bulge, etc. In some cases, polynucleotides are circular. Polynucleotides can have various lengths. For example, a polynucleotide can have a length of at least about 7 bases, 8 bases, 9 bases, 10 bases, 20 bases, 30 bases, 40 bases, 50 bases, 100 bases, 200 bases, 300 bases, 400 bases, 500 bases, 1 kilobase (kb), 2 kb, 3 kb, 4 kb, 5 kb, 10 kb, 50 kb, or more. Polynucleotides can be isolated from cells or tissues. For example, polynucleotide sequences can include isolated and purified DNA / RNA molecules, synthetic DNA / RNA molecules, and / or synthetic DNA / RNA analogs.

[0181]

[0227] A polynucleotide may contain one or more nucleotide variants, including non-standard nucleotides, non-natural nucleotides, nucleotide analogs, and / or modified nucleotides. Examples of modified nucleotides include, but are not limited to, diaminopurine, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxymethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 8-methylguanine, 9-methylguanine, 10-methylguanine, 11-methylguanine, 12-methylguanine, 13-methylguanine, 14-methylguanine, 15-methylguanine, 16-methylguanine, 17-methylguanine, 18-methylguanine, 19-methylguanine, 20-methylguanine, 21-methylguanine, 22-methylguanine, 23-methylguanine, 24-methylguanine, 25-methylguanine, 26-methylguanine, 27-methylguanine, 28-methylguanine, 29-methylguanine, 30-methylguanine, 31-methylguanine, 32-methylguanine, 33-methylguanine, 34-methylguanine, 35-methylguanine, 36-methylguanine, Examples include chiruguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueuosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxocine, pseudouracil, queuosine, 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, 2,6-diaminopurine, etc. In some cases, nucleotides may contain modifications of their phosphate moieties, including modifications to the triphosphate moiety. Non-limiting examples of such modifications include longer phosphate chains (e.g., phosphate chains having 4, 5, 6, 7, 8, 9, 10 or more phosphate moieties) and modifications with thiol moieties (e.g., alpha-thiotriphosphate and beta-thiotriphosphate). Nucleic acid molecules may also be modified at the base moiety (e.g., at one or more atoms typically available to form hydrogen bonds with a complementary nucleotide and / or at one or more atoms typically unavailable to form hydrogen bonds with a complementary nucleotide), the sugar moiety, or the phosphate backbone.Nucleic acid molecules may also contain amine-modifying groups, such as aminoallyl l-dUTP (aa-dUTP) and aminohexyl acrylamide-dCTP (aha-dCTP), which allow for the covalent attachment of amine-reactive moieties, such as N-hydroxysuccinimide ester (NHS). Substitution of standard DNA or RNA base pairs in the disclosed oligonucleotides may result in higher bit density per cubic mm, greater safety (resistance to accidental or deliberate synthesis of natural toxins), easier discrimination in light-programmed polymerases, or lower secondary structure. Such alternative base pairs compatible with natural 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 is incorporated herein by reference for all purposes.

[0182]

[0228] As used herein, the terms "polypeptide," "protein," and "peptide" are used interchangeably and refer to a polymer of amino acid residues linked through peptide bonds and may consist of two or more polypeptide chains. The terms "polypeptide," "protein," and "peptide" refer to a polymer of at least two amino acid monomers linked together through amide bonds. The amino acids may be the L or D optical isomer. More specifically, the terms "polypeptide," "protein," and "peptide" refer to a molecule composed of two or more amino acids in a specific order, e.g., an order determined by the nucleotide base sequence of a gene or RNA encoding the protein. Proteins are essential for the structure, function, and regulation of cells, tissues, and organs of the body, and each protein has a unique function. Examples are hormones, enzymes, antibodies, and any fragments thereof. In some cases, a protein may be a portion of a protein, such as 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 have been inserted, deleted, and / or substituted in the amino acid sequence of a naturally occurring (or at least known) protein. The protein or variant thereof may be naturally occurring or recombinant.

[0183]

[0229] As used herein, the terms "intercalate" or "intercalation" refer to the action of a substance (e.g., a small molecule) getting between consecutive bases of DNA. In some cases, intercalation interferes with the proper function of the DNA.

[0184]

[0230] As used herein, "complement" refers to a sequence complementary to a nucleic acid according to standard Watson-Crick pairing rules. A complementary sequence may also be a sequence of RNA complementary to a DNA sequence or its complementary sequence, or may be cDNA. A 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 a complementary or substantially complementary sequence need not hybridize along its entire length. In certain embodiments, a complementary or substantially complementary sequence may include a contiguous sequence of bases that does not hybridize to the target sequence, located 3' or 5' to the contiguous sequence of bases that hybridize to the target sequence.

[0185]

[0231] As used herein, "hybridizing" 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 ed., Cold Spring Harbor Press, Plainview, NY. Those skilled in the art know how to determine the appropriate stringency of hybridization / washing conditions so that sequences with at least the desired level of complementarity will stably hybridize, while sequences with less complementarity will not hybridize. For examples of hybridization conditions and parameters, see, e.g., Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Press, Plainview, NY; Ausubel, FM et al., 1994, Current Protocols in Molecular Biology. John Wiley & Sons, Secaucus, NJ, all of which are incorporated by reference in their entireties. In certain embodiments, hybridization can occur between nucleic acid molecules 20 to 100 nucleotides in length. In some embodiments, hybridization occurs 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, It 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 100 consecutive nucleotides.In some embodiments, hybridizing nucleic acid molecules 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 mismatches, which are tolerated.

[0186]

[0232] As used herein, the term "biological sample" refers to any biological material from which polynucleotides, polypeptides, biomarkers, and / or metabolites can be prepared and tested. Non-limiting examples include whole blood, plasma, saliva, buccal swabs, fecal specimens, urine specimens, cell clumps, or any other bodily fluid or tissue.

[0187]

[0233] As used herein, the terms "administer," "administering," "administering," "administration," and the like refer to methods that can be used to enable delivery of a compound or composition to a desired site of biological effect. 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 are familiar with 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.

[0188]

[0234] As used herein, "co-administration" and like terms are meant to encompass the administration of selected therapeutic agents to a single patient and are intended to include therapeutic regimens in which agents are administered to the same patient or by different routes of administration, or at the same time or at different times.

[0189]

[0235] As used herein, the term "effective amount" or "therapeutically effective amount" refers to a sufficient quantity of an agent or compound being administered to relieve to some extent one or more symptoms of the disease or condition being treated (e.g., reduction and / or alleviation of one or more signs, symptoms, or causes of the disease, or any other desired alteration of a biological system). For example, an "effective amount" for therapeutic purposes can be the amount of an agent that results in a clinically significant reduction in one or more disease symptoms. An appropriate "effective" amount may be determined in each individual case using techniques such as dose escalation studies.

[0190]

[0236] The terms "enhance" or "enhancing," as used herein, means to increase or prolong either in amount, potency, or duration a desired effect.

[0237] As used herein, "carbohydrate" refers to either a compound that is a carbohydrate itself, composed of one or more monosaccharide units (which may be linear, branched, or cyclic) having at least six carbon atoms with an oxygen, nitrogen, or sulfur atom bonded to each carbon atom; or a compound that has as part of its carbohydrate moiety one or more monosaccharide units (which may be linear, branched, or cyclic), each having at least six carbon atoms with an oxygen, nitrogen, or sulfur atom bonded to each carbon atom. Representative carbohydrates include sugars (monosaccharides, disaccharides, trisaccharides, and oligosaccharides containing about 4-9 monosaccharide units) and polysaccharides such as starch, glycogen, cellulose, and polysaccharide gums. Specific monosaccharides include sugars of C5 or higher (preferably C5-C8); disaccharides and trisaccharides include sugars having two or three monosaccharide units (preferably C5-C8).

[0191]

[0238] The term "monosaccharide" encompasses radicals of allose, altrose, arabinose, cladinose, erythrose, erythrulose, fructose, D-fucitol, L-fucitol, fucosamine, fucose, fuculose, galactosamine, D-galactosaminitol, N-acetyl-galactosamine, galactose, glucosamine, N-acetyl-glucosamine, glucosaminitol, glucose, glucose-6-phosphate guloseglyceraldehyde, L-glycero-D-mannos-heprose, glycerol, glycerone, guloseidose, lyxose, mannosamine, mannose, mannose-6-phosphate, psicose, quinovose, quinovosamine, rhamnitol, rhamnosamine, rhamnose, ribose, ribulose, sedoheptulose, sorbose, tagatose, talose, tartaric acid, tulose, xylose and xylulose. Monosaccharides may be in the D or L configuration. Monosaccharides may further be deoxysugars (alcoholic hydroxy group replaced with hydrogen), aminosugars (alcoholic hydroxy group replaced with amino group), thiosugars (alcoholic hydroxy group replaced with thiol, or C=O replaced with C=S, or in cyclic forms, ring oxygen replaced with sulfur), selenosugars, tellurosugars, azasugars (ring carbon replaced with nitrogen), iminosugars (ring oxygen replaced with nitrogen), phosphanosugars (ring oxygen replaced with phosphorus), phosphosugars (ring carbon replaced with phosphorus), C-substituted monosaccharides (hydrogen on a non-terminal carbon atom replaced with carbon), unsaturated monosaccharides, alditols (carbonyl group replaced with CHOH group), aldonic acids (aldehyde group replaced with carboxy group), ketoaldonic acids, uronic acids, aldaric acids, etc. The amino sugars include amino monosaccharides, preferably galactosamine, glusamine, mannosamine, fucosamine, quinabosamine, neuraminic acid, muramic acid, lactosediamine, acosamine, bacillosamine, daunosamine, desosamine, forosamine, galosamine, kanosamine, kanosamine, mycaminose, myosamine, persosamine, pneumosamine, purpurosamine, and rhodosamine. It is understood that the monosaccharides may be further substituted.

[0192]

[0239] As used herein, "N / P ratio" refers to the molar ratio of ionizable nitrogen atoms (e.g., in the physiological pH range) in a lipid (or lipids) to phosphate groups in a nucleic acid molecular entity (or nucleic acid molecular entity), e.g., in a nanoparticle composition comprising a lipid component and RNA. Ionizable nitrogen atoms can 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 can include, for example, the pH ranges of different cellular compartments (such as organs, tissues, and cells) and bodily fluids (such as blood, CSF, gastric juice, milk, bile, saliva, tears, and urine). In certain embodiments, the physiological pH range refers to the pH range of blood in mammals, e.g., 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.

[0193]

[0240] The terms "disaccharide," "trisaccharide," and "polysaccharide" refer to abequose, acrabose, amicetose, amylopectin, amylose, apiose, alkanose, ascarylose, ascorbic acid, boivinose, cellobiose, cellotriose, cellulose, chacotriose, chalcose, chitin, colitose, cyclodextrin, cymarose, dextrin, 2-deoxyribose, 2-deoxyglucose, diginose, digitalose, digitoxose, evolose, ebemitrose, fructooligosaccharides, galtooligosaccharides, gentianose, genitiobiose, glucan, glucogen, glycogen, hamamelose, heparin, inulin, isolevoglucosenone, isomaltose, isomaltotriose, isopanose, kojibiose, lactose, lactosamine, lactose, Examples of sugars include sudiamin, laminarabiose, levoglucosan, levoglucosenone, β-maltose, maltotriose, mannanoligosaccharide, amninotriose, melezitose, melibiose, muramic acid, mycarose, mycinose, neuraminic acid, mygelose, nojirimaicon, nobiose, oleandrose, panose, paratose, planteose, primverose, raffinose, rhodon, rutinose, oleandrose, panose, paratose, planteose, primverose, raffinose, rhodinose, rutinose, sarmentose, sedoheptulose, sedoheptulosan, solatriose, sophorose, stachyose, streptose, sucrose, α,α-trehalose, trahalosamine, turanose, tybelose, xylobiose, and umbelliferose. It is further understood that "disaccharides," "trisaccharides," "polysaccharides," and the like, may be further substituted. Disaccharides also include amino sugars and their derivatives, particularly mycaminose derivatized at the C-4' position or 4-deoxy-3-amino-glucose derivatized at the C-6' position.

[0194]

[0241] The term "subject" or "patient" encompasses mammals. Examples of mammals include, but are not limited to, any member of the mammalian class; humans, non-human primates, e.g., chimpanzees, and other ape and monkey species; domestic animals, such as cattle, horses, sheep, goats, and pigs; domestic animals, such as rabbits, dogs, and cats; and laboratory animals, including rodents, such as rats, mice, and guinea pigs. In one aspect, the mammal is a human. The term "animal" as used herein includes human and non-human animals. In one embodiment, the "non-human animal" is a mammal, e.g., a rodent, such as a rat or a mouse. In one embodiment, the non-human animal is a mouse or a monkey.

[0195]

[0242] As used herein, the terms "treat," "treating," or "treatment" include alleviating, ameliorating, or improving at least one symptom of a disease or condition, preventing additional symptoms, inhibiting a disease or condition, e.g., arresting the onset of a disease or condition, relieving a disease or condition, causing regression of a disease or condition, alleviating conditions caused by a disease or condition, or the prophylactic and / or therapeutic cessation of symptoms of a disease or condition. Although not excluded, it is understood that treating a disorder or condition does not require that the disorder, condition, or symptoms associated therewith be completely eliminated.

[0196]

[0243] The terms "preventing" or "prevention" of a disease state refer to keeping clinical symptoms of the disease state from occurring in a subject who may be exposed to or predisposed to the disease state but who has not yet experienced or exhibited symptoms of the condition.

[0197]

[0244] The terms "pharmaceutical composition" and "pharmaceutical formulation" (or "formulation") are used interchangeably and refer to a mixture or solution containing a therapeutically effective amount of an active pharmaceutical ingredient together with one or more pharmaceutically acceptable excipients that is administered to a subject, e.g., a human in need thereof.

[0198]

[0245] As used herein, the term "pharmaceutical combination" refers to a product obtained by mixing or combining multiple active ingredients, including both fixed and non-fixed combinations of the active ingredients. The term "fixed combination" means that both active ingredients, e.g., a compound described herein and an auxiliary agent, are administered to a patient simultaneously in the form of a single entity or dosage. The term "unfixed combination" means that the active ingredients, e.g., a compound described herein and an auxiliary agent, are administered to a patient simultaneously, concomitantly, or sequentially without a specific intervening time limit as separate entities, such that administration provides an effective level of the two compounds in the patient's body. The latter also applies to cocktail therapy, e.g., the administration of three or more active ingredients.

[0199]

[0246] The term "pharmaceutically acceptable" generally refers to the attributes of a material that is safe, non-toxic, not biologically or otherwise undesirable, and useful in preparing pharmaceutical compositions that are acceptable for veterinary and human pharmaceutical use. "Pharmaceutically acceptable" can refer to a material, such as a carrier or diluent, that does not abolish the biological activity or properties of a compound and is relatively non-toxic, e.g., the material may be administered to an individual without causing undesired biological effects or interacting in a deleterious way with any of the components of the composition in which it is included.

[0200]

[0247] The terms "pharmaceutically acceptable excipient," "pharmaceutically acceptable carrier," "pharmaceutically acceptable vehicle," and "therapeutically inactive excipient," may be used interchangeably and refer to any pharmaceutically acceptable ingredient in a pharmaceutical composition that has no therapeutic activity and is non-toxic to a subject to which it is administered, such as a disintegrant, binder, filler, solvent, buffer, isotonicity agent, stabilizer, antioxidant, surfactant, carrier, diluent, excipient, preservative, or lubricant used in formulating a pharmaceutical product.

[0201]

[0248] The terms "base editing" and "base correction" are used interchangeably to refer to a base change or mutation in a target sequence within a target gene that results in a base modification. In certain embodiments, base editing occurs at a single base in the target sequence. In preferred embodiments, base editing does not involve a double-strand break in the target sequence.

[0202]

[0249] The term "siRNA" as used herein refers to an agent that mediates targeted cleavage of RNA transcripts. These agents are associated with a cytoplasmic multiprotein complex known as the RNAi-induced silencing complex (RISC). Agents that are effective in inducing RNA interference are also referred to herein as siRNAs, RNAi agents, or iRNA agents. The term siRNA as used herein includes microRNAs and pre-microRNAs. The terms "siRNA activity" and "RNAi activity" as used herein refer to gene silencing by siRNA.

[0203]

[0250] The term "2'-O-methoxyethyl" (also referred to as 2'-MOE, 2'-O(CH2)2-OCH3, and 2'-O-(2-methoxyethyl)) refers to an O-methoxy-ethyl modification at the 2' position of the furosyl ring. A 2'-O-methoxyethyl modified sugar is a modified sugar.

[0204]

[0251] The term "2'-O-methoxyethyl nucleotide" means a nucleotide containing a 2'-O-methoxyethyl modified sugar moiety.

[0252] The term "5-methylcytosine" means a cytosine modified with a methyl group attached to the 5' position. 5-Methylcytosine is a modified nucleobase.

[0205]

[0253] The term "oxo" refers to a ═O substituent.

[0254] The term "alkyl" refers to a straight or branched hydrocarbon chain group having from 1 to 20 carbon atoms and attached to the rest of the molecule by a single bond. Alkyl groups containing up to 10 carbon atoms include C1-C 10 Similarly, for example, an alkyl containing up to 6 carbon atoms is a C1-C6 alkyl. Alkyl groups containing other numbers of carbon atoms (and other moieties defined herein) are similarly represented. Alkyl groups include, but are not limited to, C1-C 10

[0023] Examples of alkyl 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. Representative alkyl groups include, but are not limited to, 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, 1-ethyl-propyl, and the like. In some embodiments, alkyl is methyl or ethyl. In some embodiments, alkyl is -CH(CH3)2 or -C(CH3)3. Unless stated otherwise in the specification, alkyl groups may be optionally substituted as described below. "Alkylene" or "alkylene chain" refers to a straight or branched divalent hydrocarbon chain that connects the rest of the molecule to a radical group. In some embodiments, alkylene is -CH-, -CHCH-, or -CHCHCH-. In some embodiments, alkylene is -CH-. In some embodiments, alkylene is -CHCH-. In some embodiments, alkylene is -CHCHCH-. In some embodiments, alkylene is -CHCHCH-.

[0206]

[0255] The term "alkoxy" refers to a radical of formula -OR, where R is an alkyl group as defined herein. Unless stated otherwise in the specification, an alkoxy group may be optionally substituted as described below. Representative 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.

[0207]

[0256] 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 stated otherwise in the specification, an alkylamino group can be optionally substituted as described below.

[0208]

[0257] The term "alkenyl" refers to a type of alkyl group in which at least one carbon-carbon double bond is present. In one embodiment, an alkenyl group has the formula -C(R)=CR2, where R refers to the remainder of the alkenyl group and can be the same or different. In some embodiments, R is H or alkyl. In some embodiments, alkenyl is selected from ethenyl (i.e., vinyl), propenyl (i.e., allyl), butenyl, pentenyl, pentadienyl, and the like. Non-limiting examples of alkenyl groups include -CH=CH2, -C(CH3)=CH2, -CH=CHCH3, -C(CH3)=CHCH3, and -CH2CH=CH2. Depending on the structure, alkenyl groups can be monovalent or divalent (i.e., alkenylene groups).

[0209]

[0258] The term "alkynyl" refers to a type of alkyl group in which at least one carbon-carbon triple bond is present. Thus, "alkynylene" can refer to a divalent alkynyl group. In one embodiment, an alkenyl group has the formula -C≡CR, where R refers to the remainder of the alkynyl group. In some embodiments, R is H or alkyl. In some embodiments, alkynyl is selected from ethynyl, propynyl, butynyl, pentynyl, hexynyl, and the like. Non-limiting examples of alkynyl groups include -C≡CH, -C≡CCH3-C≡CCH2CH3, and -CH2C≡CH.

[0210]

[0259] The term "aryl" refers to an aromatic ring in which each of the atoms forming the ring is a carbon atom. An aryl group may be optionally substituted. Examples of aryl groups include, but are not limited to, phenyl and naphthyl. In some embodiments, an aryl is phenyl. Depending on the structure, an aryl group may be monovalent or divalent (i.e., an "arylene" group). Unless stated otherwise in this specification, the term "aryl" or the prefix "ar-" (e.g., "aralkyl") is meant to include aryl radicals that may be optionally substituted. In some embodiments, an aryl group is partially reduced to form a cycloalkyl group, as defined herein. In some embodiments, an aryl group is fully reduced to form a cycloalkyl group, as defined herein. In some embodiments, an aryl group is a C6-C 14 In some embodiments, the aryl group is a C-C 10 It is aryl.

[0211]

[0260] The term "cycloalkyl" refers to a monocyclic or polycyclic non-aromatic radical in which each of the atoms forming the ring (i.e., skeletal atoms) is a carbon atom. In some embodiments, the cycloalkyl is saturated or partially unsaturated. In some embodiments, the cycloalkyl is a spirocyclic or bridged compound. In some embodiments, the cycloalkyl is fused to an aromatic ring (in which case the cycloalkyl is attached through a non-aromatic ring carbon atom). Cycloalkyl groups include groups having 3 to 10 ring atoms. Representative cycloalkyls include, but are not limited to, cycloalkyls having 3 to 10 carbon atoms, 3 to 8 carbon atoms, 3 to 6 carbon atoms, or 3 to 5 carbon atoms. Monocyclic cycloalkyl radicals include, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. In some embodiments, the monocyclic cycloalkyl is cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl. In some embodiments, the monocyclic cycloalkyl is cyclopentenyl or cyclohexenyl. In some embodiments, the monocyclic cycloalkyl is cyclopentenyl. Polycyclic groups include, for example, adamantyl, 1,2-dihydronaphthalenyl, 1,4-dihydronaphthalenyl, tetraynyl, decalinyl, 3,4-dihydronaphthalenyl-1(2H)-one, spiro[2.2]pentyl, norbornyl, and bicyclo[1.1.1]pentyl. Unless otherwise specified in the specification, cycloalkyl groups can be optionally substituted. Depending on the structure, cycloalkyl groups can be monovalent or divalent (i.e., cycloalkylene groups).

[0212]

[0261] The term "haloalkyl" refers to an alkyl group in which at least one hydrogen atom of the alkyl group is replaced with the same or different halogen atom, particularly a fluoro atom. Examples of haloalkyl include monofluoro-, difluoro- or trifluoro-methyl, -ethyl or -propyl, such as 3,3,3-trifluoropropyl, 2-fluoroethyl, 2,2,2-trifluoroethyl, fluoromethyl, or trifluoromethyl. The term "perhaloalkyl" refers to an alkyl group in which all hydrogen atoms of the alkyl group are replaced with the same or different halogen atoms.

