SiRNA targeting and inhibiting agt gene expression and its application in treating hypertension

Abstract

The present disclosure provides a modified oligonucleotide sequence and its application. A series of siRNAs are designed based on the angiotensinogen (AGT) messenger ribonucleic acid (mRNA) sequence, and are alternately modified and modified using a specific set of modification templates. The results of cell and animal experiments show that some alternately modified and specifically template-modified oligonucleotide sequences can significantly inhibit the expression of the AGT gene, and can be used for developing drugs for treating related diseases such as hypertension.

Description

TECHNICAL FIELD

[0001] The present disclosure belongs to the technical field of nucleic acid modification, and particularly relates to a small interfering ribonucleic acid (siRNA) modified by multiple chemical methods and application thereof in preparation of a drug for a disease related to AGT gene expression, for example, an siRNA for targeted inhibition of AGT gene expression and application thereof in treatment of hypertension. BACKGROUND

[0002] Nucleic acid drugs, especially oligonucleotide drugs, are widely used due to their simple synthesis and high activity. Oligonucleotide drugs generally include antisense oligonucleotides (ASO), small interfering RNA (siRNA), microRNA (miRNA) and nucleic acid aptamer, etc.

[0003] Oligonucleotides are short DNA or RNA molecules, oligomers, which can easily bind to their respective complementary oligonucleotides, DNA or RNA in a sequence-specific manner to form duplexes, or less commonly, higher-order hybrids. This basic property makes oligonucleotides widely used in gene detection, research and medicine. In nature, oligonucleotides are usually small RNA molecules that play a role in gene expression regulation, or intermediates derived from the degradation of larger nucleic acid molecules.

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

[0005] The effective molecule of classical RNAi consists of a characteristic 19+2 nucleotide polymer structure (a double helix structure composed of a 21-nucleotide RNA molecule and a 19-nucleotide molecule corresponding to the nucleobase, containing a 2-nucleotide 3' end overhang). One strand of siRNA (guide strand or antisense strand) is complementary to the target mRNA transcript, and the other strand is designated as the passenger strand (sense strand). The siRNA (antisense strand) guides the argonaute protein (AGO2) to the target transcript and becomes part of the RNA-induced silencing complex (RISC). The complete complementarity of siRNA (antisense strand) to the target causes the target transcript to break at the 10-11 point position opposite the guide strand (antisense strand) under the catalysis of the AGO2 protein.

[0006] siRNA has natural advantages compared with small molecule and antibody drugs, because siRNA performs its function by completing Watson-Crick base pairing with mRNA, while small molecule and monoclonal antibody drugs need to recognize the complex spatial structure of specific proteins. Therefore, many diseases cannot be treated by small molecule and monoclonal antibody drugs because the target molecules have high activity and cannot recognize the molecular structure with affinity and binding specificity. The mechanism of action of siRNA drugs enables them to regulate the expression of target proteins at the genetic level, and has target specificity compared with small molecule or antibody drugs. The mechanism based on base complementary pairing principle also makes the treatment range of siRNA wider, the design simpler, and the development cycle shorter.

[0007] Oligonucleotides can bind to complementary RNA chains in a sequence-specific manner, and after hybridization, they can induce RNase H to cleave the target RNA. In natural oligonucleotides, nucleotides are connected by phosphodiester bonds, and under physiological conditions, they are particularly sensitive to nucleases, so natural, unstructured, unmodified oligonucleotide drugs are easily degraded by nucleases in vivo, have low activity, and are poor in drug development. Chemical modification of oligonucleotide structure is an effective way to improve its activity, which can improve its stability to nucleases, affinity to RNA, and better promote endocytosis and tissue targeting, thereby effectively regulating the expression of target genes.

[0008] According to the basic structure of oligonucleotides, bases, sugar rings, phosphate skeletons and ends, chemical modification can be carried out in four parts:

[0009] 1) Base modification: mainly divided into three forms of purine modification, pyrimidine modification and base replacement. Purine modification includes N6-methyladenosine, N1-methyladenosine, 7-methylguanosine modification; pyrimidine modification includes 3-methyluracil nucleoside, 5-methyluracil nucleoside, 5-methylcytosine nucleoside, N4-acetylcytosine, pseudouridine, thio-uracil nucleoside, propyne uracil nucleoside and dihydro uracil nucleoside, etc.

[0010] 2) Sugar ring modification: mainly divided into sugar ring modification and replacement. Sugar ring modification includes 2'-modification, 4'-modification, 5'-modification, isomerization modification, and combination modification of these modifications. The most common 2'-modification of siRNA is 2'-OMe (2'-methoxy) and 2'-F (2'-fluorine) modification. Compared with natural siRNA, siRNA with 2'-OMe and 2'-F modification at the same time has higher Tm value, stronger serum stability and better activity.

[0011] 3) Modification of the phosphate backbone: The main modification methods are: modification of phosphorothioate; modification by methylphosphonate, selenophosphate, boronophosphate, dithiophosphate, and replacement of the bridging oxygen atom of the phosphodiester linkage with a sulfur atom; replacement of the phosphate group between nucleotides with a group that does not contain phosphorus, such as replacement of the P atom with C, S, and N atoms, forming guanidyl, S-methyl thiourea, etc.

[0012] 4) Terminal modification: covalent conjugation of special groups at the 5' end or / and 3' end of the sense strand and 5' phosphorylation modification of the antisense strand.

[0013] Hypertension is a systemic disease characterized by elevated blood pressure, with high prevalence, low treatment rate and control rate.

[0014] Angiotensinogen (AGT) protein is a secreted protein, mainly expressed in the liver in the human body, and other tissues with AGT expression include brain, gallbladder, heart and kidney, etc. AGT protein is cleaved by renin to produce angiotensin I (Ang I), which is then cleaved by angiotensin converting enzyme (ACE) to produce physiologically active angiotensin II (Ang II), and Ang II binds to angiotensin receptor (ATR), causing vasoconstriction and elevated blood pressure.

