Modification template for improving inhibitory effect of double-stranded oligonucleotide on target gene, combination thereof and use thereof

Abstract

A modification template for improving the inhibitory effect of a double-stranded oligonucleotide on a target gene, a combination thereof and the use thereof. The double-stranded oligonucleotide is an siRNA and contains a sense strand and an antisense strand, and each nucleotide of the sense strand and antisense strand is a modified nucleotide. The double-stranded oligonucleotide is modified with a special modification template, and has a significantly improved inhibition rate on a target gene compared with a basic sequence.

Description

Modified templates for enhancing the inhibitory effect of double-stranded oligonucleotide target genes and their combination and application

[0001] This application claims the benefit of priority to Chinese Patent Application No. 2023114633757 filed on November 6, 2023, Chinese Patent Application No. 2024113570897 filed on September 26, 2024, and Chinese Patent Application No. 2024109094050 filed on July 8, 2024. The entire contents of the aforementioned Chinese patent applications are incorporated herein by reference. Technical Field

[0002] The present disclosure belongs to the field of nucleic acid modification technology, and specifically relates to modified templates for enhancing the inhibitory effect of double-stranded oligonucleotide target genes, and combinations and applications thereof. Background Art

[0003] Oligonucleotide drugs, especially oligonucleotide drugs, are widely used because they act upstream of protein expression based on the central dogma and bind to mRNA related to pathogenic protein expression according to the Watson-Crick base pairing principle. They have high specificity and strong target regulation capabilities. Oligonucleotide drugs generally include antisense oligonucleotides (ASOs), small interfering RNA (siRNA), microRNA (miRNA), and nucleic acid aptamers.

[0004] Oligonucleotides are short DNA or RNA molecules or oligomers that readily bind in a sequence-specific manner to their respective complementary oligonucleotides, DNA, or RNA, forming duplexes or, less commonly, higher-order hybrids. This fundamental property has led to their widespread application in genetic testing, research, and medicine. In nature, oligonucleotides are often small RNA molecules that play a role in regulating gene expression or intermediates derived from the degradation of larger nucleic acid molecules.

[0005] RNA interference (RNAi) is a natural defense mechanism against foreign genes. siRNA can knock out target genes by recognizing specific sequences and breaking down the target mRNA.

[0006] Classic RNAi molecules are composed of a signature 19+2 nucleotide polymer structure (a double helix consisting of a 21-nucleotide RNA molecule and 19 corresponding nucleobases, with a 2-nucleotide 3' overhang). One strand of the siRNA (the guide or antisense strand) is complementary to the target gene's mRNA transcript, while the other strand is designated the passenger (sense) strand. The siRNA (antisense strand) guides the Argonaute protein (AGO2) to complement the target transcript and becomes part of the RNA-guided silencing complex (RISC). The perfect complementarity between the siRNA (antisense strand) and the target leads to cleavage of the target transcript at positions 10-11 opposite the guide (antisense) strand, catalyzed by the AGO2 protein.

[0007] siRNA possesses inherent advantages over small molecule and antibody drugs. SiRNA performs its function by performing Watson–Crick base pairing with mRNA, whereas small molecule and monoclonal antibody drugs must recognize the complex spatial structure of specific proteins. Consequently, many diseases are resistant to small molecule and monoclonal antibody treatments because their target molecules are highly active and cannot recognize molecular structures with affinity and binding specificity. The mechanism of action of siRNA drugs enables them to regulate the expression of target proteins at the genetic level, offering greater targeting specificity than small molecule or antibody drugs. Its mechanism, based on the principle of complementary base pairing, also broadens the therapeutic scope of siRNA, simplifies design, and shortens the development cycle.

[0008] Oligonucleotides can sequence-specifically bind to complementary RNA strands, and upon hybridization, they can induce RNase H to cleave target RNA. The nucleotides in natural oligonucleotides are linked by phosphodiester bonds. Under physiological conditions, they are particularly sensitive to nucleases. Therefore, natural, unmodified, and unstructured oligonucleotide drugs are easily and rapidly degraded by nucleases in vivo, resulting in low activity and poor drugability. Chemical modification of oligonucleotide structure is an effective way to enhance their activity, improving their stability to nucleases and affinity for RNA, and better promoting cellular endocytosis and tissue targeting, thereby effectively regulating the expression of target genes.

[0009] Based on the basic structure of oligonucleotides, bases, sugar rings and phosphate backbones, chemical modifications can be performed in three parts:

[0010] 1) Base modifications: These are mainly categorized into three types: purine modifications, pyrimidine modifications, and base substitutions. Purine modifications include N6-methyladenosine, N1-methyladenosine, and 7-methylguanylate; pyrimidine modifications include 3-methyluridine, 5-methyluridine, 5-methylcytosine, N4-acetylcytidine, pseudouridine, thiouridine, propyneuridine, and dihydrouridine.

[0011] 2) Modification of sugar ring: mainly divided into modification and replacement of sugar ring. Modification of sugar ring includes 2'-modification, 4'-modification, 5'-modification, isomeric modification, and combinations of these modifications. The most common in oligonucleotides are 2'-fluoro modification, 2'-alkoxy (such as 2'-O-methoxyethyl (ie 2'-MOE), 2'-methoxy) modification, 2,4'-LNA (2,4'-locked nucleotide) modification, 2,4'-cEt (2,4'-restricted ethyl) modification, etc. Compared with natural oligonucleotides, modified oligonucleotides have higher Tm values, stronger serum stability and better activity.

[0012] 3) Modification of the phosphate backbone: mainly modification of phosphorothioate; modification with methyl phosphate, selenophosphate, methylborylphosphate, dithiophosphate, and replacement of the bridging oxygen atom in the phosphodiester bond connection region with a sulfur atom; replacement of the entire phosphate group between nucleosides with a group that does not contain a phosphorus atom, such as replacing the P atom with a C atom, S atom and / or N atom to form a guanidine group, S-methylthiourea, etc.

[0013] 4) Terminal modification: covalent conjugation of specific groups at the 5' end and / or 3' end of the sense strand and phosphorylation modification of the 5' end of the antisense strand.

[0014] All oligonucleotide drugs on the market have been chemically modified. Since the first oligonucleotide drug, Fomivirsen (Vitravene), was approved for marketing in 1998, the chemical modification technology of oligonucleotide drugs has been continuously upgraded. To date, 22 oligonucleotide drugs have been approved for marketing worldwide:

[0015] Table 1 Oligonucleotide drugs approved for marketing worldwide

[0016] There is a need in this field to further improve the chemical modification technology of oligonucleotide drugs so as to further enhance the efficacy of oligonucleotide drugs. Summary of the Invention

[0017] This disclosure is based on the basic sequences of small nucleic acid molecules, including single-stranded oligonucleotides and siRNA duplexes, and modifies these basic sequences with different modification patterns to prepare corresponding modified products. Based on this, this disclosure provides modification templates for enhancing the target gene inhibition effect of double-stranded oligonucleotides, as well as their combinations and applications.

[0018] The present disclosure solves the above technical problems through the following technical solutions.

[0019] In one aspect, the present disclosure provides a modified template for enhancing the target gene inhibition effect of a double-stranded oligonucleotide, wherein the double-stranded oligonucleotide is an siRNA, and the double-stranded oligonucleotide comprises a sense strand and an antisense strand, wherein each nucleotide of the sense strand and the antisense strand is a modified nucleotide, wherein the sense strand comprises a nucleotide sequence 1, the antisense strand comprises a nucleotide sequence 2, and the nucleotide sequence 1 and the nucleotide sequence 2 are at least partially reverse-complementary to form a double-stranded region; the length of the reverse-complementary double-stranded region is 17 to 21 bp;

[0020] From the 5' end to the 3' end, at least two nucleotides among the 2nd to 6th nucleotides of the nucleotide sequence 2 are 2'-fluoro-modified nucleotides, the 14th and / or 16th nucleotides are 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are 2'-methoxy-modified nucleotides, and the 1st, 2nd and 2nd to 3rd positions have a thiophosphate backbone.

[0021] In some embodiments of the present disclosure, at least two nucleotides in nucleotides 2-6 of the nucleotide sequence 2 are selected from one of the following combinations:

[0022] (1) Nucleotides 2 and 6;

[0023] (2) nucleotides 2, 3, 4, and 6;

[0024] (3) nucleotides 2, 4, 5, and 6;

[0025] (4) nucleotides 2, 5, and 6;

[0026] (5) nucleotides 2, 3, 5, and 6;

[0027] (6) Nucleotides 2, 3, and 6.

[0028] In some embodiments of the present disclosure, the nucleotide sequence 1 is modified in the following manner from the 5' end to the 3' end:

[0029] (1) The nucleotides at positions 6 and 16 are 2'-fluoro-modified nucleotides, at least two nucleotides among positions 8-10, preferably positions 8-10 or positions 8 and 10, are 2'-fluoro-modified nucleotides, the nucleotides at the remaining positions are 2'-methoxy-modified nucleotides, and positions 1 and 2 have a phosphorothioate backbone; or

[0030] (2) The nucleotides at positions 7 and 17 are 2'-fluoro-modified nucleotides, at least two nucleotides among the nucleotides at positions 9-11, preferably the nucleotides at positions 9-11 or the nucleotides at positions 9 and 11 are 2'-fluoro-modified nucleotides, the nucleotides at the remaining positions are 2'-methoxy-modified nucleotides, and the nucleotides at positions 1 and 2 have a thiophosphate backbone.

[0031] In some embodiments of the present disclosure, the length of the nucleotide sequence 1 is 21 or 20, and the length of the nucleotide sequence 2 is 23 or 22.

[0032] In some embodiments of the present disclosure, the length of the nucleotide sequence 1 is 20, and the length of the nucleotide sequence 2 is 22; or, the length of the nucleotide sequence 1 is 21, and the length of the nucleotide sequence 2 is 23.

[0033] In some embodiments of the present disclosure, the nucleotides in the antisense strand are modified in any of the following ways A, B, C, D, E, F and G, in the direction from the 5' end to the 3' end:

[0034] A: indicates that the antisense strand starts from the 5' end, nucleotides 1, 3, 4, 5, 7, 8, 9, 10, 11, 12, 13, 15, 17, 18, 19, 20, 21, 22, and 23 are 2'-methoxy-modified nucleotides, and nucleotides 2, 6, 14, and 16 are 2'-fluoro-modified nucleotides, and positions 1, 2, 21, and 22 have a phosphorothioate backbone;

[0035] B: Indicates that the antisense strand starts from the 5' end, nucleotides 1, 5, 7, 8, 9, 10, 11, 12, 13, 15, 17, 18, 19, 20, 21, 22, and 23 are 2'-methoxy-modified nucleotides, and nucleotides 2, 3, 4, 6, 14, and 16 are 2'-fluoro-modified nucleotides, and positions 1, 2, 21, and 22 have a phosphorothioate backbone;

[0036] C: indicates that the antisense strand starts from the 5' end, nucleotides 1, 3, 7, 8, 9, 10, 11, 12, 13, 15, 17, 18, 19, 20, 21, 22, and 23 are 2'-methoxy-modified nucleotides, and nucleotides 2, 4, 5, 6, 14, and 16 are 2'-fluoro-modified nucleotides, and positions 1, 2, 21, and 22 have a phosphorothioate backbone;

[0037] D: indicates that the antisense strand starts from the 5' end, nucleotides 1, 3, 4, 7, 8, 9, 10, 11, 12, 13, 15, 17, 18, 19, 20, 21, 22, and 23 are 2'-methoxy-modified nucleotides, and nucleotides 2, 5, 6, 14, and 16 are 2'-fluoro-modified nucleotides, and positions 1, 2, 21, and 22 have a phosphorothioate backbone;

[0038] E: indicates that the antisense strand starts from the 5' end, nucleotides 1, 4, 7, 8, 9, 10, 11, 12, 13, 15, 17, 18, 19, 20, 21, 22, and 23 are 2'-methoxy-modified nucleotides, and nucleotides 2, 3, 5, 6, 14, and 16 are 2'-fluoro-modified nucleotides, and positions 1, 2, 21, and 22 have a phosphorothioate backbone;

[0039] F: indicates that the antisense strand starts from the 5' end, and the nucleotides at positions 1, 4, 5, 7, 8, 9, 10, 11, 12, 13, 15, 17, 18, 19, 20, 21, 22, and 23 are 2'-methoxy-modified nucleotides, and the nucleotides at positions 2, 3, 6, 14, and 16 are 2'-fluoro-modified nucleotides, and the nucleotides at positions 1, 2, 21, and 22 have a thiophosphate backbone; G: indicates that the antisense strand starts from the 5' end, and the nucleotides at positions 1, 3, 4, 5, 7, 8, 9, 10, 11, 12, 13, 15, 17, 18, 19, 20, 21, 22, and 23 are 2'-methoxy-modified nucleotides, and the nucleotides at positions 2, 6, 14, and 16 are 2'-fluoro-modified nucleotides, and the nucleotides at positions 1, 2, 21, and 22 have a thiophosphate backbone;

[0040] H: indicates that the antisense strand starts from the 5' end, nucleotides 1, 3, 4, 5, 7, 8, 9, 10, 11, 12, 13, 15, 16, 17, 18, 19, 20, 21, 22, and 23 are 2'-methoxy-modified nucleotides, and nucleotides 2, 6, and 14 are 2'-fluoro-modified nucleotides, and positions 1, 2, 21, and 22 have a phosphorothioate backbone;

[0041] And / or, in the direction from the 5' end to the 3' end, the nucleotides in the sense strand are modified by any of the following methods a to d:

[0042] a: indicates that the 1st, 2nd, 3rd, 4th, 5th, 6th, 8th, 12th, 13th, 14th, 15th, 16th, 18th, 19th, 20th, and 21st nucleotides starting from the 5' end of the sense strand are 2'-methoxy-modified nucleotides, and the 7th, 9th, 10th, 11th, and 17th nucleotides are 2'-fluoro-modified nucleotides, and the 1st and 2nd nucleotides have a phosphorothioate backbone;

[0043] b: Indicates that the 1st, 2nd, 3rd, 4th, 5th, 6th, 8th, 10th, 12th, 13th, 14th, 15th, 16th, 18th, 19th, 20th, and 21st nucleotides starting from the 5' end of the sense strand are 2'-methoxy-modified nucleotides, and the 7th, 9th, 11th, and 17th nucleotides are 2'-fluoro-modified nucleotides, and the 1st and 2nd nucleotides have a thiophosphate backbone;

[0044] c: Indicates that the 1st, 2nd, 3rd, 4th, 5th, 6th, 8th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, 19th, 20th, and 21st nucleotides starting from the 5' end of the sense strand are 2'-methoxy-modified nucleotides, and the 7th, 9th, 10th, and 11th nucleotides are 2'-fluoro-modified nucleotides, and the 1st and 2nd nucleotides have a thiophosphate backbone;

[0045] d: Indicates that the 1st, 2nd, 3rd, 4th, 5th, 6th, 8th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, 19th, 20th, and 21st nucleotides starting from the 5' end of the sense strand are 2'-methoxy-modified nucleotides, and the 7th, 9th, and 10th nucleotides are 2'-fluoro-modified nucleotides, and the 1st and 2nd nucleotides have a thiophosphate backbone.

[0046] In some embodiments of the present disclosure, the nucleotides in the antisense strand are modified in any of the following ways: A', B', C', D', E', F' and G', in the direction from the 5' end to the 3' end:

[0047] A': indicates that the antisense strand starts from the 5' end, nucleotides 1, 3, 4, 5, 7, 8, 9, 10, 11, 12, 13, 15, 17, 18, 19, 20, 21, and 22 are 2'-methoxy-modified nucleotides, and nucleotides 2, 6, 14, and 16 are 2'-fluoro-modified nucleotides, and positions 1, 2, 20, and 21 have a phosphorothioate backbone;

[0048] B': indicates that the antisense strand starts from the 5' end, nucleotides 1, 5, 7, 8, 9, 10, 11, 12, 13, 15, 17, 18, 19, 20, 21, and 22 are 2'-methoxy-modified nucleotides, and nucleotides 2, 3, 4, 6, 14, and 16 are 2'-fluoro-modified nucleotides, and positions 1, 2, 20, and 21 have a phosphorothioate backbone;

[0049] C': indicates that the antisense strand starts at the 5' end, nucleotides 1, 3, 7, 8, 9, 10, 11, 12, 13, 15, 17, 18, 19, 20, 21, and 22 are 2'-methoxy-modified nucleotides, and nucleotides 2, 4, 5, 6, 14, and 16 are 2'-fluoro-modified nucleotides, and positions 1, 2, 20, and 21 have a phosphorothioate backbone;

[0050] D': indicates that the antisense strand starts at the 5' end, nucleotides 1, 3, 4, 7, 8, 9, 10, 11, 12, 13, 15, 17, 18, 19, 20, 21, and 22 are 2'-methoxy-modified nucleotides, and nucleotides 2, 5, 6, 14, and 16 are 2'-fluoro-modified nucleotides, and positions 1, 2, 20, and 21 have a phosphorothioate backbone;

[0051] E': indicates that the antisense strand starts from the 5' end, nucleotides 1, 4, 7, 8, 9, 10, 11, 12, 13, 15, 17, 18, 19, 20, 21, and 22 are 2'-methoxy-modified nucleotides, and nucleotides 2, 3, 5, 6, 14, and 16 are 2'-fluoro-modified nucleotides, and positions 1, 2, 20, and 21 have a phosphorothioate backbone;

[0052] F': indicates that the antisense strand starts at the 5' end, nucleotides 1, 4, 5, 7, 8, 9, 10, 11, 12, 13, 15, 17, 18, 19, 20, 21, and 22 are 2'-methoxy-modified nucleotides, and nucleotides 2, 3, 6, 14, and 16 are 2'-fluoro-modified nucleotides, and positions 1, 2, 20, and 21 have a phosphorothioate backbone;

[0053] G': indicates that the antisense strand starts at the 5' end, nucleotides 1, 3, 4, 5, 7, 8, 9, 10, 11, 12, 13, 15, 16, 17, 18, 19, 20, 21, and 22 are 2'-methoxy-modified nucleotides, and nucleotides 2, 6, and 14 are 2'-fluoro-modified nucleotides, and positions 1, 2, 20, and 21 have a phosphorothioate backbone;

[0054] And / or, in the direction from the 5' end to the 3' end, the nucleotides in the sense strand are modified by any of the following methods a', b', c' and d':

[0055] a': indicates that the 1st, 2nd, 3rd, 4th, 5th, 7th, 11th, 12th, 13th, 14th, 15th, 17th, 18th, 19th, and 20th nucleotides starting from the 5' end of the sense strand are 2'-methoxy-modified nucleotides, and the 6th, 8th, 9th, 10th, and 16th nucleotides are 2'-fluoro-modified nucleotides, and the 1st and 2nd nucleotides have a phosphorothioate backbone;

[0056] b': indicates that the 1st, 2nd, 3rd, 4th, 5th, 7th, 9th, 11th, 12th, 13th, 14th, 15th, 17th, 18th, 19th, and 20th nucleotides starting from the 5' end of the sense strand are 2'-methoxy-modified nucleotides, and the 6th, 8th, 10th, and 16th nucleotides are 2'-fluoro-modified nucleotides, and the 1st and 2nd nucleotides have a phosphorothioate backbone;

[0057] c': indicates that the 1st, 2nd, 3rd, 4th, 5th, 7th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, 19th, and 20th nucleotides starting from the 5' end of the sense strand are 2'-methoxy-modified nucleotides, and the 6th, 8th, 9th, and 10th nucleotides are 2'-fluoro-modified nucleotides, and the 1st and 2nd nucleotides have a thiophosphate backbone:

[0058] d': indicates that the 1st, 2nd, 3rd, 4th, 5th, 7th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, 19th, and 20th nucleotides starting from the 5' end of the sense strand are 2'-methoxy-modified nucleotides, and the 6th, 8th, and 9th nucleotides are 2'-fluoro-modified nucleotides, and the 1st and 2nd nucleotides have a thiophosphate backbone.

[0059] In some embodiments of the present disclosure, the double-stranded oligonucleotide is modified in any of the following combinations (i) to (x) from the 5' end to the 3' end:

[0060] (i) the antisense strand is modified using method A, and the sense strand is modified using method a;

[0061] (ii) the antisense strand is modified using method B, and the sense strand is modified using method a;

[0062] (iii) the antisense strand is modified using method C, and the sense strand is modified using method a;

[0063] (iv) the antisense strand is modified using method B, and the sense strand is modified using method b;

[0064] (v) the antisense strand is modified using method C, and the sense strand is modified using method b;

[0065] (vi) the antisense strand is modified using method G, and the sense strand is modified using method C;

[0066] (vi) the antisense strand is modified using method A, and the sense strand is modified using method d;

[0067] (viii) the antisense strand is modified using method D, and the sense strand is modified using method b;

[0068] (ix) the antisense strand is modified using method E, and the sense strand is modified using method b;

[0069] (x) The antisense strand is modified using method F, and the sense strand is modified using method b.

[0070] In some embodiments of the present disclosure, the double-stranded oligonucleotide is modified in the direction from the 5' end to the 3' end by any of the following combinations (1)-(8):

[0071] (1) The antisense strand is modified using method A', and the sense strand is modified using method a';

[0072] (2) the antisense strand is modified using method B', and the sense strand is modified using modification method a';

[0073] (3) the antisense strand is modified using method C', and the sense strand is modified using modification method a';

[0074] (4) the antisense strand is modified using method B', and the sense strand is modified using modification method b';

[0075] (5) The antisense strand is modified using method C', and the sense strand is modified using modification method b';

[0076] (6) The antisense strand is modified using method D', and the sense strand is modified using modification method b';

[0077] (7) The antisense strand is modified using method E', and the sense strand is modified using modification method b';

[0078] (8) The antisense strand is modified using method F', and the sense strand is modified using modification method b'.

[0079] In some embodiments of the present disclosure, the first nucleotide at the 5' end of the antisense strand is a 5'-phosphate nucleotide or a 5'-phosphate analog modified nucleotide, which includes but is not limited to the following 5'-phosphorylated groups: 5'-vinylphosphonate group (5'-E-VP); 5'-methylphosphonate group (5'-MP); 5'-C-methylphosphonate group; 5'-thiophosphate group (5'-PS); 5'-phosphate group (5'-P), the structure of which is shown as follows:

[0080] Wherein, R is hydrogen, hydroxyl, amino, C 1-4 Alkyl, aromatic, C 1-4 Alkoxy, C 1-4 alkylcarbonylamino or halogen;

[0081] The base is selected from adenine, guanine, cytosine, thymine and uracil.

[0082] In some embodiments of the present disclosure, the first nucleotide at the 5' end of the antisense strand is a 5'-vinyl-(E)-phosphonate-2'-methoxy modified nucleotide.

[0083] In some embodiments of the present disclosure, one or more nucleotides from positions 2 to 8, preferably positions 6 and / or 7, from the 5' end of the antisense strand adopt a modification group, wherein the modification group is preferably selected from UNA, GNA or DNA, wherein the structures of UNA and GNA are as follows:

[0084] The base is selected from one or more of adenine, guanine, cytosine, thymine and uracil.

[0085] Another aspect of the present invention provides a double-stranded oligonucleotide modified with the modified template described in the present disclosure.

[0086] In some embodiments of the present disclosure, the double-stranded oligonucleotide targets one of the following mRNAs: PCSK9 gene, HBV gene, and AGT gene.

[0087] Another aspect of the present disclosure provides a conjugate for reducing gene expression, wherein the conjugate comprises the double-stranded oligonucleotide as described in the present disclosure, and a ligand conjugated thereto.

[0088] In some embodiments of the present disclosure, the ligand is conjugated to the 3'-end or 5'-end of the sense strand of the double-stranded oligonucleotide.

[0089] In some embodiments of the present disclosure, the ligand is one or more GalNAc derivatives attached using a bivalent or trivalent branched linkage.

[0090] In some embodiments of the present disclosure, the ligand is:

[0091] wherein X is hydrogen or a hydroxyl protecting group or H, wherein the hydroxyl protecting group includes acetyl, benzoyl or isobutyryl; Y is an amine protecting group or H, wherein the amine protecting group is formyl, acetyl, propionyl, n-butyryl or isobutyryl; n is an integer between 0 and 20; and q, r and s are independently integers between 1 and 7.

[0092] In some embodiments of the present disclosure, the ligand is:

[0093] In some embodiments of the present disclosure, the ligand is:

[0094] Wherein, X is oxygen, nitrogen or sulfur;

[0095] Y is an alkyl group or an aromatic group;

[0096] R1 is oxygen or sulfur;

[0097] R2 is hydrogen, amino, C 1-4 Alkyl, aromatic, C 1-4 Alkoxy or halogen;

[0098] A is -(CH2) a -、-(CH2CH2O) b-、-((CH2) c NHCO) d -or-((CH2) c CONH) d -, wherein a is an integer from 1 to 15, b is an integer from 1 to 7, c is an integer from 1 to 7, and d is an integer from 1 to 5;

[0099] B is -(CH2) e -, where e is an integer from 0 to 7;

[0100] L is -CONH- or -NHCO-;

[0101] X1 is -(CH2) f -or-(CH2CH2O) f CH2-, f is an integer from 1 to 5;

[0102] X2 is -(CH2) g -, g is an integer from 1 to 6;

[0103] Y1 is 0 or 1;

[0104] Y2 is 0, 1, or 2;

[0105] Y3 is 1, 2 or 3;

[0106] m is an integer from 0 to 4;

[0107] n is an integer from 0 to 4.

[0108] In some embodiments of the present disclosure, the ligand is G4, G5, G6, G7, G101, G102, G103, G105 or G106:

[0109] In some embodiments of the present disclosure, the conjugate has the structure shown below:

[0110] In some embodiments of the present disclosure, the double-stranded oligonucleotide is conjugated to ligand G4, G5, G6 or G7.

[0111] In another aspect, the present disclosure further provides a pharmaceutical composition comprising the double-stranded oligonucleotide of the present disclosure or the conjugate of the present disclosure, and a pharmaceutically acceptable carrier.

[0112] In some embodiments of the present disclosure, the carrier is water, saline or a buffer solution.

[0113] In some embodiments of the present disclosure, the buffering agent in the buffer solution comprises acetate, citrate, prolamin, carbonate or phosphate, Tris-HCl or any combination thereof; for example, comprises phosphate.

[0114] On the other hand, the present disclosure also provides the use of the double-stranded oligonucleotide as described in the present disclosure, the conjugate as described in the present disclosure, or the pharmaceutical composition as described in the present disclosure in the preparation of a drug for regulating or treating diseases and conditions; the drug downregulates gene expression through the double-stranded oligonucleotide, the conjugate or the pharmaceutical composition to achieve the effect of regulating or treating diseases and conditions.

[0115] In some embodiments of the present disclosure, the gene is selected from one of the following genes: PCSK9 gene, HBV gene and AGT gene.

[0116] On the other hand, the present disclosure also provides a double-stranded oligonucleotide as described in the present disclosure, a conjugate as described in the present disclosure, or a pharmaceutical composition as described in the present disclosure, for use in regulating or treating diseases and disorders; the double-stranded oligonucleotide, the conjugate, or the pharmaceutical composition downregulates gene expression to achieve the effect of regulating or treating diseases and disorders.

[0117] The present disclosure also provides a method for treating or preventing diseases and disorders that can be regulated or treated by downregulating gene expression, comprising administering to a subject in need thereof an effective amount of the double-stranded oligonucleotide, the conjugate, or the pharmaceutical composition of the present disclosure.

[0118] In some embodiments of the present disclosure, the gene is selected from one of the following genes: PCSK9 gene, HBV gene and AGT gene.

[0119] In some embodiments of the present disclosure, the disease is selected from one or more of chronic hepatitis B, liver fibrosis, liver cirrhosis, liver cancer, acute hepatitis B, and HBV / hepatitis D virus co-infection-related diseases.

[0120] In some embodiments of the present disclosure, the disease is selected from hypertension, critical hypertension, essential hypertension, secondary hypertension, hypertensive crisis, hypertensive emergency, isolated systolic and diastolic hypertension, pregnancy-related hypertension, diabetic hypertension, resistant hypertension, refractory hypertension, paroxysmal hypertension, renovascular hypertension, Goldblatt's hypertension, ocular hypertension, glaucoma, pulmonary hypertension, portal hypertension, systemic venous hypertension, systolic hypertension, unstable hypertension; hypertensive heart disease, hypertensive nephropathy, atherosclerosis, arteriosclerosis vascular disease, diabetic nephropathy, diabetic retinopathy, chronic heart failure, cardiomyopathy, diabetic cardiomyopathy, glomerulosclerosis, coarctation of the aorta, aortic aneurysm, ventricular fibrosis, Cushing's syndrome and other glucocorticoid excess states (including chronic steroid therapy), pheochromocytoma, nephroblastoma, secondary aldosteronism and other mineralocorticoid excess states, sleep apnea, thyroid / parathyroid disease, heart failure, myocardial infarction, angina pectoris, stroke, diabetes mellitus, kidney disease, renal failure, systemic sclerosis, intrauterine growth restriction (IUGR), and fetal growth restriction.

[0121] In some embodiments of the present disclosure, the disease is selected from one of hypercholesterolemia, atherosclerosis, dyslipidemia and cardiovascular and cerebrovascular diseases.

[0122] On the basis of conforming to the common sense in this field, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0123] The reagents and raw materials used in the present invention are commercially available.

[0124] The positive progress effect of the present invention is:

[0125] (1) Using multiple modified sequences, the inhibition rate can reach up to 90%. In addition, the modified sequence is conjugated with a GalNAc compound, which can be efficiently delivered to the animal liver and significantly inhibit target gene expression and significantly reduce blood pressure levels.

[0126] (2) The target gene inhibition activity of each sequence modified by the modified template disclosed in the present invention was significantly improved, with the maximum improvement being 91.0%, compared with the unmodified sequence disclosed in the prior art, which had very little sequence difference. BRIEF DESCRIPTION OF THE DRAWINGS

[0127] In order to more clearly illustrate the technical solutions of the embodiments of the present disclosure, the drawings of the embodiments will be briefly introduced below. Obviously, the drawings in the following description only relate to some embodiments of the present disclosure, rather than limiting the present disclosure.

[0128] FIG1 shows the alternatively modified sequences that have a significant inhibitory effect on the PCSK9 gene, and the inhibition rates of these sequences are all higher than 50%.

[0129] FIG2 shows the alternating modified sequences with an inhibition rate of 30% to 50% on the PCSK9 gene.

[0130] FIG3 shows the alternative modified sequences with an inhibition rate of less than 30% on the PCSK9 gene.

[0131] FIG4 shows the alternative modified sequences with an inhibition rate of less than 30% on the PCSK9 gene.

[0132] FIG5 shows the unmodified sequences that have a significant inhibitory effect on the PCSK9 gene, and the inhibition rates of these sequences are all higher than 50%.

[0133] FIG6 shows that the unmodified sequence has an inhibition rate of less than 40% on the PCSK9 gene.

[0134] FIG7 shows the inhibition rate of the PCSK9 gene after basic sequence 5 is modified with different templates.

[0135] FIG8 shows the inhibition rate of the PCSK9 gene by basic sequences 5, 51, 81 and 84 using template modification, anti-off-target and / or 5'-E-VP modification sequences.

[0136] FIG9 shows the inhibition rate of PCSK9 protein in animal serum after basic sequences 5, 51, 81, 82 and 84 were modified with different schemes and conjugated with GalNAc compounds.

[0137] FIG10 shows the effects of different modification schemes of basic sequences 5, 51, 81, 82 and 84 on the reduction of low-density lipoprotein cholesterol (LDL-C) in animal serum after conjugation with GalNAc compounds.

[0138] FIG11 shows that after basic sequences 5, 51, 81, 82 and 84 are modified with different schemes and conjugated with GalNAc compounds, the total cholesterol (TC) level in animal serum is reduced.

[0139] FIG12A shows the serum HBsAg level in AAV-HBV model mice at 3 mg / kg level of candidate siRNA sequences.

[0140] Figure 12B shows the serum HBV DNA level in AAV-HBV model mice at 3 mg / kg level of candidate siRNA sequences.

[0141] Figure 12C shows the serum HBeAg level in AAV-HBV model mice at 3 mg / kg level of candidate siRNA sequences.

[0142] Figure 12D shows the serum HBsAb levels in AAV-HBV model mice at 3 mg / kg of the candidate siRNA sequence.

[0143] Figure 12E shows the serum ALT level in AAV-HBV model mice at 3 mg / kg level of candidate siRNA sequences.

[0144] Figure 12F shows the changes in body weight of AAV-HBV model mice at the 3 mg / kg level of the candidate siRNA sequence.

[0145] Figure 13A shows the serum HBsAg levels in mice after the second challenge.

[0146] Figure 13B shows the serum HBV DNA level in mice after the second challenge.

[0147] Figure 13C shows the serum HBeAg levels of mice after the second challenge.

[0148] Figure 13D shows the serum HBbAg levels of mice after the second challenge.

[0149] Figure 13E shows the serum ALT levels of mice after the second challenge.

[0150] Figure 13F shows the changes in mouse body weight after the second challenge.

[0151] Figure 14A shows the HBsAg levels in AAV-HBV model mice at 0.5 mg / kg of the candidate siRNA sequence.

[0152] Figure 14B shows the HBV DNA level in AAV-HBV model mice at 0.5 mg / kg of the candidate siRNA sequence.

[0153] Figure 14C shows the HBeAg level in AAV-HBV model mice at 0.5 mg / kg level of candidate siRNA sequences.

[0154] Figure 14D shows the HBsAb levels in AAV-HBV model mice at 0.5 mg / kg of the candidate siRNA sequence.

[0155] Figure 14E shows the ALT level in AAV-HBV model mice at 0.5 mg / kg of the candidate siRNA sequence.

[0156] Figure 14F shows the changes in body weight of AAV-HBV model mice at the 0.5 mg / kg level of the candidate siRNA sequence.

[0157] FIG15A shows the HBsAg levels in AAV-HBV model mice of candidate siRNA sequences coupled with different GalNAc.

[0158] FIG15B shows the HBV DNA levels in AAV-HBV model mice of candidate siRNA sequences conjugated with different GalNAc.

[0159] FIG15C shows the HBeAg levels in AAV-HBV model mice of candidate siRNA sequences coupled with different GalNAc.

[0160] FIG15D shows the ALT levels in AAV-HBV model mice of candidate siRNA sequences coupled with different GalNAc.

[0161] Figure 15E shows the weight changes of AAV-HBV model mice coupled with different GalNAc candidate siRNA sequences.

[0162] FIG16 shows the alternatively modified sequences that have a significant inhibitory effect on the AGT gene. The inhibition rates of these sequences are all higher than 40%.

[0163] FIG. 17 shows the alternating modified sequences with an AGT gene inhibition rate between 25% and 40%.

[0164] FIG. 18 shows alternately modified sequences with an AGT gene inhibition rate of less than 25%.

[0165] FIG19 shows the unmodified sequences that have a significant inhibitory effect on the AGT gene. The inhibition rates of these sequences are all higher than 45%.

[0166] FIG. 20 shows that the AGT gene inhibition rate of the unmodified sequence was less than 45%.

[0167] FIG21 shows the inhibition rate of the AGT gene by the unmodified sequence 1579 after modification with different templates and the inhibition rate of the sequence 579P disclosed in the prior art.

[0168] FIG22 shows the inhibition rate of the AGT gene by the unmodified sequences 1578s and 1835 using template modification and anti-off-target modification sequences.

[0169] FIG23 shows the inhibition rate of AGT protein in animal serum after the unmodified sequences 1579, 1789, 1812, 1576s, 1578s, and 1585s were modified with different schemes and conjugated with GalNAc compounds.

[0170] Figure 24 shows the inhibition rates of AGT protein in animal serum at different time points after the unmodified sequences 1579, 1789, 1812, 1576s, 1578s, and 1585s were modified with different schemes and conjugated with GalNAc compounds. DETAILED DESCRIPTION

[0171] In order to make this disclosure more easily understood, some terms are first defined. In addition, it should be noted that whenever a value or a range of values ​​for a parameter is listed, it is intended to indicate that values ​​and ranges intermediate to these cited values ​​are also part of this disclosure.

[0172] As used herein, the articles "a" and "an" refer to one or more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" refers to one element or more than one element, such as a plurality of elements.

[0173] The term "including" is used herein to mean, and is used interchangeably with, the phrase "including, but not limited to."

[0174] The term "or" is used herein to mean, and is used interchangeably with, the term "and / or," unless the context clearly dictates otherwise.

[0175] As used herein, the term "approximately" or "approximately" as applied to one or more target values ​​refers to a value similar to the reference value. In certain embodiments, unless otherwise indicated or in addition apparent from context, the term "approximately" or "approximately" refers to a value falling within 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less of the reference value in either direction (greater than or less than) or less (unless such numerals will exceed 100% of possible values).

[0176] As used herein, "PCSK9" refers to the proprotein convertase subtilisin Kexin9 gene or protein.

[0177] As used herein, "HBV" refers to hepatitis B virus, including hepatitis B virus of genotypes A, B, C, D, E, F, G, H, I, J and subtypes thereof, and is not limited to a certain genotype.

[0178] As used herein, "AGT" refers to the angiotensinogen gene or protein.

