siRNAs that target and inhibit AGT gene expression, and their use in the treatment of hypertension.

Abstract

This invention provides a modified oligonucleotide sequence that can significantly inhibit the expression of the angiotensinogen (AGT) gene and can be used in the development of drugs to treat related diseases such as hypertension. [Solution] A series of siRNAs were designed based on the angiotensinogen (AGT) messenger RNA (mRNA) sequence and modified using alternating modifications and a series of specific modification templates. Results from cell and animal experiments show that several oligonucleotide sequences that underwent alternating modifications and specific template modifications can significantly inhibit AGT gene expression and can be used in the development of pharmaceuticals to treat related diseases such as hypertension.

Description

[Technical Field]

[0001] This disclosure belongs to the field of nucleic acid modification technology, and more specifically relates to small interfering ribonucleic acid (siRNA) modified by multiple chemical methods, and its use in the manufacture of pharmaceuticals for AGT gene expression-related diseases, for example, siRNA that targets and inhibits AGT gene expression, and its use in the treatment of hypertension. [Background technology]

[0002] Nucleic acid drugs, particularly oligonucleotide drugs, are widely used due to their ease of synthesis and high activity. Oligonucleotide drugs generally include antisense oligonucleotides (ASOs), small interfering RNAs (siRNAs), microRNAs (miRNAs), and nucleic acid aptamers.

[0003] Oligonucleotides are short DNA or RNA molecules, or oligomers, that readily bind sequence-specifically to their complementary oligonucleotides, DNA, or RNA to form double helixes, with higher-order hybrids appearing even less frequently. This fundamental property makes oligonucleotides widely used in genetic testing, research, and medicine. In nature, oligonucleotides are often intermediates obtained from the degradation of small RNA molecules or larger nucleic acid molecules, playing a role in regulating gene expression.

[0004] RNA interference (RNAi) is a natural defense mechanism against exogenous genes. siRNA can knock out target genes by recognizing specific sequences and degrading target mRNA.

[0005] The effective molecules of traditional RNAi consist of an iconic 19+2 nucleotide polymer structure (a double helix structure composed of 21 nucleotide RNA molecules and 19 nucleotide molecules of corresponding nucleic acid bases, including two nucleotide 3' overhangs). One strand of siRNA (guide strand or antisense strand) is complementary to the mRNA transcript of the target gene, and the other strand is defined as the passenger strand (sense strand). The siRNA (antisense strand) induces algin (AGO2) to complement the target transcript and becomes part of an RNA-induced silencing conjugate (RISC). The perfect complementarity between the siRNA (antisense strand) and the target causes cleavage of the target transcript at 10-11 sites opposite the guide strand (antisense strand) by the catalytic action of the AGO2 protein.

[0006] siRNA functions by completing Watson-Crick base pairing with mRNA. While small molecule drugs and monoclonal antibody drugs exhibit certain effects, they have a natural advantage over small molecule drugs and antibody drugs because they need to recognize the complex spatial structure of specific proteins. Therefore, many diseases cannot be treated with small molecule and monoclonal antibodies because they cannot recognize molecular structures that have high activity, affinity, and binding specificity to the target molecule. The mechanism of action of siRNA drugs allows for the control of target protein expression at the gene level and has target specificity compared to small molecule drugs or antibody drugs. Its mechanism, based on the principle of complementary base pairing, also broadens the therapeutic range of siRNA, simplifies its design, and shortens the research and development cycle.

[0007] Oligonucleotides can sequence-specifically bind to complementary RNA strands and, after hybridization, induce RNase H to cleave the target RNA. The nucleotides of creotides are linked by phosphodiester bonds, and under physiological conditions, they are particularly sensitive to nucleases. Therefore, natural, unstructured, and unmodified oligonucleotide pharmaceuticals are easily and rapidly degraded by nucleases in the body, resulting in low activity and poor drug suitability. Chemical modification of the oligonucleotide structure is an effective way to improve its activity, increasing its stability against nucleases and affinity to RNA, thereby promoting cellular endocytosis and targeting to tissues, and thus effectively controlling the expression of target genes.

[0008] Based on the basic structure of oligonucleotides—the base, sugar ring, phosphate backbone, and terminals—chemical modifications are carried out in four separate parts. 1) Base modification: This can be mainly divided into three forms: purine modification, pyrimidine modification, and base substitution. Purine modification includes N6-methyladenosine, N1-methyladenosine, and 7-methylguanosine modification, while pyrimidine modification includes 3-methyluridine, 5-methyluridine, 5-methylcytidine, N4-acetylcytidine, pseudouridine, thiouridine, propynyluridine, and dihydrouridine.

[0009] 2) Glycation modification: This can be mainly divided into glycan modification and substitution. Glycation modification includes 2'-modification, 4'-modification, 5'-modification, isomer modification, and combinations of these modifications. The most common 2'-modifications of siRNA are 2'-OMe (2'-methoxy) and 2'-F (2'-fluoro) modifications. Compared to natural siRNA, siRNA modified using both 2'-OMe and 2'-F simultaneously has a higher Tm value, stronger serum stability, and superior activity.

[0010] 3) Modification of the phosphate backbone: The main modification methods are phosphorothioate modification; modification with methylphosphonate, selenophosphate, boranophosphate, and dithiophosphate, and substitution of bridging oxygen atoms in the phosphodiester bond connection region with sulfur atoms; for example, substitution in which the entire phosphate group between nucleosides that form guanidinyl, S-methylthiourea, etc. by substituting P atoms with C, S, and N atoms is replaced with a group that does not contain phosphorus atoms.

[0011] 4) Terminal modifications: These include covalent bonding of special groups at the 5' and / or 3' ends of the sense chain, and phosphorylation modification at the 5' end of the antisense chain. Hypertension is a systemic disease characterized by elevated blood pressure. While it has a high prevalence, its treatment and control rates are low.

[0012] Angiotensinogen (AGT) protein is a secretory protein primarily expressed in the liver of the human body. Other tissues that express AGT include the brain, gallbladder, heart, and kidneys. When AGT protein is cleaved by renin, angiotensin I (Ang I) is produced, which is then cleaved by angiotensin-converting enzyme (ACE) to produce physiologically active angiotensin II (Ang II). II binds to angiotensin receptors (ATRs), causing vasoconstriction and raising blood pressure.

[0013] Currently, there are many antihypertensive drugs to treat hypertension, but more than two-thirds of patients still have difficulty controlling their blood pressure with a single medication. Therefore, they require two or more drugs to control their blood pressure, which leads to decreased adherence to treatment, increased potential side effects, and impacts treatment effectiveness. Consequently, there is a need in this field for alternative and combination therapies for patients suffering from angiotensinogen-related disorders. [Overview of the Initiative]

[0014] This disclosure relates to double-stranded s that couple to N-acetylgalactosamine (GalNAc). This relates to iRNA drugs. When GalNAc enters the bloodstream, it can bind to the asialoglycoprotein receptor (ASGPR) on the surface of hepatocytes. Subsequently, the drug is taken up by hepatocytes and stored in the cell's endosomal structure. After being released from endosomes or lysosomes and entering the cytoplasm, the drug binds to RNA-induced silencing conjugates (RISCs). Through the mediation of antisense strands, it binds to mRNA transcribed from the AGT gene, inducing mRNA degradation and inhibiting the translation of the AGT protein. The AGT protein is an upstream protein of the renin-angiotensin-aldosterone system (RAAS), and inhibiting its expression fundamentally inhibits the blood pressure-raising effect of the RAAS system, thereby lowering blood pressure.

[0015] This disclosure describes designing a series of unique siRNA sequences targeting AGT mRNA sequences and performing alternating modifications and specific template modifications thereto. Typically, siRNA is modified using 2'-methoxy (2'-OMe) and 2'-fluoro (2'-F) groups. However, even considering only these two monomer modifications, in the case of siRNA, the sense and antisense strands have a total of 44 bases, i.e., 2 44 There are various possible combinations of species. Furthermore, when combined with different terminal thiolation configurations, the number of possible modification schemes increases even further.

[0016] Modifying the same siRNA sequence in different ways results in significantly different activity, and modifying different siRNAs in the same way also results in significantly different activity. Although there are several modification principles for designing siRNA modifications, previous research has shown that it is impossible to accurately predict activity based on the modification method; in other words, there is no clear relationship between the modification method and activity. Therefore, selecting a highly active modification scheme from among countless possible modification combinations is extremely difficult.

[0017] This disclosure describes how to select several alternating modification sequences and special modification sequences that have a significant inhibitory effect on AGT gene expression by performing chemical modifications on a designed siRNA sequence.

[0018] In one embodiment, the present disclosure provides a double-stranded RNAi agent comprising an oligonucleotide double helix consisting of a sense strand and an antisense strand pair. In another embodiment, the present disclosure provides a conjugate for reducing AGT expression, comprising the above-mentioned double-stranded RNAi agent and a ligand bound thereto.

[0019] In another embodiment, the present disclosure provides a nucleic acid protein composition comprising the double-stranded region or antisense strand of the double-stranded RNAi agent described above, and a nuclease. In another embodiment, the present disclosure provides a recombinant vector comprising a nucleic acid molecule encoding the double-stranded RNAi agent described above.

[0020] In some embodiments, the vector backbone of the recombinant vector is selected from recombinant viroid-derived circular RNA vectors, tRNAs, rRNA scaffolds, and chimeric tRNA / pre-miRNA vectors.

[0021] In another embodiment, the present disclosure provides recombinant cells that synthesize and secrete the above-described double-stranded RNAi agents. In some embodiments, the recombinant cells are selected from Thiocapsa rosepercisina and ribonuclease III-deficient Corynebacterium glutamicum.

[0022] In another embodiment, the present disclosure provides a method for producing a double-stranded RNAi agent, comprising the steps of culturing or chemically synthesizing the recombinant cells. In another embodiment, the present disclosure relates to the above-mentioned double-stranded RNAi agent, the above-mentioned conjugate, or The present invention provides a pharmaceutical composition comprising the above-described nucleic acid protein composition and a pharmaceutically acceptable carrier.

[0023] In another embodiment, the present disclosure provides a method for inhibiting the expression of an AGT gene, comprising the step of contacting a target cell with the double-stranded RNAi agent, the conjugate, the nucleic acid protein composition, or the pharmaceutical composition described above.

[0024] In some embodiments, the method is for non-diagnostic or non-therapeutic purposes. In some embodiments, the method is performed in vivo or in vitro.

[0025] In another embodiment, the present disclosure provides the use of the above-mentioned double-stranded RNAi agent, the above-mentioned conjugate, the above-mentioned nucleic acid protein composition, or the above-mentioned pharmaceutical composition in the manufacture of a pharmaceutical for treating AGT gene expression-related disease.

[0026] The aforementioned AGT gene expression-related diseases are selected from diseases caused by AGT protein overexpression, AGT gene pathogenic mutations, AGT protein metabolic disorders, and abnormal interactions between AGT and other substances.

[0027] In some embodiments, the AGT gene expression-related disorders are selected from hypertension, elevated intraocular pressure, glaucoma, pulmonary hypertension, portal hypertension, systemic venous hypertension, hypertensive heart disease, atherosclerosis, arteriosclerosis, vascular disease, diabetic nephropathy, diabetic retinopathy, chronic heart failure, cardiomyopathy, diabetic cardiomyopathy, glomerulosclerosis, aortic stenosis, aortic aneurysm, ventricular fibrosis, Cushing's syndrome and other glucocorticoid excess conditions (including chronic steroid therapy), pheochromocytoma, nephrinoma, secondary aldosteronism and other mineralocorticoid excess conditions, sleep apnea syndrome, thyroid / parathyroid disease, heart failure, myocardial infarction, angina pectoris, stroke, diabetes, kidney disease (e.g., hypertensive nephropathy), renal failure, systemic sclerosis, intrauterine growth restriction (IUGR) and fetal growth restriction.

[0028] In another embodiment, the present disclosure provides the above-mentioned double-stranded RNAi agent, the above-mentioned conjugate, the above-mentioned nucleic acid protein composition, or the above-mentioned pharmaceutical composition for use in the treatment of AGT gene expression-related disorders.

[0029] In another embodiment, the present disclosure provides a method for treating AGT gene expression-related disorders, comprising the step of administering an effective amount of the above double-stranded RNAi agent, the above conjugate, the above nucleic acid protein composition, or the above pharmaceutical composition to a subject in need.

[0030] In another embodiment, the present disclosure provides the use of the above-mentioned double-stranded RNAi agent, the above-mentioned conjugate, the above-mentioned nucleic acid protein composition, or the above-mentioned pharmaceutical composition in the manufacture of pharmaceuticals for the treatment of hypertension and cardiovascular and cerebrovascular diseases.

[0031] In another embodiment, the present disclosure provides the above-mentioned double-stranded RNAi agents, the above-mentioned conjugates, the above-mentioned nucleic acid protein compositions, or the above-mentioned pharmaceutical compositions used for the treatment of hypertension and cardiovascular and cerebrovascular diseases.

[0032] In another embodiment, the present disclosure provides a method for treating hypertension and cardiovascular and cerebrovascular diseases, comprising the step of administering an effective amount of the above double-stranded RNAi agent, the above conjugate, the above nucleic acid protein composition, or the above pharmaceutical composition to a subject in need.

[0033] The beneficial effects of this disclosure are at least as follows: (1) Unmodified sequences including unmodified sequences 1576s, 1578s, 1838, 1835, 1816, 1812, 1579, 1836, 1789, 1839, 1791, 1795 and 1585s all have a significant inhibitory effect on AGT, and the maximum inhibition rate can exceed 50%.

[0034] (2) By using multiple modified sequences, the inhibition rate can reach up to 70% or more. Furthermore, when the modified sequences are bound to a GalNAc compound, they are efficiently delivered to the liver of animals, significantly inhibiting the expression of the AGT gene and greatly lowering blood pressure levels.

[0035] (3) Sequences modified by the alternating modifications and modification templates of the present disclosure exhibit significantly increased inhibitory activity against AGT compared to unmodified sequences that show little difference from sequences disclosed in the prior art, and this increase can be up to 91.0%.

[0036] (4) This disclosure found that siRNAs with similar sequences, whether unmodified or alternatingly modified, exhibited significantly greater activity. For example, the inhibition rate of the unmodified sequence 1812 increased by 14.3% compared to the unmodified sequence 812P, and the inhibition rate of the alternatingly modified sequence B1812-AL increased by 20.4% compared to the inhibition rate of the unaltered alternatingly modified sequence 812P, demonstrating a significant improvement.

[0037] (5) The disclosure also found that different sequences had different effects on activity after alternating modification. Some inhibition rates were significantly improved; for example, in the unmodified sequence 836, the inhibition rate of alternating modification was 13.3% higher than that of the unmodified sequence. In some cases, the improvement was not significant; for example, in the unmodified sequences 994, 1279, and 1591, there was essentially no change in the inhibition rates of the alternating modified and unmodified sequences. [Brief explanation of the drawing]

[0038] To more clearly illustrate the technical solutions of the embodiments of this disclosure, the drawings of the embodiments are briefly introduced below, and it is clear that the drawings in the following description relate only to some embodiments of this disclosure and do not limit the disclosure.

[0039] [Figure 1] These are alternating modification sequences that have a significant inhibitory effect on the AGT gene, and the inhibition rate of each of these sequences exceeds 40%. [Figure 2]This is an alternating modification sequence that inhibits the AGT gene by between 25% and 40%. [Figure 3] This is an alternating modification sequence that inhibits the AGT gene by less than 25%. [Figure 4] These unmodified sequences have a significant inhibitory effect on the AGT gene, and the inhibition rate of each of these sequences exceeds 45%. [Figure 5] This is an unmodified sequence with an inhibition rate of less than 45% against the AGT gene. [Figure 6] This shows the inhibition rate of the AGT gene after modifying the unmodified sequence 1579 with a different template, and the inhibition rate of the AGT gene with sequence 579P disclosed in the prior art. [Figure 7] This shows the inhibition rates of the unmodified sequence 1578s and the sequence 1835, which underwent template modification and off-target prevention modification, against the AGT gene. [Figure 8] This describes the inhibition rate of AGT protein in animal serum after modifying the unmodified sequences 1579, 1789, 1812, 1576s, 1578s, and 1585s using different modification schemes and then binding them to a GalNAc compound. [Figure 9] This represents the inhibition rate of AGT protein in animal serum at various time points after modifying the unmodified sequences 1579, 1789, 1812, 1576s, 1578s, and 1585s using different modification schemes and then binding them with a GalNAc compound. [Modes for carrying out the invention]

[0040] To make this disclosure easier to understand, we will first define some terms. Furthermore, please note that where a range of a single number or parameter is listed, it is intended that the intermediate values ​​and ranges of those cited numbers are also part of this disclosure.

[0041] As used herein, the articles “one” and “one kind” ("a" and "an") refer to one or more (i.e., at least one) objects modified by these articles. For example, “one element” means one element, or one or more elements such as a plural element.

[0042] As used herein, the term "including" means "including, but not limited to," and can be used interchangeably. As used herein, the term "or" means "and / or" and can be used interchangeably unless otherwise clearly specified by the context.

[0043] As used herein, the terms “about” or “approximately” applied to one or more target values ​​refer to a numerical value similar to the preceding reference value. In some embodiments, unless otherwise specified or evident from the context, the terms “approximately” or “approximately” refer to a numerical value falling within a range of 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (larger or smaller) of the aforementioned reference value (provided that such a numerical value does not exceed 100% of the possible value).

[0044] As used herein, "AGT" refers to the angiotensinogen gene or protein. "G," "C," "A," and "U" represent nucleotides containing guanine, cytosine, adenine, and uracil as bases, respectively. "T," "Td," and "dT" are used interchangeably herein and refer to deoxyribonucleotides whose nucleic acid base is thymine, such as deoxyribothymine, 2'-deoxythymidine, or thymidine. However, it should be understood that the terms "ribonucleotide," "nucleotide," or "deoxyribonucleotide" may also refer to modified nucleotides (described in more detail below) or alternative substitutional portions. Those skilled in the art are well aware that guanine, cytosine, adenine, and uracil can be substituted with other portions without substantially altering the base-pairing properties of oligonucleotides (including nucleotides having such substitutional portions). For example, a nucleotide containing inosine as a base can, but is not limited to, form base pairs with a nucleotide containing adenine, cytosine, or uracil. Therefore, in the nucleotide sequences of the present disclosure, nucleotides containing uracil, guanine, or adenine may be replaced with, for example, nucleotides containing inosine. Sequences containing such substitutions are examples of the present disclosure.

[0045] The terms “iRNA,” “RNAi agent,” “iRNA agent,” and “RNA interference agent” are used interchangeably herein, and the defined terms include RNA agents that can mediate targeted cleavage of transcripts via the RNA-induced silencing complex (RISC) pathway. iRNAs induce sequence-specific degradation of mRNA through a process called RNA interference (RNAi). iRNAs regulate, for example, the expression of AGT in cells, e.g., in the cells of a subject (e.g., a mammalian subject). RNAi molecules include single-stranded RNAi molecules (Lima et al 2012 Cell 150:883), double-stranded siRNAs, and short hairpin RNA (shRNA).

[0046] The term "small interfering ribonucleotide" or "siRNA" refers to a small interfering ribonucleotide RNAi molecule. This is a type of double-stranded RNA molecule, also known in the art as short interfering RNA or silencing RNA. siRNA typically consists of a sense strand (also called a passenger strand) and an antisense strand (also called a leading strand), each strand having a length of 17–30 nucleotides and generally a length of 19–25 nucleosides, where the antisense strand is complementary to the target nucleic acid (e.g., at least 95% complementary, such as perfectly complementary) (appropriately a mature mRNA sequence), and the sense and antisense strands are complementary such that they form a double helix or double-stranded region. The siRNA strands may form a blunt-ended double helix, but preferably the 3' ends of the sense and antisense strands may form 3' overhangs (e.g., 1, 2, or 3 nucleotides) similar to Dicer products, and may form RISC substrates in vivo. Effective extended forms of the Dicer substrate are described in US8349809 and US8513207, which are incorporated herein by reference. In some embodiments, both the sense strand and the antisense strand have 2nt 3' overhanging ends. Thus, the double-stranded region may have a nucleotide length of, for example, 17 to 25 nucleotides, or for example, 21 to 23 nucleotides.

[0047] The term “antisense strand” refers to a strand of RNAi (e.g., dsRNA) containing a region substantially complementary to the target sequence. As used herein, the term “complementary region” refers to a region on the antisense strand that is substantially complementary to the sequence as defined herein (e.g., the target sequence). If the complementary region is not perfectly complementary to the target sequence, mismatches may occur in the internal or terminal regions of the molecule. Typically, the most acceptable mismatches are in the terminal regions, e.g., within 5, 4, 3, or 2 nucleotides at the 5' and / or 3' ends.

[0048] As used herein, the term "sense strand" refers to a strand of RNAi that contains a region substantially complementary to the antisense strand (as defined herein).

[0049] The term "alternating modification" refers to a fully modified siRNA sequence using alternating 2'-methoxy (2'-OMe) and 2'-fluoro (2'-F) modifications. For the antisense strand of the siRNA sequence, odd-numbered positions (i.e., positions 1, 3, 5, 7...21, 23) are modified with 2'-methoxy, and even-numbered positions (i.e., positions 2, 4, 6, 8...20, 22) are modified with 2'-fluoro. For the sense strand complementary to the antisense strand, at positions where 2'-methoxy modification is used on the antisense strand, the complementary pairing positions on the sense strand are modified with 2'-fluoro, and at positions where 2'-fluoro modification is used on the antisense strand, the complementary pairing positions on the sense strand are modified with 2'-methoxy.

[0050] As used herein, the term "inhibition" may be used interchangeably with "reduction," "silence," "downregulation," "suppression," and other similar terms, and includes all levels of inhibition.

[0051] As used herein, the term "inhibit AGT expression" includes inhibiting the expression of any AGT gene (e.g., mouse AGT gene, rat AGT gene, monkey AGT gene, or human AGT gene), and variants of AGT genes (e.g., naturally occurring variants) or mutants. Thus, the AGT gene may be a wild-type AGT gene, a mutant AGT gene, or a transgenic AGT gene in the case of genetically engineered cells, cell populations, or organisms.

[0052] "Inhibition of AGT gene expression" includes inhibition of AGT gene expression at any level, for example, at least partial inhibition of AGT gene expression, for example, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about It inhibits by 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%, and at least about 99%.

[0053] AGT gene expression can be assessed based on any variable level associated with AGT gene expression, such as AGT mRNA levels or AGT protein levels. Inhibition can be assessed by a decrease in the absolute or relative levels of one or more of these variables compared to a control level. The control level may be any type of control level used in the art, such as the baseline level before administration, or a level determined from similarly untreated or controlled (e.g., buffer-only control or inactivator control) subjects, cells, or samples.

[0054] As used herein, the terms "patient" or "subject" include humans and non-human animals, preferably mammals such as monkeys. Most preferably, the subject or patient is human.

[0055] As used herein, “AGT-related disease” includes all diseases related to the AGT gene or protein. Such diseases may be caused, for example, by overproduction of the AGT protein, mutation of the AGT gene, abnormal cleavage of the AGT protein, or abnormal interactions between AGT and other proteins or other endogenous or exogenous substances. Exemplary AGT-related diseases include hypertension, borderline hypertension, primary hypertension, secondary hypertension, hypertensive crisis, hypertensive emergency, isolated systolic and diastolic hypertension, pregnancy-related hypertension, diabetic hypertension, treatment-resistant hypertension, refractory hypertension, paroxysmal hypertension, renovascular hypertension, Goldblatt hypertension, elevated intraocular pressure, glaucoma, pulmonary hypertension, portal hypertension, systemic venous hypertension, systolic hypertension, unstable hypertension, hypertensive heart disease, hypertensive nephropathy, atherosclerosis, and arteriosclerosis. This includes vascular diseases, diabetic nephropathy, diabetic retinopathy, chronic heart failure, cardiomyopathy, diabetic cardiomyopathy, glomerulosclerosis, aortic coarctation, aortic aneurysm, ventricular fibrosis, Cushing's syndrome and other glucocorticoid excess conditions (including chronic steroid therapy), pheochromocytoma, renal cell tumor, secondary aldosteronism and other neracorticoid excess conditions, sleep apnea syndrome, thyroid / parathyroid disorders, heart failure, myocardial infarction, angina pectoris, stroke, diabetes, nephropathy, renal failure, systemic sclerosis, intrauterine growth restriction (IUGR), and fetal growth restriction.

[0056] As used herein, “therapeutic dose” is intended to include an amount of RNAi agent sufficient to treat an AGT-related disorder when administered to a patient for the treatment of the disorder (e.g., to alleviate, improve or maintain the symptoms of an existing disorder or one or more disorders). The “therapeutic dose” may vary depending on the RNAi agent, the method of administration of the reagent, the disorder and its severity, medical history, age, weight, family history, genetic makeup, stage of the pathological process mediated by AGT expression, type of previous or concurrent treatment (if any), and other personal characteristics of the patient being treated.

[0057] As used herein, “prophylactic effective dose” means an amount of RNAi agent sufficient to prevent or alleviate the disease or one or more symptoms of the disease when administered to a subject who has not yet experienced or shown symptoms of AGT-related disease but is at risk of developing the disease. Disease improvement includes slowing the progression of the disease or reducing the subsequent severity of the disease. The “prophylactic effective dose” is determined based on the RNAi agent, the method of administration of the reagent, the degree of risk of the disease, medical history, age, weight, family history, genetic makeup, previous or concurrent treatments ( The type of treatment (if applicable) and other personal characteristics of the patient being treated may vary.

[0058] The “therapeutic dose” or “preventive dose” also includes the amount of RNAi agent that produces the desired local or systemic effect at a reasonable benefit / risk ratio applicable to any treatment. The RNAi agent used in the method of this disclosure can be administered in an amount sufficient to produce a reasonable benefit / risk ratio applicable to such treatment.

[0059] As used herein, the term “sample” includes similar bodily fluids, cells, or tissues isolated from the body of a subject, as well as collections of bodily fluids, cells, or tissues present in the body of a subject. Examples of bodily fluids include blood, serum and serous fluid, plasma, cerebrospinal fluid, ocular fluid, lymph, urine, and saliva. Tissue samples may include samples from tissues, organs, or local sites. For example, a sample may originate from a specific organ, a part of an organ, or bodily fluids or cells within those organs. In some embodiments, a sample may originate from the liver (e.g., the whole liver or a specific part of the liver, or a specific type of cell within the liver, such as hepatocytes). In a preferred embodiment, “subject-derived sample” means blood or plasma taken from the subject. In other embodiments, “subject-derived sample” means liver tissue (or its subcomponents) derived from the subject.