[0015] Although there are a large number of antihypertensive drugs currently used to treat hypertension, more than two-thirds of patients are difficult to control blood pressure by one drug, and need two or more drugs to control blood pressure, which leads to decreased therapy compliance, increased potential side effects, and affects the effectiveness of therapy. Therefore, there is a need in the art for alternative therapies and combination therapies for patients with angiotensinogen-related diseases. SUMMARY

[0016] The present disclosure relates to a GalNAc-conjugated double-stranded siRNA drug. After entering the blood, GalNAc can bind to the asialoglycoprotein receptor (ASGPR) on the surface of hepatocytes, and then the drug is endocytosed into the cells by hepatocytes and stored in the endosomal structure of the cells. After the drug is released from the endosome or lysosome into the cytoplasm, it binds to the RNA-induced silencing complex (RISC) and binds to the mRNA transcribed from the AGT gene under the mediation of the antisense strand, inducing mRNA degradation and thereby inhibiting the translation of AGT protein. AGT protein is an upstream protein of the renin-angiotensin-aldosterone system (RAAS), and inhibition of its expression will fundamentally inhibit the role of the RAAS system in elevating blood pressure, thereby reducing blood pressure.

[0017] The present disclosure designs a series of unique siRNA sequences for AGT mRNA sequences, and performs alternating modification and specific template modification.

[0018] Generally, siRNA uses 2'-methoxy (2'-OMe) and 2'-fluoro (2'-F) to modify monomers. However, even if only the above two monomer modifications are combined, there are 44 bases in the sense strand and the antisense strand of siRNA, i.e. there are 2 44 possible combinations. And, with different end thio-modification layouts, the number of possible modification schemes is even more numerous.

[0019] For the same siRNA sequence, the activity has a very large difference with different modification methods. For different siRNAs, the activity also has a very large difference with the same modification method. Although there are some modification principles for siRNA modification design, studies have shown that the activity cannot be accurately predicted according to the modification method, i.e. there is no definite relationship between the modification method and the activity. Therefore, it is very difficult to screen out a modification scheme with high activity from the countless possible modification combinations.

[0020] The present disclosure screens out some alternating modification and special modification sequences with significant inhibitory effect on AGT gene expression by chemically modifying the designed siRNA sequence.

[0021] In one aspect, the present disclosure provides a double-stranded RNAi agent comprising an oligonucleotide duplex consisting of a sense strand and an antisense strand.

[0022] In another aspect, the present disclosure provides a conjugate for reducing the expression of AGT, comprising the above-mentioned double-stranded RNAi agent, and a ligand conjugated therewith.

[0023] In another aspect, the present disclosure provides a nucleic acid protein composition comprising a double-stranded region of the above-mentioned double-stranded RNAi agent or an antisense strand of the double-stranded region, and a nuclease.

[0024] In another aspect, the present disclosure provides a recombinant vector comprising a nucleic acid molecule encoding the above-mentioned double-stranded RNAi agent.

[0025] In some embodiments, the vector backbone of the recombinant vector is selected from a recombinant virus-derived circular RNA vector, a tRNA, a rRNA scaffold, and a chimeric tRNA / pre-miRNA vector.

[0026] In another aspect, the present disclosure provides a recombinant cell that synthesizes and secretes the above-mentioned double-stranded RNAi agent.

[0027] In some embodiments, the recombinant cell is selected from the group consisting of R. sulfidophilum and C. glutamicum deficient in RNase III.

[0028] In another aspect, the present disclosure provides a method for preparing a double-stranded RNAi agent, comprising culturing the above-mentioned recombinant cell, or chemical synthesis.

[0029] In another aspect, the present disclosure provides a pharmaceutical composition comprising the above-mentioned double-stranded RNAi agent, the above-mentioned conjugate, or the above-mentioned nucleic acid-protein composition, and a pharmaceutically acceptable carrier.

[0030] In another aspect, the present disclosure provides a method for inhibiting AGT gene expression, comprising contacting the above-mentioned double-stranded RNAi agent, the above-mentioned conjugate, the above-mentioned nucleic acid-protein composition, or the above-mentioned pharmaceutical composition with a target cell.

[0031] In some embodiments, the method is for non-diagnostic or non-therapeutic purposes.

[0032] In some embodiments, the method is in vivo or in vitro.

[0033] In another aspect, the present disclosure provides 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 preparation of a medicament for treating a disease associated with AGT gene expression.

[0034] The disease associated with AGT gene expression is selected from the group consisting of overexpression of AGT protein, pathogenic mutation of AGT gene, abnormal metabolism of AGT protein, and disease caused by abnormal interaction of AGT with another substance.

[0035] In some embodiments, the disease associated with AGT gene expression is selected from the group consisting of hypertension, ocular hypertension, glaucoma, pulmonary arterial hypertension, portal hypertension, systemic venous hypertension, hypertensive heart disease, atherosclerosis, arteriosclerosis, vasculopathy, diabetic nephropathy, diabetic retinopathy, chronic heart failure, cardiomyopathy, diabetic cardiomyopathy, glomerulosclerosis, coarctation of the aorta, aortic aneurysm, ventricular fibrosis, Cushing's syndrome and other glucocorticoid excess states (including chronic steroid treatment), pheochromocytoma, reninoma, secondary hyperaldosteronism and other mineralocorticoid excess states, sleep apnea, thyroid / parathyroid disease, heart failure, myocardial infarction, angina pectoris, stroke, diabetes, kidney disease (e.g., hypertensive nephropathy), kidney failure, systemic sclerosis, intrauterine growth restriction (IUGR), and fetal growth restriction.

[0036] In another aspect, the present disclosure provides 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 for treating a disease associated with AGT gene expression.

[0037] In another aspect, the present disclosure provides a method for treating a disease associated with AGT gene expression, comprising administering to a subject in need thereof an effective amount of the double-stranded RNAi agent, the conjugate, the nucleic acid-protein composition, or the pharmaceutical composition described above.

[0038] In another aspect, the present disclosure provides a use of the double-stranded RNAi agent, the conjugate, the nucleic acid-protein composition, or the pharmaceutical composition described above in the preparation of a medicament for treating hypertension and cardiovascular and cerebrovascular diseases.