[0179] "G," "C," "A," and "U" each generally represent a nucleotide comprising guanine, cytosine, adenine, and uracil as a base, respectively. "T" and "dT" are used interchangeably herein and refer to a deoxyribonucleotide in which the nucleobase is thymine, such as deoxyribothymine, 2'-deoxythymidine, or thymidine. However, it will be understood that the term "ribonucleotide" or "nucleotide" or "deoxyribonucleotide" may also refer to a modified nucleotide (as further described below) or an alternative replacement moiety. The skilled artisan will be well aware that guanine, cytosine, adenine, and uracil may be replaced by other moieties without substantially altering the base pairing properties of an oligonucleotide (including a nucleotide having such a replacement moiety). For example, without limitation, a nucleotide comprising inosine as its base may base pair with a nucleotide containing adenine, cytosine, or uracil. Thus, a nucleotide comprising uracil, guanine, or adenine may be substituted in the nucleotide sequences of the present disclosure by a nucleotide comprising, for example, inosine. Sequences comprising such replacement moieties are suitable for use in the double-stranded oligonucleotides, conjugates, pharmaceutical compositions, and methods of the present disclosure.

[0180] The terms "siRNA duplex", "siRNA", "double-stranded oligonucleotide", "RNAi agent" are used interchangeably herein. It is a class of double-stranded RNA molecules, also known in the art as short interfering RNA or silencing RNA. siRNAs typically comprise a sense strand (also known as a passenger strand) and an antisense strand (also known as a leader strand), wherein each strand is 17 to 30 nucleotides in length, typically 19 to 25 nucleosides in length, wherein the antisense strand is complementary (such as at least 95% complementary, such as fully complementary) to the target nucleic acid (suitably a mature mRNA sequence), and the sense strand is complementary to the antisense strand such that the sense strand and the antisense strand form a duplex or duplex region. The siRNA strands can form blunt-ended duplexes, or preferably, the 3' ends of the sense and antisense strands can form 3' overhangs, e.g., 1, 2, or 3 nucleosides, similar to the products produced by Dicer, which can form RISC substrates in vivo. Effective extended forms of Dicer substrates have been described in US 8,349,809 and US 8,513,207, which are incorporated herein by reference. In some embodiments, the sense strand and / or antisense strand has a 3' overhang of 2 nucleotides. Thus, the length of the duplex region can be, for example, 17 to 25 nucleotides, such as 21 to 23 nucleotides in length. In some embodiments, each nucleotide of the siRNA contains a chemical modification.

[0181] The term "antisense strand" refers to the strand of an RNAi (e.g., siRNA) that includes a region that is substantially complementary to a target sequence. As used herein, the term "region of complementarity" refers to a region on the antisense strand that is substantially complementary to a sequence defined herein (e.g., a target sequence). When the region of complementarity is not completely complementary to the target sequence, mispairing can occur in the interior or terminal regions of the molecule. Typically, the most tolerated mispairing is in the terminal regions, e.g., within 5, 4, 3, or 2 nucleotides of the 5' and / or 3' ends.

[0182] The term "sense strand," as used herein, refers to the strand of an RNAi that includes a region that is substantially complementary to a region of the antisense strand (as that term is defined herein).

[0183] As used herein, "base sequence" refers to a single-stranded oligonucleotide, siRNA duplex, or the like that does not contain modified nucleotides. In some cases, it also refers to the corresponding nucleotide sequence of an antisense oligonucleotide or siRNA. As used herein, "antisense oligonucleotide base sequence," "unmodified ASO," and "ASO base sequence" are used interchangeably; "siRNA base sequence," "unmodified siRNA," and "siRNA duplex base sequence" are used interchangeably.

[0184] In the present disclosure, “other drugs that reduce the expression of PCSK9, HBV or AGT genes” means drugs that do not contain the siRNA and / or siRNA modifications disclosed herein.

[0185] As used herein, the term "reduction" is used interchangeably with "reduction," "silencing," "downregulating," "suppressing," "inhibiting," and other similar terms, and includes any level of reduction.

[0186] As used herein, the phrase "inhibiting the expression of PCSK9" includes inhibiting the expression of any PCSK9 gene (such as, for example, a mouse PCSK9 gene, a rat PCSK9 gene, a monkey PCSK9 gene, or a human PCSK9 gene) as well as variants (e.g., naturally occurring variants) or mutants of the PCSK9 gene. Thus, the PCSK9 gene can be a wild-type PCSK9 gene, a mutant PCSK9 gene, or a transgenic PCSK9 gene in the context of a genetically manipulated cell, cell group, or organism.

[0187] "Inhibiting PCSK9 gene expression" includes any level of inhibition of the PCSK9 gene, such as at least partial inhibition of PCSK9 gene expression, such as inhibition of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%.

[0188] Based on any variable level associated with PCSK9 gene expression, such as PCSK9 mRNA level, PCSK9 protein level, or serum lipid level, the expression of the PCSK9 gene can be assessed. Inhibition can be assessed by the reduction of one or more of these variables in absolute or relative levels compared to a control level. A control level can be any type of control level utilized in the art, such as a baseline level before administration or a level determined from a similar untreated or control-treated subject, cell, or sample (e.g., a buffer-only control or an inert agent control).

[0189] As used herein, "PCSK9-related disease" is intended to include any disease associated with the PCSK9 gene or protein. Such diseases can be caused, for example, by overproduction of PCSK9 protein, by mutations in the PCSK9 gene, by abnormal cleavage of the PCSK9 protein, by abnormal interactions between PCSK9 and other proteins or other endogenous or exogenous substances. Exemplary PCSK9-related diseases include lipidemias, such as hyperlipidemia, and other forms of lipid imbalance, such as hypercholesterolemia, hypertriglyceridemia, and pathological conditions associated with these disorders, such as heart and circulatory system diseases.

[0190] As used herein, the phrase "inhibiting gene expression of HBV" includes inhibiting the expression of HBV DNA, HBV mRNA, HBsAg, HBeAg, hepatitis B core antigen (HBcAg), and the like.

[0191] "Inhibiting the expression of HBV antigens" includes inhibiting the expression of HBsAg, HBeAg, and HBcAg proteins.

[0192] "Inhibition of HBV gene expression" includes any level of inhibition of HBV DNA, HBV mRNA, HBsAg, HBeAg, HBcAg, such as at least partial inhibition of HBV DNA, HBV mRNA, HBsAg, HBeAg, HBcAg expression, such as inhibition of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%.

[0193] Based on any variable level related to HBV gene expression, such as HBV DNA level, HBV mRNA level, HBV antigen protein level, HBV virion level, the expression of HBV gene can be assessed. Suppression can be assessed by the reduction of one or more of these variables compared to the control level in terms of absolute or relative levels. The control level can be any type of control level utilized in the art, such as baseline level before administration or from a similar untreated or controlled subject, cell, or sample, such as only a buffer control or an inert agent control, treated level.

[0194] As used herein, "HBV gene expression related disease" is intended to include any disease associated with HBV genes or proteins. Such diseases can be caused, for example, by excessive production of HBV antigenic proteins, by HBV gene mutations, by abnormal cleavage of HBV antigenic proteins, by abnormal interactions between HBV antigenic proteins and other proteins or other endogenous or exogenous substances. Exemplary HBV-related diseases include hepatitis associated with HBV infection, such as chronic hepatitis B, acute hepatitis B, and HBV / hepatitis D virus (HDV) co-infection-related diseases.

[0195] The phrase "inhibiting the expression of AGT" includes inhibiting the expression of any AGT gene (such as, for example, a mouse AGT gene, a rat AGT gene, a monkey AGT gene, or a human AGT gene), as well as variants (e.g., naturally occurring variants) or mutants of an AGT gene. Thus, the AGT gene can be a wild-type AGT gene, a mutant AGT gene, or, in the case of a genetically manipulated cell, cell group, or organism, a transgenic AGT gene.

[0196] "Inhibiting AGT gene expression" includes any level of inhibition of the AGT gene, such as at least partial inhibition of the expression of the AGT gene, such as inhibition of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%.

[0197] Based on any variable level relevant to AGT gene expression, for example AGT mRNA level or AGT protein level, the expression of the AGT gene can be assessed. Inhibition can be assessed by the minimizing of the absolute or relative levels compared to the control level by one or more of these variables. The control level can be any type of control level utilized in this area, for example baseline level before administration or the level determined from a similar untreated or controlled experimenter, cell, or sample (for example, only buffer control or inert agent control) treated.

[0198] As used herein, "AGT-related disease" is intended to include any disease associated with the AGT gene or protein. Such diseases can be caused, for example, by overproduction of AGT protein, by mutations in the AGT gene, by abnormal cleavage of the AGT protein, by abnormal interactions between AGT and other proteins or other endogenous or exogenous substances. Exemplary AGT-related diseases include hypertension, critical hypertension, essential hypertension, secondary hypertension, hypertensive crisis, hypertensive urgency, isolated systolic and diastolic hypertension, pregnancy-related hypertension, diabetic hypertension, refractory hypertension, refractory hypertension, paroxysmal hypertension, renovascular hypertension, Goldblatt's hypertension, ocular hypertension, glaucoma, pulmonary hypertension, portal hypertension, systemic venous hypertension, systolic hypertension, unstable hypertension; hypertensive heart disease, hypertensive nephropathy, atherosclerosis, arteriosclerosis, blood vascular disease, diabetic nephropathy, diabetic retinopathy, chronic heart failure, cardiomyopathy, diabetic cardiomyopathy, glomerulosclerosis, coarctation of the aorta, aortic aneurysm, ventricular fibrosis, Cushing's syndrome and other glucocorticoid excess states (including chronic steroid therapy), pheochromocytoma, nephroblastoma, secondary aldosteronism and other mineralocorticoid excess states, sleep apnea, thyroid / parathyroid disease, heart failure, myocardial infarction, angina pectoris, stroke, diabetes mellitus, kidney disease, renal failure, systemic sclerosis, intrauterine growth restriction (IUGR), and fetal growth restriction.

[0199] As used herein, "patient" or "subject" is intended to include humans or non-human animals, preferably mammals, such as mice. Most preferably, the subject or patient is a human.

[0200] As used herein, "therapeutically effective amount" is intended to include an amount of a double-stranded oligonucleotide sufficient to achieve treatment of a PCSK9 / HBV infection / AGT-related disease when administered to a patient (e.g., by weakening, improving, or maintaining the existing disease or one or more disease symptoms). The "therapeutically effective amount" may vary depending on the double-stranded oligonucleotide, how the agent is administered, the disease and its severity, and medical history, age, weight, family history, genetic makeup, the stage of the pathological process mediated by PCSK9 gene / HBV gene / AGT gene expression, the type of previous or concomitant treatment (if any), and other individual characteristics of the patient to be treated.

[0201] As used herein, a "prophylactically effective amount" is intended to include an amount of a double-stranded oligonucleotide that is sufficient to prevent or improve a disease or one or more symptoms of a disease when administered to a subject who has not yet experienced or shown symptoms of a PCSK9 gene / HBV gene / AGT gene-related disease but may be susceptible to the disease. Improving a disease includes slowing the progression of the disease or reducing the severity of a subsequent disease. The "prophylactically effective amount" may vary depending on the double-stranded oligonucleotide, how the agent is administered, the degree of risk of the disease, and medical history, age, weight, family history, genetic makeup, type of previous or concomitant treatment (if any), and other individual characteristics of the patient to be treated.

[0202] "Therapeutically effective amount" or "prophylactically effective amount" also includes the amount of double-stranded oligonucleotide that produces a desired local or systemic effect at a reasonable benefit / risk ratio applicable to any treatment. The double-stranded oligonucleotide used in the methods of the present disclosure can be administered in an amount sufficient to produce a reasonable benefit / risk ratio applicable to such treatment.

[0203] As used herein, the term "sample" includes similar fluids, cells or tissues separated from a subject, and a collection of fluids, cells or tissues present in a subject. Examples of biological fluids include blood, serum and serosal fluid, plasma, cerebrospinal fluid, ocular fluid (ocular fluid), lymph, urine, saliva, etc. Tissue samples can include samples from tissues, organs or local regions. For example, samples can be derived from the fluid or cells in a particular organ, an organ part or these organs. In certain embodiments, samples can be derived from liver (for example, a part of the whole liver or the liver, or cells of some types in the liver, for example, hepatocytes). In a preferred embodiment, "sample derived from experimenter" refers to blood or plasma extracted from this experimenter. In other embodiments, "sample derived from experimenter" refers to liver tissue (or its subcomponent) derived from this experimenter.

[0204] In one aspect, the present disclosure provides a modified template for enhancing the target gene inhibition effect of a double-stranded oligonucleotide, wherein the double-stranded oligonucleotide is siRNA, and the double-stranded oligonucleotide contains a sense chain and an antisense chain, and each nucleotide of the sense chain and the antisense chain is a modified nucleotide, wherein the sense chain comprises a nucleotide sequence 1, the antisense chain comprises a nucleotide sequence 2, and the nucleotide sequence 1 and the nucleotide sequence 2 are at least partially reverse-complementary to form a double-stranded region; the length of the reverse-complementary double-stranded region is 17 to 21 bp.

[0205] In some embodiments, the modified nucleotides are selected from any one or a combination of two or more of the group consisting of deoxy-nucleotides, 3'-terminal deoxy-thymidine nucleotides, 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-deoxy-modified nucleotides, locked nucleotides, unlocked nucleotides, conformationally restricted nucleotides, restricted ethyl nucleotides, abasic nucleotides, 2'-amino-modified nucleotides, 2'-O-allyl-modified nucleotides, 2'-C-alkyl-modified nucleotides, 2'-hydroxy-modified nucleotides, 2'-O-methoxyethyl modified nucleotides, 2'-O-alkyl-modified nucleotides, morpholino nucleotides, phosphoramidates, nucleotides containing non-natural bases, tetrahydropyran-modified nucleotides, 1,5-anhydrohexitol-modified nucleotides, cyclohexenyl-modified nucleotides, nucleotides containing phosphorothioate groups, nucleotides containing methylphosphonate groups, nucleotides containing 5'-phosphates, and nucleotides containing 5'-phosphate mimetics.

[0206] In some embodiments, the siRNA is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length.

[0207] In some embodiments, the siRNA is 10-30 nucleotides in length.

[0208] In some embodiments, the siRNA is 13-25 nucleotides in length.

[0209] In some embodiments, the siRNA is 13-20 nucleotides in length.

[0210] In some embodiments, the modification comprised by the nucleotide is a chemical modification of the 2' position of the ribose sugar of the nucleotide.

[0211] In some embodiments, the chemical modification of the 2' position of the ribose of the nucleotide is selected from any one or a combination of two or more of the following groups: 2'-methoxy modification, 2'-O-methoxyethyl modification, 2'-fluoro modification, 2'-benzyloxy modification, 2'-methylcarbonylamino modification, 2'-pyridylmethoxy modification, 2,4'-locked nucleotide modification, and 2,4'-restricted ethyl nucleotide modification.

[0212] In some embodiments, the chemical modification at the 2' position of the ribose sugar of the nucleotide is a 2,4'-locked nucleotide modification.

[0213] In some embodiments, the chemical modification of the 2' position of the ribose sugar of some nucleotides of the siRNA is a 2,4'-constrained ethyl nucleotide modification.

[0214] In some embodiments, the nucleotides are linked by 3',5'-phosphodiester bonds.

[0215] In some embodiments, the 3',5'-phosphodiester bond comprises a thio modification.

[0216] In some embodiments, the 3',5'-phosphodiester bonds both comprise thio modifications and form chirally pure 3',5'-phosphothioate diester bonds.

[0217] In some embodiments, the siRNA comprises: a double-stranded region, a 3' overhanging end, and / or a 5' overhanging end.

[0218] In some embodiments, the double-stranded region consists of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 consecutive nucleotides.

[0219] In some embodiments, in the direction from the 5' end to the 3' end, at least two nucleotides among the 2nd to 6th nucleotides of the nucleotide sequence 2 are 2'-fluoro-modified nucleotides, the 14th and / or 16th nucleotides are 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are 2'-methoxy-modified nucleotides, and the 1st, 2nd and 2nd to 3rd positions have a thiophosphate backbone.

[0220] In some embodiments of the present disclosure, at least two nucleotides in nucleotides 2-6 of the nucleotide sequence 2 are selected from one of the following combinations:

[0221] (1) Nucleotides 2 and 6;

[0222] (2) nucleotides 2, 3, 4, and 6;

[0223] (3) nucleotides 2, 4, 5, and 6;

[0224] (4) nucleotides 2, 5, and 6;

[0225] (5) nucleotides 2, 3, 5, and 6;

[0226] (6) Nucleotides 2, 3, and 6.

[0227] In some embodiments of the present disclosure, the nucleotide sequence 1 is modified in the following manner from the 5' end to the 3' end:

[0228] (1) The nucleotides at positions 6 and 16 are 2'-fluoro-modified nucleotides, at least two nucleotides among positions 8-10, preferably positions 8-10 or positions 8 and 10, are 2'-fluoro-modified nucleotides, the nucleotides at the remaining positions are 2'-methoxy-modified nucleotides, and positions 1 and 2 have a phosphorothioate backbone; or

[0229] (2) The nucleotides at positions 7 and 17 are 2'-fluoro-modified nucleotides, at least two nucleotides among the nucleotides at positions 9-11, preferably the nucleotides at positions 9-11 or the nucleotides at positions 9 and 11 are 2'-fluoro-modified nucleotides, the nucleotides at the remaining positions are 2'-methoxy-modified nucleotides, and the nucleotides at positions 1 and 2 have a thiophosphate backbone.

[0230] In some embodiments of the present disclosure, the length of the nucleotide sequence 1 is 21 or 20, and the length of the nucleotide sequence 2 is 23 or 22.

[0231] In some embodiments of the present disclosure, the length of the nucleotide sequence 1 is 20, and the length of the nucleotide sequence 2 is 22; or, the length of the nucleotide sequence 1 is 21, and the length of the nucleotide sequence 2 is 23.

[0232] In some embodiments of the present disclosure, the nucleotides in the antisense strand are modified in any of the following ways A, B, C, D, E, F and G, in the direction from the 5' end to the 3' end:

[0233] A: indicates that the antisense strand starts from the 5' end, nucleotides 1, 3, 4, 5, 7, 8, 9, 10, 11, 12, 13, 15, 17, 18, 19, 20, 21, 22, and 23 are 2'-methoxy-modified nucleotides, and nucleotides 2, 6, 14, and 16 are 2'-fluoro-modified nucleotides, and positions 1, 2, 21, and 22 have a phosphorothioate backbone;

[0234] B: Indicates that the antisense strand starts from the 5' end, nucleotides 1, 5, 7, 8, 9, 10, 11, 12, 13, 15, 17, 18, 19, 20, 21, 22, and 23 are 2'-methoxy-modified nucleotides, and nucleotides 2, 3, 4, 6, 14, and 16 are 2'-fluoro-modified nucleotides, and positions 1, 2, 21, and 22 have a phosphorothioate backbone;

[0235] C: indicates that the antisense strand starts from the 5' end, nucleotides 1, 3, 7, 8, 9, 10, 11, 12, 13, 15, 17, 18, 19, 20, 21, 22, and 23 are 2'-methoxy-modified nucleotides, and nucleotides 2, 4, 5, 6, 14, and 16 are 2'-fluoro-modified nucleotides, and positions 1, 2, 21, and 22 have a phosphorothioate backbone;

[0236] D: indicates that the antisense strand starts from the 5' end, nucleotides 1, 3, 4, 7, 8, 9, 10, 11, 12, 13, 15, 17, 18, 19, 20, 21, 22, and 23 are 2'-methoxy-modified nucleotides, and nucleotides 2, 5, 6, 14, and 16 are 2'-fluoro-modified nucleotides, and positions 1, 2, 21, and 22 have a phosphorothioate backbone;

[0237] E: indicates that the antisense strand starts from the 5' end, nucleotides 1, 4, 7, 8, 9, 10, 11, 12, 13, 15, 17, 18, 19, 20, 21, 22, and 23 are 2'-methoxy-modified nucleotides, and nucleotides 2, 3, 5, 6, 14, and 16 are 2'-fluoro-modified nucleotides, and positions 1, 2, 21, and 22 have a phosphorothioate backbone;

[0238] F: indicates that the antisense strand starts at the 5' end, nucleotides 1, 4, 5, 7, 8, 9, 10, 11, 12, 13, 15, 17, 18, 19, 20, 21, 22, and 23 are 2'-methoxy-modified nucleotides, and nucleotides 2, 3, 6, 14, and 16 are 2'-fluoro-modified nucleotides, and positions 1, 2, 21, and 22 have a phosphorothioate backbone;

[0239] G: indicates that the antisense strand starts at the 5' end, nucleotides 1, 3, 4, 5, 7, 8, 9, 10, 11, 12, 13, 15, 17, 18, 19, 20, 21, 22, and 23 are 2'-methoxy-modified nucleotides, and nucleotides 2, 6, 14, and 16 are 2'-fluoro-modified nucleotides, and positions 1, 2, 21, and 22 have a phosphorothioate backbone;

[0240] H: indicates that the antisense strand starts from the 5' end, nucleotides 1, 3, 4, 5, 7, 8, 9, 10, 11, 12, 13, 15, 16, 17, 18, 19, 20, 21, 22, and 23 are 2'-methoxy-modified nucleotides, and nucleotides 2, 6, and 14 are 2'-fluoro-modified nucleotides, and positions 1, 2, 21, and 22 have a phosphorothioate backbone;

[0241] And / or, in the direction from the 5' end to the 3' end, the nucleotides in the sense strand are modified by any of the following methods a to d:

[0242] a: indicates that the 1st, 2nd, 3rd, 4th, 5th, 6th, 8th, 12th, 13th, 14th, 15th, 16th, 18th, 19th, 20th, and 21st nucleotides starting from the 5' end of the sense strand are 2'-methoxy-modified nucleotides, and the 7th, 9th, 10th, 11th, and 17th nucleotides are 2'-fluoro-modified nucleotides, and the 1st and 2nd nucleotides have a phosphorothioate backbone;

[0243] b: Indicates that the 1st, 2nd, 3rd, 4th, 5th, 6th, 8th, 10th, 12th, 13th, 14th, 15th, 16th, 18th, 19th, 20th, and 21st nucleotides starting from the 5' end of the sense strand are 2'-methoxy-modified nucleotides, and the 7th, 9th, 11th, and 17th nucleotides are 2'-fluoro-modified nucleotides, and the 1st and 2nd nucleotides have a thiophosphate backbone;

[0244] c: Indicates that the 1st, 2nd, 3rd, 4th, 5th, 6th, 8th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, 19th, 20th, and 21st nucleotides starting from the 5' end of the sense strand are 2'-methoxy-modified nucleotides, and the 7th, 9th, 10th, and 11th nucleotides are 2'-fluoro-modified nucleotides, and the 1st and 2nd nucleotides have a thiophosphate backbone;

[0245] d: Indicates that the 1st, 2nd, 3rd, 4th, 5th, 6th, 8th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, 19th, 20th, and 21st nucleotides starting from the 5' end of the sense strand are 2'-methoxy-modified nucleotides, and the 7th, 9th, and 10th nucleotides are 2'-fluoro-modified nucleotides, and the 1st and 2nd nucleotides have a thiophosphate backbone.

[0246] In some embodiments of the present disclosure, the nucleotides in the antisense strand are modified in any of the following ways: A', B', C', D', E', F' and G', in the direction from the 5' end to the 3' end:

[0247] A': indicates that the antisense strand starts from the 5' end, nucleotides 1, 3, 4, 5, 7, 8, 9, 10, 11, 12, 13, 15, 17, 18, 19, 20, 21, and 22 are 2'-methoxy-modified nucleotides, and nucleotides 2, 6, 14, and 16 are 2'-fluoro-modified nucleotides, and positions 1, 2, 20, and 21 have a phosphorothioate backbone;

[0248] B': indicates that the antisense strand starts from the 5' end, nucleotides 1, 5, 7, 8, 9, 10, 11, 12, 13, 15, 17, 18, 19, 20, 21, and 22 are 2'-methoxy-modified nucleotides, and nucleotides 2, 3, 4, 6, 14, and 16 are 2'-fluoro-modified nucleotides, and positions 1, 2, 20, and 21 have a phosphorothioate backbone;

[0249] C': indicates that the antisense strand starts at the 5' end, nucleotides 1, 3, 7, 8, 9, 10, 11, 12, 13, 15, 17, 18, 19, 20, 21, and 22 are 2'-methoxy-modified nucleotides, and nucleotides 2, 4, 5, 6, 14, and 16 are 2'-fluoro-modified nucleotides, and positions 1, 2, 20, and 21 have a phosphorothioate backbone;

[0250] D': indicates that the antisense strand starts at the 5' end, nucleotides 1, 3, 4, 7, 8, 9, 10, 11, 12, 13, 15, 17, 18, 19, 20, 21, and 22 are 2'-methoxy-modified nucleotides, and nucleotides 2, 5, 6, 14, and 16 are 2'-fluoro-modified nucleotides, and positions 1, 2, 20, and 21 have a phosphorothioate backbone;

[0251] E': indicates that the antisense strand starts from the 5' end, nucleotides 1, 4, 7, 8, 9, 10, 11, 12, 13, 15, 17, 18, 19, 20, 21, and 22 are 2'-methoxy-modified nucleotides, and nucleotides 2, 3, 5, 6, 14, and 16 are 2'-fluoro-modified nucleotides, and positions 1, 2, 20, and 21 have a phosphorothioate backbone;

[0252] F': indicates that the antisense strand starts at the 5' end, nucleotides 1, 4, 5, 7, 8, 9, 10, 11, 12, 13, 15, 17, 18, 19, 20, 21, and 22 are 2'-methoxy-modified nucleotides, and nucleotides 2, 3, 6, 14, and 16 are 2'-fluoro-modified nucleotides, and positions 1, 2, 20, and 21 have a phosphorothioate backbone;

[0253] G': indicates that the antisense strand starts at the 5' end, nucleotides 1, 3, 4, 5, 7, 8, 9, 10, 11, 12, 13, 15, 16, 17, 18, 19, 20, 21, and 22 are 2'-methoxy-modified nucleotides, and nucleotides 2, 6, and 14 are 2'-fluoro-modified nucleotides, and positions 1, 2, 20, and 21 have a phosphorothioate backbone;

[0254] And / or, in the direction from the 5' end to the 3' end, the nucleotides in the sense strand are modified by any of the following methods a', b', c' and d':

[0255] a': indicates that the 1st, 2nd, 3rd, 4th, 5th, 7th, 11th, 12th, 13th, 14th, 15th, 17th, 18th, 19th, and 20th nucleotides starting from the 5' end of the sense strand are 2'-methoxy-modified nucleotides, and the 6th, 8th, 9th, 10th, and 16th nucleotides are 2'-fluoro-modified nucleotides, and the 1st and 2nd nucleotides have a phosphorothioate backbone;

[0256] b': indicates that the 1st, 2nd, 3rd, 4th, 5th, 7th, 9th, 11th, 12th, 13th, 14th, 15th, 17th, 18th, 19th, and 20th nucleotides starting from the 5' end of the sense strand are 2'-methoxy-modified nucleotides, and the 6th, 8th, 10th, and 16th nucleotides are 2'-fluoro-modified nucleotides, and the 1st and 2nd nucleotides have a phosphorothioate backbone;

[0257] c': indicates that the 1st, 2nd, 3rd, 4th, 5th, 7th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, 19th, and 20th nucleotides starting from the 5' end of the sense strand are 2'-methoxy-modified nucleotides, and the 6th, 8th, 9th, and 10th nucleotides are 2'-fluoro-modified nucleotides, and the 1st and 2nd nucleotides have a thiophosphate backbone:

[0258] d': indicates that the 1st, 2nd, 3rd, 4th, 5th, 7th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, 19th, and 20th nucleotides starting from the 5' end of the sense strand are 2'-methoxy-modified nucleotides, and the 6th, 8th, and 9th nucleotides are 2'-fluoro-modified nucleotides, and the 1st and 2nd nucleotides have a thiophosphate backbone.

[0259] In some embodiments of the present disclosure, the double-stranded oligonucleotide is modified in the direction from the 5' end to the 3' end using any one of the following combinations (i) to (x):

[0260] (i) the antisense strand is modified using method A, and the sense strand is modified using method a;

[0261] (ii) the antisense strand is modified using method B, and the sense strand is modified using method a;

[0262] (iii) the antisense strand is modified using method C, and the sense strand is modified using method a;

[0263] (iv) the antisense strand is modified using method B, and the sense strand is modified using method b;

[0264] (v) the antisense strand is modified using method C, and the sense strand is modified using method b;

[0265] (vi) the antisense strand is modified using method G, and the sense strand is modified using method C;

[0266] (vi) the antisense strand is modified using method A, and the sense strand is modified using method d;

[0267] (viii) the antisense strand is modified using method D, and the sense strand is modified using method b;

[0268] (ix) the antisense strand is modified using method E, and the sense strand is modified using method b;

[0269] (x) The antisense strand is modified using method F, and the sense strand is modified using method b.

[0270] In some embodiments of the present disclosure, the double-stranded oligonucleotide is modified in the direction from the 5' end to the 3' end by any of the following combinations (1)-(8):

[0271] (1) The antisense strand is modified using method A', and the sense strand is modified using method a';

[0272] (2) the antisense strand is modified using method B', and the sense strand is modified using modification method a';

[0273] (3) the antisense strand is modified using method C', and the sense strand is modified using modification method a';

[0274] (4) the antisense strand is modified using method B', and the sense strand is modified using modification method b';

[0275] (5) The antisense strand is modified using method C', and the sense strand is modified using modification method b';

[0276] (6) The antisense strand is modified using method D', and the sense strand is modified using modification method b';

[0277] (7) The antisense strand is modified using method E', and the sense strand is modified using modification method b';

[0278] (8) The antisense strand is modified using method F', and the sense strand is modified using modification method b'.

[0279] In some embodiments of the present disclosure, the first nucleotide at the 5' end of the antisense strand is a 5'-phosphate nucleotide or a 5'-phosphate analog modified nucleotide, which includes but is not limited to the following 5'-phosphorylated groups: 5'-vinylphosphonate group (5'-E-VP); 5'-methylphosphonate group (5'-MP); 5'-C-methylphosphonate group; 5'-thiophosphate group (5'-PS); 5'-phosphate group (5'-P), the structure of which is shown as follows:

[0280] Wherein, R is hydrogen, hydroxyl, amino, C 1-4 Alkyl, aromatic, C 1-4 Alkoxy, C 1-4 alkylcarbonylamino or halogen;

[0281] The base is selected from adenine, guanine, cytosine, thymine and uracil.

[0282] In some embodiments of the present disclosure, the first nucleotide at the 5' end of the antisense strand is a 5'-vinyl-(E)-phosphonate-2'-methoxy modified nucleotide.

[0283] In some embodiments of the present disclosure, the antisense strand is modified in one of the following ways:

[0284] The sense strand is modified in one of the following ways:

[0285] In the above table, PS is a phosphorothioate skeleton.

[0286] The siRNA modified template with modification method A on the antisense strand and modification method a on the sense strand was named DV25;

[0287] The siRNA modified template with modification method B on the antisense strand and modification method a on the sense strand was named DV26;

[0288] The siRNA modified template with modification method C on the antisense strand and modification method a on the sense strand was named DV27;

[0289] The siRNA modification template with the antisense strand modified by method B and the sense strand modified by method b was named DV28;

[0290] The siRNA modified template with modification method C on the antisense strand and modification method b on the sense strand was named DV29.

[0291] In some embodiments of the present disclosure, the antisense strand adopts the following modified template:

[0292] The sense strand adopts the following modification template:

[0293] In the above table, PS is a phosphorothioate skeleton.

[0294] In some embodiments of the present disclosure, the antisense strand adopts the following modified template:

[0295] The positive chain adopts the following modification template:

[0296] In the above table, PS is a phosphorothioate skeleton.

[0297] In some embodiments of the present disclosure, the first nucleotide at the 5' end of the antisense strand is a 5'-phosphate nucleotide or a 5'-phosphate analog modified nucleotide, which includes but is not limited to the following 5'-phosphorylated groups: 5'-vinylphosphonate group (5'-E-VP); 5'-methylphosphonate group (5'-MP); 5'-C-methylphosphonate group; 5'-thiophosphate group (5'-PS); 5'-phosphate group (5'-P), the structure of which is shown as follows:

[0298] Wherein, R is hydrogen, hydroxyl, amino, C 1-4 Alkyl, aromatic, C 1-4 Alkoxy, C 1-4 alkylcarbonylamino or halogen;

[0299] The base is selected from adenine, guanine, cytosine, thymine and uracil.

[0300] In some embodiments of the present disclosure, the first nucleotide at the 5' end of the antisense strand is a 5'-vinyl-(E)-phosphonate-2'-methoxy modified nucleotide.

[0301] In some embodiments of the present disclosure, the antisense strand is modified in one of the following ways:

[0302] The sense strand is modified in one of the following ways:

[0303] In the above table, PS is a phosphorothioate backbone;

[0304] The siRNA modification template in which the antisense strand adopts modification method A and the sense strand adopts modification method a is named DV25P.

[0305] The siRNA modification template in which the antisense strand was modified using modification method B and the sense strand was modified using modification method a was named DV26P.

[0306] The siRNA modification template in which the antisense strand was modified using modification method C and the sense strand was modified using modification method a was named DV27P.

[0307] The siRNA modification template in which the antisense chain adopts modification method B and the sense chain adopts modification method b is named DV28P.

[0308] The siRNA modification template in which the antisense strand was modified using modification method C and the sense strand was modified using modification method b was named DV29P.

[0309] The siRNA modification template in which the antisense strand was modified using modification method D and the sense strand was modified using modification method b was named DV32P.

[0310] The siRNA modification template in which the antisense strand was modified using modification method E and the sense strand was modified using modification method b was named DV33P.

[0311] The siRNA modification template in which the antisense strand was modified using modification method F and the sense strand was modified using modification method b was named DV34P.

[0312] In some embodiments of the present disclosure, the antisense strand is modified in one of the following ways:

[0313] In the above table, 2'-OMe is 2'-methoxy; 2'-F is 2'-fluoro; PS is a phosphorothioate backbone;

[0314] The siRNA modification template with the antisense strand modified in A' and the sense strand modified in a' was named DV25SP.

[0315] The siRNA modified template with the antisense strand modified by B' and the sense strand modified by a' was named DV26SP.

[0316] The siRNA modified template with the antisense strand modified in C' and the sense strand modified in a' was named DV27SP.

[0317] The siRNA modification template in which the antisense strand is modified in the B' and the sense strand is modified in the b' is named DV28SP.

[0318] The siRNA modified template with the antisense strand modified by C' and the sense strand modified by b' was named DV29SP.

[0319] The siRNA modification template with the antisense strand modified in the D' and the sense strand modified in the b' was named DV32SP.

[0320] The siRNA modification template with the antisense strand modified in the E' and the sense strand modified in the b' was named DV33SP.

[0321] The siRNA modification template with the antisense strand modified in the F' and the sense strand modified in the b' was named DV34SP.

[0322] In some embodiments of the present disclosure, one or more nucleotides from positions 2 to 8, preferably positions 6 and / or 7, from the 5' end of the antisense strand adopt a modification group, wherein the modification group is preferably selected from UNA, GNA or DNA, wherein the structures of UNA and GNA are as follows:

[0323] The base is selected from adenine, guanine, cytosine, thymine and uracil.

[0324] In some embodiments of the present disclosure, the antisense strand adopts a modification group at the 6th and / or 7th position from the 5' end, and the modification group is DNA.

[0325] Another aspect of the present invention provides a double-stranded oligonucleotide, wherein the double-stranded oligonucleotide is modified by the modified template described in the present disclosure.

[0326] In some embodiments of the present disclosure, the double-stranded oligonucleotide targets one of the following mRNAs: PCSK9 gene, HBV gene, and AGT gene.

[0327] In some specific embodiments of the present disclosure, the nucleotide sequence of the double-stranded oligonucleotide is shown in Tables 13, 47, 49, 63-5, 79 and 85.

[0328] In another aspect, the present disclosure provides a conjugate comprising the aforementioned double-stranded oligonucleotide and a ligand conjugated to the double-stranded oligonucleotide.

[0329] In some embodiments, the ligand is conjugated to the 3'-end or the 5'-end of the sense strand of the oligonucleotide.

[0330] In some embodiments, the ligand is one or more GalNAc derivatives attached using a bivalent or trivalent branched linkage.

[0331] In some embodiments, the ligand is:

[0332] wherein X is hydrogen or a hydroxyl protecting group or H, wherein the hydroxyl protecting group includes acetyl, benzoyl or isobutyryl; Y is an amine protecting group or H, wherein the amine protecting group is formyl, acetyl, propionyl, n-butyryl or isobutyryl; n is an integer between 0 and 20; and q, r and s are independently integers between 1 and 7.

[0333] In some embodiments, the ligand is:

[0334] In some embodiments, the ligand is:

[0335] Wherein, X is oxygen, nitrogen or sulfur;

[0336] Y is an alkyl group or an aromatic group;

[0337] R1 is oxygen or sulfur;

[0338] R2 is hydrogen, amino, C 1-4 Alkyl, aromatic, C 1-4 Alkoxy or halogen;

[0339] A is -(CH2) a -、-(CH2CH2O) b -、-((CH2) c NHCO) d -or-((CH2) c CONH) d -, wherein a is an integer from 1 to 15, b is an integer from 1 to 7, c is an integer from 1 to 7, and d is an integer from 1 to 5;

[0340] B is -(CH2) e -, where e is an integer from 0 to 7;

[0341] L is -CONH- or -NHCO-;

[0342] X1 is -(CH2)f -or-(CH2CH2O) f CH2-, f is an integer from 1 to 5;

[0343] X2 is -(CH2) g -, g is an integer from 1 to 6;

[0344] Y1 is 0 or 1;

[0345] Y2 is 0, 1, or 2;

[0346] Y3 is 1, 2 or 3;

[0347] m is an integer from 0 to 4;

[0348] n is an integer from 0 to 4.