[0060] In one embodiment, the present invention is (1) A sense strand having the sequence or fragment thereof shown in Sequence ID No. 1, or a modified sequence of the sequence or fragment thereof, and an antisense strand having the sequence or fragment thereof shown in Sequence ID No. 14, or a modified sequence of the sequence or fragment thereof, (2) A sense strand having the sequence or fragment thereof shown in Sequence ID No. 2, or a modified sequence of the sequence or fragment thereof, and an antisense strand having the sequence or fragment thereof shown in Sequence ID No. 15, or a modified sequence of the sequence or fragment thereof. (3) A sense strand having the sequence or fragment thereof shown in Sequence ID No. 3, or a modified sequence of the sequence or fragment thereof, and an antisense strand having the sequence or fragment thereof shown in Sequence ID No. 16, or a modified sequence of the sequence or fragment thereof. (4) A sense strand having the sequence or fragment thereof shown in Sequence ID No. 4, or a modified sequence of the sequence or fragment thereof, and an antisense strand having the sequence or fragment thereof shown in Sequence ID No. 17, or a modified sequence of the sequence or fragment thereof, (5) A sense strand having the sequence or fragment thereof shown in Sequence ID No. 5, or a modified sequence of the sequence or fragment thereof, and an antisense strand having the sequence or fragment thereof shown in Sequence ID No. 18, or a modified sequence of the sequence or fragment thereof, (6) A sense strand having the sequence or fragment thereof shown in Sequence ID No. 6, or a modified sequence of the sequence or fragment thereof, and an antisense strand having the sequence or fragment thereof shown in Sequence ID No. 19, or a modified sequence of the sequence or fragment thereof. (7) A sense strand having the sequence or fragment thereof shown in Sequence ID No. 7, or a modified sequence of the sequence or fragment thereof, and an antisense strand having the sequence or fragment thereof shown in Sequence ID No. 20, or a modified sequence of the sequence or fragment thereof. (8) A sense strand having the sequence shown in Sequence ID No. 8 or a fragment thereof, or a modified sequence of the sequence or fragment thereof, and the sequence shown in Sequence ID No. 21 or The fragment, or an antisense chain having the sequence or a modified sequence of the fragment, (9) A sense strand having the sequence or fragment thereof shown in Sequence ID No. 9, or a modified sequence of the sequence or fragment thereof, and an antisense strand having the sequence or fragment thereof shown in Sequence ID No. 22, or a modified sequence of the sequence or fragment thereof, (10) A sense strand having the sequence or fragment thereof shown in Sequence ID No. 10, or a modified sequence of the sequence or fragment thereof, and an antisense strand having the sequence or fragment thereof shown in Sequence ID No. 23, or a modified sequence of the sequence or fragment thereof, (11) A sense strand having the sequence or fragment thereof shown in Sequence ID No. 11, or a modified sequence of the sequence or fragment thereof, and an antisense strand having the sequence or fragment thereof shown in Sequence ID No. 24, or a modified sequence of the sequence or fragment thereof, (12) A sense strand having the sequence or fragment thereof shown in Sequence ID No. 12, or a modified sequence of the sequence or fragment thereof, and an antisense strand having the sequence or fragment thereof shown in Sequence ID No. 25, or a modified sequence of the sequence or fragment thereof, (13) A sense strand having the sequence or fragment thereof shown in Sequence ID No. 13, or a modified sequence of the sequence or fragment thereof, and an antisense strand having the sequence or fragment thereof shown in Sequence ID No. 26, or a modified sequence of the sequence or fragment thereof. The present invention provides a double-stranded RNAi agent that reduces AGT expression, comprising any double-stranded oligonucleotide selected from a sense strand and antisense strand pair. In some embodiments, all nucleotides on the sense strand and antisense strand are modified nucleotides.

[0061] In some embodiments, double-stranded RNAi agents are RNAi agents used to inhibit the expression of AGT genes. In some embodiments, the sense strand has a nucleotide difference of 1 to 3 from any one of the sequences of sequence numbers 1 to 13.

[0062] In some embodiments, the antisense strand has a nucleotide difference of 1 to 3 from any one of the sequences of SEQ ID NOs. 14 to 26. In some embodiments, at least one modified nucleotide is selected from the group consisting of deoxy-nucleotides, 3'-terminal deoxythymidine (dT) nucleotides, 2'-O-methyl-modified nucleotides, 2'-fluoro-modified nucleotides, 2'-deoxy-modified nucleotides, locked nucleotides, unlocked nucleotides, conformation-restrictive nucleotides, restricted ethyl nucleotides, debasalized nucleotides, 2'-amino-modified nucleotides, 2'-O-allyl-modified nucleotides, 2'-C-alkyl-modified nucleotides, 2'-hydroxy-modified nucleotides, 2'-methoxyethyl-modified nucleotides, 2'-O-alkyl-modified nucleotides, morpholino nucleotides, phosphoramidites, nucleotides containing unnatural bases, tetrahydropyran-modified nucleotides, 1,5-anhydrohexitol-modified nucleotides, cyclohexenyl-modified nucleotides, nucleotides containing a phosphorothioate group, nucleotides containing a methylphosphonate, nucleotides containing a 5'-phosphate, and nucleotides containing a 5'-phosphate mimetic.

[0063] In some embodiments, at least one strand contains the 3' overhang of at least one nucleotide. In some embodiments, at least one strand contains the 3' overhanging ends of at least two nucleotides.

[0064] In some embodiments, the length of the double-stranded region is 15 to 30 nucleotide pairs. In some embodiments, the length of the double-stranded region is 17 to 25 nucleotide pairs.

[0065] In some embodiments, the length of the double-stranded region is 19 to 23 nucleotide pairs. In some embodiments, the length of the double-stranded region is 21 nucleotide pairs.

[0066] In some embodiments, each chain has 15 to 30 nucleotides. In some embodiments, each chain has 19 to 25 nucleotides. In some embodiments, the sense strand has 21 nucleotides and the antisense strand has 23 nucleotides.

[0067] In some embodiments, all nucleotide modifications on the sense and antisense strands are chemical modifications of the 2' position of the ribose of the nucleotide. In some embodiments, the chemical modification at the 2' position of the ribose of the nucleotide is selected from one or more of the following: 2'-methoxy, 2'-methoxyethyl, 2'-fluoro, 2'-benzyloxy, 2'-methylcarbonylamino, and 2'-pyridinylmethoxy.

[0068] In some embodiments, the chemical modification at the 2' position of ribose of each nucleotide is selected from a combination of 2'-methoxy and 2'-fluoro. In some embodiments, the chemical modification at the 2' position of ribose of each nucleotide is selected from alternating combinations of 2'-methoxy and 2'-fluoro.

[0069] In some embodiments, the chemical modification method for the 2' position of ribose in each nucleotide is such that all odd positions on the sense strand are 2'-fluoromodified and all even positions are 2'-methoxymodified, and all odd positions on the antisense strand are 2'-methoxymodified and all even positions are 2'-fluoromodified.

[0070] In some embodiments, nucleotide monomers are linked by 3',5'-phosphodiester bonds. In some embodiments, the 3',5'-phosphodiester bond linking nucleotide monomers has a thio modification.

[0071] In some embodiments, the oligonucleotide has the following modifications. The antisense chain uses one of the modification methods shown in Table 42.

[0072] [Table 1] TIFF2026059799000002.tif223163TIFF2026059799000003.tif224163TIFF2026059799000004.tif51164

[0073] The sense chain uses one of the modification methods shown in Table 43.

[0074] [Table 2]

[0075] In the table above, 2'-OMe is 2'-methoxy, 2'-F is 2'-fluoro, and PS is phosphorothioate backbone. Here, the antisense chain uses modification A, and the sense chain uses modification method a. Here, the antisense chain uses modification B, and the sense chain uses modification method a. Here, the antisense chain uses modification C, and the sense chain uses modification method a. Here, the antisense chain uses modification B, and the sense chain uses modification method b. Here, the antisense chain uses modification C, and the sense chain uses modification method b. Here, the antisense chain uses modification D, and the sense chain uses modification method b. Here, the antisense chain uses modification E, and the sense chain uses modification method b, or Here, the antisense chain uses modification method F, and the sense chain uses modification method b.

[0076] In some embodiments, the oligonucleotide has the following modifications. The antisense chain uses one of the modification methods shown in Table 44.

[0077] [Table 3] TIFF2026059799000007.tif220164TIFF2026059799000008.tif76164

[0078] The sense chain shall use one of the modification methods shown in Table 45.

[0079] [Table 4]

[0080] In the table above, 2'-OMe is 2'-methoxy, 2'-F is 2'-fluoro, and PS is phosphorothioate backbone. Here, the antisense chain uses modification A, and the sense chain uses modification method a. Here, the antisense chain uses modification B, and the sense chain uses modification method a. Here, the antisense chain uses modification C, and the sense chain uses modification method a. Here, the antisense chain uses modification B, and the sense chain uses modification method b. Here, the antisense chain uses modification C, and the sense chain uses modification method b. Here, the antisense chain uses modification D, and the sense chain uses modification method b. Here, the antisense chain uses modification E, and the sense chain uses modification method b, or Here, the antisense chain uses modification method F, and the sense chain uses modification method b.

[0081] In some embodiments, the antisense strand uses a modifying group at the 2nd to 8th positions from the 5' end, where the modifying group is selected from UNA, GNA, or DNA, where the structures of UNA and GNA are as follows:

[0082] [ka]

[0083] However, the base is selected from adenine, guanine, cytosine, thymine, and uracil. In some embodiments, the 5'-carbon atom of the glycoside of the 5'-terminal nucleotide of the modified antisense chain is phosphorylated, resulting in the following 5'-phosphorylation groups: 5'-vinylphosphonate (5'-E-VP), 5'-methylphosphonate (5'-MP), 5'-C-methylphosphate, 5'-thiophosphonate (5'-PS), and 5'-phosphonate (5'-P), and the structure is represented by the following formula.

[0084] [ka]

[0085] However, R is hydrogen, hydroxyl, amine, C 1-4 Alkyl, aryl, C 1-4 Alkoxy, C 1-4 It is an alkylcarbonylamino or halogen, The base is selected from adenine, guanine, cytosine, thymine, and uracil.

[0086] In some embodiments, the 3',5'-phosphodiester bond between the nucleotide monomers at the end of the sequence includes a thio modification to form a chiralally pure 3',5'-thiophosphodiester bond, where the 5' ends of the sense and antisense strands have 1 to 3 thio bonds, and the 3' end of the antisense strand has 1 to 3 thio bonds.

[0087] The double-stranded RNA (dsRNA) agent of this disclosure may be bound to one or more ligands. The ligand may be bound to a sense strand, an antisense strand, or both at its 3' end, 5' end, or both ends. For example, the ligand may be bound to the sense strand. In a preferred embodiment, the ligand may be ligated to the 3' end of the sense strand. In one preferred embodiment, the ligand is a single GalNAc ligand.

[0088] This disclosure provides a conjugate used to reduce AGT expression, comprising the double-stranded RNAi agent and a ligand bound thereto. In some embodiments, the ligand is bound to the 3'-terminus or 5'-terminus of the sense strand of the oligonucleotide.

[0089] In some embodiments, the ligand is a GalNAc derivative conjugated using a divalent or trivalent branched linkage. In some embodiments, the ligand is

[0090] [ka] And,

[0091] However, X is hydrogen, or a hydroxy protecting group or H, and the hydroxy protecting group includes acetyl, benzoyl, and isobutyryl; Y is an amine protecting group or H, and 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, the ligand is

[0093] [Chemical Formula] .

[0094] In some embodiments, the ligand is

[0095] [Chemical Formula] ,

[0096] where X is oxygen, nitrogen or sulfur, Y is alkyl or aryl, R1 is oxygen or sulfur, R2 is hydrogen, amine, C 1-4 alkyl, aryl, C 1-4 alkoxy or halogen, A is -(CH2) a -, -(CH2CH2O) b -, -((CH2) c NHCO) d - or -((CH2) c CONH) d -, where a is an integer from 1 to 15, b is an integer from 1 to 7, c is an integer from 1 to 7, d is an integer from 1 to 5, B is -(CH2) [[ID=5​​​​​​​​​​​​​​​​​​​​m is an integer between 0 and 4. n is an integer between 0 and 4.

[0097] In some embodiments, the ligand is G4, G5, G6, or G7.

[0098] [ka]

[0099] In some embodiments, the conjugate has a structure represented by the following formula.

[0100] [ka]

[0101] In some embodiments, the double-stranded RNAi agent is (1) The sense strand has the sequence shown in Sequence ID 423, and the antisense strand has the sequence shown in Sequence ID 488, (2) The sense strand has the sequence shown in Sequence ID No. 406, and the antisense strand has the sequence shown in Sequence ID No. 453, (3) The sense strand has the sequence shown in Sequence ID No. 404, and the antisense strand is Having the sequence shown in sequence number 450, (4) The sense strand has the sequence shown in Sequence ID 426, and the antisense strand has the sequence shown in Sequence ID 492, (5) The sense strand has the sequence shown in Sequence ID No. 412, and the antisense strand has the sequence shown in Sequence ID No. 465, (6) The sense strand has the sequence shown in Sequence ID 412, and the antisense strand has the sequence shown in Sequence ID 468, (7) The sense strand has the sequence shown in Sequence ID No. 421, and the antisense strand has the sequence shown in Sequence ID No. 483, (8) The sense strand has the sequence shown in Sequence ID No. 417, and the antisense strand has the sequence shown in Sequence ID No. 476, (9) The sense strand has the sequence shown in Sequence ID No. 419, and the antisense strand has the sequence shown in Sequence ID No. 479, (10) The sense strand has the sequence shown in Sequence ID 401, and the antisense strand has the sequence shown in Sequence ID 443, (11) The sense strand has the sequence shown in Sequence ID No. 398, and the antisense strand has the sequence shown in Sequence ID No. 433, (12) The sense strand has the sequence shown in Sequence ID No. 429, and the antisense strand has the sequence shown in Sequence ID No. 496, (13) The sense strand comprises any oligonucleotide double strand selected from the sense strand and antisense strand pair, wherein the sense strand has the sequence shown in Sequence ID No. 414 and the antisense strand has the sequence shown in Sequence ID No. 471. Here, the oligonucleotide double strand binds to ligand G4, G5, G6, or G7.

[0102] In some embodiments, the double-stranded RNAi agent is (1) The sense strand has the sequence shown in Sequence ID 423, and the antisense strand has the sequence shown in Sequence ID 488, (2) The sense strand has the sequence shown in Sequence ID 423, and the antisense strand has the sequence shown in Sequence ID 489, (3) The sense strand has the sequence shown in Sequence ID 424, and the antisense strand has the sequence shown in Sequence ID 489, (4) The sense strand has the sequence shown in Sequence ID 424, and the antisense strand has the sequence shown in Sequence ID 490, (5) The sense strand comprises any oligonucleotide double strand selected from the sense strand and antisense strand pair, wherein the sense strand has the sequence shown in Sequence ID No. 424 and the antisense strand has the sequence shown in Sequence ID No. 488. Here, the oligonucleotide double strand binds to ligand G4, G5, G6, or G7.

[0103] In some embodiments, the double-stranded RNAi agent comprises an oligonucleotide double strand consisting of a sense strand shown in SEQ ID NO: 423 and an antisense strand shown in SEQ ID NO: 488, wherein the oligonucleotide double strand binds to ligand G5.

[0104] The present invention also provides a pharmaceutical composition comprising the double-stranded RNAi agent or conjugate and a pharmaceutically acceptable carrier. In one embodiment, this specification provides a pharmaceutical composition comprising the iRNA described herein and a pharmaceutically acceptable carrier. The pharmaceutical composition comprising the iRNA can be used to treat diseases or conditions related to the expression or activity of the AGT gene, such as hypertension. Such pharmaceutical compositions are manufactured based on a delivery mode, such as subcutaneous injection (SC). Examples include compositions manufactured for systemic administration by oral administration. Another example is a composition manufactured for direct delivery to the brain parenchyma by intracerebral infusion (such as continuous pump infusion).

[0105] The pharmaceutical compositions comprising the RNAi agent of this disclosure may be, for example, a solution with or without a buffer, or a composition comprising a pharmaceutically acceptable carrier. Such compositions include, for example, aqueous or crystalline compositions, liposomal formulations, micelle formulations, emulsions, and gene therapy vectors.

[0106] In the method of this disclosure, the RNAi agent may be administered in a solution. The free RNAi agent may be administered in a non-buffered solution such as saline or water. Alternatively, the free siRNA may be administered in a suitable buffer. The buffer may contain acetate, citrate, prolamin, carbonate, or phosphate, or any combination thereof. In one preferred embodiment, the buffer is phosphate-buffered saline (PBS). The pH and volume molar osmotic concentration of the buffer containing the iRNA agent can be adjusted to suit administration to a subject.

[0107] In some embodiments, the buffer solution further includes an agent for controlling the osmolality of the solution so that the osmolality of the solution is maintained at a desired value, for example, a physiological value in human plasma. Solvents that can be added to the buffer to control the osmolality include, but are not limited to, proteins, peptides, amino acids, non-metabolic polymers, vitamins, ions, sugars, metabolites, organic acids, lipids, or salts. In some embodiments, the agent used to control the osmolality of the solution is a salt. In some embodiments, the agent used to control the osmolality of the solution is sodium chloride or potassium chloride.

[0108] The pharmaceutical compositions of this disclosure can be administered in amounts sufficient to inhibit the expression of the AGT gene. Typically, appropriate doses of the iRNAs of this disclosure range from about 0.001 to about 200.0 mg per kg of body weight of the recipient per day, and generally range from about 1 to 50 mg per kg of body weight of the recipient per day. For example, RNAi agents (e.g., dsRNA) can be administered in doses 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 per single dose. 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 0.7, 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, 19, 19.5 It can be administered at doses of 20, 20.5, 21, 21.5, 22, 22.5, 23, 23.5, 24, 24.5, 25, 25.5, 26, 26.5, 27, 27.5, 28, 28.5, 29, 29.5, 30, 31, 32, 33, 34, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or approximately 50 mg / kg.

[0109] The pharmaceutical composition can be administered once a day, or multiple times at various time intervals over a period of 1 to 365 days, or the iRNA can be administered two, three, or more times at appropriate intervals within one year. The drug can be administered in divided doses or by continuous infusion or by delivery using a sustained-release formulation. In this case, each sub-dose must contain a correspondingly smaller amount of iRNA to achieve the total daily dose. The dosing units can also be formulated to be delivered over several days, for example, using a conventional sustained-release formulation that continuously releases iRNA over several days. Sustained-release formulations are well known in the art and are particularly useful for delivering drugs to specific sites, and can therefore be used with the drugs of this disclosure. In this embodiment, the dosing units comprise a number of corresponding daily doses.

[0110] In other embodiments, a single dose of the pharmaceutical composition may be administered over a long period, with subsequent doses administered at intervals of 3, 4, or 5 days, or at intervals of 1, 2, 3, or 4 weeks. In some embodiments of the Disclosure, a single dose of the Pharmaceutical Composition of the Disclosure is administered once a week. In other embodiments of the Disclosure, a single dose of the Pharmaceutical Composition of the Disclosure is administered once every two months.

[0111] Those skilled in the art will understand that certain factors, including but not limited to the severity of the disease or symptoms, previous treatments, the subject's overall health and / or age, and other existing illnesses, may influence the dosage and schedule required to effectively treat the subject. Furthermore, treating a subject with a therapeutically effective dose of the composition may include a single treatment or a series of treatments. As described elsewhere in this specification, the effective dose and in vivo half-life of each iRNA contained herein can be estimated using conventional methods or based on in vivo studies using appropriate animal models.

[0112] The pharmaceutical compositions of this disclosure can be administered in a variety of ways, depending on whether topical or systemic treatment is preferred and the area to be treated. Administration may be topical (e.g., by skin patch); pulmonary administration, e.g., by inhalation or blowing of powder or aerosol including by spray; intratracheal; intranasal; epidermal, transdermal, oral, or parenteral. Parenteral administration includes intravenous, intra-arterial, subcutaneous, intraperitoneal, or intramuscular injection or infusion; e.g., subcutaneous via an implantable device; or intracranial administration, e.g., intracerebral, intrathecal, or intraventricular administration.

[0113] The iRNAs used in the compositions and methods of this disclosure can be formulated to be delivered in membrane-bound molecular assemblies such as liposomes or micelles. As used herein, the term “liposome” refers to a vesicle composed of amphiphilic lipids arranged in at least one bilayer (e.g., one or more bilayers). A liposome comprises a monolayer or multilayer vesicle having a membrane formed from one lipophilic material and one aqueous interior. The aqueous portion contains the iRNA composition. The lipophilic material typically separates the aqueous interior from the aqueous exterior, which does not contain the iRNA composition (although it may be included in some embodiments). Liposomes are useful for transporting and delivering active ingredients to the site of action. This is because the liposome membrane is structurally similar to a biological membrane, so when a liposome is used in a tissue, the liposome bilayer fuses with the cell membrane bilayer. As the liposome-cell fusion progresses, the aqueous contents of the interior containing the iRNA are delivered into the cell, where the iRNA can specifically bind to a target RNA and mediate RNAi. In some cases, these liposomes are target-specific, such as inducing iRNAs to specific cell types.

[0114] Liposomes containing RNAi agents can be manufactured in various ways. In one example, the lipid component of the liposome is dissolved in a detergent, and micelles are formed from the lipid component. For example, the lipid component may be an amphiphilic cationic lipid or a lipid conjugate. The detergent may have a high critical micelle concentration and may be nonionic. Exemplary detergents include cholates, CHAPS, octyl glucoside, deoxycholate, and lauroyl sarcosine. Next, The RNAi agent formulation is added to micelles containing the lipid component. The cation groups on the lipid interact with the RNAi agent and condense around the RNAi agent to form liposomes. After condensation, the detergent is removed by dialysis or the like to obtain a liposomal formulation of the RNAi agent.

[0115] iRNAs such as the dsRNAs of this disclosure can be completely encapsulated within lipid products (e.g., LNPs or other nucleic acid-lipid particles). As used herein, the term "LNP" refers to stable nucleic acid-lipid particles. An LNP comprises one cationic lipid, one non-cationic lipid, and one lipid that prevents aggregation of the particles (e.g., a PEG-lipid conjugate). LNPs are very useful for synthetic applications because they exhibit a long circulating lifetime after intravenous (iv) injection and accumulate at distal sites (e.g., sites physically distant from the injection site).

[0116] In one embodiment, the ratio of lipid to drug (mass / mass ratio) (e.g., the ratio of lipid to dsRNA) is in the range of approximately 1:1 to approximately 50:1, approximately 1:1 to approximately 25:1, approximately 3:1 to approximately 15:1, approximately 4:1 to approximately 10:1, approximately 5:1 to approximately 9:1, or approximately 6:1 to approximately 9:1.

[0117] In some preferred embodiments, the lipid nanoparticles include cationic lipids, neutral lipids, structural lipids, and polymer-conjugated lipids. In some preferred embodiments, the cationic lipid is a compound of the structure of formula (I), or its N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer, where G1 is C 1~6 It is alkylene, and G2 is C2~8 It is alkylene, and G3 is C 1~3 It is alkylene, and L1 is C 6~15 It is a linear alkyl group, and L2 is C 12~25 It is a branched alkyl group. For example, YK-009 has the structure of formula (II) (see patent CN114044741B).

[0118] [ka]

[0119] In some preferred embodiments, the cationic lipid is a compound of the structure of formula (II), or its N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer, where G1 is C 2~8 It is alkylene, and G2 is C 2~8 It is an alkylene, L1 is -C(O)O- or -OC(O)-, L2 is -C(O)O- or -OC(O)-, and R1 is C 6~25 It is a linear or branched alkyl group, and R2 is C 6~25 It is a linear or branched alkyl group, G3 is HO(CH2)2- or HO(CH2)3-, and G4 is H The molecule is O(CH2)2- or HO(CH2)3-, and L is (CH2)2-, -(CH2)3-, or -(CH2)4-. For example, YK-401 has the structure of formula (II-I), and YK-402 has the structure of formula (II-II) (see patent CN115784921B).

[0120] [ka]

[0121] In some preferred embodiments, the cationic lipid is a compound of the structure of formula (III), or its N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer, where G1 is C 1~6 It is alkylene, and G2 is C 2~8 It is alkylene, and R1 is C 6~20 It is a linear or branched alkyl group, and R2 is C 12~25These are branched alkyl groups, and G3 is modified. For example, YK-201 has the structure of formula (III-I), and YK-202 has the structure of formula (III-II) (see patent CN115677518B).

[0122] [ka]

[0123] In some preferred embodiments, the cationic lipid is a compound of the structure of formula (IV), or its N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer, where G1 is C 1~8 It is alkylene, and G2 is C 2~8 It is alkylene, and R1 is C 6~25 It is a linear or branched alkyl group, and R2 is C 12~25 The alkyl group is linear or branched, and G3 is HO(CH2)2N(R3)CH2CH(OH)CH2-, where R3 is -CH3, -CH2CH3, or -CH2CH2OH. For example, YK-305 has the structure of formula (IV-I), and YK-310 has the structure of formula (IV-II) (see patent CN115745820B).

[0124] [ka]

[0125] In some preferred embodiments, the cationic lipid is a compound of the structure of formula (V), or its N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer, where G 1 and G 2 Each of these is an independent, unsubstituted C6~C 10 It is alkylene, G 3 This is unsubstituted C1~C 12 It is alkylene, R 1 and R 2 Each is independently C6~C 24 Alkyl or C6-C 24 It is an alkenyl, R 3 is OR 5, N, -C(=O)OR 4 -OC(=O)R 4 or -NR 5 C(=O)R 4 And R 4 is C1~C 12 It is a hydrocarbon group, R 5 is H or a C1-C6 hydrocarbon group, for example, ALC0315 of the structure of formula (VI) (see patent CN108368028B).

[0126] [ka]

[0127] In some preferred embodiments, the cationic lipid is a compound of the structure of formula (VI), or its N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer, where R4 is -(CH2) n Q and -(CH2) n Selected from CHQR, where Q is -OR, -OH, -O(CH2) n Selected from the group consisting of N(R)2, -OC(O)R, -CX3, -CN, -N(R)C(O)R, -N(H)C(O)R, -N(R)S(O)2R, -N(H)S(O)2R, -N(R)C(O)N(R)2, -N(H)C(O)N(R)2, -N(H)C(O)N(H)(R), -N(R)C(S)N(R)2, -N(H)C(S)N(R)2, -N(H)C(S)N(H)(R), -N(R)S(O)2R8 and heterocyclyl, where n is 1, 2 or 3, for example, SM102 of the (VI-I) structure (see patent CN110520409A).

[0128] [ka]

[0129] In some preferred embodiments, the cationic lipid is a compound of the structure of formula (VII), or its N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer (see Patent CN102625696B, DLIN-MC3-DMA).

[0130] [ka]

[0131] In some preferred embodiments, the cationic lipids include YK-009, YK-401, YK-305, ALC0315, SM102, and DLIN-MC3-DMA.

[0132] In some preferred embodiments, the molar ratio of the cationic lipid to the neutral lipid is 1:1 to 10:1. In some preferred embodiments, the molar ratio of the cationic lipid to the structural lipid is 1:1 to 5:1.

[0133] In some preferred 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).

[0134] In some preferred 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).

[0135] In some preferred 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.

[0136] In some preferred embodiments, the neutral lipid includes one or more of phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, ceramide, sterol, and derivatives thereof.

[0137] In some preferred embodiments, the neutral lipid is 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-3-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-diundecanoyl-sn-glycero-3-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl-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 16.0 PE), 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-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleoylphosphatidylethanolamine (POPE), distearoylphosphatidyl One or more of the following are selected: dilethanolamine (DSPE), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), 1-stearoyl-2-oleoyl-stearoylethanolamine (SOPE), 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC), sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine (LPE), or mixtures thereof.

[0138] In some preferred embodiments, the neutral lipid is DOPE and / or DSPC. In some preferred embodiments, the structural lipid is selected from one or more of the following: cholesterol, nonsterols, sitosterol, ergosterol, campesterol, stigmasterol, brassinosteroids, tomatine, ursolic acid, α-tocopherol, and corticosteroids.

[0139] In some preferred embodiments, the structural lipid is cholesterol. In some preferred 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.

[0140] In some preferred embodiments, the polymer-conjugated lipid is selected from one or more of the following: distearoylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000), 1,2-dimiristoyl-rac-glycero-methoxypolyethylene glycol-2000 (DMG-PEG2000), and methoxypoly(ethylene glycol)ditetradecylacetamide (ALC-0159).

[0141] The pharmaceutical compositions of this disclosure include, but are not limited to, solutions, emulsions, and liposome-containing formulations. These compositions can be prepared from a variety of components, including, but are not limited to, pre-formed liquids, self-emulsifying solids, and self-emulsifying semi-solids. When treating liver diseases (e.g., liver cancer), formulations targeting the liver are particularly preferred.

[0142] The pharmaceutical formulations of this disclosure (which may be available in convenient unit dose forms) can be manufactured according to the prior art well known in the pharmaceutical industry. Such art includes, but is not limited to, the step of combining the active ingredients with these pharmaceutical carriers or excipients. Generally, these formulations are manufactured by homogeneously and closely mixing these active ingredients with a liquid carrier or a finely divided solid carrier, or both, and forming the product as needed.

[0143] The compositions of this disclosure can be formulated into any of a number of possible dosage forms, not limited to tablets, capsules, gel capsules, liquid syrups, soft gelatin capsules, suppositories, and enemas. The compositions of this disclosure can also be prepared as suspensions in aqueous, non-aqueous, or mixed media. The aqueous suspension may further contain substances that increase the viscosity of the suspension, such substances including, for example, sodium carboxymethylcellulose, sorbitol, and / or dextran. The suspension may further contain stabilizers.