[0039] In another aspect, the present disclosure provides the double-stranded RNAi agent, the conjugate, the nucleic acid-protein composition, or the pharmaceutical composition described above for use in treating hypertension and cardiovascular and cerebrovascular diseases.

[0040] In another aspect, the present disclosure provides a method for treating hypertension and cardiovascular and cerebrovascular diseases, comprising administering to a subject in need thereof an effective amount of the double-stranded RNAi agent, the conjugate, the nucleic acid-protein composition, or the pharmaceutical composition described above.

[0041] The beneficial effects achieved by the present disclosure are at least as follows:

[0042] (1) The unmodified sequences, including unmodified sequences 1576s, 1578s, 1838, 1835, 1816, 1812, 1579, 1836, 1789, 1839, 1791, 1795, and 1585s, all have significant inhibitory effects on AGT, with the highest inhibition rate being more than 50%.

[0043] (2) The use of multiple modified sequences can achieve an inhibition rate of more than 70%. Moreover, the modified sequences are conjugated with GalNAc compounds, which can be efficiently delivered to the liver of animals and significantly inhibit AGT gene expression, thereby significantly reducing blood pressure levels.

[0044] (3) The present disclosure uses each sequence modified by alternating modification and the template modification of the present disclosure, and the unmodified sequences with little difference from the sequences disclosed in the prior art have significantly improved inhibitory activity on AGT, with an increase of up to 91.0%.

[0045] (4) The present disclosure finds that, whether it is an unmodified sequence or an alternating modified sequence, siRNAs with similar sequences have very large differences in activity. For example, the inhibition rate of unmodified sequence 1812 is increased by 14.3% compared to unmodified sequence 812P, and the inhibition rate of alternating modified sequence B1812-AL is increased by 20.4% compared to unmodified sequence 812P, which is significantly improved.

[0046] (5) The disclosure also found that different sequences after alternating modification, the effect on the activity is not consistent. Some significantly improved inhibition rate, for example, no modification sequence 836, alternating modification than no modification sequence inhibition rate increased by 13.3%; some are not obvious, for example, no modification sequence 994, 1279 and 1591, alternating modification sequence and no modification sequence inhibition rate is basically no change. BRIEF DESCRIPTION OF DRAWINGS

[0047] In order to more clearly illustrate the technical solutions of the embodiments of the disclosure, the drawings of the embodiments will be briefly introduced below. Obviously, the drawings described below only relate to some embodiments of the disclosure, not limit the disclosure.

[0048] Figure 1 The alternating modification sequence with significant inhibition effect on AGT gene is shown, and the inhibition rate of these sequences is higher than 40%.

[0049] Figure 2 The alternating modification sequence with inhibition rate between 25% and 40% on AGT gene is shown.

[0050] Figure 3 The alternating modification sequence with inhibition rate lower than 25% on AGT gene is shown.

[0051] Figure 4 The no modification sequence with significant inhibition effect on AGT gene is shown, and the inhibition rate of these sequences is higher than 45%.

[0052] Figure 5 The no modification sequence with inhibition rate lower than 45% on AGT gene is shown.

[0053] Figure 6 The inhibition rate of no modification sequence 1579 on AGT gene after using different templates for modification, and the inhibition rate of prior art sequence 579P on AGT gene are shown.

[0054] Figure 7 The inhibition rate of no modification sequence 1578s and 1835 on AGT gene after using templates for modification, and the inhibition rate of off-target prevention modification sequence on AGT gene are shown.

[0055] Figure 8 The inhibition rate of no modification sequence 1579, 1789, 1812, 1576s, 1578s, 1585s on AGT protein in animal serum after using different modification schemes and conjugating with GalNAc compound.

[0056] Figure 9The inhibition of AGT protein in serum of animals at different time points after the unmodified sequences 1579, 1789, 1812, 1576s, 1578s, 1585s were subjected to different modification schemes and conjugated with GalNAc compounds. DETAILED DESCRIPTION

[0057] To make the disclosure more readily understood, certain terms are first defined. Additionally, it should be noted that whenever a value or a range of values of a parameter is recited, it is intended that the intervening technically discernible values or ranges be encompassed, as well.

[0058] The articles "a" and "an" as used herein mean one or more than one (i.e., at least one) of the grammatical article's object. By way of example, "an element" means one element or more than one element, e.g., multiple elements.

[0059] The term "including" is used herein to mean, and is used interchangeably with, the phrase "including but not limited to".

[0060] The term "or" as used herein is used to mean, and is used interchangeably with, the term "and / or", unless context clearly indicates otherwise.

[0061] As used herein, the term "about" or "approximately," as applied to one or more values of a quantity, refers to a value that is similar to a stated reference value. In certain embodiments, unless otherwise stated, or otherwise apparent from context, the term "approximately" or "about" means falling within 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 (greater than or less than) of the stated reference value (unless this number would exceed 100% of the possible values).

[0062] As used herein, "AGT" refers to the angiotensinogen gene or protein.

[0063] “G,”“C,”“A,” and“U” each generally represent a nucleotide comprising, respectively, guanine, cytosine, adenine, and uracil as the base. “T,”“Td,” and“dT” are used interchangeably herein and refer to a deoxyribonucleotide in which the nucleobase is thymine, such as deoxyribothymine, 2’-deoxythymidine, or thymidine. However, it is understood that the term“ribonucleotide” or“nucleotide” or“deoxyribonucleotide” can also refer to a modified nucleotide (as further detailed below) or an alternative substitution moiety. The skilled artisan will be well aware that guanine, cytosine, adenine, and uracil can be substituted with other moieties without substantially altering the base pairing properties of an oligonucleotide, including a nucleotide having such substitution moieties. For example, without limitation, a nucleotide comprising inosine as its base can base pair with a nucleotide containing adenine, cytosine, or uracil. Thus, a nucleotide comprising uracil, guanine, or adenine can be substituted in a nucleotide sequence of the disclosure with a nucleotide comprising, for example, inosine. Sequences comprising such substitution moieties are embodiments of the disclosure.