[0213]

[0262] The term "heteroalkylene" refers to an alkyl group as defined above in which one or more carbon atoms of the alkyl have been replaced with an O, N, or S atom. A "heteroalkylene" or "heteroalkylene chain" refers to a straight or branched divalent heteroalkyl chain connecting the remainder of the molecule to a radical group. Unless stated otherwise in the specification, a heteroalkyl or heteroalkylene group may be optionally substituted as described below. Representative heteroalkylene groups include, but are not limited to, -OCH2CHO-, -OCH2CHOCH2CHO-, or -OCH2CHOCH2CHOCH2CHO-.

[0214]

[0263] The term "heterocycloalkyl" refers to a cycloalkyl group containing at least one heteroatom selected from nitrogen, oxygen, and sulfur. Unless otherwise specified herein, a heterocycloalkyl group can be a monocyclic or bicyclic ring system, which can include fused (when fused to an aryl or heteroaryl ring, the heterocycloalkyl is attached through a non-aromatic ring atom) or bridged ring systems. The nitrogen, carbon, or sulfur atoms in a heterocyclyl group can be optionally oxidized. The nitrogen atom can be optionally quaternized. The heterocycloalkyl group can be partially or fully saturated. Examples of heterocycloalkyl groups include, but are not limited to, dioxolanyl, thienyl[1,3]dithianyl, 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, trithianyl, tetrahydropyranyl, thiomorpholinyl, thiamorpholinyl, 1-oxo-thiomorpholinyl, and 1,1-dioxo-thiomorpholinyl. The term heterocycloalkyl also includes all ring forms of carbohydrates, including, but not limited to, monosaccharides, disaccharides, and oligosaccharides. Unless otherwise specified, heterocycloalkyls have 2-12 carbons in the ring. In some embodiments, heterocycloalkyls have 2-10 carbons in the ring. In some embodiments, heterocycloalkyls have 2-10 carbons and 1 or 2 N atoms in the ring. In some embodiments, heterocycloalkyls have 2-10 carbons and 3 or 4 N atoms in the ring. In some embodiments, heterocycloalkyls have 2-12 carbons, 0-2 N atoms, 0-2 O atoms, 0-2 P atoms, and 0-1 S atoms in the ring.In some embodiments, a heterocycloalkyl has 2 to 12 carbons, 1 to 3 N atoms, 0 to 1 O atoms, and 0 to 1 S atoms in the ring. When referring to the number of carbon atoms in a heterocycloalkyl, it is understood that the number of carbon atoms in the heterocycloalkyl is not the same as the total number of atoms (including heteroatoms) comprising the heterocycloalkyl (i.e., skeletal atoms of the heterocycloalkyl ring). Unless otherwise specified in this specification, a heterocycloalkyl group may be optionally substituted. As used herein, the term "heterocycloalkylene" may refer to a divalent heterocycloalkyl group.

[0215]

[0264] The term "heteroaryl" refers to an aryl group containing one or more ring heteroatoms selected from nitrogen, oxygen, and sulfur. Heteroaryls are 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, furazanyl, indolizine, indole, benzofuran, benzothiophene, indazole, benzimidazole, purine, quinolizine, 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 furazanyl. Specific examples of bicyclic heteroaryls include indolizine, indole, benzofuran, benzothiophene, indazole, benzimidazole, purine, quinolizine, quinoline, isoquinoline, cinnoline, phthalazine, quinazoline, quinoxaline, 1,8-naphthyridine, and pteridine. In some embodiments, heteroaryl is pyridinyl, pyrazinyl, pyrimidinyl, thiazolyl, thienyl, thiadiazolyl, or furyl. In some embodiments, heteroaryls contain 0 to 6 N atoms in the ring. In some embodiments, the heteroaryl contains 1-4 N atoms in the ring. In some embodiments, the heteroaryl contains 4-6 N atoms in the ring. In some embodiments, the heteroaryl contains 0-4 N atoms, 0-1 O atoms, 0-1 P atoms, and 0-1 S atoms in the ring. In some embodiments, the heteroaryl contains 1-4 N atoms, 0-1 O atoms, and 0-1 S atoms in the ring. In some embodiments, the heteroaryl is a C1-C9 heteroaryl.In some embodiments, the monocyclic heteroaryl is a C1-C5 heteroaryl. In some embodiments, the monocyclic heteroaryl is a 5- 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 fully reduced to form a heterocycloalkyl group, as defined herein. Depending on the structure, heteroaryl groups can be monovalent or divalent (i.e., "heteroarylene" groups).

[0216]

[0265] The terms "substituted," "substituent," and the like, unless otherwise indicated, may individually and independently refer to the replacement of one or more hydrogen radicals in a given structure with the radical of the specified substituent, including, but not limited to, the following: D, halogen, -CN, -NH, -NH(alkyl), -N(alkyl), -OH, -COH, -COalkyl, -C(=O)NH, -C(=O)NH(alkyl), -C(=O)N(alkyl), -S(=O)NH, -S(=O)NH(alkyl), -S(=O)N(alkyl), alkyl, cycloalkyl, fluoroalkyl, heteroalkyl, alkoxy, fluoroalkoxy, heterocycloalkyl, aryl, heteroaryl, aryloxy, alkylthio, arylthio, alkylsulfoxide, arylsulfoxide, alkylsulfone, and arylsulfone. In some other embodiments, the optional substituents are independently selected from D, halogen, —CN, —NH, —NH(CH), —N(CH), —OH, —COH, —CO(C-C alkyl), —C(═O)NH, —C(═O)NH(C-C alkyl), —C(═O)N(C-C alkyl), —S(═O)NH, —S(═O)NH(C-C alkyl), —S(═O)N(C-C alkyl), C-C alkyl, C-C cycloalkyl, C-C fluoroalkyl, C-C heteroalkyl, C-C alkoxy, C-C fluoroalkoxy, —SC-C alkyl, —S(═O)C-C alkyl, and —S(═O)(C-C alkyl). In some embodiments, optional substituents are independently selected from D, halogen, -CN, -NH, -OH, -NH(CH), -N(CH), -NH(cyclopropyl), -CH, -CHCH, -CF, -OCH, and -OCF. In some embodiments, substituents are substituted with one or two of the foregoing groups. In some embodiments, optional substituents on aliphatic carbon atoms (acyclic or cyclic) include oxo (=O).

[0217]

[0266] The term "unsubstituted" means that a particular group has no substituents. The term "optionally substituted" means that a particular group is unsubstituted or substituted with one or more substituents independently selected from a 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 replacement of one hydrogen to the replacement of all hydrogens by a substituent.

[0218]

[0267] "About" means within ±10% of the value. For example, a statement that "a marker can be increased by about 50%" means that the marker can be increased by between 45% and 55%.

[0219]

[0268] "Active agent" means one or more substances in a pharmaceutical composition that produce a therapeutic effect when administered to an individual.

[0269] "Dosage unit" refers to the form in which a pharmaceutical product is provided, such as a pill, tablet, or other dosage form known in the art. In certain embodiments, the dosage form is a vial containing lyophilized antisense oligonucleotide. In certain embodiments, the dosage form is a vial containing reconstituted antisense oligonucleotide.

[0220]

[0270] "Dose" refers to a specific amount of a pharmaceutical agent provided in a single administration or over a specific period of time. 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, the desired dose requires an amount that cannot be easily accommodated by a single injection, so two or more injections may be used to achieve the desired dose. In certain embodiments, a pharmaceutical agent is administered by infusion over an extended period of time or continuously. A dose may be expressed as the amount of pharmaceutical agent per hour, day, week, or month. A dosage may also be expressed in mg / kg or g / kg.

[0221]

[0271] "Modified internucleoside linkage" refers to a substitution or any change from a naturally occurring internucleoside linkage. For example, a phosphorothioate linkage is a modified internucleoside linkage.

[0222]

[0272] "Modified nucleobase" refers to any nucleobase other than adenine, cytosine, guanine, thymidine, or uracil. For example, 5-methylcytosine is a modified nucleobase. "Unmodified nucleobase" refers to the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (U).

[0223]

[0273] "Modified nucleoside" means a nucleoside having at least one modified sugar moiety and / or modified nucleobase.

[0274] "Modified nucleotide" means a nucleotide having at least one modified sugar moiety, modified internucleoside linkage, and / or modified nucleobase.

[0224]

[0275] "Modified oligonucleotide" means an oligonucleotide containing at least one modified nucleotide.

[0276] A "modified sugar" refers to a substitution or variation from a natural sugar. For example, a 2'-O-methoxyethyl modified sugar is a modified sugar.

[0225]

[0277] By "motif" is meant a pattern of chemically distinct regions in an antisense compound.

[0278] "Statin" refers to a drug that inhibits the activity of HMG-CoA reductase.

[0226]

[0279] "Symptoms of a cardiovascular disease or disorder" means phenomena resulting from or accompanying a cardiovascular disease or disorder and serving as indicators thereof. For example, angina, chest pain, dyspnea, palpitations, weakness, dizziness, nausea, sweating, tachycardia, bradycardia, arrhythmia, atrial fibrillation, swelling of the legs, cyanosis, fatigue, fainting, numbness in the face, numbness in the hands and feet, claudication or muscle cramps, abdominal distension, or fever are symptoms of a cardiovascular disease or disorder.

[0227]

[0280] "Target nucleic acid" and "target sequence" refer to a nucleic acid that can be targeted by a genome editing composition. For example, a target DNA sequence within or adjacent to the ANGPTL3 gene can be targeted by a guide nucleotide associated with a Cas9 nuclease.

[0228]

[0281] Methods for detecting and / or measuring polypeptides in biological materials are well known in the art, including, but not limited to, Western blotting, flow cytometry, ELISA, RIA, and various proteomics techniques. An exemplary method for measuring or detecting polypeptides is an immunoassay 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. An exemplary assay for detecting and / or measuring polypeptides is described in Harlow, E. and Lane, D. Antibodies: A Laboratory Manual, (1988), Cold Spring Harbor Laboratory Press.

[0229]

[0282] Methods for detecting and / or measuring RNA in biological materials are well known in the art, including but not limited to Northern blotting, RNA protection assay, RT PCR.Suitable methods are described in Molecular Cloning: A Laboratory Manual (4th Edition) by Michael R. Green, Joseph Sambrook, Peter MacCallum 2012, 2, 028pp, ISBN 978-1-936113-42-2.

[0230]

[0283] Ribonucleoprotein (RNP) refers to a nuclear protein containing RNA. RNPs may be complexes of ribonucleic acid and RNA-binding proteins. Such combinations are sometimes called protein-RNA complexes. These complexes can function in several biological functions, including, but not limited to, DNA replication, DNA modification, gene expression, RNA metabolism and modification, and pre-mRNA splicing.

[0231]

[0284] As used herein, the term "nucleobase editor (BE)" or "base editor (BE)" refers to a composition, e.g., a fusion protein, comprising a polypeptide capable of performing nucleobase modifications and a Cas protein. In some embodiments, the fusion protein comprises a nuclease-inactive Cas9 (dCas9) fused to a deaminase. In some embodiments, the fusion protein comprises a Cas9 nickase fused to a deaminase. In some embodiments, the fusion protein comprises a wild-type Cas9 sequence, e.g., the D10X or H840X mutation of Cas9 numbered in SEQ ID NO: 1 (which allows Cas9 to cleave only one strand of a nucleic acid duplex). In some embodiments, the base editor comprises a programmable DNA nuclease domain fused or linked to a deaminase domain (e.g., an adenosine deaminase domain or a cytidine deaminase domain). Details of base editors are described in International PCT Application Nos. PCT / 2017 / 045381 (WO2018 / 027078) and PCT / US2016 / 058344 (WO2017 / 070632), each of which is incorporated by reference in its entirety.Also, 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, Joung JK. An APOBEC3A-Cas9 base editor with minimized bystander and off-target activities. Nat Biotechnol. 2018 Nov;36(10):977-982.

[0232]

[0285] As used herein, the terms "biomarker" or "marker" are used interchangeably to refer to any biochemical, serological, genetic, or other clinical or sonographic feature that can be used to classify a sample from a patient as being associated with a pathological condition, such as a cardiovascular disease or disorder.

[0233]

[0286] As used herein, the term "antibody" includes, but is not limited to, a population of immunoglobulin molecules, which may be polyclonal or monoclonal, and may be of any class and isotype, or fragments of immunoglobulin molecules. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, several of which can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1 (human), IgA2 (human), IgAa (dog), IgAb (dog), IgAc (dog), and IgAd (dog). Such fragments generally contain the portion of an antibody molecule that specifically binds to an antigen. For example, fragments of immunoglobulin molecules known in the art as Fab, Fab', or F(ab')2 are included within the meaning of the term antibody.

[0234]

[0287] The term "label" as used herein refers to a detectable compound, composition, or solid support that can be directly or indirectly conjugated (e.g., covalently or non-covalently, alone or encapsulated) to a monoclonal antibody or protein. The label may be detectable by itself (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 of 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 phosphatase; glucose-6-phosphate dehydrogenase ("G6PDH"); horseradish peroxidase (HRP); chemiluminescent agents such as isoluminol, fluorescent agents such as fluorescein and rhodamine compounds; ribozymes; and dyes. A label may also be a specific binding molecule that is itself detectable (e.g., biotin, avidin, streptavidin, digoxigenin, maltose, oligohistidine, e.g., hexahistidine (SEQ ID NO: 114), 2,4-dinitrobenzene, phenylarsenate, ssDNA, dsDNA, etc.). Use of a label generates a signal that can be detected, and optionally measured, by means such as detection of electromagnetic radiation or direct visualization.

[0235]

[0288] "Substantial binding" or "substantially binds" refers to the amount of specific binding or affinity between molecules in an assay mixture under specific assay conditions. In its broadest aspect, substantial binding refers to the difference between the inability of a first molecule to bind or recognize a second molecule and the inability of a first molecule to bind or recognize a third molecule, a difference sufficient to enable a meaningful assay to be performed to identify specific binding under a specific set of assay conditions, including the relative concentrations of the molecules and the incubation time and temperature. In another aspect, a molecule is substantially unable to bind or recognize another molecule in the sense of cross-reactivity if the first molecule exhibits less than 25%, e.g., less than 10%, e.g., less than 5%, of the reactivity it exhibits with a third molecule under a specific set of assay conditions, including, for example, the relative concentrations of the molecules and incubation. Specific binding can be tested using several widely known methods, such as immunohistochemistry, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), or Western blot assay.

[0236]

[0289] As used herein, the term "substantially the same amino acid sequence" includes amino acid sequences that are similar but not identical to a naturally occurring amino acid sequence. For example, an amino acid sequence, e.g., a polypeptide, having substantially the same amino acid sequence as a flagellin protein may have one or more modifications, such as addition, deletion, or substitution 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 "percentage similarity" 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 compared, multiplied by 100. In this regard, conserved residues in a sequence are residues that are physically or functionally similar to the corresponding reference residues, e.g., have similar size, shape, charge, chemical properties, such as the ability to form covalent or hydrogen bonds.

[0237]

[0290] The term "targeting moiety" refers to any molecule that provides enhanced affinity to selected targets, such as cells, cell types, tissues, organs, body regions, or compartments, such as cells, tissues, or organ compartments.Some exemplary targeting moieties include, but are not limited to, antibodies, antigens, carbohydrate base moieties, folate, 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, multivalent galactose, N-acetyl-D-galactosamine (GalNAc), multivalent GalNAc, such as GalNAc2 and GalNAc3; D-mannose, multivalent mannose, multivalent lactose, N-acetyl-glucosamine, multivalent fucose, glycosylated polyamino acids, and lectins. The term multivalent indicates the presence of multiple monosaccharide units. Such monosaccharide subunits may be linked to each other via glycosidic bonds or to a scaffold molecule.

[0238]

[0291] The term "heterologous" refers to any two or more nucleic acid or polypeptide sequences that are not normally found in the same relationship to each other in nature. For example, heterologous nucleic acids are typically produced recombinantly, having two or more sequences from unrelated genes arranged to create a new functional nucleic acid, such as, for example, a promoter from one source and a coding region from another source. Similarly, a heterologous polypeptide often refers to two or more subsequences that are not found in the same relationship to each other in nature (e.g., a fusion protein).

[0239]

[0292] As used herein, the term "fragment" includes a peptide, polypeptide or protein segment of amino acids of the full-length protein, provided that the fragment retains reactivity with at least one antibody in the serum of a disease patient.

[0240]

[0293] An "epitope" is an antigenic determinant on a polypeptide that is recognized for binding by an antibody specific for the polypeptide, eg, a paratope on an IBD-associated antibody.

[0294] The term "clinical factor" includes the symptoms of patients related to cardiovascular disease. Examples of clinical factors include, but are not limited to, angina pectoris; chest pain; shortness of breath; palpitations; weakness; dizziness; nausea; sweating; tachycardia; bradycardia; arrhythmia; atrial fibrillation; swelling of the lower extremities; cyanosis; fatigue; fainting; facial numbness; numbness of the extremities; claudication or muscle cramps; 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 a statistical algorithm and determining whether the patient has one or more clinical factors.

[0241]

[0295] The term "prognosis" includes the prediction of the likely course and outcome of a pathological condition, such as cardiovascular disease, or recovery from the disease. In some embodiments, a prognosis of a patient's cardiovascular disease is obtained by using a statistical algorithm. For example, the prognosis may be surgery, the occurrence of one or more clinical factors, or recovery from the disease.

[0242]

[0296] The term "RNA" in an LNP generally refers to the total RNA payload present, encapsulated, or used to prepare said LNP. The total RNA payload can include any nucleic acid entity, such as mRNA, gRNA, DNA, antisense oligonucleotides, siRNA, microRNA, antagomir, aptamer, prime-editing guide RNA (pegRNA), and any combination thereof.

[0243]

[0297] Provided herein are methods and compositions for targeted delivery of therapeutic agents, such as nucleic acid agents. The therapeutic agents used herein can be linked or associated with a targeting moiety to aid in targeted delivery. For example, the therapeutic agent and the targeting moiety can form a conjugate. The therapeutic agent can include a nucleic acid-guided programmable nuclease system complexed with a nucleic acid, such as a guide RNA. In some embodiments, the guide RNA can be chemically modified. In some embodiments, the modified guide RNA can be used to prepare a medicament for treating any gene-related disease, disorder, or condition in which genes can be altered, manipulated, edited, and modified by inserting or deleting DNA. According to a further aspect of the present disclosure, the modified guide RNA can be used to alter genes by deleting, replacing, repairing, or inserting DNA. This can be done in microorganisms or animals, particularly mammals, and more specifically, humans. Human cells or tissues can be genetically altered or corrected in vitro using the guide RNAs disclosed herein and CRISPR / Cas systems known in the art, and then inoculated into a patient in need thereof. In another aspect of the present disclosure, there is provided a pharmaceutical composition comprising the modified guide RNA of the present disclosure, a CRISPR-Cas system, and a pharmaceutically acceptable carrier or excipient.This pharmaceutical composition can comprise a vector or cell with the modified guide RNA of the present disclosure.In another aspect of the present disclosure, there is provided a composition comprising modified guide RNA and at least one delivery means selected from GalNAc, polymer, liposome, peptide, aptamer, antibody, viral vector, folate or transferrin.

[0244] Nuclease System

[0298] Provided herein are compositions and methods for targeted delivery of active or therapeutic agents, including nucleic acids, polynucleotides, or oligonucleotides. The active agent may be a pharmaceutical composition, a drug, a polynucleotide, an oligonucleotide, an RNP, a lipid nanoparticle, or a protein-RNA complex. The targeted delivery described herein can guide the active agent to a specific desired location, such as a specific in vivo location, a cell, tissue, or organ, a recognition location within an intracellular matrix, or a specific location within a cell. In some embodiments, the active agent comprises a guide RNA associated with a nuclease, such as a CRISPR nuclease. In some embodiments, the active agent comprises a nuclease system that can alter the activity and / or function of one or more target genes, such as the PCSK9 or ANGPTL3 gene.

[0245]

[0299] In some embodiments, the active agent comprises a genome editing composition comprising a nuclease system. In some embodiments, the genome editing composition is a target-specific genome editing composition. In some embodiments, the genome editing composition comprises a nucleic acid-guided programmable nuclease or a portion thereof. In some embodiments of the present 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 heterologous to the DNA-binding domain. In certain embodiments, the nuclease is a DNA endonuclease and can cleave single-stranded or double-stranded DNA. In certain embodiments, the nuclease can cleave RNA.

[0246]

[0300] In some embodiments, the nuclease system may include a Cas protein domain (also referred to as a "Cas nuclease") from a 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 a guide RNA. The guide RNA interacts with the Cas protein and the target sequence such 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-guided (RNA-guided) nuclease. In some embodiments, the 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.

[0247]

[0301] In some embodiments, a CRISPR / Cas system may include components of type I, type II, or type III systems, or any orthologs thereof. An updated classification scheme for CRISPR / Cas loci defines class 1 and class 2 CRISPR / Cas systems, with types I-V and 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 2 CRISPR / Cas systems have a single protein effector. Type II, V, and VI Cas proteins may be single-protein RNA-guided endonucleases, referred to herein as "class 2 Cas nucleases." Class 2 Cas nucleases include, for example, Cas9, Cpf1, C2c1, C2c2, and C2c3 proteins. The Cpf1 protein, Zetsche et al., Cell, 163:1-13 (2015), is homologous to Cas9 and contains the RuvC-like nuclease domain, S3.

[0248]

[0302] In some embodiments, the Cas protein can be from a type II CRISPR / Cas system, i.e., a Cas9 protein from a CRISPR / Cas9 system. In some embodiments, the Cas protein can be from a class 2 CRISPR / Cas system, i.e., a single-protein Cas nuclease, such as a Cas9 protein or a 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.

[0249]

[0303] Type II CRISPR / Cas system components can be derived from type IIA, type IIB, or type IIC systems. The structure and sequence of Cas9 nuclease are known to those skilled in the art (Jinek et al. Science 2012, 337:816-821; Delcheva et al. Nature 2011, 471:602-607, incorporated herein by reference). In some embodiments, wild-type Cas9 corresponds to Streptococcus pyogenes Cas9 (NCBI reference number NC_002737.2, SEQ ID NO: 2) and Uniprot reference Q99ZW2 (SEQ ID NO: 1).

[0250]

[0304] Streptococcus pyogenes Cas9 (wild type) protein sequence (SEQ ID NO: 1)

[0305]

[0251]

[0306] Streptococcus pyogenes Cas9 (wild type) nucleotide sequence (SEQ ID NO: 2)

[0252]

[0307] The Cas9 strain contains a specific strain of the genus 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., 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.