[0144] Certain compositions of this disclosure also incorporate carrier compounds in the formulation. As used herein, “carrier compound” or “carrier” refers to a nucleic acid or analogue that is inactive (i.e., not biologically active itself) but is recognized as a nucleic acid in an in vivo process that reduces the bioavailability of biologically active nucleic acids by degrading them or facilitating their removal from circulation. Co-administration of nucleic acids and carrier compounds (generally in excess of the latter) may significantly reduce the amount of nucleic acid recovered in the liver, kidneys, or other extracirculating storage, which is thought to be a result of competition between the carrier compound and the nucleic acid for a common receptor. For example, co-administration with polyinosinic acid, dextran sulfate, polycytidylic acid, or 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid may reduce the recovery of partially phosphorothioated dsRNA in liver tissue (Miyao et al., DsRNA Res. Dev., 1995, 5, 115-121; Takakura et al., DsRNA & Nucl. Acid Drug Dev., 1996, 6, 177-183).

[0145] In contrast to carrier compounds, a “pharmaceutical carrier” or “excipient” is a pharmaceutically acceptable solvent, suspension, or any other pharmaceutically inert medium used to deliver one or more nucleic acids to an animal. The excipient may be liquid or solid and is selected to provide a desired volume, viscosity, etc., when combined with the nucleic acid and other components of a particular pharmaceutical composition, with reference to the intended mode of administration. Common pharmaceutical carriers include, but are not limited to, binders (e.g., pre-gelatinized corn starch, polyvinylpyrrolidone, or hydroxypropyl methylcellulose), fillers (e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethylcellulose, polyacrylate, or calcium hydrogen phosphate), lubricants (e.g., magnesium stearate, talc, silicon dioxide, colloidal silicon dioxide, stearic acid, metallic stearate, hydrogenated vegetable oil, corn starch, polyethylene glycol, sodium benzoate, sodium acetate), disintegrants (e.g., starch, sodium starch glycolate), and wetting agents (e.g., sodium lauryl sulfate).

[0146] Nucleic acids suitable for parenteral administration and pharmaceutically acceptable organic or inorganic excipients that do not cause toxic reactions can also be used to prepare the compositions of this disclosure. Suitable pharmaceutically acceptable carriers include, but are not limited to, water, saline, alcohol, polyethylene glycol, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, and polyvinylpyrrolidone.

[0147] Nucleic acid topical formulations include sterile or non-sterile aqueous solutions, non-aqueous solutions, or solutions of nucleic acids in a liquid or solid oily base in a common solvent such as alcohol. These solutions may further contain buffers, diluents, and other suitable additives. Suitable for parenteral administration, pharmaceutically acceptable organic or inorganic excipients that do not react toxicly with nucleic acids may be used.

[0148] Appropriate pharmaceutically acceptable excipients include water, saline solution, alcohol, and polyethylene glycol. This includes, but is not limited to, kohl, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, and polyvinylpyrrolidone.

[0149] This disclosure also provides methods for treating or preventing diseases and conditions that can be regulated by downregulating AGT gene expression. For example, hypertension, borderline hypertension, primary hypertension, secondary hypertension, hypertensive crisis, hypertensive emergency, isolated systolic and diastolic hypertension, pregnancy-related hypertension, diabetic hypertension, treatment-resistant hypertension, refractory hypertension, paroxysmal hypertension, renovascular hypertension, Goldblatt hypertension, increased intraocular pressure, glaucoma, pulmonary hypertension, portal hypertension, systemic venous hypertension, systolic hypertension, unstable hypertension, hypertensive heart disease, hypertensive nephropathy, atherosclerosis, arteriosclerosis, and vascular diseases. It treats diabetic nephropathy, diabetic retinopathy, chronic heart failure, cardiomyopathy, diabetic cardiomyopathy, glomerulosclerosis, aortic coarctation, aortic aneurysm, ventricular fibrosis, Cushing's syndrome and other glucocorticoid excess conditions (including chronic steroid therapy), pheochromocytoma, renal cell tumor, secondary aldosteronism and other neracorticoid excess conditions, sleep apnea syndrome, thyroid / parathyroid disease, heart failure, myocardial infarction, angina pectoris, stroke, diabetes, nephropathy, renal failure, systemic sclerosis, intrauterine growth restriction (IUGR), and fetal growth restriction.

[0150] The RNAi agents of this disclosure can be administered to a subject using any method of administration known in the art, including but not limited to subcutaneous, intravenous, intramuscular, intraocular, intrabronchial, intrapleural, intraperitoneal, intraarterial, translymphatic, transcerebrospinal, and any combination thereof. In preferred embodiments, these agents are administered subcutaneously.

[0151] In another embodiment, siRNA is administered in combination with other therapeutic agents. siRNA and other therapeutic agents may be administered together in the same composition, for example, by parenteral administration, or the other therapeutic agent may be administered as part of another composition, or by other methods described herein.

[0152] Other embodiments of therapeutic agents include agents used to treat well-known hypertension or agents used to treat well-known cardiovascular and cerebrovascular diseases. For example, other agents for the treatment of 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.), and β-receptor blockers (e.g., propranolol, bisoprolol, metoprolol tartrate, metoprolol succinate, etc.).

[0153] In one embodiment, the iRNA agent is administered to the patient, followed by the administration of another therapeutic agent (or vice versa). In another embodiment, the iRNA agent and the other therapeutic agent are administered simultaneously.

[0154] The following examples are provided to illustrate the present invention and do not limit its scope. Unless otherwise noted, the technical solutions used in the examples are conventional methods well known to those skilled in the art, and all raw materials used are commercially available.

[0155] The nucleotide abbreviations used herein are as follows: A = Adenosine-3'-phosphate Am=2'-Methoxyadenosine-3'-phosphate Ams = 2'-methoxyadenosine-3'-phosphorothioate Af = 2'-Fluoradenosine-3'-phosphate Afs = 2'-fluoroadenosine-3'-phosphorothioate G = Guanosine-3'-phosphate Gm = 2'-Methoxyguanosine-3'-phosphate Gms = 2'-methoxyguanosine-3'-phosphorothioate Gf = 2'-Fluoroguanosine-3'-phosphate Gfs = 2'-fluoroguanosine-3'-phosphorothioate C = cytidine-3'-phosphate Cm=2'-Methoxycytidine-3'-phosphate Cms = 2'-methoxycytidine-3'-phosphorothioate Cf = 2'-fluorocytidine-3'-phosphate Cfs = 2'-fluorocytidine-3'-phosphorothioate U = Uridine-3'-phosphate Um = 2'-Methoxyuridine-3'-phosphate Ums = 2'-methoxyuridine-3'-phosphorothioate Uf = 2'-Fluorouridine-3'-phosphate Ufs = 2'-fluorouridine-3'-phosphorothioate AmsEVP = 5'-ethenyl-(E)-phosphonate-2'-methoxyadenosine-3'-phosphorothioate UmsEVP = 5'-ethenyl-(E)-phosphonate-2'-methoxyuridine-3'-phosphorothioate Agna = Adenosine Glycol Nucleic Acid Cgna = Cytidine-glycol nucleic acid Ggna = Guanosine Glycol Nucleic Acid Tgna = Thymidine-glycol nucleic acid Ugna = Uridine-glycol nucleic acid The corresponding English names for the modifications used in this specification are as follows:

[0156] 2'-Methoxy:2'-O-methyl 2'-Fluoro:2'-Fluor 3'-thiophosphate 5'-Ethenyl-(E)-phosphonate 2'-deoxy: 2'-deoxyl The numbering of the compounds used herein is as follows:

[0157] An unmodified sequence is an unmodified RNA sequence. For sequences with sense and antisense strands of 21 / 23 nucleotides in length, the number indicates the position of the sequence on AGT mRNA (e.g., compound 1579). For sequences with sense and antisense strands of 20 / 22 nucleotides in length, "s" is added after the position number to distinguish them (e.g., compound 1578s).

[0158] In sequences alternatingly modified with 2'-methoxy (2'-OMe) and 2'-fluoro (2'-F), a "B" is added before the number of the unmodified sequence, and "-AL" is added after the number. For example, the number of the alternatingly modified 1578s sequence is B1578s-AL.

[0159] The compounds produced in Example 4 use sequences modified with the DV25P-29P, DV32P-34P templates disclosed herein, or the DV22 template disclosed in the prior art, with a "C" added before the number of the unmodified sequence and the corresponding template name added after the number. For example, a 1578s sequence modified using the DV29P template would have the number C1578s-DV29P.

[0160] The compounds produced in Example 6 are modified using the DV25P-29P / DV32P-34P template disclosed herein or the DV22 template disclosed in the prior art, and may also include modification sequences to prevent off-target effects. The first 1 of the unmodified sequence number is removed, "D" is added before the number, and the corresponding template name and anti-thio off-target modification name are added after the number in order. For example, the number of the unmodified sequence 1578s modified using the DV29P template and containing the anti-thio off-target modification d7B is D578s-DV29Pd7B.

[0161] The compound obtained in Example 7 is modified using the DV25P-29P template disclosed herein and may also contain an anti-off-target modification sequence, with GalNAc ligated to the 3' end of the sense strand to achieve liver-targeted delivery, and its numbering is based on the numbering in Example 6, with G5 added to the end. For example, the D578s-DV29Pd7B sequence ligated to G5 is numbered D578s-DV29Pd7BG5.

[0162] [Table 5]

[0163] In the following examples, if the experimental data for intergroup comparisons shows a P-value < 0.05, the difference is statistically significant. Examples Example 1: Synthesis of alternatingly modified low molecular weight interfering oligonucleotides Ninety-nine unmodified siRNA sequences were designed based on the AGT mRNA sequence (NM_001384479.1). To improve the inhibitory efficiency and stability of the sequences, the unmodified sequences were alternately modified with 2'-methoxy (2'-OMe) and 2'-fluoro (2'-F), and the ends were thiolated. For sequences with an odd sense strand length, all odd positions on the sense strand were modified with 2'-F, and all even positions were modified with 2'-OMe. Conversely, for sequences with an even sense strand length, all odd positions on the sense strand were modified with 2'-OMe, and all even positions were modified with 2'-F. Similarly, all odd positions on the antisense strand were modified with 2'-OMe, and all even positions were modified with 2'-F. Furthermore, there were two thio modifications at the 5' end of the sense strand, and two thio modifications at the 5' and 3' ends of the antisense strand, respectively. The alternately modified siRNA sequences are shown in Table 2.

[0164] 1. Synthesis of alternating modification sequence B1579-AL Unmodified sequences of low molecular weight interfering ribonucleic acids with sequence number B1579-AL in Table 1: Sense chain: 5'-CCUUUUCUUCUAAUGAGUCGA-3' (Sequence ID 4) Antisense chain: 5'-UCGACUCAUUAGAAGAAAAGGUG-3' (Sequence ID 17) All odd-numbered positions on the sense strand and even-numbered positions on the antisense strand used 2'-F modifications, while all other positions used 2'-OMe modifications. Furthermore, there were two thio modifications at the 5' end of the sense strand, and two thio modifications at both the 5' and 3' ends of the antisense strand.

[0165] Instrument and reagents: The 192 P model DNA / RNA automated synthesizer from Beijing Tsingke Biotech Co., Ltd. uses cross-linked polystyrene beads as its solid-phase carrier, and the model supports 5G primer. It's the Unylinker 350 (by Cytiva).

[0166] Manufacturing method: Solutions of the following nucleotide monomers were prepared in acetonitrile according to 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), and 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).

[0167] [ka]

[0168] It was manufactured using the following steps. The solid-phase carrier was loaded into the designated position in the synthesis apparatus, and the corresponding fully protected product was obtained through several synthesis cycles. The synthesis cycles included (1) deprotection, (2) coupling, (3) oxidation / sulfidation, and (4) hydroxy protection. The cycle process and reagents used are as follows.

[0169] (1) Deprotection A 3% dichloroacetic acid-toluene solution was used as a deprotecting agent to remove the DMT protecting group, and then the material was washed with acetonitrile.

[0170] (2) Coupling Each nucleotide monomer was coupled with an acetonitrile solution using 0.25 M 5-(ethylthio)-1H-tetrazole as an activator, and then rinsed with acetonitrile.

[0171] (3) Oxidation / Sulfidation Oxidation: Oxidation was performed using a 0.05 M iodine-pyridine / water (90 / 10) solution as the oxidizing agent, followed by washing with acetonitrile.

[0172] Sulfidation: Sulfidation was performed using a 3% pyridine solution of xanthan hydride as the sulfidating agent, followed by washing with acetonitrile. (4) Hydroxyprotection Hydroxyprotection was performed using a 10% anhydrous tetrahydrofuran solution (CAP A) and tetrahydrofuran / pyridine / N-methylimidazole 74 / 10 / 16 (v / v / v) (CAP B) as hydroxyprotection reagents, followed by washing with acetonitrile.

[0173] The above operation was repeated, and the cycle was performed according to the set sequence to obtain a fully protected product. (5) After removing the DMT protecting group of the last nucleotide using a 3% dichloroacetic acid-toluene solution as a deprotection agent, the nucleotides were washed with acetonitrile.

[0174] (6) Ammonium-mediated cracking and purification The solid-phase carrier was transferred to a reactor, concentrated aqueous ammonia (25 - 28%) was added, and after maintaining ammonolysis at 60 °C for 12 hours, the reaction system was cooled to room temperature. The mixture was transferred to a filter press, rinsed with a mixed solution of purified water and ethanol, the filtrates were combined, subjected to a chromatography column, concentrated, and lyophilized to obtain the product.

[0175] (7) Annealing After mixing the purified sense strand and antisense strand in a 1:1 ratio, they were heated to 95 °C and maintained for 3 minutes, and then slowly cooled to room temperature to form a double strand.

[0176] The purity of B1579-AL: 90.61%, measured molecular weight: 14437.86. 2. Synthesis of Other Sequences The other sequences listed in Table 1 were synthesized according to the above method, and there were a total of 99 siRNA sequences.

[0177]

Table 6

[0178] Example 2: Inhibitory Effect of Reciprocal Modification on the AGT Gene After introducing the 2'-OMe and 2'-F reciprocal modification siRNA sequences synthesized in Example 1 into HepG2 cells via lipid nanoparticles (LNP), the inhibitory effects of each sequence on the AGT gene were detected by qPCR technology.

[0179] 1. Experimental Materials Test sample: Interacting small molecule siRNA sequences with alternating modifications shown in Table 1 (synthesized in Example 1). Cell type: HepG2 cell line Drug solvent: Sterilized enzyme-free water, Gibco Opti-MEM.

[0180] 2. Experimental Method The inhibitory effect of the sample on AGT gene mRNA expression in the HepG2 cell line was detected using qRT-PCR.

[0181] 2.1 Cell Culture The HepG2 cell line was taken out of the subcultured cell line and the cells in the logarithmic growth phase were mixed with 10% fetal bovine serum. Cells were cultured in DMEM medium (supplemented with 100× penicillin and 10 μL / mL of 100× streptomycin) and placed in a cell culture incubator containing 5% CO2 at 37°C, with the medium changed once daily. After digestion with 0.25% trypsin, the cells were subcultured, centrifuged at 800 r / min for 3 minutes, the supernatant was discarded, and fresh medium was added for further subculture.

[0182] 2.2 Cell transfection Preparation of transfection mixture: Lipofectamine RNAiMAX and Opti-MEM were mixed in a 2:98 ratio and vortexed to ensure uniform mixing.

[0183] Preparation of transfection reagent: A siRNA solution diluted with Opti-MEM was added to 65 μL of transfection mixture in a 1:1 (v / v) ratio. The mixture was homogeneously mixed by vortexing and left at room temperature for 15 minutes to obtain lipid nanoparticles (LNPs). A 12.5 μL sample was taken to detect encapsulation efficiency.

[0184] For the blank control group, transfection reagent: 65 μL of the prepared transfection mixture was added to 65 μL of Opti-MEM. The mixture was vortexed to ensure uniform mixing and left at room temperature for 15 minutes.

[0185] The prepared transfection reagent was added to a 24-well cell culture plate (100 μL per well) to achieve a final siRNA concentration of 0.07 nM in each well. 500 μL of cell suspension (1.5 × 10⁶ per mL) 5 (containing individual cells) was added. After homogeneous mixing using the crosswise method, the cells were placed in a cell incubator at 37°C and 5% CO2 and cultured for 40 hours.

[0186] 2.3 Detection of AGT mRNA 1) RNA extraction a. The culture medium was aspirated from the 12-well plate, and 0.5 mL of 1×PBS was added to each well to wash the cells, then the PBS was aspirated. 0.5 mL of TRIzol reagent was added to the wells, and the cells were completely lysed by blowing them out with a pipette tip. The cells were then transferred to 1.5 mL of RNase-free EP tubes and left at room temperature for 5 minutes.

[0187] 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 and 12000 × g for 15 minutes, and transfer 200 μL of the supernatant to a new EP tube.

[0188] c. Add the same volume of isopropanol, invert the tube to mix gently, let it stand at -20°C for 10 minutes, then centrifuge at 4°C and 12000×g for 15 minutes, and discard the supernatant. d. Add 0.5 mL of 75% ethanol and gently wash the RNA precipitate. Centrifuge at 12000 × g at 4°C for 5 minutes and remove the supernatant. Repeat rinsing once, centrifuge at 12000 × g at 4°C for 1 minute, and remove any remaining ethanol using a micropipette tip.

[0189] e. The residual ethanol was dried at room temperature for 2-3 minutes, and then dissolved in 40 μL of RNase-free ddH2O. 2) Detection of RNA concentration RNA concentration was measured using nanodrop. 2 μL of RNase-free ddH2O was used as a blank control, and 2 μL of RNA sample was used for detection each time. Sample concentrations were recorded.

[0190] 3) Reverse transcription of AGT mRNA The reverse transcription reagent, RNA solution and water were mixed at a volume ratio of 2:5:3, reacted at 37 °C for 15 minutes and 85 °C for 5 seconds in a PCR device, and finally maintained at 4 °C. The cDNA obtained after the reaction was diluted 5-fold with sterile enzyme-free water.

[0191] 4) Quantitative detection of AGT mRNA In a 15 mL centrifuge tube, qPCR reagent, upstream primer and downstream primer were added at a volume ratio of 5:0.1:0.1, mixed uniformly, and marked as solution A.

[0192] A 1.5 mL EP tube was marked, and the diluted cDNA and water were added at a volume ratio of 1:3.8 and mixed uniformly, marked as solution B. 5.2 μL of solution A + 4.8 μL of solution B were added to each well of a 96-well PCR plate. It was covered with a sealing film, centrifuged at 3000 rpm for 1 minute, and detected with an analyzer.

[0193] * This step was performed on ice to maintain a low temperature state. The plate was placed in a qPCR device and run with the following program. Initial denaturation: 95 °C, 30 seconds, Cycling reaction: 95 °C, 5 seconds; 60 °C, 34 seconds; 40 cycles, Melting curve: 95 °C, 15 seconds; 60 °C, 60 seconds; 95 °C, 15 seconds.

[0194] The running time was about 2 hours. The experimental results were analyzed and 2 -ΔΔCt was calculated. 2.4 Data processing Calculation formula for AGT mRNA expression rate (%): Expression rate = (AGT mRNA expression level / AGT mRNA expression level of the blank control group) × 100%, AGT gene expression inhibition rate = 100% - expression rate (%).

[0195] 3. Experimental results The inhibition rates in this example are the average values ​​of three experiments, and the inhibition rates of each sequence against AGT mRNA expression in HepG2 cells are shown in Tables 2-5.

[0196] The experimental results show that several sequences, including the B1789-AL sequence, which are alternatingly modified with 2'-OMe and 2'-F, have a significant inhibitory effect on AGT mRNA expression in HepG2 cells, with inhibition rates exceeding 45% for all of them. Among these, the inhibition rate of B1576s-AL exceeded 60%.

[0197] Other sequences showed weaker inhibitory effects on AGT mRNA expression, with inhibition rates all below 45%. (1) Of the 99 sequences designed, 33 showed a significant inhibitory effect on the AGT gene, with inhibition rates exceeding 40% for all of them. Among these, some sequences showed inhibition rates exceeding 60%.

[0198] [Table 7] TIFF2026059799000054.tif96165

[0199] As can be seen from Table 2, the alternating modification sequences in the above table (the specific sequences are shown in Table 1) had a significant inhibitory effect on AGT mRNA expression, with inhibition rates of 40% or more for all of them, and the top 10 sequences had inhibition rates exceeding 60% (Figure 1).

[0200] The sequences of the compounds in Table 2 are shown in Table 3 below.

[0201] [Table 8] TIFF2026059799000056.tif225165TIFF2026059799000057.tif224164TIFF2026059799000058.t if224164TIFF2026059799000059.tif225165TIFF2026059799000060.tif225165TIFF2026059799 000061.tif224164TIFF2026059799000062.tif225165TIFF2026059799000063.tif225164TIFF20 26059799000064.tif225165TIFF2026059799000065.tif225165TIFF2026059799000066.tif91164

[0202] (2) The inhibition rates of the 23 sequences against the AGT gene ranged from 25% to 40%. For example, the inhibition rates of B734-AL and B994-AL were 39.2% and 37.3%, respectively.

[0203] [Table 9]

[0204] The inhibitory effects of the 22 sequences shown in Table 4 on the AGT gene were weak, with inhibition rates all below 40%, ranging from 25% to 40%. For example, the inhibition rates of B734-AL and B994-AL were 39.2% and 37.3%, respectively (Figure 2).

[0205] (3) The inhibition rates of the 43 sequences against the AGT gene were 25% or less. For example, the inhibition rate of B1017-AL was only 23.1%.

[0206] [Table 10] TIFF2026059799000069.tif154165

[0207] The 43 sequences listed in Table 5 showed very low inhibitory effects on AGT mRNA expression, with inhibition rates of less than 25% for all of them. For example, the inhibition rate of B1017-AL was only 23.1% (Figures 3 and 4).

[0208] (4) Even siRNAs with similar sequences exhibited significantly different activities. For example, the inhibition rate of B1365-AL increased by 40.4% compared to B1367-AL, showing a significant improvement.

[0209] Table 6 shows a comparison of the inhibitory effects of siRNAs with similar sequences on AGT mRNA.

[0210] [Table 11]

[0211] As can be seen from Table 6, several siRNAs with similar sequences show very large differences in their inhibitory effects on AGT mRNA expression. Unmodified sequence 1367 differs from unmodified sequence 1365 by only two terminal bases. In the sense strand, the 5' end of unmodified sequence 58 is GA, and the 3' end of unmodified sequence 59 is GU, with the rest of the sequence being identical. In the antisense strand, the 3' end of unmodified sequence 144 is AC, and the 5' end of unmodified sequence 145 is AC, with the rest of the sequence being identical. However, the inhibition rate of the alternating modified sequence B1365-AL was significantly improved, increasing by 40.4% compared to B1367-AL.

[0212] Unmodified sequence 1815 differs from unmodified sequence 1816 by only one terminal base. In the sense strand, the 5' end of unmodified sequence 86 is U, the 3' end of sequence 2 is U, and the other sequences are identical. In the antisense strand, the 3' end of unmodified sequence 172 is G, the 5' end of unmodified sequence 15 is A, and the other sequences are identical. However, the inhibition rate of the alternating modified sequence B1816-AL was significantly improved, increasing by 30.5% compared to B1815-AL.

[0213] Unmodified sequence 729 differs from unmodified sequence 734s by only five terminal bases. In the sense strand, the 5' end of unmodified sequence 97 is AGAAC, and the 3' end of unmodified sequence 101 is GAAA, with the rest of the sequence being identical. In the antisense strand, the 3' end of unmodified sequence 183 is UCUGU, and the 5' end of unmodified sequence 187 is UUUC, with the rest of the sequence being identical. However, the inhibition rate of the alternatingly modified B734s-AL was significantly improved, increasing by 40.0% compared to B729-AL.

[0214] Therefore, selecting sequences with significant inhibitory activity from the vast number of oligonucleotide sequences designed to target AGT mRNA sequences is not easy and requires a great deal of creative work.

[0215] summary: (1) Of the 99 sequences designed, 33 showed a significant inhibitory effect on the AGT gene, with inhibition rates exceeding 40%. Of these, 10 showed inhibition rates exceeding 60%.

[0216] (2) 23 sequences showed inhibition rates of 25% to 40% against the AGT gene. For example, the inhibition rates of B734-AL and B994-AL were 39.2% and 37.3%, respectively.

[0217] (3) 43 sequences showed inhibition rates of 25% or less against the AGT gene. For example, the inhibition rate of B1017-AL was only 23.1%. (4) Even siRNAs with similar sequences exhibited significantly different activities. For example, the inhibition rate of B1365-AL increased by 40.4% compared to B1367-AL, representing a significant improvement. Therefore, selecting sequences with significant inhibitory activity from the vast number of oligonucleotide sequences designed to target AGT mRNA sequences is not easy and requires a great deal of creative work.

[0218] Example 3: Inhibitory effect of unmodified sequences on the AGT gene In this example, several unmodified sequences corresponding to the modified sequence in Example 2 were synthesized, transfected into HepG2 cells via lipid nanoparticles (LNPs), and the inhibitory effect of each unmodified sequence on the AGT gene was detected using qPCR technology, and the unmodified siRNA sequence exhibiting superior inhibitory effect was selected.

[0219] 1. Experimental materials Test sample: The unmodified siRNA sequences are shown in Table 7.

[0220] 2. Synthesis of 1579 unmodified sequences Table 7 shows the siRNA sequence for sequence number 1579: Sense chain: 5'-CCUUUUCUUCUAAUGAGUCGA-3' (Sequence ID 4) Antisense chain: 5'-UCGACUCAUUAGAAGAAAAGGUG-3' (Sequence ID 17) Equipment and reagents: Beijing Tsingke Biotech Co., Ltd. 192 P model DNA / RNA automated synthesizer, its solid-phase carrier is cross-linked polysulfide Chilenbees is a common carrier, and the model supports 5G as its primer. It's the Unylinker 350 (by Cytiva).

[0221] Manufacturing method: The following nucleotide monomer solutions were prepared in acetonitrile according to a monomer concentration of 0.15 M: DMT-A-2'-O-TBDMS phosphoramidite monomer (Formula 9), DMT-C-2'-O-TBDMS phosphoramidite monomer (Formula 10), DMT-G-2'-O-TBDMS phosphoramidite monomer (Formula 11), and DMT-U-2'-O-TBDMS phosphoramidite monomer (Formula 12).

[0222] [ka]

[0223] It was manufactured using the following steps. The solid-phase carrier is loaded into the designated position in the synthesis apparatus, and the corresponding fully protected product is obtained through several synthesis cycles. The synthesis cycles include (1) deprotection, (2) coupling, (3) oxidation / sulfidation, and (4) hydroxy protection. The cycle process and reagents used are as follows.

[0224] (1) Deprotection A 3% dichloroacetic acid-toluene solution was used as a deprotection reagent to remove the DMT protecting group, and then the sample was washed with acetonitrile.

[0225] (2) Coupling Each nucleotide monomer was coupled with an acetonitrile solution using 0.25 M 5-(ethylthio)-1H-tetrazole as an activator, and then washed with acetonitrile.

[0226] (3) Oxidation / Sulfidation Oxidation: Oxidation was performed using a 0.05 M iodine-pyridine / water (90 / 10) solution as the oxidizing agent, followed by washing with acetonitrile.

[0227] Sulfidation: Sulfidation was performed using a 3% pyridine solution of xanthan hydride as the sulfiding agent, followed by washing with acetonitrile. (4) Hydroxyprotection Hydroxyprotection was performed using a 10% anhydrous tetrahydrofuran solution (CAP A) and tetrahydrofuran / pyridine / N-methylimidazole 74 / 10 / 16 (v / v / v) (CAP B) as hydroxyprotection reagents, followed by washing with acetonitrile.

[0228] The above operation was repeated, and the cycle was performed according to the set sequence to obtain a fully protected product. (5) After removing the DMT protecting group of the last nucleotide using a 3% dichloroacetic acid-toluene solution as a deprotection agent, the nucleotides were washed with acetonitrile.