[0064] The terms“iRNA,”“RNAi agent,”“iRNA agent,”“RNA interference agent” are used interchangeably herein, the defined terms include RNA agents, and can mediate transcript targeted cleavage through the RNA-induced silencing complex (RISC) pathway. iRNAs direct the sequence-specific degradation of mRNA through a process known as RNA interference (RNAi). iRNAs modulate, e.g., inhibit, expression of AGT in a cell, such as a cell in a subject, such as a mammalian subject, in vivo. RNAi molecules include single-stranded RNAi molecules (Lima et al 2012 Cell 150:883) and double-stranded siRNAs, as well as short hairpin RNAs (shRNAs).

[0065] The term "small interfering ribonucleic acid" or "siRNA" refers to a small interfering ribonucleic acid RNAi molecule. It is a class of double-stranded RNA molecules, also known in the art as short interfering RNA or silencing RNA. The siRNA typically comprises a sense strand (also referred to as a passenger strand) and an antisense strand (also referred to as a guide strand), each strand being 17 to 30 nucleotides in length, typically 19 to 25 nucleotides in length, wherein the antisense strand is complementary (such as at least 95% complementary, such as fully complementary) to a target nucleic acid (suitably a mature mRNA sequence), and the sense strand is complementary to the antisense strand, such that the sense and antisense strands form a duplex or duplex region. The siRNA strands can form a blunt end duplex, or preferably, the 3' ends of the sense and antisense strands can form 3' overhangs, for example 1, 2 or 3 nucleosides, similar to the Dicer produced product, which can form a RISC substrate in vivo. Efficient extended versions of the Dicer substrate have been described in US 8349809 and US 8513207, incorporated herein by reference. In some embodiments, both the sense and antisense strands have a 2nt 3' overhang. Thus, the duplex region can be, for example, 17 to 25 nucleotides in length, such as 21 to 23 nucleotides in length.

[0066] The term "antisense strand" refers to the strand of an RNAi (e.g., dsRNA) that comprises a region of substantial complementarity to a target sequence. As used herein, the term "region of complementarity" refers to a region on the antisense strand that is substantially complementary to a sequence defined herein (e.g., a target sequence). When the region of complementarity is not perfectly complementary to the target sequence, mismatches can be in the internal or terminal regions of the molecule. Generally, the most tolerated mismatches are in the terminal regions, e.g., within 5, 4, 3, or 2 nucleotides of the 5' and / or 3' terminus.

[0067] The term "sense strand" as used herein refers to the strand of an RNAi that comprises a region of substantial complementarity to a region of an antisense strand (as the term is defined herein).

[0068] The term "alternating modification" refers to a fully modified siRNA sequence with 2'-methoxy (2'-OMe) and 2'-fluoro (2'-F) alternating modifications. For the antisense strand of the siRNA sequence, odd positions (i.e., positions 1, 3, 5, 7...21, 23) are modified with 2'-methoxy and even positions (i.e., positions 2, 4, 6, 8...20, 22) are modified with 2'-fluoro. For the sense strand, which is complementary paired to the antisense strand, the position on the sense strand that is complementary paired to a position on the antisense strand modified with 2'-methoxy is modified with 2'-fluoro and the position on the sense strand that is complementary paired to a position on the antisense strand modified with 2'-fluoro is modified with 2'-methoxy.

[0069] The term "inhibit," as used herein, can be used interchangeably with "reduce," "silence," "down-regulate," "suppress," and other similar terms, and includes inhibition at any level.

[0070] As used herein, the phrase "inhibit expression of AGT" includes inhibiting expression of any AGT gene (such as, for example, a mouse AGT gene, a rat AGT gene, a monkey AGT gene, or a human AGT gene) as well as variants (e.g., naturally occurring variants) or mutants of AGT genes. Thus, the AGT gene can be a wild-type AGT gene, a mutant AGT gene, or a transgenic AGT gene in the context of a genetically manipulated cell, cell population, or organism.

[0071] "Inhibit expression of an AGT gene" includes inhibition of an AGT gene at any level, such as at least partial inhibition of expression of an AGT gene, such as inhibition of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%.

[0072] Expression of an AGT gene can be assessed based on any variable level associated with AGT gene expression, such as AGT mRNA level or AGT protein level. Inhibition can be assessed by a decrease in the absolute or relative level of one or more of these variables compared to a control level. The control level can be any type of control level utilized in the art, such as a pre-dose baseline level or a level determined from a similar untreated or control (e.g., buffer control or inert agent control) treated subject, cell, or sample.

[0073] As used herein, "patient" or "subject" is intended to include a human or non-human animal, preferably a mammal, such as a monkey. Most preferably, the subject or patient is a human.

[0074] As used herein, "AGT-associated disease" is intended to include any disease associated with the AGT gene or protein. Such a disease can be caused, for example, by overproduction of the AGT protein, by mutation of the AGT gene, by abnormal cleavage of the AGT protein, by abnormal interactions between AGT and other proteins or other endogenous or exogenous agents. Exemplary AGT-associated diseases include hypertension, borderline hypertension, essential hypertension, secondary hypertension, hypertensive crisis, hypertensive emergency, isolated systolic and diastolic hypertension, pregnancy-associated hypertension, diabetic hypertension, resistant hypertension, refractory hypertension, paroxysmal hypertension, renovascular hypertension, Goldblatt hypertension, ocular hypertension, glaucoma, pulmonary hypertension, portal hypertension, systemic venous hypertension, systolic hypertension, labile hypertension; hypertensive heart disease, hypertensive nephropathy, atherosclerosis, arteriosclerosis, vasculopathy, diabetic nephropathy, diabetic retinopathy, chronic heart failure, cardiomyopathy, diabetic cardiomyopathy, glomerulosclerosis, coarctation of the aorta, aortic aneurysm, ventricular fibrosis, Cushing's syndrome and other glucocorticoid excess states (including chronic steroid treatment), pheochromocytoma, reninoma, secondary hyperaldosteronism and other mineralocorticoid excess states, sleep apnea, thyroid / parathyroid disease, heart failure, myocardial infarction, angina, stroke, diabetes, kidney disease, kidney failure, systemic sclerosis, intrauterine growth restriction (IUGR), and fetal growth restriction.