[0349] In some embodiments, the ligand is G4, G5, G6, G7, G101, G102, G103, G105, or G106:

[0350] In some embodiments, the conjugate has the structure shown below:

[0351] In other embodiments, the conjugate has the structure shown below:

[0352] In some embodiments, the conjugate is used to inhibit PCSK9, HBV, or AGT gene expression.

[0353] In another aspect, the present disclosure provides a pharmaceutical composition comprising the aforementioned double-stranded oligonucleotide or the aforementioned conjugate, and a pharmaceutically acceptable carrier.

[0354] In one embodiment, provided herein is a pharmaceutical composition containing a double-stranded oligonucleotide as described herein and a pharmaceutically acceptable carrier. Pharmaceutical compositions containing double-stranded oligonucleotides can be used to treat diseases or conditions related to the expression or activity of PCSK9, HBV or AGT genes, such as lipid disorders, hypercholesterolemia, chronic hepatitis B or hypertension. Such pharmaceutical compositions are formulated based on delivery models. One example is a composition formulated to be systemically administered by parenteral delivery, such as by intravenous (IV) or subcutaneous injection (SC). Another example is the following composition, which is formulated for direct delivery to the brain parenchyma, such as by infusion into the brain, such as by continuous pump infusion.

[0355] The pharmaceutical composition comprising the double-stranded oligonucleotide of the present disclosure can be, for example, a solution with or without a buffer or a composition containing a pharmaceutically acceptable carrier. Such compositions include, for example, aqueous or crystalline compositions, liposomal formulations, micellar formulations, emulsions, and gene therapy vectors.

[0356] In the method of the present disclosure, the double-stranded oligonucleotide can be given in a solution. A free double-stranded oligonucleotide can be given in a non-buffered solution, for example, in normal saline or in water. Alternatively, the free double-stranded oligonucleotide can also be given in a suitable buffer solution. The buffer solution can include acetate, citrate, prolamin, carbonate or phosphate, or any combination thereof. In a preferred embodiment, the buffer solution is phosphate buffered saline (PBS). The pH and volume molar osmotic pressure concentration of the buffer comprising the double-stranded oligonucleotide can be adjusted so that it is suitable for giving to the subject.

[0357] In some embodiments, the buffer solution further comprises an agent for controlling the osmolality of the solution so that the osmolality is maintained at a desired value, such as the physiological value of human plasma. Solutes that can be added to the buffer solution to control osmolality include, but are not limited to, proteins, peptides, amino acids, non-metabolizable polymers, vitamins, ions, sugars, metabolites, organic acids, lipids, or salts. In some embodiments, the agent for controlling the osmolality of the solution is a salt. In certain embodiments, the agent for controlling the osmolality of the solution is sodium chloride or potassium chloride.

[0358] Pharmaceutical compositions of the present invention can be given in a dosage sufficient to inhibit the expression of PCSK9, HBV or AGT genes. Typically, the suitable dosage of the double-stranded oligonucleotide of the present invention is in the range of about 0.001 to about 200.0 mg per kilogram of body weight per day, typically in the range of about 0.1 to 50 mg per kilogram of body weight per day. For example, double-stranded oligonucleotides (e.g., siRNA) can be given in an amount of about 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7. 6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 48, 49, or about 50 mg / kg.

[0359] This pharmaceutical composition can be given once on the day, or this double-stranded oligonucleotide can be given twice, three times or more subdose with appropriate interval in one day, or even can use continuous infusion or sending and give by controlled release formulation.In this case, the double-stranded oligonucleotide contained in each subdose must be correspondingly still less, so as to realize total dose every day.Also can the dosage unit composite be used for sending in a few days, for example, use the conventional sustained release formulation that lasting double-stranded oligonucleotide is provided to discharge within a few days timeframe.Sustained release formulation is well known in the art and is particularly useful for delivering reagent at a specific site, can use with reagent of the present disclosure thus.In this embodiment, this dosage unit comprises corresponding multiple daily doses.

[0360] In other embodiments, a single dose of the pharmaceutical composition can be long-lasting, such that subsequent doses are administered at intervals of no more than 3, 4, or 5 days, or at intervals of no more than 1, 2, 3, or 4 weeks. In some embodiments of the present disclosure, a single dose of the pharmaceutical composition of the present disclosure is administered once per week. In other embodiments of the present disclosure, a single dose of the pharmaceutical composition of the present disclosure is administered once per month.

[0361] Those skilled in the art will appreciate that certain factors may influence the dosage and timing required to effectively treat a subject, including, but not limited to, the severity of the disease or condition, previous treatment, the subject's overall health and / or age, and other existing diseases. In addition, treating a subject with a therapeutically effective dose of a composition may include a single treatment or a series of treatments. As described elsewhere herein, the effective dosage and in vivo half-life of each double-stranded oligonucleotide encompassed by the present disclosure may be estimated using conventional methods or based on in vivo testing using appropriate animal models.

[0362] The pharmaceutical compositions of the present disclosure can be administered in a number of ways, depending on whether local or systemic treatment is desired and on the area to be treated. Administration can be topical (e.g., via a skin patch); pulmonary; e.g., by inhalation or insufflation of a powder or aerosol, including by nebulizer; intratracheal; intranasal; epidermal, and transdermal, oral, or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal, or intramuscular injection or infusion; subdermal, e.g., via an implant device; or intracranial, e.g., intraparenchymal, intrathecal, or intraventricular administration.

[0363] The double-stranded oligonucleotide used in the compositions and methods of the present disclosure can be formulated to deliver, for example, liposomes or micelles in a membranous molecular assembly. As used herein, the term "liposome" refers to a vesicle composed of amphiphilic lipids arranged in at least one double layer (e.g., a double layer or multiple double layers). Liposomes include single-layer or multi-layer vesicles having a film formed from a lipophilic material and an aqueous interior. The aqueous portion comprises the double-stranded oligonucleotide composition. The lipophilic material is separated from the aqueous exterior (although in some instances, it may include) that typically does not include the double-stranded oligonucleotide composition. Liposomes are useful for transferring active ingredients and delivering them to the site of action. Because the liposome membrane is similar in structure to the biological membrane, when liposomes are applied to a tissue, the double-layer of the liposome fuses with the double layer of the cell membrane. As the fusion of liposomes and cells proceeds, the internal aqueous contents comprising the double-stranded oligonucleotide are delivered to the cell, wherein the double-stranded oligonucleotide can specifically bind to a target RNA and can mediate RNAi. In some cases, the liposomes are also specifically targeted, for example to direct the double-stranded oligonucleotide to a particular cell type.

[0364] Liposomes comprising double-stranded oligonucleotides can be prepared by a variety of methods. In one example, the lipid component of the liposome is dissolved in a detergent so as to form micelles with the lipid component. For example, the lipid component can be an amphipathic cationic lipid or a lipid conjugate. The detergent can have a high critical micelle concentration and can be nonionic. Exemplary detergents include cholate, CHAPS, octyl glucoside, deoxycholate, and lauroyl sarcosine. The double-stranded oligonucleotide preparation is then added to the micelle comprising the lipid component. The cationic group on the lipid interacts with the double-stranded oligonucleotide and condenses around the double-stranded oligonucleotide to form liposomes. After condensation, the detergent is removed, for example, by dialysis to obtain a liposome preparation of the double-stranded oligonucleotide.

[0365] Double-stranded oligonucleotides, such as siRNAs of the present disclosure, can be fully encapsulated in lipid formulations (eg, LNPs or other nucleic acid-lipid particles).

[0366] As used herein, the term "lipid nanoparticle (LNP)" refers to a stable nucleic acid-lipid particle. LNP contains a cationic lipid, a non-cationic lipid, and a lipid (e.g., a PEG-lipid conjugate) that prevents the particle from aggregating. LNP is extremely useful for synthetic applications because they demonstrate a circulation life that is prolonged after intravenous (iv) injection and accumulate at distal sites (e.g., at a site physically separated from the site of administration).

[0367] In one embodiment, the lipid to drug ratio (mass / mass ratio) (e.g., lipid to siRNA ratio) will be in the range of from about 1:1 to about 50:1, from about 1:1 to about 25:1, from about 3:1 to about 15:1, from about 4:1 to about 10:1, from about 5:1 to about 9:1, or about 6:1 to about 9:1.

[0368] In some embodiments, the double-stranded oligonucleotide or the conjugate is administered in an unbuffered solution.

[0369] In some embodiments, the non-buffered solution is saline or water.

[0370] In some embodiments, the double-stranded oligonucleotide or the conjugate is administered with a buffer solution.

[0371] In some embodiments, the buffer solution comprises acetate, citrate, prolamine, carbonate, or phosphate, or any combination thereof.

[0372] In some embodiments, the buffer solution is phosphate buffered saline.

[0373] In some embodiments, the double-stranded oligonucleotide or the conjugate is formulated into a lipid formulation for delivery in a membranous molecular assembly.

[0374] In some embodiments, the lipid formulation is a nucleic acid-lipid particle, such as a lipid nanoparticle.

[0375] In some embodiments, the mass / mass ratio of lipid to the double-stranded oligonucleotide or the conjugate is 1:1-50:1, 1:1-25:1, 3:1-15:1, 4:1-10:1, 5:1-9:1, or 6:1-9:1.

[0376] In some embodiments, the lipid nanoparticles include cationic lipids, neutral lipids, structured lipids, and polymer-conjugated lipids.

[0377] In some embodiments, the cationic lipid is a compound of formula (I), or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof,

[0378] G1 is C 1～6 Alkylene; G2 is C 2～8 Alkylene; G3 is C 1～3 Alkylene; L1 is C 6～15 Straight chain alkyl; L2 is C 12～25In some embodiments, the cationic lipid is YK-009 (see patent CN114044741B) of formula (II):

[0379] In some embodiments, the cationic lipid is a compound of formula (II), or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof,

[0380] G1 is C 2～8 Alkylene; G2 is C 2～8 Alkylene; L1 is -C(O)O- or -OC(O)-; L2 is -C(O)O- or -OC(O)-; R1 is C 6～25 Straight or branched alkyl; R2 is C 6～25 Straight chain or branched chain alkyl; G3 is HO(CH2)2- or HO(CH2)3-; G4 is HO(CH2)2- or HO(CH2)3-; L is (CH2)2- or -(CH2)3- or -(CH2)4-.

[0381] In some embodiments, the cationic lipid is YK-401 of formula (II-I) or YK-402 of formula (II-II) (see patent CN115784921B):

[0382] In some embodiments, the cationic lipid is a compound of formula (III), or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof,

[0383] G1 is C 1～6 Alkylene; G2 is C 2～8 Alkylene; R1 is C 6～20 Straight or branched alkyl; R2 is C 12～25 Branched alkyl; G3 is: HO(CH2)2N(CH3)(CH2)2-, HO(CH2)2N(CH2CH3)(CH2)2-, (HO(CH2)2)2N(CH2)2-, CH3O(CH2)2N(CH3)(C H2)2-, (CH3)2N(CH2)3SC(O)O(CH2)2-, (CH3)2N(CH2)3SC(O)-, CH3NH(CH2)2N(CH3)(CH2)2- or CH3CH2NH(CH2)2-.

[0384] In some embodiments, the cationic lipid is YK-201 of formula (III-I) or YK-202 of formula (III-II) (see patent CN115677518B):

[0385] In some embodiments, the cationic lipid is a compound of formula (IV), or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof,

[0386] G1 is C 1～8 Alkylene; G2 is C 2～8 Alkylene; R1 is C 6～25 Straight or branched alkyl; R2 is C 12～25 Straight chain or branched alkyl; G3 is: HO(CH2)2N(R3)CH2CH(OH)CH2-, wherein R3 is -CH3 or -CH2CH3 or -CH2CH2OH.

[0387] In some embodiments, the cationic lipid is YK-305 of formula (IV-I) or YK-310 of formula (IV-II) (see patent CN115745820B):

[0388] In some embodiments, the cationic lipid is a compound of formula (V), or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof,

[0389] G 1 and G 2 Each independently is an unsubstituted C6-C 10 Alkylene; G 3 For unsubstituted C1-C 12 Alkylene; R 1 and R 2 Each independently is C6-C 24 Alkyl or C6-C 24 Alkenyl; R 3 OR 5 、N、-C(=O)OR 4 、-OC(=O)R 4 or -NR 5 C(=O)R 4 ; R 4 C1-C 12 hydrocarbon group; and R 5 is H or a C1-C6 hydrocarbon group.

[0390] In some embodiments, the cationic lipid is ALC0315 (see patent CN108368028B) of formula (VI):

[0391] In some embodiments, the cationic lipid is a compound of formula (VI), or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof,

[0392] R4 is selected from -(CH2) n Q and -(CH2) n CHQR; Q is selected from the group consisting of: -OR, -OH, -O(CH2) n N(R), -OC(O)R, -CX, -CN, -N(R)C(O)R, -N(H)C(O)R, -N(R)S(O)R, -N(H)S(O)R, -N(R)C(O)N(R), -N(H)C(O)N(R), -N(H)C(O)N(H)(R), -N(R)C(S)N(R), -N(H)C(S)N(R), -N(H)C(S)N(H)(R), -N(R)S(O)R and heterocycle; n is 1, 2 or 3.

[0393] In some embodiments, the cationic lipid is SM102 (see patent CN110520409A) of formula (VI-I):

[0394] In some embodiments, the cationic lipid is a compound of formula (VII) DLIN-MC3-DMA (see CN102625696B), or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof,

[0395] In some embodiments, the cationic lipid comprises one or more selected from the group consisting of: YK-009, YK-401, YK-305, ALC0315, SM102, and DLIN-MC3-DMA.

[0396] In some embodiments, the molar ratio of the cationic lipid to the neutral lipid is 1:1 to 10:1.

[0397] In some embodiments, the molar ratio of the cationic lipid to the structural lipid is 1:1 to 5:1.

[0398] In some embodiments, the molar ratio of the cationic lipid, the neutral lipid, the structural lipid, and the polymer-conjugated lipid is (25-65):(5-25):(25-70):(0.5-5).

[0399] In some embodiments, the molar ratio of the cationic lipid, the neutral lipid, the structural lipid, and the polymer-conjugated lipid is (25-65):(5-25):(25-45):(0.5-5).

[0400] In some embodiments, the molar ratio of the cationic lipid, the neutral lipid, the structural lipid, and the polymer-conjugated lipid is 50:10:38.5:1.5 or 49:10:39.5:1.5.

[0401] In some embodiments, the neutral lipids include one or more selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, ceramide, sterol, and derivatives thereof.

[0402] In some embodiments, the neutral lipid is selected from one or more of the following: 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-diondecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0Diether PC), 1-oleoyl-2-cholesteryl hemisuccinyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dialinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-bisdocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleoyl phosphatidylethanolamine (POPE), distearoyl-phosphatidyl-ethanolamine (DSPE), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), 1-stearoyl-2-oleoyl-stearoylethanolamine (SOPE), 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC), sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine (LPE), and mixtures thereof.

[0403] In some embodiments, the neutral lipid is 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine and / or 1,2-distearoyl-sn-glycero-3-phosphocholine.

[0404] In some embodiments, the structured lipid is selected from one or more of the following: cholesterol, non-sterols, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, ursolic acid, α-tocopherol, and corticosteroids.

[0405] In some embodiments, the structured lipid is cholesterol.

[0406] In some embodiments, the polymer-conjugated lipid is selected from one or more of the following: PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, and PEG-modified dialkylglycerol.

[0407] In some embodiments, the polymer-conjugated lipid is selected from one or more of the following: distearoylphosphatidylethanolamine polyethylene glycol 2000 (DSPE-PEG2000), dimyristoylglycerol-3-methoxy polyethylene glycol 2000 (DMG-PEG2000) and methoxy polyethylene glycol ditetradecanoyl acetamide (ALC-0159).

[0408] Pharmaceutical compositions of the present disclosure include, but are not limited to, solutions, emulsions, and liposome-containing formulations. These compositions can be produced from a variety of components, including, but not limited to, preformed liquids, self-emulsifying solids, and self-emulsifying semisolids. Particularly preferred are formulations that target the liver when treating liver disorders (e.g., liver cancer).

[0409] The pharmaceutical compositions of the present invention (which may conveniently be in unit dosage form) can be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the following steps: combining the active ingredients with the pharmaceutical carrier(s) or excipient(s). Generally speaking, the pharmaceutical compositions are prepared by uniformly and finely combining the active ingredients with a liquid carrier or a finely dispersed solid carrier or both, and, if desired, shaping the product.

[0410] Pharmaceutical compositions of the present disclosure can be formulated into any one of many possible dosage forms, such as but not limited to tablets, capsules, gel capsules, liquid syrups, soft capsules, suppositories and enemas. Pharmaceutical compositions of the present disclosure can also be formulated into suspensions in aqueous, non-aqueous or mixed media. Aqueous suspensions can further include substances that increase the viscosity of the suspension, such substances including, for example, sodium carboxymethylcellulose, sorbitol and / or dextran. The suspension can also include a stabilizer.

[0411] Certain pharmaceutical compositions of the present disclosure also incorporate carrier compounds into the formulations. As used herein, a "carrier compound" or "carrier" may refer to a nucleic acid or its analogues that is inert (i.e., not biologically active per se), but is recognized as a nucleic acid during in vivo processes, such as by degrading biologically active nucleic acids or promoting their removal from circulation, thereby reducing the bioavailability of biologically active nucleic acids. Co-administration of nucleic acids and carrier compounds (generally in excess of the latter) can result in a substantial reduction in the amount of nucleic acid recovered in the liver, kidneys, or other external circulation reservoirs, presumably due to competition between the carrier compound and the nucleic acid for common receptors. For example, the recovery of partially phosphorothioated siRNA in liver tissue can be reduced when co-administered with polyinosinic acid, dextran sulfate, polycytidylic acid, or 4-acetamido-4'isothiocyanatostilbene-2,2'-disulfonic acid (Miyao et al., DsRNA Res. Dev., 1995, 5, 115-121; Takakura et al., DsRNA & Nucl. Acid Drug Dev., 1996, 6, 177-183).

[0412] In contrast to carrier compounds, a "pharmaceutical carrier" or "excipient" is a pharmaceutically acceptable solvent, suspending agent, or any other pharmaceutically inert vehicle for delivering one or more nucleic acids to an animal. The excipient can be liquid or solid and is selected to provide the desired volume, consistency, etc. when combined with the nucleic acid and other components of a particular pharmaceutical composition, with reference to the intended mode of administration. Typical pharmaceutical carriers include, but are not limited to, binders (e.g., pregelatinized corn starch, polyvinyl pyrrolidone, or hydroxypropyl methylcellulose, etc.); fillers (e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylates, or calcium hydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc, silicon dioxide, colloidal silicon dioxide, stearic acid, metallic stearates, hydrogenated vegetable oils, corn starch, polyethylene glycol, sodium benzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodium starch glycolate, etc.); and wetting agents (e.g., sodium lauryl sulfate, etc.).

[0413] Pharmaceutically acceptable organic or inorganic excipients suitable for non-parenteral administration, which do not react toxically with nucleic acids, can also be used to formulate the pharmaceutical compositions of the present disclosure. Suitable pharmaceutically acceptable carriers include, but are not limited to, water, saline solutions, alcohol, polyethylene glycol, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethyl cellulose, polyvinyl pyrrolidone, and the like.

[0414] The preparation that is used for local administration of nucleic acid can comprise the aseptic or non-sterile aqueous solution, the non-aqueous solution in common solvent such as alcohol, or the nucleic acid solution in liquid or solid oil matrix.These solutions can also comprise buffer, diluent and other suitable additives.Can use be suitable for non-parenteral administration and not with nucleic acid generation toxic reaction, pharmaceutically acceptable organic or inorganic excipient.

[0415] Suitable pharmaceutically acceptable excipients include, but are not limited to, water, saline solution, alcohol, polyethylene glycol, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinyl pyrrolidone, and the like.

[0416] The dosage form, carrier compound, pharmaceutical carrier, excipient, etc. of the above-mentioned pharmaceutical composition are described in U.S. Patent No. 10125369B2, which is incorporated herein by reference.

[0417] In another aspect, the present disclosure provides a kit of parts, comprising kit A, wherein kit A comprises one or more of the aforementioned double-stranded oligonucleotides, conjugates, or pharmaceutical compositions.

[0418] In some embodiments, the kit further comprises a kit B, which contains one or both of the following:

[0419] (1) Other drugs that reduce PCSK9, HBV or AGT gene expression or compositions containing such drugs that reduce PCSK9, HBV or AGT gene expression;

[0420] (2) One or more of the group consisting of hormone agents, targeted small molecule agents, proteasome inhibitors, imaging agents, diagnostic agents, chemotherapeutic agents, oncolytic drugs, cytotoxic agents, cytokines, activators of co-stimulatory molecules, inhibitors of inhibitory molecules, and vaccines.

[0421] The "other drugs for reducing PCSK9, HBV or AGT gene expression or compositions containing the drugs for reducing PCSK9, HBV or AGT gene expression" described herein refer to drugs that do not contain the double-stranded oligonucleotides, conjugates or pharmaceutical compositions provided by the present disclosure.

[0422] In another aspect, the present disclosure provides use of the aforementioned double-stranded oligonucleotide, conjugate or pharmaceutical composition in the preparation of a drug for preventing and / or treating diseases related to PCSK9, HBV or AGT gene expression.

[0423] In some embodiments, the PCSK9 expression-related disease is hypercholesterolemia, atherosclerosis, dyslipidemia or cardiovascular and cerebrovascular diseases.

[0424] In some embodiments, the HBV gene expression-related disease is selected from the following disease types: chronic hepatitis B, liver fibrosis, cirrhosis, liver cancer, acute hepatitis B, and HBV / hepatitis D virus co-infection-related diseases.

[0425] In some embodiments, the AGT gene expression-related disease is selected from hypertension, critical hypertension, essential hypertension, secondary hypertension, hypertensive crisis, hypertensive emergency, isolated systolic and diastolic hypertension, pregnancy-related hypertension, diabetic hypertension, resistant hypertension, refractory hypertension, paroxysmal hypertension, renovascular hypertension, Goldblatt's hypertension, ocular hypertension, glaucoma, pulmonary hypertension, portal hypertension, systemic venous hypertension, systolic hypertension, unstable hypertension; hypertensive heart disease, hypertensive nephropathy, atherosclerosis, Atherosclerosis, vascular disease, diabetic nephropathy, diabetic retinopathy, chronic heart failure, cardiomyopathy, diabetic cardiomyopathy, glomerulosclerosis, coarctation of the aorta, aortic aneurysm, ventricular fibrosis, Cushing's syndrome and other glucocorticoid excess states (including chronic steroid therapy), pheochromocytoma, nephroblastoma, secondary aldosteronism and other mineralocorticoid excess states, sleep apnea, thyroid / parathyroid disease, heart failure, myocardial infarction, angina pectoris, stroke, diabetes mellitus, kidney disease, renal failure, systemic sclerosis, intrauterine growth restriction (IUGR), and fetal growth restriction.

[0426] In another aspect, the present disclosure provides a method for reducing PCSK9, HBV or AGT gene expression or inhibiting PCSK9, HBV or AGT replication, comprising administering one or more of the aforementioned double-stranded oligonucleotides, conjugates, pharmaceutical compositions and kits to a sample.

[0427] In some embodiments, the method is for non-prophylactic and / or therapeutic purposes.

[0428] The "non-preventive and / or therapeutic purposes" mentioned herein refers to reducing PCSK9, HBV or AGT gene expression or inhibiting PCSK9, HBV or AGT replication for research purposes, such as in the laboratory.

[0429] In another aspect, the present disclosure provides a method for preventing and / or treating a subject suffering from a disease related to PCSK9, HBV or AGT gene expression, the method comprising administering to the subject a preventively or therapeutically effective amount of the aforementioned double-stranded oligonucleotide, conjugate or pharmaceutical composition.

[0430] The methods are used to treat and / or prevent diseases and conditions that can be modulated by downregulating PCSK9, HBV, or AGT gene expression. For example, the double-stranded oligonucleotides described herein can be used to treat diseases associated with PCSK9, HBV, or AGT gene expression. These methods comprise administering to the subject a therapeutically effective amount or a prophylactically effective amount of a double-stranded oligonucleotide, conjugate, or pharmaceutical composition of the present disclosure.

[0431] In some embodiments, the double-stranded oligonucleotides described herein can be used to treat lipid disorders, such as hyperlipidemia, and other forms of lipid imbalance, such as hypercholesterolemia, hypertriglyceridemia, and pathological conditions associated with these disorders, such as heart and circulatory system diseases. Other diseases and conditions that can be regulated by downregulating PCSK9 gene expression include lysosomal storage diseases, including but not limited to Niemann-Pick disease, Tay-Sachs disease, lysosomal acid lipase deficiency, and Gaucher disease. The RNAi agents described herein can be used to treat cardiovascular diseases such as coronary heart disease (CHD), cerebrovascular disease (CVD), aortic valve stenosis, peripheral vascular disease, atherosclerosis, arteriosclerosis, myocardial infarction (heart attack), cerebrovascular disease (stroke), transient ischemic attack (TIA), angina (stable and unstable), atrial fibrillation, arrhythmia, valvular disease and / or congestive heart failure or any other trait. These methods include administering to the subject a therapeutically effective amount or a prophylactically effective amount of a double-stranded oligonucleotide, conjugate or composition of the present disclosure. In some embodiments, the method includes administering a therapeutic amount of PCSK9 siRNA to a patient with a heterozygous LDLR genotype.

[0432] The double-stranded oligonucleotides of the present invention can be administered to a subject using any mode of administration known in the art, including but not limited to subcutaneous, intravenous, intramuscular, intraocular, intrabronchial, intrapleural, intraperitoneal, intraarterial, lymphatic, cerebrospinal, and any combination thereof. In a preferred embodiment, these agents are administered subcutaneously.

[0433] In other embodiments, the siRNA is administered in combination with another therapeutic agent. The siRNA and the additional therapeutic agent can be administered in combination in the same composition, for example, parenterally, or the additional therapeutic agent can be administered as part of a separate composition or by another method described herein.

[0434] Examples of additional therapeutic agents include agents known to treat hepatitis B. For example, additional drugs for treating chronic hepatitis B are selected from nucleoside (acid) analogs (such as entecavir, tenofovir disoproxil fumarate, tenofovir alafenamide fumarate, and tenofovir alafenamide), and interferon alpha (such as pegylated interferon alpha).

[0435] Examples of additional therapeutic agents include agents known to treat hypertension or agents known to treat cardiovascular and cerebrovascular diseases. For example, additional agents for treating hypertension are selected from angiotensin converting enzyme inhibitors (e.g., captopril, enalapril, benazepril, perindopril, etc.), angiotensin II receptor antagonists (e.g., losartan, losartan hydrochlorothiazide, valsartan, valsartan hydrochlorothiazide, telmisartan, telmisartan hydrochlorothiazide, olmesartan medoxomil, etc.), beta-blockers (e.g., propranolol, bisoprolol, metoprolol tartrate, metoprolol succinate, etc.).

[0436] Examples of other therapeutic agents include drugs known to treat lipid disorders such as hypercholesterolemia, atherosclerosis, dyslipidemia, or cardiovascular and cerebrovascular diseases. For example, other drugs for the treatment of hyperlipidemia are selected from fibrates, statins, bile acid sequestrants, and nicotinic acids. Other drugs for the treatment of cardiovascular and cerebrovascular diseases are selected from angiotensin-converting enzyme inhibitors (e.g., captopril, enalapril, benazepril, perindopril, etc.), angiotensin II receptor antagonists (e.g., losartan, losartan hydrochlorothiazide, valsartan, valsartan hydrochlorothiazide, telmisartan, telmisartan hydrochlorothiazide, olmesartan medoxomil, etc.), beta-blockers (e.g., propranolol, bisoprolol, metoprolol tartrate, metoprolol succinate, etc.).

[0437] In one embodiment, the double-stranded oligonucleotide is administered to a patient and subsequently the additional therapeutic agent is administered to the patient (or vice versa). In another embodiment, the double-stranded oligonucleotide and the additional therapeutic agent are administered simultaneously.

[0438] In some embodiments, the subject is a primate or a rodent.

[0439] In some embodiments, the subject is a human.

[0440] In some embodiments, the double-stranded oligonucleotide is administered at a dose of 0.01 mg / kg-10 mg / kg or 0.5 mg / kg-50 mg / kg.

[0441] In some embodiments, the double-stranded oligonucleotide is administered at a dose of about 1-5 mg / kg.

[0442] In some embodiments, the double-stranded oligonucleotide is administered subcutaneously.

[0443] In some embodiments, the double-stranded oligonucleotide is administered intravenously.

[0444] In some embodiments, the double-stranded oligonucleotide is administered in two or more doses.

[0445] In some embodiments, the double-stranded oligonucleotide is administered in a dosing regimen comprising a loading phase followed by a maintenance phase.

[0446] The loading phase includes administration of 2 mg / kg, 1 mg / kg or 0.5 mg / kg five times a week, and

[0447] The maintenance phase includes administering a dose of 2 mg / kg, 1 mg / kg, or 0.5 mg / kg once a week, twice a week, three times a week, once every two weeks, once every three weeks, once a month, once every two months, once every three months, once every four months, once every five months, or once every six months.

[0448] In some embodiments, the double-stranded oligonucleotide is administered at intervals selected from the group consisting of once every 7 days, once every 14 days, once every 35 days, once every 42 days, and once every 49 days.

[0449] In some embodiments, the HBV gene expression-related disease is selected from the following disease types: chronic hepatitis B, liver fibrosis, cirrhosis, liver cancer, acute hepatitis B, and HBV / hepatitis D virus co-infection-related diseases.

[0450] The nucleotide abbreviations herein are as follows:

[0451] A = adenosine 3'-phosphate

[0452] Am = 2'-methoxyadenosine-3'-phosphate

[0453] Ams = 2'-methoxyadenosine-3'-phosphorothioate

[0454] Af = 2'-fluoroadenosine-3'-phosphate

[0455] Afs = 2'-fluoroadenosine-3'-phosphorothioate

[0456] G = guanosine 3'-phosphate

[0457] Gm=2'-methoxyguanosine-3'-phosphate

[0458] Gms = 2'-methoxyguanosine-3'-phosphorothioate

[0459] Gf = 2'-fluoroguanosine-3'-phosphate

[0460] Gfs = 2'-fluoroguanosine-3'-phosphorothioate

[0461] C = cytidine-3'-phosphate

[0462] Cm=2'-methoxycytidine-3'-phosphate

[0463] Cms = 2'-methoxycytidine-3'-phosphorothioate

[0464] Cf = 2'-fluorocytidine-3'-phosphate

[0465] Cfs = 2'-fluorocytidine-3'-phosphorothioate

[0466] U = uridine-3'-phosphate

[0467] Um = 2'-methoxyuridine-3'-phosphate

[0468] Ums = 2'-methoxyuridine-3'-phosphorothioate

[0469] Uf = 2'-fluorouridine-3'-phosphate

[0470] Ufs = 2'-fluorouridine-3'-phosphorothioate

[0471] AmsEVP = 5'-vinyl-(E)-phosphonate-2'-methoxyadenosine-3'-phosphorothioate

[0472] UmsEVP = 5'-vinyl-(E)-phosphonate-2'-methoxyuridine-3'-phosphorothioate

[0473] A(gna) = adenosine diol nucleic acid

[0474] C(gna) = cytidine-diol nucleic acid

[0475] G (gna) = guanosine diol nucleic acid

[0476] T(gna) = thymidine diol nucleic acid

[0477] U(gna)=uridine-diol nucleic acid

[0478] dA = deoxyadenosine 3'-phosphate

[0479] dG = deoxyguanosine 3'-phosphate

[0480] dC = deoxycytidine-3'-phosphate

[0481] dT = deoxythymidine 3'-phosphate

[0482] The following examples are used to illustrate the present disclosure but are not intended to limit the scope of the present disclosure. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0483] Example

[0484] Example 1: Synthesis of alternatively modified small interfering oligonucleotides

[0485] 84 siRNA base sequences were designed based on the PCSK9 mRNA sequence. To enhance the inhibitory efficiency and stability of the sequences, the base sequences were alternatingly modified with 2'-methoxy (2'-OMe) and 2'-fluoro (2'-F) groups, and the termini were thiolated. The sense strand had 2'-F modifications at all odd-numbered positions and 2'-OMe modifications at all even-numbered positions; the antisense strand had 2'-OMe modifications at all odd-numbered positions and 2'-F modifications at all even-numbered positions. Furthermore, the sense strand had two thiolation modifications at the 5' end, and the antisense strand had two thiolation modifications at both the 5' and 3' ends. The alternatingly modified siRNA sequences are shown in Table 2.

[0486] 1. Synthesis of the Alternately Modified Sequence P92-si5

[0487] The basic sequence of the small interfering RNA numbered P92-si5 in Table 2 is:

[0488] Sense strand: 5'-AAGAUCCUGCAUGUCUUCCAU-3' (SEQ ID NO. 1)

[0489] Antisense strand: 5'-AUGGAAGACAUGCAGGAUCUUGG-3' (SEQ ID NO. 13)

[0490] The sense strand has 2'-F modifications at all odd-numbered positions and 2'-OMe modifications at all even-numbered positions. The antisense strand has 2'-OMe modifications at all odd-numbered positions and 2'-F modifications at all even-numbered positions. Additionally, the sense strand has two thiolate modifications at the 5' end, while the antisense strand has two thiolate modifications at both the 5' and 3' ends.

[0491] Instruments and reagents: Qingke 192P automatic DNA / RNA synthesizer, whose solid phase support is a universal support of cross-linked polystyrene beads, model number is Primer support 5G Unylinker 350 (Cytiva manufacturer).

[0492] Preparation method:

[0493] The following nucleotide monomer solutions were prepared using acetonitrile at a monomer concentration of 0.15 M: DMT-A-OMe phosphoramidite monomer (Formula 1), DMT-C-OMe phosphoramidite monomer (Formula 2), DMT-G-OMe phosphoramidite monomer (Formula 3), DMT-U-OMe phosphoramidite monomer (Formula 4), DMT-AF phosphoramidite monomer (Formula 5), ​​DMT-CF phosphoramidite monomer (Formula 6), DMT-GF phosphoramidite monomer (Formula 7), and DMT-UF phosphoramidite monomer (Formula 8).

[0494] Prepared by the following steps:

[0495] (1) Deprotection

[0496] A 3% dichloroacetic acid toluene solution was used as a deprotection agent to remove the DMT protecting group, followed by washing with acetonitrile.

[0497] (2) Coupling

[0498] Each nucleotide monomer was coupled in acetonitrile using 0.25 M 5-ethylthiotetrazolium as an activating agent, followed by rinsing with acetonitrile.

[0499] (3) Oxidation or sulfidation

[0500] Oxidation: Oxidation was performed using a 0.05 M iodine solution in pyridine / water (90 / 10) as an oxidant, followed by rinsing with acetonitrile.

[0501] Sulfurization: 3% hydrogenated xanthan gum in pyridine was used as a sulfurizing agent for sulfurization, followed by rinsing with acetonitrile.

[0502] (4) Hydroxyl protection

[0503] Hydroxyl group protection was performed using 10% acetic anhydride tetrahydrofuran solution (CAP A) and tetrahydrofuran / pyridine / nitromethylimidazole 74 / 10 / 16 (v / v / v) (CAP B) as hydroxyl group protecting reagents, followed by rinsing with acetonitrile.

[0504] Repeat the above steps and cycle according to the set sequence to obtain a fully protected product.

[0505] (5) Use 3% dichloroacetic acid toluene solution as a deprotection reagent to remove the DMT protecting group of the last nucleotide, and then use acetonitrile to wash.

[0506] (6) Ammonolysis and purification

[0507] The solid phase carrier is transferred to a reactor, concentrated ammonia water (25-28%) is added, and after maintaining aminolysis at 60°C for 12 hours, the system is cooled to room temperature and the mixture is transferred to a filter press. It is eluted with a mixed solution of purified water and ethanol, the filtrate is combined, passed through a chromatography column, concentrated, and freeze-dried to obtain the product.

[0508] (7) Annealing

[0509] The purified sense and antisense strands were mixed in a 1:1 molar ratio, heated to 95°C for 3 min, and then slowly cooled to room temperature to form a double strand.

[0510] The purity of P92-si5 is 97.4%; the measured molecular weight is 14493.52.

[0511] 2. Synthesis of other sequences

[0512] The other sequences listed in Table 2 were synthesized according to the above method.

[0513] Table 2 Alternative modified small interfering RNA sequences

[0514] Example 2: Inhibitory effect of alternating modified sequences on the PCSK9 gene

[0515] After the 2'-OMe and 2'-F alternating modified siRNA sequences synthesized in Example 1 were transfected into HELA cells via lipid nanoparticles (LNP), the inhibitory effect of each sequence on the PCSK9 gene was detected using qPCR technology.

[0516] 1. Experimental Materials

[0517] Test sample: the alternatively modified small interfering RNA sequence P92-si1-84 listed in Table 2 (synthesized in Example 1).

[0518] Cell type: HeLa cell line HELA cells

[0519] Drug solvent: sterile enzyme-free water, Gibco Opti-MEM.