[0253]

[0308] In some embodiments, a Cas protein may contain two or more nuclease domains. For example, a 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 DSBs into a target sequence. In some embodiments, a Cas9 protein may be modified to contain only one functional nuclease domain. For example, a Cas9 protein may be modified so that one of the nuclease domains is mutated or completely or partially deleted to reduce its nucleic acid cleavage activity. In some embodiments, a Cas9 protein may be modified to not contain a functional RuvC-like nuclease domain. In other embodiments, a Cas9 protein may be modified to not contain a functional HNH-like nuclease domain. In some embodiments in which only one of the nuclease domains is functional, the Cas9 protein may be a nickase that can introduce 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 comprise an amino acid substitution in the RuvC-like nuclease domain. Exemplary amino acid substitutions in the RuvC-like nuclease domain include D10A (based on the S. pyogenes Cas9 protein). In some embodiments, the nickase may comprise an amino acid substitution in the HNH-like nuclease domain. Exemplary amino acid substitutions in the HNH-like nuclease domain include E762A, H840A, N863A, H983A, and D986A (based on the S. pyogenes Cas9 protein). In some embodiments, the nuclease system described herein may comprise a nickase and a pair of guide RNAs complementary to the sense and antisense strands of a target sequence, respectively.The guide RNA can direct the nickase to the target and introduce DSB by generating a nick on the opposite strand of the target sequence (i.e., double nicking). Chimeric Cas9 proteins can also be used, in which one domain or region of the protein is replaced by a part of a different protein. For example, the Cas9 nuclease domain can be replaced with a domain from a different nuclease, such as Fok1. The Cas9 protein can also be a modified nuclease.

[0254]

[0309] The wild-type Cas9 and Cas9 sequences from various species can be aligned to determine corresponding homologous amino acid residues, for example, the amino acid residues D10 and H840 of SEQ ID NO: 1 can be determined and / or modified to allow the generation of Cas9 variants with corresponding mutations of homologous amino acid residues.Alignment methods are known to those skilled in the art.For example, alignment can be performed using the NCBI Constraint-based Multiple Alignment Tool (COBALT, accessible at st-va.ncbi.nlm.nih.gov / tools / cobalt).

[0255]

[0310] In alternative embodiments, the Cas protein may be from a type I CRISPR / Cas system. In some embodiments, the Cas protein may be a component of a cascade complex of a type I CRISPR / Cas system. For example, the Cas protein may be a Cas3 protein. In some embodiments, the Cas protein may be from a type III CRISPR / Cas system. In some embodiments, the Cas protein may be from a type IV CRISPR / Cas system. In some embodiments, the Cas protein may be from a type V CRISPR / Cas system. In some embodiments, the Cas protein may be from a type VI CRISPR / Cas system. In some embodiments, the Cas protein may have RNA cleavage activity.

[0256] fusion proteins

[0311] Provided herein are 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. 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 in 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-guided programmable nuclease domain. In some embodiments, the nucleic acid-guided programmable nuclease may comprise at least one DNA-binding domain and at least one nuclease domain. In some embodiments, the nuclease domain may be heterologous to the DNA-binding domain. In some embodiments, the nuclease domain may be modified such that it is mutated to reduce its nuclease cleavage activity. In some embodiments, the nuclease activity is completely eliminated. In some embodiments, the 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 a 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 nickase domain.In some embodiments, the modified Cas9 domain comprises 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, or Argonaute protein domain.

[0257]

[0312] In some embodiments, the fusion proteins described herein contain one or more functional domains other than a nuclease domain. At least one protein domain can 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 a repressor domain, an activator domain, a methyltransferase domain, and a demethylase domain. In some embodiments, the functional domain comprises 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 cellular repair mechanisms to form a UA base pair, followed by a TA base pair, thereby creating a C→T substitution. Cytidine deaminase domains 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 an adenine deaminase domain. For example, an adenine deaminase domain can deaminate adenosine to generate inosine, which base pairs with cytidine and is subsequently modified to guanine by cellular repair mechanisms, thereby converting A to G. An exemplary adenosine deaminase fusion protein is described in Gaudelli et al., Nature 2017 551(7681):464-471, the entire contents of which are incorporated herein by reference.

[0258]

[0313] In some embodiments, the fusion proteins described herein contain a nuclear localization signal (NLS). In some embodiments, the fusion proteins may contain 2, 3, 4, or 5 NLSs. In some embodiments, the fusion proteins may contain 1 to 10 NLSs. The NLS sequence may be fused at the N-terminus and / or C-terminus of the fusion protein. In some embodiments, the NLS may be a single-part sequence, such as the SV40 NLS, PKKKRKV (SEQ ID NO: 3) or PKKKRRV (SEQ ID NO: 4). In some embodiments, the NLS may be a two-part sequence, such as the nucleoplasmin NLS, 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 contains the sequence of ABE7.10 (SEQ ID NO: 6).

[0259]

[0314] In some embodiments, the fusion protein may further comprise a tag domain. In some embodiments, the tag domain may comprise a fluorescent tag, a purification tag, an epitope tag, or a reporter gene tag. In some embodiments, the tag domain may comprise 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), and the like. Green, 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 In some embodiments, the tag domain may comprise a purification tag and / or an epitope tag.Non-limiting examples of 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, 6xHis (SEQ ID NO: 114), biotin carboxyl carrier protein (BCCP), and calmodulin. In some embodiments, the tag domain may comprise a reporter gene domain. Non-limiting exemplary reporter genes include glutathione-S-transferase (GST), horseradish peroxidase (HRP), chloramphenicol acetyltransferase (CAT), beta-galactosidase, beta-glucuronidase, luciferase, or fluorescent protein.

[0260]

[0315] 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.

[0261]

[0316] 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 can be selected from the LAGLIDADG family, the GIY-YIG family, the HNH family, the His-Cys box family, and the PD-(D / E)XK family. In some embodiments, the DNA-binding domain of a meganuclease can be engineered to recognize and bind to a sequence other than its cognate target sequence. In some embodiments, the DNA-binding domain of a meganuclease can be fused to a heterologous nuclease domain. In some embodiments, meganucleases, such as homing endonucleases, can be fused to TAL modules to create hybrid proteins, such as "megaTAL" proteins. MegaTAL proteins may have improved DNA target specificity by recognizing the target sequence of both the meganuclease DNA-binding domain and the TAL module.

[0262]

[0317] 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 the ZF repeats combine to recognize the DNA target sequence and provide sufficient affinity. Thus, engineered ZF repeats can be combined to recognize longer DNA sequences, such as 9 bp, 12 bp, 15 bp, or 18 bp. In some embodiments, ZFNs can contain a ZF fused to a nuclease domain derived from a restriction endonuclease. For example, the restriction endonuclease can be FokI. In some embodiments, the nuclease domain may include a dimerization domain, such as 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 half targets recognized by each ZF repeat on opposite strands of a DNA molecule, with an interconnecting sequence (sometimes referred to in the literature as a spacer) between them. For example, the interconnecting sequence may be 5-7 bp in length. Upon binding of both ZFNs in a pair, the nuclease domains may dimerize and introduce a DSB within the interconnecting sequence. In some embodiments, the dimerization domain of the nuclease domain may include a knob-into-hole motif to promote dimerization. For example, the ZFN may include a knob-into-hole motif in the dimerization domain of FokI.

[0263]

[0318] The DNA-binding domain of a TALEN typically contains a variable number of 34 or 35 amino acid repeats ("modules" or "TAL modules"), each of which binds to a single DNA base pair: A, T, G, or C. The adjacent residues at positions 12 and 13 of each module (the "repeated variable dinucleotides" or RVD) specify the single DNA base pair to which the module binds. While the 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 customization of DNA-binding domains to recognize specific target sequences. In some embodiments, a TALEN may contain a nuclease domain derived from a restriction endonuclease. For example, the restriction endonuclease may be FokI. In some embodiments, the nuclease domains may dimerize to become active, and a pair of TALENs may be designed to target a target sequence containing two half target sequences recognized by each DNA-binding domain on opposite strands of a DNA molecule with an interconnecting sequence between them. For example, each half-target sequence can be in the range of 10-20 bp, and the interconnecting sequence can be 12-19 bp in length. Upon binding of both TALENs of a pair, the nuclease domains can dimerize and introduce a DSB within the interconnecting sequence. In some embodiments, the dimerization domain of the nuclease domain can contain a knob-into-hole motif that promotes dimerization. For example, a TALEN can contain a knob-into-hole motif in the dimerization domain of FokI.

[0264]

[0319] Certain embodiments of the present disclosure also provide a nucleic acid encoding the nuclease system 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.

[0265]

[0320] In some embodiments, the nucleic acid encoding the nuclease can be codon optimized for efficient expression in one or more eukaryotic cell types. In some embodiments, the nucleic acid encoding the nuclease can be codon optimized for efficient expression in one or more mammalian cells. In some embodiments, the nucleic acid encoding the nuclease can be codon optimized for efficient expression in human cells. Methods of codon optimization, including codon usage tables and codon optimization algorithms, are available in the art.

[0266] Guide polynucleotide

[0321] In some embodiments of the present disclosure, the CRISPR / Cas nuclease system includes at least one guide polynucleotide, e.g., a guide RNA. In some embodiments, the guide RNA and the Cas protein may form a ribonucleoprotein (RNP), e.g., 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.

[0267]

[0322] The guide nucleic acid (e.g., guide RNA) can bind to the Cas protein and target the Cas protein to a specific location within the target polynucleotide. The guide nucleic acid may include a nucleic acid targeting segment and a Cas protein binding segment.

[0268]

[0323] A guide nucleic acid may refer to a nucleic acid that can hybridize to another nucleic acid, for example, a target polynucleotide in the genome of a cell. A guide nucleic acid may be RNA, for example, a guide RNA. A guide nucleic acid may be DNA. A guide nucleic acid may include DNA and RNA. A guide nucleic acid may be single-stranded. A guide nucleic acid may be double-stranded. A guide nucleic acid may include nucleotide analogs. A guide nucleic acid may include modified nucleotides. A guide nucleic acid may be programmed or designed to bind to a nucleic acid sequence in a site-specific manner.

[0269]

[0324] The guide nucleic acid may contain one or more modifications to provide the nucleic acid with new or improved characteristics. The guide nucleic acid may include a nucleic acid affinity tag. The guide nucleic acid may include synthetic nucleotides, synthetic nucleotide analogs, nucleotide derivatives, and / or modified nucleotides.

[0270]

[0325] The guide nucleic acid may include a nucleic acid target region (e.g., a spacer region) complementary to a 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 through hybridization (base pairing). The protospacer sequence may be located 5' or 3' of the protospacer adjacent motif (PAM) of the target polynucleotide. The nucleotide sequence of the spacer region may vary and determine the location within the target nucleic acid with 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.

[0271]

[0326] A guide nucleic acid may comprise two separate nucleic acid molecules, which may be referred to as a dual guide nucleic acid. A guide nucleic acid may comprise a single nucleic acid molecule, which may be referred to as a single guide nucleic acid (e.g., sgRNA). In some embodiments, a guide nucleic acid is a single guide nucleic acid comprising a fused CRISPR RNA (crRNA) and a trans-activating crRNA (tracrRNA). In some embodiments, a guide nucleic acid is a single guide nucleic acid comprising a crRNA. In some embodiments, a guide nucleic acid is a single guide nucleic acid comprising a crRNA but lacking a tracrRNA. In some embodiments, a 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.

[0272]

[0327] The crRNA may include a nucleic acid target segment (e.g., a spacer region) of the guide nucleic acid and a stretch of nucleotides that can form one half of a double-stranded duplex of the Cas protein binding segment of the guide nucleic acid.

[0273]

[0328] The tracrRNA may comprise a stretch of nucleotides that forms the other half of the double-stranded duplex of the Cas protein-binding segment of the gRNA. The stretch of nucleotides of the crRNA is complementary to and can hybridize with the stretch of nucleotides of the tracrRNA to form the double-stranded duplex of the Cas protein-binding domain of the guide nucleic acid.

[0274]

[0329] The crRNA and tracrRNA can hybridize to form a guide nucleic acid. The 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, including the spacer region, can be designed to be specific for the species in which the guide nucleic acid is used.

[0275]

[0330] Guide RNAs in the CRISPR / Cas9 system typically include CRISPR RNA (crRNA) and tracr RNA (tracr). Guide RNAs in 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 resemble the structure of naturally occurring crRNA transcribed from bacterial CRISPR loci, in which the targeting sequence acts as a spacer in the CRISPR / Cas9 system.

[0276]

[0331] The guide RNA can target any sequence of interest 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 can 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 can be 100% complementary. In other embodiments, the targeting sequence of the guide RNA and the target sequence on the target nucleic acid molecule can contain at least one mismatch. For example, the targeting sequence of the guide RNA and the target sequence on the target nucleic acid molecule can 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 can 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.

[0277]

[0332] The length of the targeting sequence may depend on the CRISPR / Cas9 system and components used. For example, different Cas9 proteins from different bacterial species have various optimal targeting sequence lengths. Thus, the targeting sequence may comprise 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 nucleotides in length. In some embodiments, the targeting sequence may comprise 18-30 nucleotides in length. In some embodiments, the targeting sequence may comprise 19-24 nucleotides in length. In some embodiments, the targeting sequence may comprise 20 nucleotides in length.

[0278]

[0333] The crRNA and tracr may comprise any sequences that are sufficiently complementary to promote the formation of a functional CRISPR / Cas9 complex. In some embodiments, the complementary sequence between the crRNA and tracr may comprise all or a portion of the sequence of a naturally occurring crRNA that is complementary to the tracr RNA in the same CRISPR / Cas9 system (also referred to as a "tag" or "handle"). In some embodiments, the complementary sequence may comprise all or a portion of a repeat sequence from a naturally occurring CRISPR / Cas9 system. In some embodiments, the complementary sequence may comprise a truncated or modified tag or handle sequence. In some embodiments, the degree of complementarity between the tracr RNA and the portion of the complementary portion that hybridizes to the tracr RNA along the length of the shorter of the two sequences may be about 40%, 50%, 60%, 70%, 80%, or more, but less than 100%. In some embodiments, due to the presence of one or more bulge structures above tracr and / or wobble base pairing, the tracr RNA and the portion hybridizing to the tracr RNA are not 100% complementary along the length of 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 comprise 10 to 50 nucleotides, or more than 50 nucleotides. In some embodiments, the complementary portion may comprise 15 to 40 nucleotides in length. In other embodiments, the complementary portion may comprise 20 to 30 nucleotides in length. In still other embodiments, the complementary portion may comprise 22 nucleotides in length. For example, when using dual guide RNAs, there may be no upper limit to the length of the complementary portion.

[0279]

[0334] In some embodiments, the tracr RNA may comprise all or a portion of the wild-type tracr RNA sequence 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 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 nucleotides in length. In certain embodiments, the tracr RNA is at least 26 nucleotides in length. In additional embodiments, the tracr RNA is at least 40 nucleotides in length. In some embodiments, the tracr RNA may comprise a specific secondary structure, such as, for example, one or more hairpin or stem-loop structures, or one or more bulges.

[0280]

[0335] In some embodiments, a guide RNA may comprise two RNA molecules, referred to herein as a "dual guide RNA" or "dgRNA." In some embodiments, a dgRNA may comprise a first RNA molecule comprising a crRNA and a second RNA molecule comprising a tracrRNA. The first and second RNA molecules may form an RNA duplex via base pairing between a flagpole on the crRNA and the tracrRNA.

[0281]

[0336] In some embodiments, the guide RNA may comprise a single RNA molecule, referred to herein as a "single guide RNA" or "sgRNA." In some embodiments, the sgRNA may comprise a crRNA covalently linked to a tracr RNA. In some embodiments, the crRNA and tracr RNA may be covalently linked via a linker. In some embodiments, the single-molecule guide RNA may comprise a stem-loop structure via base pairing between a flagpole on the crRNA and the tracr RNA.

[0282]

[0337] Certain embodiments of the present disclosure also provide nucleic acids, e.g., vectors, encoding the guide RNAs 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 comprise a nucleotide sequence encoding a crRNA. In some embodiments, the nucleotide sequence encoding the crRNA comprises a targeting sequence flanked by all or part of repeat sequences from a naturally occurring CRISPR / Cas system. In some embodiments, the nucleic acid may comprise a nucleotide sequence encoding a tracr RNA. In some embodiments, the crRNA and tracr RNA may be encoded by two separate nucleic acids. In some embodiments, the crRNA and tracr RNA may be encoded by a single nucleic acid. In some embodiments, the crRNA and tracr RNA may be encoded by opposite strands of a single nucleic acid. In other embodiments, the crRNA and tracr RNA may be encoded by the same strand of a single nucleic acid.

[0283]

[0338] In certain embodiments, two or more guide RNAs may be used with a CRISPR / Cas nuclease system. Each guide RNA may contain a different targeting sequence, such 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 a different vector. The promoters used to drive expression of two or more guide RNAs may be the same or different.

[0284]

[0339] The method of selecting guide RNA for efficient targeting with high specificity and low off-target effect is known to those skilled in the art.For programmable base editing, the selection of genome sequence containing target sequence can be as described in Komor et al., Nature, 533, 420-424 (2016), which is incorporated herein by reference.The preference of guide RNA sequence and PAM defines the genome target sequence of programmable nuclease domain (for example, Cas9, dCas9, Cas9n, Cpfl, NgAgo domain). 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 Biol. 2016, 17:148) are incorporated herein by reference. The possibility of bulge formation between the guide RNA and the target DNA, as well as other parameters that may affect target sequence binding, may also be considered, as described by Bae et al. (Bioinformatics, 2014, 30, 1473-5), and Housden et al. (Science Signaling, 2015, 8(393):rs9) and Farboud et al. (Genetics, 2015, 199(4):959-71), which are also incorporated herein by reference.

[0285] RNA modification

[0340] Provided herein are modified RNA molecules suitable for targeted ex vivo and in vivo delivery systems. Modified RNA molecules can contain two or more linked ribonucleic acid subunits. Non-limiting examples of modified RNA include CRISPR guide RNA, short interfering RNA (siRNA), microRNA (miRNA), short hairpin RNA (shRNA), small nuclear RNA (snRNA), messenger RNA (mRNA), precursor mRNA (pre-mRNA), antisense RNA (asRNA), and heteronuclear RNA (hnRNA). 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 can comprise a length of at least about 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100, 200, 300, 400, or 500 bases. The modified RNA may comprise a length of at least about 1 kilobase (kb), 2 kb, 3 kb, 4 kb, 5 kb, 10 kb, 20 kb, 50 kb, or more. In some embodiments, the modified RNA is a CRISPR guide RNA (gRNA). The gRNA may be a single guide RNA or a dual guide RNA. In some embodiments, the modified RNA is an mRNA. In some embodiments, the mRNA may be isolated from a cell or tissue. In some embodiments, the mRNA may be transcribed from DNA. In some embodiments, the mRNA may be chemically synthesized.

[0286]

[0341] In certain embodiments, the modified RNA molecules provided herein are resistant to degradation by RNase 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., TLR, RIG-I) compared to unmodified RNA. RNA modifications are as described 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 in their entirety.

[0287]

[0342] In addition to "unmodified" or "natural" nucleobases, such as the purine nucleobases adenine (A) and guanine (G) and the pyrimidine nucleobases thymine (T), cytosine (C), and uracil (U), many modified nucleobases or nucleobase mimics known to those skilled in the art are suitable for use in the compounds described herein. Unmodified or natural nucleobases may be modified or replaced to provide oligonucleotides with improved properties. For example, nuclease-resistant oligonucleotides may be prepared using these bases, or using synthetic and natural nucleobases (e.g., inosine, xanthine, hypoxanthine, nubularine, isoguanisine, or tubercidin) and any one of the oligomer 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 with an unnatural and / or universal base, the nucleotide is said herein to contain a modified nucleobase and / or nucleobase modification. Modified nucleobases and / or nucleobase modifications also include natural, unnatural, and universal bases, including conjugate moieties, such as the ligands described herein. Preferred conjugate moieties for conjugation to nucleobases include cationic amino groups, which can be conjugated to the nucleobase via a linker having a suitable alkyl, alkenyl, or amide bond.