[0229] (6) Ammonium-mediated cracking and purification The solid-phase carrier was transferred to the reactor, concentrated aqueous ammonia (25-28%) was added, and ammonium lysis was maintained at 60°C for 12 hours. After cooling the reaction system to room temperature, the mixture was transferred to a filter press, rinsed with a mixed solution of purified water and ethanol, the filtrates were combined, passed through a chromatography column, concentrated, and freeze-dried to obtain a product protected with 2'-O-TBDMS.

[0230] (7) Discontinuation of TBDMS DMSO and triethylamine hydrofluoric acid were added to the obtained product, and the mixture was reacted at 60°C for 2 hours. Then, aqueous ammonium acetate solution was added to the reaction solution, and the mixture was shaken to mix homogeneously. Anhydrous ethanol was added, and the mixture was shaken to mix homogeneously. The mixture was then crystallized at -20°C for 8 to 12 hours. After centrifugation, the supernatant was discarded, and the precipitate was rinsed with anhydrous ethanol to obtain the unmodified single-chain product.

[0231] (8) Annealing The obtained unmodified sense strands and antisense strands were mixed in a 1:1 ratio, heated to 95°C and maintained at that temperature for 3 minutes, and then slowly cooled to room temperature to form unmodified double helix.

[0232] Purity of unmodified sequence 1579: 97.4%, measured molecular weight: 13990.9. 3. Combination of other sequences Other sequences listed in Table 7 were synthesized according to the method described above.

[0233] [Table 12] TIFF2026059799000073.tif219165TIFF2026059799000074.tif219165TIFF2026059799000075.tif219165TIFF20260597990 00076.tif218165TIFF2026059799000077.tif222164TIFF2026059799000078.tif223165TIFF2026059799000079.tif162165

[0234] Cell type: Hepatoma cell line HepG2 cells. Drug solvent: Sterilized enzyme-free water, Gibco Opti-MEM. 4. Experimental Method The experimental steps and siRNA concentrations were as described in Example 2.

[0235] 5. Experimental Results The inhibition rates of each unmodified sequence against AGT mRNA expression are shown in Tables 8 and 9.

[0236] Unmodified sequences 1576s, 1578s, 1838, 1835, 1816, 1812, 1579, 1836, 1789, 1839, 1791, 1795, and 1585s showed significant inhibitory effects on AGT mRNA expression in HepG2 cells, with inhibition rates of 45% or higher for all of them. Among these, the inhibition rates of 1576s, 1578s, 1838, 1835, 1812, 1579, and 1789 exceeded 50%. A comparison of the inhibitory effects of the unmodified sequences in this disclosure and sequences of the prior art can be found in Table 29.

[0237] (1) 13 unaccounted for, including 1576s, 1578s, 1838, 1835, 1816, 1812, 1579, 1836, 1789, 1839, 1791, 1795 and 1585s The modified sequences had a significant inhibitory effect on the AGT gene, with inhibition rates exceeding 45% in all cases. Among these, inhibition of 1576s, 1578s, 1838, 1835, 1812, 1579, and 1789 exceeded 50%.

[0238] [Table 13]

[0239] Each of the unmodified sequences listed in Table 8, including 1576s, 1578s, 1838, 1835, 1816, 1812, 1579, 1836, 1789, 1839, 1791, 1795, and 1585s, had a significant inhibitory effect on AGT mRNA expression, with inhibition rates exceeding 45% for all of them. Among these, inhibition of 1576s, 1578s, 1838, 1835, 1812, 1579, and 1789 exceeded 50% (Figure 5).

[0240] (2) The inhibition rates of some unmodified formulations of the AGT gene were all less than 45%. For example, the inhibition rates of 731 and 1011 were only 1.3% and 2.1%, respectively.

[0241] [Table 14] TIFF2026059799000082.tif78165

[0242] Each sequence in Table 9 showed very low inhibitory effects on AGT mRNA expression, with inhibition rates of less than 45% for all. For example, the inhibition rates for 731 and 1011 were only 1.3% and 2.1%, respectively (Figure 5).

[0243] (3) Even siRNAs with similar unmodified sequences exhibited significantly different activity. For example, the inhibition rate of unmodified sequence 1812 increased by 32.3% compared to that of unmodified sequence 1810, showing a significant improvement.

[0244] Table 10 shows a comparison of the inhibitory effects of several siRNAs with similar sequences on AGT mRNA.

[0245] [Table 15] TIFF2026059799000084.tif77165

[0246] As can be seen from Table 10, several siRNAs with similar sequences have a very strong inhibitory effect on AGT mRNA expression. Unmodified sequence 1608 differs from unmodified sequence 1612 by only four terminal bases. In the sense strand, the 5' end of unmodified sequence 76 is UGGA, and the 3' end of unmodified sequence 77 is UGGU, with the rest of the sequence being identical. In the antisense strand, the 3' end of unmodified sequence 162 is CAGC, and the 5' end of unmodified sequence 163 is ACCA, with the rest of the sequence being identical. However, the inhibition rate of unmodified sequence 1608 was significantly improved, increasing by 29.7% compared to 1612.

[0247] Unmodified sequence 2025 differs from unmodified sequence 2030 by only six terminal bases. For the sense strand, the 5' end of unmodified sequence 92 is GUUAU and the 3' end is A, and the 3' end of sequence 93 is GUAAUA, with the rest of the sequence being exactly the same. For the antisense strand, the 3' end of unmodified sequence 178 is AACCA and the 5' end is U, and the 5' end of unmodified sequence 179 is UAUUAC, with the rest of the sequence being exactly the same. However, the inhibition rate of unmodified sequence 2030 was significantly improved, increasing by 29.1% compared to 2025.

[0248] Unmodified sequence 731 differs from unmodified sequence 734 by only three terminal bases. In the sense strand, the 5' end of unmodified sequence 98 is AAC, and the 3' end of unmodified sequence 100 is AGA, with the rest of the sequence being identical. In the antisense strand, the 3' end of unmodified sequence 184 is UCU, and the 5' end of unmodified sequence 186 is UCU, with the rest of the sequence being identical. However, the inhibition rate of unmodified sequence 734 was significantly improved, increasing by 41.7% compared to 731.

[0249] Unmodified sequence 1812 differs from unmodified sequence 1810 by only two terminal bases. In the sense strand, the 3' end of unmodified sequence 3 is AA, and the 5' end of unmodified sequence 83 is UC; the rest of the sequence is identical. In the antisense strand, the 5' end of unmodified sequence 16 is UU, and the 3' end of unmodified sequence 169 is AC; the rest of the sequence is identical. However, the inhibition rate of unmodified sequence 1812 was significantly improved, increasing by 32.3% compared to 1810.

[0250] Therefore, there are a great many oligonucleotides designed to target AGT mRNA sequences. Selecting unmodified sequences with significant inhibitory activity from cytoplasmic sequences is not easy and requires a great deal of creative work.

[0251] (4) The effect on activity improvement after alternating modification of different sequences was inconsistent. In some cases, the inhibition rate was significantly improved; for example, in the unmodified sequence 731, the inhibition rate of the alternating modification increased by 25.1% compared to the inhibition rate of the unmodified sequence. In some cases, the improvement was not significant; for example, in the unmodified sequences 994, 1279, and 1591, the inhibition rates of the alternating modified sequence and the unmodified sequence were essentially unchanged.

[0252] [Table 16] TIFF2026059799000086.tif227164TIFF2026059799000087.tif184164

[0253] Table 11 shows that the effect of alternating modifications of different sequences on AGT mRNA inhibition rates is not entirely consistent. Some showed significant improvement; for example, the inhibition rate of alternating modification increased by 25.1% compared to the unmodified sequence in the unmodified sequence 731. Some did not show significant improvement; for example, the inhibition rates of alternating and unmodified sequences were essentially unchanged in the unmodified sequences 994, 1279, and 1591.

[0254] Therefore, not all unmodified sequences can significantly improve their activity after alternating modification, and the effect of alternating modification on the activity of different sequences is inconsistent. summary: (1) Thirteen unmodified gene sequences, including 1576s, 1579, 1812, 1578s, 1789, 1835, 1838, 1795, 1585s, 1816, 1791, 1836, and 1839, showed significant inhibitory effects on the AGT gene, with inhibition rates exceeding 45% in all cases. Among these, 576s, 1578s, 1812, 1579, 1789, 1835, and 18 The inhibition rate for 38 exceeded 50%.

[0255] (2) Other unmodified sequences inhibited the AGT gene by less than 45%. For example, the inhibition rates of 731 and 1011 were only 1.3% and 2.1%, respectively. (3) Even siRNAs with similar sequences exhibited significantly different activity. For example, the inhibition rate of unmodified sequence 734 increased by 41.7% compared to unmodified sequence 731, showing a significant improvement.

[0256] (4) The effects on activity after alternating modification of different sequences were inconsistent. Some inhibition rates were significantly improved; for example, the inhibition rate of the unmodified sequence 731 increased by 25.1% with alternating modification compared to the inhibition rate of the unmodified sequence. Some were not significant; for example, the inhibition rates of the unmodified sequences 994, 1279, and 1591 were essentially unchanged compared to the alternating modified and unmodified sequences.

[0257] Example 4: Inhibitory effect of template-modified sequences on the AGT gene In this embodiment, the sequences selected in Example 3, namely the unmodified sequences 1576s, 1578s, 1838, 1835, 1816, 1812, 1579, 1836, 1789, 1839, 1791, 1795, and 1585s, a total of 13 sequences, were modified using modification templates. Among these, DV25P, DV26P, DV27P, DV28P, DV29P, DV32P, DV33P, and DV34P are novel modification templates designed in this disclosure, while DV22 is a previously disclosed Advanced ESC modification template (Foster, DJ, et al. (2018). “Advanced siRNA Designs Further Improve In Vivo Performance of GalNAc-siRNA Conjugates.” Mol Ther 26(3): 708-717.)

[0258] Natural 5'-terminus phosphorylation or simple direct 5'-terminus phosphorylation can be dephosphorylated intracellularly; oligonucleotide chains with directly phosphorylated 5' terminus are 90% dephosphorylated after 2 hours of circulation in the blood and completely disappear after 24 hours. The 5'-terminus phosphorylation design (5'-E-VP) uses E-vinyl phosphonate to replace the crosslinking oxygen, resulting in improved phosphorylation effect and stability. The 5' terminus of the antisense chains of the modified templates DV25-29P and DV32-34P in this disclosure all had the 5'-E-VP phosphorylation design.

[0259] 1. Experimental materials Test sample: Table 12 shows the sequences 1576s, 1578s, 1838, 1835, 1816, 1812, 1579, 1836, 1789, 1839, 1791, 1795, and 1585s of Example 3 modified using different templates (DV25P, DV26P, DV27P, DV28P, DV29P, DV30P, DV32P, DV33P, DV34P, and DV22), and the corresponding sequences. The alternating modification sequences B1576s-AL, B1578s-AL, B1838-AL, B183B1576s-AL, B1578s-AL, B1838-AL, B1835-AL, B1816-AL, B1812-AL, B1579-AL, B1836-AL, B1789-AL, B1839-AL, B1791-AL, B1795-AL, and B1585s-AL (synthesized in Example 1) are shown.

[0260] Array composition: Referring to Example 1, when synthesizing the siRNA sequence and the 5' terminal base (the last base) of the antisense strand, a monomer containing a phosphate group at the 5' end was used, for example, vinyl-(E)-phosphonate-A-OMe phosphoramidite monomer (Formula 9) and vinyl-(E)-phosphonate-U-OMe phosphoramidite monomer (Formula 10). Its structure is as shown below.

[0261] [ka]

[0262] The modification principle of the modification template in this disclosure is as follows: DV22, DV25-34P: The antisense chain was modified using one of the modification methods shown in Table 46.

[0263] [Table 17] TIFF2026059799000090.tif227164TIFF2026059799000091.tif227164TIFF2026059799000092.tif227164TIFF2026059799000093.tif27164

[0264] The sense chain was modified using one of the modification methods shown in Table 47.

[0265] [Table 18] TIFF2026059799000095.tif53164

[0266] In the table above, PS represents the phosphorothioate backbone. We named the siRNA modification template DV25P, which used modification method A for the antisense strand and modification method a for the sense strand.

[0267] We named the siRNA modification template DV26P, which used modification method B for the antisense strand and modification method a for the sense strand. The siRNA modification template, which uses modification method C for the antisense strand and modification method a for the sense strand, was named DV27P.

[0268] The siRNA modification template, which uses modification method B for the antisense strand and modification method b for the sense strand, was named DV28P. We named the siRNA modification template DV29P, which used modification method C for the antisense strand and modification method b for the sense strand.

[0269] The siRNA modification template, which uses modification method D for the antisense strand and modification method b for the sense strand, was named DV32P. We named the siRNA modification template DV33P, which used modification method E for the antisense strand and modification method b for the sense strand.

[0270] We named the siRNA modification template DV34P, which used modification method F for the antisense strand and modification method b for the sense strand. We named the siRNA modification template DV22, which used modification method G for the antisense strand and modification method c for the sense strand.

[0271] For compounds where the antisense and antisense chains each consist of 20 / 22 bases, one of the modification methods shown in Table 44 was used for the antisense chain.

[0272] [Table 19] TIFF2026059799000097.tif225164TIFF2026059799000098.tif147164

[0273] The sense strand was modified using one of the modification methods shown in Table 48.

[0274] [Table 20]

[0275] In the table above, 2'-OMe is 2'-methoxy, 2'-F is 2'-fluoro, and PS is phosphorothioate backbone. We named the siRNA modification template DV25P, which used modification method A for the antisense strand and modification method a for the sense strand.

[0276] We named the siRNA modification template DV26P, which used modification method B for the antisense strand and modification method a for the sense strand. The siRNA modification template, which uses modification method C for the antisense strand and modification method a for the sense strand, was named DV27P.

[0277] The siRNA modification template, which uses modification method B for the antisense strand and modification method b for the sense strand, was named DV28P. We named the siRNA modification template DV29P, which used modification method C for the antisense strand and modification method b for the sense strand.

[0278] The siRNA modification template, which uses modification method D for the antisense strand and modification method b for the sense strand, was named DV32P. We named the siRNA modification template DV33P, which used modification method E for the antisense strand and modification method b for the sense strand.

[0279] We named the siRNA modification template DV34P, which used modification method F for the antisense strand and modification method b for the sense strand. Details of each template modification sequence are shown in Table 12, and the synthesis method for each sequence is the same as in Example 1.

[0280] [Table 21] TIFF2026059799000101.tif231164TIFF2026059799000102.tif231164TIFF2026059799000103.tif225164TIFF2026059799000104.tif229164TIFF2026059799000105.tif227164TIFF2026059799000106.tif231164TIFF2026059799000107.tif231164TIFF2026059799000108.tif225164TIFF2026059799000109.tif231164TIFF2026059799000110.tif220164TIFF2026059799000111.tif229164TIFF2026059799000112.tif229164TIFF2026059799000113.tif229164TIFF2026059799000114.tif229165TIFF2026059799000115.tif228164TIFF2026059799000116.tif231164TIFF2026059799000117.tif231164TIFF2026059799000118.tif231164TIFF2026059799000119.tif231164TIFF2026059799000120.tif219164TIFF2026059799000121.tif230164TIFF2026059799000122.tif229164TIFF2026059799000123.tif230164TIFF2026059799000124.tif230165TIFF2026059799000125.tif226164TIFF2026059799000126.tif230164TIFF2026059799000127.tif229163TIFF2026059799000128.tif230163TIFF2026059799000129.tif218164TIFF2026059799000130.tif230164TIFF2026059799000131.tif230164TIFF2026059799000132.tif231164TIFF2026059799000133.tif230164TIFF2026059799000134.tif230164TIFF2026059799000135.tif186164.

[0281] Cell type: HepG2 cell line Drug solvent: Sterilized enzyme-free water, Gibco Opti-MEM. 2. Experimental Method QRT-PCR was used to detect the inhibitory effect of the test sample on AGT gene mRNA expression in the HepG2 cell line.

[0282] 2.1 Cell Culture HepG2 cell lines were extracted from the subcultured cells, and cells in the logarithmic growth phase were cultured in 10% fetal bovine serum DMEM medium (with 100 μL / mL penicillin and streptomycin added, respectively). The cells were placed in a cell incubator at 37°C with 5% CO2, and the medium was changed once daily. The cells were digested with 0.25% trypsin, subcultured, and centrifuged at 1000 r / min for 5 minutes. The supernatant was discarded, and fresh medium was added for subculture.

[0283] 2.2 Cell transfection Preparation of transfection mixture: Lipofectamine RNAiMAX and Opti-MEM were mixed in a 2:98 ratio and vortexed to ensure uniform mixing.

[0284] Preparation of transfection reagent: A siRNA solution diluted with 60 μL of Opti-MEM was added to 60 μL of transfection mixture in a 1:1 (v / v) ratio, and the mixture was homogeneously mixed by vortexing. The mixture was left at room temperature for 15 minutes to obtain lipid nanoparticles (LNPs). A 12.5 μL sample was taken to detect the encapsulation efficiency.

[0285] For the blank control group, transfection reagent: 60 μL of the prepared transfection mixture was added to 60 μL of Opti-MEM. The mixture was vortexed to ensure uniform mixing and left at room temperature for 15 minutes.

[0286] The prepared transfection reagent was added to a 24-well cell culture plate (100 μL per well) to achieve a final siRNA concentration of 0.02 nM in each well. 500 μL of cell suspension (1.5 × 10⁶ per mL) 5 (containing individual cells) was added. After homogeneous mixing using the crosswise method, the cells were placed in a cell incubator at 37°C and 5% CO2 and cultured for 40 hours.

[0287] 2.3 AGT mRNA detection The steps are the same as in "2.3 AGT mRNA detection" in Example 2. 2.4 Data Processing Formula for calculating AGT mRNA expression rate (%): Expression rate = (AGT mRNA expression level / AGT mRNA expression level in the blank control group) × 100% AGT gene expression inhibition rate = 100% - expression rate (%).

[0288] 2.5 IC 50 experiment In this experiment, the EC of each sequence 50 The experimental siRNA concentration was started at 1.0 nM and diluted fourfold to a total of eight concentration points (1.0 nM, 0.25 nM, 0.0625 nM, 15.6 pM, 3.9 pM, 0.977 pM, 0.244 pM, and 0.061 pM). The inhibition rate of each sequence at each concentration was measured and plotted, and the IC of each sequence was calculated. 50 The concentration was calculated.

[0289] 3. Experimental Results The three experiments were repeated, and the inhibition rates of each modification sequence against the AGT gene in HepG2 cells are shown in Tables 13-25.

[0290] Activity detection experiments revealed that 13 candidate sequences modified with the modification templates DV25P-29P and DV32P-34P designed in this disclosure (unmodified sequences 576s, 1578s, 1838, 1835, 1816, 1812, 1579, 1836, 1789, 1839, 1791, 1795, and 1585s) had a significant inhibitory effect on the AGT gene, with inhibition rates of 40% or higher for all of them. Among these, the inhibition rates for C1812-DV25P, C1579-DV33P, and C1789-DV26P reached 74.4%, 74.0%, and 73.6%, respectively.

[0291] Sequences modified using the modification templates DV25P-29P and DV32P-34P of this disclosure showed significantly improved inhibition rates of AGT gene expression compared to alternatingly modified sequences. For example, the inhibition rate of sequence C1579-DV33, which was modified from unmodified sequence 1579 using template DV33P, increased by 12.9% compared to alternatingly modified sequences, and the inhibition rate of sequence C1812-DV25P, which was modified from unmodified sequence 1812 using template DV25P, increased by 12.9%. This increased by 11.5% compared to the alternating modification sequence.

[0292] Furthermore, the activity of the same siRNA sequence after modification with different modification templates varied significantly. For example, when the unmodified sequence 1579 was modified using the modification template DV33P of this disclosure, the inhibition rate increased by 29.8% compared to the sequence modified using the modification template DV28P of this disclosure. Compared to the sequence modified using the conventionally disclosed Advanced ESC template DV22, the inhibition rate increased by 18.4%.

[0293] I C 50 The experiment involved the IC of an array modified using the modification template designed in this disclosure. 50 The values ​​were shown to be between 0.0019 nM and 0.0615 nM. For example, the ICs C1789-DV25P, C1812-DV25P, and C1789-DV26P. 50The values ​​were 0.0019 nM, 0.0022 nM, and 0.0026 nM, respectively. These sequences were able to effectively inhibit AGT gene expression at low concentrations.

[0294] (i) Inhibitory effect on each unmodified sequence of the AGT gene after modification with a template. (1) Unmodified sequence 1576s

[0295] [Table 22]

[0296] I. DV25P-29P template of this disclosure When the unmodified sequence 1576s was modified using the modification templates DV25P, DV26P, and DV29P designed in this disclosure, the inhibition rate against the AGT gene exceeded 70% in all cases, and compared to alternating methoxy and fluoromodification sequences at the 2' position, the AGT gene The inhibition rate against was significantly improved. For example, after modification using templates DV25P and DV29P, the inhibition rates of C1576s-DV25P and C1576s-DV29P increased by 8.7% and 14.7%, respectively.

[0297] II. Prior art disclosed the Advanced ESC template DV22 (fluoro moieties: antisense chain positions 2, 6, 14 and 16, sense chain positions 7, 9, 10 and 11).

[0298] After modifying the unmodified sequence 1576s with template DV22, the inhibition rate of C1576s-DV22 against the AGT gene was 63.9%, which was 0.7% lower than the inhibition rate of the alternating modification sequence.

[0299] By modifying the sequences using the modification templates DV25P and DV29P of this disclosure, the inhibition rates of C1576s-DV25P and C1576s-DV29P were significantly improved, increasing by 9.4% and 15.4%, respectively, compared to C1576s-DV22.

[0300] This reveals the following: 1) Sequences modified using the modification templates DV25-29 of this disclosure, in which the unmodified sequence 1576s was modified, showed a significantly improved inhibitory effect on the AGT gene compared to sequences with alternating modifications. For example, sequences modified using DV26, in which the unmodified sequence 1576s was modified, showed an 8.8% increase in inhibition compared to sequences with alternating modifications.

[0301] 2) Even with the same siRNA sequence, modification with different modification templates resulted in significantly different activity. For example, sequences modified using the modification templates DV25P and DV29P of this disclosure of the unmodified sequence 1576s showed a significant improvement, with inhibition rates increasing by up to 20.5% compared to sequences modified using the modification template DV28P of this disclosure. Compared to sequences modified using the conventionally disclosed Advanced ESC template DV22, the inhibition rates increased by up to 15.4%, also showing a significant improvement.

[0302] 3) The activity of siRNA sequences modified with different templates varied significantly, with differences of up to 20.5%. Therefore, it remains unclear which template modification can produce high activity in siRNA sequences.

[0303] (2) Unmodified sequence 1578s

[0304] [Table 23]

[0305] I. DV25P-29P template of this disclosure When the unmodified sequence 1578s was modified using the modification templates DV25P, DV27P, and DV29P designed in this disclosure, the inhibition rate against the AGT gene exceeded 70% in all cases, and the inhibition rate against the AGT gene was significantly improved compared to sequences with alternating methoxy and fluoro modifications at the 2' position. For example, after modification using templates DV25P and DV29P, the inhibition rates of C1578s-DV25P and C1578s-DV29P increased by 11.9% and 13.2%, respectively.

[0306] II. Prior art disclosed the Advanced ESC template DV22 (fluoro moieties: antisense chain positions 2, 6, 14 and 16, sense chain positions 7, 9, 10 and 11).

[0307] After modifying the unmodified sequence 1578s with template DV22, the inhibition rate of C1578s-DV22 against the AGT gene was 61.1%, which was 2.8% lower compared to the inhibition rate of the alternating modification sequence.

[0308] By modifying the sequences using the modification templates DV25P and DV29P of this disclosure, the inhibition rates of C1578s-DV25P and C1578s-DV29P were significantly improved, increasing by 14.7% and 16.0%, respectively, compared to C1578s-DV22.

[0309] This reveals the following: 1) The unmodified sequence 1578s modified using the modification templates DV25P-29P of this disclosure showed a significantly improved inhibitory effect on the AGT gene compared to the alternatingly modified sequence. For example, the sequence 1578s modified using DV27P showed a 9.2% increase in inhibition compared to the alternatingly modified sequence.

[0310] 2) Even with the same siRNA sequence, modification with different modification templates resulted in significantly different activity. For example, sequences modified using the modification templates DV25P and DV29P of this disclosure of the unmodified sequence 1578s showed a significant improvement, with inhibition rates increasing by up to 20.2% compared to sequences modified using the modification template DV28P of this disclosure. Compared to sequences modified using the conventionally disclosed Advanced ESC template DV22, the inhibition rates increased by up to 16.0%, also showing a significant improvement.

[0311] 3) The activity of siRNA sequences modified with different templates varied significantly, with differences of up to 20.2%. Therefore, it remains unclear which template modification can produce high activity in siRNA sequences.

[0312] (3) Unmodified sequence 1838

[0313] [Table 24]

[0314] I. DV25P-29P template of this disclosure When the unmodified sequence 1838 was modified using the modification templates DV27P and DV29P designed in this disclosure, the inhibition rate against the AGT gene exceeded 70% in both cases, and the 2' position Compared to alternating methoxy and 2' fluoromodification sequences, the inhibition rate against the AGT gene was significantly improved. For example, after modification with templates DV27P and DV29P, the inhibition rates of C1838-DV27P and C1838-DV29P increased by 8.7% and 12.0%, respectively.

[0315] II. Prior art disclosed the Advanced ESC template DV22 (fluoro moieties: antisense chain positions 2, 6, 14 and 16, sense chain positions 7, 9, 10 and 11).

[0316] After modifying the unmodified sequence 1838 with template DV22, the inhibition rate of C1838-DV22 against the AGT gene was 62.7%, which was 0.9% lower compared to the inhibition rate of the alternating modification sequence.

[0317] Sequences modified using the modification templates DV27P and DV29P of this disclosure showed significant improvements, with inhibition rates increased by 9.6% and 12.9%, respectively, compared to C1838-DV27P and C1838-DV29P.

[0318] This reveals the following: 1) Sequences modified using the modification templates DV25P-29P of this disclosure, in which the unmodified sequence 1838 was modified, showed a significantly improved inhibitory effect on the AGT gene compared to sequences with alternating modifications. For example, sequences modified using DV27P in which the unmodified sequence 1838 was modified showed an 8.7% increase in inhibition compared to sequences with alternating modifications.

[0319] 2) Even with the same siRNA sequence, modification with different modification templates resulted in significantly different activity. For example, sequences modified using the modification templates DV27P and DV29P of this disclosure of the unmodified sequence 1838 showed a significant improvement, with inhibition rates increasing by up to 15.3% compared to sequences modified using the modification template DV28P of this disclosure. Compared to sequences modified using the conventionally disclosed Advanced ESC template DV22, the inhibition rates increased by up to 12.9%, also showing a significant improvement.

[0320] 3) The activity of siRNA sequences modified with different templates varied significantly, with differences of up to 15.3%. Therefore, it remains unclear which template modification can produce high activity in siRNA sequences.

[0321] (4) Unmodified sequence 1579

[0322] [Table 25]

[0323] I. DV25P-29P template of this disclosure When the unmodified sequence 1579 was modified using the modification templates DV25P, DV26P, and DV29P designed in this disclosure, the inhibition rate against the AGT gene exceeded 70% in all cases, and the inhibition rate against the AGT gene was significantly improved compared to sequences with alternating methoxy and fluoro modifications at the 2' position. For example, after modification with templates DV25P and DV29P, the inhibition rates of C1579-DV25P and C1579-DV29P increased by 11.1% and 10.6%, respectively (Figure 7).

[0324] II. Other Qualifying Templates of the Disclosure After modifying the unmodified sequence 1579 with templates DV32P, DV33P, and DV34P, the inhibition rates of C1579-DV32P, C1579-DV33P, and C1579-DV34P against the AGT gene were 68.9%, 74.0%, and 63.9%, respectively, representing increases of 7.8%, 12.9%, and 2.8% compared to the corresponding alternating modification sequences.