[0520] 2. Experimental Methods

[0521] qRT-PCR was used to detect the inhibitory effect of the samples on the expression of PCSK9 gene mRNA in HELA cell lines.

[0522] 2.1 Cell culture

[0523] Take subcultured HELA cells and culture them in RPMI 1640 medium (10% fetal bovine serum supplemented with 10 μL / mL of 100× penicillin and streptomycin) at logarithmic growth. Place the cells in a 37°C cell culture incubator with 5% CO2, changing the medium daily. Digest the cells with 0.25% trypsin, centrifuge at 1000 rpm for 5 minutes, discard the supernatant, and add fresh medium for subculture.

[0524] 2.2 Cell transfection

[0525] Preparation of transfection mixture: Mix Lipofectamine RNAiMAX and Opti-MEM at a ratio of 1.5:98.5 and vortex to mix thoroughly.

[0526] Transfection reagent preparation: Add 60 μL of siRNA solution diluted in Opti-MEM to 60 μL of transfection mixture at a 1:1 (v / v) ratio. Vortex to mix thoroughly and let stand at room temperature for 15 minutes to obtain lipid nanoparticles (LNPs). 12.5 μL of the solution was used for encapsulation efficiency testing.

[0527] For the blank control group, add 60 μL of the prepared transfection mixture to 60 μL of Opti-MEM. Vortex to mix thoroughly and let stand at room temperature for 15 minutes.

[0528] Add the prepared transfection reagent to a 24-well cell culture plate (100 μL per well) to make the final siRNA concentration in each well 1 nM. Add 500 μL of cell suspension (6×10 cells per ml) 4 After mixing using the cross method, the cells were placed in a 37°C, 5% CO2 cell incubator and cultured for 40 h.

[0529] 2.3 PCSK9 mRNA detection

[0530] 1) RNA extraction

[0531] a. Aspirate the culture medium from the 12-well plate and wash the cells by adding 0.5 mL of 1× PBS to each well. Aspirate the PBS. Add 0.5 mL of TRIzol reagent to each well and thoroughly lyse the cells by pipetting. Transfer the cells to a 1.5 mL RNase-free EP tube and let them stand at room temperature for 5 minutes.

[0532] b. Add 0.1 mL of chloroform to each tube, shake vigorously for 15 seconds, and let stand at room temperature for 5 minutes. Centrifuge at 4°C, 12,000 × g for 15 minutes, and transfer 200 μL of the supernatant to a new EP tube.

[0533] c. Add an equal volume of isopropanol and gently mix the contents by inverting the tube. Incubate at -20°C for 10 min. Centrifuge at 4°C at 12,000 × g for 15 min and discard the supernatant.

[0534] d. Add 0.5 mL of 75% ethanol to gently wash the RNA pellet. Centrifuge at 12,000 × g at 4°C for 5 minutes, and aspirate the supernatant. Repeat the rinse cycle once more, centrifuging at 12,000 × g at 4°C for 1 minute. Remove any remaining ethanol using a micropipette.

[0535] e. Allow the remaining ethanol to dry at room temperature for 2-3 minutes, then add 40 μL of RNase-free ddH2O to dissolve.

[0536] 2) RNA concentration detection

[0537] RNA concentration was measured using a nanodrop. A blank control of 2 μL of RNase-free ddH2O was used, and 2 μL of RNA sample was used for each test. The sample concentration was recorded.

[0538] 3) PCSK9 mRNA quantitative detection

[0539] In a 15 mL centrifuge tube, add the remaining components except primers and template in 7.5×84=630 portions, marked as A.

[0540] Label 1.5 mL EP tubes (84 pieces), add 77 μL of A and 420 ng of total RNA to each tube (7 portions), mix well and set aside (labeled as B).

[0541] The upstream and downstream primers of the internal reference gene GAPDH and the target gene PCSK9 were mixed and set aside (marked as C).

[0542] Add 1 μL of B1 and 1 μL of C to each well of the PCR plate. Cover with sealing film, centrifuge at 3000 rpm for 1 min, and transfer to the PCR machine.

[0543] *This step is performed on ice to maintain low temperature conditions.

[0544] Place the plate in the qPCR instrument and run the following program:

[0545] Reverse transcription: 55°C, 15 min;

[0546] Pre-denaturation: 95°C, 30 sec;

[0547] Cyclic reaction: 95°C, 10 sec; 60°C, 35 sec; 40 cycles;

[0548] Melting curve: 95°C, 15 sec; 60°C, 60 sec; 95°C, 15 sec.

[0549] The running time is about 2 hours. Analyze the experimental results and calculate 2 -ΔΔCt .

[0550] 2.4 Data Processing

[0551] The calculation formula for PCSK9 mRNA expression rate (%) is:

[0552] Expression rate = (PCSK9 mRNA expression level / PCSK9 mRNA expression level in blank control group) × 100%;

[0553] PCSK9 gene expression inhibition rate = 1-expression rate (%).

[0554] 3. Experimental Results

[0555] The inhibition rate in this example is the average of four experiments. The inhibition rate of each sequence on PCSK9 mRNA expression in HELA cells is shown in Tables 4-6.

[0556] Experimental results showed that some sequences modified with alternating 2'-OMe and 2'-F, including P92-si5, P92-si51, P92-si81, P92-si82, P92-si84, P92-si3, P92-si8, P92-si9, P92-si21, P92-si31, P92-si73, and P92-si83 (see Table 3 for specific sequences), significantly inhibited PCSK9 mRNA expression in HELA cells, with inhibition rates exceeding 50%. Among them, P92-si5, P92-si81, P92-si82, P92-si3, P92-si8, and P92-si31 had inhibition rates exceeding 70%.

[0557] The other sequences had poor inhibitory effects on PCSK9 mRNA expression, with inhibition rates lower than 50%.

[0558] Table 3 siRNA sequences with significant inhibitory effects on PCSK9 gene

[0559] The inhibition rate of each sequence on PCSK9 mRNA expression in HELA cells is shown in Tables 4-6.

[0560] (1) Among the 84 designed sequences, 12 sequences showed significant inhibitory effects on the PCSK9 gene, with inhibition rates exceeding 50%, including P92-si5, P92-si51, P92-si81, P92-si82, P92-si84, P92-si3, P92-si8, P92-si9, P92-si21, P92-si31, P92-si73, and P92-si83. Among them, the inhibition rates of P92-si5, P92-si81, P92-si82, P92-si3, P92-si8, and P92-si31 exceeded 70%.

[0561] Table 4 Alternative modified sequences with significant inhibitory effect on PCSK9 gene (inhibition rate higher than 50%)

[0562] As can be seen from Table 4, the alternatively modified sequences P92-si5, P92-si51, P92-si81, P92-si82, P92-si84, P92-si3, P92-si8, P92-si9, P92-si21, P92-si31, P92-si73, and P92-si83 (see Table 3 for specific sequences), a total of 12 sequences, have a significant inhibitory effect on PCSK9 mRNA expression, with inhibition rates of more than 50%. Among them, the inhibition rates of P92-si5, P92-si81, P92-si82, P92-si3, P92-si8, and P92-si31 exceeded 70% (Figure 1). These 12 sequences can be used as candidate sequences.

[0563] (2) The inhibition rates of the 26 sequences on the PCSK9 gene ranged from 30% to 50%. For example, the inhibition rates of P92-si6 and P92-si7 were 38.3% and 35.7%, respectively.

[0564] Table 5 Alternative modified sequences with 30%-50% inhibition rate on PCSK9 gene

[0565] The 26 sequences listed in Table 5 had poor inhibitory effects on the PCSK9 gene, with inhibition rates below 50%, ranging from 30% to 50%. For example, the inhibition rates of P92-si6 and P92-si7 were 38.3% and 35.7%, respectively (Figure 2).

[0566] (3) The inhibition rates of 46 sequences on the PCSK9 gene were below 30%. For example, the inhibition rates of P92-si1 and P92-si20 were only 9.2% and 6.0%, respectively.

[0567] Table 6 Alternative modified sequences with PCSK9 gene inhibition rate below 30%

[0568] The 46 sequences listed in Table 6 had very poor inhibitory effects on PCSK9 mRNA expression, with inhibition rates all below 30%. For example, the inhibition rates of P92-si1 and P92-si20 were only 9.2% and 6.0%, respectively (Figures 3 and 4).

[0569] (4) siRNAs with similar sequences have very different activities. For example, the inhibition rate of P92-si5 is 61.8% higher than that of P92-si4, which is a significant improvement.

[0570] The comparison of the inhibitory effects of some siRNAs with similar sequences on PCSK9 mRNA is shown in Table 7.

[0571] Table 7 Comparison of the inhibition rate of PCSK9 gene expression by siRNAs with similar sequences

[0572] As can be seen from Table 7, some siRNAs with similar sequences have very different inhibitory effects on PCSK9 mRNA expression.

[0573] Base sequence 4 differs from base sequence 5 only by one base at the end. The sense strand has an additional C at the 5' end of base sequence 4, and an additional U at the 3' end of base sequence 5; the rest of the sequences are identical. The antisense strand has an additional U at the 3' end of base sequence 4, and an additional A at the 5' end of base sequence 5; the rest of the sequences are identical. However, the inhibition rate of the alternatively modified sequence P92-si5 was significantly higher, increasing by 61.8% compared to P92-si4.

[0574] Base sequences 50 and 51 differ only in the terminal six bases. The sense strand has GAUUAA at the 5' end and CUGGAU at the 3' end of 50, with the rest of the sequence identical. The antisense strand has AAUCAG at the 3' end and AUCCAG at the 5' end of 51, with the rest of the sequence identical. However, the inhibition rate of the alternatively modified sequence P92-si51 was significantly higher, reaching 46.8% compared to P92-si50.

[0575] Base sequence 2 differs from base sequence 3 only in the terminal six bases. The sense strand: base sequence 2 has a 5' end of AUACCU, while base sequence 3 has a 3' end of UGUCUU; the rest of the sequence is identical. The antisense strand: base sequence 2 has a 3' end of GUAUCC, while base sequence 3 has a 5' end of AAGACA; the rest of the sequence is identical. However, the inhibition rate of the alternatively modified sequence P92-si3 was significantly higher, by 45.9%, than that of P92-si2.

[0576] Base sequences 9 and 10 differ only in their terminal two bases. The sense strand of base sequence 9 has a GG at its 5' end, while base sequence 10 has an AG at its 3' end; the rest of the sequence is identical. The antisense strand of base sequence 9 has a UA at its 3' end, while base sequence 10 has a CU at its 5' end; the rest of the sequence is identical. However, the inhibition rate of the alternating sequence P92-si9 was significantly higher, increasing by 53.1% compared to that of P92-si10.

[0577] Base sequence 21 differs from base sequence 22 only at the two terminal bases. The sense strand has a CA at the 5' end of base sequence 21 and a GA at the 3' end of base sequence 22, with the remaining sequences identical. The antisense strand has a CA at the 3' end of base sequence 21 and a UC at the 5' end of base sequence 22, with the remaining sequences identical. However, the inhibition rate of the alternatively modified sequence P92-si21 was significantly higher, increasing by 65.6% compared to P92-si22.

[0578] Base sequences 30 and 31 differ only in one base at the end. The sense strand has an A at the 5' end of base sequence 30 and a G at the 3' end of base sequence 31, with the remaining sequences identical. The antisense strand has a G at the 3' end of base sequence 30 and a C at the 5' end of base sequence 31, with the remaining sequences identical. However, the inhibition rate of the alternatively modified sequence P92-si31 was significantly higher, by 39.4%, compared to P92-si30.

[0579] Base sequences 73 and 74 differ only in one terminal base. The sense strand has a G at the 5' end of base sequence 73, while the 3' end of base sequence 74 has a G, with the remaining sequences identical. The antisense strand has an A at the 3' end of base sequence 73, while the 5' end of base sequence 74 has a C, with the remaining sequences identical. However, the inhibition rate of the alternatively modified sequence P92-si73 was significantly higher, increasing by 62.2% compared to P92-si74.

[0580] Therefore, it is not easy to screen out sequences with significant inhibitory activity from a very large number of oligonucleotide sequences designed against the PCSK9 mRNA sequence, and it requires a lot of creative work.

[0581] Summarize:

[0582] (1) Among the 84 designed sequences, 12 sequences showed significant inhibitory effects on the PCSK9 gene, with inhibition rates exceeding 50%, including P92-si5, P92-si51, P92-si81, P92-si82, P92-si84, P92-si3, P92-si8, P92-si9, P92-si21, P92-si31, P92-si73, and P92-si83. Among them, the inhibition rates of P92-si5, P92-si81, P92-si82, P92-si3, P92-si8, and P92-si31 exceeded 70%.

[0583] (2) The inhibition rates of the 26 sequences on the PCSK9 gene ranged from 30% to 50%. For example, the inhibition rates of P92-si6 and P92-si7 were 38.3% and 35.7%, respectively.

[0584] (3) The inhibition rates of 46 sequences on the PCSK9 gene were below 30%. For example, the inhibition rates of P92-si1 and P92-si20 were only 9.2% and 6.0%, respectively.

[0585] (4) siRNAs with similar sequences have very different activities. For example, the inhibition rate of P92-si5 was 61.8% higher than that of P92-si4, which is a significant improvement. Therefore, it is not easy to screen out sequences with significant inhibitory activity from a large number of oligonucleotide sequences designed against the PCSK9 mRNA sequence, and a lot of creative work is required.

[0586] Example 3: Inhibitory effect of unmodified sequences on the PCSK9 gene

[0587] In this example, some unmodified sequences (i.e., basic sequences) corresponding to the modified sequences in Example 2 were synthesized and transfected into HELA cells via lipid nanoparticles (LNPs). The inhibitory effect of each unmodified sequence on the PCSK9 gene was detected using qPCR technology, and unmodified siRNA sequences with better inhibitory effects were screened out.

[0588] 1. Experimental Materials

[0589] Test samples:

[0590] The unmodified small interfering RNA sequences are listed in Table 8. All sequences were synthesized according to the method in Example 1.

[0591] Table 8 Unmodified siRNA sequences

[0592] Cell type: HeLa cell line HELA cells

[0593] Drug solvent: sterile enzyme-free water, Gibco Opti-MEM.

[0594] 2. Experimental Methods

[0595] Refer to Example 2.

[0596] 3. Experimental Results

[0597] The inhibition rates of PCSK9 mRNA expression by each unmodified sequence are shown in Tables 9 and 10.

[0598] The unmodified sequences si5, si51, si81, si82, si84, si3, si8, si9, si21, si31, si73 and si83 had significant inhibitory effects on the expression of PCSK9 mRNA in HELA cells, with inhibition rates all above 50%, among which the inhibition rates of si5, si81, si82, si3 and si8 exceeded 60%.

[0599] (1) 12 unmodified sequences showed significant inhibitory effects on the PCSK9 gene, with inhibition rates exceeding 50%, including si5, si51, si81, si82, si84, si3, si8, si9, si21, si31, si73, and si83. Among them, si5, si81, si82, si3, and si8 showed inhibition rates exceeding 60%.

[0600] Table 9 Unmodified sequences with significant inhibitory effect on PCSK9 gene (inhibition rate greater than 50%)

[0601] The unmodified sequences listed in Table 9, including si5, si51, si81, si82, si84, si3, si8, si9, si21, si31, si73, and si83, significantly inhibited PCSK9 mRNA expression, with inhibition rates exceeding 50%. Among them, si5, si81, si82, si3, and si8 exhibited inhibition rates exceeding 60% ( FIG5 ). These 12 sequences can be used as candidate sequences.

[0602] (2) The inhibition rates of other unmodified sequences on the PCSK9 gene were all lower than 40%. For example, the inhibition rates of si52 and si74 were only 0.5% and 2.2% respectively.

[0603] Table 10: Unmodified sequences with an inhibition rate of less than 40% on the PCSK9 gene

[0604] The sequences in Table 10 had very poor inhibitory effects on PCSK9 mRNA expression, with inhibition rates all below 40%. For example, the inhibition rates of si52 and si74 were only 0.5% and 2.2% ( FIG. 6 ).

[0605] (3) siRNAs with similar sequences have very different activities. For example, the inhibition rate of base sequence 5 is 51.2% higher than that of base sequence 4, which is a significant improvement.

[0606] The comparison of the inhibitory effects of some siRNAs with similar sequences on PCSK9 mRNA is shown in Table 11.

[0607] Table 11 Comparison of the inhibition rate of PCSK9 gene expression by unmodified sequences with similar sequences

[0608] As can be seen from Table 11, some siRNAs with similar sequences have very different inhibitory effects on PCSK9 mRNA expression.

[0609] Base sequence 4 differs from base sequence 5 only by one base at the end. The sense strand has an additional C at the 5' end of base sequence 4, while the 3' end of base sequence 5 has an additional U; the rest of the sequences are identical. The antisense strand has an additional U at the 3' end of base sequence 4, while the 5' end of base sequence 5 has an additional A; the rest of the sequences are identical. However, base sequence 5 significantly increases its inhibition rate by 51.2% compared to base sequence 4.

[0610] Base sequence 50 differs from base sequence 51 only in the terminal six bases. The sense strand of base sequence 50 has a 5' end of GAUUAA, while the 3' end of base sequence 51 has a CUGGAU, with the rest of the sequence identical. The antisense strand of base sequence 50 has a 3' end of AAUCAG, while the 5' end of base sequence 51 has a 5' end of AUCCAG, with the rest of the sequence identical. However, the inhibition rate of base sequence 51 was significantly higher, by 42.6%, compared to base sequence 50.

[0611] Base sequence 2 differs from base sequence 3 only in the terminal six bases. The sense strand: base sequence 2 has a 5' end of AUACCU, while base sequence 3 has a 3' end of UGUCUU; the rest of the sequence is identical. The antisense strand: base sequence 2 has a 3' end of GUAUCC, while base sequence 3 has a 5' end of AAGACA; the rest of the sequence is identical. However, base sequence 3 significantly increased its inhibition rate by 42.7% compared to base sequence 2.

[0612] Base sequences 9 and 10 differ only in their terminal two bases. The sense strand of base sequence 9 has a GG at its 5' end, while base sequence 10 has an AG at its 3' end; the rest of the sequence is identical. The antisense strand of base sequence 9 has a UA at its 3' end, while base sequence 10 has a CU at its 5' end; the rest of the sequence is identical. However, the inhibition rate of base sequence 9 was significantly higher, by 41.2%, compared to base sequence 10.

[0613] Base sequence 21 differs from base sequence 22 only in the two terminal bases. The sense strand of base sequence 21 has a CA at its 5' end, while the 3' end of base sequence 22 has a GA, with the remaining sequences identical. The antisense strand of base sequence 21 has a CA at its 3' end, while the 5' end of base sequence 22 has a UC, with the remaining sequences identical. However, the inhibition rate of base sequence 21 was significantly higher, by 53.2%, compared to base sequence 22.

[0614] Base sequence 30 differs from base sequence 31 only at the terminal end. The sense strand of base sequence 30 has an A at its 5' end, while base sequence 31 has a G at its 3' end; the rest of the sequences are identical. The antisense strand of base sequence 30 has a G at its 3' end, while base sequence 31 has a C at its 5' end; the rest of the sequences are identical. However, base sequence 31 significantly increased its inhibition rate by 32.5% compared to base sequence 30.

[0615] Base sequence 73 differs from base sequence 74 only by a single terminal base. The sense strand of base sequence 73 has a G at its 5' end, while base sequence 74 has a G at its 3' end; the rest of the sequence is identical. The antisense strand of base sequence 73 has an A at its 3' end, while base sequence 74 has a C at its 5' end; the rest of the sequence is identical. However, the inhibition rate of base sequence 73 was significantly higher, by 50.2%, compared to base sequence 74.

[0616] Therefore, it is not easy to screen out sequences with significant inhibitory activity from a very large number of oligonucleotide sequences designed against the PCSK9 mRNA sequence, and it requires a lot of creative work.

[0617] (4) The effects of alternating modifications on the activity of different sequences were not consistent. Some showed significant increases in inhibition rate, such as the base sequence 5, where the inhibition rate of alternating modifications was 12.4% higher than that of the unmodified sequence. Others showed little difference, such as the base sequences 4, 22, and 52, where the inhibition rates of the alternating modified sequences and the unmodified sequences remained essentially unchanged.

[0618] Table 12 Comparison of the inhibition rate of PCSK9 gene by alternating modified sequences and unmodified sequences

[0619] As can be seen from Table 12, the effects of alternating modifications on PCSK9 mRNA inhibition rates were inconsistent across different sequences. Some showed significant improvements, such as a 12.4% increase in inhibition rate for base sequence 5 alternating modification compared to the unmodified sequence, and a 12.2% increase in inhibition rate for base sequence 82 alternating modification compared to the unmodified sequence. Other improvements were less significant, such as the inhibition rates for base sequences 4, 22, and 52 alternating modification sequences compared to the unmodified sequence, which showed essentially no difference.

[0620] Therefore, not all unmodified sequences can have their activity improved after alternating modification, and the effects of alternating modification on the activities of different sequences are not consistent.

[0621] Summarize:

[0622] (1) 12 unmodified sequences showed significant inhibitory effects on the PCSK9 gene, with inhibition rates exceeding 50%, including si5, si51, si81, si82, si84, si3, si8, si9, si21, si31, si73, and si83. Among them, si5, si81, si82, si3, and si8 showed inhibition rates exceeding 60%.

[0623] (2) The inhibition rate of other unmodified sequences on the PCSK9 gene was less than 40%. For example, the inhibition rates of si52 and si74 were only 0.5% and 2.2% respectively.

[0624] (3) siRNAs with similar sequences have very different activities. For example, the inhibition rate of base sequence 5 is 51.2% higher than that of base sequence 4, which is a significant improvement.

[0625] (4) The effects of alternating modifications on the activity of different sequences were not consistent. Some showed significant increases in inhibition rate, such as the base sequence 5, where the inhibition rate of alternating modifications was 12.4% higher than that of the unmodified sequence. Others showed little difference, such as the base sequences 4, 22, and 52, where the inhibition rates of the alternating modified sequences and the unmodified sequences remained essentially unchanged.

[0626] Example 4: Inhibition of PCSK9 gene using template-modified sequences

[0627] In this example, 12 sequences, namely the unmodified sequences si5, si51, si81, si82, si84, si3, si8, si9, si21, si31, si73, and si83, screened in Example 3, were modified using a modification template. DV25, DV26, DV27, DV28, DV29, DV30, and DV31 are new modification templates designed in this disclosure, while DV21 and DV22 are previously published Advanced ESC modification templates.

[0628] 1. Experimental Materials

[0629] Test samples:

[0630] Table 13 lists the sequences si5, si51, si81, si82, si84, si3, si8, si9, si21, si31, si73 and si83 in Example 3 modified using different templates (DV25, DV26, DV27, DV28, DV29, DV30, DV31, DV21 and DV22), and the corresponding alternatively modified sequences P92-si5, P92-si51, P92-si81, P92-si82, P92-si84, P92-si3, P92-si8, P92-si9, P92-si21, P92-si31, P92-si73 and P92-si83 (synthesized in Example 1).

[0631] The modification principles of the disclosed modified templates are as follows: DV25 to DV29:

[0632] The antisense strand is modified in one of the following ways:

[0633] The sense strand is modified in one of the following ways:

[0634] In the above table, PS is a phosphorothioate skeleton.

[0635] The siRNA modified template with modification method A on the antisense strand and modification method a on the sense strand was named DV25;

[0636] The siRNA modified template with modification method B on the antisense strand and modification method a on the sense strand was named DV26;

[0637] The siRNA modified template with modification method C on the antisense strand and modification method a on the sense strand was named DV27;

[0638] The siRNA modification template with the antisense strand modified by method B and the sense strand modified by method b was named DV28;

[0639] The siRNA modified template with modification method C on the antisense strand and modification method b on the sense strand was named DV29.

[0640] DV30:

[0641] Antisense strand:

[0642] Chain of Justice:

[0643] In the above table, PS is a phosphorothioate skeleton.

[0644] DV31:

[0645] Antisense strand:

[0646] Chain of Justice:

[0647] In the above table, PS is a phosphorothioate skeleton.

[0648] DV21:

[0649] Antisense strand:

[0650] Chain of Justice:

[0651] In the above table, PS is a phosphorothioate skeleton.

[0652] DV22:

[0653] Antisense strand:

[0654] Chain of Justice:

[0655] In the above table, PS is a phosphorothioate skeleton.

[0656] The modified sequences of each template are detailed in Table 13, and the synthesis methods of each sequence are the same as in Example 1.

[0657] Table 13 Sequences modified using different modification templates

[0658] Cell type: HEP3B cell line

[0659] Drug solvent: sterile enzyme-free water, Gibco DMEM (purchased from Thermo Fisher Scientific; catalog number: 10569010)

[0660] 2. Experimental Methods

[0661] qRT-PCR was used to detect the inhibitory effect of the test samples on PCSK9 mRNA expression in the HEP3B cell line. Hep3B cells were provided by Shanghai WuXi AppTec Pharmaceutical Development Co., Ltd. (ATCC-tings-1618164).

[0662] 2.1 Cell culture

[0663] Take the subcultured HEP3B cell line and culture the exponentially growing cells in RPMI 1640 medium (supplemented with 100 μL / mL each of penicillin and streptomycin) with 10% fetal bovine serum in a 37°C cell culture incubator with 5% CO2. Change the medium daily. Digest the cells with 0.25% trypsin, centrifuge at 1000 rpm for 5 minutes, discard the supernatant, and add fresh medium for subculture.

[0664] 2.2 Cell transfection

[0665] Preparation of transfection mixture: Mix Lipofectamine RNAiMAX and Opti-MEM at a ratio of 1.5:98.5 and vortex to mix thoroughly.

[0666] Transfection reagent preparation: Add 60 μL of siRNA solution diluted in Opti-MEM to 60 μL of transfection mixture at a 1:1 (v / v) ratio. Vortex to mix thoroughly and let stand at room temperature for 15 minutes to obtain lipid nanoparticles (LNPs). 12.5 μL of the solution was used for encapsulation efficiency testing.

[0667] For the blank control group, add 60 μL of the prepared transfection mixture to 60 μL of Opti-MEM. Vortex to mix thoroughly and let stand at room temperature for 15 minutes.

[0668] Add the prepared transfection reagent to a 24-well cell culture plate (100 μL per well) to make the final siRNA concentration in each well 0.05 nM. Add 500 μL of cell suspension (6×10 cells per ml) 4 After mixing using the cross method, the cells were placed in a 37°C, 5% CO2 cell incubator and cultured for 40 h.

[0669] 2.3 PCSK9 mRNA detection

[0670] The steps are the same as those in “2.3 PCSK9 mRNA detection” in Example 2.

[0671] 2.4 Data Processing

[0672] The calculation formula for PCSK9 mRNA expression rate (%) is:

[0673] Expression rate = (PCSK9 mRNA expression level / PCSK9 mRNA expression level in blank control group) × 100%;

[0674] PCSK9 gene expression inhibition rate = 1-expression rate (%).

[0675] 2.5 EC 50 experiment

[0676] In this experiment, each sequence EC 50The siRNA concentration in the experiment was selected from 0.2nM and diluted 4-fold, with a total of 8 concentration points (0.2nM, 0.05nM, 0.0125nM, 3.13pM, 0.78pM, 0.2pM, 0.05pM and 0.01pM). The inhibition rate of each sequence at each concentration was measured, and the graph was plotted to calculate the EC of each sequence. 50 concentration.

[0677] 3. Experimental Results

[0678] After three repeated experiments, the inhibition rates of each modified sequence on the PCSK9 gene in Hep3B cells are shown in Tables 14-25.

[0679] Activity assay results showed that 12 candidate sequences (unmodified sequences si5, si51, si81, si82, si84, si3, si8, si9, si21, si31, si73, and si83) modified with the disclosed modified template DV25-29 exhibited significant inhibition against the PCSK9 gene, with inhibition rates exceeding 70%. Among them, P92-si81-DV26, P92-si82-DV27, and P92-si82-DV29 achieved inhibition rates of 89.3%, 89.3%, and 89.1%, respectively.

[0680] Using the modified templates DV25 to DV29 of the disclosed disclosure significantly improved the inhibition rate of PCSK9 gene expression compared to the alternating modified sequences. For example, the inhibition rate of base sequence 51 using the template DV27 modified sequence P92-si51-DV27 was 29.1% higher than that of the alternating modified sequence, and the inhibition rate of base sequence 81 using the template DV26 modified sequence P92-si81-DV26 was 26.9% higher than that of the alternating modified sequence.

[0681] Furthermore, the activity of the same siRNA sequence modified with different templates varied significantly. For example, when base sequence 51 was modified with the disclosed template DV27, the inhibition rate increased by 22.8% compared to when it was modified with the disclosed template DV31. Furthermore, the inhibition rate increased by 21.3% compared to when it was modified with the previously disclosed Advanced ESC template DV21.

[0682] EC 50 Experiments have shown that sequences modified using the disclosed design templates have EC50 values ​​ranging from 0.0001 to 0.005 nM. For example, the EC50 values ​​for P92-si81-DV27, P92-si84-DV26, and P92-si82-DV27 were 0.0003 nM, 0.0004 nM, and 0.0006 nM, respectively. These sequences can effectively inhibit PCSK9 gene expression at low concentrations.

[0683] (i) Effect of template modification on PCSK9 gene inhibition of each basic sequence

[0684] (1) Basic sequence 5

[0685] Table 14 PCSK9 gene inhibition rate after template modification of basic sequence 5

[0686] I. DV25-DV29 templates disclosed herein

[0687] Modification of base sequence 5 with the disclosed modification templates DV25 to DV29 resulted in PCSK9 gene inhibition rates exceeding 80%, significantly improving PCSK9 gene inhibition compared to alternating 2'-methoxy and 2'-fluorinated modifications. For example, modification with templates DV26 and DV27 increased the inhibition rates of P92-si5-DV26 and P92-si5-DV27 by 19.5% and 16.6%, respectively (Figure 7).

[0688] II. Other modified templates disclosed herein

[0689] After the basic sequence 5 was modified with templates DV30 and DV31, the inhibition rates of P92-si5-DV30 and P92-si5-DV31 on the PCSK9 gene were 65.3% and 62.4%, respectively, which were 0.1% and 3.0% lower than those of the corresponding alternatively modified sequences.

[0690] Using the modified templates DV26 and DV27 modified sequences disclosed herein, the inhibition rates of P92-si5-DV26 and P92-si5-DV27 were significantly improved by 19.6% and 16.7% compared to P92-si5-DV30, and by 22.5% and 19.6% compared to P92-si5-DV31, respectively.

[0691] III. Prior Art Advanced ESC templates DV21 (fluorinated sites: antisense strand positions 2, 6, 14, and 16, sense strand positions 7, 9, 10, 11, 16, and 17) and DV22 (fluorinated sites: antisense strand positions 2, 6, 14, and 16, sense strand positions 7, 9, 10, and 11) have been disclosed.

[0692] After the basic sequence 5 was modified with templates DV21 and DV22, the inhibition rates of P92-si5-DV21 and P92-si5-DV22 on the PCSK9 gene were 68.2% and 69.1%, respectively, which were 2.8% and 3.7% higher than those of the alternatively modified sequences.

[0693] Using the modified templates DV26 and DV27 modified sequences disclosed herein, the inhibition rates of P92-si5-DV26 and P92-si5-DV27 were significantly improved by 16.7% and 13.8% compared to P92-si5-DV21, and by 15.8% and 12.9% compared to P92-si5-DV22, respectively.

[0694] From this we can see that:

[0695] 1) Base sequence 5, modified with the disclosed templates DV25 to DV29, exhibited significantly enhanced PCSK9 gene inhibition compared to the alternatively modified sequences. For example, base sequence 5, modified with DV26, exhibited a 19.5% higher inhibition rate than the alternatively modified sequences.

[0696] 2) The activity of the same siRNA sequence modified with different templates varied significantly. For example, when base sequence 5 was modified with the disclosed templates DV26 and DV27, the inhibition rate was significantly increased by up to 22.5% compared to when modified with the disclosed templates DV30 and DV31. Furthermore, when modified with the disclosed Advanced ESC templates DV21 and DV22, the inhibition rate was significantly increased by up to 16.7%.

[0697] 3) The activity of siRNA sequences modified with different templates varies greatly, up to 20%. Therefore, it is uncertain which template modification siRNA sequence will have the highest activity.

[0698] (2) Basic sequence 51

[0699] Table 15 PCSK9 gene inhibition rate after template modification of basic sequence 51

[0700] I. DV25-29 template of the present disclosure

[0701] After base sequence 51 was modified with the templates DV25 to DV29 designed in this disclosure, the inhibition rate against the PCSK9 gene exceeded 75%. This was significantly improved compared to the modification with alternating 2'-methoxy and 2'-fluoro groups. For example, after modification with templates DV26 and DV27, the inhibition rates of P92-si51-DV26 and P92-si51-DV27 increased by 27.7% and 29.1%, respectively.

[0702] II. Other modified templates disclosed herein

[0703] After the basic sequence 51 was modified with templates DV30 and DV31, the inhibition rates of P92-si51-DV30 and P92-si51-DV31 on the PCSK9 gene were 63.5% and 62.8%, respectively, which were only 7.0% and 6.3% higher than those of the corresponding alternatively modified sequences.

[0704] Using the modified templates DV26 and DV27 modified sequences disclosed herein, the inhibition rates of P92-si51-DV26 and P92-si51-DV27 were significantly improved by 20.7% and 22.1% compared with P92-si51-DV30, and by 21.4% and 22.8% compared with P92-si51-DV31, respectively.

[0705] III. Prior Art Advanced ESC templates DV21 (fluorinated sites: antisense strand positions 2, 6, 14, and 16, sense strand positions 7, 9, 10, 11, 16, and 17) and DV22 (fluorinated sites: antisense strand positions 2, 6, 14, and 16, sense strand positions 7, 9, 10, and 11) have been disclosed.

[0706] After the basic sequence 51 was modified with the published Advanced ESC templates DV21 and DV22, the inhibition rates of P92-si51-DV21 and P92-si51-DV22 on the PCSK9 gene were 64.3% and 65.1%, respectively, which were 7.8% and 8.6% higher than those of the alternatively modified sequences.

[0707] Using the modified templates DV26 and DV27 modified sequences disclosed herein, the inhibition rates of P92-si51-DV26 and P92-si51-DV27 were significantly improved by 19.9% ​​and 21.3% compared with P92-si51-DV21, and by 19.1% and 20.5% compared with P92-si5-DV22, respectively.

[0708] From this we can see that:

[0709] 1) Base sequence 51, using the disclosed modified templates DV25 to DV29, significantly improved PCSK9 gene inhibition compared to the alternatively modified sequences. For example, base sequence 51, using the DV27 modified sequence, showed a 29.1% higher inhibition rate than the alternatively modified sequences.

[0710] 2) The activity of the same siRNA sequence modified with different templates varied significantly. For example, when base sequence 51 was modified with the disclosed templates DV26 and DV27, the inhibition rate was significantly increased by up to 22.8% compared to when modified with the disclosed templates DV30 and DV31. Furthermore, when modified with the disclosed Advanced ESC templates DV21 and DV22, the inhibition rate was significantly increased by up to 21.3%.

[0711] 3) The activity of siRNA sequences modified with different templates varies greatly, up to 20%. Therefore, it is uncertain which template modification siRNA sequence will have the highest activity.

[0712] (3) Basic sequence 81

[0713] Table 16 PCSK9 gene inhibition rate after template modification of basic sequence 81

[0714] I. Modification using the disclosed DV25-DV29 templates

[0715] Modification of base sequence 81 with templates DV25 to DV29, designed in this disclosure, resulted in PCSK9 gene inhibition rates exceeding 80%. This significantly improved PCSK9 gene inhibition compared to alternating 2'-methoxy and 2'-fluorinated modifications. For example, modification with templates DV26 and DV27 increased the inhibition rates of P92-si81-DV26 and P92-si81-DV27 by 26.9% and 24.7%, respectively.

[0716] II. Other modified templates disclosed herein

[0717] After the basic sequence 81 was modified with templates DV30 and DV31, the inhibition rates of P92-si81-DV30 and P92-si81-DV31 on the PCSK9 gene were 71.6% and 73.5%, respectively, which were 9.2% and 11.1% higher than those of the corresponding alternatively modified sequences.

[0718] Using the modified templates DV26 and DV27 modified sequences disclosed herein, the inhibition rates of P92-si81-DV26 and P92-si81-DV27 were significantly improved by 17.7% and 15.5% compared to P92-si81-DV30, and by 15.8% and 13.6% compared to P92-si81-DV31, respectively.

[0719] III. Prior Art Advanced ESC templates DV21 (fluorinated sites: antisense strand positions 2, 6, 14, and 16, sense strand positions 7, 9, 10, 11, 16, and 17) and DV22 (fluorinated sites: antisense strand positions 2, 6, 14, and 16, sense strand positions 7, 9, 10, and 11) have been disclosed.

[0720] After the basic sequence 81 was modified with templates DV21 and DV22, the inhibition rates of P92-si81-DV21 and P92-si81-DV22 on the PCSK9 gene were 78.3% and 75.1%, respectively, which were 15.9% and 12.7% higher than those of the alternatively modified sequences.

[0721] Using the modified templates DV26 and DV27 modified sequences disclosed herein, the inhibition rates of P92-si81-DV26 and P92-si81-DV27 were significantly improved by 11.0% and 8.8% compared with P92-si81-DV21, and by 14.2% and 12.0% compared with P92-si81-DV22, respectively.