[0288]

[0343] As used herein, "unmodified" or "natural" nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U).Exemplary modified nucleobases include, but are not limited to, other synthetic and natural nucleobases, such as inosine, xanthine, hypoxanthine, nuvalalin, isoguanisine, tubercidin, 2-(halo)adenine, 2-(alkyl)adenine, 2-(propyl)adenine, 2-(amino)adenine, 2-(aminoalkyl)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-(adenine (alkyl)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-(guanidiniumaralkyl)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-(methoxycarbonylmethyl)uracil, 5-(propynyl)uracil, 5-(propynyl)uracil, 5-(trifluoromethyl)uracil, 6-(azo)uracil, dihydrouracil, N-(methyl)uracil, 5-uracil (i.e., pseudouracil), 2-(thio)pseudouracil, 4-(thio)pseudouracil, 2,4-(dithio)pseudouracil Uracil, 5-(alkyl)pseudouracil, 5-(methyl)pseudouracil, 5-(alkyl)-2-(thio)pseudouracil, 5-(methyl)-2-(thio)pseudouracil, 5-(alkyl)-4-(thio)pseudouracil, 5-(methyl)-4-(thio)pseudouracil, 5-(alkyl)-2,4-(dithio)pseudouracil, 5-(methyl)-2,4-(dithio)pseudouracil, 1-methylpseudouracil (N1-methylpseudouracil), 1-substituted pseudouracil, 1-substituted 2(thio)-pseudouracil, 1- substituted 4-(thio)pseudouracil, 1-substituted 2,4-(dithio)pseudouracil, 1-(aminocarbonylethylenyl)-pseudouracil, 1-(aminocarbonylethylenyl)-2(thio)-pseudouracil, 1-(aminocarbonylethylenyl)-4-(thio)pseudouracil, 1-(aminocarbonylethylenyl)-2,4-(dithio)pseudouracil, 1-(aminoalkylaminocarbonylethylenyl)-pseudouracil, 1-(aminoalkylamino-carbonylethylenyl)-2(thio)-pseudouracil,1-(aminoalkylaminocarbonylethylenyl)-4-(thio)pseudouracil, 1-(aminoalkylaminocarbonylethylenyl)-2,4-(dithio)pseudouracil, 1,3-(diaza)-2-(oxo)-phenoxazin-1-yl, 1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl, 1,3-(diaza)-2-(oxo)-phenthiazin-1-yl, 1-(aza)-2-(thio)-3-(aza)-phenthiazin-1-yl, 7-substituted 1,3-(diaza)-2-(oxo)-phenoxazin-1-yl 7-substituted-1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl, 7-substituted 1,3-(diaza)-2-(oxo)-phenthiazin-1-yl, 7-substituted 1-(aza)-2-(thio)-3-(aza)-phenthiazin-1-yl, 7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl, 7-(aminoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl, 7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl phenthiazin-1-yl, 7-(aminoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenthiazin-1-yl, 7-(guanidiniumaralkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl, 7-(guanidiniumaralkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl, 7-(guanidiniumaralkylhydroxy)-1,3-(diaza)-2-(oxo)-phenthiazin-1-yl, 7-(guanidiniumaralkylhydroxy)-1-(aza)-2- (thio)-3-(aza)-phenthiazin-1-yl, 1,3,5-(triaza)-2,6-(dioxa)-naphthalene, inosine, xanthine, hypoxanthine, nubularine, tubercidin, isoguanisine, inosinyl, 2-aza-inosinyl, 7-deaza-inosinyl, nitroimidazolyl, nitropyrazolyl, nitrobenzimidazolyl, nitroindazolyl, aminoindolyl, pyrrolopyrimidinyl, 3-(methyl)isocarbostyrilyl, 5-(methyl)isocarbostyrilyl, 3-(methyl)-7-(propynyl)isocarbostyrilyl,7-(aza)indolyl, 6-(methyl)-7-(aza)indolyl, imidizopyridinyl, 9-(methyl)-imidizopyridinyl, pyrrolopyridinyl, isocarbostyrilyl, 7-(propynyl)isocarbostyrilyl, 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, pyrimidine para-substituted-6-phenyl-pyrrolo-pyrimidin-2-one-3-yl, ori / zo-substituted-6-phenyl-pyrrolo-pyrimidin-2-one-3-yl, bis-ori / zo-substituted-6-phenyl-pyrrolo-pyrimidin-2-one-3-yl, para-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-one-3-yl , ori / zo-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-one-3-yl, bis-ori / zo-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-one-3-yl, pyridopyrimidin-3-yl, 2-oxo-7-amino-pyridopyrimidin-3-yl, 2-oxo-pyridopyrimidin-3-yl, or any O- or N-alkylated derivative thereof. Alternatively, substituted or modified analogs of any of the above bases and "universal bases" may be used. A universal nucleobase is any nucleobase that can base pair with all four naturally occurring nucleobases without substantially affecting the melting behavior, recognition by intracellular enzymes, or activity of the oligonucleotide duplex. Some exemplary universal nucleobases include, but are not limited to:2,4-Difluorotoluene, Nitropyrrolyl, Nitroindolyl, 8-Aza-7-deazadenine, 4-Fluoro-6-methylbenzimidazuryl, 4-Methylbenzimidazuryl, 3-Methylisocarbostyrilyl, 5-Methylisocarbostyrilyl, 3-Methyl-7-propynylisocarbostyrilyl, 7-Azaindolyl, 6-Methyl-7-azaindolyl, Imidizopyridinyl, 9-Methyl-imidizopyridinyl, Pyrrolopyridinyl, Imidizopyridinyl Isocarbostyrilyl, 7-propynylisocarbostyrilyl, propynyl-7-azaindolyl, 2,4,5-trimethylphenyl, 4-methylinolyl, 4,6-dimethylindolyl, phenyl, naphthalenyl, anthracenyl, phenanthracenyl, pyrenyl, stilbenyl, tetracenyl, pentacenyl, and structural derivatives thereof (see, e.g., Loakes, 2001, Nucleic Acids Research, 29, 2437-2447, which is incorporated herein by reference in its entirety). Further nucleobases include those disclosed in U.S. Pat. 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, pages 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, pages 289-302, edited by Crooke, ST and Lebleu, B., CRC Press, 1993. All of the above are incorporated herein by reference.

[0289]

[0344] In some embodiments, the modified RNAs described herein are modified to attach a delivery moiety and / or a targeting moiety, such as GalNAc. Suitably, GalNAc may be attached to the 3' end, 5' end, or both of the RNA. In some embodiments, GalNAc is attached to the 3' end. In some embodiments, the modified RNAs exhibit improvements compared to their unmodified counterparts. 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 immunogenic or immunostimulatory properties. In some embodiments, the modified RNAs exhibit improved properties that enable efficient transfection into cells and / or delivery and maintenance of the RNA within an organism, tissue, body fluid, or cell, such that the function of the RNA, e.g., guide RNA, can occur. Methods for measuring these improved properties compared to their unmodified counterparts are known to those of skill in the art and include methods described herein. Accordingly, in some embodiments, modified RNAs are provided herein that exhibit increased stability compared to their unmodified counterparts. An unmodified equivalent refers to an RNA, e.g., a guide RNA, that targets the same specific gene sequence, interacts with the same Cas9 or CRISPR nuclease, and contains natural nucleotides. Improved stability includes improved stability or resistance to enzymes, such as nucleases, that may be present in cells, tissues, or bodily fluids and that may otherwise contribute to the degradation of RNA, resulting in reduced functionality. In certain embodiments, increased stability includes increased serum stability. In some embodiments, provided herein are modified guide RNAs with increased CRISPR activity compared to their unmodified counterparts. Methods for measuring CRISPR activity are described herein. In some embodiments, provided herein are modified guide RNAs with reduced immunostimulatory activity compared to their unmodified counterparts. Methods for measuring immunostimulation are described herein.

[0290]

[0345] Provided herein are modified mRNA molecules for targeted delivery. For example, mRNA encoding CRISPR enzymes, such as Cas9, Cas12b, or base editors (BEs), may be modified to target specific tissues. The mRNA may have at least one nucleotide modified at the 2' position and / or at the backbone modification. In some embodiments, the nucleotide in the mRNA may include a thioate modification. In some embodiments, the mRNA may include one or more modifications of 2'-OMe, 2'-F, N-1-methyl-pseudouridine, 5-methyluridine, 5-methoxyuridine, and 5-ethoxyuridine.

[0291]

[0346] In certain embodiments, the mRNA sequences provided herein include fully modified or partially modified mRNA. In some embodiments, the mRNA contains chemical modifications in fragments or multiple fragments of the full length. Non-limiting exemplary modifications and modification patterns of mRNA nucleotides or segments thereof are shown in Tables 2 and 3.

[0292]

[0347] Provided herein are modified guide RNAs for use in CRISPR / Cas systems, which can be modified by chemical and / or backbone modification of at least one nucleotide at the 2' position. The backbone modification can include a thioate modification. In certain embodiments, the modified nucleotide is selected from a group of nucleotides that interact with a Cas amino acid in a Cas protein to result in binding of the guide RNA to Cas. In certain embodiments, the modification may include one in which the 2'-OH on the nucleotide is replaced with at least one of H, -OR, -R, -O-Ci-C6-alkylene-OR, -O-Ci-C6-alkylene-OH, halo, -SH, -SR, -NH, -NHR, -N(R), -Ci-C6-alkylene-NH, -Ci-C6-alkylene-NHR, -Ci-C6-alkylene-N(R), or CN, where each R is independently Ci-C6 alkyl, C2-C6 alkenyl, or C2-C6 alkynyl and halo is F, CI, Br, or I. In some instances, the modification is 2'-O-methyl and / or 2'-F. In some embodiments, the modification includes one or more of 2'-F, phosphorothioate internucleotide linkage modifications, acyclic nucleotides, LNA, HNA, CeNA, 2'-methoxyethyl, 2'-O-methyl, 2'-O-allyl, 2'-C-allyl, 2'-deoxy, 2'-ON-methylacetamido (2'-O-NMA), 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE), 2'-O-aminopropyl (2'-O-AP), and 2'-ara-F modifications. In some embodiments, the modification includes 2'-MOE. In some embodiments, the modification includes a phosphorothioate internucleotide linkage modification. In some embodiments, the modification includes 4-O-alkylribosugars, such as 4'-methoxy and 4'-ethoxy modifications.

[0293]

[0348] Preferably, the modified guide RNA can be compatible with the CRISPR / Cas system of S. pyogenes or any other CRISPR / Cas system, such as the RNA found in Staphylococcus aureus or Staphylococcus haemolyticus. This modification or a similar modification pattern may also be performed to guide the RNA of Cpf1 from Lachnospiraceae bacterium ND2006 or Cpf1 from Acidominococcus species BV3L6.

[0294]

[0349] In certain embodiments, the guide RNA sequence comprises a fully modified single guide RNA. In some embodiments, the guide RNA comprises a chemical modification in the tracr RNA portion. Non-limiting exemplary modifications and modification patterns of nucleotides in guide RNAs according to the present disclosure are shown in Tables 2 and 3.

[0295]

[0350] The modified guide RNAs described herein can be used in a CRISPR / Cas system or complex with a CRISPR / Cas enzyme to introduce changes into target genes or DNA sequences. The CRISPR / Cas enzyme can include a CRISPR nuclease such as Cas9, Cpf1, C2c1, C2c2, or C2c3. In some embodiments, the CRISPR / Cas enzyme can 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 generate a Cas9 nickase or a nuclease-inactive Cas9. Exemplary inactivating mutations in Cas9 include changes at positions D10, E762, H840, N854, N863, or D986 of 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 inactivity. The D10A mutation in the RuvC subdomain or the H840A mutation in the HNH subdomain of Cas9 generates a Cas9 nickase. Additional amino acid substitutions in Cas9 are discussed in WO 15 / 89354, which is incorporated herein in its entirety.

[0296]

[0351] The modified guide RNA may share sequence identity with or hybridize to 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 to the target nucleotide of the gene or target DNA.

[0297]

[0352] The nucleotides described herein may be synthetic or chemically modified. For example, the guide RNAs provided herein may be synthetic or chemically modified guide RNAs. The modified nucleotides in the guide RNA may correspond to one or more nucleotides in the binding region between the guide RNA and Cas9 and / or the nucleotides in the binding region between the guide RNA and target DNA. The remaining unmodified nucleotides in the guide RNA may be nucleotides that must be specified to minimize Cas9 binding to the 2'-OH position of the base. In some embodiments, the nucleotide 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 with a group selected from H, OR, R, halo, SH, SR, H2, NHR, N(R)2, or CN, where R is C1-C6 alkyl, alkenyl, or alkynyl, and halo is F, Cl, Br, or I. Other modifications include inverted (deoxy) abasic, amino, fluoro, chloro, bromo, CN, CF, methoxy, imidazole, carboxylate, thioate, C1 to C10 (CI to CIO) lower alkyl, substituted lower alkyl, alkaryl or aralkyl, heterocycloalkyl; heterocycloalkaryl; aminoalkylamino; polyalkylamino, or substituted silyl. Methods for generating 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, the entire contents of which are incorporated herein by reference.

[0298]

[0353] 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 the sequence and modification. 2'-O-methyl modification has been shown to increase the effectiveness or efficiency of coupling during RNA synthesis, thus increasing the yield of chemically synthesized RNA. Additionally, nucleotides may be modified with phosphorothioates. Phosphothioate (PS) bonds replace the non-bridging oxygen of the phosphate backbone of an oligonucleotide with a sulfur atom. Thus, exemplary nucleotides of the present 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, e.g., LNA with a β-D-ribo configuration, a-LNA (a diastereomer of LNA) with an α-L-ribo configuration, 2′-amino-LNA with a 2′-amino functionalization, and 2′-amino-a-LNA with a 2′-amino functionalization), or hybrids thereof.

[0299] Conjugates for targeted delivery

[0354] Provided herein are conjugates suitable for targeted delivery of agents such as mRNA, guide RNA, miRNA, siRNA, DNA, peptides, or other small molecules or macromolecules. The conjugates may include one or more aptamers, ligands, or moieties for targeted delivery ex vivo or in vivo. In some embodiments, the conjugates include a targeting moiety (or ligand), a linker, and an active agent (or payload) connected to the targeting moiety. The active agent may be a therapeutic agent, a preventive agent, or a diagnostic / prognostic agent. The active agent may have the ability to manipulate physiological functions (e.g., gene expression) in a subject. The active agent may be a guide RNA, mRNA, miRNA, siRNA, DNA, or peptide. The active agent may be linked to the targeting moiety via a linker, a non-covalent bond, nucleobase pairing, or any combination thereof. In some embodiments, the conjugate may be a conjugate between a single active agent and a single targeting moiety having the formula (I): XYZ, where X is a targeting moiety; Y is a linker; and Z is a guide RNA. In certain embodiments, one targeting ligand may be conjugated to two or more active agents, and the conjugate has the formula: X-(YZ)n. For example, the conjugate may include a guide RNA and an mRNA. In certain embodiments, one active agent may be linked to two or more targeting ligands, and the conjugate has the formula: (XY)nZ. In other embodiments, one or more targeting moieties may be linked to one or more active payloads, whose conjugate formula 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 a targeting moiety; Y is a linker; and Z is an active agent, such as a guide RNA. The number of each moiety in the conjugate can vary depending on the type of drug, the size of the conjugate, the delivery target, the particles used to package the conjugate, other active agents (e.g., immunological adjuvants), and the route of administration.Each occurrence of X, Y, and Z can be the same or different, e.g., a conjugate can include multiple types of targeting moieties, multiple types of linkers, and / or multiple types of active agents, where n is an integer greater than or equal to 1. 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 can 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.

[0300]

[0355] In some embodiments, active agents, such as guide RNA, can be delivered to cells and tissues using viruses, polymers and liposome preparations, cell-penetrating peptides, aptamers, ligands, or conjugates and antibody approaches. A moiety or ligand can direct guide RNA to a specific organ, tissue, or cell, such as hepatocytes in the liver, and can be referred to as a targeting moiety. In some embodiments, a targeting moiety modifies one or more properties of the attached molecule (e.g., mRNA or guide RNA), including, but not limited to, pharmacodynamics, pharmacokinetics, binding, absorption, cellular distribution, cellular uptake, charge, and clearance.

[0301]

[0356] Exemplary moieties that may be attached to the active agents described herein include, but are not limited to, intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, thioethers, polyethers, cholesterol, thiocholesterol, cholate moieties, folate, lipids, phospholipids, biotin, phenazine, phenanthridine, anthraquinone, adamantane, acridine, fluorescein, rhodamine, coumarin, dyes, 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-tritylthiol (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-1,2-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); adamantaneacetic acid (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 moieties (Crooke et al.,J. Pharmacol. Exp. Ther., 1996, 277, 923 (all citations are incorporated herein by reference in their entirety). Targeting moieties include naturally occurring or recombinant or synthetic molecules, including, but not limited to, GalNAc or a derivative thereof (e.g., a dimer, trimer, or tetramer of GalNAc or a derivative thereof), polylysine (PLL), poly-L-aspartic acid, poly-L-glutamic acid, styrene-maleic anhydride copolymer, poly(L-lactide-co-glycosylated) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamido copolymer (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, polyphosphazine, polyethyleneimine, cationic group, spermine, spermidine, polyamine, pseudopeptide-polyamine, peptidomimetic polyamine, dextran Drimer polyamines, arginine, amidine, protamine, cationic lipids, cationic porphyrins, quaternary salts of polyamines, thyrotropin, melanotropin, lectins, glycoproteins, surfactant protein A, mucins, glycosylated polyamino acids, transferrin, bisphosphonates, polyglutamic acid, polyaspartic acid, aptamers, asialofetuin, hyaluronic acid, procollagen, immunoglobulins (e.g., antibodies), insulin, transferrin, albumin, glycoalbumin conjugates esters, intercalating agents (e.g., acridine), crosslinking agents (e.g., psoralen, mitomycin C), porphyrins (e.g., TPPC4, texaphyrin, sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g., EDTA), lipophilic molecules (e.g., steroids, bile acids, cholesterol, cholic acid, adamantaneacetic acid, 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)cholenoic acid, dimethoxytrityl, or phenoxazine), peptides (e.g., α-helical peptides, amphipathic peptides, RGD peptides, cell-penetrating peptides, endosomolytic / fusogenic peptides), alkylating agents, phosphate, amino, mercapto, polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes, haptens (e.g., biotin), transport / absorption promoters accelerators (e.g., naproxen, aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, bisimidazole, histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+ complexes of tetraazamacrocycles), dinitrophenyl, HRP, AP, antibodies, hormones and hormone receptors, lectins, carbohydrates, polyvalent 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 Activators of MAP kinases, activators of NF-κB, taxon, vincristine, vinblastine, cytochalasin, nocodazole, japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, myoservin, 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-penetrating agents (e.g., helical cell-penetrating agents), peptides and peptidomimetics. peptides, e.g., 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 a peptide bond is replaced by one or more urea, thiourea, carbamate, or sulfonylurea bonds; or cyclic peptides; amphipathic peptides, such as, but not limited to, cecropins, lycotoxins, paradaxins, buforins, CPFs, bombinin-like peptides (BLPs), cathelicidins,Examples include ceratotoxin, S. clava peptide, hagfish intestinal antimicrobial peptide (HFIAP), magainin, brevinin-2, dermaseptin, melittin, pleurocidin, H2A peptide, Xenopus laevis peptide, esculentinis-1, and caerin. Peptidomimetics (also referred to herein as oligopeptidomimetics) are molecules that can fold into defined three-dimensional structures similar to natural peptides. Peptide or peptidomimetic ligands or moieties can be about 5-50 amino acids in length, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids in length. In some embodiments, the targeting moiety may be other peptides, e.g., somatostatin, octeotide, LHRH (luteinizing hormone-releasing hormone), epidermal growth factor receptor (EGFR)-binding peptides, aptides or bidentate peptides, RGD-containing peptides, protein scaffolds, e.g., fibronectin domains, single-domain antibodies, stable scFvs, or other homing peptides. As non-limiting examples, protein- or peptide-based targeting moieties may be proteins, e.g., thrombospondin, tumor necrosis factor (TNF), annexin V, interferons, 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, It may be an antibody fragment, an RGD peptide, folate, 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 (dAbs), nanobodies, single-chain variable fragments (scFvs), antigen-binding fragments (Fabs), avibodies, minibodies, CH2D domains, Fcabs, and bispecific T-cell engager (BiTE) molecules. In some embodiments, the scFv is a stable scFv, wherein the scFv has ultra-stable properties. In some embodiments,Nanobodies can be derived from camelid antibody single variable domains (VHHs).

[0302]

[0357] In some embodiments, the targeting moiety recognizes or binds to a target cell, a marker, or a molecule that is present exclusively or predominantly on the surface of a particular cell. For example, the targeting moiety may bind to a tumor antigen and direct an activator, such as a guide RNA-Cas complex, to malignant cells. In some embodiments, the targeting moiety recognizes an intracellular protein. In some embodiments, the targeting moiety directs the conjugate to a particular tissue, cell, or subcellular location. The targeting moiety may direct the conjugate to culture or to a whole organism, or both. In either case, the targeting moiety may bind to a receptor present on the surface or inside the target cell, and the targeting moiety binds to the receptor with effective specificity, affinity, and avidity. In other embodiments, the targeting moiety targets the conjugate to a particular tissue, such as the liver, kidney, lung, or pancreas. In other cases, the targeting moiety may direct the conjugate to cells of the reticuloendothelium or lymphatic system, or to professional phagocytes, such as macrophages or eosinophils. In some embodiments, the targeting moiety may recognize an RTK receptor, an EGF receptor, a serine or threonine kinase, a G protein-coupled receptor, a methyl-CpG binding protein, a cell surface glycoprotein, a cancer stem cell antigen or marker, carbonic anhydrase, a cytolytic T lymphocyte antigen, a DNA methyltransferase, an exoenzyme, a glycosylphosphatidylinositol-anchored co-receptor, a glypican-related integral membrane proteoglycan, a heat shock protein, a hypoxia-inducible protein, a multidrug resistance transporter, a tumor-associated macrophage marker, a tumor-associated carbohydrate antigen, a TNF receptor family member, a transmembrane protein, a tumor necrosis factor receptor superfamily member, a tumor differentiation antigen, a zinc-dependent metalloexopeptidase, a zinc transporter, a sodium-dependent transmembrane transport protein, a member of the SIGLEC family of lectins, or a matrix metalloproteinase.

[0303]

[0358] In some embodiments, a conjugate described herein, e.g., a 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 moiety comprises at least one N-acetyl-galactosamine (GalNAc), N-Ac-glucosamine (GluNAc), or mannose (e.g., mannose-6-phosphate).

[0304]

[0359] In some embodiments, the conjugates described herein comprise one or more targeting moieties comprising N-acetylgalactosamine (GalNAc) or GalNAc derivatives. Such conjugates are also referred to herein as GalNAc conjugates. In some embodiments, the conjugates target RNA to specific cells, such as liver cells, e.g., hepatocytes. In some embodiments, the GalNAc derivatives can be linked via a linker, such as a bivalent or trivalent branched linker.

[0305]

[0360] In some embodiments, the conjugates described herein are carbohydrate conjugates. In some embodiments, the carbohydrate conjugates comprise 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, a lectin primarily expressed in hepatocytes in the liver.

[0306]

[0361] 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 the guide RNA to specific cells. In some embodiments, the GalNAc conjugate targets the guide RNA to liver cells (e.g., hepatocytes), for example, by serving as a ligand for the asialoglycoprotein receptor of liver cells (e.g., hepatocytes). In some embodiments, the carbohydrate conjugate comprises one or more GalNAc derivatives. The GalNAc derivatives can be attached via a linker, e.g., a bivalent 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 active agent (e.g., the 3' end of the guide RNA) via a linker, e.g., a linker described herein. In some other embodiments, the GalNAc ligand is conjugated to the active agent (e.g., to the 5' end of the guide RNA) via a linker, e.g., a linker described herein.

[0307]

[0362] In some embodiments, GalNAc ligands can be conjugated to shorter oligonucleotides via linkers and spacers, where the shorter oligonucleotide conjugates are complementary to RNA segments.RNA encompasses all lengths, structures, and forms of RNA molecules, including, for example, target mRNA and target guide RNA.In some embodiments, shortmer-GalNAc conjugates and RNA constitute pharmaceutical compositions.For example, shorter GalNAc-linked oligonucleotides and RNA, such as coupling sequences, can be combined to form pharmaceutical compositions via complementary nucleotide WCH bonds.Shorter oligonucleotide conjugates can include 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 nucleotides in length. In some embodiments, the coupling sequence may comprise 15 to 40 nucleotides in length. In some embodiments, the coupling sequence may comprise 19 to 30 nucleotides in length. In some embodiments, the coupling sequence may comprise 20 to 24 nucleotides in length.