[0325] The inhibition rates of sequences C1579-DV25P and C1579-DV29P modified using the modification templates DV25P and DV29P of this disclosure were increased by 3.3% and 2.8% compared to C1579-DV32P, decreased by 1.8% and 2.3% compared to C1579-DV33P, and increased by 8.3% and 7.8% compared to C1579-DV34P.

[0326] III. Prior art disclosed the Advanced ESC template DV22 (fluoro moieties: antisense chain positions 2, 6, 14 and 16, sense chain positions 7, 9, 10 and 11).

[0327] After modifying the unmodified sequence 1579 with template DV22, the inhibition rate of C1579-DV22 against the AGT gene was 55.6%, which was 5.5% lower than the inhibition rate of the alternating modification sequence.

[0328] The inhibition rates of sequences C1579-DV25P and C1579-DV29P modified using the modification templates DV25P and DV29P of this disclosure were significantly improved, increasing by 16.6% and 16.2%, respectively, compared to C1579-DV22.

[0329] This reveals the following: 1) Sequences modified using the modification template DV25-29P of this disclosure, in which the unmodified sequence 1579 was modified, showed a significantly improved inhibitory effect on the AGT gene compared to sequences with alternating modifications. For example, sequences modified using DV26P in which the unmodified sequence 1579 was modified showed a 9.7% increase in inhibition compared to sequences with alternating modifications.

[0330] 2) Even with the same siRNA sequence, modification with different modification templates resulted in significantly different activity. For example, sequences modified using the modification templates DV25P and DV29P of this disclosure of the unmodified sequence 1579 showed a significant improvement, with the inhibition rate increasing by up to 28.0% compared to sequences modified using the modification template DV28P of this disclosure. Compared to sequences modified using the conventionally disclosed Advanced ESC template DV22, the inhibition rate increased by up to 16.6%, also showing a significant improvement.

[0331] 3) The activity of siRNA sequences modified with different templates varied significantly, with differences of up to 28.0%. Therefore, it remains unclear which template modification can produce high activity in siRNA sequences.

[0332] (5) Unmodified sequence 1585s

[0333] [Table 26]

[0334] I. DV26P-29P template of this disclosure When the unmodified sequence 1585s was modified using the modification templates DV26P, DV27P, and DV29P designed in this disclosure, the inhibition rate against the AGT gene exceeded 50%, and the inhibition rate against the AGT gene was significantly improved compared to sequences with alternating methoxy and fluoro modifications at the 2' position. For example, after modification using templates DV26P and DV27P, the inhibition rates of C1585s-DV26P and C1585s-DV27P increased by 3.3% and 5.3%, respectively.

[0335] II. Other Qualifying Templates of the Disclosure After modifying the unmodified sequence 1585s with templates DV32P and DV34P, the inhibition rates of C1585s-DV32P and C1585s-DV34P against the AGT gene were 61.8% and 54.1%, respectively, which were increased by 2.5% and -5.2% compared to the inhibition rates of the corresponding alternating modification sequences.

[0336] The inhibition rates of sequences C1585s-DV26P and C1585s-DV27P modified using the modification templates DV26P and DV27P of this disclosure increased by 0.8% and 2.8% compared to C1585s-DV32P, and by 8.5% and 0.5% compared to C1585s-DV34P.

[0337] III. Prior art disclosed the Advanced ESC template DV22 (fluoro moieties: antisense chain positions 2, 6, 14 and 16, sense chain positions 7, 9, 10 and 11).

[0338] After modifying the unmodified sequence 1585s with template DV22, the inhibition rate of C1585s-DV22 against the AGT gene was 55.2%, which was 4.1% lower compared to the inhibition rate of the alternating modification sequence.

[0339] The inhibition rates of sequences C1585s-DV26P and C1585s-DV27P modified using the modification templates DV26P and DV27P of this disclosure were significantly improved, increasing by 7.4% and 9.4%, respectively, compared to C1585s-DV22.

[0340] This reveals the following: 1) The unmodified sequence 1585s modified using the modification templates DV26P-29P of this disclosure showed a significantly improved inhibitory effect on the AGT gene compared to the alternatingly modified sequence. For example, the sequence 1585s modified using DV27P showed a 5.3% increase in inhibition compared to the alternatingly modified sequence.

[0341] 2) Even with the same siRNA sequence, modification with different modification templates resulted in significantly different activity. For example, the unmodified sequence 51 modified using the modification template DV27P of this disclosure showed a significant improvement, with the inhibition rate increasing by up to 15.1% compared to using the modification template DV28P of this disclosure. Compared to sequences modified using the ESC template DV22, the inhibition rate increased by up to 9.4%, showing a significant improvement.

[0342] 3) The activity of siRNA sequences modified with different templates varied significantly, with differences of up to 15.1%. Therefore, it remains unclear which template modification can produce high activity in siRNA sequences.

[0343] (6) Unmodified sequence 1789

[0344] [Table 27]

[0345] I. DV25P-29P template of this disclosure When the unmodified sequence 1789 was modified using the modification templates DV25P-29P designed in this disclosure, the inhibition rate against the AGT gene exceeded 60%, and the inhibition rate against the AGT gene was significantly improved compared to sequences with alternating methoxy and fluoro modifications at the 2' position. For example, after modification with templates DV25P and DV26P, the inhibition rates of C1789-DV25P and C1789-DV26P increased by 5.5% and 10.1%, respectively.

[0346] II. Other Qualifying Templates of the Disclosure After modifying the unmodified sequence 1789 with templates DV32P–DV34P, the inhibition rates of C1789-DV32P, C1789-DV33P, and C1789-DV34P against the AGT gene were 61.8%, 57.9%, and 54.1%, respectively, which were 1.7%, 5.6%, and 6.6% lower, respectively, than the inhibition rates of the corresponding alternating modification sequences.

[0347] The inhibition rates of sequences C1789-DV25P and C1789-DV26P modified using the modification templates DV25P and DV26P of this disclosure were increased by 7.2% and 11.8%, respectively, compared with C1789-DV32P; increased by 11.1% and 15.7%, respectively, compared with C1789-DV33P; and increased by 12.1% and 16.7%, respectively, compared with C1789-DV34P.

[0348] III. Prior art disclosed the Advanced ESC template DV22 (fluoro moieties: antisense chain positions 2, 6, 14 and 16, sense chain positions 7, 9, 10 and 11).

[0349] After modifying the unmodified sequence 1789 with template DV22, the inhibition rate of C1789-DV22 against the AGT gene was 60.3%, which was 3.2% lower compared to the alternating modification sequence.

[0350] The inhibition rates of sequences C1789-DV25P and C1789-DV26P modified using the modification templates DV25P and DV26P of this disclosure were significantly improved, increasing by 8.7% and 13.3%, respectively, compared to C1789-DV22.

[0351] This reveals the following: 1) The unmodified sequence 1789 modified using the modification templates DV25P-29P of this disclosure showed a significantly improved inhibitory effect on the AGT gene compared to the alternatingly modified sequence. For example, modifying the unmodified sequence 81 using DV26P increased the inhibition rate by 10.1% compared to the alternatingly modified sequence.

[0352] 2) Even with the same siRNA sequence, modification with different modification templates resulted in significantly different activity. For example, sequences modified using the modification templates DV25P and DV26P of this disclosure of the unmodified sequence 1789 showed a significant improvement in inhibition rate, up to 16.7%, compared to sequences modified using the modification templates DV32P-34P of this disclosure. Compared to sequences modified using the conventionally disclosed Advanced ESC template DV22, the inhibition rate increased by up to 13.3%, also showing a significant improvement.

[0353] 3) The activity of siRNA sequences modified with different templates varied significantly, with differences of up to 16.7%. Therefore, it remains unclear which template modification can produce high activity in siRNA sequences.

[0354] (7) Unmodified sequence 1791

[0355] [Table 28]

[0356] I. DV26P-29P template of this disclosure When the unmodified sequence 1791 was modified using the modification templates DV26P-29P designed in this disclosure, the inhibition rate against the AGT gene exceeded 55%, which was a significant improvement in inhibition rate against the AGT gene compared to sequences with alternating methoxy and fluoro modifications at the 2' position. For example, after modification with templates DV26P and DV28P, the inhibition rates of C1791-DV26P and C1791-DV28P increased by 8.5% and 2.8%, respectively.

[0357] II. Other Qualifying Templates of the Disclosure After modifying the unmodified sequence 1791 with templates DV32P and DV34P, the inhibition rates of C1791-DV32P and C1791-DV34P against the AGT gene were 65.9% and 66.7%, respectively, which were 4.1% and 4.9% higher, respectively, compared to sequences with alternating methoxy and fluoro modifications at the 2' position.

[0358] The inhibition rate of the sequence C1791-DV26P modified using the modification template DV26P of this disclosure was increased by 4.4% compared to C1791-DV32P and by 3.6% compared to C1791-DV34P.

[0359] This reveals the following: 1) The unmodified sequence 1791 modified using the modification template DV26-29P of this disclosure showed a significantly improved inhibitory effect on the AGT gene compared to the alternatingly modified sequence. For example, the sequence 1791 modified using DV26P showed an 8.5% increase in inhibition compared to the alternatingly modified sequence.

[0360] 2) Even with the same siRNA sequence, modification with different modification templates resulted in significantly different activity. For example, the unmodified sequence 1791 modified using the modification template DV26P of this disclosure showed a 10.9% increase in inhibition rate, a significant improvement, compared to the sequence modified using the modification template DV29P of this disclosure.

[0361] 3) The activity of siRNA sequences modified with different templates varied significantly, with differences of up to 10.9%. Therefore, it remains unclear which template modification can produce high activity in siRNA sequences.

[0362] (8) Unmodified sequence 1795

[0363] [Table 29]

[0364] I. Template DV25-29P of this disclosure When the unmodified sequence 1795 was modified using the modification templates DV26-29P designed in this disclosure, the inhibition rate against the AGT gene exceeded 50%, and the inhibition rate against the AGT gene was significantly improved compared to sequences with alternating methoxy and fluoro modifications at the 2' position. For example, after modification with templates DV27P and DV29P, the inhibition rates of C1795-DV27P and C1797-DV29P improved by 5.8% and 4.1%, respectively.

[0365] II. Other Qualifying Templates of the Disclosure After modifying the unmodified sequence 1795 with templates DV32P and DV34P, the AGT gene The inhibition rates of C1795-DV32P and C1795-DV34P against the gene were 55.8% and 55.0%, respectively, which were 1.7% and 2.5% lower compared to the corresponding alternating modification sequences.

[0366] The inhibition rates of sequences C1795-DV27P and C1795-DV29P modified using the modification templates DV27P and DV29P of this disclosure were increased by 7.5% and 5.8% compared to C1795-DV32P, and by 8.3% and 6.6% compared to C1795-DV34P.

[0367] This reveals the following: 1) The sequence modified using the modification template DV25-29P of this disclosure, in which the unmodified sequence 1795 was modified, showed a significantly improved inhibitory effect on the AGT gene compared to the alternatingly modified sequence. For example, the sequence modified using DV27P, in which the unmodified sequence 84 was modified, showed a 5.8% increase in inhibition compared to the alternatingly modified sequence.

[0368] 2) Even with the same siRNA sequence, modification with different modification templates resulted in significantly different activity. For example, the unmodified sequence 1795 modified using the modification template DV27P of this disclosure showed a significantly improved inhibition rate of 11.4% compared to the sequence modified using the modification template DV28P of this disclosure.

[0369] 3) The activity of siRNA sequences modified with different templates varied significantly, with a difference of up to 11.4%. Therefore, it remains unclear which template modification can produce high activity in siRNA sequences.

[0370] (9) Unmodified sequence 1812

[0371] [Table 30]

[0372] I. DV25P-29P template of this disclosure When the unmodified sequence 1812 was modified using the modification templates DV25P-29P designed in this disclosure, the inhibition rate against the AGT gene exceeded 55%, which was a significant improvement in inhibition rate against the AGT gene compared to sequences with alternating methoxy and fluoro modifications at the 2' position. For example, after modification with template DV25P, the inhibition rates of C1812-DV25P increased by 11.5%, respectively.

[0373] II. Other Qualifying Templates of the Disclosure After modifying the unmodified sequence 1812 with templates DV32P-DV34P, C1812-DV32P, C1812-DV33P, and C1812-DV34 are applied to the AGT gene. The inhibition rates for P were 63.6%, 54.2%, and 60.7%, respectively, which were increased by 0.7%, -8.7%, and -2.2% compared to the inhibition rates for the corresponding alternating modification sequences.

[0374] The inhibition rate of the sequence C1816-DV25P modified using the modification template DV25P of this disclosure was significantly improved, increasing by 10.8% compared to C1812-DV32P, by 20.2% compared to C1812-DV33P, and by 13.7% compared to C1812-DV34P.

[0375] III. Prior art disclosed the Advanced ESC template DV22 (fluoro moieties: antisense chain positions 2, 6, 14 and 16, sense chain positions 7, 9, 10 and 11).

[0376] After modifying the unmodified sequence 1812 with template DV22, the inhibition rate of C1812-DV22 against the AGT gene was 63.6%, an increase of 0.7% compared to the alternating modification sequence.

[0377] The inhibition rate of the sequence C1812-DV25P modified using the modification template DV25P of this disclosure was significantly improved, increasing by 10.8% compared to C1812-DV22.

[0378] This reveals the following: 1) Sequences modified using the modification template DV25-29P of this disclosure, in which the unmodified sequence 1812 was modified, showed a significantly improved inhibitory effect on the AGT gene compared to sequences with alternating modifications. For example, sequences modified using DV25P in which the unmodified sequence 1812 was modified showed an 11.5% increase in inhibition rate compared to sequences with alternating modifications.

[0379] 2) Even with the same siRNA sequence, modification with different modification templates resulted in significantly different activity. For example, the unmodified sequence 1812 modified using the modification template DV25P of this disclosure showed a significantly improved inhibition rate, up to 20.2% higher, compared to the sequence modified using the modification template DV33P of this disclosure. Compared to the sequence modified using the conventionally disclosed Advanced ESC template DV22, the inhibition rate increased by up to 10.8%, also significantly improving.

[0380] 3) The activity of siRNA sequences modified with different templates varied significantly, with differences of up to 20.2%. Therefore, it remains unclear which template modification can produce high activity in siRNA sequences.

[0381] (10) Unmodified sequence 1816

[0382] [Table 31]

[0383] I. Modification using the DV25P-29P template of this disclosure When the unmodified sequence 1816 was modified using the modification templates DV25P, DV26P, DV27P, and DV29P designed in this disclosure, the inhibition rate against the AGT gene exceeded 55%, which was a significant improvement in inhibition rate against the AGT gene compared to sequences with alternating methoxy and fluoro modifications at the 2' position. For example, after modification with template DV29P, the inhibition rate of C1812-DV25P increased by 7.6%.

[0384] II. Other Qualifying Templates of the Disclosure After modifying the unmodified sequence 1816 with templates DV32P-DV34P, C1816-DV32P, C1816-DV33P, and C1816-DV34 were applied to the AGT gene. The inhibition rates for P were 58.6%, 48.4%, and 38.5%, respectively, which were increased by 2.3%, 12.5%, and 22.4% compared to the inhibition rates for the corresponding alternating modification sequences.

[0385] The inhibition rate of the sequence C1816-DV25P modified using the modification template DV29P of this disclosure was significantly improved, increasing by 9.9% compared to C1816-DV32P, by 20.1% compared to C1816-DV33P, and by 30.3% compared to C1816-DV34P.

[0386] III. Prior art disclosed the Advanced ESC template DV22 (fluoro moieties: antisense chain positions 2, 6, 14 and 16, sense chain positions 7, 9, 10 and 11).

[0387] After modifying the unmodified sequence 1816 with template DV22, the inhibition rate of C1816-DV22 against the AGT gene was 66.7%, which was 5.8% lower compared to the alternating modification sequence.

[0388] The inhibition rate of the sequence C1816-DV29P modified using the modification template DV29P of this disclosure was increased by 1.8% compared to C1816-DV22. This reveals the following:

[0389] 1) The unmodified sequence 1816 modified using the modification template DV25P-29P of this disclosure showed a significantly improved inhibitory effect on the AGT gene compared to the alternatingly modified sequence. For example, the sequence modified using DV29P showed a 7.6% increase in inhibition rate compared to the alternatingly modified sequence.

[0390] 2) Even with the same siRNA sequence, modification with different modification templates resulted in significantly different activity. For example, the unmodified sequence 1816 modified using the modification template DV29P of this disclosure showed a significant improvement in inhibition rate, up to 30.0% higher, compared to the sequence modified using the modification template DV34P of this disclosure. The inhibition rate increased by 1.8% compared to the sequence modified using the conventionally disclosed Advanced ESC template DV22.

[0391] 3) The activity of siRNA sequences modified with different templates varied significantly, with differences of up to 30.0%. Therefore, it remains unclear which template modification can produce high activity in siRNA sequences.

[0392] (11) Unmodified sequence 1835

[0393] [Table 32]

[0394] I. Modification using the DV25P-29P template of this disclosure When the unmodified sequence 1835 was modified using the templates DV25P-DV29P designed in this disclosure, the inhibition rate against the AGT gene exceeded 50%, and with the exception of DV27P, the inhibition rate against the AGT gene was significantly improved compared to sequences with alternating methoxy and fluoro modifications at the 2' position. For example, after modification with template DV26P, the inhibition rate of C1835-DV26P increased by 9.5%.

[0395] II. Other Qualifying Templates of the Disclosure After modifying the unmodified sequence 1835 with templates DV32P-DV34P, C1835-DV32P, C1835-DV33P, and C1835-DV34 were applied to the AGT gene. The inhibition rates for P were 53.3%, 64.9%, and 71.8%, respectively, which were increased by -9.0%, 2.6%, and 9.5% compared to the inhibition rates for the corresponding alternating modification sequences.

[0396] The inhibition rate of the sequence C1835-DV26P modified using the modification template DV26P of this disclosure was increased by 18.5% compared to C1835-DV32P, by 10.7% compared to C1835-DV33P, and by 0.0% compared to C1835-DV34P.

[0397] III. Prior art disclosed the Advanced ESC template DV22 (fluoro moieties: antisense chain positions 2, 6, 14 and 16, sense chain positions 7, 9, 10 and 11).

[0398] After modifying the unmodified sequence 1835 with template DV22, the inhibition rate of C1835-DV22 against the AGT gene was 58.2%, which was 4.1% lower compared to the alternating modification sequence.

[0399] The inhibition rate of the sequence C1835-DV26P modified using the modification template DV25P of this disclosure was significantly improved, increasing by 13.6% compared to C1835-DV22.

[0400] This reveals the following: 1) Sequences modified using the modification templates DV25-29P of this disclosure, in which the unmodified sequence 1835 was modified, showed a significantly improved inhibitory effect on the AGT gene compared to sequences with alternating modifications. For example, sequences modified using DV26P in which the unmodified sequence 1835 was modified showed a 9.5% increase in inhibition compared to sequences with alternating modifications.

[0401] 2) Even with the same siRNA sequence, modification with different modification templates resulted in significantly different activity. For example, the unmodified sequence 1835 modified using the modification template DV26P of this disclosure showed a 20.6% increase in inhibition rate, a significant improvement, compared to the sequence modified using the modification template DV27P of this disclosure. Compared to the sequence modified using the conventionally disclosed Advanced ESC template DV22, the inhibition rate increased by up to 13.6%, a significant improvement.

[0402] 3) The activity of siRNA sequences modified with different templates varied significantly, with differences of up to 20.6%. Therefore, it remains unclear which template modification can produce high activity in siRNA sequences.

[0403] (12) Unmodified sequence 1836

[0404] [Table 33]

[0405] I. Modification using the DV26-29P template of this disclosure When the unmodified sequence 1836 was modified using the templates DV26P-DV29P designed in this disclosure, the inhibition rate against the AGT gene exceeded 55%, which was a significant improvement in inhibition rate against the AGT gene compared to sequences with alternating methoxy and fluoro modifications at the 2' position. For example, after modification with templates DV26P and DV28P, the inhibition rates of C1836-DV26P and C1836-DV28P increased by 4.0%.

[0406] II. Other Qualifying Templates of the Disclosure After modifying the unmodified sequence 1836 with templates DV32P and DV34P, the inhibition rates of C1836-DV32P and C1836-DV34P against the AGT gene were 49.2% and 63.0%, respectively, which were reduced by 2.9% and 1.06% compared to the corresponding alternating modification sequences.

[0407] The inhibition rates of sequences C1836-DV26P and C1836-DV28P modified using the modification templates DV26P and DV28P of this disclosure were increased by 14.6% compared to C1836-DV32P and by 0.8% compared to C1836-DV34P.

[0408] This reveals the following: 1) Sequences modified using the modification templates DV26-29P of this disclosure, in which the unmodified sequence 1836 was modified, showed a significantly improved inhibitory effect on the AGT gene compared to sequences with alternating modifications. For example, sequences modified using DV26P and DV28P in which the unmodified sequence 1836 was modified showed a 4.0% increase in inhibition rate compared to sequences with alternating modifications.

[0409] 2) Even with the same siRNA sequence, modification with different modification templates resulted in significantly different activity. For example, sequences modified using the modification templates DV26P and DV28P of this disclosure of the unmodified sequence 1836 showed a 14.6% increase in inhibition rate, a significant improvement, compared to sequences modified using the modification template DV32P of this disclosure.

[0410] 3) The activity of siRNA sequences modified with different templates varied significantly, with differences of up to 14.6%. Therefore, it remains unclear which template modification can produce high activity in siRNA sequences.

[0411] (13) Unmodified sequence 1839

[0412] [Table 34]

[0413] I. Modification using the DV26-29P template of this disclosure The unmodified sequence 1839 is modified using templates DV26P-DV29P of this disclosure. The results showed that the inhibition rate against the AGT gene exceeded 50% in all cases, and the inhibition rate against the AGT gene was significantly improved compared to alternating methoxy and fluoromodification sequences at the 2' position. For example, after modification with template DV29P, the inhibition rate of C1839-DV29P increased by 5.1% in each case.

[0414] II. Other Qualifying Templates of the Disclosure After modifying the unmodified sequence 1839 with templates DV32P and DV34P, the inhibition rates of C1839-DV32P and C1839-DV34P against the AGT gene were 63.7% and 55.9%, respectively, which were increased by 4.6% and -3.2% compared to the inhibition rates of the corresponding alternating modification sequences.

[0415] The inhibition rate of the sequence C1839-DV29P modified using the modification template DV29P of this disclosure was increased by 0.5% compared to C1839-DV32P and by 8.3% compared to C1839-DV34P.

[0416] This reveals the following: 1) The unmodified sequence 1839 modified using the modification template DV26-29P of this disclosure showed a significantly improved inhibitory effect on the AGT gene compared to the alternatingly modified sequence. For example, the sequence modified using DV29P showed a 5.1% increase in inhibition rate compared to the alternatingly modified sequence.

[0417] 2) Even with the same siRNA sequence, modification with different modification templates resulted in significantly different activity. For example, the unmodified sequence 1839 modified using the modification template DV29P of this disclosure showed a 15.6% increase in inhibition rate, a significant improvement, compared to the sequence modified using the modification template DV26P of this disclosure.

[0418] 3) The activity of siRNA sequences modified with different templates varied significantly, with differences of up to 15.6%. Therefore, it remains unclear which template modification can produce high activity in siRNA sequences.

[0419] summary: (1) The selected unmodified sequences 1576s, 1578s, 1838, 1835, 1816, 1812, 1579, 1836, 1789, 1839, 1791, 1795, and 1585s were modified using the modification template DV25P-29P designed in this disclosure. These modifications showed a significant inhibitory effect on the discovery of AGT genes, with inhibition rates of 40% or higher in all cases. Among these, the inhibition rates of C1812-DV25P, C1579-DV33P, and C1789-DV26P reached 74.4%, 74.0%, and 73.6%, respectively.

[0420] (2) The sequences modified using the modification templates DV25P-29P of this disclosure, which were the 12 sequences described above, showed significantly improved inhibition rates against the AGT gene compared to the alternatingly modified sequences. For example, the inhibition rate of sequence C1812-DV25P, in which the unmodified sequence 1812 was modified using template DV25P, increased by 11.5% compared to the alternatingly modified sequences, and the inhibition rate of sequence C1791-DV26P, in which the unmodified sequence 1791 was modified using template DV26P, increased by 8.5% compared to the alternatingly modified sequences.

[0421] (3) When the same sequence was modified using the modification templates DV25P-29P of this disclosure, the inhibition rate against the AGT gene was significantly increased compared to when using the modification templates disclosed in the prior art. For example, the sequence modified using template DV25P from the unmodified sequence 1579 showed a 16.6% increase in inhibition rate compared to the sequence modified with the Advanced ESC template DV22 disclosed in the prior art.

[0422] (4) Modification of the same sequence using the modification templates DV25P-29P of the Disclosure significantly improved the inhibition rate against the AGT gene compared to modification using the other modification templates DV32-34P of the Disclosure. For example, the sequence modified with the modification template DV25P of the Disclosure of the unmodified sequence 1812 showed a 20.2% increase in inhibition rate compared to the sequence modified with the modification template DV33P of the Disclosure.

[0423] (5) The activity of sequences modified with different templates varied considerably. For example, modifying the unmodified sequence 1816 with DV29P increased the inhibition rate by 30.0% compared to modification with DV34P. Modifying the unmodified sequence 1579 with DV33P increased the inhibition rate by 29.8% compared to modification with DV28P. Therefore, it remains unclear which template modification can produce high activity in siRNA sequences.

[0424] (ii) IC 50 experiment C1816-DV29P, C1816-DV25P, C1812-DV25P, C1579-DV32P, C1789-DV26P, C1789-DV25P, C1839-DV 29P, C1795-DV27P, C1585s-DV27P, C1835-DV26P, C1835-DV25P, C1579-DV25P, C1836-DV26P, C17 Select a template modification sequence containing 24 sequences: 91-DV26P, C1585s-DV29P, C1604-DV29P, C1606-DV29P, C1607-DV29P, C1835-DV29P, C1838-DV29P, C1579-DV29P, C1576s-DV29P, C1578s-DV29P, and C1579s-DV29P, and IC 50 The experiment was carried out, and the IC of each array 50 The concentration was measured. The experimental results are shown in Table 26.

[0425] [Table 35]

[0426] As can be seen from Table 26, these 24 arrays of ICs 50 The values ​​were in the range of 1.884 pM to 61.498 pM. Among them, the ICs C1789-DV25P, C1812-DV25P, and C1789-DV26P 50 The values ​​were 1.884 pM, 2.160 pM, and 2.643 pM, respectively. This indicates that these sequences can effectively inhibit AGT gene expression at low concentrations.

[0427] Example 5: Comparison of inhibitory effects of sequences disclosed in the prior art on the AGT gene In this embodiment, the unmodified sequences 1579, 1789, 1812, and 157 disclosed in this application are used. The inhibition rates against the AGT gene were compared by comparing 6s, 1578s, 1585s, 1816, 1835, 1836, 1838, 1839, and 1791 with similar unmodified sequences disclosed in the prior art, as well as with the alternatingly modified sequences disclosed in this application and sequences modified with the DV25P-29P template.

[0428] 1. Experimental materials Test sample: (1) The sequence disclosed in the prior art is as shown in Table 27.

[0429] [Table 36] TIFF2026059799000151.tif105164

[0430] (2) An siRNA sequence with terminal thiolation modification using the alternating 2'-OMe and 2'-F modification from Example 2. (3) siRNA sequences modified using the modification template DV25-29P; see Table 28 for details.

[0431] [Table 37] TIFF2026059799000153.tif226164TIFF2026059799000154.tif226164TIFF2026059799000155.tif226164TIFF2026059799000156.tif226164 TIFF2026059799000157.tif226164TIFF2026059799000158.tif226164TIFF2026059799000159.tif226164TIFF2026059799000160.tif226164 TIFF2026059799000161.tif226164TIFF2026059799000162.tif226164TIFF2026059799000163.tif226164TIFF2026059799000164.tif226164 TIFF2026059799000165.tif226164TIFF2026059799000166.tif226164TIFF2026059799000167.tif226164TIFF2026059799000168.tif175164

[0432] Cell type: HepG2 cells, provided by Cyagen (H1-1701). HepG2 cells were cultured in DMEM medium (ATCC-30-2003) containing 10% fetal bovine serum (FBS, ExCell Bio-FSP500) and 1% penicillin-streptomycin (HyClone-SV30010).