[0075] As used herein, "therapeutically effective amount" is intended to include the amount of an RNAi agent that, when administered to a patient for treating an AGT-associated disease, is sufficient to effect treatment (e.g., by diminishing, ameliorating or maintaining the existing disease or one or more symptoms of the disease). The "therapeutically effective amount" can vary depending on the RNAi agent, how the agent is administered, the disease and its severity, and the history, age, weight, family history, genetic makeup, stage of pathological processes mediated by AGT expression, type of previous or concomitant treatments, if any, and other individual characteristics of the patient to be treated.

[0076] As used herein, "prophylactically effective amount" refers to an amount of an RNAi agent that, when administered to a subject who does not yet exhibit or display symptoms of an AGT-associated disease, but who is at risk of developing the disease, is sufficient to prevent or ameliorate the disease or one or more symptoms of the disease. Ameliorating a disease includes slowing the progression of the disease or reducing the severity of the disease that develops later. The "prophylactically effective amount" can vary depending on the RNAi agent, how the agent is administered, the degree of risk of the disease, and the history, age, weight, family history, genetic makeup, type of previous or concomitant treatments, if any, and other individual characteristics of the patient to be treated.

[0077] A "therapeutically effective amount" or "prophylactically effective amount" also includes an amount of an RNAi agent that produces some desired local or systemic effect at a reasonable benefit / risk ratio applicable to any treatment. The RNAi agents used in the methods of the disclosure can be administered in an amount sufficient to produce a reasonable benefit / risk ratio applicable to such treatment.

[0078] As used herein, the term "sample" includes a similar fluid, cell, or tissue isolated from a subject, as well as a collection of fluids, cells, or tissues present in a subject. Examples of biological fluids include blood, serum and plasma, cerebrospinal fluid, ocular fluid, lymphatic fluid, urine, saliva, and the like. A tissue sample can include a sample from a tissue, organ, or a localized region. For example, a sample can be derived from a particular organ, organ portion, or fluid or cells within these organs. In certain embodiments, a sample can be derived from a liver (e.g., the entire liver or certain segments of the liver, or certain types of cells in the liver, e.g., hepatocytes). In preferred embodiments, a "sample derived from a subject" refers to blood or plasma drawn from the subject. In other embodiments, a "sample derived from a subject" refers to liver tissue (or a sub- constituent thereof) derived from the subject.

[0079] In one aspect, provided herein is a double stranded RNAi agent for reducing the expression of AGT, comprising any of the following oligonucleotide duplexes, wherein the sense strand is paired with the antisense strand:

[0080] (1) the sense strand has a sequence as set forth in SEQ ID NO: 1, or a fragment thereof, or a modified sequence of the sequence or fragment thereof; and the antisense strand has a sequence as set forth in SEQ ID NO: 14, or a fragment thereof, or a modified sequence of the sequence or fragment thereof;

[0081] (2) the sense strand has a sequence as set forth in SEQ ID NO: 2, or a fragment thereof, or a modified sequence of the sequence or fragment thereof; and the antisense strand has a sequence as set forth in SEQ ID NO: 15, or a fragment thereof, or a modified sequence of the sequence or fragment thereof;

[0082] (3) the sense strand has a sequence as set forth in SEQ ID NO: 3, or a fragment thereof, or a modified sequence of the sequence or fragment thereof; and the antisense strand has a sequence as set forth in SEQ ID NO: 16, or a fragment thereof, or a modified sequence of the sequence or fragment thereof;

[0083] (4) the sense strand has a sequence as set forth in SEQ ID NO: 4, or a fragment thereof, or a modified sequence of the sequence or fragment thereof; and the antisense strand has a sequence as set forth in SEQ ID NO: 17, or a fragment thereof, or a modified sequence of the sequence or fragment thereof;

[0084] (5) the sense strand has a sequence as set forth in SEQ ID NO: 5, or a fragment thereof, or a modified sequence of the sequence or the fragment thereof; and the antisense strand has a sequence as set forth in SEQ ID NO: 18, or a fragment thereof, or a modified sequence of the sequence or the fragment thereof;

[0085] (6) the sense strand has a sequence as set forth in SEQ ID NO: 6, or a fragment thereof, or a modified sequence of the sequence or the fragment thereof; and the antisense strand has a sequence as set forth in SEQ ID NO: 19, or a fragment thereof, or a modified sequence of the sequence or the fragment thereof;

[0086] (7) the sense strand has a sequence as set forth in SEQ ID NO: 7, or a fragment thereof, or a modified sequence of the sequence or the fragment thereof; and the antisense strand has a sequence as set forth in SEQ ID NO: 20, or a fragment thereof, or a modified sequence of the sequence or the fragment thereof;

[0087] (8) the sense strand has a sequence as set forth in SEQ ID NO: 8, or a fragment thereof, or a modified sequence of the sequence or the fragment thereof; and the antisense strand has a sequence as set forth in SEQ ID NO: 21, or a fragment thereof, or a modified sequence of the sequence or the fragment thereof;

[0088] (9) the sense strand has a sequence as set forth in SEQ ID NO: 9, or a fragment thereof, or a modified sequence of the sequence or the fragment thereof; and the antisense strand has a sequence as set forth in SEQ ID NO: 22, or a fragment thereof, or a modified sequence of the sequence or the fragment thereof;

[0089] (10) the sense strand has a sequence as set forth in SEQ ID NO: 10, or a fragment thereof, or a modified sequence of the sequence or the fragment thereof; and the antisense strand has a sequence as set forth in SEQ ID NO: 23, or a fragment thereof, or a modified sequence of the sequence or the fragment thereof;

[0090] (11) the sense strand has a sequence as set forth in SEQ ID NO: 11, or a fragment thereof, or a modified sequence of the sequence or the fragment thereof; and the antisense strand has a sequence as set forth in SEQ ID NO: 24, or a fragment thereof, or a modified sequence of the sequence or the fragment thereof;

[0091] (12) the sense strand has a sequence as set forth in SEQ ID NO: 12, or a fragment thereof, or a modified sequence of the sequence or the fragment thereof; and the antisense strand has a sequence as set forth in SEQ ID NO: 25, or a fragment thereof, or a modified sequence of the sequence or the fragment thereof;

[0092] (12) the sense strand has a sequence as shown in SEQ ID NO: 13, or a fragment thereof, or a modification sequence of the sequence or fragment thereof; and the antisense strand has a sequence as shown in SEQ ID NO: 26, or a fragment thereof, or a modification sequence of the sequence or fragment thereof. In some embodiments, all nucleotides on the sense and antisense strands are modified nucleotides.