[0308]

[0363] In some embodiments, provided herein is a pharmaceutical composition comprising one or more GalNAc-conjugated shortmer oligonucleotides and one or more RNAs. In some embodiments, a single (single) GalNAc-conjugated shortmer oligonucleotide, such as a GalNAc-conjugated RNA, can be complementary to multiple oligonucleotide segments within the RNA. For example, a single (single) GalNAc-conjugated shortmer may contain coupling sequences complementary to multiple segments within the RNA. In some embodiments, multiple GalNAc-conjugated shortmer oligonucleotides complementary to multiple oligonucleotide segments within the RNA can constitute a pharmaceutical composition.

[0309]

[0364] In certain embodiments, the targeting moiety of the conjugates described herein comprises a ligand having the structure shown in Table 1 below.

[0310] [Table 1-1]

[0311] [Table 1-2]

[0312] [Table 1-3]

[0313] [Table 1-4]

[0314] [Table 1-5]

[0315] [Table 1-6]

[0316] [Table 1-7]

[0317] [Table 1-8]

[0318] [Table 1-9]

[0319]

[0365] 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. Thus, it should be understood that in some embodiments of the compounds in Table 1, t is contemplated herein to be 0 to 10. In some embodiments, t is 1 to 5. In some embodiments, t is 10-20. 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 of Table 1, m is 0-10. In some embodiments, m is 1-5. In some embodiments, m is 10-20. 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 of Table 1, n is 0-10. In some embodiments, n is 1-5. In some embodiments, n is 10-20. 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 of Table 1, p is 0-10. In some embodiments, p is 1-5. In some embodiments, p is 10-20. 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 of Table 1, q is 0-10. 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 or 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) in 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.

[0320]

[0366] Targeting moiety can be conjugated to nucleic acid base, sugar moiety, or internucleoside bond of nucleic acid, for example, guide RNA or mRNA.Conjugation to purine nucleic acid base or its derivative can occur at any position, including endocyclic and exocyclic atoms.In some embodiments, the 2nd, 6th, 7th, or 8th position of purine nucleic acid base is bound to a moiety.Conjugation to pyrimidine nucleic acid base or its derivative can also occur at any position.In some embodiments, the 2nd, 5th, and 6th positions of pyrimidine nucleic acid base can be replaced with a moiety.When a moiety is conjugated to nucleic acid base, the preferred position is one that does not interfere with hybridization, i.e., does not interfere with the hydrogen bond interaction required for base pairing.

[0321]

[0367] Conjugation to the sugar moiety of a nucleoside can occur at any carbon atom. Examples of carbon atoms of the sugar moiety that can be bound to the conjugate moiety include the 2', 3', and 5' carbon atoms. The gamma position can also be bound to a conjugate moiety such as an abasic residue. An internucleoside linkage can also have a conjugate moiety. In the case of phosphorus-containing linkages (e.g., phosphodiester, phosphorothioate, phosphorodithioate, phosphoramidate, etc.), the conjugate moiety can be directly bound to the phosphorus atom or to an O, N, or S atom bound to the phosphorus atom. In the case of internucleoside linkages containing amines or amides (e.g., PNA), the conjugate moiety can be bound to the nitrogen atom of the amine or amide or to the carbon atom adjacent to it.

[0322]

[0368] There are many methods for preparing oligonucleotide conjugates. Generally, oligonucleotides are linked to conjugate moieties by contacting a reactive group (e.g., OH, SH, amine, carboxyl, aldehyde, etc.) on the oligonucleotide with a reactive group on the conjugate moiety. In some embodiments, one reactive group is electrophilic and the other is nucleophilic. For example, the electrophilic group can be a carbonyl-containing functional group, and the nucleophilic group can be an amine or thiol. Methods for conjugating nucleic acids and related oligomeric compounds with or without linking groups are well described in the literature, for example, Manoharan in Antisense Research and Application, Crooke and LeBleu (eds.), CRC Press, Boca Raton, FL, 1993, Chapter 17, which is incorporated herein by reference in its entirety.

[0323]

[0369] The targeting moiety can be attached to an active agent or therapeutic nucleic acid described herein, such as a guide RNA, via RNA-RNA or RNA-DNA base pairing and hybridization. Without intending to be bound by any theory, the targeting moiety may include a coupling sequence that can recognize or bind to an active agent, such as a guide RNA or mRNA. In some embodiments, the targeting moiety includes a coupling sequence that can hybridize to the 5', 3', or middle portion of the guide RNA. The guide RNA that hybridizes with the coupling sequence may include an extension. For example, the coupling sequence can hybridize to the extension sequence of the guide RNA, thereby directing the guide RNA to a desired in vivo, ex vivo, intercellular, or intracellular location, while leaving guide RNA function, such as interaction with a CRISPR enzyme or binding to a target sequence, unaffected. In some embodiments, the guide RNA includes an extension that includes a polynucleotide tail. In some embodiments, the guide nucleic acid comprises a poly(A) tail, a poly(U) tail, or a poly(T) tail that can hybridize with a poly(U) tail, a poly(A) tail, or a poly(A) tail of the coupling sequence, respectively. In some embodiments, the guide nucleic acid can be a guide RNA comprising an (A)n or (U)n sequence. In some embodiments, the guide nucleic acid can comprise DNA and can comprise an (A)n or (T)n sequence. In some embodiments, the coupling sequence can comprise an (A)n (SEQ ID NO: 115), (U)n (SEQ ID NO: 116), or (T)n (SEQ ID NO: 117) sequence. As will be used immediately, n can be any integer from 1 to 200.

[0324]

[0370] The coupling sequence may share sequence identity or complementarity with a nucleic acid active agent 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 a guide RNA described herein or a portion of such a guide RNA. 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 a guide RNA described herein, or the complementarity of a portion of such a guide RNA.In some embodiments, 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, at least at least 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 contiguous nucleobases, or the complementarity thereof.

[0325]

[0371] In some embodiments, the targeting moiety may comprise or be associated with a chemically modified coupling sequence. In some embodiments, the coupling sequence comprises an extension that hybridizes with a therapeutic nucleic acid, such as a guide RNA, or a portion thereof. In some embodiments, the extension of the coupling sequence may be chemically modified. In some embodiments, the therapeutic nucleic acid, such as a guide RNA, may comprise the extension. In some embodiments, the extension of the guide RNA may be chemically modified. Non-limiting examples of guide RNA extensions and complementary or substantially complementary coupling sequence extensions are shown in Table 2 below.

[0326] [Table 2-1]

[0327] [Table 2-2]

[0328]

[0372] As used in Table 2, capital letters A, C, G, and U refer to ribonucleotides having the nucleobases adenine, cytosine, guanidine, and uracil, respectively. Lowercase letters a, c, g, and u refer to modified (e.g., 2'-OMe or 2'-MOE) ribonucleotides containing the nucleobases adenine, cytosine, guanidine, and uracil, respectively. The letter "T" refers to thymidine or deoxythymidine. The letter "s" refers to a phosphorus-containing linkage (such as a phosphorothioate (PS), phosphodiester, or phosphorodithioate linkage). As used in Table 2, "(GalNAc)" refers to a targeting moiety, such as a targeting moiety comprising GalNAc or a derivative thereof. As used in Table 2, "(GalNAc)" also encompasses targeting moieties comprising multiple GalNAc structures or derivatives thereof, e.g., GalNAc dimers, trimers, tetramers, or derivatives thereof (including the GalNAc structures listed in Table 1). In some embodiments, "s" represents a phosphorothioate (PS) linkage. As disclosed herein, the nucleotide sequences and modification patterns encompass all lengths, structures, and types of RNA or fragments thereof, such as CRISPR guide RNAs, e.g., sgRNAs, dual guide RNAs, or mRNAs. For example, the nucleotide sequences and modification patterns listed in Table 2 above may represent RNA sequences and modification patterns in single guide RNAs, dual guide RNAs, nuclease mRNAs, or any fragment or segment thereof.

[0329]

[0373] Non-limiting examples of guide RNAs conjugated to receptor targeting moieties and coupling sequences containing targeting moieties are shown in Table 3 below. The (GalNAc) conjugate moiety is covalently linked to the 3' and / or 5' end of the guide RNA and / or is covalently linked to the 3' and / or 5' end of the guide RNA, with an additional nucleotide spacer between the ligand and the guide RNA. Guide RNA conjugates 3-1 and 3-2 (Table 3) are representative examples of direct conjugation of a GalNAc ligand to a guide RNA. Guide RNAs are conjugated to 3-10 through 3-21, with the GalNAc ligand conjugated to the 3' / 5' end of an additional 3' and / or 5' nucleotide spacer. The guide RNA strand is extended by the desired number of nucleotides to the 3' or 5' end, or both ends. GalNAc is conjugated to the 3', 5', or both ends of an oligonucleotide complementary to the extended nucleotide of a guide RNA strand, forming a complementary duplex leading to a single chemical entity. In this conjugate design, 3-3 through 3-8 are constructed from an extended nucleotide spacer and a spacer complementary strand bearing a GalNAc ligand. As used in Table 3, capital letters A, C, G, and U refer to ribonucleotides with the nucleobases adenine, cytosine, guanidine, and uracil, respectively. Lowercase letters a, c, g, and u refer to modified (e.g., 2'-OMe or 2'-MOE) ribonucleotides with the nucleobases adenine, cytosine, guanidine, and uracil, respectively. The letter "T" refers to thymidine or deoxythymidine. The letter "s" refers to a phosphate linkage (e.g., phosphorothioate (PS), phosphodiester, or phosphorodithioate). In some embodiments, "s" represents a PS linkage. As used in Table 3, "(GalNAc)" refers to a targeting moiety, such as one comprising GalNAc or a derivative thereof. As used in Table 3, "(GalNAc)" also encompasses targeting moieties comprising multiple GalNAc structures or derivatives thereof, such as GalNAc dimers, trimers, tetramers, or derivatives thereof.

[0330] [Table 3-1]

[0331] [Table 3-2]

[0332] [Table 3-3]

[0333]

[0374] As disclosed herein, the nucleotide sequences and modification patterns include all lengths, structures, and types of RNA or fragments thereof, CRISPR guide RNA, such as sgRNA, dual guide RNA, or mRNA.For example, the nucleotide sequences and modification patterns listed in Table 3 above may represent the RNA sequences and modification patterns in single guide RNA, dual guide RNA, nuclease mRNA, or any fragment or segment thereof.

[0334]

[0375] The targeting moiety can be attached to the nucleic acid described herein via a carrier. The carrier may include (i) at least one "backbone attachment point," preferably two "backbone attachment points," and (ii) at least one "tethering attachment point." As used herein, a "backbone attachment point" refers to a functional group, such as a hydroxyl group, or generally a bond that is available and suitable for incorporation of a carrier monomer into the backbone of an oligonucleotide, such as a phosphate or modified phosphate, such as a sulfur-containing backbone. A "tethering attachment point" (TAP) refers to an atom of the carrier monomer to which the selected moiety is attached, such as a carbon atom or heteroatom (different from the atom that provides the backbone attachment point). The selected moiety may be, for example, a carbohydrate, such as a monosaccharide, disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, or polysaccharide. Optionally, the selected moiety is attached to the carrier monomer by an intervening tether. Thus, carriers often contain functional groups, such as amino groups, or generally provide bonds suitable for incorporation or tethering of another chemical entity, such as a ligand, to a constituent atom. Representative United States patents that teach the preparation of nucleic acid conjugates include, but are not limited to, U.S. Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717; 5,580,731; 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 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 ,208; 6,300,319; 6,335,434; 6,335,437; 6,395,437; 6,444,806; 6,486,308; 6,525,031; 6,528,631; 6,559,279.

[0335]

[0376] The targeting moiety can be linked to an active agent, such as a guide RNA, via a linker. The linker can be linked to one or more active agents and a targeting moiety ligand to form a conjugate, which releases at least one active agent, such as a guide RNA or a guide RNA-Cas complex, upon delivery to a target cell. The linker can be linked to the targeting moiety and the active agent by a functional group independently selected from an ester bond, a disulfide, an amide, an acylhydrazone, an ether, a carbamate, a carbonate, and a urea. Alternatively, the linker can be linked to either the targeting moiety or the active agent by a non-cleavable group, such as that provided by conjugation between a thiol and a maleimide, or an azide and an alkyne. In some embodiments, the targeting moiety includes one or more linkers. In some embodiments, the one or more linkers described herein connect one portion of the targeting moiety to a different portion of the targeting moiety. For example, the targeting moiety can include 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 conjugates described herein comprise an active agent directly attached to the sugar moiety of the targeting moiety.

[0336]

[0377] The linker may each independently comprise one or more functional groups selected from the group consisting of ethylene glycol, propylene glycol, amide, ester, ether, alkyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl, wherein each of the alkyl, alkenyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl groups is each independently selected from halogen, cyano, nitro, hydroxyl, carboxyl, carbamoyl, ether, alkoxy, aryloxy, amino, amido, carbamate, alkyl, alkenyl, alkynyl, aryl, arylalkyl, cycloalkyl, heteroaryl, and heterocyclyl. In some embodiments, the linker independently comprises a phosphate, a phosphorothioate, an amide, an ether, an oxime, a hydrazine, or a carbamate. As contemplated herein, in some embodiments, the targeting conjugate of formula (V), (VI), (VIa), or (VIb) should be understood to include a linker as described herein. For example, the groups R and L may be independently selected from the group consisting of carboxyl, carbamoyl, ether, alkoxy, aryloxy, amino, amido, carbamate, alkyl, alkenyl, alkynyl, aryl, arylalkyl, cycloalkyl, heteroaryl, and heterocyclyl. 1 ~L 12 Any of the above may include one or more linkers.

[0337]

[0378] In some embodiments, the linker is C1-C 10 Straight chain alkyl, C1-C 10 Straight chain O-alkyl, C1-C 10 Straight chain substituted alkyl, C1-C 10 Straight-chain substituted O-alkyl, C4-C13 Branched chain alkyl, C4-C 13 Branched chain O-alkyl, C2-C 12 Straight chain alkenyl, C2-C 12 Straight chain O-alkenyl, aralkyl, C3-C 12 Straight-chain substituted alkenyl, C3-C 12 The linker may independently include linear substituted O-alkenyl, polyethylene glycol, polylactic acid, polyglycolic acid, poly(lactide-co-glycolide), polycaprolactone, polycyanoacrylate, ketone, aryl, heterocycle, succinic acid ester, amino acid, aromatic group, ether, crown ether, urea, thiourea, amide, purine, pyrimidine, bipyridine, indole derivative acting as a crosslinker, chelating agent, aldehyde, ketone, bisamine, bisalcohol, heterocyclic structure, azirine, 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 can be optionally interrupted by one or more atoms or groups selected from -O-, -C(=O)-, -NR, -OC(=O)-NR-, -S-, and -SS-.

[0338]

[0379] In some embodiments, the linker can be cleavable, and can be cleaved to release the active agent. The cleavable functional group can be hydrolyzed in vivo, or can be designed to be enzymatically hydrolyzed, for example, by cathepsin B. As used herein, the term "cleavable" linker refers to any linker that can be physically or chemically cleaved. Examples of physical cleavage include cleavage by light, radioactive radiation, or heat, and examples of chemical cleavage include cleavage by redox reaction, hydrolysis, and pH-dependent cleavage.

[0339]

[0380] A linker can be a direct bond or an atom such as oxygen or sulfur, a unit such as N(R), C(O), C(O)NH, SO, SO, SONH, or a chain of atoms, including, but not limited to, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl, heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, alkylarylalkyl, alkylarylalkenyl, alkylarylalkynyl, alkenylarylalkyl, alkenylarylalkenyl, alkenylarylalkynyl, alkynylarylalkyl, alkynylarylalkenyl, alkynylarylalkynyl, alkylheteroarylalkyl, alkylheteroarylalkenyl, alkylheteroarylalkynyl, alkenylheteroaryl and R' is hydrogen, acyl, aliphatic, or substituted or unsubstituted aryl; wherein R' is hydrogen, acyl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclic; and R' is hydrogen, acyl, aliphatic, or substituted or unsubstituted heterocyclic; and R' is hydrogen, acyl, aliphatic, or substituted or unsubstituted heterocyclic; and R' is hydrogen, acyl, aliphatic, or substituted aliphatic ... substituted or unsubstituted heterocyclic; and R' is substituted or unsubstituted hetero 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.

[0340]

[0381] In one embodiment, the linker is -[(PQ"-R)qX-(P'Q'"-R')q']q"-T-, wherein P, R, T, P', R', and T, for each occurrence, are each independently selected from the group consisting of: absent, CO, NH, O, S, OC(O), NHC(O), CH, CHNH, CHO; NHCH(Ra)C(O), -C(O)-CH(Ra)-NH-, CH-NO,

[0341] [ka]

[0342] or heterocyclyl; Q" and Q'" are, for each occurrence, each independently absent, -(CH2)n-, -C(R1)(R2)(CH2)n-, -(CH2)nC(R1)(R2)-, -(CH2CHO)mCH2CH2-, or -(CH2CHO)mCH2CH2NH-; X is absent or a cleavable linking group; Ra is H or an amino acid side chain; R1 and R2 are each independently, for each occurrence, H, CH3, OH, SH, or N(RN)2; R, for each occurrence, is independently H, methyl, ethyl, propyl, isopropyl, butyl, or benzyl; q, q', and q" are, for each occurrence, each independently 0 to 20, where the repeating units can be the same or different; n is, for each occurrence, independently 1 to 20; and m is, for each occurrence, independently 0 to 50.

[0343]

[0382] In one embodiment, the linker comprises at least one cleavable linking group. In certain embodiments, the linker is a branched linker. The branch point of the branched linker may be at least trivalent, but may also be a tetravalent, pentavalent, or hexavalent atom or group exhibiting such multiple valencies. In certain embodiments, the branch 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, for each occurrence, is independently H or an optionally substituted alkyl. In other embodiments, the branch point is glycerol or a glycerol derivative.

[0344]

[0383] In one embodiment, the linker can be cleaved by an enzyme. As a non-limiting example, the linker can be a polypeptide moiety that is 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 the AA in the linker can be a dipeptide, tripeptide, or tetrapeptide, such as Ala-Leu, Leu-Ala-Leu, and Ala-Leu-Ala-Leu. In another example, the cleavable linker can be a branched peptide. A branched peptide linker can contain two or more amino acid moieties that provide an enzyme cleavage site. Any of the branched peptide linkers disclosed in WO1998019705 to Dubowchik (the contents of which are incorporated herein by reference in their entirety) can be used as a linker in the conjugates of the present disclosure. As another example, the linker may comprise a lysosomal-cleavable polypeptide as disclosed in U.S. Patent No. 8,877,901 to Govindan et al., the contents of which are incorporated herein by reference in their entirety. As another example, the linker may comprise a protein peptide sequence that is selectively enzymatically cleavable by a tumor-associated protease, such as any of the Y and Z structures disclosed in U.S. Patent No. 6,214,345 to Firestone et al., the contents of which are incorporated herein by reference in their entirety.

[0345]

[0384] In some embodiments, the linker may include a cleavable linking group. A cleavable linking group is a group that is sufficiently stable outside a cell but is cleaved upon entry into a target cell to release the two moieties held together by the linker. In a preferred embodiment, the cleavable linking group is cleaved at least 10 times faster, preferably at least 100 times faster, within a target cell or under a first reference condition (e.g., which may be selected to mimic or represent intracellular conditions) or under a second reference condition (e.g., which may be selected to mimic or represent conditions found in blood or serum) than in the subject's blood. The cleavable linking group may be susceptible to cleavage agents, such as pH, redox potential, or the presence of degrading molecules. Generally, cleavage agents are found more preferentially or at higher levels or activity within cells than in serum or blood. Examples of such degrading agents include redox agents that are selective for a particular substrate or have no substrate specificity, such as oxidases or reductases present in cells or reducing agents such as mercaptans (which can degrade the redox-cleavable linking group by reduction); esterases; endosomes or agents that can create an acidic environment, e.g., pH 5 or below; enzymes that can hydrolyze or degrade the acid-cleavable linking group by acting as general acids, peptidases (which can be substrate-specific), and phosphatases.

[0346]

[0385] Cleavable linking groups, such as disulfide bonds, can be sensitive to pH. While the pH of human serum is 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 have cleavable linking groups that are cleaved at a preferred pH, thereby releasing the cationic lipid from its ligand intracellularly or to a desired compartment within the cell.

[0347]

[0386] Linker can comprise a cleavable linking group that can be cleaved by specific enzyme.The type of cleavable linking group incorporated into linker can depend on the cell to be targeted.For example, liver targeting ligand can be linked to cationic lipid via a linker that comprises an ester group.Because liver cells are rich in esterase, linker is more efficiently cleaved in liver cells than in cell types that are not rich in esterase.Other cell types that are rich in esterase include lung, renal cortex and testicular cells.

[0348]

[0387] One class of cleavable linking groups is redox-cleavable linking groups, which are cleaved by reduction or oxidation. An example of a reductively cleavable linking group is a 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, methods described herein may be examined. For example, candidates 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 preferred embodiments, candidate compounds are cleaved up to 10% in blood. In preferred embodiments, useful candidate compounds are degraded at least 2, 4, 10, or 100 times faster in cells (or under in vitro conditions selected to mimic intracellular conditions) than in blood (or under in vitro conditions selected to mimic extracellular conditions). The rate of cleavage of a candidate compound may be determined using standard enzyme kinetic assays under conditions selected to mimic the intracellular medium and compared to conditions selected to mimic the extracellular medium.

[0349]

[0388] In some embodiments, the linker may include a phosphate-based cleavable linker that is cleaved by an agent that degrades or hydrolyzes the phosphate group. An example of an agent that cleaves phosphate groups within a cell is an enzyme such as an intracellular phosphatase. Examples of phosphate-based linking groups (i.e., phosphorus-containing linkages or 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 group is -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)-S-, -SP(S)(OH)-O-, -OP(O)(H)-O-, -OP(S)(H)-O-, -SP(O)(H)-O-, -SP(S)(H)-S-, -OP(S)(H)-SS-. In some embodiments, the phosphate-based linker is -OP(O)(OH)-O-.

[0350]

[0389] 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 a preferred embodiment, 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 an agent, such as an enzyme, that can act as a general acid. Within a cell, certain low-pH organelles, such as endosomes and lysosomes, may provide a cleavage environment for the acid-cleavable linking group. Examples of acid-cleavable linking groups include, but are not limited to, hydrazones, esters, and esters of amino acids. Acid-cleavable groups may have the general formula -C=NN-, C(O)O, or -OC(O). In a preferred embodiment, the carbon attached 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 can be evaluated using methods similar to those described above.