[0433] Drug solvent: Sterilized enzyme-free water, Gibco Opti-MEM. 2. Experimental Method The inhibitory effect of the test sample on mRNA expression of the AGT gene in HepG2 cell lines was detected using qRT-PCR.

[0434] 2.1 Cell Culture HepG2 cell lines were extracted from the subcultured cells, and cells in the logarithmic growth phase were cultured in 10% fetal bovine serum DMEM medium (with 100 μL / mL penicillin and streptomycin added, respectively). The cells were placed in a cell incubator containing 5% CO2 at 37°C, and the medium was changed once daily. The cells were digested with 0.25% trypsin, subcultured, and incubated at 1000 r / min for 5 minutes. The heart was separated, the supernatant was discarded, and fresh culture medium was added for subculturing.

[0435] 2.2 Cell transfection Preparation of transfection mixture: Lipofectamine RNAiMAX and Opti-MEM were mixed in a 2:98 ratio and vortexed to ensure uniform mixing.

[0436] Preparation of transfection reagent: A siRNA solution diluted with 60 μL of Opti-MEM was added to 60 μL of transfection mixture in a 1:1 (v / v) ratio, homogeneously mixed by vortexing, and left at room temperature for 15 minutes to obtain lipid nanoparticles (LNPs). 12.5 μL was taken and the encapsulation efficiency was detected.

[0437] For the blank control group, transfection reagent was added to 60 μL of Opti-MEM. The mixture was vortexed to ensure homogeneity and left at room temperature for 15 minutes.

[0438] The prepared transfection reagent was added to a 24-well cell culture plate (100 μL per well) to achieve a final siRNA concentration of 0.02 nM in each well. 500 μL of cell suspension (1.5 × 10⁶ per mL) 5 (containing individual cells) was added. After homogeneous mixing using the crosswise method, the cells were placed in a cell incubator at 37°C and 5% CO2 and cultured for 40 hours.

[0439] 2.3 Detection of AGT mRNA 1) RNA extraction The steps are the same as in "2.3 AGT mRNA detection, '1) RNA extraction'" in Example 2.

[0440] 2) Detection of RNA concentration The steps are the same as in "2.3 Detection of RNA concentration in AGT mRNA detection" of Example 2.

[0441] 3) Quantitative detection of AGT mRNA The remaining components, excluding the primer and template, were added to a 15 mL centrifuge tube for 7.5 × 48 = 360 tests, and the tube was marked with "A".

[0442] 1.5 mL EP tubes were marked, and 77 μL of A and 420 ng of total RNA were added to each tube and mixed uniformly for use. The upstream and downstream primers for the internal reference gene GAPDH and the target gene AGT were homogeneously mixed for use.

[0443] 11 μL of B and 1 μL of C were added to each well of the PCR plate. The plates were covered with sealing film, centrifuged at 3000 rpm for 1 minute, and then placed in the apparatus. *This step was performed on ice to maintain a low temperature.

[0444] The plate was placed in a qPCR instrument and run using the following program. Reverse transfer: 55°C, 15 minutes, Initial denaturation: 95°C, 30 seconds, Circulating reaction: 95°C for 10 seconds; 60°C for 35 seconds; 40 cycles. Melting curve: 95°C, 15 seconds; 60°C, 60 seconds; 95°C, 15 seconds.

[0445] The execution time was approximately 2 hours. Analyzing the experimental results, 2 -ΔΔCt I calculated it. 2.4 Data Processing Formula for calculating AGT mRNA expression rate (%): Expression rate = (AGT mRNA expression level / AGT mRNA expression level in the blank control group) × 100% AGT gene expression inhibition rate = 100% - expression rate (%).

[0446] 3. Experimental Results The specific experimental results are shown in Table 29. Experimental results showed that the alternating modified sequences and sequences modified with specific modification templates of this disclosure exhibited significantly improved inhibitory activity against AGT compared to similar unmodified sequences disclosed in the prior art. For example, comparing sequence 812P disclosed in the prior art with sequence 1812 of this disclosure, the sequences were identical except for one less A base at the 3' end of the sense strand and one less U base at the 5' end of the antisense strand. However, the inhibition rate of the unmodified sequence 1812 of this disclosure increased by 14.3% compared to 812P, the inhibition rate of the alternating modified sequence was 62.9%, an increase of 20.4% compared to 812P, and after modification using the modification template DV25 of this disclosure, the inhibition rate reached 74.4%, an increase of 31.9% compared to 31P. (1) Compared to similar unmodified sequences disclosed in the prior art, the unmodified sequences, alternatingly modified sequences, and sequences modified with specific modification templates of this disclosure significantly improved the inhibition rate against the AGT gene, increasing it to up to 91.0%.

[0447] The unmodified sequences, alternatingly modified sequences, and sequences modified with specific modification templates of this disclosure showed significantly improved inhibition rates against the AGT gene compared to similar sequences disclosed in the prior art. For example, the inhibition rate of the unmodified sequence 812P was 42.5%, the inhibition rate of the unmodified sequence 1812, which is similar to the sequence of this disclosure, was 56.8%, an increase of 14.3% compared to 812P, the inhibition rate of the alternatingly modified sequence was 62.9%, an increase of 20.4% compared to 812P, and after modification with the modification template DV25 of this disclosure, the inhibition rate reached 74.4%, an increase of 31.9% compared to 812P.

[0448] [Table 38] TIFF2026059799000170.tif217165TIFF2026059799000171.tif217165TIFF2026059799000172.tif217165 TIFF2026059799000173.tif217165TIFF2026059799000174.tif217165TIFF2026059799000175.tif118165

[0449] I. Comparison of Sequence Structures The sequences disclosed in the prior art shown in Table 30 are very similar to the sequences of this disclosure, with only small differences, and a specific comparison is shown in the table below.

[0450] [Table 39] TIFF2026059799000177.tif227165TIFF2026059799000178.tif162165

[0451] The following can be seen from Table 30. Comparing sequence 579P disclosed in the prior art with sequence 1579 of this disclosure, the sense strand has one more A at the 5' end and one more U at the 3' end, the antisense strand has one more A at the 5' end and one more G at the 3' end.

[0452] Comparing sequence 789P disclosed in the prior art with sequence 1789 of this disclosure, the sense strand has one less base G at the 5' end and one different base A at the 3' end, the antisense strand has one different base U at the 5' end and one less base A at the 3' end.

[0453] Comparing sequence 812P disclosed in the prior art with sequence 1812 of this disclosure, the sense strand has one less base A at its 3' end, and the antisense strand has one less base U at its 5' end.

[0454] Comparing sequence 576P disclosed in the prior art with sequence 1576s of this disclosure, we find that the sense strand differs by two bases GU at its 3' end and the antisense strand differs by two bases AC at its 5' end.

[0455] Comparing sequence 578P disclosed in the prior art with sequence 1578s disclosed in this disclosure, there is one more base C at the 5' end of the sense strand and one more base G at the 3' end of the antisense strand.

[0456] Comparing sequence 585P disclosed in the prior art with sequence 1585s of this disclosure, the sense strand has one less base C at its 5' end, and the antisense strand has three fewer bases CAA at its 3' end.

[0457] Comparing sequence 816P disclosed in the prior art with sequence 1816 of this disclosure, the sense strand has one less base U at its 5' end, the antisense strand has one different base U at its 5' end, and the antisense strand has one less base A at its 3' end.

[0458] Comparing sequence 835P disclosed in the prior art with sequence 1835 of this disclosure, the sense strand has two fewer UU bases at its 5' end, and the antisense strand has two fewer UU bases at its 3' end.

[0459] Comparing sequence 836P disclosed in the prior art with sequence 1836 of this disclosure, the sense strand has two fewer bases UG at its 5' end, and the antisense strand has two fewer bases AU at its 3' end.

[0460] Comparing sequence 839P disclosed in the prior art with sequence 1839 of this disclosure, the sense strands are identical, and the antisense strand has two fewer bases UG at its 5' end. Compared with sequence 1838 of this disclosure, the sense strand has one fewer base G at its 5' end, the antisense strand has two different bases CA at its 3' end, the antisense strand has one fewer base U at its 5' end, and the antisense strand has one fewer base A at its 3' end.

[0461] Comparing sequence 791P disclosed in the prior art with sequence 1791 of this disclosure, we find that the sense strand differs by one base U at its 3' end, the antisense strand has two fewer bases UA at its 5' end, and two different bases UU at its 3' end.

[0462] II. Comparison of Activity 1) The activity of the unmodified sequences disclosed herein was significantly improved compared to similar unmodified sequences disclosed in the prior art. For example, the inhibition rate of the unmodified sequence 1576s disclosed herein increased by 20.9% compared to the structurally similar 576P (Figure 6).

[0463] The unmodified sequence 1579 disclosed herein showed an inhibition rate of 58.2% against the AGT gene, which is a significant improvement of 11.2% compared to a similar sequence 579P disclosed in the prior art (inhibition rate: 47%).

[0464] The unmodified sequence 1789 disclosed herein showed an inhibition rate of 54.5% against the AGT gene, which is a 2.6% increase compared to a similar sequence 789P disclosed in the prior art (inhibition rate: 51.9%).

[0465] The unmodified sequence 1812 disclosed herein showed an inhibition rate of 56.8% against the AGT gene, which is a significant improvement of 14.3% compared to a similar sequence 812P disclosed in the prior art (inhibition rate: 42.5%).

[0466] The unmodified sequence 1576s disclosed herein showed an inhibition rate of 59.1% against the AGT gene, which is a significant improvement of 20.9% compared to a similar sequence 576P disclosed in the prior art (inhibition rate: 38.2%).

[0467] The unmodified sequence 1578s disclosed herein exhibits an inhibition rate of 55.2% against the AGT gene, which is 21.6% lower than the similar sequence 578P disclosed in the prior art (inhibition rate: 33.6%). It increased by a percentage, showing a significant improvement.

[0468] The unmodified sequence 1585s disclosed herein showed an inhibition rate of 48.9% against the AGT gene, which is a 1.57% increase compared to a similar sequence 585P disclosed in the prior art (inhibition rate: 47.33%).

[0469] The unmodified sequence 1816 of this disclosure showed an inhibition rate of 46.8% against the AGT gene, which is a significant improvement of -17.7% compared to a similar sequence 816P disclosed in the prior art (inhibition rate: 64.5%).

[0470] The unmodified sequence 1835 disclosed herein showed an inhibition rate of 51.6% against the AGT gene, which is a significant improvement of 37.7% compared to a similar sequence 835P disclosed in the prior art (inhibition rate: 13.9%).

[0471] The unmodified sequence 1836 of this disclosure showed an inhibition rate of 46.5% against the AGT gene, which is a 4.6% increase compared to a similar sequence 836P disclosed in the prior art (inhibition rate: 41.9%).

[0472] The unmodified sequence 1839 disclosed herein showed an inhibition rate of 45.7% against the AGT gene, which is a significant improvement of 61.1% compared to a similar sequence 839P disclosed in the prior art (inhibition rate: -15.4%).

[0473] The unmodified sequence 1838 disclosed herein showed an inhibition rate of 50.6% against the AGT gene, which is a significant improvement of 66.0% compared to a similar sequence 839P disclosed in the prior art (inhibition rate: -15.4%).

[0474] The unmodified sequence 1791 disclosed herein showed an inhibition rate of 46.5% against the AGT gene, which is a 4.3% increase compared to a similar sequence 791P disclosed in the prior art (inhibition rate: 50.8%).

[0475] 2) The activity of the alternating modified sequences disclosed herein was significantly improved compared to similar unmodified sequences disclosed in the prior art. For example, the inhibition rate of the alternating modified sequence B1579-AL disclosed herein increased by 14.1% compared to sequence 579P.

[0476] After alternating modification of the unmodified sequence 1579 of this disclosure, the inhibition rate of B1579-AL against the AGT gene was 61.1%, which is a significant improvement, representing a 14.1% increase compared to the unmodified sequence 579P disclosed in the prior art.

[0477] After alternating modification of the unmodified sequence 1789 of this disclosure, the inhibition rate of B1789-AL against the AGT gene was 63.5%, which is an 11.6% increase compared to the unmodified sequence 789P disclosed in the prior art, and represents a significant improvement.

[0478] After alternating modification of the unmodified sequence 1812 of this disclosure, the inhibition rate of B1812-AL against the AGT gene was 62.9%, which is a significant improvement of 20.4% compared to the inhibition rate of the unmodified sequence 812P disclosed in the prior art.

[0479] After alternating modification of the unmodified sequence 1576s disclosed herein, the inhibition rate of B1576s-AL against the AGT gene was 64.6%, which is a significant improvement, representing a 26.4% increase compared to the unmodified sequence 576P disclosed in the prior art.

[0480] After alternating modification of the unmodified sequence 1578s disclosed herein, the inhibition rate of B1578s-AL against the AGT gene was 63.9%, which is a significant improvement, representing a 30.3% increase compared to the unmodified sequence 578P disclosed in the prior art.

[0481] After alternating modification of the unmodified sequence 1585s disclosed herein, the inhibition rate of B1585s-AL against the AGT gene was 59.3%, which is an 11.97% increase compared to the unmodified sequence 585P disclosed in the prior art, and represents a significant improvement.

[0482] After alternating modification of the unmodified sequence 1816 of this disclosure, the inhibition rate of B1816-AL against the AGT gene was 60.9%, which is a 3.6% decrease compared to the unmodified sequence 816P disclosed in the prior art.

[0483] After alternating modification of the unmodified sequence 1835 of this disclosure, the inhibition rate of B1835-AL against the AGT gene was 62.3%, which is a significant improvement, representing a 48.4% increase compared to the unmodified sequence 835P disclosed in the prior art.

[0484] After alternating modification of the unmodified sequence 1836 of this disclosure, the inhibition rate of B1836-AL against the AGT gene was 59.8%, which is a significant improvement, representing a 17.9% increase compared to the unmodified sequence 836P disclosed in the prior art.

[0485] After alternating modification of the unmodified sequence 1839 of this disclosure, the inhibition rate of B1839-AL against the AGT gene was 59.1%, which is a significant improvement, representing a 74.5% increase compared to the unmodified sequence 839P disclosed in the prior art.

[0486] After alternating modification of the unmodified sequence 1838 of this disclosure, the inhibition rate of B1838-AL against the AGT gene was 63.6%, which is a significant improvement, representing a 79% increase compared to the unmodified sequence 839P disclosed in the prior art.

[0487] After alternating modification of the unmodified sequence 1791 of this disclosure, the inhibition rate of B1791-AL against the AGT gene was 61.8%, which is an 11% increase compared to the unmodified sequence 791P disclosed in the prior art, and represents a significant improvement.

[0488] 3) The activity of sequences modified using the modification templates of this disclosure was significantly improved compared to similar unmodified sequences disclosed in the prior art. For example, the inhibition rate of the modified sequence C1579-DV26P of this disclosure increased by 25.2% compared to sequence 579P.

[0489] After modifying the unmodified compound 1579 of this disclosure with the modification templates DV25P-29P, the inhibition rate increased by more than 20% compared to the unmodified compound 579P disclosed in the prior art. For example, C1579-DV26P showed a 25.2% increase, which was a significant improvement.

[0490] After modifying the unmodified compound 1789 of this disclosure with the modification templates DV25P-29P, the inhibition rate increased by more than 20% compared to the unmodified compound 789P disclosed in the prior art. For example, C1789-DV26P showed a 21.7% increase, which was a significant improvement.

[0491] After modifying the unmodified compound 1812 of this disclosure with the modification template DV25P-29P, the inhibition rate increased by more than 30% compared to the unmodified compound 812P disclosed in the prior art. For example, C1812-DV25P showed a 31.9% increase, which was a significant improvement.

[0492] After modifying the unmodified compound 1576s of this disclosure with the modification template DV25P-29P, the inhibition rate increased by more than 40% compared to the unmodified compound 576P disclosed in the prior art. For example, C1576s-DV29P increased by 41.1%, showing a significant improvement.

[0493] After modifying the unmodified compound 1578s of this disclosure with the modification template DV25P-29P, the inhibition rate increased by more than 40% compared to the unmodified compound 578P disclosed in the prior art. For example, C1578s-DV29P showed a 43.5% increase, which was a significant improvement.

[0494] After modifying the unmodified compound 1585s of this disclosure with the modification templates DV25P-29P, the inhibition rate increased by more than 10% compared to the unmodified compound 585P disclosed in the prior art. For example, C1585s-DV27P showed a 17.27% increase, which was a significant improvement.

[0495] When the unmodified compound 1816 of this disclosure was modified with the modification template DV25P-29P and then compared to the unmodified compound 816P disclosed in the prior art, the increase in inhibition rate was not significant.

[0496] After modifying the unmodified compound 1835 of this disclosure with the modification templates DV25P-29P, the inhibition rate increased by more than 50% compared to the unmodified compound 835P disclosed in the prior art. For example, C1835-DV26P showed a 57.9% increase, which was a significant improvement.

[0497] After modifying the unmodified compound 1836 of this disclosure with the modification templates DV25P-29P, the inhibition rate increased by more than 20% compared to the unmodified compound 836P disclosed in the prior art. For example, C1836-DV26P showed a 21.9% increase, which was a significant improvement.

[0498] After modifying the unmodified compound 1839 of this disclosure with the modification template DV25P-29P, the inhibition rate increased by more than 70% compared to the unmodified compound 839P disclosed in the prior art. For example, C1839-DV29P showed a 79.6% increase, which was a significant improvement.

[0499] After modifying the unmodified compound 1838 of this disclosure with the modification template DV25P-29P, the inhibition rate increased by more than 90% compared to the unmodified compound 839P disclosed in the prior art. For example, C1838-DV29P showed a 91% increase, which was a significant improvement.

[0500] After modifying the unmodified compound 1791 of this disclosure with the modification templates DV25P-29P, the inhibition rate increased by more than 10% compared to the unmodified compound 791P disclosed in the prior art. For example, C1791-DV26P showed a 19.5% increase, which was a significant improvement.

[0501] Compared to similar unmodified sequences of the prior art, the unmodified sequence, the alternatingly modified sequence, and the template-modified sequence of this disclosure were found to significantly improve inhibitory activity against AGT, increasing the inhibition rate by up to 91%.

[0502] summary: Compared to similar sequences disclosed in the prior art, the unmodified sequences, alternatingly modified sequences, and sequences modified using specific modification templates in this disclosure all significantly improved the inhibitory effect on the AGT gene. For example, comparing the unmodified sequence 1839 in this disclosure with a similar sequence 839P disclosed in the prior art resulted in a 61.1% increase in inhibition. Comparing the alternatingly modified sequence B1835-AL of the disclosed sequence 1835 with the unmodified sequence 835P, which is similar to the 1835 sequence disclosed in the prior art, resulted in a 48.4% increase in inhibition. Comparing the sequence C1812-DV25P, which is sequence 1812 modified with the modification template DV25P in this disclosure, with the unmodified sequence 812P, which is similar to the 1812 sequence disclosed in the prior art, resulted in a 31.9% increase in inhibition.

[0503] Example 6: Experiment on off-target effects of modified sequences In the practical applications of siRNA, the guide strand (antisense strand) is only partially complementary. In many cases, the expression of non-target mRNA is inhibited. Research by Alnylam has shown that the hepatotoxicity of n-acetylgalactosamine (GalNAc)-binding siRNA is mainly due to off-target effects, where recognition mechanisms such as microRNA (miRNA) cause gene inhibition at the wrong targets.

[0504] To investigate whether the sequences of this disclosure produce off-target effects, this example examined the inhibitory efficiency of the more active sequence from Example 4 on potential off-target genes in HepG2 cells and compared it with Zilebesiran, a drug in Phase II clinical trials. For 13 sequences, including C1576s-DV29P, C1578s-DV29P, C1579-DV25P, C1789-DV25P, C1789-DV26P, C1791-DV26P, C1795-DV27P, C1812-DV25P, C1816-DV25P, C1835-DV26P, C1838-DV29P, and C1585s-DV27P, potential off-target genes with high sequence similarity were detected by blast. No highly similar genes were found for C1585s-DV27P, so it was not detected. The remaining 12 sequences all showed significant inhibitory effects on AGT at concentrations ranging from 16 pM to 10 nM. The sequences, excluding C1578s-DV29P and C1835-DV26P, did not show significant inhibitory effects on potential off-target genes (IC). 50 The difference is more than 200 times.

[0505] 1. Experimental materials 1) Test sample: The 12 sequences with higher activity in Example 4 and the positive control sequence APC-ZL (Table 31) are shown. APC-ZL is a zilebesiran sequence that is not linked to a GalNAc delivery vector.

[0506] [Table 40] TIFF2026059799000180.tif228165TIFF2026059799000181.tif227165TIFF2026059799000182.tif77165

[0507] Cell type: HepG2 cells, provided by Cyagen (H1-1701). HepG2 cells were cultured in DMEM medium containing 10% fetal bovine serum (FBS, ExCell Bio-FSP500) and 1% penicillin-streptomycin (HyClone-SV30010).

[0508] Drug solvent: Sterilized enzyme-free water, Gibco Opti-MEM. 2. Experimental Method Using qRT-PCR, we detected the inhibitory effect of the test sample on the mRNA expression of the AGT gene in the HepG2 cell line.

[0509] 2.1 Cell Culture HepG2 cell lines were extracted from the subcultured cells, and cells in the logarithmic growth phase were cultured in DMEM medium containing 10% fetal bovine serum (with 100 μL / mL penicillin and streptomycin added, respectively). The cells were placed in a cell incubator at 37°C with 5% CO2, and the medium was changed once daily. The cells were digested with 0.25% trypsin, subcultured, centrifuged at 1000 r / min for 5 minutes, the supernatant was discarded, and fresh culture medium was added for subculture.

[0510] 2.2 Cell transfection Preparation of transfection mixture: Lipofectamine RNAiMAX and Opti-MEM were mixed in a 2:98 ratio and vortexed to ensure uniform mixing.

[0511] Preparation of transfection reagent: A siRNA solution diluted with 60 μL of Opti-MEM was added to 60 μL of transfection mixture in a 1:1 (v / v) ratio, and the mixture was homogeneously mixed by vortexing. The mixture was left at room temperature for 15 minutes to obtain lipid nanoparticles (LNPs). A 12.5 μL sample was taken to detect the encapsulation efficiency.

[0512] For the blank control group, transfection reagent: 60 μL of the prepared transfection mixture was added to 60 μL of Opti-MEM. The mixture was vortexed to ensure uniform mixing and left at room temperature for 15 minutes.

[0513] The prepared transfection reagent was added to a 24-well cell culture plate (100 μL per well) so that the final siRNA concentrations in each well were 0.016 nM / 0.08 nM / 0.4 nM / 2 nM / 10 nM. 500 μL of cell suspension (1.5 × 10⁶ per mL) 5 (Individual cells) were added. After homogeneous mixing using the crosswise method, the cells were heated to 37°C. The cells were placed in a 5% CO2 cell incubator and cultured for 40 hours.

[0514] 2.3 RNA Extraction and Reverse Transcription 24 hours after transfection, the culture medium was removed and cells were collected for RNA extraction. Total RNA was extracted using the RNeasy® 96 Kit (QIAGEN-74182) according to the kit instructions. Subsequently, cDNA was synthesized using the FastKing RT Kit (With gDNase) (TIANGEN-KR116-02) according to the instructions.

[0515] 2.4 RT-qPCR Refer to "2.3 Quantitative Detection of mRNA" in Example 2. 2.5 Data Processing The RNA expression levels of target genes in each sample were calculated based on the Ct value of each sample using the ΔΔCt relative quantification method. -ΔΔCT It was represented as follows.

[0516] Calculation formula: ΔCT = average Ct value of target gene - average Ct value of internal reference gene. ΔΔCT = ΔCT (drug-treated group) - ΔCT (RNAiMAX control group) Relative expression level of target gene mRNA = 2 -ΔΔCT Inhibition rate = (1 - relative expression level of the sample / average expression level of the RNAiMAX control group) × 100%.

[0517] 3. Experimental Results 3.1 Each sequence had a significant inhibitory effect on the AGT gene. For example, C1576s-DV29P achieved an inhibition rate of 83.8% against AGT at 10 nM.

[0518] The inhibition rates of each sequence against AGT at different concentrations are shown in Table 32 below. Each sequence had a significant inhibitory effect on AGT. For example, C1576s-DV29P achieved an inhibition rate of 83.8% against AGT at 10 nM.

[0519] [Table 41]

[0520] 3.2 The sequences C1578s-DV29P and C1835-DV26P exhibited the expected off-target effects, but the off-target effects of the other sequences were not significant. Inhibitory efficacy of each sequence against AGT and potential off-target genes at each concentration, IC 50 This is as shown in Table 33 below.

[0521] [Table 42] TIFF2026059799000185.tif215165TIFF2026059799000186.tif218165TIFF2026059799000187.tif219165TIFF20260597990 00188.tif219165TIFF2026059799000189.tif219165TIFF2026059799000190.tif229164TIFF2026059799000191.tif223164

[0522] The following can be seen from the table above. (1) Some sequences showed low inhibitory efficiency against potential off-target genes. For example, C1789-DV25P showed only 2.7% inhibition of MRPL42 at 10 nM, and the inhibitory effect was not significant.

[0523] (2) Some sequences exhibit a predetermined inhibitory effect on potential off-target genes at high concentrations. It possessed IC, which inhibits potential off-target genes. 50 IC that inhibits AGT 50 It was much higher. For example, C1579-DV25P had an inhibition rate of 42.3% against GLTP at 10nM, but IC's inhibition rate against GLTP was much higher. 50 While the IC is greater than 10 nM, the IC for AGT 50 Since the value was 0.011 nM and its ratio was much larger than 200, the off-target effect was not significant.

[0524] (3) At 10nM, the inhibition rate of C1578s-DV29P against ERRFI1 was 66.9%, IC 50 It is 0.021nM, and the IC for this array relative to AGT 50 It is 0.004nM, IC 50 The ratio was only 5.3. The inhibition rate of C1835-DV26P against CCNA2 at 10nM was 74.6, and IC 50 It is 1.766 nM, and the IC for this array relative to AGT 50 It is less than 0.016 nM, IC 50 The minimum ratio was 110.4. Since the two sequences mentioned above are thought to have a predetermined off-target effect, it was necessary to reduce the inhibition rate against the off-target gene by anti-off-target modification.

[0525] summary: (1) Each sequence showed a significant inhibitory effect on AGT. For example, C1576s-DV29P achieved an inhibitory efficiency of 83.8% on AGT at 10 nM.

[0526] (2) C1578s-DV29P and C1835-DV26P showed the expected off-target effects, but the off-target effects of the other sequences were not significant. Example 7: Inhibitory effect of sequences on AGT and off-target genes using a specific anti-off-target effect design In practical applications of siRNA, the expression of non-target mRNAs that are only partially complementary to the guide strand (antisense strand) is often inhibited. Alnylam's research showed that the hepatotoxicity of n-acetylgalactosamine (GalNAc)-conjugated siRNA is mainly due to off-target effects, such as the inhibition of the wrong target gene through recognition mechanisms like microRNA (miRNA).

[0527] To address this issue, Alnylam incorporated a glycol nucleic acid (GNA) modification at the 7th position of the siRNA antisense strand in its latest 5th generation template design. This disrupts the seed region of the antisense strand, significantly reducing off-target effects and mitigating hepatotoxicity. Such modifications can inhibit off-target effects by influencing the binding of siRNA to undesigned targets through the recognition of the seed region.

[0528] Furthermore, a 2008 study revealed that replacing all eight base pairs at the 5' end of the antisense strand of a double-stranded siRNA with DNA significantly reduces the off-target effects of siRNA without affecting siRNA activity. Studies on mRNA cleavage sites have shown that DNA substitution does not affect mRNA cleavage by the RISC conjugate between nucleotides 10 and 11 of the antisense strand, but does affect mRNA cleavage at secondary sites. The mechanism is thought to be that DNA substitution inhibits nonspecific cleavage of double-stranded RNA by RNase.