[0722] From this we can see that:

[0723] 1) Base sequence 81, using the disclosed modified template DV25-29, significantly improved its inhibitory effect on the PCSK9 gene compared to the alternating modified sequence. For example, base sequence 81, using the DV26 modified sequence, showed a 26.9% higher inhibition rate than the alternating modified sequence.

[0724] 2) The activity of the same siRNA sequence modified with different templates varied significantly. For example, when the base sequence 81 was modified with the disclosed templates DV26 and DV27, the inhibition rate was significantly increased by up to 17.7% compared to when it was modified with the disclosed templates DV30 and DV31. Furthermore, when it was modified with the disclosed Advanced ESC templates DV21 and DV22, the inhibition rate was significantly increased by up to 14.2%.

[0725] 3) The activity of siRNA sequences modified with different templates varies greatly, up to 20%. Therefore, it is uncertain which template modification siRNA sequence will have the highest activity.

[0726] (4) Basic sequence 82

[0727] Table 17 PCSK9 gene inhibition rate after template modification of basic sequence 82

[0728] I. DV25-DV29 templates disclosed herein

[0729] Modification of base sequence 82 with the disclosed modification templates DV25-29 resulted in PCSK9 gene inhibition rates exceeding 80%. This significantly improved PCSK9 gene inhibition compared to alternating 2'-methoxy and 2'-fluoro modifications. For example, modification with templates DV27 and DV29 increased the inhibition rates of P92-si82-DV27 and P92-si82-DV29 by 19.6% and 19.4%, respectively.

[0730] II. Other modified templates disclosed herein

[0731] After the basic sequence 82 was modified with templates DV30 and DV31, the inhibition rates of P92-si82-DV30 and P92-si82-DV31 on the PCSK9 gene were 70.2% and 69.7%, respectively, which were comparable to the corresponding alternatively modified sequences.

[0732] Using the modified templates DV27 and DV29 modified sequences disclosed herein, the inhibition rates of P92-si82-DV27 and P92-si82-DV29 were significantly improved by 19.1% and 18.9% compared to P92-si82-DV30, and by 19.6% and 19.4% compared to P92-si82-DV31, respectively.

[0733] III. Prior Art Advanced ESC templates DV21 (fluorinated sites: antisense strand positions 2, 6, 14, and 16, sense strand positions 7, 9, 10, 11, 16, and 17) and DV22 (fluorinated sites: antisense strand positions 2, 6, 14, and 16, sense strand positions 7, 9, 10, and 11) have been disclosed.

[0734] After the basic sequence 82 was modified with templates DV21 and DV22, the inhibition rates of P92-si82-DV21 and P92-si82-DV22 on the PCSK9 gene were 76.5% and 75.4%, respectively, which were 6.8% and 5.7% higher than those of the alternatively modified sequences.

[0735] Using the modified templates DV27 and DV29 modified sequences disclosed herein, the inhibition rates of P92-si82-DV27 and P92-si82-DV29 were significantly improved by 12.8% and 12.6% compared to P92-si82-DV21, and by 13.9% and 13.7% compared to P92-si82-DV22, respectively.

[0736] From this we can see that:

[0737] 1) Base sequence 82, using the disclosed modified templates DV25-DV29, significantly improved PCSK9 gene inhibition compared to the alternatively modified sequences. For example, base sequence 82, using the DV27 modified sequence, showed a 19.6% higher inhibition rate than the alternatively modified sequences.

[0738] 2) The activity of the same siRNA sequence modified with different templates varied significantly. For example, when the base sequence 82 was modified with the disclosed templates DV27 and DV29, the inhibition rate was significantly increased by up to 19.6% compared to when it was modified with the disclosed templates DV30 and DV31. Furthermore, when it was modified with the disclosed Advanced ESC templates DV21 and DV22, the inhibition rate was significantly increased by up to 13.9%.

[0739] 3) The activity of siRNA sequences modified with different templates varies greatly, up to 20%. Therefore, it is uncertain which template modification siRNA sequence will have the highest activity.

[0740] (5) Basic sequence 84

[0741] Table 18 PCSK9 gene inhibition rate after template modification of basic sequence 84

[0742] I. DV25-29 template of the present disclosure

[0743] Modification of base sequence 84 with the disclosed modification templates DV25 to DV29 resulted in PCSK9 gene inhibition rates exceeding 85%. This significantly improved PCSK9 gene inhibition compared to alternating 2'-methoxy and 2'-fluorinated modifications. For example, modification with templates DV28 and DV29 increased the inhibition rates of P92-si84-DV28 and P92-si84-DV29 by 26.8% and 25.9%, respectively.

[0744] II. Other modified templates disclosed herein

[0745] After the basic sequence 84 was modified with templates DV30 and DV31, the inhibition rates of P92-si84-DV30 and P92-si84-DV31 on the PCSK9 gene were 70.3% and 72.4%, respectively, which were 8.7% and 10.8% higher than those of the corresponding alternatively modified sequences.

[0746] Using the modified templates DV28 and DV29 modified sequences disclosed herein, the inhibition rates of P92-si84-DV28 and P92-si84-DV29 were significantly improved by 18.1% and 17.2% compared to P92-si84-DV30, and by 16.0% and 15.1% compared to P92-si84-DV31, respectively.

[0747] III. Prior Art Advanced ESC templates DV21 (fluorinated sites: antisense strand positions 2, 6, 14, and 16, sense strand positions 7, 9, 10, 11, 16, and 17) and DV22 (fluorinated sites: antisense strand positions 2, 6, 14, and 16, sense strand positions 7, 9, 10, and 11) have been disclosed.

[0748] After the basic sequence 84 was modified with templates DV21 and DV22, the inhibition rates of P92-si84-DV21 and P92-si84-DV22 on the PCSK9 gene were 77.7% and 75.1%, respectively, which were 16.1% and 13.5% higher than those of the alternatively modified sequences.

[0749] Using the modified templates DV28 and DV29 modified sequences disclosed herein, the inhibition rates of P92-si84-DV28 and P92-si84-DV29 were significantly improved by 10.7% and 9.8% compared to P92-si84-DV21, and by 13.3% and 12.4% compared to P92-si84-DV22, respectively.

[0750] From this we can see that:

[0751] 1) Base sequence 84, using the disclosed modified templates DV25-DV29, showed significantly enhanced inhibition of the PCSK9 gene compared to the alternatively modified sequences. For example, base sequence 84, using the DV28 modified sequence, showed a 26.8% higher inhibition rate than the alternatively modified sequences.

[0752] 2) The activity of the same siRNA sequence modified with different templates varied significantly. For example, when base sequence 84 was modified with the disclosed templates DV28 and DV29, the inhibition rate was significantly increased by up to 18.1% compared to when it was modified with the disclosed templates DV30 and DV31. Furthermore, when it was modified with the disclosed Advanced ESC templates DV21 and DV22, the inhibition rate was significantly increased by up to 13.3%.

[0753] 3) The activity of siRNA sequences modified with different templates varies greatly, up to 20%. Therefore, it is uncertain which template modification siRNA sequence will have the highest activity.

[0754] (6) Basic sequence 3

[0755] Table 19 PCSK9 gene inhibition rate after template modification of basic sequence 3

[0756] 1) Modification of base sequence 3 with the disclosed modification templates DV25-29 resulted in PCSK9 gene inhibition rates exceeding 75%. This significantly improved PCSK9 gene inhibition compared to alternating 2'-methoxy and 2'-fluoro modifications. For example, modification with templates DV28 and DV29 increased the inhibition rates of P92-si3-DV28 and P92-si3-DV29 by 24.0% and 24.7%, respectively.

[0757] 2) It can be seen that the basic sequence 3 using the modified template DV25-29 modified sequence disclosed herein has significantly improved the inhibitory activity on the PCSK9 gene compared to using the alternating modification.

[0758] (7) Basic sequence 8

[0759] Table 20 PCSK9 gene inhibition rate after template modification of basic sequence 8

[0760] 1) Modification of base sequence 8 with the disclosed modification templates DV25-29 resulted in PCSK9 gene inhibition rates exceeding 75%. This significantly improved PCSK9 gene inhibition compared to alternating 2'-methoxy and 2'-fluoro modifications. For example, modification with templates DV27 and DV28 increased the inhibition rates of P92-si8-DV27 and P92-si8-DV28 by 19.7% and 20.2%, respectively.

[0761] 2) It can be seen that the basic sequence 8 modified with the disclosed modification template DV25-29 has a significantly improved inhibitory activity on the PCSK9 gene compared to the alternating modification.

[0762] (8) Basic sequence 9

[0763] Table 21 PCSK9 gene inhibition rate after template modification of basic sequence 9

[0764] 1) Modification of base sequence 9 with the disclosed modification templates DV25-29 resulted in PCSK9 gene inhibition rates exceeding 70%. This significantly improved PCSK9 gene inhibition compared to alternating 2'-methoxy and 2'-fluoro modifications. For example, modification with templates DV27 and DV28 increased the inhibition rates of P92-si9-DV27 and P92-si9-DV28 by 27.2% and 25.4%, respectively.

[0765] 2) It can be seen that the basic sequence 9 modified with the disclosed modification template DV25-29 has a significantly improved inhibitory activity on the PCSK9 gene compared to the alternating modification.

[0766] (9) Basic sequence 21

[0767] Table 22 PCSK9 gene inhibition rate after template modification of basic sequence 21

[0768] 1) Modification of base sequence 21 with the disclosed modification templates DV25-29 resulted in PCSK9 gene inhibition rates exceeding 70%. This significantly improved PCSK9 gene inhibition compared to alternating 2'-methoxy and 2'-fluoro modifications. For example, modification with templates DV25 and DV26 increased the inhibition rates of P92-si21-DV25 and P92-si21-DV26 by 24.4% and 26.5%, respectively.

[0769] 2) It can be seen that the base sequence 21 modified with the disclosed modification template DV25-29 has a significantly improved inhibitory activity on the PCSK9 gene compared to the alternating modification.

[0770] (10) Basic sequence 31

[0771] Table 23 PCSK9 gene inhibition rate after template modification of base sequence 31

[0772] 1) Modification of base sequence 31 with the disclosed modification templates DV25-29 resulted in PCSK9 gene inhibition rates exceeding 70%. This significantly improved PCSK9 gene inhibition compared to alternating 2'-methoxy and 2'-fluoro modifications. For example, modification with templates DV25 and DV26 increased the inhibition rates of P92-si31-DV25 and P92-si31-DV26 by 22.9% and 22.6%, respectively.

[0773] 2) It can be seen that the base sequence 31 modified with the disclosed modification template DV25-29 has a significantly improved inhibitory activity on the PCSK9 gene compared to the alternating modification.

[0774] (11) Basic sequence 73

[0775] Table 24 PCSK9 gene inhibition rate after template modification of basic sequence 73

[0776] 1) Modification of base sequence 73 with the disclosed modification templates DV25-29 resulted in PCSK9 gene inhibition rates exceeding 70%. This significantly improved PCSK9 gene inhibition compared to alternating 2'-methoxy and 2'-fluoro modifications. For example, modification with templates DV28 and DV29 increased the inhibition rates of P92-si73-DV28 and P92-si73-DV29 by 30.6% and 32.7%, respectively.

[0777] 2) It can be seen that the basic sequence 73 modified with the modified template DV25-29 disclosed herein has a significantly improved inhibitory activity on the PCSK9 gene compared to the alternating modification.

[0778] (12) Basic sequence 83

[0779] Table 25 PCSK9 gene inhibition rate after template modification of basic sequence 83

[0780] 1) Modification of base sequence 83 with the disclosed modification templates DV25-29 resulted in PCSK9 gene inhibition rates exceeding 75%. This significantly improved PCSK9 gene inhibition compared to alternating 2'-methoxy and 2'-fluoro modifications. For example, modification with templates DV27 and DV29 increased the inhibition rates of P92-si83-DV27 and P92-si83-DV29 by 30.9% and 29.7%, respectively.

[0781] 2) It can be seen that the basic sequence 83 modified with the disclosed modification template DV25-29 has a significantly improved inhibitory activity on the PCSK9 gene compared to the alternating modification.

[0782] Summarize:

[0783] (1) The selected base sequences 5, 51, 81, 82, 84, 3, 8, 9, 21, 31, 73, and 83, after modification with the modified templates DV25-29 designed in the present disclosure, showed significant inhibitory effects on PCSK9 gene expression, with inhibition rates exceeding 70%. Among them, the inhibition rates of P92-si81-DV26, P92-si82-DV27, and P92-si82-DV29 reached 89.3%, 89.3%, and 89.1%, respectively.

[0784] (2) The 12 sequences described above, modified with the disclosed template DV25-29, showed significantly improved inhibition of PCSK9 gene expression compared to the alternatively modified sequences. For example, the inhibition of base sequence 51 modified with template DV27, P92-si51-DV27, was 29.1% higher than that of the alternatively modified sequence. The inhibition of base sequence 81 modified with template DV26, P92-si81-DV26, was 26.9% higher than that of the alternatively modified sequence.

[0785] (3) Modification of the same sequence with the disclosed modified templates DV25-29 significantly improved the inhibition rate of PCSK9 gene expression compared to modification templates disclosed in the prior art. For example, when base sequence 51 was modified with the disclosed modified template DV27, the inhibition rate increased by 21.3% compared to modification with the disclosed Advanced ESC template DV21.

[0786] (4) The same sequence modified with the disclosed modification templates DV25-29 showed significantly improved inhibition of PCSK9 gene expression compared to the modified templates DV30 and DV31. For example, the base sequence 51 modified with the disclosed modification template DV27 showed a 22.8% increase in inhibition compared to the modified template DV31.

[0787] (5) The activity of different template modification sequences varies greatly. For example, the inhibition rate of base sequence 5 modified with DV26 increased by 22.5% compared to that of base sequence 5 modified with DV31; the inhibition rate of base sequence 51 modified with DV27 increased by 20.5% compared to that of base sequence 51 modified with DV22. Therefore, it is uncertain which modification template can achieve high activity for siRNA sequences.

[0788] (ii)EC 50 experiment

[0789] Some template modification sequences were selected, including P92-si5-DV26, P92-si51-DV25, P92-si81-DV27, P92-si82-DV27, P92-si84-DV26, P92-si3-DV28, P92-si8-DV29, P92-si9-DV28, P92-si21-DV25, P92-si31-DV27, P92-si73-DV28 and P92-si83-DV26, a total of 12 sequences, for E C50 Experiment, determination of EC of each sequence 50 The experimental results are shown in Table 26.

[0790] Table 26 EC50 experimental results of each modified sequence

[0791] As can be seen from Table 26, the EC of these 12 sequences 50 The values ​​ranged from 0.0001 nM to 0.005 nM. The EC values ​​of P92-si81-DV27, P92-si84-DV26, and P92-si82-DV27 were 50 The values ​​were 0.0003nM, 0.0004nM and 0.0006nM, respectively, indicating that these sequences can effectively inhibit PCSK9 gene expression at low concentrations.

[0792] Example 5: Comparison of the inhibitory effect of the sequence disclosed in the prior art on the PCSK9 gene

[0793] This example compares the inhibition efficiency of the PCSK9 gene by using the unmodified sequences disclosed in the prior art that are identical or similar to the basic sequences 3, 5, 8, 9, 31, 81, 82, 83 and 84 of the present disclosure, the alternatively modified sequences disclosed in the present disclosure, and the sequences modified using the DV25-29 template.

[0794] 1. Experimental Materials

[0795] Test samples:

[0796] (1) Prior art public sequence

[0797] Table 27 Prior Art Disclosed Sequences Note: The sequences disclosed in the above patent applications are all unmodified sequence RNA.

[0798] (2) siRNA sequences modified with alternating 2'-OMe and 2'-F and terminal thiolation in Example 2: P92-si5, P92-si81, P92-si82, P92-si84, P92-si3, P92-si8, P92-si9, P92-si31, and P92-si83;

[0799] (3) The modified template DV25-29 was used to modify the siRNA sequence. The specific sequence is shown in Table 28.

[0800] Table 28 Sequences modified using DV25-29 modification template

[0801] Cell type: Hep3B cells, provided by Shanghai WuXi AppTec Co., Ltd. (ATCC-tings-1618164).

[0802] Hep3B cells were cultured in EMEM medium (ATCC-30-2003) containing 10% fetal bovine serum (FBS, ExCell Bio-FSP500), 1% penicillin-streptomycin (HyClone-SV30010), 1% non-essential amino acid solution (Gibco-11140-050), and 1% GlutaMAX supplement (Gibco-35050-061).

[0803] Drug solvent: sterile enzyme-free water, Gibco DMEM.

[0804] 2. Experimental Methods

[0805] qRT-PCR was used to detect the inhibitory effect of the test samples on the expression of PCSK9 gene mRNA in HEP3B cell line.

[0806] 2.1 Cell culture

[0807] Take subcultured Hep3B cells and culture them in RPMI 1640 medium (supplemented with 100 μL / mL each of penicillin and streptomycin) at logarithmic growth. Place the cells in a 37°C cell culture incubator with 5% CO2, changing the medium daily. Digest the cells with 0.25% trypsin, centrifuge at 1000 rpm for 5 minutes, discard the supernatant, and add fresh culture medium for subculture.

[0808] 2.2 Cell transfection

[0809] Add 2 μL of siRNA (1 μg / μL) to 18 μL of sterile, enzyme-free water at a 1:0.06 (wt / wt) ratio, mix thoroughly, and then add 22 μL of the prepared LNPs. Mix thoroughly and let stand at room temperature for 15 minutes. Aspirate 12.5 μL for encapsulation efficiency testing. The final transfection volume per well is (1 μg theoretical sample size / siRNA purity) / drug loading concentration, for a final siRNA concentration of 0.05 nM.

[0810] Negative control group: Add 22 μL of prepared LNPs to 20 μL of sterile, enzyme-free water. Mix thoroughly and let stand at room temperature for 15 minutes.

[0811] After mixing using the cross method, the cells were placed in a 37°C, 5% CO2 cell culture incubator and cultured for 40 h.

[0812] 2.3 PCSK9 mRNA detection

[0813] 1) RNA extraction

[0814] The steps are the same as those in “1) RNA extraction” in “2.3 PCSK9 mRNA detection” in Example 2.

[0815] 2) RNA concentration detection

[0816] The steps are the same as those in "2) RNA concentration detection" in "2.3 PCSK9 mRNA detection" in Example 2.

[0817] 3) PCSK9 mRNA quantitative detection

[0818] In a 15 mL centrifuge tube, add the remaining components except primers and template in 7.5×48=360 portions, marked as A.

[0819] Label 1.5 mL EP tubes and add 77 μL of A and 420 ng of total RNA to each tube. Mix well and set aside.

[0820] The upstream and downstream primers of the internal reference gene GAPDH and the target gene PCSK9 were mixed and set aside.

[0821] Add 1 μL of B1 and 1 μL of C to each well of the PCR plate. Cover with sealing film, centrifuge at 3000 rpm for 1 min, and transfer to the PCR machine.

[0822] *This step is performed on ice to maintain low temperature conditions.

[0823] Place the plate in the qPCR instrument and run the following program:

[0824] Reverse transcription: 55°C, 15 min;

[0825] Pre-denaturation: 95°C, 30 sec;

[0826] Cyclic reaction: 95°C, 10 sec; 60°C, 35 sec; 40 cycles;

[0827] Melting curve: 95°C, 15 sec; 60°C, 60 sec; 95°C, 15 sec.

[0828] The running time is about 2 hours. Analyze the experimental results and calculate 2 -ΔΔCt .

[0829] 2.4 Data Processing

[0830] The PCSK9 mRNA expression rate (%) was calculated as follows:

[0831] Expression rate = (PCSK9 mRNA expression in the experimental group / PCSK9 mRNA expression in the blank control group) × 100%;

[0832] PCSK9 gene expression inhibition rate = 1-expression rate (%).

[0833] 3. Experimental Results

[0834] The specific experimental results are shown in Tables 29 and 30.

[0835] Experimental results demonstrate that the alternatively modified sequences disclosed herein and sequences modified with specific modification templates exhibit significantly enhanced PCSK9 inhibitory activity compared to identical or similar unmodified sequences disclosed in the prior art. For example, while the identical unmodified sequence si84 exhibited an inhibition rate of 38.6%, the alternatively modified sequence exhibited an inhibition rate of 61.6%, a 23.0% improvement over the unmodified sequence si84. After modification with the disclosed modification template DV26, the inhibition rate reached 86.8%, a 48.2% improvement over the unmodified sequence.

[0836] Compared with the sequence si31 disclosed in the prior art, the only difference between the sequence 31P disclosed in the present invention and the sequence si31 is that the 3' end of the antisense strand is missing two bases UG, and the rest are exactly the same. However, the inhibition rate of the unmodified sequence si31 disclosed in the present invention is 10.4% higher than that of 31P, and the inhibition rate of the alternatively modified sequence is 59.5%, which is 21.2% higher than that of 31P. After modification with the modified template DV26 disclosed in the present invention, the inhibition rate reached 80.2%, which is 41.9% higher than that of 31P.

[0837] (1) Compared with the identical unmodified sequences disclosed in the prior art, the alternating modified sequences disclosed in the present invention and the modified sequences using specific modification templates significantly improve the inhibition rate of the PCSK9 gene, which can be increased by up to 40%.

[0838] Compared to identical sequences disclosed in the prior art, the alternatively modified sequences disclosed herein and sequences modified with specific modification templates significantly improved the inhibition rate of the PCSK9 gene. For example, the inhibition rate of the unmodified sequence si84 was 38.6%, while the inhibition rate after alternative modification was 61.6%, a 23.0% increase over the unmodified sequence si84. After modification with the disclosed modification template DV26, the inhibition rate reached 86.8%, a 48.2% increase over the unmodified sequence.

[0839] Table 29 Existing technology identical sequence inhibition rate of PCSK9 gene

[0840] The unmodified sequences si81, si84, si3, si8, si9, and si83 disclosed in the prior art in Table 29 are identical to the sequences disclosed herein. After modification with alternating modifications and the modified template disclosed herein, the inhibitory effect on PCSK9 was significantly improved. For example, the inhibition rate of the unmodified sequence si84 was 38.6%. After alternating modifications, the inhibition rate of P92-si84 was 61.6%, which was 23.0% higher than that of the unmodified sequence si84. After modification with the modified template DV25-29 disclosed herein, the inhibition rates of P92-si84-DV25, P92-si84-DV26, P92-si84-DV27, P92-si84-DV28, and P92-si84-DV29 reached 84.9%, 86.8%, 84.5%, 86.2%, and 86.4%, respectively, all of which were more than 40% higher than those of the unmodified sequence si84.

[0841] (2) Compared with the unmodified sequences disclosed in the prior art, which have only minor differences, the unmodified sequences, the alternatively modified sequences, and the sequences modified with specific modification templates disclosed in the present invention significantly improve the inhibition rate of the PCSK9 gene, which can be increased by up to 40%.

[0842] Compared to sequences disclosed in the prior art with only minor differences, the unmodified sequences, alternatively modified sequences, and sequences modified with specific modification templates disclosed herein significantly improved the inhibition rate of the PCSK9 gene. For example, the unmodified sequence 31P had an inhibition rate of 38.3%, while the similar unmodified sequence si31 disclosed herein had an inhibition rate of 48.7%, a 10.4% increase over 31P. The alternatively modified sequence had an inhibition rate of 59.5%, a 21.2% increase over 31P. After modification with the disclosed modification template DV26, the inhibition rate reached 80.2%, a 41.9% increase over 31P.

[0843] Table 30: PCSK9 gene inhibition rate of similar sequences in the prior art and the present disclosure

[0844] I. Sequence Alignment

[0845] The sequences of the prior art disclosed sequences 5P, 31P and 82P in Table 30 are very close to the sequences si5, si31 and si82 disclosed in the present invention, with only minor differences. Specific comparisons are shown in the table below:

[0846] Table 31 Comparison of sequences disclosed in the prior art and sequences disclosed herein

[0847] As can be seen from Table 31, the sequence 5P disclosed in the prior art is only missing two bases GG at the 3' end of the antisense chain compared with the sequence si5 disclosed in the present invention; the sequence 31P is only missing two bases UG at the 3' end of the antisense chain compared with the sequence si31 disclosed in the present invention; the sequence 82P is only missing two bases AA at the 3' end of the antisense chain compared with the sequence si82 disclosed in the present invention, and the other sequences are exactly the same.

[0848] II. Activity Comparison

[0849] 1) The unmodified sequences disclosed in this paper showed significantly improved activity compared to similar unmodified sequences disclosed in the prior art. For example, the unmodified sequence si31 disclosed in this paper showed a 10.4% higher inhibition rate than the structurally similar 31P.

[0850] The unmodified sequence si5 disclosed in the present invention has an inhibition rate of 50.1% on the PCSK9 gene, which is 8.3% higher than the similar sequence 5P disclosed in the prior art (inhibition rate of 41.8%), a significant improvement.

[0851] The unmodified sequence si82 disclosed in the present invention has an inhibition rate of 53.8% on the PCSK9 gene, which is 7.1% higher than the similar sequence 82P disclosed in the prior art (inhibition rate of 46.7%), a significant improvement.

[0852] The unmodified sequence si31 disclosed in the present invention has an inhibition rate of 48.7% on the PCSK9 gene, which is 10.4% higher than the similar sequence 31P disclosed in the prior art (inhibition rate of 38.3%), a significant improvement.

[0853] 2) The alternatively modified sequences disclosed in this disclosure have significantly improved activity compared to similar unmodified sequences disclosed in the prior art. For example, compared to sequence 5P, the inhibition rate of the alternatively modified sequence P92-si5 disclosed in this disclosure increased by 22.0%.

[0854] After the basic sequence 5 disclosed in the present invention is alternately modified, the inhibition rate of P92-si5 on the PCSK9 gene is 63.8%, which is 22.0% higher than that of the unmodified sequence 5P disclosed in the prior art, a significant improvement.

[0855] After the basic sequence 82 disclosed in the present invention is alternately modified, the inhibition rate of P92-si82 on the PCSK9 gene is 68.3%, which is 21.6% higher than that of the unmodified sequence 82P disclosed in the prior art, a significant improvement.

[0856] After the basic sequence 31 disclosed in the present invention is alternately modified, the inhibition rate of P92-si31 on the PCSK9 gene is 59.5%, which is 21.2% higher than that of the unmodified sequence 31P disclosed in the prior art, a significant improvement.

[0857] 3) Sequences modified with the disclosed modified templates showed significantly improved activity compared to similar unmodified sequences disclosed in the prior art. For example, compared to sequence 82P, the disclosed alternatively modified sequence P92-si82-DV29 showed a 42.8% higher inhibition rate.

[0858] After modification of the disclosed basic sequence 5 with modified templates 25-29, the inhibition rate was increased by more than 30% compared to the unmodified sequence 5P disclosed in the prior art. For example, P92-si5-DV27 increased by 40.8%, a significant improvement.

[0859] After modification of the disclosed base sequence 82 with the modified templates 25-29, the inhibition rate was increased by more than 30% compared to the unmodified sequence 82P disclosed in the prior art. For example, P92-si82-DV29 increased the inhibition rate by 42.8%, a significant improvement.

[0860] After modification of the base sequence 31 with the modified templates 25-29, the inhibition rate was increased by more than 30% compared to the unmodified sequence 31P disclosed in the prior art. For example, P92-si31-DV26 increased by 41.9%, a significant improvement.

[0861] It can be seen that compared with similar unmodified sequences in the prior art, the unmodified sequences disclosed herein, and the alternatingly modified and template-modified sequences disclosed herein can significantly enhance the inhibitory activity against PCSK9, with the inhibition rate being increased by up to 40%.

[0862] Summarize:

[0863] (1) Compared with identical sequences disclosed in the prior art, the alternatively modified sequences and sequences modified with specific modification templates disclosed herein significantly enhance the inhibitory effect on the PCSK9 gene. For example, the inhibition rate of the unmodified sequence si84 was 38.6%, while the inhibition rate after alternative modification was 61.6%, a 23.0% increase over the unmodified sequence si84. After modification with the modified template DV26 disclosed herein, the inhibition rate reached 86.8%, a 48.2% increase over the unmodified sequence.

[0864] (2) Compared with similar sequences disclosed in the prior art, the unmodified sequences, alternatively modified sequences, and sequences modified using a specific modification template disclosed in the present invention have significantly improved inhibitory effects on the PCSK9 gene. For example, the unmodified sequence si31 disclosed in the present invention has an inhibition rate increased by 10.4% compared with the 31P sequence disclosed in the prior art, which is similar to the sequence disclosed in the prior art. The sequence si5 disclosed in the present invention is alternatively modified to the sequence P92-si5, and the inhibition rate is increased by 22.0% compared with the unmodified sequence 5P disclosed in the prior art, which is similar to the sequence si5. The sequence si82 disclosed in the present invention is modified using the modified template DV29 disclosed in the present invention, the sequence P92-si82-DV29, and the inhibition rate is increased by 42.8% compared with the unmodified sequence 82P disclosed in the prior art, which is similar to the sequence si82.

[0865] Example 6: Inhibitory effect of PCSK9 and off-target genes using sequences designed to prevent off-target effects

[0866] In practical applications of siRNA, the expression of non-target mRNAs that are only partially complementary to the guide strand (antisense strand) is often suppressed. Research by Alnylam has shown that the hepatotoxicity of n-acetylgalactosamine (GalNAc)-conjugated siRNAs is primarily due to off-target effects, resulting in gene suppression of the wrong target through a recognition mechanism similar to that of microRNA (miRNA).

[0867] To address this issue, Alnylam's latest fifth-generation template design incorporates a glycol nucleic acid (GNA) modification at position 7 of the siRNA antisense strand to disrupt the antisense strand's seed region, significantly mitigating off-target effects and alleviating hepatotoxicity. This modification can influence siRNA binding to non-targets through recognition in the seed region, thereby suppressing off-target effects.

[0868] Natural or simple direct 5' phosphorylation may lead to dephosphorylation within cells. Direct 5' phosphorylated oligonucleotides can be 90% dephosphorylated after 2 hours of circulation in the blood, with complete dephosphorylation occurring after 24 hours. The 5'-phosphorylated design (5'-E-VP) utilizes E-vinylphosphonate in place of the bridging oxygen, resulting in optimal phosphorylation and higher stability.

[0869] In summary, in order to reduce the off-target effect of the sequence and examine the effect of 5' end phosphorylation on off-target efficacy, this example uses the basic sequences 5, 51, 81 and 84 modified with DV25-29, and adds 5'-E-VP modification and 7-position GNA anti-off-target modification at the 5' end of the antisense strand.

[0870] In addition, the sequences P92-si3, P92-si8, P92-si21, P92-si31, P92-si73 and P92-si83 modified with alternating 2'-methoxy and 2'-fluoro groups, and the above sequences P92-si3+, P92-si8+, P92-si21+, P92-si31+, P92-si73+ and P92-si83+ containing the antisense chain 7-position GNA anti-off-target modification design were compared to detect off-target effects.

[0871] The experimental results showed that at a concentration of 10 nM, the sequences using the anti-off-target design had a significant inhibitory effect on the target gene PCSK9, while the inhibitory effect on off-target genes was significantly reduced. This indicates that the anti-off-target design does not affect the inhibitory effect of the disclosed alternating modified and template modified sequences on the PCSK9 gene, but can significantly suppress off-target effects.

[0872] 1. Experimental Materials

[0873] 1) Test sample:

[0874] Alternating modified sequences: Alternating modified sequences P92-si3, P92-si8, P92-si9, P92-si21, P92-si31, P92-si73 and P92-si83 were used.

[0875] Sequences of alternating modifications and anti-off-target design: P92-si3+, P92-si8+, P92-si9+, P92-si21+, P92-si31+, P92-si73+, and P92-si83+ (Table 32).

[0876] The basic sequences 5, 84, 81 and 51 used template modified sequences, sequences using template modification and 5'-E-VP modification, sequences using template modification and anti-off-target design, and sequences using template modification, 5'-E-VP modification and anti-off-target design (Table 33).

[0877] 2) Sequence synthesis:

[0878] I. Alternating Modified Sequences and Template Modified Sequences

[0879] The synthesis method is as in Example 1.

[0880] II. siRNA sequences containing 5'-E-VP

[0881] Referring to Example 1, the siRNA sequence was synthesized. When synthesizing the base at the 5' end of the antisense strand (the last base), a monomer containing a phosphonic acid group at the 5' end was used, for example, a vinyl-(E)-phosphonate-A-OMe phosphoramidite monomer (Formula 9) or a vinyl-(E)-phosphonate-U-OMe phosphoramidite monomer (Formula 10). Examples of their structures are as follows:

[0882] III. siRNA sequences with anti-off-target design

[0883] The siRNA sequence was synthesized with reference to Example 1. When synthesizing the antisense strand from the 5' end to the 7th base, a GNA monomer was used to synthesize the sequence. The structure of the GNA monomer is as follows:

[0884] Table 32 Alternative modification plus anti-off-target modification sequence

[0885] The above sequences all adopt the alternating modification scheme of 2'-methoxy (2'-OMe) and 2'-fluoro (2'-F) at the 2' position.

[0886] (1) All odd-numbered positions of the sense strand are modified with 2'-F, and all even-numbered positions are modified with 2'-OMe;

[0887] (2) The odd-numbered positions of the antisense strand are all modified with 2'-OMe, and the even-numbered positions are all modified with 2'-F;

[0888] (3) There are two thiolation modifications at the 5' end of the sense strand and at both the 5' and 3' ends of the antisense strand;

[0889] (4) The 7th base of the antisense strand (5'-3' direction) was modified with GNA.

[0890] Table 33 Template modification, 5'-E-VP modification, and anti-off-target design sequences

[0891] Cell type: Hep3B cells, provided by Shanghai WuXi AppTec Co., Ltd. (ATCC-tings-1618164).

[0892] Hep3B cells were cultured in EMEM medium (ATCC-30-2003) containing 10% fetal bovine serum (FBS, ExCell Bio-FSP500), 1% penicillin-streptomycin (HyClone-SV30010), 1% non-essential amino acid solution (Gibco-11140-050), and 1% GlutaMAX supplement (Gibco-35050-061).

[0893] Drug solvent: sterile enzyme-free water, Gibco DMEM.

[0894] 2. Experimental Methods

[0895] 1) Compound dilution

[0896] For off-target experiments, the test samples were diluted to a concentration of 10 nM and tested in triplicate wells.

[0897] Hep3B cell digestion and counting:

[0898] Hep3B cells were taken out when the density reached 80%, rinsed with Dulbecco's phosphate buffered saline (DPBS), and digested with 0.05% trypsin for 3-10 min. After the cells were collected, they were counted using Countstar Rigel S2 and the number of cells was adjusted to 2×10 5 / mL density.

[0899] 2) Preparation of transfection reagent

[0900] Prepare RNAiMAX transfection reagent at a ratio of 3:97 RNAiMAX:Opti-MEM in a 15 mL centrifuge tube. Vortex for 15 seconds to mix thoroughly, and incubate at room temperature for 15 minutes. Add 40 μL of RNAiMAX Opti-MEM to each well of the corresponding position. Add 40 μL of the diluted compound of the corresponding concentration to the corresponding wells of the dilution plate, mix thoroughly, and incubate for 15 minutes.

[0901] 3) Cell plating

[0902] Hep3B cells (2×10 4 cells / well) to a 96-well cell plate, and the siRNA compound was mixed with RNAiMAX Opti-MEM transfection reagent and then added to the cells in each well. At the same time, a control group containing RNAiMAX Opti-MEM without siRNA compound was set up.

[0903] Off-target prevention assay: Add the compound and RNAiMAX Opti-MEM mixture from the dilution plate to a 96-well cell culture plate (20 μL / well). Subsequently, add 100 μL / well of cells to the 96-well plate for a final volume of 120 μL per well. After plating, incubate in a 5% CO2, 37°C incubator for 24 h.

[0904] 4) RNA extraction and reverse transcription

[0905] 24 hours after transfection, the culture medium was removed and the cells were collected for RNA extraction. Total RNA was extracted using QIAGEN 96 Kit (QIAGEN-74182), and cDNA was synthesized using FastKing RT Kit (with gDNase) (TIANGEN-KR116-02) according to the manufacturer's instructions.

[0906] 5) RT-qPCR

[0907] Target cDNA was detected by qPCR, and GAPDH cDNA was detected in parallel as an internal control. 8 μL of the prepared qPCR reaction solution and 2 μL of sample cDNA were added to 384-well plates. The TaqMan qPCR reaction program was as follows: heating at 95°C for 10 minutes, followed by cycling at 95°C for 15 seconds, followed by 60°C for 1 minute, for a total of 40 cycles. The SYBR qPCR reaction program was as follows: heating at 50°C for 2 minutes, heating at 95°C for 10 minutes, then cycling at 95°C for 15 seconds, followed by 60°C for 1 minute, for a total of 40 cycles. The final melting curve was performed by heating at 95°C for 15 seconds, 60°C for 1 minute, and 95°C for 15 seconds.

[0908] 6) Data Analysis

[0909] The target gene RNA expression level in each sample was calculated based on the Ct value of each sample using the ΔΔCt relative quantification method. The relative expression of the target gene was calculated using 2 -ΔΔCT express.

[0910] The calculation formula is as follows:

[0911] ΔCT = average Ct value of target gene - average Ct value of reference gene;

[0912] ΔΔCT = ΔCT (drug-treated group) - ΔCT (RNAiMAX control group);

[0913] Relative expression of target gene mRNA = 2-ΔΔCT

[0914] Inhibition rate = (1-relative expression level of sample / average expression level of RNAiMAX control) × 100%

[0915] 3. Experimental Results

[0916] (1) Different modified sequences all had significant inhibitory effects on the PCSK9 gene. For example, the inhibition rate of the alternating modification P92-si3 reached 75.6%, and the inhibition rate of the template modification and 5'-E-VP modification sequence D84-DV27P was as high as 94.4%.