[0351]

[0390] In some embodiments, the linker may include an ester-based linking group. Ester-based cleavable linking groups are cleaved by enzymes such as intracellular esterases and amidases. Examples of ester-based cleavable linking groups include, but are not limited to, esters of alkylene, alkenylene, and alkynylene groups. Ester-cleavable linking groups have the general formula -C(O)O- or -OC(O)-. These candidates can be evaluated using methods similar to those described above.

[0352]

[0391] In some embodiments, the linker may include a peptide-based linking group. Peptide-based cleavable linking groups are cleaved by enzymes such as intracellular peptidases and proteases. Peptide-based cleavable linking groups are peptide bonds formed between amino acids to generate oligopeptides (e.g., dipeptides, tripeptides, etc.) and polypeptides. Peptide-based cleavable 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 generate peptides and proteins. Peptide-based cleaving groups are generally limited to peptide bonds (i.e., amide bonds) formed between amino acids and proteins to generate peptides, but do not include the entire amide functionality. Peptide-based cleavable linking groups have the general formula —NHCHRAC(O)NHCHRBC(O)—, where R and R are the R groups of two adjacent amino acids.

[0353]

[0392] Linkers containing peptide bonds may be used when targeting cell types rich in peptidases, such as liver cells and synovial cells.

[0393] In general, the suitability of a candidate cleavable linking group can be evaluated by testing the ability of a degradation agent (or condition) to cleave the candidate linking group. It may also be desirable to test candidate cleavable linking groups for their ability to resist cleavage when in blood or other non-target tissues. Thus, if a first condition is selected to exhibit cleavage in target cells and a second condition is selected to exhibit cleavage in other tissues or body fluids, such as blood or serum, the relative susceptibility to cleavage between the first and second conditions can be determined. Evaluation can be performed in a cell-free system, in cells, in cell culture, in organ or tissue culture, or in a whole animal. It may be useful to perform initial evaluations in cell-free or culture conditions and confirm with further evaluations in a whole animal. In preferred embodiments, useful candidate compounds are cleaved at least 2, 4, 10, or 100 times faster in cells (or under in vitro conditions selected to mimic intracellular conditions) than in blood or serum (or under in vitro conditions selected to mimic extracellular conditions).

[0354]

[0394] In some embodiments, the conjugates described herein have the structure of formula (I):

[0355] [ka]

[0356] Including, wherein each X is independently H or a protecting group, and W represents an activator or a coupling sequence. One or more linkers of formula (I) each independently comprise a linker described in the present disclosure. In some embodiments, each of the protecting groups of formula (I) independently comprises 4-acetoxy-2,2-dimethylbutanoyl (ADMB), 3-(2-hydroxyphenyl)-3,3-dimethylpropanoate (DMBPP), 3-(2-hydroxy-4,6-dimethylphenyl)-3,3-dimethylpropanoic acid (TMBPP), methylsulfonylethoxycarbonyl (Msc), 2,2-dimethyltrimethylene (DMTM) phosphate, 2-pyridylmethyl, ethyl mandelate, (phenylthiomethyl)benzyl, pentaerythritol, methyl methyl ester ... The protecting group is selected from tetrafluoropropionyl (PFP), benzoyl (Bz), acetyl (Ac), bacillosamine (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 entire contents of which are 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 active agent. In some embodiments, W is a nucleic acid. In some embodiments, W is a 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 nucleobase may comprise any chemical modification described herein. In some embodiments, the nucleobase comprises a 2'-OH or 2'-OMe modification.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, 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 modified nucleobases. In some embodiments, the modified RNA comprises one or more modified nucleobases near the 5' end, near the 3' end, or in the middle of the sequence. The modified nucleobases within the modified RNA may or may not be contiguous. In some embodiments, the modified RNA comprises one or more 2'-OMe modifications interspersed along the length of the sequence. In some embodiments, the modified RNA comprises one or more 2'OH modifications interspersed along the length of the sequence. In some embodiments, the modified RNA comprises 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'-OMeA), and u is 2'-O-methyluridine (2'-OMe-U). In some embodiments, W comprises (u)n, where n is an integer between 3 and 50 (SEQ ID NO: 118). In some embodiments, W comprises (u)n, where n is an integer between 3 and 20 (SEQ ID NO: 119) or between 3 and 15 (SEQ ID NO: 120). In some embodiments, W comprises one or more nucleotide sequences complementary to a 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 cytidines or guanines in the coupling sequence. In some embodiments, the guanines or cytidines are at the terminus of W or the coupling sequence.Without intending to be bound by any theory, it is believed that the guanine-cytidine pairing forms a "GC lock" or "CG lock," which increases binding affinity. The guanines and / or cytidines in the W or coupling sequence may or may not be contiguous and may include any one of the chemical modifications described herein, e.g., 2'-OMe or 2'-OH modifications.

[0357]

[0395] In some embodiments, the conjugate of formula (I) has the structure of formula (Ia):

[0358] [ka]

[0359] Includes.

[0396] In some embodiments, the conjugate of formula (I) has the structure of formula (Ib):

[0360] [ka]

[0361] Includes.

[0397] In some embodiments, the conjugates described herein have the structure of formula (II)

[0362] [ka]

[0363] Including, wherein each X is independently H or a protecting group, Z is a modified or unmodified C5 or C6 monosaccharide, and W represents an active agent or a coupling sequence. One or more linkers of formula (II) may each independently comprise a linker described herein. In some embodiments, each protecting group of formula (II) is independently selected from 4-acetoxy-2,2-dimethylbutanoyl (ADMB), 3-(2-hydroxyphenyl)-3,3-dimethylpropanoate (DMBPP), 3-(2-hydroxy-4,6-dimethylphenyl)-3,3-dimethylpropanoic acid (TMBPP), methylsulfonylethoxycarbonyl (Msc), 2,2-dimethyltrimethylene (DMTM) phosphate, 2-pyridylmethyl, ethyl mandelate, (phenylthiomethyl)benzyl, phenylmethyl ... In some embodiments, X is selected from pentafluoropropionyl (PFP), benzoyl (Bz), acetyl (Ac), bacillosamine (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 active agent. In some embodiments, W is a nucleic acid. In some embodiments, W is a 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'-OMeA), and u is 2'-O-methyluridine (2'-OMe-U).In some embodiments, W comprises (u)n, where n is an integer between 3 and 50 (SEQ ID NO: 118). In some embodiments, W comprises (u)n, where n is an integer between 3 and 20 (SEQ ID NO: 119) or between 3 and 15 (SEQ ID NO: 120).

[0364]

[0398] In some embodiments, the conjugate of formula (II) has the structure of formula (IIa)

[0365] [ka]

[0366] Includes.

[0399] In some embodiments, the conjugate of formula (II) has the structure of formula (IIb)

[0367] [ka]

[0368] Includes.

[0400] In some embodiments, the conjugate of formula (II) has the structure of formula (IIc)

[0369] [ka]

[0370] Includes.

[0401] In some embodiments, the conjugates described herein have the structure of formula (III)

[0371] [ka]

[0372] Including, wherein each X is independently H or a protecting group, Z is a modified or unmodified C5 or C6 monosaccharide, and W represents an active agent or a coupling sequence. One or more linkers of formula (III) each independently comprise a linker described herein. In some embodiments, each of the protecting groups of formula (III) independently comprises 4-acetoxy-2,2-dimethylbutanoyl (ADMB), 3-(2-hydroxyphenyl)-3,3-dimethylpropanoate (DMBPP), 3-(2-hydroxy-4,6-dimethylphenyl)-3,3-dimethylpropanoic acid (TMBPP), methylsulfonylethoxycarbonyl (Msc), 2,2-dimethyltrimethylene (DMTM) phosphate, 2-pyridylmethyl, ethyl mandelate, (phenylthiomethyl)benzyl, phenylmethyl ... In some embodiments, X is selected from pentafluoropropionyl (PFP), benzoyl (Bz), acetyl (Ac), bacillosamine (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 active agent. In some embodiments, W is a nucleic acid. In some embodiments, W is a 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'-OMeA), and u is 2'-O-methyluridine (2'-OMe-U).In some embodiments, W comprises (u)n, where n is an integer between 3 and 50 (SEQ ID NO: 118). In some embodiments, W comprises (u)n, where n is an integer between 3 and 20 (SEQ ID NO: 119) or between 3 and 15 (SEQ ID NO: 120).

[0373]

[0402] In some embodiments, the conjugate of formula (III) has the structure of formula (IIIa)

[0374] [ka]

[0375] Includes.

[0403] In some embodiments, the conjugate of formula (III) has the structure of formula (IIIb)

[0376] [ka]

[0377] Includes.

[0404] In some embodiments, the conjugate of formula (III) has the structure of formula (IIIc)

[0378] [ka]

[0379] Including, wherein Y is O or S.

[0405] In some embodiments, the conjugate of formula (III) has the structure of formula (IIId):

[0380] [ka]

[0381] Including, wherein Y is O or S.

[0406] In some embodiments, the conjugate of formula (III) has the structure of formula (IIIe):

[0382] [ka]

[0383] Including, wherein Y is O or S.

[0407] In some embodiments, the conjugates described herein have the structure of formula (IV)

[0384] [ka]

[0385] Including, wherein each X is independently H or a protecting group; R A is -OX or -NHAc, Y is O or S, and W represents an activator or a coupling sequence. One or more linkers of formula (IV) may each independently comprise a linker described in the present disclosure. In some embodiments, each of the protecting groups of formula (IV) independently comprises 4-acetoxy-2,2-dimethylbutanoyl (ADMB), 3-(2-hydroxyphenyl)-3,3-dimethylpropanoate (DMBPP), 3-(2-hydroxy-4,6-dimethylphenyl)-3,3-dimethylpropanoic acid (TMBPP), methylsulfonylethoxycarbonyl (Msc), 2,2-dimethyltrimethylene (DMTM) phosphate, 2-pyridylmethyl, ethyl mandelate, (phenylthiomethyl)benzyl, phenylmethyl ... In some embodiments, X is selected from pentafluoropropionyl (PFP), benzoyl (Bz), acetyl (Ac), bacillosamine (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, RA is -OX. In some embodiments, R A is —OH. In some embodiments, R A is -NHAc. In some embodiments of Formula (IV), W is an active agent. In some embodiments, W is a nucleic acid. In some embodiments, W is a 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'-OMeA), and u is 2'-O-methyluridine (2'-OMe-U). In some embodiments, W comprises (u)n, where n is an integer between 3 and 50 (SEQ ID NO: 118). In some embodiments, W comprises (u)n, where n is an integer between 3 and 20 (SEQ ID NO: 119) or between 3 and 15 (SEQ ID NO: 120).

[0386]

[0408] In some embodiments, the conjugate of formula (IV) comprises structure 1-1, 1-2, 1-5, 1-6, 1-9, 1-10, 1-11, or 1-12, as shown in Table 1.

[0387]

[0409] 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), the "one or more linkers" referenced in the boxes of the preceding formulas comprise a structure selected from the group consisting of:

[0388] [ka]

[0389] and each linker is independently. 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), wherein each of the linkers is independently -(L 1 ) k1 -(L 2 ) k2 -(L 3 ) k3 -(L 4 ) k4 wherein each of k1, k2, k3, and k4 is independently 0, 1, or 2; and L 1 , L 2 , L 3 and L 4 each independently represents oxo, ester, amide, amino, C1-C3 alkylene, and -(CH2-CH2-O) 1~3 In some embodiments, the sum of k1, k2, k3, and k4 is an integer greater than or equal to 1. In some embodiments, the sum of k1, k2, k3, and k4 is an integer greater than or equal to 2. As one of ordinary skill in the art will recognize, "N" refers to nitrogen; "

[0390] [ka]

[0391] " denotes a point of attachment.

[0410] 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 is independently -(L 1 ) k1 -(L 2 ) k2 -(L 3 ) k3 -(L 4 ) k4wherein 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 the groups independently represents -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 arylene, and C5-C9 heteroarylene, wherein alkylene, alkenylene, alkynylene, cycloalkylene, cycloalkylene, arylene, or heteroarylene is substituted or unsubstituted, and wherein each R L are 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 Lare independently H, substituted or unsubstituted C1-C6 alkyl, —OCH3, substituted or unsubstituted C1-C6 haloalkyl, substituted or unsubstituted C1-C6 heteroalkyl, substituted or unsubstituted C3-C8 cycloalkyl, or substituted or unsubstituted C2-C7 heterocycloalkyl.

[0392]

[0411] 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 is independently one of the following:

[0393] [ka]

[0394] wherein 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.

[0395]

[0412] 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), wherein the "one or more linkers" referred to in the box in the preceding formula are:

[0396] [ka]

[0397] [ka]

[0398] [ka]

[0399] [ka]

[0400] [ka]

[0401] wherein 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.

[0402]

[0413] 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 nucleobase may comprise any chemical modification described herein. In some embodiments, the nucleobase comprises a 2'-OH or 2'-OMe modification. 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, a modified RNA, e.g., a gRNA or mRNA, 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 modified nucleobases. In some embodiments, the modified RNA comprises one or more modified nucleobases near the 5' end, near the 3' end, or in the middle of the sequence. The modified nucleobases within the modified RNA may or may not be contiguous. In some embodiments, the modified RNA comprises one or more 2'-OMe modifications interspersed along the length of the sequence. In some embodiments, the modified RNA comprises one or more 2'OH modifications interspersed along the length of the sequence. In some embodiments, the modified RNA comprises 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'-OMeA), and u is 2'-O-methyluridine (2'-OMe-U). In some embodiments, W comprises (u)n, where n is an integer between 3 and 50 (SEQ ID NO: 118). In some embodiments, W comprises (u)n, where n is an integer between 3 and 20 (SEQ ID NO: 119) or between 3 and 15 (SEQ ID NO: 120). In some embodiments, W comprises one or more nucleotide sequences complementary to a 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 cytidines or guanines in the coupling sequence. In some embodiments, the guanines or cytidines are at the terminus of W or the coupling sequence. Without intending to be bound by any theory, it is believed that guanine-cytidine pairings form a "GC lock" or "CG lock," which increases binding affinity. The guanines and / or cytidines in W or the coupling sequence may be contiguous or non-contiguous and may include any one of the chemical modifications described herein, such as 2'-OMe or 2'-OH modifications.

[0403] Receptor-targeting conjugates

[0414] The key to achieving nucleic acid-based therapeutics is the safe and effective delivery of payloads to specific cell types and tissues. Lipid nanoparticles (LNPs) currently represent the most advanced nonviral drug delivery technology platform. LNPs can physically penetrate blood vessels and reach hepatocytes [Am. J. Pathol. 2010, 176, 14-21]. Furthermore, apolipoprotein E (ApoE) protein has been shown to bind to LNPs in a nearly neutral state in the bloodstream after PEG-lipid diffusion from the LNP surface and function as an endogenous ligand for hepatocytes, representing the low-density lipoprotein receptor (LDLr) [Mol. Ther. 2010, 18, 1357-1364]. Therefore, two key factors controlling efficient liver delivery of LNPs are postulated to be 1) effective PEG-lipid shedding from the LNP surface in serum and 2) ApoE binding to LNPs. It is conceivable that the endogenous ApoE-mediated LDLr-dependent LNP delivery pathway described above is not an effective route for achieving LNP-based hepatic gene delivery to the LDLr-deficient patient population.

[0404]

[0415] In one aspect, LNPs comprising receptor-targeting conjugates are described herein. In some aspects, receptor-targeting conjugates are described herein. LNPs with targeting conjugates are configured to have receptor-targeting moieties on the surface or periphery of the particle. In one aspect, a low mol% of receptor-targeting conjugate is used in constructing the targeted LNP to achieve a low surface density of the targeting moiety on the surface / periphery of the particle. In another aspect, a high mol% of receptor-targeting conjugate is used in constructing the targeted LNP to achieve a high surface density of the targeting moiety on the surface / periphery of the particle. In another aspect, a desired mol% of 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 GalNAc or a derivative GalNAc that targets ASGPR. In one aspect, the receptor-targeting conjugate comprises one GalNAc moiety or a derivative thereof. In another aspect, the receptor-targeting conjugate comprises two GalNAc moieties or derivatives thereof. In another aspect, the receptor-targeting conjugate comprises three GalNAc moieties or derivatives thereof. In another aspect, 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.

[0405]

[0416] The present disclosure provides efficient tissue-specific LNP delivery to hepatocytes in an LDLr-independent manner. The trivalent GalNAc moiety developed by the present disclosure is linked to a hydrophobic glycerol-based dialkyl lipid chain, a sterol (e.g., cholesterol), and a hydrophobic α-tocopherol via different PEG spacers. These GalNAc-conjugated lipids are then formulated with various excipients to produce LNPs with low to high surface densities of GalNAc ligands custom-designed to target the asialoglycoprotein receptor (ASGPR), which is highly expressed on the surface of hepatocytes.

[0406]

[0417] Ligands on the surface of engineered LNPs promote ASGPR-mediated tissue-specific uptake into hepatocytes. Different GalNAc-LNPs are configured to avoid ApoE binding and allow GalNAc-ASGPR interactions to facilitate clathrin-mediated hepatocyte uptake. By using the PEG-lipids described herein in combination with GalNAc-lipids with various PEG tethers to modulate PEG shedding kinetics and adjust the net surface charge density of GalNAc-LNP particles, GalNAc-LNPs lacking endogenous ApoE binding properties can be obtained, delivering particles bearing specific RNA payloads to hepatocytes in preclinical animal models of LDLR deficiency at safe and effective doses. Further dose optimization in preclinical animal models will advance GalNAc-LNPs (or LNPs) into clinical development to treat LDLR-deficient patient populations and induce genome editing at therapeutically viable, safe, and effective doses.

[0407]

[0418] Thus, in one aspect, disclosed herein is a compound of formula (V):

[0408] [ka]

[0409] a receptor-targeting conjugate comprising: During the ceremony, the plurality of A groups collectively comprise a receptor-targeting ligand; L 1 , L 2 , L 3 , L 4 , L 5 , L 6 , L 7 , L 8 , L 9 , L 10 and L 12 each independently represents a substituted or unsubstituted C1 to C 12 Alkylene, substituted or unsubstituted C1-C 12 Heteroalkylene, substituted or unsubstituted C2-C 12 Alkenylene, 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)N(R 1 )-, -N(R 1 )C(=O)-, -OC(=O)N(R 1 )-, -N(R 1 )C(=O)O-, -N(R 1 )C(=O)N(R 1 )-, -C(=O)N(R 1 )C(=O)-, -S(=O)2N(R 1 )-, -N(R 1 )S(=O)2-, -N(R 1 )-, -N(OR 1 )-, -O[(P=O)O - ]O- or -O[(P=O)S - ]O- or bond; L 11 is a substituted or unsubstituted -(CH2CH2O) n -, substituted or unsubstituted -(OCH2CH2) n -, substituted or unsubstituted -(CH2) n - or in conjunction; Each R1 are independently H or substituted or unsubstituted C1-C6 alkyl; R is a lipid, a nucleic acid, an amino acid, a protein, or a lipid nanoparticle; m is an integer selected from 1 to 10; n is an integer selected from 1 to 200.

[0410]

[0419] In some embodiments, the receptor-targeting conjugate is a compound of formula (V):

[0411] [ka]

[0412] Including, During the ceremony, the plurality of A groups collectively comprise a receptor-targeting ligand; L 1 , L 2 , L 3 , L 4 , L 5 , L 6 , L 7 , L 8 , L 9 , L 10 and L 12 each independently represents a substituted or unsubstituted C1 to C 12 Alkylene, substituted or unsubstituted C1-C 12 Heteroalkylene, substituted or unsubstituted C2-C 12 Alkenylene, 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)N(R1)-, -N(R 1 )C(=O)-, -OC(=O)N(R1)-, -N(R 1 )C(=O)O-, -N(R1 )C(=O)N(R 1 )-, -C(=O)N(R 1 )C(=O)-, -S(=O)2N(R 1 )-, -N(R 1 )S(=O)2-, -N(R 1 )-, or -N(OR 1 )-and; L 11 is a substituted or unsubstituted -(CH2CH2O) n - or substituted or unsubstituted -(OCH2CH2) n - and; Each R 1 are independently H or substituted or unsubstituted C1-C6 alkyl; R is a lipid, a nucleic acid, an amino acid, a protein, or a lipid nanoparticle; m is an integer selected from 1 to 10; n is an integer selected from 1 to 200.

[0413]

[0420] In some embodiments, L 11 is -(CH2CH2O) n -or-(OCH2CH2) n -It is.

[0421] 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-acetylgalactosamine (GalNAc) or GalNAc derivatives.

[0414]

[0422] 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 or includes galactose.

[0415]

[0423] In some embodiments of compounds of Formula (V), L 1 , L 4 , and L7 are each independently substituted or unsubstituted C1 to C 12 In some embodiments of the compound of Formula (V), each L 1 , L 4 , and L 7 is independently a substituted or unsubstituted C2-C6 alkylene. In some embodiments of the compound of Formula (V), each L 1 , L 4 , and L 7 is a C4 alkylene.

[0416]

[0424] In some embodiments of the compound of Formula (V), each L 2 , L 5 , and L 8 are independently -C(=O)N(R 1 )-, -N(R 1 )C(=O)-, -OC(=O)NR 1 N(R 1 )-, -N(R 1 )C(=O)O-, -N(R 1 )C(=O)N(R 1 )-, or -C(=O)N(R 1 In some embodiments of the compound of Formula (V), each L 2 , L 5 , and L 8 are independently -C(=O)N(R 1 )- or -N(R 1 In some embodiments of the compound of Formula (V), each L 2 , L 5 , and L 8 is -C(=O)NH-.

[0417]

[0425] In some embodiments of the compound of Formula (V), each L 3 , L 6 , and L 9 are independently substituted or unsubstituted C1 to C 12 In some embodiments of the compound of Formula (V), each L 3is a substituted or unsubstituted C2-C6 alkylene. In some embodiments of the compound of Formula (V), L 3 is a C4 alkylene. In some embodiments of compounds of Formula (V), L 6 and L 9 are each independently substituted or unsubstituted C2 to C 10 In some embodiments of the compound of Formula (V), each L 6 and L 9 is independently a substituted or unsubstituted C2-C6 alkylene. In some embodiments of the compound of Formula (V), each L 6 and L 9 is a C3 alkylene.

[0418]

[0426] In some embodiments of compounds of Formula (V), R 1 is H. In some embodiments, R 1 is a substituted or unsubstituted C1-C6 alkyl. In some embodiments, R 1 is methyl.