[0093] In some embodiments, the double stranded RNAi agent is an RNAi agent for inhibiting expression of an AGT gene.

[0094] In some embodiments, the sense strand differs from any one of SEQ ID NOs: 1-13 by 1-3 nucleotides.

[0095] In some embodiments, the antisense strand differs from any one of SEQ ID NOs: 14-26 by 1-3 nucleotides.

[0096] In some embodiments, at least one modified nucleotide is selected from the group consisting of: a deoxy-nucleotide, a 3'-terminal deoxy-thymine (dT) nucleotide, a 2'-O-methyl modified nucleotide, a 2'-fluoro modified nucleotide, a 2'-deoxy-modified nucleotide, a locked nucleotide, a non-locked nucleotide, a conformationally restricted nucleotide, a constrained ethyl nucleotide, an abasic nucleotide, a 2'-amino-modified nucleotide, a 2'-O-allyl-modified nucleotide, a 2'-C-alkyl-modified nucleotide, a 2'-hydroxyl-modified nucleotide, a 2'-methoxyethyl modified nucleotide, a 2'-O-alkyl-modified nucleotide, a morpholino nucleotide, a phosphoramidate, a non-natural base containing nucleotide, a tetrahydropyran modified nucleotide, a 1,5- anhydrohexitol modified nucleotide, a cyclohexenyl modified nucleotide, a nucleotide containing a phosphorothioate group, a nucleotide containing a methylphosphonate group, a nucleotide containing a 5'-phosphate, and a nucleotide containing a 5'-phosphate mimic.

[0097] In some embodiments, at least one strand comprises a 3' overhang of at least 1 nucleotide.

[0098] In some embodiments, at least one strand comprises a 3' overhang of at least 2 nucleotides.

[0099] In some embodiments, the double stranded region is 15-30 pairs of nucleotides in length.

[0100] In some embodiments, the double stranded region is 17-25 pairs of nucleotides in length.

[0101] In some embodiments, the double stranded region is 19-23 pairs of nucleotides in length.

[0102] In some embodiments, the double-stranded region is 21 pairs of nucleotides in length.

[0103] In some embodiments, each strand has 15-30 nucleotides.

[0104] In some embodiments, each strand has 19-25 nucleotides.

[0105] In some embodiments, the sense strand has 21 nucleotides and the antisense strand has 23 nucleotides.

[0106] In some embodiments, all nucleotide modifications on the sense and antisense strands are chemical modifications at the 2' position of the ribose sugar of the nucleotide.

[0107] In some embodiments, the chemical modification at the 2' position of the ribose sugar of the nucleotide is selected from any one or a combination of several of 2'-methoxy, 2'-methoxy ethyl, 2'-fluoro, 2'-benzyloxy, 2'-methylcarbonylamino, and 2'-pyridyimethoxy.

[0108] In some embodiments, the chemical modification at the 2' position of the ribose sugar of each nucleotide is selected from a combination of 2'-methoxy and 2'-fluoro.

[0109] In some embodiments, the chemical modification at the 2' position of the ribose sugar of each nucleotide is selected from an alternating combination of 2'-methoxy and 2'-fluoro.

[0110] In some embodiments, the chemical modification at the 2' position of the ribose sugar of each nucleotide is such that: the odd positions of the sense strand are all 2'-fluoro modified, and the even positions of the sense strand are all 2'-methoxy modified; the odd positions of the antisense strand are all 2'-methoxy modified, and the even positions of the antisense strand are all 2'-fluoro modified.

[0111] In some embodiments, the linkage between nucleotide monomers is a 3',5'-phosphodiester bond.

[0112] In some embodiments, the linkage between nucleotide monomers is a 3',5'-phosphorothioate bond.

[0113] In some embodiments, the foregoing oligonucleotide has the following modification: the antisense strand has one of the modifications shown in Table 42:

[0114] Table 42: Modifications of Antisense Strand

[0115]

[0116]

[0117] the sense strand has one of the modifications shown in Table 43:

[0118] Table 43: Modifications of Sense Strand

[0119]

[0120] 2'-OMe = 2'-methoxy; 2'-F = 2'-fluoro; PS = phosphorothioate backbone

[0121] wherein the antisense strand is modified with Modification A and the sense strand is modified with modification type a;

[0122] wherein the antisense strand is modified with Modification B and the sense strand is modified with modification type a;

[0123] wherein the antisense strand is modified with Modification C and the sense strand is modified with modification type a;

[0124] wherein the antisense strand is modified with Modification B and the sense strand is modified with modification type b;

[0125] wherein the antisense strand is modified with Modification C and the sense strand is modified with modification type b;

[0126] wherein the antisense strand is modified with Modification D and the sense strand is modified with modification type b;

[0127] wherein the antisense strand is modified with Modification E and the sense strand is modified with modification type b;

[0128] wherein the antisense strand is modified with Modification F and the sense strand is modified with modification type b.