[0917] The inhibition rates of PCSK9 gene by all tested samples are shown in Tables 34-36.

[0918] All the alternatively modified sequences had significant inhibitory effects on PCSK9 at a concentration of 10 nM. For example, the inhibition rate of the alternatively modified sequence P92-si3 reached 75.6%.

[0919] I. The alternating modified sequences have a significant inhibitory effect on the PCSK9 gene, with inhibition rates exceeding 60%, with P92-si3 reaching 75.6%.

[0920] Table 34 Inhibition rate of alternating modified sequence PCSK9 gene

[0921] As can be seen from the above table, at a concentration of 10 nM, the alternatively modified sequences P92-si3, P92-si8, P92-si9, P92-si21, P92-si31, P92-si51, P92-si73 and P92-si83 all had significant inhibitory effects on the PCSK9 gene, with inhibition rates exceeding 60%. Among them, P92-si3 had the highest inhibition rate, reaching 75.6%.

[0922] II. Template modification sequences employing off-target design and / or 5'-E-VP modification have a significant inhibitory effect on the PCSK9 gene. For example, the template modification and 5'-E-VP modification sequence D84-DV27P achieved an inhibition rate of up to 94.4%.

[0923] Table 35 Template modification and inhibition rate of PCSK9 gene using anti-off-target or / and 5'-E-VP modified sequence

[0924] 1) After modification with the disclosed modification templates, base sequences 5, 51, 81, and 84 exhibited significant inhibitory effects on the PCSK9 gene, with inhibition rates exceeding 90%. Furthermore, the inhibitory effects were further enhanced compared to those of the alternatively modified sequences. For example, after the base sequence 51 was alternatively modified, the inhibition rate of P92-si51 was 70.1% (see Table 34). After modification with template DV26, the inhibition rate of D51-DV26 reached 90.7%, a significant 20.6% increase compared to the alternatively modified sequences (Figure 8).

[0925] 2) After template modification and anti-off-target design, the researchers demonstrated significant inhibitory activity against PCSK9 gene expression. For example, when the base sequence 84 was modified with the template DV26 and then subjected to anti-off-target design, the inhibition rate of D84-DV26+ reached 90.1%.

[0926] 3) Template modification followed by 5'-E-VP modification can enhance the inhibitory effect on the PCSK9 gene. For example, when base sequence 84 was modified with template DV27, the inhibition rate of D84-DV27 was 91.5%. After 5'-E-VP modification, the inhibition rate of D84-DV27P was 94.4%, an increase of 2.9%.

[0927] 4) After template modification, followed by off-target protection and 5'-E-VP modification, inhibitory activity against the PCSK9 gene can reach over 90%. For example, base sequence 84 was modified with template DV27, followed by off-target protection and 5'-E-VP modification, and the inhibition rate of D84-DV27+P reached 90.4%.

[0928] This demonstrates that using the disclosed modified template DV25-29 to modify a sequence can further enhance the inhibitory effect compared to alternatively modified sequences. Furthermore, sequences modified with the disclosed modified template DV25-29, followed by off-target design and / or 5'-E-VP modification, significantly inhibit PCSK9 gene expression, with inhibition rates exceeding 90%.

[0929] (2) After adopting any one or more of the various modification methods disclosed in the present invention, anti-off-target design is performed, which has a significant anti-off-target effect on off-target genes and reduces the inhibitory effect on off-target genes.

[0930] The experimental results show that:

[0931] 1) The alternating modification or template modification sequence disclosed herein, with an anti-off-target design, can reduce the inhibitory effect on off-target genes, i.e., has a significant anti-off-target effect;

[0932] 2) By using the template-modified sequence, anti-off-target design and 5'-E-VP modification, the inhibition rate of off-target genes was significantly reduced, indicating a significant anti-off-target effect.

[0933] I. The alternating modified sequence disclosed herein adopts an anti-off-target design, which has a significant anti-off-target effect and can reduce the inhibition rate of off-target genes by 20%.

[0934] Table 36 Inhibition rate of off-target genes by alternating modification and its anti-off-target modification sequence

[0935] As can be seen from Table 36, after the alternate modified sequences of the present disclosure adopt an anti-off-target design, the inhibition rate of off-target genes is reduced, showing an anti-off-target effect. For example, the alternate modified sequence P92-si3+ adopting an anti-off-target design has a 20.0% lower inhibition rate for the off-target gene AARSD1 and a 20.5% lower inhibition rate for the off-target gene ACAP2 than the sequence P92-si3 without an anti-off-target design, which is a significant reduction.

[0936] II. Using the disclosed modification template to modify the sequence using an anti-off-target design has a significant anti-off-target effect, and the inhibition rate of off-target genes can be reduced by up to 73.6%.

[0937] Table 37 Template modification sequence and its anti-off-target and / or 5'-E-VP modification inhibition rate on off-target genes

[0938] 1) Only use the anti-off-target design (indicated by "+" in the sequence number)

[0939] After the template-modified sequence was designed to prevent off-target effects, the inhibitory effect on off-target genes was significantly reduced. For example, the inhibition rate of D84-DV26+ on the MXD1 gene was reduced by 39.9%, the inhibition rate of D81-DV25+ on the PCYOX1 gene was reduced by 54.1%, the inhibition rate of D51-DV26+ on the NEPRO gene was reduced by 25.9%, and the inhibition rate of D5-DV25+ on the DTWD1 gene was reduced by 10.9%.

[0940] It shows that after the basic sequences 5, 51, 81 and 84 are modified with the modification template designed in the present disclosure and subjected to the anti-off-target design, they all have significant anti-off-target effects.

[0941] 2) Simultaneous use of off-target protection and 5'-E-VP modification (indicated by "+P" in the sequence numbering)

[0942] The use of both template-modified and 5'-E-VP-modified sequences significantly reduced the inhibition rate of off-target genes, demonstrating a significant anti-off-target effect. For example, D84-DV26+P reduced the inhibition rate of the off-target gene MXD1 by 73.6%, D81-DV27+P reduced the inhibition rate of the off-target gene PCYOX1 by 31.1%, and D51-DV26+P reduced the inhibition rate of the off-target gene KIF1B by 41.6%, significantly reducing the inhibition rate.

[0943] It shows that after the basic sequences 5, 51, 81 and 84 are modified with the modification template designed by the present disclosure, and the anti-off-target design and 5'-E-VP modification are simultaneously adopted, there is a significant off-target effect.

[0944] Summarize:

[0945] 1. All modified sequences showed significant inhibitory effects on the PCSK9 gene. For example, the alternating modification P92-si3 achieved an inhibition rate of 75.6%, while the template-modified and 5'-E-VP modified sequence D84-DV27P achieved an inhibition rate of 94.4%.

[0946] (1) The alternating modified sequences had a significant inhibitory effect on the PCSK9 gene, with inhibition rates exceeding 60%, with P92-si3 reaching 75.6%.

[0947] (2) Template modification sequences using an anti-off-target design and / or 5'-E-VP modification have a significant inhibitory effect on the PCSK9 gene. For example, the template modification and 5'-E-VP modification sequence D84-DV27P has an inhibition rate of up to 94.4%.

[0948] 2. After adopting any one or more of the various modification methods disclosed in the present invention, anti-off-target design is performed, which has a significant anti-off-target effect on off-target genes and reduces the inhibitory effect on off-target genes.

[0949] (1) The alternating modified sequence disclosed herein adopts an anti-off-target design, which has a significant anti-off-target effect and can reduce the inhibition rate of off-target genes by 20%.

[0950] (2) The modified sequence of the modified template disclosed herein adopts an anti-off-target design, which has a significant anti-off-target effect and can reduce the inhibition rate of off-target genes by up to 73.6%.

[0951] Example 7: Effects of sequences modified with the disclosed modified template on PCSK9, low-density lipoprotein cholesterol (LDL-C) and total cholesterol (TC) in mouse serum

[0952] In this example, several sequences, including base sequences 5, 51, 81, 82, and 84, were modified using, for example, template modification alone, template modification combined with an anti-off-target design, template modification combined with a 5'-E-VP design, and template modification, 5'-E-VP, and an anti-off-target design. Transgenic mice expressing the human PCSK9 gene were used to test the inhibitory effects of these sequences on serum PCSK9, low-density lipoprotein cholesterol (LDL-C), and total cholesterol (TC) at different time points using ELISA.

[0953] 1. Experimental Materials

[0954] Test drug:

[0955] The sequences in Table 38, including sequences using only template modification, sequences using template modification and 5'-E-VP modification, sequences using template modification and anti-off-target design, and sequences using template modification, 5'-E-VP and anti-off-target design simultaneously, have the GalNAc ligand G4, G5, G6 or G7 coupled to the 3' end of the sense strand:

[0956] The method for coupling the oligonucleotide to the ligand G4, G5, G6, or G7 is the same as the method for preparing conjugates 4, 5, 6, and 7 in Example 3 of patent application CN116854754A, namely, coupling YK-GAL-304, YK-GAL-305, YK-GAL-306, and YK-GAL-307 to the oligonucleotide. The synthesis method of YK-GAL-304, YK-GAL-305, YK-GAL-306, and YK-GAL-307 is the same as that described in Example 1 of the same patent application.

[0957] The oligonucleotide forms a conjugate with the ligand as shown below:

[0958] The specific sequences of each sequence are shown in Table 38. G4, G5, G6 and G7 in the sequence number indicate that the sequence is coupled with the GalNAc ligand G4, G5, G6 or G7.

[0959] Table 38 Sequences in animal experiments

[0960] Preparation of test drug:

[0961] Drug solvent: PBS buffer

[0962] Preparation conditions: sterile environment

[0963] Labeling method: The prepared drug preparation shall be labeled with the special number, name, concentration, quantity, preparation date, preparer and storage conditions on the outer packaging;

[0964] Storage conditions: Prepare immediately before use and store remaining samples at -80℃.

[0965] Experimental animal information:

[0966] Species / Strain: hPCSK9 transgenic mice

[0967] Rating: SPF

[0968] Gender: Male

[0969] Quantity: 214

[0970] Age: 4 to 7 weeks

[0971] Weight: 18~20g

[0972] Source: Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd.

[0973] Production license number: SCXK(Su)2018-0008

[0974] Laboratory Animal Ethics Review (IACUC):

[0975] The experimental animals were housed at Youji (Tianjin) Pharmaceutical Technology Co., Ltd., license number: SYXK (Jinbin) 2019-0002. This project has been reviewed by the Experimental Animal Ethics Committee of Youji (Tianjin) Pharmaceutical Technology Co., Ltd., and the experimental procedures were conducted in strict accordance with IACUC requirements to ensure animal welfare.

[0976] Feeding and management:

[0977] Animal husbandry: Experimental animals were housed at Youji (Tianjin) Pharmaceutical Technology Co., Ltd. (license number: SYXK (Jinbin) 2019-0002). Cages were maintained with dimensions of 29.0 cm (length × width × height) × 18.5 cm × 13.0 cm. The temperature was set between 20 and 26°C, the humidity between 40% and 70%, and the air was exchanged at least 15 times per hour with fresh air. Artificial lighting was maintained with a 12-hour light and 12-hour dark cycle.

[0978] The breeding environment conditions are based on the National Standard of the People's Republic of China GB14925-2010, and the environment is controlled by a modular air-conditioning unit.

[0979] Animals were allowed to eat and drink freely. The drinking water bottles and the drinking water in the bottles should be changed at least twice a week. After use, the drinking water bottles should be sterilized in a pulse vacuum sterilizer before reuse.

[0980] Animal cages and bedding should be changed at least once a week. All animal cages and bedding should be sterilized under high pressure in a pulsating vacuum sterilizer before being used in a barrier environment. Animal cages should be cleaned, disinfected and wiped at least once a week.

[0981] The animal breeding and observation room is cleaned and disinfected every day, including the flat racks, floors, tables, etc.

[0982] The disinfectants used in the barrier environment include: 6.67% chlorhexidine solution, 0.5% 84 disinfectant, 75% disinfectant, and 0.08% Baidusai. The four disinfectants should be used in turn and cannot be mixed.

[0983] Experimental animal feed: SPF rat maintenance feed: produced by Sibeifu (Beijing) Biotechnology Co., Ltd., animal feed production license number is SCXK (Beijing) 2019-0010, issued by Beijing Municipal Science and Technology Commission.

[0984] Feed Testing: Each batch of feed is certified for quality. Microbiological testing is conducted quarterly by our company, and every six months, the feed supplier provides a recent third-party feed testing report. Feed nutrient content testing is conducted in accordance with the National Standard of the People's Republic of China GB14924.3-2010, while contaminant index testing is conducted in accordance with the National Standard of the People's Republic of China GB14924.2-2001.

[0985] Drinking water for experimental animals: Drinking water: sterile water prepared by the filtration system, directly filled in drinking bottles.

[0986] Drinking Water Testing: We conduct microbiological testing quarterly and send water to a third-party testing agency for annual water quality testing. Drinking water testing is conducted in accordance with the National Standard of the People's Republic of China, GB5749-2006.

[0987] Animal bedding: Corn cob bedding: Produced by Sibefor (Beijing) Biotechnology Co., Ltd., animal bedding production license number is SCXK(Beijing)2019-0004, issued by Beijing Municipal Science and Technology Commission.

[0988] Litter Testing: We conduct microbiological testing quarterly, and the bedding supplier provides at least one third-party bedding testing report every six months. Bedding testing is conducted in accordance with the National Standard of the People's Republic of China, GB14924.2-2001.

[0989] 2. Experimental Methods

[0990] Dosage design and grouping

[0991] Definition of experimental day: The day on which the animals were administered with the vehicle or the test drug was defined as day 0.

[0992] Grouping and Dosing: After the experimental animals were acclimated to feeding, they were randomly divided into a negative control group and a test drug group based on serum PSCK9 protein levels, with 5 animals in each group. The drug was administered via a single subcutaneous injection at a dose of 6 mg / kg in a volume of 1 mL / kg at a concentration of 6 mg / mL. The day of administration was designated as day 0.

[0993] Individual animals are identified using ear tags. Cages are identified by hanging cage labels. A laboratory sign is hung at the laboratory entrance. Grouping information is detailed in Table 39.

[0994] Table 39

[0995] Detection indicators

[0996] (1) General observation

[0997] The subjects were observed once a day from one week before administration to the end of the experiment.

[0998] Observation content: Observe the animal's death or dying, mental state, behavioral activities, fecal characteristics, feed and water supply, etc. beside the cage.

[0999] Test animals: all animals in the negative control group and the test drug group.

[1000] (2) Expression of PCSK9 protein in serum

[1001] Testing time: before dosing on day 3 (day-3), 1 week after dosing on day 7 (1w), 2 weeks after dosing on day 14 (2w), 3 weeks after dosing on day 21 (3w), 4 weeks after dosing on day 28 (4w), and 5 weeks after dosing on day 35 (5w).

[1002] PCSK9 protein level detection method: ELISA kit is used for detection; serum samples should be kept away from repeated freezing and thawing.

[1003] Test animals: all animals in the negative control group and the test drug group.

[1004] (3) Blood lipid measurement

[1005] Testing time: Before dosing on day 3 (day 3), one week after dosing on day 7 (1 week), two weeks after dosing on day 14 (2 weeks), three weeks after dosing on day 21 (3 weeks), four weeks after dosing on day 28 (4 weeks), and five weeks after dosing on day 35 (5 weeks). Approximately 200 μL of blood was collected from the inner canthus of the eye. The whole blood sample was temporarily stored in an ice box before centrifugation. Centrifuge at 2-8°C and a centrifugal force of approximately 3000 g for 10 minutes. The sample was aliquoted into two tubes (one aliquot of approximately 60 μL, with the remaining serum in the other tube) and stored at -70-86°C for lipid testing.

[1006] (4) Data processing and statistical analysis

[1007] The experimental data are expressed as mean ± standard deviation (mean ± SD) and analyzed using GraphPad Prism 8.3 software. Homogeneity of variance was tested using the LSD test, while unequal variance was tested using the Dunnett T3 test. P < 0.05 was considered statistically significant.

[1008] 3. Experimental Results

[1009] Specific experimental results are shown in Tables 40-42.

[1010] It can be seen that these sequences can significantly and continuously inhibit PCSK9 protein in serum and significantly reduce serum low-density lipoprotein cholesterol (LDL-C) and serum total cholesterol (TC) levels. For example, the D81-DV25G7 group achieved an inhibition rate of 83.1%, 82.2%, and 70.3% on days 7, 14, and 28, respectively; the D82-DV27PG5 group reduced serum LDL-C levels by 32.6%, 35.8%, and 21.7% on days 7, 14, and 28, respectively; and the D82-DV29PG7 group reduced serum TC levels by 32.2%, 26.8%, and 19.6% on days 7, 14, and 28, respectively.

[1011] (1) Sequences disclosed herein, such as base sequences 5, 51, 81, 82, and 84, with various modifications, exhibited significant inhibitory effects on PCSK9 protein expression in serum. For example, D81-DV25G7 achieved inhibition rates of 83.1%, 82.2%, and 70.3% on days 7, 14, and 28, respectively.

[1012] Table 40 Different sequences inhibit PCSK9 protein in serum

[1013] As can be seen from Table 40, the conjugates formed by conjugating each sequence with a GalNAc compound have a significant inhibitory effect on PCSK9 protein expression in serum. For example, D81-DV25G7 achieved inhibition rates of 83.1%, 82.2%, and 70.3% on days 7, 14, and 28, respectively ( FIG9 ).

[1014] It shows that the basic sequences designed in the present invention, such as 5, 51, 81, 82 and 84, are modified with the template of the present invention, or simultaneously modified with 5'-E-VP and / or anti-off-target design, and conjugated with GalNAc compounds, can be efficiently delivered to the animal liver and significantly inhibit PCSK9 gene expression.

[1015] (2) Sequences disclosed herein, such as base sequences 5, 51, 81, 82, and 84, with various modifications, can significantly reduce serum low-density lipoprotein cholesterol (LDL-C) levels. For example, the D82-DV27PG5 group achieved reductions of 32.6%, 35.8%, and 21.7% on days 7, 14, and 28, respectively.

[1016] Table 41 Effects of different sequences on the reduction of serum low-density lipoprotein cholesterol (LDL-C)

[1017] As can be seen from Table 41, serum LDL-C levels were significantly reduced in all experimental groups. For example, in the D82-DV27PG5 group, serum LDL-C levels were reduced by 32.6%, 35.8%, and 21.7% on days 7, 14, and 28, respectively ( FIG10 ).

[1018] It shows that the basic sequences designed in the present invention, such as 5, 51, 81, 82 and 84, are modified with the template of the present invention, or simultaneously modified with 5'-E-VP and / or anti-off-target design, and conjugated with GalNAc compounds, can be efficiently delivered to the liver of animals and significantly reduce the level of low-density lipoprotein cholesterol (LDL-C) in serum.

[1019] (3) Sequences disclosed herein, such as base sequences 5, 51, 81, 82, and 84, with various modifications, can significantly reduce serum total cholesterol (TC) levels. For example, D82-DV29PG7 reduced total cholesterol by 32.2%, 26.8%, and 19.6% on days 7, 14, and 28, respectively.

[1020] Table 42 Different sequences reduce serum total cholesterol (TC) levels

[1021] As can be seen from Table 42, serum total cholesterol (TC) was significantly reduced in all experimental groups. For example, in the D82-DV29PG7 group, TC was reduced by 32.2%, 26.8% and 19.6% on days 7, 14 and 28, respectively ( FIG11 ).

[1022] It shows that the basic sequences designed in the present invention, such as 5, 51, 81, 82 and 84, are modified with the template of the present invention, or simultaneously modified with 5'-E-VP and / or anti-off-target design, and conjugated with GalNAc compounds, can be efficiently delivered to the liver of animals and significantly reduce the total cholesterol (TC) level in serum.

[1023] Summarize:

[1024] The basic sequences designed in the present invention, such as 5, 51, 81, 82 and 84, are modified using the template of the present invention, or simultaneously modified with 5'-E-VP and / or anti-off-target design, and conjugated with a GalNAc compound. They can be efficiently delivered to the liver of animals and can significantly and sustainably inhibit PCSK9 protein expression in serum, thereby reducing serum low-density lipoprotein cholesterol (LDL-C) and serum total cholesterol (TC) levels.

[1025] For example, the inhibition rate of PCSK9 protein expression in the serum of the D81-DV25G7 group reached 83.1%, 82.2% and 70.3% on the 7th, 14th and 28th days, respectively; the serum low-density lipoprotein cholesterol (LDL-C) level of the D82-DV27PG5 group decreased by 32.6%, 35.8% and 21.7% on the 7th, 14th and 28th days, respectively; the serum total cholesterol (TC) level of the D82-DV29PG7 group decreased by 32.2%, 26.8% and 19.6% on the 7th, 14th and 28th days, respectively.

[1026] in conclusion:

[1027] This paper designs a series of siRNAs based on the PCSK9 mRNA sequence, modifies them alternately and with a set of specific modification templates, and also performs off-target design on some of these sequences with 5'-E-VP modification. The results show that:

[1028] (1) Unmodified sequences, including basic sequences 5, 51, 81, 82, 84, 3, 8, 9, 21, 31, 73, and 83, all have significant inhibitory effects on PCSK9, with the highest inhibition rate exceeding 60%.

[1029] (2) Using multiple modified sequences, the inhibition rate can reach up to 90%. Moreover, the modified sequence is conjugated with a GalNAc compound and can be efficiently delivered to the animal liver, significantly inhibiting PCSK9 gene expression, significantly reducing serum low-density lipoprotein cholesterol (LDL-C) levels, and significantly reducing serum total cholesterol (TC) levels.

[1030] The details are as follows:

[1031] 1. The disclosed alternative modified sequence has a significant inhibitory effect on the PCSK9 gene

[1032] (1) Among the 84 designed sequences, 12 sequences showed significant inhibitory effects on the PCSK9 gene, with inhibition rates exceeding 50%, including P92-si5, P92-si51, P92-si81, P92-si82, P92-si84, P92-si3, P92-si8, P92-si9, P92-si21, P92-si31, P92-si73, and P92-si83. Among them, the inhibition rates of P92-si5, P92-si81, P92-si82, P92-si3, P92-si8, and P92-si31 exceeded 70%.

[1033] (2) The inhibition rates of the 26 sequences on the PCSK9 gene ranged from 30% to 50%. For example, the inhibition rates of P92-si6 and P92-si7 were 38.3% and 35.7%, respectively.

[1034] (3) The inhibition rates of 46 sequences on the PCSK9 gene were below 30%. For example, the inhibition rates of P92-si1 and P92-si20 were only 9.2% and 6.0%, respectively.

[1035] (4) siRNAs with similar sequences have very different activities. For example, the inhibition rate of P92-si5 is 61.8% higher than that of P92-si4, which is a significant improvement.

[1036] 2. The unmodified sequence disclosed herein has a significant inhibitory effect on the PCSK9 gene

[1037] (1) 12 unmodified sequences showed significant inhibitory effects on the PCSK9 gene, with inhibition rates exceeding 50%, including si5, si51, si81, si82, si84, si3, si8, si9, si21, si31, si73, and si83. Among them, si5, si81, si82, si3, and si8 showed inhibition rates exceeding 60%.

[1038] (2) The inhibition rates of other unmodified sequences on the PCSK9 gene were all lower than 40%. For example, the inhibition rates of si52 and si74 were only 0.5% and 2.2% respectively.

[1039] (3) siRNAs with similar sequences have very different activities. For example, the inhibition rate of base sequence 5 is 51.2% higher than that of base sequence 4, which is a significant improvement.

[1040] (4) The effects of alternating modifications on the activity of different sequences were not consistent. Some showed significant increases in inhibition rate, such as the base sequence 5, where the inhibition rate of alternating modifications was 12.4% higher than that of the unmodified sequence. Others showed little difference, such as the base sequences 4, 22, and 52, where the inhibition rates of the alternating modified sequences and the unmodified sequences remained essentially unchanged.

[1041] 3. The sequence modified by the modified template disclosed herein has a significant inhibitory effect on the PCSK9 gene

[1042] (1) Base sequences 5, 51, 81, 82, 84, 3, 8, 9, 21, 31, 73, and 83, after modification with the modified templates DV25-29 designed in this disclosure, showed significant inhibitory effects on PCSK9 gene expression, with inhibition rates exceeding 70%. Among them, P92-si81-DV26, P92-si82-DV27, and P92-si82-DV29 achieved inhibition rates of 89.3%, 89.3%, and 89.1%, respectively.

[1043] (2) The 12 sequences described above, modified with the disclosed template DV25-29, showed significantly improved inhibition of PCSK9 gene expression compared to the alternatively modified sequences. For example, the inhibition of base sequence 51 modified with template DV27, P92-si51-DV27, was 29.1% higher than that of the alternatively modified sequence. The inhibition of base sequence 81 modified with template DV26, P92-si81-DV26, was 26.9% higher than that of the alternatively modified sequence.

[1044] (3) Modification of the same sequence with the disclosed modified templates DV25-29 significantly improved the inhibition rate of PCSK9 gene expression compared to modification templates disclosed in the prior art. For example, when base sequence 51 was modified with the disclosed modified template DV27, the inhibition rate increased by 21.3% compared to modification with the disclosed Advanced ESC template DV21.

[1045] (4) The same sequence modified with the disclosed modification templates DV25-29 showed significantly improved inhibition of PCSK9 gene expression compared to the modified templates DV30 and DV31. For example, the base sequence 51 modified with the disclosed modification template DV27 showed a 22.8% increase in inhibition compared to the modified template DV31.

[1046] (5) The EC50 values ​​of the above 12 sequences ranged from 0.0001 nM to 0.005 nM. Among them, the EC50 values ​​of P92-si81-DV27, P92-si84-DV26, and P92-si82-DV27 were 0.0003 nM, 0.0004 nM, and 0.0006 nM, respectively. This indicates that these sequences can effectively inhibit PCSK9 gene expression at low concentrations.

[1047] 4. The sequences modified by alternating modification and modification template disclosed in the present invention have significantly improved PCSK9 inhibitory activity compared with the unmodified sequences disclosed in the prior art that are completely identical or have very little difference.

[1048] (1) Compared with the identical unmodified sequences disclosed in the prior art, the alternating modified sequences disclosed in the present invention and the modified sequences using specific modification templates significantly improve the inhibition rate of the PCSK9 gene, which can be increased by up to 40%.

[1049] (2) Compared with the unmodified sequences disclosed in the prior art, which have only minor differences, the unmodified sequences, the alternatively modified sequences, and the sequences modified with specific modification templates disclosed in the present invention significantly improve the inhibition rate of the PCSK9 gene, which can be increased by up to 40%.

[1050] I. The unmodified sequences disclosed in this disclosure have significantly improved activity compared to similar unmodified sequences disclosed in the prior art. For example, the unmodified sequence si31 disclosed in this disclosure has a 10.4% higher inhibition rate than the structurally similar 31P.

[1051] II. The alternatively modified sequences disclosed herein have significantly improved activity compared to similar unmodified sequences disclosed in the prior art. For example, compared to sequence 5P, the inhibition rate of the alternatively modified sequence P92-si5 disclosed herein was increased by 22.0%.

[1052] III. Sequences modified with the disclosed modified templates showed significantly improved activity compared to similar unmodified sequences disclosed in the prior art. For example, compared to sequence 82P, the disclosed alternatively modified sequence P92-si82-DV29 showed a 42.8% higher inhibition rate.

[1053] 5. The alternating modified sequence and template modified sequence disclosed herein adopt a specific anti-off-target design, which has a significant inhibitory effect on the PCSK9 gene and significantly reduces the inhibitory effect on off-target genes.

[1054] (1) Different modified sequences all had significant inhibitory effects on the PCSK9 gene. For example, the inhibition rate of the alternating modification P92-si3 reached 75.6%, and the inhibition rate of the template modification and 5'-E-VP modification sequence D84-DV27P was as high as 94.4%.

[1055] I. The alternating modified sequences have a significant inhibitory effect on the PCSK9 gene, with inhibition rates exceeding 60%, with P92-si3 reaching 75.6%.

[1056] II. Template modification sequences employing off-target design and / or 5'-E-VP modification have a significant inhibitory effect on the PCSK9 gene. For example, the template modification and 5'-E-VP modification sequence D84-DV27P achieved an inhibition rate of up to 94.4%.

[1057] (2) After adopting any one or more of the various modification methods disclosed in the present invention, anti-off-target design is performed, which has a significant anti-off-target effect on off-target genes and reduces the inhibitory effect on off-target genes.

[1058] I. The alternating modified sequence disclosed herein adopts an anti-off-target design, which has a significant anti-off-target effect and can reduce the inhibition rate of off-target genes by 20%.

[1059] II. Using the disclosed modification template to modify the sequence using an anti-off-target design has a significant anti-off-target effect, and the inhibition rate of off-target genes can be reduced by up to 73.6%.

[1060] 6. The modified sequences using the disclosed modified templates significantly inhibited PCSK9 expression in mouse serum and significantly reduced low-density lipoprotein cholesterol (LDL-C) and total cholesterol (TC) levels.

[1061] (1) Different modifications of the disclosed base sequences 5, 51, 81, 82, and 84 significantly inhibited PCSK9 protein expression in serum. For example, D81-DV25G7 achieved inhibition rates of 83.1%, 82.2%, and 70.3% on days 7, 14, and 28, respectively.

[1062] (2) Different modifications of the disclosed base sequences 5, 51, 81, 82, and 84 significantly reduced serum low-density lipoprotein cholesterol (LDL-C) levels. For example, the D82-DV27PG5 group reduced LDL-C by 32.6%, 35.8%, and 21.7% on days 7, 14, and 28, respectively.

[1063] (3) Different modifications of the disclosed base sequences 5, 51, 81, 82, and 84 significantly reduced serum total cholesterol (TC). For example, D82-DV29PG7 reduced TC by 32.2%, 26.8%, and 19.6% on days 7, 14, and 28, respectively.

[1064] Example 8: Synthesis of small interfering oligonucleotides modified using DV29P template

[1065] A total of 36 siRNA basic sequences were designed, with Alnylam's VIR-2218 as the Yang Shen (patent number: CN108064294B), and the sequence number in this example is APC-VIR; ANC-DV29P is used as a negative control. In order to improve the inhibition efficiency and stability of the sequence, the basic sequence is modified using a DV29P or DV29SP template. Positions 7, 9, 11, and 17 of the sense chain are 2'-F modified, and the remaining positions are 2'-OMe modified; positions 2, 4, 5, 6, 14, and 16 of the antisense chain are 2'-F modified, and the remaining positions are 2'-OMe modified. In addition, there are two thio modifications at the 5' end of the sense chain; there are two thio modifications at the 5' and 3' ends of the antisense chain; and there is an EVP modification at the 5' end of the antisense chain. The siRNA sequences modified with the DV29P template are shown in Table 43.

[1066] 1. Synthesis of DV29P template modified sequence A194-DV29P

[1067] The basic sequence of the small interfering RNA numbered A194-DV29P in Table 43 is:

[1068] Sense strand: 5'-UCGUGUUACAGGCGGGGUUUU-3' (SEQ ID NO: 463)

[1069] Antisense strand: 5'-AAAACCCCGCCUGUAACACGAGA-3' (SEQ ID NO: 495)

[1070] The sense strand has 2'-F modifications at positions 7, 9, 11, and 17, and 2'-OMe modifications at the remaining positions. The antisense strand has 2'-F modifications at positions 2, 4, 5, 6, 14, and 16, and 2'-OMe modifications at the remaining positions. Additionally, the sense strand has two thiolate modifications at the 5' end, the antisense strand has two thiolate modifications at the 5' and 3' ends, and the antisense strand has one EVP modification at the 5' end.

[1071] Instruments and reagents: Qingke 192P automatic DNA / RNA synthesizer, whose solid phase support is a universal support of cross-linked polystyrene beads, model number is Primer support 5G Unylinker 350 (Cytiva manufacturer).

[1072] Preparation method:

[1073] The following nucleotide monomer solutions were prepared using acetonitrile at a monomer concentration of 0.15 M: DMT-A-OMe phosphoramidite monomer (Formula 1), DMT-C-OMe phosphoramidite monomer (Formula 2), DMT-G-OMe phosphoramidite monomer (Formula 3), DMT-U-OMe phosphoramidite monomer (Formula 4), DMT-AF phosphoramidite monomer (Formula 5), ​​DMT-CF phosphoramidite monomer (Formula 6), DMT-GF phosphoramidite monomer (Formula 7), DMT-UF phosphoramidite monomer (Formula 8), vinyl-(E)-phosphonate-A-OMe phosphoramidite monomer (Formula 11), and vinyl-(E)-phosphonate-U-OMe phosphoramidite monomer (Formula 12).

[1074] The antisense strand of the APC-VIR sequence of Yangshen contains a diol nucleic acid (GNA) modification at the 7th base from the 5' end. The structure of the GNA monomer is as follows:

[1075] Prepared by the following steps:

[1076] (1) Deprotection

[1077] A 3% dichloroacetic acid solution in toluene was used as a deprotection agent to remove the DMT protecting group, followed by washing with acetonitrile.

[1078] (2) Coupling

[1079] The acetonitrile solution of each nucleotide monomer was coupled using a 0.25 M 5-ethylthiotetrazolium solution in acetonitrile as an activating agent, followed by rinsing with acetonitrile.

[1080] (3) Oxidation / sulfurization

[1081] Oxidation: Oxidation was performed using a 0.05 M iodine solution in pyridine / water (90 / 10) as an oxidant, followed by rinsing with acetonitrile.

[1082] Sulfurization: 3% hydrogenated xanthan gum in pyridine was used as a sulfurizing agent for sulfurization, followed by rinsing with acetonitrile.

[1083] (4) Hydroxyl protection

[1084] Hydroxyl protection was performed using 10% acetic anhydride in tetrahydrofuran (CAP A) and tetrahydrofuran / pyridine / nitromethylimidazole 74 / 10 / 16 (v / v / v) (CAP B) as hydroxyl protecting reagents, followed by rinsing with acetonitrile.

[1085] Repeat the above steps and cycle according to the set sequence to obtain a fully protected product.

[1086] (5) Use 3% dichloroacetic acid in toluene as a deprotection reagent to remove the DMT protecting group of the last nucleotide, and then use acetonitrile to wash.

[1087] (6) Ammonolysis and purification

[1088] The solid phase carrier was transferred to a reactor, concentrated ammonia water (25-28%) was added, and after maintaining aminolysis at 60°C for 12 hours, the system was cooled to room temperature, and the mixture was filtered and rinsed with a mixed solution of purified water and ethanol. The filtrate was combined, passed through a chromatography column, concentrated, and freeze-dried to obtain the product.

[1089] (7) Annealing

[1090] The purified sense chain and antisense chain were mixed in a 1:1 ratio, heated to 95°C and maintained for 3 minutes, and then slowly cooled to room temperature to form a double chain.

[1091] The purity of A194-DV29P is 90.52%; the measured molecular weight is 14727.09.

[1092] 2. Synthesis of other sequences

[1093] The other sequences listed in Table 43 were synthesized according to the above method.

[1094] Table 43 DV29P template modified siRNA sequence

[1095] Example 9: Inhibitory effect of DV29P template modified sequence on HBsAg and HBeAg

[1096] After the DV29P or DV29SP template-modified siRNA sequence synthesized in Example 8 was transfected into HepG2.2.15 cells via lipid nanoparticles (LNP), the inhibitory effect of each sequence on HBsAg and HBeAg was detected using ELISA technology.

[1097] 1. Experimental Materials

[1098] Test samples: DV29P or DV29SP template-modified siRNA sequences listed in Table 43 (synthesized in Example 8).

[1099] Cell type: HepG2.2.15 cells

[1100] Drug solvent: sterile enzyme-free water, Gibco Opti-MEM (Thermo Fisher Scientific).

[1101] 2. Experimental Methods

[1102] ELISA was used to detect the inhibition of samples on HBsAg and HBeAg in HepG2.2.15 cell lines.

[1103] 2.1 Cell culture

[1104] Subculture: HepG2.2.15 cells were subcultured in DMEM / F12 medium supplemented with 10% fetal bovine serum, 370 μg / ml GENETICN, 1% L-glutamine, 1% non-essential amino acids, and 1% penicillin-streptomycin in a 37°C, 5% CO2 incubator. Cells were subcultured every three days. Cells were digested with 0.25% trypsin, centrifuged at 800 rpm for 3 minutes, the supernatant discarded, and fresh medium added for subculture.

[1105] Plating culture: HepG2.2.15 cells were plated and cultured in DMEM / F12 medium containing 10% fetal bovine serum, 1% L-glutamine, 1% non-essential amino acids, and 1% penicillin-streptomycin.

[1106] 2.2 Cell transfection

[1107] Preparation of transfection mixture: Lipofectamine TM RNAiMAX (Thermo Fisher Scientific) and Opti-MEM were mixed at a ratio of 2:98 and vortexed.

[1108] Preparation of transfection complex: Add 30 μL of siRNA solution diluted in Opti-MEM to 30 μL of transfection mixture at a ratio of 1:1 (v / v), vortex to mix, and let stand at room temperature for 15 minutes to obtain transfection complex.