[0529] In summary, to reduce off-target effects of the sequence, in this embodiment, DV25-29P was used to modify unmodified sequences 1578 and 1835 by substituting the nucleotide at position 7 or 6,7 of the antisense strand with DNA, and simultaneously substituting the nucleotide at the complementary pairing position of the sense strand with DNA to reduce off-target effects of the sequence. These two anti-off-target modifications were compared with an anti-off-target design in which position 7 of the antisense strand was substituted with (S)-GNA.

[0530] The experimental results showed that at concentrations of 10 nM to 16 pM, the anti-off-target designed sequences exhibited a significant inhibitory effect on the target gene AGT, and the inhibitory effect of some sequences on off-target genes was significantly reduced. This indicates that the anti-off-target design does not affect the inhibitory effect of the alternating modification and template modification sequences of this disclosure on AGT genes, but can inhibit the off-target effects of some sequences.

[0531] 1. Experimental materials 1) Test sample: Unmodified sequences 1578s, 1835, and 1838 were used as template-modified sequences, and sequences with template modification and anti-off-target design were used (Table 4).

[0532] 2) Array composition: I. Template-modified array The synthesis method followed the synthesis portion of the alternating modification sequence in Example 1.

[0533] II. siRNA sequences containing anti-off-target designed DNA Referring to Example 1, an siRNA sequence was synthesized, and when synthesizing the nucleotide at position 6 or 7 of the 5' end of the antisense strand, the DNA monomers DMT-dA phosphoramidite monomer (Formula 15), DMT-dT phosphoramidite monomer (Formula 16), DMT-dC phosphoramidite monomer (Formula 17), and DMT-dG phosphoramidite monomer (Formula 18) were used, and their structures are as follows.

[0534] [ka]

[0535] III. Anti-off-target designed siRNA sequences containing GNAs Refer to Example 1 to synthesize the siRNA sequence, and use the GNA monomer synthesis sequence when synthesizing the 7-position base at the 5' end of the antisense strand. The structure of the GNA monomer is as follows.

[0536] [ka]

[0537] [Table 43] TIFF2026059799000195.tif231165TIFF2026059799000196.tif62165

[0538] Cell type: HepG2 liver cancer cell line, provided by Cyagen (H1-1701). HepG2 cells were cultured in DMEM medium (ATCC-30-2003) containing 10% fetal bovine serum (FBS, ExCell Bio-FSP500) and 1% penicillin-streptomycin (HyClone-SV30010).

[0539] Drug solvent: Sterilized enzyme-free water, Gibco Opti-MEM. 2. Experimental Method Using qRT-PCR, we detected the inhibitory effect of the test samples on the expression of AGT and potential off-target gene mRNA in HepG2 cell lines.

[0540] 2.1 Cell Culture HepG2 cell lines were extracted from the subcultured cells, and cells in the logarithmic growth phase were cultured in DMEM medium containing 10% fetal bovine serum (with 100 μL / mL each of penicillin and streptomycin added). The cells were placed in a cell incubator at 37°C with 5% CO2, and the medium was changed once daily. The cells were digested with 0.25% trypsin, subcultured, centrifuged at 1000 r / min for 5 minutes, the supernatant was discarded, and fresh culture medium was added for subculture.

[0541] 2.2 Cell transfection Preparation of transfection mixture: Lipofectamine RNAiMAX and Opti-MEM were mixed in a 2:98 ratio and vortexed to ensure uniform mixing.

[0542] Preparation of transfection reagent: A siRNA solution diluted with 60 μL of Opti-MEM was added to 60 μL of transfection mixture in a 1:1 (v / v) ratio, and the mixture was homogeneously mixed by vortexing. The mixture was left at room temperature for 15 minutes to obtain lipid nanoparticles (LNPs). A 12.5 μL sample was taken to detect the encapsulation efficiency.

[0543] For the blank control group, transfection reagent: 60 μL of the prepared transfection mixture was added to 60 μL of Opti-MEM. The mixture was vortexed to ensure uniform mixing and left at room temperature for 15 minutes.

[0544] The prepared transfection reagent was added to a 24-well cell culture plate (100 μL per well) to achieve final siRNA concentrations of 16 pM / 80 pM / 400 pM / 2 nM / 10 nM in each well. 500 μL of cell suspension (1.5 × 10⁶ per mL) 5 (containing individual cells) was added. After mixing using the crosswise method, the cells were placed in a cell incubator at 37°C and 5% CO2 and cultured for 40 hours.

[0545] 2.3 RNA Extraction and Reverse Transcription 24 hours after transfection, the culture medium was removed and cells were collected for RNA extraction. Total RNA was extracted using the RNeasy® 96 Kit (QIAGEN-74182) according to the kit instructions. Subsequently, cDNA was synthesized using the FastKing RT Kit (With gDNase) (TIANGEN-KR116-02) according to the instructions.

[0546] 2.4 RT-qPCR Target cDNA was detected by qPCR, and GAPDH cDNA was detected simultaneously as an internal control. 8 μL of prepared qPCR reaction solution and 2 μL of sample cDNA were added to 384 wells. TaqMan qPCR reaction program: Heat at 95°C for 10 minutes, then enter cycling mode, heat at 95°C for 15 seconds, followed by heating at 60°C for 1 minute, for a total of 40 cycles. SYBR qPCR reaction program: Heat at 50°C for 2 minutes, heat at 95°C for 10 minutes, then enter cycling mode, heat at 95°C for 15 seconds, followed by heating at 60°C for 1 minute, for a total of 40 cycles. The final melting curve was obtained by heating at 95°C for 15 seconds, 60°C for 1 minute, and 95°C for 15 seconds.

[0547] 2.5 Data Processing The RNA expression levels of target genes in each sample were calculated based on the Ct value of each sample using the ΔΔCt relative quantification method. -ΔΔCT It was represented as follows.

[0548] Calculation formula: ΔCT = average Ct value of target gene - average Ct value of reference gene ΔΔCT = ΔCT (drug-treated group) - ΔCT (RNAiMAX control group) Relative expression level of target gene mRNA = 2 -ΔΔCT Inhibition rate = (1 - relative expression level of the sample / average expression level of the RNAiMAX control group) × 100%.

[0549] 3. Experimental Results (1) All different modification sequences had a significant inhibitory effect on the AGT gene. For example, the inhibition rate of the template modification sequence D578s-DV29P reached 85.7%, and the inhibition rate of the template modification and anti-off-target modification sequence D578s-DV29Pd7B was high at 81.3%.

[0550] The inhibition rates of the AGT gene for all tested samples are shown in Table 35. All template-modified sequences except D838-DV29P+ showed a significant inhibitory effect on AGT at a concentration of 10 nM; for example, the inhibition rate of the template-modified sequence D578s-DV29P reached 85.7%.

[0551] [Table 44]

[0552] After template modification, anti-off-target designation resulted in significant inhibitory activity against AGT gene expression. For example, the unmodified sequence 1578s was modified using template DV29P, and the inhibition rate of the anti-off-target designed sequence D578s-DV29Pd7B was high at 81.3%.

[0553] (2) After using one or more of the various modification methods of this disclosure, anti-off-target design had a significant anti-off-target effect on some off-target genes and reduced the inhibitory effect on off-target genes.

[0554] As shown in Figure 36, the experimental results are as follows: The template-modified sequences of this disclosure utilize an anti-off-target design and can reduce the inhibitory effect on off-target genes, i.e., they possess an anti-off-target effect. Sequences modified using the modification templates of this disclosure utilize an anti-off-target design, possess an anti-off-target effect, and can reduce the inhibition rate on off-target genes by up to 72.535%.

[0555] [Table 45]

[0556] 1) Design using only template modifications Some template-modified sequences exhibited predetermined off-target effects. For example, the sequence D578s-DV29P, which was created by modifying the unmodified sequence 1578s with the DV29P modification template, showed a 66.894% inhibition rate against the potential off-target gene ERRFI1. The sequence D835-DV26P, which was created by modifying the unmodified sequence 1835 with the DV26P modification template, showed a 74.619% inhibition rate against the potential off-target gene CCNA2.

[0557] 2) Simultaneously use an anti-off-target design (sequence number is denoted as "d67B" or "d7B") Some sequences using both template modifications and anti-off-target modifications significantly reduced the inhibition rate against off-target genes, demonstrating a significant anti-off-target effect. For example, D578s-DV25Pd7B reduced the inhibition rate against ERRFI1 by 72.535%. Furthermore, D835-DV26Pd7B significantly reduced the inhibition rate against CCNA2, with a 53.666% decrease.

[0558] summary: 1. Various modified sequences all showed significant inhibitory effects on the AGT gene. For example, the inhibition rate of the template-modified sequence D578s-DV29P reached 85.7%, and the inhibition rate of the template-modified and anti-off-target modified sequence D578s-DV29Pd7B was high at 81.3%.

[0559] 2. After modification using one or more of the various modification methods of this disclosure, further anti-off-target modifications resulted in a significant anti-off-target effect against some off-target genes and reduced the inhibitory effect against off-target genes.

[0560] 1) Some of the template-modified sequences have a predetermined off-target effect. For example, for the sequence D578s-DV29P obtained by modifying the unmodified sequence 1578s with the DV29P modification template, the inhibition rate against the potential off-target gene ERRFI1 was 66.894%. For the sequence D835-DV26P obtained by modifying the unmodified sequence 1835 with the DV26P modification template, the inhibition rate against the potential off-target gene CCNA2 was 74.619%.

[0561] 2) When the template-modified sequences of the present disclosure are designed against off-targets, the inhibitory effect on some off-target genes can be reduced, that is, they have an anti-off-target effect, and the inhibition rate against off-target genes can be reduced by up to 72.535%.

[0562] Example 8: Inhibitory effect on AGT in primary human hepatocytes of the sequences modified with the modified template of the present disclosure In this example, the inhibitory efficiency of the more active sequences of Example 4 on potential off-target genes in primary human hepatocytes was studied and compared with Zilebesiran, a drug that has entered clinical phase II. For five sequences such as C1576s-DV29P, C1578s-DV29P, C1579-DV25P, C1812-DV25P, and C1585s-DV27P, their respective modification templates were used, or the modification template and anti-off-target modification were used simultaneously, and at the same time, they were coupled with GalNAc to achieve free uptake by hepatocytes. Primary human hepatocytes were treated with the sequences in Table 37 by free uptake within the concentration range of 0.0064 nM to 100 nM, and the dose-response curves were fitted to calculate the IC 50 of each sequence. The results showed that the IC 50 of some sequences was better than that of the positive control Zilebesiran. For example, the IC 50 of D576s-DV29PG5 was 4.698 nM, which was better than 7.071 nM of the positive control Zilebesiran.

[0563] 1. Experimental materials 1) Test samples: Each sequence in Table 37 is either a sequence using template modification or a sequence using both template modification and anti-off-target design, with GalNAc ligand G5 coupled to the 3' end of the sense strand of these sequences.

[0564] [ka]

[0565] The coupling method for oligonucleotides and ligand G5 was the same as the method for producing conjugates 4, 5, 6, and 7 in Example 3 of Chinese Patent CN116854754A, that is, YK-GAL-304, YK-GAL-305, YK-GAL-306, and YK-GAL-307 were coupled with oligonucleotides. The synthesis methods for YK-GAL-304, YK-GAL-305, YK-GAL-306, and YK-GAL-307 were the same as in Example 1 of CN116854754A.

[0566] Oligonucleotides and ligands form the following conjugates.

[0567] [ka]

[0568] Each sequence can be specifically identified by referring to Table 7. The sequence number G5 indicates that the sequence is coupled with GalNAc ligand G5, and GL indicates that the sequence is coupled with GalNAc ligand L96.

[0569] [Table 46] TIFF2026059799000202.tif229165TIFF2026059799000203.tif229165TIFF2026059799000204.tif229165TIFF2026059799000205.tif176165

[0570] Cell type: Primary human hepatocytes, provided by Liver Biotechnology (Shenzhen) Co., Ltd (LV-PHH001). Drug solvent: Sterilized enzyme-free water, primary human hepatocyte maintenance medium (LV-WEM001).

[0571] 2. Experimental Method QRT-PCR was used to detect the inhibitory effect of the test sample on AGT gene mRNA expression in primary human hepatocytes.

[0572] 2.1 Thawing and culturing of primary human hepatocytes The culture medium was preheated at a constant temperature of 37°C for at least 30 minutes. 300 μL of PBS was added to each well of a collagen-coated 24-well plate, shaken several times, and then the PBS was aspirated and discarded. The cell cryopreservation tubes were removed from the liquid nitrogen tank and placed in a 37°C water bath. They were gently shaken until only a small amount of ice crystals remained in the tubes, and the cell suspension was poured all at once into the thawing medium. 1 mL of thawing medium was pipetted and the inner wall of the cryopreservation tube was rinsed 2-3 times. The cell suspension was inverted into the thawing medium and mixed. The rising speed was set to 5 and the decelerating speed to 3. Then, the cells were centrifuged at 50g for 5 minutes at room temperature. The cells were then placed in plating medium in a 3x10⁶ layer. 5 The solution was diluted to cells / mL, inoculated into 24-well plates at a rate of 0.5 mL / well, and incubated in an incubator for 24 hours.

[0573] 2.2 Free uptake by primary human hepatocytes The test product was diluted to 100-0.0064 nM in primary human hepatocyte maintenance medium. Cultured primary human hepatocytes were removed, the culture medium was aspirated, 500 μL of the diluted test product was added to each well, and the cells were returned to a 37°C, 5% CO2 cell culture incubator and cultured for 40 hours.

[0574] 2.3 RNA Extraction and Reverse Transcription 24 hours after transfection, the culture medium was removed and cells were collected for RNA extraction. Total RNA was extracted using the RNeasy® 96 Kit (QIAGEN-74182) according to the kit instructions. Subsequently, cDNA was synthesized using the FastKing RT Kit (With gDNase) (TIANGEN-KR116-02) according to the instructions.

[0575] 2.4 RT-qPCR Refer to section 2.3 of Example 2, which describes the quantitative mRNA analysis. 2.5 Data Analysis The RNA expression levels of target genes in each sample were calculated based on the Ct value of each sample using the ΔΔCt relative quantification method. -ΔΔCT It was represented as follows.

[0576] Calculation formula: ΔCT = average Ct value of target gene - average Ct value of reference gene ΔΔCT = ΔCT (drug-treated group) - ΔCT (RNAiMAX control group) Relative expression level of target gene mRNA = 2 -ΔΔCT Inhibition rate = (1 - relative expression level of the sample / average expression level of RNAiMAX control) × 100%.

[0577] 3. Experimental Results The inhibitory efficiencies of each sequence against AGT at different concentrations are shown in Table 38 below. Each sequence showed a significant inhibitory effect against AGT. For example, the inhibitory efficiency of D576s-DV29PG5 against AGT reached 83.1% at 100 nM. Furthermore, the IC50 of some sequences 50It is superior to the positive control Zilebesiran, for example, in IC576s-DV29PG5 50 The result was 4.698 nM, which was superior to the positive control's 7.071 nM.

[0578] [Table 47] TIFF2026059799000207.tif31165

[0579] Example 9: Inhibitory effect of sequences modified with the modification template of this disclosure on AGT in mouse serum. This embodiment uses unmodified sequences 1576s, 1578s, 1579, 1585s, 1789, A series of sequences including 1791, 1795, 1812, 1816, 1835, 1838, and 1839 were selected as examples and modified, for example, by template-only modification and by simultaneous template modification and anti-off-target design. Zilebesiran, an siRNA drug currently in Phase II clinical trials, was used as a positive control. Using transgenic mice expressing the human AGT gene, the inhibitory effects of the above sequences on AGT in serum at different time points were detected by ELISA.

[0580] 1. Experimental materials Test reagents: Each sequence in Table 39 was either a sequence using template modification or a sequence using both template modification and anti-off-target design, with GalNAc ligand G5 coupled to the 3' end of the sense strand of these sequences.

[0581] [ka]

[0582] The coupling method for oligonucleotides and ligand G5 was the same as the method for producing conjugates 4, 5, 6, and 7 in Example 3 of Chinese Patent CN116854754A, that is, YK-GAL-304, YK-GAL-305, YK-GAL-306, and YK-GAL-307 were coupled with oligonucleotides. The synthesis methods for YK-GAL-304, YK-GAL-305, YK-GAL-306, and YK-GAL-307 were the same as in Example 1 of CN116854754A.

[0583] Oligonucleotides and ligands form the following conjugates.

[0584] [ka]

[0585] The specific sequences of each sequence are shown in Table 39. In the sequence number, G5 indicates coupling with GalNAc ligand G5, and GL indicates coupling with GalNAc ligand L96.

[0586] The structure of G5 is as follows:

[0587] [ka]

[0588] [[(1R,2R,3R,4R)-1-[[28-[[2-(acetylamino)-2-deoxy-β-D-galactopyranosyl]oxy]-13,13-bis[[3-[[3-[[5-[[2-(acetylamino)-2-deoxy-β-D-galactopyranosyl]oxy]-1 -oxopentyl]amino]propyl]amino]-3-oxopropoxy]methyl]-11,18,24-trioxo-15-oxa-12,19,23-triazaoctacosan-1-yl]oxy]-2-O-methyl-5-β-D-ribofuranosyl]hydrogen phosphate] [[(1R,2R,3R,4R)-1-[[28-[[2-(acetylamino)-2-deoxy-β-D-galactopyranosyl]oxy]-13,13-bis[[3-[[3-[[5-[[2-(acetylamino)-2-deoxy-β-D-galactopyranosyl]oxy]-1-oxopentyl]amino]propyl]amino]-3-oxopropoxy]methyl]-11,18,24-trioxo-15-oxa-12,19,23-triazaoctacosacin-1-yl]oxy]-2-O-methyl-5-β-D-ribofuranosyl]hydrogen phosphate.

[0589] GL structure:

[0590] [ka]

[0591] [[(2S,4R)-1-[29-[[2-(acetylamino)-2-deoxy-β-D-galactopyranosyl]oxy]-14,14-bis[[ 3-[[3-[[5-[[2-(acetylamino)-2-deoxy-β-D-galactopyranosyl]oxy]-1-oxopentyl]amino]propyl]amino]-3-oxopr opoxy]methyl]-1,12,19,25-tetraoxo-16-oxa-13,20,24-triazanonacos-1-yl]-4-hydroxy-2-pyrrolidinyl]methyl hydrogen phosphate] [[(2S,4R)-1-[29-[[2-(acetylamino)-2-deoxy-β-D-galactopyranosyl]oxy]-14,14-bis[[3-[[3-[[5-[[2-(acetylamino)-2-deoxy-β-D-galactopyranosyl]oxy]-1-oxopentyl]amino]propyl]amino]-3-oxopropoxy]methyl]-1,12,19,25-tetraoxo-16-oxa-13,20,24-triazanonacos-1-yl]-4-hydroxy-2-pyrrolidinyl]methylhydrogenphosphate

[0592] [Table 48] TIFF2026059799000213.tif227165TIFF2026059799000214.tif233165TIFF202 6059799000215.tif228165TIFF2026059799000216.tif228166TIFF2026059799 000217.tif228165TIFF2026059799000218.tif228165TIFF2026059799000219. tif228165TIFF2026059799000220.tif228165TIFF2026059799000221.tif57165

[0593] Manufacturing of test reagents: Pharmaceutical solvent: Physiological saline Manufacturing conditions: sterile environment Marking method: Tags were attached to each manufactured dosage form, and the topic number, name, concentration, quantity, manufacturing date, manufacturer, storage conditions, etc. were described on the outer box.

[0594] Storage conditions: Manufactured immediately before use, and the remaining samples were stored at -80 °C. Experimental animal information: Species / strain: hAGT transgenic mice Grade: SPF Gender: Male Quantity: 150 Age: 6 - 8 weeks old Weight: 18 - 28 g Supplier: GemPharmatech Co., Ltd Production license number: SCXK(Su)2018 - 0008 Animal Experiment Committee (IACUC): After receiving the experimental animals, they were bred by BrightShines, and the project approval number is SYXK(Beijing)2022 - 0025. This project was reviewed by the Animal Experiment Committee of BrightShines, and the test process was carried out strictly in accordance with the requirements of IACUC to ensure animal welfare.

[0595] Breeding and management: Breeding conditions: After receiving the experimental animals, they were bred by BrightShines, and the project approval number is SYXK(Beijing)2022 - 0025. The animals were bred in a breeding cage with length × width × height = 29.0 cm × 18.5 cm × 13.0 cm. The set temperature range is 20 - 26 °C, the set humidity range is 40% - 70%, the ventilation frequency is more than 15 times / hour of fresh air, and the artificial lighting repeated alternately for 12 hours of light period and 12 hours of dark period.

[0596] The breeding environmental conditions comply with the national standard GB14925 - 2010 of the People's Republic of China, and the environment was controlled by a modular air - conditioning system. The animals had free access to food and water. The bottles and the drinking water inside needed to be replaced at least twice a week, and after use, the bottles were sterilized under high pressure in a pulsating vacuum sterilizer before being reused.

[0597] Animal cages and bedding were changed at least once a week, and all animal cages and pads were high-pressure sterilized in a pulsating vacuum sterilizer before being moved to the barrier environment. Animal cages were cleaned and disinfected at least once a week.

[0598] The animal breeding and observation room, including the shelves, floor, and tables, was cleaned and disinfected daily. The disinfectants used in barrier environments include a 6.67% benzalkonium bromide solution, a 0.5% 84 disinfectant, a 75% disinfectant, and a 0.08% didecyldimethylammonium bromide. These four disinfectants must be used sequentially and cannot be mixed.

[0599] Laboratory animal feed: SPF rat maintenance feed: Manufactured by SPF(Beijing) Biotechnology Co.,Ltd., with animal feed production license number SCXK(Beijing)2019-0010, issued by the Beijing Municipal Science and Technology Commission.

[0600] Feed Inspection: Each batch of feed comes with a quality certificate, microbiological testing is conducted quarterly at our company, and the feed supplier provides an updated third-party feed inspection report every six months. Feed nutrient content testing complies with the People's Republic of China National Standard GB14924.3-2010, and contamination index testing complies with the People's Republic of China National Standard GB14924.2-2001.

[0601] Drinking water for laboratory animals: This is sterile water produced using a filtration system and directly filled into drinking bottles. Drinking water testing: We conduct microbiological testing in-house once a quarter and commission a third-party testing agency to perform water quality testing once a year. Drinking water testing complies with the People's Republic of China National Standard GB5749-2006.

[0602] Animal pads: Cone Titanium pads: SPF (Beijing) Biotechnology Co., Ltd., Animal pad production license number SCXK (Beijing) 2019-0004, issued by the Beijing Municipal Science and Technology Commission.

[0603] Pad Testing: Microbial testing is conducted quarterly at our company, and pad suppliers provide at least one third-party bedding inspection report every six months. Pad material testing complies with the People's Republic of China National Standard GB14924.2-2001.

[0604] 2. Experimental Method Dosage design and group classification Definition of experimental day: The day on which the animal was administered the solvent or test drug was defined as day 0.

[0605] Grouping and administration: After a 3-day acclimatization period, experimental animals were divided according to the AGT protein content in their serum. Then, the animals were randomly divided into groups of 6 each into a negative control group and an investigational drug group. A single subcutaneous injection of 3 mg / kg, 5 mL / kg, and 0.6 mg / mL was administered, and the day of administration was recorded as day 0.

[0606] Each animal was identified using an ear tag. Cage cards were used to identify the enclosures. A laboratory sign was hung at the entrance of the laboratory. Detailed grouping information is shown in Table 40 below.

[0607] [Table 49]

[0608] Measuring index (1) General observations The subjects were observed once a day from one week before administration until the end of the experiment.

[0609] Observations: The animals were observed next to their cages for death or near-death status, mental state, behavior, fecal characteristics, and feeding and watering conditions. Test animals: All animals in the negative control group and the test drug administration group.

[0610] (2) Expression of AGT protein in serum Detection times: Pre-administration to 3 days later (-3 days), 1 week after administration to 7 days later (1w), 2 weeks after administration to 14 days later (2w), 3 weeks after administration to 21 days later (3w), 4 weeks after administration to 28 days later (4w), and 5 weeks after administration to 35 days later (5w).

[0611] AGT protein level measurement method: An ELISA kit was used for detection, and serum samples were stored in a way that prevented repeated freezing and thawing. Test animals: All animals in the negative control group and the test drug administration group.

[0612] (4) Data processing and statistical analysis Experimental data were presented as mean ± standard deviation (Mean ± SD), and GraphPad Prism 8.3 analysis software was used for data analysis. Statistical analysis was performed using two-way ANOVA and post-hoc tests. The LSD test was used to assess the homogeneity of variances, and the Dunnett T3 test was used to assess the heterogeneity of variances. A p-value of < 0.05 was considered statistically significant.

[0613] 3. Experimental Results The specific experimental results are shown in Table 41. These sequences were found to be able to continuously and significantly inhibit the expression of AGT proteins in serum. For example, the inhibition rates of D576s-DV29PG5 reached 84.09%, 82.11%, and 85.25% on days 7, 14, and 28, respectively.

[0614] (1) The sequences of this disclosure, for example, the unmodified sequences 1579, 1789, 1812, 1576s, 1578s, and 1585s, using different modifications, showed significant inhibitory effects on the expression of AGT protein in serum. For example, the inhibition rates of D576s-DV29PG5 reached 84.09%, 82.11%, and 85.25% on days 7, 14, and 21, respectively (Figure 8).

[0615] [Table 50] TIFF2026059799000224.tif220165TIFF2026059799000225.tif220165TIFF2026059799000226.tif57164

[0616] As can be seen from Table 41, the conjugates formed by linking each sequence with a GalNAc compound had a significant inhibitory effect on the expression of AGT protein in serum. For example, the inhibition rates of D576s-DV29PG5 reached 84.09%, 82.11%, and 85.25% on days 7, 14, and 21, respectively (Figure 9).

[0617] This demonstrates that unmodified sequences such as 1579, 1789, 1812, 1576s, 1578s, and 1585s, designed in this disclosure, can be efficiently delivered to the liver of animals and significantly inhibit the expression of AGT genes by modifying them using the template modifications of this disclosure, or simultaneously using anti-off-target designs, and then conjugating them with GalNAc compounds.

[0618] summary: We have shown that unmodified sequences designed in this disclosure, such as 1579, 1789, 1812, 1576s, 1578s, and 1585s, can be efficiently delivered to the liver of animals and continuously and significantly inhibit AGT gene expression by being modified using the template modifications of this disclosure, or simultaneously using anti-off-target designs, and then conjugated with a GalNAc compound.

[0619] For example, the inhibition rates of D576s-DV29PG5 reached 84.09%, 82.11%, and 85.25% on days 7, 14, and 28, respectively. summary: This disclosure describes designing a series of siRNAs based on AGT mRNA sequences, modifying them using alternating modifications and a specific set of modification templates, and designing anti-off-target siRNAs for some of the sequences. The results show the following: (1) Thirteen unmodified sequences, including 1576s, 1579, 1812, 1578s, 1789, 1835, 1838, 1795, 1585s, 1816, 1791, 1836, and 1839, showed a significant inhibitory effect on the AGT gene, with inhibition rates exceeding 45%.

[0620] (2) By using multiple modified sequences, the inhibition rate could be reached at over 70%. Furthermore, the modified sequences bound to the GalNAc compound were efficiently delivered to the liver of animals and were able to significantly inhibit the expression of the AGT gene.

[0621] The specifics are as follows: 1. The alternating modification sequences described herein had a significant inhibitory effect on the AGT gene. (1) Of the 99 sequences designed, 33 showed a significant inhibitory effect on the AGT gene, with inhibition rates exceeding 40%. Of these, 10 showed inhibition rates exceeding 60%.

[0622] (2) The inhibition rates of the 23 sequences against the AGT gene ranged from 25% to 40%. For example, the inhibition rates for B734-AL and B994-AL were 39.2% and 37.3%, respectively.