[0419]

[0427] In another aspect, disclosed herein is a receptor-targeting conjugate having the formula (VI):

[0420] [ka]

[0421] and During the ceremony, the plurality of A groups collectively comprise a receptor-targeting ligand; L 1 , L 2 , L 3 , L 4 , L 5 , L 6 , L 7 , L 8 , L 9 , L 10 and L 12 each independently represents a substituted or unsubstituted C1 to C 12Alkylene, substituted or unsubstituted C1-C 12 Heteroalkylene, substituted or unsubstituted C2-C 12 Alkenylene, 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)N(R 1 )-, -N(R 1 )C(=O)-, -OC(=O)N(R 1 )-, -N(R 1 )C(=O)O-, -N(R 1 )C(=O)N(R 1 )-, -C(=O)N(R 1 )C(=O)-, -S(=O)2N(R 1 )-, -N(R 1 )S(=O)2-, -N(R 1 )-, -N(OR 1 )-, -O[(P=O)O - ]O-, -O[(P=O)S - ]O-, -N(R 1 )-, -N(OR 1 )-, -(CH2) p -O-, -O-(CH2) p -O-, -O-(CH2) p -, -SS- or a bond; L 11 is a substituted or unsubstituted -(CH2CH2O) n -, substituted or unsubstituted -(OCH2CH2) n -, substituted or unsubstituted -(CH2) n - or in conjunction; Each R 1 are independently H or substituted or unsubstituted C1-C6 alkyl; R is a lipid, a nucleic acid, an amino acid, a protein, or a lipid nanoparticle; m is an integer selected from 1 to 10; n is an integer selected from 1 to 200.

[0422]

[0428] In some embodiments, disclosed herein are receptor-targeting conjugates having the formula (VI):

[0423] [ka]

[0424] and During the ceremony, the plurality of A groups collectively comprise a receptor-targeting ligand; L 1 , L 2 , L 3 , L 4 , L 5 , L 6 , L 7 , L 8 , L 9 , L 10 and L 12 each independently represents a substituted or unsubstituted C1 to C 12 Alkylene, substituted or unsubstituted C1-C 12 Heteroalkylene, substituted or unsubstituted C2-C 12 Alkenylene, substituted or unsubstituted C2-C 12 Alkynylene, -(CH2CH2O) m -, -(OCH2CH2) m -, -O-, -S-, -S(=O)-, -S(=O)2-, -S(=O)(=N(R1))-, -C(=O)-, -C(=N-OR 1 )-, -C(=O)O-, -OC(=O)-, -C(=O)C(=O)-, -C(=O)N(R 1 )-, -N(R 1 )C(=O)-, -OC(=O)N(R 1 )-, -N(R 1 )C(=O)O-, -N(R 1 )C(=O)N(R 1 )-, -C(=O)N(R 1 )C(=O)-, -S(=O)2N(R 1)-, -N(R1)S(=O)2-, -N(R 1 )-, -N(OR 1 )-, -O[(P=O)O - ]O-, -O[(P=O)S - ]O-, -N(R 1 )-, -N(OR 1 )-, -(CH2) p -O-, -O-(CH2) p -O-, -O-(CH2) p -, -SS- or a bond; L 11 is a substituted or unsubstituted -(CH2CH2O) n - or substituted or unsubstituted -(OCH2CH2) n -, substituted or unsubstituted -(CH2) n - or in conjunction; Each R 1 are independently H or substituted or unsubstituted C1-C6 alkyl; R is a lipid, a nucleic acid, an amino acid, a protein, or a lipid nanoparticle; m is an integer selected from 1 to 10; n is an integer selected from 1 to 200; p is an integer selected from 0 to 10.

[0425]

[0429] In some embodiments, L 11 is -(CH2CH2O) n -or-(OCH2CH2) n -It is.

[0430] 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-acetylgalactosamine (GalNAc) or GalNAc derivatives.

[0426]

[0431] In some embodiments of the compound of Formula (VI), A is N-acetylgalactosamine (GalNAc) or a derivative thereof. In some embodiments, A is GalNAc.

[0427]

[0432] In some embodiments of the compound of Formula (VI), each L 1 , L 4 , and L 7 are independently substituted or unsubstituted C1 to C 12 Alkylene or substituted or unsubstituted C1-C 12 It is heteroalkylene.

[0428]

[0433] In some embodiments of the compound of Formula (VI), each L 1 , L 4 , and L 7 are independently substituted or unsubstituted C1 to C 12 It is heteroalkylene.

[0434] In some embodiments of the compound of Formula (VI), each L 1 , L 4 , and L 7 are independently substituted or unsubstituted C1-C containing 1-10 O atoms 12 It is heteroalkylene.

[0429]

[0435] In some embodiments of the compound of Formula (VI), each L 1 , L 4 , and L 7 are independently -(CH2CH2O) p1 -(CH2) q1 - in which p1 is 1 to 8; and q1 is 1 to 6.

[0430]

[0436] In some embodiments of the compound of Formula (VI), each L 1 , L 4 , and L 7 is -(CH2CH2O)3-(CH2)2-.

[0437] In some embodiments of the compound of Formula (VI), each L 1 , L 4, and L 7 are independently substituted or unsubstituted C1 to C 12 It is alkylene.

[0431]

[0438] In some embodiments of the compound of Formula (VI), each L 1 , L 4 , and L 7 are independently substituted or unsubstituted C2 to C6 alkylene.

[0439] In some embodiments of the compound of Formula (VI), each L 1 , L 4 , and L 7 is a C4 alkylene.

[0432]

[0440] In some embodiments of the compound of Formula (VI), each L 2 , L 5 , and L 8 are independently -C(=O)N(R 1 )-, -N(R 1 )C(=O)-, -OC(=O)N(R 1 )-, -N(R 1 )C(=O)O-, -N(R 1 )C(=O)N(R 1 )-, or -C(=O)N(R 1 )C(=O)-.

[0433]

[0441] In some embodiments of the compound of Formula (VI), each L 2 , L 5 , and L 8 are independently -C(=O)N(R 1 )- or -N(R 1 )C(=O)-.

[0442] In some embodiments of the compound of Formula (VI), each L 2 , L 5 , and L 8 is -NHC(=O)-.

[0434]

[0443] In some embodiments of the compound of Formula (VI), each L 2 , L 5, and L 8 is -C(=O)NH-.

[0444] In some embodiments of the compound of Formula (VI), each L 3 , L 6 , and L 9 are independently substituted or unsubstituted C1 to C 12 It is heteroalkylene.

[0435]

[0445] In some embodiments of the compound of Formula (VI), each L 3 , L 6 , and L 9 are independently substituted or unsubstituted C1-C containing 1-10 O atoms 12 It is heteroalkylene.

[0436]

[0446] In some embodiments of the compound of Formula (VI), each L 3 , L 6 , and L 9 are independently -(CH2CH2O) p2 -(CH2CH2CH2O) q2 -; where p2 is 1 to 8; and 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.

[0437]

[0447] In some embodiments of the compound of Formula (VI), each L 3 , L 6 , and L 9 is -(CH2CH2O)-(CH2CH2CH2O)-.

[0448] In some embodiments of the compound of Formula (VI), each L 3 , L 6 , and L 9 are independently -(CH2CH2CH2O) q3 -; wherein 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.

[0438]

[0449] In some embodiments of the compound of Formula (VI), each L 3 , L 6 , and L 9 is -(CH2CH2CH2O)2-.

[0450] In some embodiments, the compound of formula (VI) has formula (VIa):

[0439] [ka]

[0440] having the structure During the ceremony, Each q4 is 1 to 10.

[0441]

[0451] In some embodiments of compounds 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.

[0442]

[0452] In some embodiments of the compound of Formula (V) or Formula (VI), L 10 is a substituted or unsubstituted C1-C 12 In some embodiments, L is alkylene.10 is a substituted or unsubstituted C1-C4 alkylene. In some embodiments, L 10 is a C2 alkylene.

[0443]

[0453] In some embodiments, the compound of formula (VI) has formula (VIb):

[0444] [ka]

[0445] having the structure During the ceremony, r is 1 to 4.

[0446]

[0454] 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.

[0447]

[0455] In some embodiments of the compound of Formula (V), Formula (VI), Formula (VIa), or Formula (VIb), L 11 is -(OCH2CH2) nIn some embodiments, n is -. 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.

[0448]

[0456] In some embodiments of the compound of Formula (V), Formula (VI), Formula (VIa), or Formula (VIb), L 12 are -O-, -C(=O)O-, -C(=O)N(R 1 )-, -N(R 1 )C(=O)-, or -N(R 1 )C(═O)O—. In some embodiments, L 12 is -C(=O)O- or -N(R1 )C(═O)O—. In some embodiments, L 12 is —C(═O)O—. In some embodiments, L 12 is —NHC(═O)O—. In some embodiments, L 12 is -NHC(=O)-.

[0449]

[0457] In some embodiments of the compound of Formula (VI), Formula (VIa), or Formula (VIb), R 1 is H. In some embodiments, R 1 is a substituted or unsubstituted C1-C6 alkyl. In some embodiments, R 1 is methyl.

[0450]

[0458] According to the above-mentioned formula, in some embodiments of compounds of formula (V) or (VI), L 1 is a substituted or unsubstituted C1-C 12 Alkylene, substituted or unsubstituted C1-C 12 Heteroalkylene, substituted or unsubstituted C2-C 12 Alkenylene, substituted or unsubstituted C2-C 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)N(R 1 )-, -N(R 1 )C(=O)-, -OC(=O)N(R 1 )-, -N(R 1 )C(=O)O-, -N(R 1 )C(=O)N(R 1 )-, -C(=O)N(R 1 )C(=O)-, -S(=O)2N(R 1 )-, -N(R 1 )S(=O)2-, -N(R 1)-, -N(OR1)-, -O[(P=O)O-]O-, -O[(P=O)S-]O-, or a bond. 1 is a substituted or unsubstituted C1-C 12 In some embodiments, L is alkylene. 1 is a substituted or unsubstituted C1-C 12 In some embodiments, L is heteroalkylene. 1 is a substituted or unsubstituted C2 to C 12 In some embodiments, L is alkenylene. 1 is a substituted or unsubstituted C2 to C 12 In some embodiments, L is alkynylene. 1 is -(CH2CH2O) m -or-(OCH2CH2) m In some embodiments, L 1 is —O—. In some embodiments, L 1 is -S-. In some embodiments, L 1 is -S(=O)-. In some embodiments, L 1 is -S(=O)2-. In some embodiments, L 1 is -S(=O)(=NR1)-. In some embodiments, L 1 is —C(═O)—. In some embodiments, L 1 is -C(=N-OR1)-. In some embodiments, L 1 is —C(═O)O—. In some embodiments, L 1 is OC(=O)-. In some embodiments, L 1 is —C(═O)C(═O)—. In some embodiments, L 1 is —C(═O)N(R)—. In some embodiments, L 1 is —N(R)C(═O)—. In some embodiments, L 1 is —OC(═O)N(R)—. In some embodiments, L 1 is —N(R)C(═O)O—. In some embodiments, L 1is —N(R1)C(═O)N(R1)—. In some embodiments, L 1 is —C(═O)N(R)C(═O)—. In some embodiments, L 1 is —S(═O)N(R)—. In some embodiments, L 1 is —N(R)S(═O)—. In some embodiments, L 1 is -N(R1)-. 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 is a bond.

[0451]

[0459] According to the above-mentioned formula, in some embodiments of compounds of formula (V) or (VI), L 2 is a substituted or unsubstituted C1-C 12 Alkylene, substituted or unsubstituted C1-C 12 Heteroalkylene, substituted or unsubstituted C2-C 12 Alkenylene, 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)N(R 1 )-, -N(R 1 )C(=O)-, -OC(=O)N(R 1 )-, -N(R 1 )C(=O)O-, -N(R 1 )C(=O)N(R 1 )-, -C(=O)N(R 1 )C(=O)-, -S(=O)2N(R 1 )-, -N(R 1)S(=O)2-, -N(R 1 )-, -N(OR 1 )-, -O[(P=O)O - ]O-, -O[(P=O)S - ]O—, or a bond. In some embodiments, L 2 is a substituted or unsubstituted C1-C 12 In some embodiments, L is alkylene. 2 is a substituted or unsubstituted C1-C 12 In some embodiments, L is heteroalkylene. 2 is a substituted or unsubstituted C2 to C 12 In some embodiments, L is alkenylene. 2 is a substituted or unsubstituted C2 to C 12 In some embodiments, L is alkynylene. 2 is -CCH2CH2O) m -or-(OCH2CH2) m In some embodiments, L 2 is —O—. In some embodiments, L 2 is -S-. In some embodiments, L 2 is -S(=O)-. In some embodiments, L 2 is -S(=O)2-. In some embodiments, L 2 is -S(=O)(=NR 1 In some embodiments, L 2 is —C(═O)—. In some embodiments, L 2 is -C(=N-OR 1 In some embodiments, L 2 is —C(═O)O—. In some embodiments, L 2 is OC(=O)-. In some embodiments, L 2 is —C(═O)C(═O)—. In some embodiments, L 2 is -C(=O)N(R 1 In some embodiments, L 2 is -N(R 1 )C(=O)-. In some embodiments, L2 is -NRHC(=O)-. In some embodiments, L 2 is -OC(=O)N(R 1 In some embodiments, L 2 is -N(R 1 )C(═O)O—. In some embodiments, L 2 is -N(R 1 )C(=O)N(R 1 In some embodiments, L 2 is -C(=O)N(R 1 )C(=O)-. In some embodiments, L 2 is -S(=O)2N(R 1 In some embodiments, L 2 is -N(R 1 )S(=O)2-. In some embodiments, L 2 is -N(R-)-. 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 is a bond.

[0452]

[0460] According to the formulae mentioned above, in some embodiments of compounds of formula (V) or (VI), L 3 is a substituted or unsubstituted C1-C 12 Alkylene, substituted or unsubstituted C1-C 12 Heteroalkylene, substituted or unsubstituted C2-C 12 Alkenylene, 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)N(R 1 )-, -N(R 1 )C(=O)-, -OC(=O)N(R 1 )-, -N(R 1 )C(=O)O-, -N(R 1 )C(=O)N(R 1 )-, -C(=O)N(R 1 )C(=O)-, -S(=O)2N(R 1 )-, -N(R 1 )S(=O)2-, -N(R 1 )-, -N(OR 1 )-, -O[(P=O)O - ]O-, -O[(P=O)S - ]O—, or a bond. In some embodiments, L 3 is a substituted or unsubstituted C1-C 12 In some embodiments, L is alkylene. 3 is the unsubstituted C 3~4 In some embodiments, L is alkylene. 3 is the unsubstituted C 1~4 In some embodiments, L is alkylene. 3 is a substituted or unsubstituted C1-C 12 In some embodiments, L is heteroalkylene. 3 is a substituted or unsubstituted C2 to C 12 In some embodiments, L is alkenylene. 3 is a substituted or unsubstituted C2 to C 12 In some embodiments, L is alkynylene. 3 is -(CH2CH2O) m -or-(OCH2CH2) m In some embodiments, L 3 is —O—. In some embodiments, L 3 is -S-. In some embodiments, L 3 is -S(=O)-. In some embodiments, L 3 is -S(=O)2-. In some embodiments, L 3 is -S(=O)(=NR1 In some embodiments, L 3 is —C(═O)—. In some embodiments, L 3 is -C(=N-OR 1 In some embodiments, L 3 is —C(═O)O—. In some embodiments, L 3 is OC(=O)-. In some embodiments, L 3 is —C(═O)C(═O)—. In some embodiments, L 3 is -C(=O)N(R 1 In some embodiments, L 3 is -N(R 1 )C(=O)-. In some embodiments, L 3 is -OC(=O)N(R 1 In some embodiments, L 3 is -N(R 1 )C(═O)O—. In some embodiments, L 3 is -N(R 1 )C(=O)N(R 1 In some embodiments, L 3 is -C(=O)N(R 1 )C(=O)-. In some embodiments, L 3 is -S(=O)2N(R 1 In some embodiments, L 3 is -N(R 1 )S(=O)2-. In some embodiments, L 3 is -N(R 1 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 is a bond.

[0453]

[0461] According to the formulae mentioned above, in some embodiments of compounds of formula (V) or (VI), L 4 is a substituted or unsubstituted C1-C 12 Alkylene, substituted or unsubstituted C1-C 12 Heteroalkylene, substituted or unsubstituted C2-C 12 Alkenylene, 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)N(R 1 )-, -N(R 1 )C(=O)-, -OC(=O)N(R 1 )-, -N(R 1 )C(=O)O-, -N(R 1 )C(=O)N(R 1 )-, -C(=O)N(R 1 )C(=O)-, -S(=O)2N(R 1 )-, -N(R 1 )S(=O)2-, -N(R 1 )-, -N(OR 1 )-, -O[(P=O)O - ]O-, -O[(P=O)S - ]O—, or a bond. In some embodiments, L 4 is a substituted or unsubstituted C1-C 12 In some embodiments, L is alkylene. 4 is an unsubstituted C4 alkylene. In some embodiments, L 4 is a substituted or unsubstituted C1-C 12 In some embodiments, L is heteroalkylene. 4 is a substituted or unsubstituted C2 to C 12 In some embodiments, L is alkenylene. 4 is a substituted or unsubstituted C2 to C 12 In some embodiments, L is alkynylene.4 is -(CH2CH2O) m -or-(OCH2CH2) m In some embodiments, L 4 is —O—. In some embodiments, L 4 is -S-. In some embodiments, L 4 is -S(=O)-. In some embodiments, L 4 is -S(=O)2-. In some embodiments, L 4 is -S(=O)(=NR 1 In some embodiments, L 4 is —C(═O)—. In some embodiments, L 4 is -C(=N-OR 1 In some embodiments, L 4 is —C(═O)O—. In some embodiments, L 4 is OC(=O)-. In some embodiments, L 4 is —C(═O)C(═O)—. In some embodiments, L 4 is -C(=O)N(R 1 In some embodiments, L 4 is -N(R 1 )C(=O)-. In some embodiments, L 4 is -OC(=O)N(R 1 In some embodiments, L 4 is -N(R 1 )C(═O)O—. In some embodiments, L 4 is -N(R 1 )C(=O)N(R 1 In some embodiments, L 4 is -C(=O)N(R 1 )C(=O)-. In some embodiments, L 4 is -S(=O)2N(R 1 In some embodiments, L 4 is -N(R 1 )S(=O)2-. In some embodiments, L 4 is -N(R1 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 is a bond.

[0454]

[0462] According to the formulae mentioned above, in some embodiments of compounds of formula (V) or (VI), L 5 is a substituted or unsubstituted C1-C 12 Alkylene, substituted or unsubstituted C1-C 12 Heteroalkylene, substituted or unsubstituted C2-C 12 Alkenylene, 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)N(R 1 )-, -N(R 1 )C(=O)-, -OC(=O)N(R 1 )-, -N(R 1 )C(=O)O-, -N(R 1 )C(=O)N(R 1 )-, -C(=O)N(R 1 )C(=O)-, -S(=O)2N(R 1 )-, -N(R 1 )S(=O)2-, -N(R 1 )-, -N(OR 1 )-, -O[(P=O)O - ]O-, -O[(P=O)S - ]O—, or a bond. In some embodiments, L 5 is a substituted or unsubstituted C1-C 12In some embodiments, L is alkylene. 5 is a substituted or unsubstituted C1-C 12 In some embodiments, L is heteroalkylene. 5 is a substituted or unsubstituted C2 to C 12 In some embodiments, L is alkenylene. 5 is a substituted or unsubstituted C2 to C 12 In some embodiments, L is alkynylene. 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)N(R 1 In some embodiments, L 5 is -N(R 1 )C(=O)-. In some embodiments, L 2 is -NRHC(=O)-. In some embodiments, L 5 is -OC(=O)N(R 1 In some embodiments, L 5 is -N(R 1 )C(═O)O—. In some embodiments, L 5is -N(R 1 )C(=O)N(R 1 In some embodiments, L 5 is —C(═O)N(R)C(═O)—. In some embodiments, L 5 is -S(=O)2N(R 1 In some embodiments, L 5 is -N(R 1 )S(=O)2-. In some embodiments, L 5 is -N(R 1 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 is a bond.

[0455]

[0463] According to the formulae mentioned above, in some embodiments of compounds of formula (V) or (VI), L 6 is a substituted or unsubstituted C1-C 12 Alkylene, substituted or unsubstituted C1-C 12 Heteroalkylene, substituted or unsubstituted C2-C 12 Alkenylene, 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)N(R 1 )-, -N(R 1 )C(=O)-, -OC(=O)N(R 1 )-, -N(R 1 )C(=O)O-, -N(R 1 )C(=O)N(R1 )-, -C(=O)N(R 1 )C(=O)-, -S(=O)2N(R 1 )-, -N(R 1 )S(=O)2-, -N(R 1 )-, -N(OR 1 )-, -O[(P=O)O - ]O-, -O[(P=O)S - ]O—, or a bond. In some embodiments, L 6 is a substituted or unsubstituted C1-C 12 In some embodiments, L is alkylene. 6 is the unsubstituted C 3-4 In some embodiments, L is alkylene. 6 is the unsubstituted C 1~4 In some embodiments, L is alkylene. 6 is a substituted or unsubstituted C1-C 12 In some embodiments, L is heteroalkylene. 6 is a substituted or unsubstituted C2 to C 12 In some embodiments, L is alkenylene. 6 is a substituted or unsubstituted C2 to C 12 In some embodiments, L is alkynylene. 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, L6 is OC(=O)-. In some embodiments, L 6 is —C(═O)C(═O)—. In some embodiments, L 6 is -C(=O)N(R 1 In some embodiments, L 6 is -N(R 1 )C(=O)-. In some embodiments, L 6 is -OC(=O)N(R 1 In some embodiments, L 6 is -N(R 1 )C(═O)O—. In some embodiments, L 6 is -N(R 1 )C(=O)N(R 1 In some embodiments, L 6 is -C(=O)N(R 1 )C(=O)-. In some embodiments, L 6 is -S(=O)2N(R 1 In some embodiments, L 6 is -N(R 1 )S(=O)2-. In some embodiments, L 6 is -N(R 1 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 is a bond.