[1109] Transfection control group transfection reagent preparation: Add 15 μL of the prepared transfection mixture to 15 μL of Opti-MEM. Vortex to mix thoroughly and let stand at room temperature for 15 minutes.

[1110] The prepared transfection complex was added to a 96-well cell culture plate (15 μL per well, 3 replicates per sequence) to make the final siRNA concentration in each well 0.3 nM. 135 μL of cell suspension (containing 2.25×10 4 After mixing using the cross method, the cells were cultured in a 37°C, 5% CO2 cell incubator.

[1111] 2.3 Detection of HBsAg and HBeAg in cell supernatants

[1112] 1) Collection of cell supernatant

[1113] a. On the third day after cell transfection, change the medium, discard the cell supernatant, and add 150 μL / well of fresh medium to continue culturing.

[1114] b. On the 6th day after transfection, the cell supernatant was collected for detection of HBsAg and HBeAg levels.

[1115] 2) Quantitative detection of HBsAg and HBeAg

[1116] HBsAg and HBeAg concentrations were detected using the Hepatitis B Virus e Antigen Detection Kit (Antu Biotech CL0310) and the Hepatitis B Virus Surface Antigen Detection Kit (Antu Biotech CL0312). The specific steps are as follows:

[1117] a. Return the test kit and the sample to room temperature.

[1118] b. Add 50 μL of the sample to be tested, standard, negative control, and Yang Shen into the well plate.

[1119] c. Add 50 μL of enzyme conjugate to each well, mix thoroughly, and incubate at 37°C for 60 minutes.

[1120] d. Remove the liquid from the well plate and wash it with detergent 5 times. Finally, pat the well plate dry on absorbent paper.

[1121] e. Mix luminescent substrates A and B in equal proportions, add 50 μL / well, and react at room temperature in the dark for 3 minutes.

[1122] f. Measure the luminescence value on an enzyme-labeled instrument.

[1123] 2.4 Data Processing

[1124] The formula for calculating HBsAg and HBeAg inhibition rates is as follows:

[1125] HBsAg inhibition rate (%) = (1-HBsAg expression level of sample / HBsAg expression level of transfection control group in the same plate) × 100%;

[1126] HBeAg inhibition rate (%) = (1-HBeAg expression level of sample / HBeAg expression level of transfection control group in the same plate) × 100%.

[1127] The inhibition rate of HBsAg relative to Yang Shen (the inhibition rate of Yang Shen is set to 1) = sample HBsAg inhibition rate (%) / Yang Shen inhibition rate (%) in the same plate; it should be understood that a value greater than 1 indicates that the sequence has a better inhibitory effect on HBsAg than Yang Shen, and a value less than 1 indicates that the sequence has a lower inhibitory efficiency on HBsAg than Yang Shen.

[1128] The inhibition rate of HBeAg relative to Yang Shen (the inhibition rate of Yang Shen is set to 1) = sample HBeAg inhibition rate (%) / Yang Shen inhibition rate (%) in the same plate; it should be understood that a value greater than 1 indicates that the sequence has a better inhibitory effect on HBeAg than Yang Shen, and a value less than 1 indicates that the sequence has a lower inhibitory efficiency on HBeAg than Yang Shen.

[1129] 2.5 EC 50 experiment

[1130] In this experiment, the concentration of each sequence was set to start from 10nM and diluted 4 times, with a total of 8 concentration points (10nM, 2.5nM, 0.625nM, 0.15625nM, 39.06pM, 9.77pM, 2.44pM and 0.61pM). The inhibition rate of each sequence at each concentration was measured, and the graph was plotted to calculate the EC of each sequence and Yangshen. 50 concentration.

[1131] 2.6 Cytotoxicity assay

[1132] In progress EC 50 During the experiment, the cell supernatant was collected and the Cell viability was determined using a Promega kit. The method was briefly described as follows: CellTiter-Glo reagent was mixed with culture medium in a 1:1 ratio, 100 μL was added to each well, and the cells were incubated at room temperature for 10 min. The luminescence signal was then detected using a microplate reader.

[1133] The cell viability was calculated as follows: cell viability (%) = (sample signal value - blank control average) / (transfection control average - blank control average) × 100%.

[1134] 3. Experimental Results

[1135] The experimental results showed that the sequences B207S-DV29SP, A1550-DV29P, B1572-DV29P, B1575-DV29P, A1573-DV29P, B418S-DV29SP, and A416-DV29P had significantly better inhibitory effects on HBsAg and HBeAg than other sequences.

[1136] Example 10: Inhibitory effect of unmodified sequences on HBV genes

[1137] In this example, some unmodified sequences corresponding to the modified sequences in Example 8 were synthesized and transfected into HepG2.2.15 cells via lipid nanoparticles (LNP). The inhibitory effects of each sequence on HBsAg and HBeAg were detected using ELISA technology, and unmodified siRNA sequences with better inhibitory effects were screened out.

[1138] 1. Experimental Materials

[1139] Test samples:

[1140] The unmodified small interfering RNA sequences are listed in Table 44. All sequences were synthesized according to the method in Example 8.

[1141] Table 44 Unmodified siRNA sequences

[1142] Cell type: HepG2.2.15 cells

[1143] Drug solvent: sterile enzyme-free water, Gibco Opti-MEM.

[1144] 2. Experimental Methods

[1145] Refer to Example 2.

[1146] 3. Experimental Results

[1147] The experimental results showed that the sequences B207S, A1550, B1572, B1575, A1573, B418S, and A416 had significantly better inhibitory effects on HBsAg and HBeAg than other sequences.

[1148] Example 11: Inhibitory effects of sequences modified with different templates on HBsAg and HBeAg

[1149] In this example, 15 sequences, including the unmodified sequences A205, B208, B207S, A261, B262, B264, A1550, B1556, B1560, B1575, A1573, B1572, B418S, A416, and B416S, were modified using the eight modified templates DV25P, DV26P, DV27P, DV28P, DV29P, DV32P, and DV33 designed in the present invention. The researchers used 154 sequences modified with DV3P, DV34P, and DV36SP (the shorter sequences were modified with DV25SP, DV26SP, DV27SP, DV28SP, DV29SP, DV32SP, DV33SP, and DV34SP, respectively), as well as existing publicly available templates modified with DV30P, DV31P, DV21P, DV22P, or DV30SP, DV31SP, DV21SP, and DV22SP. After lipid nanoparticle (LNP) transfection into HepG2.2.15 cells, the inhibitory effects of each sequence on HBsAg and HBeAg were tested using ELISA.

[1150] 1. Experimental Materials

[1151] Test samples: Listed in Table 47 are sequences modified from 15 siRNA basic sequences using different templates (DV25P, DV26P, DV27P, DV28P, DV29P, DV32P, DV33P, DV34P, DV30P, DV31P, DV21P, DV22P, DV25SP, DV26SP, DV27SP, DV28SP, DV29SP, DV32SP, DV33SP, DV34SP and DV22SP) (refer to Example 8 for the synthesis method).

[1152] Cell type: HepG2.2.15 cells

[1153] Drug solvent: sterile enzyme-free water, Gibco Opti-MEM

[1154] The modification principles of the disclosed modified template are as follows:

[1155] For the modified templates DV25P~DV29P, DV32P~DV34P

[1156] The antisense strand adopts one of the modifications shown in Table 45-1:

[1157] Table 45-1 Antisense Strand Modification

[1158] The positive chain adopts one of the modification methods shown in Table 45-2:

[1159] Table 45-2 Sense Chain Modification

[1160] In the above table, 2'-OMe is 2'-methoxy; 2'-F is 2'-fluoro; PS is a phosphorothioate backbone; EVP is 5'-vinyl-(E)-phosphonate;

[1161] The siRNA modification template in which the antisense strand adopts modification method A and the sense strand adopts modification method a is named DV25P.

[1162] The siRNA modification template in which the antisense strand was modified using modification method B and the sense strand was modified using modification method a was named DV26P.

[1163] The siRNA modification template in which the antisense strand was modified using modification method C and the sense strand was modified using modification method a was named DV27P.

[1164] The siRNA modification template in which the antisense chain adopts modification method B and the sense chain adopts modification method b is named DV28P.

[1165] The siRNA modification template in which the antisense strand was modified using modification method C and the sense strand was modified using modification method b was named DV29P.

[1166] The siRNA modification template in which the antisense strand was modified using method D and the sense strand was modified using method b was named DV32P.

[1167] The siRNA modification template in which the antisense strand was modified using modification method E and the sense strand was modified using modification method b was named DV33P.

[1168] The siRNA modification template in which the antisense strand was modified using modification method F and the sense strand was modified using modification method b was named DV34P.

[1169] The siRNA modification template with the antisense strand modified by modification method A and the sense strand modified by modification method C was named DV22P.

[1170] For modified templates DV25SP~DV29SP, DV32SP~DV34SP

[1171] The antisense strand adopts one of the modifications shown in Table 46-1:

[1172] Table 46-1 Antisense Strand Modification

[1173] The positive chain adopts one of the modification methods shown in Table 46-2:

[1174] Table 46-2 Sense Chain Modification

[1175] In the above table, 2'-OMe is 2'-methoxy; 2'-F is 2'-fluoro; PS is a phosphorothioate backbone;

[1176] The siRNA modification template with the antisense strand modified in A' and the sense strand modified in a' was named DV25SP.

[1177] The siRNA modified template with the antisense strand modified by B' and the sense strand modified by a' was named DV26SP.

[1178] The siRNA modified template with the antisense strand modified in C' and the sense strand modified in a' was named DV27SP.

[1179] The siRNA modification template in which the antisense strand is modified in the B' and the sense strand is modified in the b' is named DV28SP.

[1180] The siRNA modified template with the antisense strand modified by C' and the sense strand modified by b' was named DV29SP.

[1181] The siRNA modification template with the antisense strand modified in the D' and the sense strand modified in the b' was named DV32SP.

[1182] The siRNA modification template with the antisense strand modified in the E' and the sense strand modified in the b' was named DV33SP.

[1183] The siRNA modification template with the antisense strand modified in the F' and the sense strand modified in the b' was named DV34SP.

[1184] The siRNA modification template with the antisense strand modified by A' and the sense strand modified by C' was named DV22SP.

[1185] For DV30SP:

[1186] Antisense strand:

[1187] Chain of Justice:

[1188] In the above table, PS is a phosphorothioate skeleton.

[1189] For DV31SP:

[1190] Antisense strand:

[1191] In the above table, PS is a phosphorothioate skeleton.

[1192] The synthesis methods of each sequence are the same as in Example 8.

[1193] Table 47 Sequences modified using different modification templates

[1194] 2. Experimental Methods

[1195] Referring to Example 9, the concentration of the single-concentration screening experiment was set to 0.3 nM.

[1196] 3. Experimental Results

[1197] The experimental results showed that the sequences B207S-DV32SP, A1550-DV27P, B1572-DV27P, B1575-DV27P, B207S-DV29SP, A1573-DV29P, B1575-DV29P, B418S-DV34SP, and A416-DV27P had significantly better inhibitory effects on HBsAg than the Yangshen sequence, and significantly better inhibitory effects on HBeAg than the Yangshen sequence.

[1198] Example 12: Comparison of the inhibitory effect of the sequences disclosed in the prior art on the HBV gene

[1199] In this example, the unmodified sequences disclosed in the prior art that are identical or similar to the basic sequences of the present disclosure, B207S, B1575, B1550, B1572, A416, B1573, and B418S, are compared with the sequences modified with the existing modified templates DV30P, DV31P, DV21P, and DV22P (or DV30SP, DV31SP, DV21SP, and DV22SP), and sequences modified with the modified templates DV25P, DV26P, DV27P, DV28P, DV29P, DV32P, DV33P, and DV34P (or DV25SP, DV26SP, DV27SP, DV28SP, DV29SP, DV32SP, DV33SP, and DV34SP) disclosed herein to compare the inhibition efficiency of the HBV gene.

[1200] 1. Experimental Materials

[1201] Test samples:

[1202] (1) Prior art disclosed sequences, see Table 48.

[1203] Table 48 Prior Art Disclosed Sequences Note: The sequences disclosed in the above patent applications are all unmodified sequence RNA.

[1204] (2) In Example 11, the modified templates DV25P, DV26P, DV27P, DV28P, DV29P, DV32P, DV33P, DV34P, DV30P, DV30P, DV31P, DV21P, DV22P, DV25SP, DV26SP, DV27SP, DV28SP, DV29SP, DV32SP, DV33SP, DV34SP, DV30SP, DV31SP, and DV21SP were used to modify the siRNA sequence.

[1205] Cell type: HepG2.2.15 cells

[1206] Drug solvent: sterile enzyme-free water, Gibco Opti-MEM

[1207] 2. Experimental Methods

[1208] Referring to Example 9, the concentration of the single-concentration screening experiment was set to 0.3 nM.

[1209] 3. Experimental Results

[1210] Experimental results show that the inhibition rates of the disclosed basic sequences B207S, A1550, B1572, B1575, A1573, B418S, and A416 against HBsAg and HBeAg are significantly better than those of the sequence-similar P206, P413, P1551, and APC. After template modification, the inhibition rates are further significantly improved.

[1211] Example 13: Inhibitory effect of HBV and off-target genes using sequences designed to prevent off-target effects

[1212] In practical applications of siRNA, the expression of non-target mRNAs that are only partially complementary to the guide strand (antisense strand) is often suppressed. Alnylam's research indicates that the hepatotoxicity of n-acetylgalactosamine (GalNAc)-conjugated siRNAs is primarily due to off-target effects caused by gene suppression of the wrong target through a microRNA (miRNA)-like recognition mechanism.

[1213] To address this issue, thermolabile nucleotide modifications, such as glycolated nucleic acid (GNA), unblocking nucleotide (UNA), or DNA, can be made at positions 6 and 7 of the antisense strand seed region. This disrupts the antisense strand seed region, thereby affecting the binding of siRNA to non-target mRNAs through seed region recognition, significantly mitigating off-target effects and alleviating hepatotoxicity. In its latest fifth-generation template design, Alnylam employs a GNA modification at position 7 of the siRNA antisense strand to reduce off-target effects.

[1214] In order to reduce the off-target effect of the sequence, the sequences B418S-DV34SP, B416-DV27P, B1550-DV27P, B207S-DV29SP, and B207S-DV32SP were used in this example, and DNA or GNA anti-off-target modification was performed at positions 6 and 7 of the antisense strand, and the activity and off-target effect were compared with those of the sequences without anti-off-target modification.

[1215] 1. Experimental Materials

[1216] Test samples: template modified sequences and sequences designed to prevent off-target effects listed in Table 49 (see Example 8 for the synthesis method).

[1217] Cell type: HepG2.2.15 cells

[1218] Drug solvent: sterile enzyme-free water, Gibco Opti-MEM.

[1219] Table 49 Template modification and anti-off-target design sequences

[1220] 2. Experimental Methods

[1221] The qRT-PCR method was used to detect the inhibition of samples on HBV and off-target genes in HepG2.2.15 cells.

[1222] 2.1 Cell culture

[1223] Subculture: HepG2.2.15 cells were subcultured in DMEM / F12 medium supplemented with 10% fetal bovine serum, 370 μg / ml GENETICN, 1% L-glutamine, 1% non-essential amino acids, and 1% penicillin-streptomycin in a 37°C, 5% CO2 incubator. Cells were subcultured every three days. Cells were digested with 0.25% trypsin, centrifuged at 800 rpm for 3 minutes, the supernatant discarded, and fresh medium added for subculture.

[1224] Plating culture: HepG2.2.15 cells were plated and cultured in DMEM / F12 medium containing 10% fetal bovine serum, 1% L-glutamine, 1% non-essential amino acids, and 1% penicillin-streptomycin.

[1225] 2.2 Cell transfection

[1226] Transfection reagent preparation: Mix Lipofectamine RNAiMAX and Opti-MEM at a ratio of 2:98 and vortex to mix thoroughly.

[1227] Preparation of transfection complex: Add 30 μL of siRNA solution diluted in Opti-MEM to 30 μL of transfection mixture at a ratio of 1:1 (v / v), vortex to mix, and let stand at room temperature for 15 minutes to obtain transfection complex.

[1228] Transfection control group transfection reagent preparation: Add 15 μL of the prepared transfection mixture to 15 μL of Opti-MEM. Vortex to mix thoroughly and let stand at room temperature for 15 minutes.

[1229] The prepared transfection complex was added to a 96-well cell culture plate (15 μL per well, 3 replicates per well for each sequence) to make the final siRNA concentrations in each well 0.065, 0.14, and 0.28 nM, respectively. Three concentrations of each siRNA were set, and 3 replicates were set for each concentration. 135 μL of cell suspension (containing 2.25×10 4 After mixing using the cross method, the cells were cultured in a 37°C, 5% CO2 cell incubator.

[1230] 2.3 RNA extraction and reverse transcription

[1231] 48 h after transfection, the culture medium was removed and the cells were collected for RNA extraction. Total RNA was extracted using QIAGEN-74182 RT-PCR Kit and reverse transcribed into cDNA using HiScript III RT SuperMix for qPCR (Vazyme) according to the manufacturer's instructions.

[1232] 2.4 RT-qPCR

[1233] HBV and off-target gene cDNAs were detected by qPCR, with GAPDH cDNA used in parallel as an internal control. 8 μL of the prepared qPCR reaction solution and 2 μL of sample cDNA were added to a 384-well plate. The SYBR qPCR reaction program was as follows: heating at 50°C for 2 minutes, 95°C for 2 minutes, followed by cycling at 95°C for 5 seconds, then 60°C for 30 seconds, for a total of 40 cycles. Finally, the melting curve was performed by heating at 95°C for 15 seconds, 60°C for 1 minute, and 95°C for 15 seconds.

[1234] 2.5 Data Analysis

[1235] The target gene RNA expression level in each sample was calculated based on the CT value of each sample using the ΔΔCT relative quantification method. The relative expression of the target gene was calculated using 2 -ΔΔCT express.

[1236] The calculation formula is as follows:

[1237] ΔCT = average CT value of target gene - average CT value of GAPDH;

[1238] ΔΔCT = ΔCT (experimental group) - ΔCT (transfection control group);

[1239] Relative expression of target gene mRNA = 2 -ΔΔCT

[1240] Gene expression inhibition rate (%) = (1-sample mRNA expression level / transfection control group mRNA expression level) × 100%

[1241] 3. Experimental Results

[1242] Experimental results showed that sequences B207S-DV32SP, A1550-DV27P, and B207S-DV29SP had no off-target effects. Sequences B418S-DV34SP and A416-DV27P, after adopting an anti-off-target design, significantly reduced their inhibitory effect on off-target genes without affecting the inhibitory effect on the target gene HBV. This indicates that the anti-off-target design does not affect the inhibitory effect of the disclosed template modified sequence on the HBV gene, but can significantly suppress off-target effects.

[1243] Example 14: Effects of sequences modified with the disclosed modified template on HBsAg, HBeAg, and HBV DNA in mouse serum and on reconstitution of adaptive immune function in mice (high dose)

[1244] In this embodiment, some sequences are selected as examples, including the basic sequences B418S, A416, B1550, B207S, B1575, B1572, and B1573, and these sequences are modified, for example, only template modification, template modification and anti-off-target design are used at the same time, and the GalNAc ligand is coupled. The in vivo efficacy of these RNAi agents is analyzed using the AAV-HBV mouse model. After infection with a recombinant adeno-associated virus (AAV) carrying a replicable HBV genome, this mouse model can continuously produce HBV virus particles and HBV antigens and has no serum conversion for more than one year, reproducing some of the immunological characteristics of clinical chronic hepatitis B patients. Therefore, this model is also used to evaluate new immune-based therapies and antiviral treatments. In this embodiment, the AAV-HBV mouse model was used to detect the inhibitory effects of the above-mentioned sequences on HBsAg, HBeAg, and HBV DNA in serum at different time points, as well as the effect of each sequence on the reconstruction of adaptive immune function in mice at a specific time point.

[1245] 1. Experimental Materials

[1246] Test drug:

[1247] Each sequence in Table 50, including sequences using only template modification, sequences using template modification and anti-off-target design, has the GalNAc ligand G5 coupled to the 3' end of the sense strand of these sequences. The structural formula of G5 is as follows:

[1248] The coupling method of oligonucleotide and ligand G5 refers to the preparation method in Example 10 of patent application CN116854754A.

[1249] The oligonucleotide forms a conjugate with ligand G5 as shown below:

[1250] The specific sequences of each sequence are shown in Table 50. In the sequence number, G5 indicates that the sequence is coupled with the GalNAc ligand G5, and GL indicates that the sequence is coupled with the GalNAc ligand L96.

[1251] The structural formula of L96 is as follows:

[1252] Table 50 siRNA sequences in animal experiments

[1253] Preparation of test drug:

[1254] Drug solvent: PBS buffer

[1255] Preparation conditions: sterile environment

[1256] Labeling method: The prepared drug preparation shall be labeled with the special number, name, concentration, quantity, preparation date, preparer and storage conditions on the outer packaging;

[1257] Storage conditions: Prepare immediately before use and store remaining samples at -80℃.

[1258] Experimental animal information:

[1259] Species / Strain: AAV-HBV mice

[1260] Rating: SPF

[1261] Gender: Male

[1262] Quantity: 100

[1263] Age: 7 weeks

[1264] Weight: 19~24g

[1265] Source: Guangdong Zhiyuan Biopharmaceutical Technology Co., Ltd.

[1266] Production license number: SCXK (Guangdong) 2021-0057

[1267] Laboratory Animal Ethics Review (IACUC):

[1268] The experimental animals were housed at Guangzhou Genio Biotechnology Co., Ltd. under license number JENNIO (Jinbin) 2019-0002. This project has been reviewed by the Laboratory Animal Ethics Committee of Guangzhou Genio Biotechnology Co., Ltd., with IACUC number JENNIO-IACUC-2024-A005. The experimental procedures were conducted in strict accordance with IACUC guidelines to ensure animal welfare.

[1269] Feeding and management:

[1270] Animals were housed at Guangzhou Geneo Biotechnology Co., Ltd. in cages measuring 29.0 cm (length × width × height) × 18.5 cm (height × height) × 13.0 cm (height × height). The temperature was set between 20 and 26°C, the humidity between 40% and 70%, and the lighting was automated with a 12-hour light-dark cycle.

[1271] The breeding environment conditions are in accordance with the National Standard of the People's Republic of China GB14925-2010.

[1272] Animals had free access to food and water. The feed was irradiated and sterilized, provided by Jiangsu Collaborative Pharmaceutical Bioengineering Co., Ltd., with production license number Su Feed Certificate (2019) 01008. Feed nutrient content testing followed the National Standard of the People's Republic of China (GB14924.3-2010), and contaminant content testing followed the National Standard of the People's Republic of China (GB14924.2-2001). The feed supplier provided a test report for each batch. Drinking water was reverse osmosis water, supplied in drinking bottles. Drinking water testing followed the National Standard of the People's Republic of China (GB5750-2006), and was tested annually by a third-party testing agency.

[1273] The animal bedding is corn cob bedding, provided by Guangzhou Saibono Biotechnology Co., Ltd. The animal bedding production license number is SCXK(Beijing)2019-0004. The bedding pollutant content test refers to the National Standard of the People's Republic of China GB14924.2-2001, and the bedding supplier provides a test report for each batch.

[1274] Animal cages and bedding should be changed at least once a week. All animal cages and bedding should be sterilized under high pressure in a pulsating vacuum sterilizer before being used in a barrier environment. Animal cages should be cleaned, disinfected and wiped at least once a week.

[1275] The animal breeding and observation room is cleaned and disinfected every day, including the flat racks, floors, tables, etc.

[1276] The disinfectants used in the barrier environment include: 6.67% chlorhexidine solution, 0.5% 84 disinfectant, 75% disinfectant, and 0.08% Baidusai. The four disinfectants should be used in turn and cannot be mixed.

[1277] 2. Experimental Methods

[1278] 2.1 AAV-HBV mouse model

[1279] Definition of experimental day: The day on which the animals were administered with the vehicle or the test drug was defined as day 0.

[1280] 220 SPF-grade C57BL / 6 male mice were adaptively housed in a barrier facility for 7 days and observed daily to confirm that the mice were healthy and normal before modeling. The mice were randomly divided into two batches. In one batch, 180 mice were injected with rAAV8-1.3HBV (Guangzhou Paizhen Biotechnology Co., Ltd., name: AAV8[HBV-D,ayw](D#2012), batch number: HBV101-6) through the tail vein. Each mouse was injected with 1×10 11GC / 100 μL, as the low baseline group animals; the remaining 40 mice were injected with rAAV-HBV1.3-mer WT replicon (Wuhan Shumi Brain Science Technology Co., Ltd., batch number: ayw1-P4-230516) through the tail vein, and each mouse was injected with 1×10 10 vg / 100μL, as the high baseline group animals. After modeling, blood was collected from animals on the 5th and 6th weeks (D-14 and D-7), centrifuged and plasma was collected and tested for HBV DNA, HBsAg and HBeAg. Modeling criteria: HBV DNA>10 5 IU / mL with a fluctuation of no more than 2 log 10 HBsAg fluctuates within 1 log 10 The average HBsAg level of animals in the low baseline group reached 2000-4000 IU / mL, and the average HBsAg level of animals in the high baseline group reached 10,000-15,000 IU / mL.

[1281] 2.2 Animal grouping and drug administration

[1282] Definition of experimental day: The day on which the animals were administered with the vehicle or the test drug was defined as day 0 (D0).

[1283] Grouping was performed based on modeling indicators at week 6 (D-7). Seventy-two animals from the successful low-baseline group were randomly divided into 12 groups of six animals each based on HBsAg levels. Eighteen animals from the successful high-baseline group were randomly divided into three groups of six animals each based on HBsAg levels. The mean HBsAg levels were maintained at the same level across groups, and no statistical differences in HBV DNA or HBeAg levels were observed between groups. After grouping, drug administration was initiated at week 7 (D 0) of modeling. Grouping and drug administration details are shown in Table 51.

[1284] Table 51 Group setting and drug administration details Note: sc: subcutaneous injection.

[1285] 2.3 Secondary challenge experiment

[1286] On D 60 of drug administration, two groups of animals were selected, with 3 mice in each group, totaling 6 mice, and 8 μg of pAAV-HBV 1.2 plasmid (Fenghui Bio) was hydrodynamically injected into the tail vein at an injection volume of 100 μL / g for virus challenge. The remaining 6 mice were not treated.

[1287] 2.4 Observation and indicator monitoring

[1288] (1) General observation

[1289] During the modeling and experimental periods, the animals were observed and recorded daily. The observation contents included: whether they were dead or dying, feed and water intake, external injuries, feces, appearance and coat, mental state, activity status, etc.

[1290] (2)Weight

[1291] Acclimation period: The animals were weighed and recorded upon receipt, and the animals were weighed and recorded on the day the acclimation period ended.

[1292] Experimental period: During the experimental period, the animals were weighed and recorded every week. If medication or blood sampling was required on the same day, the animals were weighed before the operation and before being sacrificed.

[1293] (3) Determination of serum HBsAg, HBeAg, HBV DNA, HBsAb, and ALT levels

[1294] Blood samples (200 μL) were collected from the medial canthus of the eye at 4 weeks (D-14) and 5 weeks (D-7) after model establishment, before dosing (D 0), and weekly thereafter. Plasma was collected after centrifugation at 1000 g for 10 minutes. Twelve mice in the two challenged groups underwent the challenge on D60. Blood samples (200 μL) were collected from the medial canthus of the eye at D63, D67, D74, and D81. Plasma was collected after centrifugation at 1000 g for 10 minutes. Twenty μL of plasma was added to 980 μL of PBS and vortexed for HBsAg, HBV DNA, HBeAg, and ALT assays. Another 15 μL of plasma was added to 210 μL of PBS and vortexed for HBsAb assays. All processed samples were tested by Guangzhou Huayin Medical Testing Center Co., Ltd., and the remaining plasma was stored at -80℃.

[1295] 2.5 In vitro immune index detection

[1296] Liver and spleen mononuclear cells were isolated at fixed time points for immunological testing. The IFN-γ secretion level in liver mononuclear cells was detected by ELISpot method, the IFN-γ, IFN-α, and IL-2 secretion levels in spleen mononuclear cells were detected by ELISpot method, and the HBsAg-specific IgG secretion level in effector B cells and memory B cells in spleen mononuclear cells was detected by ELISpot method. The proportion of HBsAg-specific memory B cells in the spleen was detected by flow cytometry, and the proportions of CD4+ T cells, CD8+ T cells, and the proportions of cells secreting IFN-γ, IFN-α, and IL-2 in the spleen were detected by flow cytometry.

[1297] 2.6 Data processing and statistical analysis

[1298] The experimental data are expressed as mean ± standard deviation (mean ± SD) and analyzed using GraphPad Prism 8.3 software. Data conforming to a normal distribution and homogeneity of variance were tested using one-way ANOVA. When the variances were unequal, the Welch ANOVA method was used. When the data did not conform to a normal distribution, the nonparametric Kruskal-Wallis H method was used. A P value of less than 0.05 was considered statistically significant.

[1299] 3. Experimental Results

[1300] The experimental results showed that compared with the vehicle control group and the negative control group, the sequences B207S-DV32SP, A1550-DV27P, B1572-DV27P, B1575-DV27P, B207S-DV29SP, A1573-DV29P, B1575-DV29P, B418S-DV34SPd7B, and A416-DV27Pd7B could be targetedly delivered to the liver after coupling with conjugate G5, and continuously and significantly inhibited the levels of HBsAg, HBeAg, and HBV DNA in the serum.

[1301] Among them, sequences C207S-DV32SPG5, C207S-DV29SPG5, and C418S-DV34SPd7BG5 showed superior effects in lowering HBsAg, HBV DNA, and HBeAg, and were significantly better than the Yangshen sequence VIR-2218-GL. In addition, sequences C207S-DV32SPG5 and C207S-DV29SPG5 eventually achieved negative conversion of HBsAg and HBV DNA in all mice in the group, and both produced high levels of antibodies. In the C418S-DV34SPd7BG5 group, 3 / 6 mice eventually achieved negative conversion of HBsAg and also produced high levels of antibodies.

[1302] The results of the secondary challenge experiment showed that HBsAg, HBeAg and HBV DNA appeared in the blank group (not infected with HBV virus before D56) after the challenge, indicating that the challenge method was effective. After the challenge, the HBV DNA of the mice in the C207S-DV32SPG5 group first increased and then decreased to the lower limit of detection. At the same time, the serum HBsA antibody increased significantly, and HBsAg remained below the detection limit, proving that the challenge was effective, and the hepatitis B mice treated with this small nucleic acid sequence were able to avoid re-attack by the HBV virus. It was proved that the hepatitis B mice treated with this small nucleic acid sequence were able to avoid re-attack by the HBV virus.

[1303] (i) Changes in HBsAg, HBeAg, HBV DNA, HBsAb, and ALT levels in mice in each sequence group over time

[1304] The changes in HBsAg, HBeAg, HBV DNA, HBsAb, ALT and body weight levels of mice in each group over time are shown in Table 51-1 to Table 51-9 and Figure 12A to Figure 12F. It can be seen from the table that sequences C207S-DV32SPG5, C207S-DV29SPG5, and C418S-DV34SPd7BG5 showed excellent effects in reducing HBsAg, HBV DNA, and HBeAg, which were significantly better than the Yangshen sequence VIR-2218-GL. In addition, sequences C207S-DV32SPG5 and C207S-DV29SPG5 eventually achieved negative conversion of HBsAg and HBV DNA in all mice in the group, and all produced high levels of antibodies. Among them, the C207S-DV32SPG5 group achieved negative conversion of HBsAg and HBV DNA in all mice in the group on the 14th and 21st days after administration, respectively. DNA conversion to negative was maintained until the end of the experiment, indicating that all mice achieved functional cure. Three out of six mice in the C418S-DV34SPd7BG5 group ultimately achieved HBsAg conversion to negative, while also producing high levels of antibodies. These data demonstrate the superior efficacy of the C207S-DV32SPG5, C207S-DV29SPG5, and C418S-DV34SPd7BG5 sequences.

[1305] Table 51-1 Changes in plasma HBsAg levels in mice in each sequence group Note: "-" indicates that the test was stopped; " / " indicates that the C207S-DV32SPG5 group was selected as the challenge test animal on D56. Compared with the PBS group, * indicates P < 0.05; ** indicates P < 0.01. Compared with the positive control group VIR-2218-GL group, # indicates P < 0.05; ## Indicates P < 0.01.

[1306] Table 51-2 Changes in plasma HBV DNA levels in mice in each sequence group Note: "-" indicates that the test was stopped; " / " indicates that the C207S-DV32SPG5 group was selected as the challenge test animal on D56. Compared with the PBS group, * indicates P < 0.05; ** indicates P < 0.01. Compared with the positive control group VIR-2218-GL group, # indicates P < 0.05; ## Indicates P < 0.01.

[1307] Table 51-3 Changes in plasma HBeAg levels in mice in each sequence group Note: "-" indicates that the test was stopped; " / " indicates that the C207S-DV32SPG5 group was selected as the challenge test animal on D56. Compared with the PBS group, * indicates P < 0.05; ** indicates P < 0.01. Compared with the positive control group VIR-2218-GL group, # indicates P < 0.05; ## Indicates P < 0.01.

[1308] Table 51-4 Changes in the number of mice with HBsAg negative results in each sequence group

[1309] Table 51-5 Changes in the number of mice with HBV DNA negative results in each sequence group

[1310] Table 51-6 Changes in the number of mice producing antibodies in each sequence group

[1311] Table 51-7 Changes in plasma HBsAb levels in mice in each sequence group

[1312] Table 51-8 Changes in plasma ALT levels in mice in each sequence group

[1313] Table 51-9 Changes in body weight of mice in each sequence group

[1314] (ii) Protective effect of sequence CS7S-DV32PG5 against secondary challenge in mice

[1315] The mice in the CS7S-DV32SPG5 group were injected with HBV plasmids via tail vein high-pressure hydrodynamic injection on D60 to simulate secondary infection with HBV. The experimental results are shown in Figures 13A-13F, showing that HBsAg, HBeAg and HBV DNA appeared in the blank group (not infected with HBV virus before D56) after high-pressure hydrodynamic injection of HBV plasmids, indicating that the attack method is effective. After the attack, the HBV DNA of the mice in the C207S-DV32SPG5 group first increased and then decreased to the lower limit of detection. At the same time, the serum HBsA antibodies increased significantly, and HBsAg remained below the detection limit. This result proves that the attack is effective, and the hepatitis B mice treated with this small nucleic acid sequence can be protected from re-attack by the HBV virus.

[1316] Example 15: Inhibitory effect of sequences modified with the disclosed modified template (low dose) on HBV in mice

[1317] In this example, some sequences were selected as examples, such as the sequences C207S-DV32SPG5, C207S-DV29SPG5, C418S-DV34SPd7BG5 and C1575-DV27PG5 in Example 14, see Table 49, and the in vivo efficacy of these RNAi agents at low doses, such as 0.5 mg / kg, was analyzed using the AAV-HBV mouse model.

[1318] 1. Experimental Materials

[1319] The sequences C207S-DV32SPG5, C207S-DV29SPG5, C418S-DV34SPd7BG5, C1575-DV27PG5 and VIR-2218-GL in Example 14 are shown in Table 50, as well as entecavir (ETV).

[1320] 2. Experimental Methods

[1321] Same as Example 7, the grouping and dosing details are shown in Table 51-10.

[1322] Table 51-10 Group setting and drug administration details Note: sc: subcutaneous injection; ig: intragastric administration.

[1323] 3. Experimental Results

[1324] The changes in HBsAg, HBeAg, HBV DNA, HBsAb, and ALT levels of mice in each group over time are shown in Tables 51-11 to 51-18 and Figures 14A to 14F. The experimental results showed that sequences C207S-DV32SPG5 and C207S-DV29SPG5 were significantly better than the Yangshen sequence VIR-2218-GL in inhibiting HBsAg, HBV DNA, and HBeAg. Among them, C207S-DV32SPG5 had the best effect, which could reduce HBsAg by about 2.9 log10 on the 56th day, which was about 1.4 log10 higher than C207S-DV29SPG5. In addition, on the 56th day, 3 mice turned HBsAg negative and 4 mice produced antibodies. There was no negative-turning mouse in the C207S-DV29SPG5 group. The above data once again prove the superiority of sequence C207S-DV32SPG5 in terms of efficacy.

[1325] Table 51-11 Changes in plasma HBsAg levels in mice in each sequence group Note: Compared with the PBS group, * indicates P < 0.05; ** indicates P < 0.01.

[1326] Table 51-12 Changes in plasma HBV DNA levels in mice in each sequence group Note: Compared with the PBS group, * indicates P < 0.05; ** indicates P < 0.01

[1327] Table 51-13 Changes in plasma HBeAg levels in mice in each sequence group Note: Compared with the PBS group, * indicates P < 0.05; ** indicates P < 0.01.

[1328] Table 51-14 Number of mice with negative HBsAg expression in each sequence group

[1329] Table 51-15 Number of mice with negative HBV DNA in plasma in each sequence group

[1330] Table 51-16 Number of mice producing HBsAb in each sequence group

[1331] Table 51-17 Changes in plasma HBsAb levels in mice in each sequence group

[1332] Table 51-18 Changes in plasma ALT levels in mice in each sequence group

[1333] Example 16: Inhibitory effect of modified sequences of the disclosed delivery ligands on HBV in mice

[1334] In this example, we selected several sequences, including the base sequences B418S and B207S, and modified these sequences using, for example, template-only modification, template modification combined with off-target design, and coupled delivery with different ligands. The in vivo efficacy of these RNAi agents was analyzed using an AAV-HBV mouse model.