[0129] In some embodiments, the foregoing oligonucleotide has the following modification: the antisense strand is modified with one of the modification types indicated in Table 44:

[0130] Table 44: Antisense strand modifications

[0131]

[0132] the sense strand is modified with one of the modification types indicated in Table 45:

[0133] Table 45: Sense strand modifications

[0134]

[0135] 2'-OMe = 2'-methoxy; 2'-F = 2'-fluoro; PS = phosphorothioate backbone; wherein the antisense strand is modified with Modification A and the sense strand is modified with modification type a;

[0136] wherein the antisense strand is modified with Modification B and the sense strand is modified with modification type a;

[0137] wherein the antisense strand is modified with Modification C and the sense strand is modified with modification type a;

[0138] wherein the antisense strand is modified with Modification B and the sense strand is modified with modification type b;

[0139] wherein the antisense strand is modified with modification C and the sense strand is modified with modification b;

[0140] wherein the antisense strand is modified with modification D and the sense strand is modified with modification b;

[0141] wherein the antisense strand is modified with modification E and the sense strand is modified with modification b;

[0142] wherein the antisense strand is modified with modification F and the sense strand is modified with modification b.

[0143] In some embodiments, the antisense strand is modified with a modification group at the second through eighth positions from the 5' end, wherein the modification group is selected from UNA, GNA, or DNA, wherein the UNA and GNA structures are as follows:

[0144]

[0145] wherein the base is selected from adenine, guanine, cytosine, thymine, and uracil.

[0146] In some embodiments, the 5' position carbon atom of the glycoside of the 5' terminal nucleotide of the modified antisense strand is phosphorylated, including but not limited to the following 5' phosphorylation groups: 5'-vinyl phosphonate group (5'-E-VP); 5'-methyl phosphonate group (5'-MP); 5'-C-methyl phosphate group; 5'-phosphorothioate group (5'-PS); 5'-phosphate group (5'-P), which are illustrated as follows:

[0147]

[0148] wherein R is hydrogen, hydroxyl, amine, C 1-4 alkyl, aryl, C 1-4 alkoxy, C 1-4 alkylcarbonylamino, or halogen;

[0149] wherein the base is selected from adenine, guanine, cytosine, thymine, and uracil.

[0150] In some embodiments, the 3', 5'-phosphodiester linkages between the nucleotide monomers at the end of the sequence shown are modified with a sulfur and form a chiral pure 3', 5'-phosphorothioate linkage, wherein the 5' end of the sense strand and the antisense strand contain 1-3 sulfur modifications and the 3' end of the antisense strand contains 1-3 sulfur modifications.

[0151] The double stranded RNA (dsRNA) agents of the present disclosure can be optionally conjugated to one or more ligands. The ligand can be attached to the sense strand, the antisense strand, or both strands at the 3’ end, the 5’ end, or both ends. For example, the ligand can be conjugated to the sense strand. In preferred embodiments, the ligand is bound to the 3’ end of the sense strand. In a preferred embodiment, the ligand is a GalNAc ligand.

[0152] The present disclosure provides a conjugate for reducing expression of AGT, comprising the double stranded RNAi agent described, and a ligand conjugated thereto.

[0153] In some embodiments, the ligand is conjugated at the 3'-terminal or 5'-terminal end of the oligonucleotide sense strand.

[0154] In some embodiments, the ligand is one or more GalNAc derivatives attached using a divalent or trivalent branched linker.

[0155] In some embodiments, the ligand is:

[0156]

[0157] wherein X is hydrogen or a hydroxyl protecting group comprising acetyl, benzoyl, or isobutyryl; Y is an amine protecting group or H, the amine protecting group being formyl, acetyl, propionyl, n-butyryl, or isobutyryl; n is an integer between 0-20; q, r, and s are independently integers between 1-7.

[0158] In some embodiments, the ligand is:

[0159]

[0160] In some embodiments, the ligand is:

[0161]

[0162] wherein X is oxygen, nitrogen, or sulfur;

[0163] Y is an alkyl or aryl group;

[0164] R1is oxygen or sulfur;

[0165] R2is hydrogen, an amine group, a C 1-4 alkyl, aryl, C 1-4 alkoxy, or halogen;

[0166] A is -(CH2) a -, -(CH2CH2O) b -, -((CH2) c NHCO) d- -((CH2) c CONH) d - wherein a is an integer from 1 to 15, b is an integer from 1 to 7, c is an integer from 1 to 7, and d is an integer from 1 to 5;

[0167] B is -(CH2) e - wherein e is an integer from 0 to 7;

[0168] L is -CONH- or -NHCO-;

[0169] X1is -(CH2) f - or -(CH2CH2O) f CH2-, and f is an integer from 1 to 5;

[0170] X2is -(CH2) g - and g is an integer from 1 to 6;

[0171] Y1is 0 or 1;

[0172] Y2is 0, 1, or 2;

[0173] Y3is 1, 2, or 3;

[0174] m is an integer from 0 to 4;

[0175] n is an integer from 0 to 4.

[0176] In some embodiments, the ligand is G4, G5, G6, or G7:

[0177]

[0178]

[0179] In some embodiments, the conjugate has the structure shown below:

[0180]

[0181]

[0182] In some embodiments, the double stranded RNAi agent comprises any of the following oligonucleotide duplexes selected from the pair of sense and antisense strands:

[0183] (1) the sense strand has the sequence as shown in SEQ ID NO: 423; and the antisense strand has the sequence as shown in SEQ ID NO: 488;

[0184] (2) the sense strand has the sequence as shown in SEQ ID NO: 406; and the antisense strand has the sequence as shown in SEQ ID NO: 453;

[0185] (3) the sense strand has the sequence of SEQ ID NO: 404; and the antisense strand has the sequence of SEQ ID NO: 450;

[0186] (4) the sense strand has the sequence of SEQ ID NO: 426; and the antisense strand has the sequence of SEQ ID NO: 492;

[0187] (5) the sense strand has the sequence of SEQ ID NO: 412; and the antisense strand has the sequence of SEQ ID NO: 465;

[0188] (6) the sense strand has the sequence of SEQ ID NO: 412; and the antisense strand has the sequence of SEQ ID NO: 468;

[0189] (7) the sense strand has the sequence of SEQ ID NO: 421; and the antisense strand has the sequence of SEQ ID NO: 483;

[0190] (8) the sense strand has the sequence of SEQ ID NO: 417; and the antisense strand has the sequence of SEQ ID NO: 476;

[0191] (9) the sense strand has the sequence of SEQ ID NO: 419; and the antisense strand has the sequence of SEQ ID NO: 479;

[0192] (10) the sense strand has the sequence of SEQ ID NO: 401; and the antisense strand has the sequence of SEQ ID NO: 443;

[0193] (11) the sense strand has the sequence of SEQ ID NO: 398; and the antisense strand has the sequence of SEQ ID NO: 433;

[0194] (12) the sense strand has the sequence of SEQ ID NO: 429; and the antisense strand has the sequence of SEQ ID NO: 496;

[0195] (13) the sense strand has the sequence of SEQ ID NO: 414; and the antisense strand has the sequence of SEQ ID NO: 471;

[0196] wherein the oligonucleotide duplex is conjugated to ligand G4, G5, G6, or G7.