[0456]

[0464] According to the formulae mentioned above, in some embodiments of compounds of formula (V) or (VI), L 7 is a substituted or unsubstituted C1-C 12 Alkylene, substituted or unsubstituted C1-C 12 Heteroalkylene, substituted or unsubstituted C2-C 12Alkenylene, 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)N(R 1 )-, -N(R 1 )C(=O)-, -OC(=O)N(R 1 )-, -N(R 1 )C(=O)O-, -N(R 1 )C(=O)N(R 1 )-, -C(=O)N(R 1 )C(=O)-, -S(=O)2N(R 1 )-, -N(R 1 )S(=O)2-, -N(R 1 )-, -N(OR 1 )-, -O[(P=O)O - ]O-, -O[(P=O)S - ]O—, or a bond. In some embodiments, L 7 is a substituted or unsubstituted C1-C 12 In some embodiments, L is alkylene. 7 is an unsubstituted C alkylene. In some embodiments, L 7 is a substituted or unsubstituted C1-C 12 In some embodiments, L is heteroalkylene. 7 is a substituted or unsubstituted C2 to C 12 In some embodiments, L is alkenylene. 7 is a substituted or unsubstituted C2 to C 12 In some embodiments, L is alkynylene. 7 is -(CH2CH2O) m -or-(OCH2CH2) m In some embodiments, L 7 is —O—. In some embodiments, L 7 is -S-. In some embodiments, L7 is -S(=O)-. In some embodiments, L 7 is -S(=O)2-. In some embodiments, L 7 is -S(=O)(=NR 1 In some embodiments, L 7 is —C(═O)—. In some embodiments, L 7 is -C(=N-OR 1 In some embodiments, L 7 is —C(═O)O—. In some embodiments, L 7 is OC(=O)-. In some embodiments, L 7 is —C(═O)C(═O)—. In some embodiments, L 7 is -C(=O)N(R 1 In some embodiments, L 7 is -N(R 1 )C(=O)-. In some embodiments, L 7 is -OC(=O)N(R 1 In some embodiments, L 7 is -N(R 1 )C(═O)O—. In some embodiments, L 7 is -N(R 1 )C(=O)N(R 1 In some embodiments, L 7 is -C(=O)N(R 1 )C(=O)-. In some embodiments, L 7 is -S(=O)2N(R 1 In some embodiments, L 7 is -N(R 1 )S(=O)2-. In some embodiments, L 7 is -N(R 1 In some embodiments, L 7 is -N(OR 1 In some embodiments, L 7 is -O[(P=O)O - ]O-. In some embodiments, L 7is -O[(P=O)S - ]O-. In some embodiments, L 7 is a bond.

[0457]

[0465] According to the formulae mentioned above, in some embodiments of compounds of formula (V) or (VI), L 8 is a substituted or unsubstituted C1-C 12 Alkylene, substituted or unsubstituted C1-C 12 Heteroalkylene, substituted or unsubstituted C2-C 12 Alkenylene, 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)N(R 1 )-, -N(R 1 )C(=O)-, -OC(=O)N(R 1 )-, -N(R 1 )C(=O)O-, -N(R 1 )C(=O)N(R 1 )-, -C(=O)N(R 1 )C(=O)-, -S(=O)2N(R 1 )-, -N(R 1 )S(=O)2-, -N(R 1 )-, -N(OR 1 )-, -O[(P=O)O - ]O-, -O[(P=O)S - ]O— or a bond. In some embodiments, L 8 is a substituted or unsubstituted C1-C 12 In some embodiments, L is alkylene. 8 is a substituted or unsubstituted C1-C 12 In some embodiments, L is heteroalkylene. 8 is a substituted or unsubstituted C2 to C 12 In some embodiments, L is alkenylene.8 is a substituted or unsubstituted C2 to C 12 In some embodiments, L is alkynylene. 8 is -(CH2CH2O) m -or-(OCH2CH2) m In some embodiments, L 8 is —O—. In some embodiments, L 8 is -S-. In some embodiments, L 8 is -S(=O)-. In some embodiments, L 8 is -S(=O)2-. In some embodiments, L 8 is -S(=O)(=NR 1 In some embodiments, L 8 is —C(═O)—. In some embodiments, L 8 is -C(=N-OR 1 In some embodiments, L 8 is —C(═O)O—. In some embodiments, L 8 is OC(=O)-. In some embodiments, L 8 is —C(═O)C(═O)—. In some embodiments, L 8 is -C(=O)N(R 1 In some embodiments, L 8 is -N(R 1 )C(=O)-. In some embodiments, L 8 is -OC(=O)N(R 1 In some embodiments, L 8 is -N(R 1 )C(═O)O—. In some embodiments, L 2 is -NRHC(=O)-. In some embodiments, L 8 is -N(R 1 )C(=O)N(R 1 In some embodiments, L 8 is -C(=O)N(R 1 )C(=O)-. In some embodiments, L 8 is -S(=O)2N(R1 In some embodiments, L 8 is -N(R 1 )S(=O)2-. In some embodiments, L 8 is -N(R 1 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 is a bond.

[0458]

[0466] According to the formulae mentioned above, in some embodiments of compounds of formula (V) or (VI), L 9 is a substituted or unsubstituted C1-C 12 Alkylene, substituted or unsubstituted C1-C 12 Heteroalkylene, substituted or unsubstituted C2-C 12 Alkenylene, 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)N(R 1 )-, -N(R 1 )C(=O)-, -OC(=O)N(R 1 )-, -N(R 1 )C(=O)O-, -N(R 1 )C(=O)N(R 1 )-, -C(=O)N(R 1 )C(=O)-, -S(=O)2N(R 1 )-, -N(R 1 )S(=O)2-, -N(R 1 )-, -N(OR 1)-, -O[(P=O)O - ]O-, -O[(P=O)S - ]O—, or a bond. In some embodiments, L 9 is a substituted or unsubstituted C1-C 12 In some embodiments, L is alkylene. 9 is the unsubstituted C 3~4 In some embodiments, L is alkylene. 9 is the unsubstituted C 1~4 In some embodiments, L is alkylene. 9 is a substituted or unsubstituted C1-C 12 In some embodiments, L is heteroalkylene. 9 is a substituted or unsubstituted C2 to C 12 In some embodiments, L is alkenylene. 9 is a substituted or unsubstituted C2 to C 12 In some embodiments, L is alkynylene. 9 is -(CH2CH2O) m -or-(OCH2CH2) m In some embodiments, L 9 is —O—. In some embodiments, L 9 is -S-. In some embodiments, L 9 is -S(=O)-. In some embodiments, L 9 is -S(=O)2-. In some embodiments, L 9 is -S(=O)(=NR 1 In some embodiments, L 9 is —C(═O)—. In some embodiments, L 9 is -C(=N-OR 1 In some embodiments, L 9 is —C(═O)O—. In some embodiments, L 9 is OC(=O)-. In some embodiments, L 9 is —C(═O)C(═O)—. In some embodiments, L 9 is -C(=O)N(R 1In some embodiments, L 9 is -N(R 1 )C(=O)-. In some embodiments, L 9 is -OC(=O)N(R 1 In some embodiments, L 9 is -N(R 1 )C(═O)O—. In some embodiments, L 9 is -N(R 1 )C(=O)N(R 1 In some embodiments, L 9 is -C(=O)N(R 1 )C(=O)-. In some embodiments, L 9 is -S(=O)2N(R 1 In some embodiments, L 9 is -N(R 1 )S(=O)2-. In some embodiments, L 9 is -N(R 1 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 is a bond.

[0459]

[0467] According to the formulae mentioned above, in some embodiments of compounds of formula (V) or (VI), L 10 is a substituted or unsubstituted C1-C 12 Alkylene, substituted or unsubstituted C1-C 12 Heteroalkylene, substituted or unsubstituted C2-C 12 Alkenylene, substituted or unsubstituted C2-C 12 Alkynylene, -(CH2CH2O) m -, -(OCH2CH2) m -, -O-, -S-, -S(=O)-, -S(=O)2-, -S(=O)(=NR1 )-, -C(=O)-, -C(=N-OR 1 )-, -C(=O)O-, -OC(=O)-, -C(=O)C(=O)-, -C(=O)N(R 1 )-, -N(R 1 )C(=O)-, -OC(=O)N(R 1 )-, -N(R 1 )C(=O)O-, -N(R 1 )C(=O)N(R 1 )-, -C(=O)N(R 1 )C(=O)-, -S(=O)2N(R 1 )-, -N(R 1 )S(=O)2-, -N(R 1 )-, -N(OR 1 )-, -O[(P=O)O - ]O-, -O[(P=O)S - ]O—, or a bond. In some embodiments, L 10 is a substituted or unsubstituted C1-C 12 In some embodiments, L is alkylene. 10 is a substituted or unsubstituted C1-C 12 In some embodiments, L is heteroalkylene. 10 is a substituted or unsubstituted C2 to C 12 In some embodiments, L is alkenylene. 10 is a substituted or unsubstituted C2 to C 12 In some embodiments, L is alkynylene. 10 is -(CH2CH2O) m -or-(OCH2CH2) m In some embodiments, L 10 is —O—. In some embodiments, L 10 is -S-. In some embodiments, L 10 is -S(=O)-. In some embodiments, L 10 is -S(=O)2-. In some embodiments, L 10 is -S(=O)(=NR 1 In some embodiments, L 10is —C(═O)—. In some embodiments, L 10 is -C(=N-OR 1 In some embodiments, L 10 is —C(═O)O—. In some embodiments, L 10 is OC(=O)-. In some embodiments, L 10 is —C(═O)C(═O)—. In some embodiments, L 10 is -C(=O)N(R 1 In some embodiments, L 10 is -N(R 1 )C(=O)-. In some embodiments, L 10 is —OC(═O)N(R)—. In some embodiments, L 10 is —N(R)C(═O)O—. In some embodiments, L 10 is -N(R 1 )C(=O)N(R 1 In some embodiments, L 10 is -C(=O)N(R 1 )C(=O)-. In some embodiments, L 10 is -S(=O)2N(R 1 In some embodiments, L 10 is -N(R 1 )S(=O)2-. In some embodiments, L 10 is -N(R 1 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 is a substituted or unsubstituted C1-C6 alkylene. In some embodiments, L 10 is a substituted or unsubstituted C1-C3 alkylene. In some embodiments, L 10is a substituted or unsubstituted C2-C3 alkylene. In some embodiments, L 10 is -CHCH-. In some embodiments, L 10 is a bond.

[0460]

[0468] According to the formulae mentioned above, in some embodiments of compounds of formula (V) or (VI), L 11 is a substituted or unsubstituted -(CH2CH2O) n -, substituted or unsubstituted -(OCH2CH2) n -, substituted or unsubstituted -(CH2) n -, or a bond. In some embodiments, L 11 is a substituted or unsubstituted -(CH2CH2O) n In some embodiments, L 11 is a substituted or unsubstituted -(OCH2CH2) n In some embodiments, L 11 is a substituted or unsubstituted -(CH2) n In some embodiments, L 11 is a bond. In some embodiments, n is 30 to 50. In some embodiments, n is 30 to 40. In some embodiments, n is 40 to 50.

[0461]

[0469] According to the formulae mentioned above, in some embodiments of compounds of formula (V) or (VI), L 12 is a substituted or unsubstituted C1-C 12 Alkylene, substituted or unsubstituted C1-C 12 Heteroalkylene, substituted or unsubstituted C2-C 12 Alkenylene, 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)N(R 1 )-, -N(R 1 )C(=O)-, -OC(=O)N(R 1 )-, -N(R 1 )C(=O)O-, -N(R 1 )C(=O)N(R 1 )-, -C(=O)N(R 1 )C(=O)-, -S(=O)2N(R 1 )-, -N(R 1 )S(=O)2-, -N(R 1 )-, -N(OR 1 )-, -O[(P=O)O - ]O-, -O[(P=O)S - ]O—, or a bond. In some embodiments, L 12 is a substituted or unsubstituted C1-C 12 In some embodiments, L is alkylene. 12 is a substituted or unsubstituted C1-C 12 In some embodiments, L is heteroalkylene. 12 is a substituted or unsubstituted C2 to C 12 In some embodiments, L is alkenylene. 12 is a substituted or unsubstituted C2 to C 12 In some embodiments, L is alkynylene. 12 is -(CH2CH2O) m -or-(OCH2CH2) m In some embodiments, L 12 is —O—. In some embodiments, L 12 is -S-. In some embodiments, L 12 is -S(=O)-. In some embodiments, L 12 is -S(=O)2-. In some embodiments, L 12 is -S(=O)(=NR 1 In some embodiments, L 12 is —C(═O)—. In some embodiments, L 12 is -C(=N-OR 1In some embodiments, L 12 is —C(═O)O—. In some embodiments, L 12 is OC(=O)-. In some embodiments, L 12 is —C(═O)C(═O)—. In some embodiments, L 12 is -C(=O)N(R 1 In some embodiments, L 12 is -N(R 1 )C(=O)-. In some embodiments, L 12 is -OC(=O)N(R 1 In some embodiments, L 12 is —N(R)C(═O)O—. In some embodiments, L 12 is -N(R 1 )C(=O)N(R1)-. In some embodiments, L 12 is —C(═O)N(R)C(═O)—. In some embodiments, L 12 is -S(=O)2N(R 1 In some embodiments, L 12 is -N(R 1 )S(=O)2-. In some embodiments, L 12 is -N(R 1 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 is a substituted or unsubstituted C1-C6 alkylene. In some embodiments, L 12 is a substituted or unsubstituted C1-C3 alkylene. In some embodiments, L 12 is a substituted or unsubstituted C2-C3 alkylene. In some embodiments, L 12In some embodiments of compounds of Formula (V) or (VI), L 12 is -N(R 1 )C(═O)O—. In some embodiments, L 12 is a bond. In some embodiments, L 12 is the residue of an organic molecule that intercalates with group R. In some embodiments, L 12 may interact with the base pair ionically / electrostatically or covalently bond to the base pair. Some non-limiting examples of organic molecule residues that intercalate with the group R include berberine, ethidium bromide, daunomycin, thalidomide, doxorubicin (adriamycin), aflatoxin B1, amsacrine, acridines (e.g., proflavine, quinacrine, acridine orange, pyrazoloacridine), acriflavine, 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 ellipticine.

[0462]

[0470] In some embodiments of a 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.

[0463]

[0471] In some embodiments of a 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.

[0464]

[0472] In some embodiments of a compound 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.

[0465]

[0473] In some embodiments of a 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.

[0466]

[0474] In some embodiments of the compound of Formula (V), Formula (VI), Formula (VIa), or Formula (VIb), each R 1 is independently H or -CH. In some embodiments, R 1 is H.

[0467]

[0475] In some embodiments of a compound of Formula (V), Formula (VI), Formula (VIa), or Formula (VIb), R comprises one or more fatty alcohols, fatty acids, glycerolipids, glycerophospholipids, sphingolipids, saccharolipids, polyketides, sterol lipids, and prenol lipids. 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-C6 26 In some embodiments, the fatty alcohol comprises two or more C2-C 26The fatty alcohols include 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)-docos-13-en-1-yl alcohol, docosanyl alcohol, (E)-octadec-9-en-1-yl alcohol, icosanyl 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 comprises one or more sterol lipids. In some embodiments, R comprises one or more vitamins. In some embodiments, each vitamin is independently vitamin A, vitamin D, vitamin E, or vitamin K.

[0468]

[0476] In some embodiments, the R group of Formula (V), Formula (VI), Formula (VIa), or Formula (VIb) comprises a payload described herein. In some embodiments, the R group of Formula (V), Formula (VI), Formula (VIa), or Formula (VIb) comprises a lipid.

[0469]

[0477] In some embodiments, the R group from Formula (V), Formula (VI), Formula (VIa), or Formula (VIb) comprises a nucleic acid. In some embodiments, the 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, the nucleic acid is a double-stranded nucleic acid. In some embodiments, the double-stranded nucleic acid is DNA. In some embodiments, the double-stranded nucleic acid is RNA. In some embodiments, the double-stranded nucleic acid is a DNA-RNA hybrid. In some embod...

Claims

1. 1. A method for preparing a formulation comprising GalNAc-lipid nanoparticles (GalNAc-LNPs), wherein the nanoparticles comprise (i) one or more nucleic acid active agents, (ii) one or more lipid excipients selected from a sterol or derivative thereof, a phospholipid, a stealth lipid, and an amino lipid, and (iii) a GalNAc-lipid; a. providing a first solution comprising one or more nucleic acid active agents in an aqueous buffer; b. Providing a second solution comprising (i) at least one of the one or more lipid excipients and (ii) at least a portion of the GalNAc-lipid in a water-miscible organic solvent; c. optionally, combining an antioxidant with the first solution; d. mixing the first solution with the second solution; e. incubating the mixture of the first and second solutions to form GalNAc-LNPs; f. optionally, performing one or more processes selected from dilution, buffer exchange, concentration, filtration, freezing, thawing, incubation, and GalNAc-LNP evaluation; A method comprising:

2. 10. The method of claim 1, wherein the first solution is divided into two feeds before mixing with the second solution, the two feeds being positioned opposite each other.

3. 10. The method of claim 1, wherein the first solution is divided into two solutions for feeding before mixing with the second solution, and the two feeds are positioned opposite each other.

4. 10. The method of claim 1, wherein step d comprises: (i) adding a first solution to the mixer through a first channel; and (ii) adding a second solution to the mixer through a second channel, wherein steps (i) and (ii) can be performed in any order.

5. 5. The method of claim 4, wherein the first solution and the second solution do not contact each other before entering the mixer.

6. 15. The method of claim 4, wherein the first channel and the second channel are oriented orthogonal to each other as shown in FIG.

7. The method of any one of claims 1 to 6, further comprising dissolving one or more nucleic acid active agents in an aqueous buffer.

8. 7. The method of claim 1, wherein step d comprises: (i) introducing a first solution into the mixer through first and second supply lines, the first and second supply lines being positioned opposite each other on the mixer; and (ii) introducing a second solution into the mixer through a third supply line, the third supply line being positioned perpendicular to the first and second supply lines on the mixer.

9. The method of claim 8, wherein the mixer is a cross mixer.

10. The method of any one of claims 1 to 6, further comprising the step of pumping a first solution into two supply lines and separately pumping a second solution into a third supply line.

11. 7. The method of any one of claims 1 to 6, further comprising the step of diluting GalNAc-lipid in an aqueous solution to prepare a diluted GalNAc-LNP solution, further comprising the step of diluting GalNAc-LNP in the solution one or more times.

12. 7. The method of any one of claims 1 to 6, further comprising exchanging the water-miscible organic solvent with a buffer solution one or more times.

13. The method of any one of claims 1 to 6, further comprising concentrating the GalNAc-LNP, wherein the concentration comprises passing the GalNAc-LNP through a membrane.

14. The method of any one of claims 1 to 6, further comprising filtering the GalNAc-LNP through a membrane.

15. The method of any one of claims 1 to 6, further comprising a second incubation after step e for about 1 minute to about 120 minutes.

16. 7. The method of any one of claims 1-6, further comprising the steps of: (i) thawing stored GalNAc-LNP; (ii) pooling the GalNAc-LNP; (iii) diluting the GalNAc-LNP in solution; and (iv) filtering the GalNAc-LNP through a membrane prior to administering the dose of GalNAc-LNP to the subject, wherein the order of performing steps (iii) and (iv) may be reversed.

17. The method of any one of claims 1 to 6, wherein at least a portion of the GalNAc-lipid is combined with one or more lipids prior to the mixing step.

18. The method of any one of claims 1 to 6, wherein at least a portion of the GalNAc-lipid is combined with one or more lipids after the mixing step.

19. The method according to any one of claims 1 to 6, wherein the mixing is carried out in an in-line mixer, a cross mixer, or a T-mixer apparatus.

20. The method according to any one of claims 1 to 6, wherein the mixing is carried out in a cross mixer.

21. 18. The method of claim 17, wherein the first solution and the second solution are arranged to be introduced orthogonally into the mixer.

22. The method of any one of claims 1 to 6, wherein the mixing comprises laminar mixing, vortex mixing, turbulent mixing, or a combination thereof.

23. The method of any one of claims 1 to 6, further comprising concentrating or buffer exchanging the GalNAc-LNP using a tangential flow filtration (TFF) process.

24. 7. The method of any one of claims 1 to 6, further comprising performing buffer exchange using a chromatography, dialysis, or TFF process.

25. The method of any one of claims 1 to 6, wherein the GalNAc-lipid comprises one or more N-acetylgalactosamine (GalNAc) or GalNAc derivatives.

26. 26. The method of claim 25, wherein the GalNAc-lipid is selected from the structures identified in Table 4.

27. 7. The method of claim 1, wherein the mixing is performed by an in-line mixing device having a first mixing chamber comprising a first port through which the first solution is separately introduced into the first mixing chamber and a second port through which the second solution is separately and simultaneously introduced into the first mixing chamber.

28. 7. The method of any one of claims 1 to 6, wherein the mixing comprises a mixing chamber, a first port for separately introducing a first solution into the mixing chamber, and a second port for separately introducing a second solution into the mixing chamber.

29. 30. The method of claim 28, wherein the first solution and the second solution are introduced into the mixing chamber simultaneously.

30. 10. The method of claim 1, wherein the first solution comprises RNA.

31. 31. The method of Claim 30, wherein the RNA comprises a guide RNA (gRNA) and an mRNA encoding a gene editor nuclease or base editor.

32. 32. The method of claim 31, wherein the weight ratio of gNRA to mRNA is from about 0.1 to about 10.

33. 32. The method of claim 31, wherein the weight ratio of gNRA to mRNA is from about 0.5 to about 2.

34. The concentration (mol %) of GalNAc-lipid is from about 0.01 mol % to about 10 mol %. The method according to any one of claims 1 to 6.

35. The neutral lipid is distearoylphosphatidylcholine (DSPC), the stealth lipid is polyethylene glycol-dimyristoylglycerol (PEG-DMG), or 7. The method of claim 1, wherein the concentration of stealth lipids in the second solution is from 0 mol% to about 5 mol%.

36. 7. The method of any one of claims 1 to 6, wherein the concentration of the nucleic acid agent is about 0.1 to about 5 mg / mL (e.g., about 0.1, 0.25, 0.5, 0.75, 1, 1.5, 2, 2.5, 3, 4, or 5 mg / mL).

37. The method of any one of claims 1 to 6, wherein the mixture is incubated for about 1 minute to about 24 hours.

38. 7. The method of any one of claims 1 to 6, further comprising introducing GalNAc-lipid into the second solution at a concentration of at least 0.01 (e.g., at least 0.01, 0.05, 0.1, or 0.5) mol% relative to the total volume.

39. 39. The method of claim 38, further comprising introducing GalNAc-lipids into the second solution at a concentration of at least 1 mol% by total volume, at least 3 mol% by total volume, at least 5 mol% by total volume, at least 7 mol% by total volume, at least 9 mol% by total volume, or at least 10 mol% by total volume.

Images