[1335] 1. Experimental Materials

[1336] Test drug:

[1337] The sequences in this example include sequences using only template modification, sequences using template modification and anti-off-target design, and the 3' end of the sense strand of these sequences is coupled to the delivery ligand G5, G101 or G103, and the structural formula is as follows:

[1338] The coupling method of oligonucleotide and ligand G5, G101 or G103 refers to the preparation method in Example 3 of patent application CN116854754A. The oligonucleotide and ligand G5, G101 or G103 form a conjugate as shown below:

[1339] Among the sequences in this example, six siRNA sequences were designed for the basic sequence B207S based on the modified templates DV25SP to DV34SP, specifically including: C207S-DV29SPG5, C207S-DV29SPG101 and C207S-DV29SPG103 designed based on the D29SP modified template, and C207S-DV32SPG5, C207S-DV32SPG101 and C207S-DV32SPG103 designed based on the D32SP modified template (see Table 50 for the sequences of C207S-DV29SP and C207S-DV32SP). For the basic sequence B418S, three siRNA sequences of C418S-DV34SPd7BG5, C418S-DV34SPd7BG101 and C418S-DV34SPd7BG103 were designed based on the D34SP modification template (the sequence of C418S-DV34SPd7B is shown in Table 50). The present embodiment also adopts VIR-2218-GL for the Yangshen basic sequence APC and CNC-G5 for the basic sequence ANC (see Table 50). In the present embodiment, G5 represents the coupling of sequence to delivery ligand G5, G101 represents the coupling of sequence to delivery ligand G101, G103 represents the coupling of sequence to delivery ligand G103, and GL represents the coupling of sequence to delivery ligand L96. The structural formula of L96 is as follows:

[1340] Preparation of test drug:

[1341] Same as Example 14.

[1342] Experimental animal information:

[1343] Species / Strain: AAV-HBV mice

[1344] Rating: SPF

[1345] Gender: Male

[1346] Quantity: 82

[1347] Age: 5 weeks

[1348] Weight: 19~24g

[1349] Source: Beijing Weitonglihua Laboratory Animal Technology Co., Ltd.

[1350] Production license number: SCXK (Beijing) 2021-0006

[1351] Laboratory Animal Ethics Review (IACUC):

[1352] After the experimental animals were received, they were raised in Beijing Weitongda Biotechnology Co., Ltd. The license number for use is: SCXK(Beijing)2022-0013. This project has passed the review of the Experimental Animal Ethics Committee of Beijing Weitongda Biotechnology Co., Ltd. The IACUC number is VST-SY-24062701. The test process was carried out strictly in accordance with the requirements of IACUC to ensure animal welfare.

[1353] Feeding and management:

[1354] The animals were raised in a negative pressure barrier environment and fed in a plastic (polycarbonate) box (volume 370×157×180mm) IVC. Since the experimental animals are male and aggressive, they were raised individually in cages. The feeding of the animals is the responsibility of Beijing Weitongda Biotechnology Co., Ltd.

[1355] The use and detection of animal feed, bedding and drinking water all comply with the specifications of GB14925-2010 "Experimental Animal Environment and Facilities". The control of the animal feeding environment, including temperature and humidity, pressure difference, noise, illuminance, air change rate, ammonia concentration, etc., is temporarily carried out in accordance with GB50447-2008 "Technical Code for the Construction of Experimental Animal Facilities". The temperature of the animal room is controlled at 20-26°C (daily temperature difference ≤ 4°C); the relative humidity is controlled at 40-70%; artificial lighting is used for lighting, with 12 / 12 hours of day and night light and dark alternation. Compressed wood shavings bedding was purchased from Beijing Keao Xieli Feed Co., Ltd. (batch number: 23109613), and the growth and reproduction feed for mice and rats was purchased from Beijing Keao Xieli Feed Co., Ltd. (batch number: 23103313). During the experiment, the relevant records generated by the animal feeding management and environment control are stored in Beijing Weitongda Biotechnology Co., Ltd.

[1356] 2. Experimental methods

[1357] 2.1 AAV-HBV mouse model establishment

[1358] Definition of test date: The day when the animals were administered the vehicle or the test drug was defined as day 0. <​​​​​​​​​​

[1361] 2.2 Animal grouping and drug administration

[1362] Definition of experimental day: The day on which the animals were administered with the vehicle or the test drug was defined as day 0 (D0).

[1363] On day -2, all mice underwent submandibular bleeding to collect plasma. The collected blood samples were anticoagulated with EDTA and centrifuged at 5000 rpm for 10 minutes. The supernatant was used for HBV model testing. On day 0, 72 animals were divided into 12 groups of 6 animals each based on the test results on day -2. Grouping and dosing details are shown in Table 51-19.

[1364] Table 51-19 Group setting and drug administration details Note: sc: subcutaneous injection.

[1365] 2.4 Observation and indicator monitoring

[1366] (1) General observation

[1367] During the modeling and experimental periods, the animals were observed and recorded daily. The observation contents included: whether they were dead or dying, feed and water intake, external injuries, feces, appearance and coat, mental state, activity status, etc.

[1368] (2)Weight

[1369] Acclimation period: The animals were weighed and recorded upon receipt, and the animals were weighed and recorded on the day the acclimation period ended.

[1370] Experimental period: During the experimental period, the animals were weighed and recorded every week. If medication or blood sampling was required on the same day, the animals were weighed before the operation and before being sacrificed.

[1371] (3) Determination of serum HBsAg, HBeAg, HBV DNA, and ALT levels

[1372] All animals were bled submandibularly to collect plasma on days -2, 6, 13, 20, 27, and 34. The collected blood samples were anticoagulated with EDTA, centrifuged at 5000rpm for 10 minutes, and the serum was separated. After blood collection, the serum was separated, diluted with PBS solution, and sent for testing. 10μL of serum was taken from each sample, diluted to 500μL (50-fold dilution) with PBS solution, and sent to Beijing Dian Medical Testing Laboratory Co., Ltd. for testing of serum HBV DNA, HBeAg, and HBsAg. 30μL of serum was taken from each sample, diluted to 120μL (4-fold dilution) with PBS solution, and sent to Beijing Dian Medical Testing Laboratory Co., Ltd. for testing of serum ALT. The remaining plasma for testing was stored at -80°C.

[1373] 3. Experimental Results

[1374] The changes in HBsAg, HBeAg, HBV DNA, ALT levels and body weight of mice in each group over time are shown in Tables 51-20 and 51-24 and Figures 15A to 15E. The experimental results showed that the efficacy of all sequences was better than that of the Yangshen sequence VIR-2218-GL. Among them, C207S-DV29SPG101, C207S-DV29SPG103, C207S-DV32SPG101, and C207S-DV32SPG103 showed superior effects in lowering HBsAg, HBV DNA, and HBeAg, which were significantly better than the Yangshen sequence VIR-2218-GL. On the 28th day after administration, they could still reduce HBsAg by more than 3 log10 and HBV DNA by more than 3 log10, and no rebound had occurred, showing excellent anti-HBV efficacy.

[1375] Table 51-20 Changes in plasma HBsAg levels in mice in each sequence group

[1376] Table 51-21 Changes in plasma HBV DNA levels in mice in each sequence group

[1377] Table 51-22 Changes in plasma HBeAg levels in mice in each sequence group

[1378] Table 51-23 Changes in plasma ALT levels in mice in each sequence group

[1379] Table 51-24 Changes in body weight of mice in each sequence group

[1380] Example 17: Synthesis of alternatively modified small interfering oligonucleotides

[1381] 99 unmodified siRNA sequences were designed based on the AGT mRNA sequence (NM_001384479.1). To improve the inhibition efficiency and stability of the sequence, the unmodified sequence was modified alternately with 2'-methoxy (2'-OMe) and 2'-fluoro (2'-F), and the ends were thiolated. For sequences with an odd-numbered sense chain, the odd-numbered positions of the sense chain were all modified with 2'-F, and the even-numbered positions were all modified with 2'-OMe; however, for sequences with an even-numbered sense chain, the odd-numbered positions of the sense chain were all modified with 2'-OMe, and the even-numbered positions were all modified with 2'-F; the odd-numbered positions of the antisense chain were all modified with 2'-OMe, and the even-numbered positions were all modified with 2'-F. In addition, there are two thiolation modifications at the 5' end of the sense chain; and there are two thiolation modifications at the 5' and 3' ends of the antisense chain. The alternatingly modified siRNA sequences are shown in Table 52.

[1382] 1. Synthesis of the Alternately Modified Sequence B1579-AL

[1383] The unmodified sequence of the small interfering RNA with sequence number B1579-AL in Table 52 is:

[1384] Sense strand: 5'-CCUUUUCUUCUAAUGAGUCGA-3' (SEQ ID NO: 788)

[1385] Antisense strand: 5'-UCGACUCAUUAGAAGAAAAGGUG-3' (SEQ ID NO: 801)

[1386] Odd-numbered sites on the sense strand and even-numbered sites on the antisense strand are modified with 2'-F, while all other sites are modified with 2'-OMe. Additionally, there are two thiolate modifications at the 5' end of the sense strand and two thiolate modifications at the 5' and 3' ends of the antisense strand.

[1387] Instruments and reagents: Qingke 192P automatic DNA / RNA synthesizer, whose solid phase support is a universal support of cross-linked polystyrene beads, model number is Primer support 5G Unylinker 350 (Cytiva manufacturer).

[1388] Preparation method:

[1389] The following nucleotide monomer solutions were prepared using acetonitrile at a monomer concentration of 0.15 M: DMT-A-OMe phosphoramidite monomer (Formula 1), DMT-C-OMe phosphoramidite monomer (Formula 2), DMT-G-OMe phosphoramidite monomer (Formula 3), DMT-U-OMe phosphoramidite monomer (Formula 4), DMT-AF phosphoramidite monomer (Formula 5), ​​DMT-CF phosphoramidite monomer (Formula 6), DMT-GF phosphoramidite monomer (Formula 7), and DMT-UF phosphoramidite monomer (Formula 8).

[1390] Prepared by the following steps:

[1391] The solid phase carrier is loaded into the designated position of the synthesizer, and the corresponding fully protected product is obtained after several synthesis cycles. The synthesis cycle includes (1) deprotection, (2) coupling, (3) oxidation / sulfurization and (4) hydroxyl protection. The cycle process and the reagents used are described as follows:

[1392] (1) Deprotection

[1393] A 3% dichloroacetic acid toluene solution was used as a deprotection agent to remove the DMT protecting group, followed by washing with acetonitrile.

[1394] (2) Coupling

[1395] Each nucleotide monomer was coupled in acetonitrile using 0.25 M 5-ethylthiotetrazolium as an activating agent, followed by rinsing with acetonitrile.

[1396] (3) Oxidation / sulfurization

[1397] Oxidation: Oxidation was performed using a 0.05 M iodine solution in pyridine / water (90 / 10) as an oxidant, followed by rinsing with acetonitrile.

[1398] Sulfurization: 3% hydrogenated xanthan gum in pyridine was used as a sulfurizing agent for sulfurization, followed by rinsing with acetonitrile.

[1399] (4) Hydroxyl protection

[1400] Hydroxyl group protection was performed using 10% acetic anhydride tetrahydrofuran solution (CAP A) and tetrahydrofuran / pyridine / nitromethylimidazole 74 / 10 / 16 (v / v / v) (CAP B) as hydroxyl group protecting reagents, followed by rinsing with acetonitrile.

[1401] Repeat the above steps and cycle according to the set sequence to obtain a fully protected product.

[1402] (5) Use 3% dichloroacetic acid toluene solution as a deprotection reagent to remove the DMT protecting group of the last nucleotide, and then use acetonitrile to wash.

[1403] (6) Ammonolysis and purification

[1404] The solid phase carrier is transferred to a reactor, concentrated ammonia water (25-28%) is added, and after maintaining aminolysis at 60°C for 12 hours, the system is cooled to room temperature and the mixture is transferred to a filter press. It is eluted with a mixed solution of purified water and ethanol, the filtrate is combined, passed through a chromatography column, concentrated, and freeze-dried to obtain the product.

[1405] (7) Annealing

[1406] The purified sense chain and antisense chain were mixed in a 1:1 ratio, heated to 95°C for 3 minutes, and then slowly cooled to room temperature to form a double chain.

[1407] The purity of B1579-AL is 90.61%; the measured molecular weight is 14437.86.

[1408] 2. Synthesis of other sequences

[1409] The other sequences listed in Table 52 were synthesized according to the above method, for a total of 99 siRNA sequences.

[1410] Table 52 Alternative Modified Small Interfering RNA Sequences

[1411] Example 18: Inhibitory effect of alternating modified sequences on the AGT gene

[1412] After the 2'-OMe and 2'-F alternating modified siRNA sequences synthesized in Example 17 were transfected into HepG2 cells via lipid nanoparticles (LNP), the inhibitory effect of each sequence on the AGT gene was detected using qPCR technology.

[1413] 1. Experimental Materials

[1414] Test samples: the alternatively modified small interfering RNA sequences listed in Table 52 (synthesized in Example 17).

[1415] Cell type: HepG2 cell line

[1416] Drug solvent: sterile enzyme-free water, Gibco Opti-MEM.

[1417] 2. Experimental Methods

[1418] qRT-PCR was used to detect the inhibitory effect of the samples on the expression of AGT gene mRNA in HepG2 cell lines.

[1419] 2.1 Cell culture

[1420] Subcultured HepG2 cells were cultured in logarithmically growing DMEM (supplemented with 10% fetal bovine serum, 100× penicillin, and 100× streptomycin, 10 μL / mL) at 37°C in a 5% CO2 incubator, with the medium changed daily. The cells were then digested and subcultured using 0.25% trypsin. After centrifugation at 800 rpm for 3 minutes, the supernatant was discarded and fresh culture medium was added for subculture.

[1421] 2.2 Cell transfection

[1422] Preparation of transfection mixture: Mix Lipofectamine RNAiMAX and Opti-MEM at a ratio of 2:98 and vortex to mix thoroughly.

[1423] Transfection reagent preparation: Add 65 μL of the diluted siRNA solution in Opti-MEM to 65 μL of the transfection mixture at a 1:1 (v / v) ratio. Vortex to mix thoroughly and let stand at room temperature for 15 minutes to obtain lipid nanoparticles (LNPs). 12.5 μL of the solution was used for encapsulation efficiency testing.

[1424] For the blank control group, add 65 μL of the prepared transfection mixture to 65 μL of Opti-MEM. Vortex to mix thoroughly and let stand at room temperature for 15 minutes.

[1425] Add the prepared transfection reagent to a 24-well cell culture plate (100 μL per well) to make the final siRNA co...

Claims

1. A modified template for enhancing the inhibitory effect of a double-stranded oligonucleotide target gene, wherein the double-stranded oligonucleotide is siRNA, the double-stranded oligonucleotide contains a sense strand and an antisense strand, and each nucleotide of the sense strand and the antisense strand is a modified nucleotide, characterized in that: The sense strand comprises nucleotide sequence 1, the antisense strand comprises nucleotide sequence 2, and the nucleotide sequence 1 and the nucleotide sequence 2 are at least partially reverse-complemented to form a double-stranded region; the length of the reverse-complementary double-stranded region is 17 to 21 bp; In the direction from the 5' end to the 3' end, at least two nucleotides among the 2nd to 6th nucleotides of the nucleotide sequence 2 are 2'-fluoro-modified nucleotides, the 14th and / or 16th nucleotides are 2'-fluoro-modified nucleotides, the nucleotides at the remaining positions are 2'-methoxy-modified nucleotides, and the 1st, 2nd and 2nd to 3rd positions have a thiophosphate backbone.

2. The modified template according to claim 1, characterized in that At least two nucleotides in the nucleotides 2-6 of the nucleotide sequence 2 are selected from one of the following combinations: (1) Nucleotides 2 and 6; (2) nucleotides 2, 3, 4, and 6; (3) nucleotides 2, 4, 5, and 6; (4) nucleotides 2, 5, and 6; (5) nucleotides 2, 3, 5, and 6; (6) nucleotides 2, 3, and 6; And / or, in the direction from the 5' end to the 3' end, the nucleotide sequence 1 is modified in the following manner: (1) the nucleotides at positions 6 and 16 are 2'-fluoro-modified nucleotides, at least two nucleotides among the nucleotides at positions 8 to 10, preferably the nucleotides at positions 8 to 10 or the nucleotides at positions 8 and 10 are 2'-fluoro-modified nucleotides, the nucleotides at the remaining positions are 2'-methoxy-modified nucleotides, and the nucleotides at positions 1 and 2 have a phosphorothioate backbone; or (2) The nucleotides at positions 7 and 17 are 2'-fluoro-modified nucleotides, at least two nucleotides among the nucleotides at positions 9-11, preferably the nucleotides at positions 9-11 or the nucleotides at positions 9 and 11 are 2'-fluoro-modified nucleotides, the nucleotides at the remaining positions are 2'-methoxy-modified nucleotides, and the nucleotides at positions 1 and 2 have a thiophosphate backbone.

3. The modified template according to claim 1 or 2, characterized in that: The length of the nucleotide sequence 1 is 21 or 20, and the length of the nucleotide sequence 2 is 23 or 22; Preferably, the length of the nucleotide sequence 1 is 20, and the length of the nucleotide sequence 2 is 22; or, the length of the nucleotide sequence 1 is 21, and the length of the nucleotide sequence 2 is 23.

4. The modified template according to any one of claims 1 to 3, characterized in that: From the 5' end to the 3' end, the nucleotides in the antisense strand are modified in any of the following ways A, B, C, D, E, F and G: A: indicates that the antisense strand starts from the 5' end, the nucleotides at positions 1, 3, 4, 5, 7, 8, 9, 10, 11, 12, 13, 15, 17, 18, 19, 20, 21, 22, and 23 are 2'-methoxy-modified nucleotides, and the nucleotides at positions 2, 6, 14, and 16 are 2'-fluoro-modified nucleotides, and the nucleotides at positions 1, 2, 21, and 22 have a phosphorothioate backbone; B: indicates that the antisense strand starts from the 5' end, the nucleotides at positions 1, 5, 7, 8, 9, 10, 11, 12, 13, 15, 17, 18, 19, 20, 21, 22, and 23 are 2'-methoxy-modified nucleotides, and the nucleotides at positions 2, 3, 4, 6, 14, and 16 are 2'-fluoro-modified nucleotides, and the nucleotides at positions 1, 2, 21, and 22 have a phosphorothioate backbone; C: indicates that the antisense strand starts from the 5' end, the nucleotides at positions 1, 3, 7, 8, 9, 10, 11, 12, 13, 15, 17, 18, 19, 20, 21, 22, and 23 are 2'-methoxy-modified nucleotides, and the nucleotides at positions 2, 4, 5, 6, 14, and 16 are 2'-fluoro-modified nucleotides, and the nucleotides at positions 1, 2, 21, and 22 have a phosphorothioate backbone; D: indicates that the antisense strand starts from the 5' end, the nucleotides at positions 1, 3, 4, 7, 8, 9, 10, 11, 12, 13, 15, 17, 18, 19, 20, 21, 22, and 23 are 2'-methoxy-modified nucleotides, and the nucleotides at positions 2, 5, 6, 14, and 16 are 2'-fluoro-modified nucleotides, and the nucleotides at positions 1, 2, 21, and 22 have a phosphorothioate backbone; E: indicates that the antisense strand starts from the 5' end, the nucleotides at positions 1, 4, 7, 8, 9, 10, 11, 12, 13, 15, 17, 18, 19, 20, 21, 22, and 23 are 2'-methoxy-modified nucleotides, and the nucleotides at positions 2, 3, 5, 6, 14, and 16 are 2'-fluoro-modified nucleotides, and the nucleotides at positions 1, 2, 21, and 22 have a phosphorothioate backbone; F: indicates that the antisense strand starts from the 5' end, and the nucleotides at positions 1, 4, 5, 7, 8, 9, 10, 11, 12, 13, 15, 17, 18, 19, 20, 21, 22, and 23 are 2'-methoxy-modified nucleotides, and the nucleotides at positions 2, 3, 6, 14, and 16 are 2'-fluoro-modified nucleotides, and the nucleotides at positions 1, 2, 21, and 22 have thiophosphate backbones; G: indicates that the antisense strand starts from the 5' end, and the nucleotides at positions 1, 3, 4, 5, 7, 8, 9, 10, 11, 12, 13, 15, 17, 18, 19, 20, 21, 22, and 23 are 2'-methoxy-modified nucleotides, and the nucleotides at positions 2, 6, 14, and 16 are 2'-fluoro-modified nucleotides, and the nucleotides at positions 1, 2, 21, and 22 have thiophosphate backbones; G: indicates that the antisense strand starts from the 5' end, the nucleotides at positions 1, 3, 4, 5, 7, 8, 9, 10, 11, 12, 13, 15, 17, 18, 19, 20, 21, 22, and 23 are 2'-methoxy-modified nucleotides, and the nucleotides at positions 2, 6, 14, and 16 are 2'-fluoro-modified nucleotides, and the nucleotides at positions 1, 2, 21, and 22 have a phosphorothioate backbone; And / or, in the direction from the 5' end to the 3' end, the nucleotides in the sense strand are modified by any of the following schemes a to d: a: indicates that the 1st, 2nd, 3rd, 4th, 5th, 6th, 8th, 12th, 13th, 14th, 15th, 16th, 18th, 19th, 20th, and 21st nucleotides starting from the 5' end of the sense strand are 2'-methoxy-modified nucleotides, and the 7th, 9th, 10th, 11th, and 17th nucleotides are 2'-fluoro-modified nucleotides, and the 1st and 2nd nucleotides have a thiophosphate backbone; b: indicates that the 1st, 2nd, 3rd, 4th, 5th, 6th, 8th, 10th, 12th, 13th, 14th, 15th, 16th, 18th, 19th, 20th, and 21st nucleotides starting from the 5' end of the sense strand are 2'-methoxy-modified nucleotides, and the 7th, 9th, 11th, and 17th nucleotides are 2'-fluoro-modified nucleotides, and the 1st and 2nd nucleotides have a thiophosphate backbone; c: indicates that the 1st, 2nd, 3rd, 4th, 5th, 6th, 8th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, 19th, 20th, and 21st nucleotides starting from the 5' end of the sense strand are 2'-methoxy-modified nucleotides, and the 7th, 9th, 10th, and 11th nucleotides are 2'-fluoro-modified nucleotides, and the 1st and 2nd nucleotides have a thiophosphate backbone; d: Indicates that the 1st, 2nd, 3rd, 4th, 5th, 6th, 8th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, 19th, 20th, and 21st nucleotides starting from the 5' end of the sense strand are 2'-methoxy-modified nucleotides, and the 7th, 9th, and 10th nucleotides are 2'-fluoro-modified nucleotides, and the 1st and 2nd nucleotides have a thiophosphate backbone.

5. The modified template according to any one of claims 1 to 4, characterized in that: From the 5' end to the 3' end, the nucleotides in the antisense strand are modified in any of the following ways: A', B', C', D', E', F' and G': A': indicates that the antisense strand starts from the 5' end, the nucleotides at positions 1, 3, 4, 5, 7, 8, 9, 10, 11, 12, 13, 15, 17, 18, 19, 20, 21, and 22 are 2'-methoxy-modified nucleotides, and the nucleotides at positions 2, 6, 14, and 16 are 2'-fluoro-modified nucleotides, and the nucleotides at positions 1, 2, 20, and 21 have a phosphorothioate backbone; B': indicates that the antisense strand starts from the 5' end, the nucleotides at positions 1, 5, 7, 8, 9, 10, 11, 12, 13, 15, 17, 18, 19, 20, 21, and 22 are 2'-methoxy-modified nucleotides, and the nucleotides at positions 2, 3, 4, 6, 14, and 16 are 2'-fluoro-modified nucleotides, and the nucleotides at positions 1, 2, 20, and 21 have a phosphorothioate backbone; C': indicates that the antisense strand starts from the 5' end, the nucleotides at positions 1, 3, 7, 8, 9, 10, 11, 12, 13, 15, 17, 18, 19, 20, 21, and 22 are 2'-methoxy-modified nucleotides, and the nucleotides at positions 2, 4, 5, 6, 14, and 16 are 2'-fluoro-modified nucleotides, and the nucleotides at positions 1, 2, 20, and 21 have a phosphorothioate backbone; D': indicates that the antisense strand starts from the 5' end, the nucleotides at positions 1, 3, 4, 7, 8, 9, 10, 11, 12, 13, 15, 17, 18, 19, 20, 21, and 22 are 2'-methoxy-modified nucleotides, and the nucleotides at positions 2, 5, 6, 14, and 16 are 2'-fluoro-modified nucleotides, and the nucleotides at positions 1, 2, 20, and 21 have a phosphorothioate backbone; E': indicates that the antisense strand starts from the 5' end, the nucleotides at positions 1, 4, 7, 8, 9, 10, 11, 12, 13, 15, 17, 18, 19, 20, 21, and 22 are 2'-methoxy-modified nucleotides, and the nucleotides at positions 2, 3, 5, 6, 14, and 16 are 2'-fluoro-modified nucleotides, and the nucleotides at positions 1, 2, 20, and 21 have a phosphorothioate backbone; F': indicates that the antisense strand starts from the 5' end, the nucleotides at positions 1, 4, 5, 7, 8, 9, 10, 11, 12, 13, 15, 17, 18, 19, 20, 21, and 22 are 2'-methoxy-modified nucleotides, and the nucleotides at positions 2, 3, 6, 14, and 16 are 2'-fluoro-modified nucleotides, and the nucleotides at positions 1, 2, 20, and 21 have a phosphorothioate backbone; G': indicates that the antisense strand starts from the 5' end, the nucleotides at positions 1, 3, 4, 5, 7, 8, 9, 10, 11, 12, 13, 15, 16, 17, 18, 19, 20, 21, and 22 are 2'-methoxy-modified nucleotides, and the nucleotides at positions 2, 6, and 14 are 2'-fluoro-modified nucleotides, and the nucleotides at positions 1, 2, 20, and 21 have a phosphorothioate backbone; And / or, in the direction from the 5' end to the 3' end, the nucleotides in the sense strand are modified in any of the following ways a', b', c' and d': a': indicates that the 1st, 2nd, 3rd, 4th, 5th, 7th, 11th, 12th, 13th, 14th, 15th, 17th, 18th, 19th, and 20th nucleotides starting from the 5' end of the sense strand are 2'-methoxy-modified nucleotides, and the 6th, 8th, 9th, 10th, and 16th nucleotides are 2'-fluoro-modified nucleotides, and the 1st and 2nd nucleotides have a phosphorothioate backbone; b': indicates that the 1st, 2nd, 3rd, 4th, 5th, 7th, 9th, 11th, 12th, 13th, 14th, 15th, 17th, 18th, 19th, and 20th nucleotides starting from the 5' end of the sense strand are 2'-methoxy-modified nucleotides, and the 6th, 8th, 10th, and 16th nucleotides are 2'-fluoro-modified nucleotides, and the 1st and 2nd nucleotides have a thiophosphate backbone; c': indicates that the 1st, 2nd, 3rd, 4th, 5th, 7th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, 19th, and 20th nucleotides at the 5' end of the sense strand are 2'-methoxy-modified nucleotides, and the 6th, 8th, 9th, and 10th nucleotides are 2'-fluoro-modified nucleotides, and the 1st and 2nd nucleotides have a thiophosphate backbone: d': indicates that the 1st, 2nd, 3rd, 4th, 5th, 7th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, 19th, and 20th nucleotides starting from the 5' end of the sense strand are 2'-methoxy-modified nucleotides, and the 6th, 8th, and 9th nucleotides are 2'-fluoro-modified nucleotides, and the 1st and 2nd nucleotides have a thiophosphate backbone; Preferably, in the direction from the 5' end to the 3' end, the double-stranded oligonucleotide is modified by any one of the following combinations (i)-(x): (i) the antisense strand is modified using method A, and the sense strand is modified using method a; (ii) the antisense strand is modified using method B, and the sense strand is modified using method a; (iii) the antisense strand is modified using method C, and the sense strand is modified using method a; (iv) the antisense strand is modified using method B, and the sense strand is modified using method b; (v) the antisense strand is modified using method C, and the sense strand is modified using method b; (vi) the antisense strand is modified using method G, and the sense strand is modified using method C; (vi) the antisense strand is modified using method A, and the sense strand is modified using method d; (viii) the antisense strand is modified using method D, and the sense strand is modified using method b; (ix) the antisense strand is modified using method E, and the sense strand is modified using method b; (x) the antisense strand is modified using method F, and the sense strand is modified using method b; Alternatively, in the direction from the 5' end to the 3' end, the double-stranded oligonucleotide is modified by any of the following combinations (1)-(8): (1) The antisense strand is modified using method A', and the sense strand is modified using method a'; (2) the antisense strand is modified using method B', and the sense strand is modified using modification method a'; (3) the antisense strand is modified using method C', and the sense strand is modified using modification method a'; (4) the antisense strand is modified using method B', and the sense strand is modified using modification method b'; (5) the antisense strand is modified using method C', and the sense strand is modified using modification method b'; (6) the antisense strand is modified using method D', and the sense strand is modified using modification method b'; (7) the antisense strand is modified using method E', and the sense strand is modified using modification method b'; (8) The antisense strand is modified using method F', and the sense strand is modified using modification method b'.

6. The modified template according to any one of claims 1 to 5, characterized in that: The first nucleotide at the 5' end of the antisense strand is a 5'-phosphate nucleotide or a 5'-phosphate analog modified nucleotide, which includes but is not limited to the following 5'-position phosphorylation groups: 5'-vinyl phosphonate group (5'-E-VP); 5'-methyl phosphonate group (5'-MP); 5'-C-methyl phosphate group; 5'-thiophosphate group (5'-PS); 5'-phosphate group (5'-P), and its structure is shown as follows: Wherein, R is hydrogen, hydroxyl, amine, C 1-4 Alkyl, aromatic, C 1-4 Alkoxy, C 1-4 Alkylcarbonylamino or halogen; The base is selected from adenine, guanine, cytosine, thymine and uracil; Preferably, the first nucleotide at the 5' end of the antisense strand is a 5'-vinyl-(E)-phosphonate-2'-methoxy modified nucleotide.

7. The modified template according to any one of claims 1 to 6, characterized in that: One or more nucleotides from the 2nd to 8th position from the 5' end of the antisense strand, preferably the 6th and / or 7th position, adopt a modification group, wherein the modification group is preferably selected from UNA, GNA or DNA, wherein the structures of UNA and GNA are as follows: The bases in the double-stranded oligonucleotide are selected from one or more of adenine, guanine, cytosine, thymine and uracil.

8. A double-stranded oligonucleotide, characterized in that The double-stranded oligonucleotide is modified by a modification template as described in any one of claims 1 to 7; Preferably, the double-stranded oligonucleotide targets one of the mRNAs of the following genes: PCSK9 gene, HBV gene and AGT gene.

9. A conjugate for reducing gene expression, characterized in that The conjugate comprises the double-stranded oligonucleotide according to claim 8, and a ligand conjugated thereto.

10. The conjugate according to claim 9, characterized in that The ligand is conjugated to the 3'-end or 5'-end of the sense strand of the double-stranded oligonucleotide; Preferably, the ligand is one or more GalNAc derivatives attached using a divalent or trivalent branched linkage; More preferably, the double-stranded oligonucleotide is conjugated to the ligand L96, G4, G5, G6 or G7.

11. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the double-stranded oligonucleotide according to claim 8 or the conjugate according to claim 9 or 10, and a pharmaceutically acceptable carrier.

12. Use of the double-stranded oligonucleotide according to claim 8, the conjugate according to claim 9 or 10, or the pharmaceutical composition according to claim 11 in the preparation of a drug for regulating or treating diseases and conditions; the drug achieves the effect of regulating or treating diseases and conditions by down-regulating gene expression through the double-stranded oligonucleotide, the conjugate or the pharmaceutical composition; Preferably, the gene is selected from one of the following genes: PCSK9 gene, HBV gene and AGT gene; More preferably, the disease is selected from one or more of chronic hepatitis B, liver fibrosis, liver cirrhosis, liver cancer, acute hepatitis B, and HBV / hepatitis D virus co-infection related diseases; The disease is selected from hypertension, critical hypertension, essential hypertension, secondary hypertension, hypertensive crisis, hypertensive emergency, isolated systolic and diastolic hypertension, pregnancy-related hypertension, diabetic hypertension, resistant hypertension, refractory hypertension, paroxysmal hypertension, renovascular hypertension, Goldblatt's hypertension, ocular hypertension, glaucoma, pulmonary hypertension, portal hypertension, systemic venous hypertension, systolic hypertension, unstable hypertension; hypertensive heart disease, hypertensive nephropathy, atherosclerosis, arteriosclerosis, vascular disease, diabetes Diabetic nephropathy, diabetic retinopathy, chronic heart failure, cardiomyopathy, diabetic cardiomyopathy, glomerulosclerosis, coarctation of the aorta, aortic aneurysm, ventricular fibrosis, Cushing's syndrome and other glucocorticoid excess states (including chronic steroid therapy), pheochromocytoma, nephrinoma, secondary aldosteronism and other mineralocorticoid excess states, sleep apnea, thyroid / parathyroid disease, heart failure, myocardial infarction, angina pectoris, stroke, diabetes mellitus, kidney disease, renal failure, systemic sclerosis, intrauterine growth retardation (IUGR), and fetal growth restriction; or The disease is selected from one of hypercholesterolemia, atherosclerosis, dyslipidemia and cardiovascular and cerebrovascular diseases.

13. The double-stranded oligonucleotide according to claim 8, the conjugate according to claim 9 or 10, or the pharmaceutical composition according to claim 11, for use in regulating or treating diseases and disorders; the double-stranded oligonucleotide, the conjugate, or the pharmaceutical composition downregulates gene expression to achieve the effect of regulating or treating diseases and disorders; Preferably, the gene is selected from one of the following genes: PCSK9 gene, HBV gene and AGT gene; More preferably, the disease is selected from one or more of chronic hepatitis B, liver fibrosis, liver cirrhosis, liver cancer, acute hepatitis B, and HBV / hepatitis D virus co-infection related diseases; The disease is selected from hypertension, critical hypertension, essential hypertension, secondary hypertension, hypertensive crisis, hypertensive emergency, isolated systolic and diastolic hypertension, pregnancy-related hypertension, diabetic hypertension, resistant hypertension, refractory hypertension, paroxysmal hypertension, renovascular hypertension, Goldblatt's hypertension, ocular hypertension, glaucoma, pulmonary hypertension, portal hypertension, systemic venous hypertension, systolic hypertension, unstable hypertension; hypertensive heart disease, hypertensive nephropathy, atherosclerosis, arteriosclerosis, vascular disease, diabetes Diabetic nephropathy, diabetic retinopathy, chronic heart failure, cardiomyopathy, diabetic cardiomyopathy, glomerulosclerosis, coarctation of the aorta, aortic aneurysm, ventricular fibrosis, Cushing's syndrome and other glucocorticoid excess states (including chronic steroid therapy), pheochromocytoma, nephrinoma, secondary aldosteronism and other mineralocorticoid excess states, sleep apnea, thyroid / parathyroid disease, heart failure, myocardial infarction, angina pectoris, stroke, diabetes mellitus, kidney disease, renal failure, systemic sclerosis, intrauterine growth retardation (IUGR), and fetal growth restriction; or The disease is selected from one of hypercholesterolemia, atherosclerosis, dyslipidemia and cardiovascular and cerebrovascular diseases.

14. A method for treating or preventing diseases and disorders that can be regulated or treated by downregulating gene expression, characterized in that: The method comprises administering an effective amount of the double-stranded oligonucleotide of claim 8, the conjugate of claim 9 or 10, or the pharmaceutical composition of claim 11 to a subject in need thereof; Preferably, the gene is selected from one of the following genes: PCSK9 gene, HBV gene and AGT gene; More preferably, the disease is selected from one or more of chronic hepatitis B, liver fibrosis, liver cirrhosis, liver cancer, acute hepatitis B, and HBV / hepatitis D virus co-infection related diseases; The disease is selected from hypertension, critical hypertension, essential hypertension, secondary hypertension, hypertensive crisis, hypertensive emergency, isolated systolic and diastolic hypertension, pregnancy-related hypertension, diabetic hypertension, resistant hypertension, refractory hypertension, paroxysmal hypertension, renovascular hypertension, Goldblatt's hypertension, ocular hypertension, glaucoma, pulmonary hypertension, portal hypertension, systemic venous hypertension, systolic hypertension, unstable hypertension; hypertensive heart disease, hypertensive nephropathy, atherosclerosis, arteriosclerosis, vascular disease, diabetes Diabetic nephropathy, diabetic retinopathy, chronic heart failure, cardiomyopathy, diabetic cardiomyopathy, glomerulosclerosis, coarctation of the aorta, aortic aneurysm, ventricular fibrosis, Cushing's syndrome and other glucocorticoid excess states (including chronic steroid therapy), pheochromocytoma, nephrinoma, secondary aldosteronism and other mineralocorticoid excess states, sleep apnea, thyroid / parathyroid disease, heart failure, myocardial infarction, angina pectoris, stroke, diabetes mellitus, kidney disease, renal failure, systemic sclerosis, intrauterine growth retardation (IUGR), and fetal growth restriction; or The disease is selected from one of hypercholesterolemia, atherosclerosis, dyslipidemia and cardiovascular and cerebrovascular diseases.

Images