[0623] (3) The inhibition rates of the AGT gene for 43 sequences were 25% or less. For example, the inhibition rate for B1017-AL was only 23.1%. (4) Even siRNAs with similar sequences exhibited significantly different activity. For example, the inhibition rate of B1365-AL increased by 40.4% compared to B1367-AL, a significant improvement, but the only difference between the two was the last two bases. The inhibition rate of B1816-AL increased by 30.5% compared to B1815-AL, a significant improvement, but the only difference between the two was the last single base. Therefore, selecting sequences with significant inhibitory activity from the very large number of oligonucleotide sequences designed for AGT mRNA sequences is not easy and requires a lot of creative work.

[0624] 2. The unmodified sequences of this disclosure had a significant inhibitory effect on the AGT gene. (1) Thirteen unmodified sequences, including 1576s, 1579, 1812, 1578s, 1789, 1835, 1838, 1795, 1585s, 1816, 1791, 1836, and 1839, showed significant inhibitory effects on the AGT gene, with inhibition rates exceeding 45%. Of these, 1576s, 1578s, 1812, 1579, 1789, 1835, and 1838 showed inhibition rates exceeding 50%.

[0625] (2) The inhibition rates of other unmodified sequences against the AGT gene were less than 45%. For example, the inhibition rates of 731 and 1011 were only 1.3% and 2.1%, respectively. (3) Even siRNAs with similar sequences exhibited significantly different activity. For example, the inhibition rate of unmodified sequence 734 increased by 41.7% compared to unmodified sequence 731, showing a significant improvement.

[0626] (4) The effects on activity after alternating modification of different sequences were inconsistent. Some inhibition rates were significantly improved; for example, the inhibition rate of the unmodified sequence 731 was 25.1% higher with alternating modification than with the unmodified sequence. Some were not significant; for example, the inhibition rates of the unmodified sequences 994, 1279, and 1591 were essentially unchanged between alternating modification and the unmodified sequence.

[0627] 3. Sequences modified with the modification templates of this disclosure had a significant inhibitory effect on the AGT gene. (1) The selected unmodified sequences 1576s, 1578s, 1838, 1835, 1816, 1812, 1579, 1836, 1789, 1839, 1791, 1795, and 1585s, after modification with the modification template DV25P-29P designed in this disclosure, showed a significant inhibitory effect on AGT gene expression, with inhibition rates exceeding 40%. Of these, the inhibition rates for C1812-DV25P, C1579-DV33P, and C1789-DV26P reached 74.4%, 74.0%, and 73.6%, respectively.

[0628] (2) The above 12 arrays were modified using the modified template DV25-29P of the present disclosure, and the inhibition rate against AGT gene expression was significantly improved as compared with the alternating modified arrays. For example, the inhibition rate of the array C1812-DV25P obtained by modifying the unmodified array 1812 with the template DV25P increased by 11.5% as compared with the alternating modified array, and the inhibition rate of the array C1791-DV26P obtained by modifying the unmodified array 1791 with the template DV26P increased by 8.5% as compared with the alternating modified array.

[0629] (3) When the same array was modified with the modified template DV25-29P of the present disclosure, the inhibition rate against AGT gene expression was significantly increased as compared with the case of modification with the modified template disclosed in the prior art. For example, when the modified array 1579 was modified with the modified template DV25P of the present disclosure, the inhibition rate increased by 16.6% as compared with the case of modification with the Advanced ESC template DV22 disclosed in the prior art.

[0630] (4) When the same array was modified with the modified template DV25-29P of the present disclosure, the inhibition rate against AGT gene expression was significantly increased as compared with the case of modification with another modified template DV32-34P of the present disclosure. For example, the inhibition rate of the array obtained by modifying the unmodified array 1812 with the modified template DV25P of the present disclosure increased by 20.2% as compared with the array modified with the modified template DV33P of the present disclosure.

[0631] (5) The activities of different template-modified arrays are very different. For example, the inhibition rate of the unmodified array 1816 modified with DV29P increased by 30.0% as compared with the inhibition rate modified with DV34. The inhibition rate of the unmodified array 1579 modified with DV33P increased by 29.8% as compared with the inhibition rate modified with DV28P. Therefore, it is unclear which modified template should be used to modify the siRNA sequence to produce high activity.

[0632] (6) The IC of the above 24 arrays 50The values ​​were in the range of 1.884 pM to 61.498 pM. Among them, the ICs C1789-DV25P, C1812-DV25P, and C1789-DV26P 50 The values ​​were 1.884 pM, 2.160 pM, and 2.643 pM, respectively. This indicates that these sequences can effectively inhibit AGT gene expression at low concentrations.

[0633] 4. Sequences modified with alternating modifications and the modification templates of this disclosure showed significantly improved inhibitory activity against AGT compared to unmodified sequences that were virtually identical to those disclosed in the prior art.

[0634] Compared to unmodified sequences with only minor differences disclosed in the prior art, the unmodified sequences, alternatingly modified sequences, and sequences modified with specific modification templates in this disclosure significantly improved the inhibition rate against the AGT gene, increasing it to up to 91%.

[0635] I. The activity of the unmodified sequences of this disclosure was significantly improved compared to similar unmodified sequences disclosed in the prior art. For example, the inhibition rate of unmodified sequence 1835 of this disclosure increased by 37.7% compared to the structurally similar 835P.

[0636] II. The activity of the alternating modified sequences disclosed herein was significantly improved compared to similar unmodified sequences disclosed in the prior art. For example, the inhibition rate of the alternating modified sequence B1812-AL disclosed herein increased by 20.4% compared to sequence 812P.

[0637] III. The activity of sequences modified with the modification template of this disclosure was significantly improved compared to similar unmodified sequences disclosed in the prior art. For example, compared to sequence 576P, the activity of the modified sequence of this disclosure The inhibition rate of the alternating modification sequence C1576s-DV29P increased by 41.1%.

[0638] 5. The alternating modification sequences and template modification sequences of this disclosure utilize specific anti-off-target designs, exhibiting significant inhibitory effects on AGT genes and significantly reducing inhibitory effects on off-target genes.

[0639] (1) All different modification sequences had a significant inhibitory effect on the AGT gene. For example, the inhibition rate of the template modification sequence D576s-DV29P reached 83.8%, and the inhibition rate of the template modification and anti-off-target modification sequence D576s-DV29Pd7B was high at 85.4%.

[0640] (2) After using one or more of the modification methods of the Disclosure, anti-off-target design was performed, which had a significant anti-off-target effect on some off-target genes and reduced the inhibitory effect on off-target genes, i.e., it had an anti-off-target effect and reduced the inhibition rate on off-target genes by up to 72.535%.

[0641] 6. Sequences modified with the modification templates of this disclosure significantly inhibited AGT expression in mouse serum. The unmodified sequences designed in this disclosure, e.g., 1579, 1789, 1812, 1576s, 1578s, and 1585s, were modified using the templates of the present invention and subsequently conjugated with a GalNAc compound using an anti-off-target design. These were efficiently delivered to the liver of animals and were able to sustainably and significantly inhibit the expression of AGT protein in serum.

[0642] For example, the inhibition rates of D576s-DV29PG5 reached 84.09%, 82.11%, and 85.25% on days 7, 14, and 21, respectively.

[0643] [Table 51] TIFF2026059799000228.tif230164TIFF2026059799000229.tif228164TIFF2026059799000230.tif228164TIFF2026059799000231.tif229164TIFF2026059799000232.tif229164TIFF2026059799000233.tif228164TIFF2026059799000234.tif228164TIFF2026059799000235.tif229164TIFF2 026059799000236.tif228164TIFF2026059799000237.tif228164TIFF2026059799000238.tif229164TIFF2026059799000239.tif229164TIFF2026059799000240.tif229164TIFF2026059799000241.tif228164TIFF2026059799000242.tif228164TIFF2026059799000243.tif223164TIFF202605 9799000244.tif227164TIFF2026059799000245.tif227164TIFF2026059799000246.tif222164TIFF2026059799000247.tif226164TIFF2026059799000248.tif220164TIFF2026059799000249.tif220164TIFF2026059799000250.tif226164TIFF2026059799000251.tif226164TIFF20260597990 00252.tif221164TIFF2026059799000253.tif229164TIFF2026059799000254.tif229164TIFF2026059799000255.tif221164TIFF2026059799000256.tif228164TIFF2026059799000257.tif228164TIFF2026059799000258.tif228164TIFF2026059799000259.tif228164TIFF2026059799000260.tif221164TIFF2026059799000261.tif228164TIFF2026059799000262.tif228164TIFF2026059799000263.tif222164TIFF2026059799000264.tif229164TIFF2026059799000265.tif229164TIFF2026059799000266.tif229164TIFF2026059799000267.tif229164TIFF2026059799000268.tif2221 64TIFF2026059799000269.tif227164TIFF2026059799000270.tif227164TIFF2026059799000271.tif219164TIFF2026059799000272.tif228164TIFF2026059799000273.tif228164TIFF2026059799000274.tif222164TIFF2026059799000275.tif227164TIFF2026059799000276.tif226164TIFF20 26059799000277.tif221164TIFF2026059799000278.tif229164TIFF2026059799000279.tif229164TIFF2026059799000280.tif222164TIFF2026059799000281.tif226164TIFF2026059799000282.tif226164TIFF2026059799000283.tif221164TIFF2026059799000284.tif225164TIFF202605979 9000285.tif226164TIFF2026059799000286.tif219164TIFF2026059799000287.tif228164TIFF2026059799000288.tif222164TIFF2026059799000289.tif228164TIFF2026059799000290.tif228164TIFF2026059799000291.tif228164TIFF2026059799000292.tif215164TIFF2026059799000293.tif227164TIFF2026059799000294.tif219164TIFF2026059799000295.tif219164TIFF2026059799000296.tif230164TIFF2026059799000297.tif230164TIFF2026059799000298.tif230164TIFF2026059799000299.tif230164TIFF2026059799000300.tif230164TIFF2026059799000301.tif2 30164TIFF2026059799000302.tif222164TIFF2026059799000303.tif227164TIFF2026059799000304.tif227164TIFF2026059799000305.tif223164TIFF2026059799000306.tif228164TIFF2026059799000307.tif228164TIFF2026059799000308.tif221164TIFF2026059799000309.tif89164.

Claims

1. A double-stranded RNAi agent comprising any double-stranded oligonucleotide selected from the group consisting of the following sense strand and antisense strand pairs: (1) A sense strand having the sequence or fragment thereof shown in Sequence ID No. 11, or a modified sequence of the sequence or fragment thereof, and an antisense strand having the sequence or fragment thereof shown in Sequence ID No. 24, or a modified sequence of the sequence or fragment thereof, (2) A sense strand having the sequence or fragment thereof shown in Sequence ID No. 4, or a modified sequence of the sequence or fragment thereof, and an antisense strand having the sequence or fragment thereof shown in Sequence ID No. 17, or a modified sequence of the sequence or fragment thereof, (3) A sense chain having the sequence or fragment thereof shown in Sequence ID No. 3, or a modified sequence of the sequence or fragment thereof, and an antisense chain having the sequence or fragment thereof shown in Sequence ID No. 16, or a modified sequence of the sequence or fragment thereof, (4) A sense chain having the sequence or fragment thereof shown in Sequence ID No. 12, or a modified sequence of the sequence or fragment thereof, and an antisense chain having the sequence or fragment thereof shown in Sequence ID No. 25, or a modified sequence of the sequence or fragment thereof, (5) A sense strand having the sequence or fragment thereof shown in Sequence ID No. 6, or a modified sequence of the sequence or fragment thereof, and an antisense strand having the sequence or fragment thereof shown in Sequence ID No. 19, or a modified sequence of the sequence or fragment thereof. (6) A sense strand having the sequence or fragment thereof shown in Sequence ID No. 10, or a modified sequence of the sequence or fragment thereof, and an antisense strand having the sequence or fragment thereof shown in Sequence ID No. 23, or a modified sequence of the sequence or fragment thereof, (7) A sense strand having the sequence or fragment thereof shown in Sequence ID No. 1, or a modified sequence of the sequence or fragment thereof, and an antisense strand having the sequence or fragment thereof shown in Sequence ID No. 14, or a modified sequence of the sequence or fragment thereof, (8) A sense strand having the sequence or fragment thereof shown in Sequence ID No. 2, or a modified sequence of the sequence or fragment thereof, and an antisense strand having the sequence or fragment thereof shown in Sequence ID No. 15, or a modified sequence of the sequence or fragment thereof, (9) A sense strand having the sequence or fragment thereof shown in Sequence ID No. 5, or a modified sequence of the sequence or fragment thereof, and an antisense strand having the sequence or fragment thereof shown in Sequence ID No. 18, or a modified sequence of the sequence or fragment thereof, (10) A sense strand having the sequence or fragment thereof shown in Sequence ID No. 7, or a modified sequence of the sequence or fragment thereof, and an antisense strand having the sequence or fragment thereof shown in Sequence ID No. 20, or a modified sequence of the sequence or fragment thereof. (11) A sense strand having the sequence or fragment thereof shown in Sequence ID No. 8, or a modified sequence of the sequence or fragment thereof, and an antisense strand having the sequence or fragment thereof shown in Sequence ID No. 21, or a modified sequence of the sequence or fragment thereof, (12) A sense strand having the sequence or fragment thereof shown in Sequence ID No. 9, or a modified sequence of the sequence or fragment thereof, and the sequence or thereof shown in Sequence ID No. 22 an antisense chain having the fragment, or the sequence or modified sequence of the fragment, (13) A sense strand having the sequence or fragment thereof shown in Sequence ID No. 13, or a modified sequence of the sequence or fragment thereof, and an antisense strand having the sequence or fragment thereof shown in Sequence ID No. 26, or a modified sequence of the sequence or fragment thereof.

2. The double-stranded RNAi agent according to claim 1, wherein the double-stranded RNAi agent satisfies any one of the following conditions: (1) The condition that the nucleotide sequence of the sense strand has a difference of 1 to 3 nucleotides from any one of the sequences of SEQ ID NOs: 1 to 13, and (2) The condition that the nucleotide sequence of the antisense strand has a difference of 1 to 3 nucleotides from any one of the sequences of SEQ ID NOs. 14 to 26.

3. The sense strand or antisense strand comprises at least one modified nucleotide, wherein the modified nucleotide is selected from deoxynucleotides, 2'-deoxythymidine (dT) nucleotides, 2'-O-methyl-modified nucleotides, 2'-fluoro-modified nucleotides, 2'-deoxy-modified nucleotides, locked nucleotides, unlocked nucleotides, conformation-restrictive nucleotides, restricted ethyl nucleotides, debasic nucleotides, 2'-amino-modified nucleotides, 2'-O-allyl-modified nucleotides, 2'-C-alkyl-modified nucleotides, 2'-hydroxyl-modified nucleotides, 2'-methoxyethyl-modified nucleotides, 2'-O-alkyl-modified nucleotides, morpholino nucleotides, phosphoramidites, nucleotides containing unnatural bases, tetrahydropyran-modified nucleotides, 1,5-anhydrohexitol-modified nucleotides, cyclohexenyl-modified nucleotides, nucleotides containing a phosphorothioate group, nucleotides containing a methylphosphonate group, nucleotides containing a 5'-phosphate, and nucleotides containing a 5'-phosphate mimetic, and combinations thereof. Alternatively, the double-stranded RNAi agent according to claim 1 or 2, wherein the nucleotide monomers are linked by 3',5'-phosphodiester bonds.

4. The double-stranded RNAi agent according to any one of claims 1 to 3, wherein the double-stranded RNAi agent satisfies any one of the following conditions: (1) The condition that at least one strand contains the 3' overhang of at least one or at least two nucleotides, (2) The condition that the length of the double-stranded region of the double-stranded RNAi agent is 15 to 30 nucleotide pairs, or the length of the double-stranded region is 17 to 25 nucleotide pairs, or the length of the double-stranded region is 19 to 23 nucleotide pairs, or the length of the double-stranded region is 21 nucleotide pairs, (3) Each chain has 15 to 30 nucleotides, or 19 to 25 nucleotides, (4) The sense strand has 21 nucleotides and the antisense strand has 23 nucleotides, (5) The condition that all nucleotide modifications on the sense strand and antisense strand are chemical modifications at the 2' position of the ribose of the nucleotide, (6) The condition that the 3',5'-phosphodiester bond linked between the nucleotide monomers has a thio modification.

5. The double-stranded RNAi agent according to any one of claims 1 to 4, wherein the double-stranded RNAi agent satisfies one or more of the following conditions: (1) Chemical modification of the 2' position of the ribose of the nucleotide is 2'-methoxy, 2'-methoxy Conditions selected from one or more of the following: ethyl, 2'-fluoro, 2'-benzyloxy, 2'-methylcarbonylamino, and 2'-pyridinemethoxy, (2) Conditions under which the chemical modification of the 2' position of the ribose of each nucleotide is selected from a combination of 2'-methoxy and 2'-fluoro, (3) Conditions under which the chemical modification of the 2' position of the ribose of each nucleotide is selected from an alternating combination of 2'-methoxy and 2'-fluoro, (4) The chemical modification method of the 2' position of ribose of each nucleotide is such that all odd positions on the antisense strand are 2'-methoxy modified, all even positions are 2'-fluoro modified, the relative positions of the 2'-methoxy modifications on the sense strand and antisense strand are 2'-fluoro modified, and the relative positions of the 2'-fluoro modifications are 2'-methoxy modified.

6. The double-stranded RNAi agent according to any one of claims 1 to 5, further comprising the following modifications: (1) The antisense chain is modified using one of the modification methods shown in Table 42. The sense strand is modified using one of the modification methods shown in Table 43. Here, the antisense chain uses modification A, and the sense chain uses modification a. Here, the antisense chain uses modification B, and the sense chain uses modification a. Here, the antisense chain uses modification C, and the sense chain uses modification a. Here, the antisense chain uses modification B, and the sense chain uses modification b. Here, the antisense chain uses modification C, and the sense chain uses modification b. Here, the antisense chain uses modification D, and the sense chain uses modification b. Here, the antisense chain uses modification E, and the sense chain uses modification b, or, Here, the antisense chain uses modification F, and the sense chain uses modification b, or (2) The antisense chain is modified using one of the modification methods shown in Table 44. The sense chain is modified using one of the modification methods shown in Table 45. Here, the antisense chain uses modification A, and the sense chain uses modification a. Here, the antisense chain uses modification B, and the sense chain uses modification a. Here, the antisense chain uses modification C, and the sense chain uses modification a. Here, the antisense chain uses modification B, and the sense chain uses modification b. Here, the antisense chain uses modification C, and the sense chain uses modification b. Here, the antisense chain uses modification D, and the sense chain uses modification b. Here, the antisense chain uses modification E, and the sense chain uses modification b, or, Here, the antisense chain uses modification F, and the sense chain uses modification b.

7. The double-stranded RNAi agent according to any one of claims 1 to 6, wherein the double-stranded RNAi agent satisfies one or more of the following conditions: (1) The antisense strand modifies the nucleotides using a modifying group at positions 2 to 8 from the 5' end, where the modifying group is selected from UNA, GNA, and DNA, and the structures of UNA and GNA are as follows: 【Chemistry 1】 However, the base must be selected from adenine, guanine, cytosine, thymine, and uracil. (2) The 5' carbon atom of the glycoside of the 5' terminal nucleotide of the modified antisense chain is phosphorylated, and the phosphorylation of the 5' carbon atom is selected from the following 5' phosphorylation groups: 5'-vinylphosphonate (5'-E-VP), 5'-methylphosphonate (5'-MP), 5'-C-methylphosphate ester, 5'-thiophosphonate (5'-PS), and 5'-phosphonate (5'-P), and the structure of each is represented by the following formula, 【Chemistry 2】 However, R is hydrogen, hydroxyl, amine, C １－４ Alkyl, aryl, C １－４ Alkoxy, C １－４ It is an alkylcarbonylamino or halogen, and The base is selected from adenine, guanine, cytosine, thymine, and uracil, and (3) The 3',5'-phosphodiester bond between nucleotides at the end of the sequence contains a thio modification, forming a chiral pure 3',5'-thiophosphodiester bond, wherein the 5' end of the sense and antisense strands contains 1 to 3 thio bonds, and the 3' end of the antisense strand contains 1 to 3 thio bonds.

8. The double-stranded RNAi agent according to claim 1 or 2, wherein the double-stranded RNAi agent comprises any double-stranded oligonucleotide selected from the group consisting of the following sense strand and antisense strand pairs: (1) The sense strand has the sequence shown in Sequence ID No. 209, and the antisense strand has the sequence shown in Sequence ID No.

222. (2) The sense strand has the sequence shown in Sequence ID No. 202, and the antisense strand has the sequence shown in Sequence ID No.

215. (3) The sense strand has the sequence shown in Sequence ID No. 201, and the antisense strand has the sequence shown in Sequence ID No.

214. (4) The sense strand has the sequence shown in Sequence ID 210, and the antisense strand has the sequence shown in Sequence ID 223. (5) The sense strand has the sequence shown in Sequence ID No. 204, and the antisense strand has the sequence shown in Sequence ID No.

217. (6) The sense strand has the sequence shown in Sequence ID No. 208, and the antisense strand has the sequence shown in Sequence ID No.

221. (7) The sense strand has the sequence shown in Sequence ID 199, and the antisense strand has the sequence shown in Sequence ID 212. (8) The sense strand has the sequence shown in Sequence ID No. 200, and the antisense strand has the sequence shown in Sequence ID No.

213. (9) The sense strand has the sequence shown in Sequence ID No. 203, and the antisense strand has the sequence shown in Sequence ID No.

216. (10) The sense strand has the sequence shown in Sequence ID No. 205, and the antisense strand has the sequence shown in Sequence ID No.

218. (11) The sense strand has the sequence shown in Sequence ID No. 206, and the antisense strand has the sequence shown in Sequence ID No. 219, (12) The sense strand has the sequence shown in Sequence ID No. 207, and the antisense strand has the sequence shown in Sequence ID No.

220. (13) The sense strand has the sequence shown in sequence number 211, and the antisense strand has the sequence shown in sequence number 224.

9. A conjugate for reducing AGT expression, comprising a double-stranded RNAi agent according to any one of claims 1 to 8 and a ligand bound thereto.

10. The double-stranded RNAi agent according to claim 9, wherein the conjugate satisfies one or more of the following conditions. (1) Conditions under which the ligand binds to the 3'-terminus or 5'-terminus of the sense strand of the oligonucleotide, (2) The condition that the ligand is a GalNAc derivative attached using a divalent or trivalent branched bond, (3) The ligand is of the following formula: 【Transformation 3】 However, X is hydrogen, a hydroxy protecting group or H, and the hydroxy protecting group is selected from acetyl, benzoyl and isobutyryl; Y is an amine protecting group or H, and the amine protecting group is selected from formyl, acetyl, propionyl, n-butyryl and isobutyryl; n is an integer between 0 and 20; and q, r and s are independently integers between 1 and 7. (4) The ligand is given by the following formula: 【Chemistry 4】 However, X is oxygen, nitrogen, or sulfur. Y is an alkyl or aryl, R １ It is oxygen or sulfur, R ２ is hydrogen, amine, C １－４ Alkyl, aryl, C １－４ It is an alkoxy or halogen, A is -(CH ２ ) ａ -, -(CH ２ CH ２ O) ｂ -, -((CH ２ ) ｃ NHCO) ｄ - or -((CH ２ ) ｃ CONH) ｄ -, where a is an integer from 1 to 15, b is c is an integer from 1 to 7, d is an integer from 1 to 7, B is - (CH ２ ) ｅ - and here e is an integer from 0 to 7, L is -CONH- or -NHCO-, X １ ha- (CH ２ ) ｆ - or - (CH ２ CH ２ O) ｆ CH ２ - and f is an integer from 1 to 5, X ２ ha- (CH ２ ) ｇ - and g is an integer from 1 to 6, Y １ is 0 or 1, Y ２ is 0, 1, or 2, Y ３ is 1, 2, or 3, m is an integer between 0 and 4. The condition is that n is an integer between 0 and 4.

11. The conjugate according to claim 9 or 10, wherein the conjugate satisfies one or more of the following conditions: (1) The ligand is given by the following formula, 【Transformation 5】 (2) The ligand is G4, G5, G6, or G7, and its structure is represented by the following formula, and 【Transformation 6】 (3) The condition that the conjugate has a structure represented by the following formula. 【Transformation 7】

12. The double-stranded RNAi agent comprises any double-stranded oligonucleotide selected from the group consisting of the following sense strand and antisense strand pairs, (1) The sense strand has the sequence shown in Sequence ID No. 423, and the antisense strand has the sequence shown in Sequence ID No.

488. (2) The sense strand has the sequence shown in Sequence ID No. 406, and the antisense strand has the sequence shown in Sequence ID No.

453. (3) The sense strand has the sequence shown in Sequence ID 404, and the antisense strand has the sequence shown in Sequence ID 450. (4) The sense strand has the sequence shown in Sequence ID No. 426, and the antisense strand has the sequence shown in Sequence ID No.

492. (5) The sense strand has the sequence shown in Sequence ID No. 412, and the antisense strand has the sequence shown in Sequence ID No.

465. (6) The sense strand has the sequence shown in Sequence ID No. 412, and the antisense strand has the sequence shown in Sequence ID No.

468. (7) The sense strand has the sequence shown in Sequence ID No. 421, and the antisense strand has the sequence shown in Sequence ID No.

483. (8) The sense strand has the sequence shown in Sequence ID No. 417, and the antisense strand has the sequence shown in Sequence ID No.

476. (9) The sense strand has the sequence shown in Sequence ID No. 419, and the antisense strand has the sequence shown in Sequence ID No. 479, (10) The sense strand has the sequence shown in Sequence ID No. 401, and the antisense strand has the sequence shown in Sequence ID No. 443, (11) The sense strand has the sequence shown in Sequence ID No. 398, and the antisense strand has the sequence shown in Sequence ID No. 433, (12) The sense strand has the sequence shown in Sequence ID No. 429, and the antisense strand has the sequence shown in Sequence ID No. 496, (13) The sense strand has the sequence shown in Sequence ID No. 414, and the antisense strand has the sequence shown in Sequence ID No. 471, Here, the oligonucleotide double strand binds to ligand G5, Alternatively, the double-stranded RNAi agent comprises any double-stranded oligonucleotide selected from the group consisting of the following sense strand and antisense strand pairs, (14) The sense strand has the sequence shown in Sequence ID No. 554, and the antisense strand has the sequence shown in Sequence ID No. 488, (15) The sense strand has the sequence shown in Sequence ID 530, and the antisense strand has the sequence shown in Sequence ID 457, (16) The sense strand has the sequence shown in Sequence ID 531, and the antisense strand has the sequence shown in Sequence ID 570, (17) The sense strand has the sequence shown in Sequence ID 541, and the antisense strand has the sequence shown in Sequence ID 450, (18) The sense strand has the sequence shown in Sequence ID No. 557, and the antisense strand has the sequence shown in Sequence ID No. 492, (19) The sense strand has the sequence shown in Sequence ID 533, and the antisense strand has the sequence shown in Sequence ID 465, (20) The sense strand has the sequence shown in Sequence ID No. 560, and the antisense strand has the sequence shown in Sequence ID No.

483. Here, the oligonucleotide double strand binds to ligand G5, Alternatively, the conjugate according to any one of claims 9 to 11, wherein the double-stranded RNAi agent comprises an oligonucleotide double strand consisting of a sense strand shown in SEQ ID NO: 423 and an antisense strand pair shown in SEQ ID NO: 488, and the oligonucleotide double strand is bound to G5.

13. A double-stranded RNAi agent according to any one of claims 1 to 8, comprising a double-stranded region and a nuclease, Alternatively, a nucleic acid protein composition comprising the antisense strand of the double-stranded region of the double-stranded RNAi agent and a nuclease.

14. A pharmaceutical composition comprising a double-stranded RNAi agent according to any one of claims 1 to 8, a conjugate according to any one of claims 9 to 12, or a nucleic acid protein composition according to claim 13, and a pharmaceutically acceptable carrier.

15. A double-stranded RNAi agent according to any one of claims 1 to 8, a conjugate according to any one of claims 9 to 12, a nucleic acid protein composition according to claim 13, or a pharmaceutical composition according to claim 14, used for the treatment of AGT gene expression-related diseases, or for the treatment of hypertension and cardiovascular / cerebrovascular diseases.

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