[0197] In some embodiments, the double stranded RNAi agent comprises any of the following oligonucleotide duplexes paired by a sense strand and an antisense strand selected from the group consisting of:

[0198] (1) the sense strand has the sequence as shown in SEQ ID NO: 423; and the antisense strand has the sequence as shown in SEQ ID NO: 488;

[0199] (2) the sense strand has the sequence as shown in SEQ ID NO: 423; and the antisense strand has the sequence as shown in SEQ ID NO: 489;

[0200] (3) the sense strand has the sequence as shown in SEQ ID NO: 424; and the antisense strand has the sequence as shown in SEQ ID NO: 489;

[0201] (3) the sense strand has the sequence as shown in SEQ ID NO: 424; and the antisense strand has the sequence as shown in SEQ ID NO: 490;

[0202] (3) the sense strand has the sequence as shown in SEQ ID NO: 424; and the antisense strand has the sequence as shown in SEQ ID NO: 488;

[0203] wherein the oligonucleotide duplex is conjugated to Ligand G4, G5, G6 or G7.

[0204] In some embodiments, the double stranded RNAi agent comprises an oligonucleotide duplex paired by a sense strand as set forth in SEQ ID NO: 423 and an antisense strand as set forth in SEQ ID NO: 488, which is conjugated to Ligand G5.

[0205] The present disclosure also provides a pharmaceutical composition comprising the double stranded RNAi agent or conjugate described, and a pharmaceutically acceptable carrier.

[0206] In one embodiment, provided herein is a pharmaceutical composition comprising an iRNA as described herein and a pharmaceutically acceptable carrier. Pharmaceutical compositions comprising an iRNA can be used to treat a disease or disorder associated with expression or activity of the AGT gene, such as hypertension. Such pharmaceutical compositions are formulated based on the delivery model. One example is a composition formulated for systemic administration by parenteral delivery, e.g., by subcutaneous injection (S.C.). Another example is a composition formulated for direct delivery to the brain parenchyma, e.g., by infusion into the brain, e.g., by continuous pump infusion.

[0207] Pharmaceutical compositions comprising the RNAi agents of the disclosure can be, for example, solutions with or without a buffer or compositions containing a pharmaceutically acceptable carrier. Such compositions include, for example, aqueous or crystalline compositions, liposome formulations, micelle formulations, emulsions, and gene therapy vectors.

[0208] In the methods of the disclosure, the RNAi agent can be administered in a solution. A free RNAi agent can be administered in a non-buffered solution, for example, in physiological saline or in water. Alternatively, the free siRNA can also be administered in a suitable buffered solution. The buffered solution can include acetate, citrate, prolamine, carbonate, or phosphate, or any combination thereof. In a preferred embodiment, the buffered solution is phosphate buffered saline (PBS). The pH and the osmolality of the buffer solution comprising the iRNA agent can be adjusted such that it is suitable for administration to a subject.

[0209] In some embodiments, the buffered solution further comprises an agent for controlling the osmolality of the solution such that the osmolality is maintained at a desired value, for example, at the physiological value of human plasma. Solutes that can be added to the buffered solution to control the osmolality include, but are not limited to, proteins, peptides, amino acids, non-metabolizable polymers, vitamins, ions, sugars, metabolites, organic acids, lipids, or salts. In some embodiments, the agent for controlling the osmolality of the solution is a salt. In certain embodiments, the agent for controlling the osmolality of the solution is sodium chloride or potassium chloride.

[0210] The pharmaceutical compositions of the disclosure can be administered in a dosage sufficient to inhibit expression of the AGT gene. Generally, a suitable dosage of the iRNA of the disclosure is in the range of about 0.001 to about 200.0 milligrams per kilogram of body weight of the recipient per day, typically in the range of about 1 to 50 mg per kilogram of body weight per day. For example, an RNAi agent (e.g., dsRNA) can be administered at about 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, 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 about 50 mg / kg per single dose.

[0211] The pharmaceutical composition can be administered once daily, or multiple times at various intervals from 1 to 365 days, or the iRNA can be administered in two, three or more sub-doses at appropriate intervals throughout the year, or even continuously via a controlled release formulation using a continuous infusion or delivery. In this case, the iRNA contained in each sub-dose must be correspondingly less so as to achieve the total daily dose. Dose units can also be compounded for delivery over several days, for example using conventional sustained release formulations that provide a sustained release of iRNA over a period of several days. Sustained release formulations are well known in the art and are particularly useful for delivering agents at a particular site, and can be used with the agents of the present disclosure. In this embodiment, the dose unit comprises a corresponding plurality of daily doses.

[0212] In other embodiments, a single dose of the pharmaceutical composition can be sustained for a long duration, such that subsequent doses are administered at intervals of no more than 3, 4, or 5 days or at intervals of no more than 1, 2, 3, or 4 weeks. In some embodiments of the disclosure, a single dose of the pharmaceutical composition of the disclosure is administered once per week. In other embodiments of the disclosure, a single dose of the pharmaceutical composition of the disclosure is administered once per month.

[0213] Those of skill in the art will appreciate that certain factors can influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or condition, previous treatments, the general health and / or age of the subject, and other existing diseases. In addition, treatment of a subject with a therapeutically effective dose of the composition can include a single treatment or a series of treatments. As described elsewhere herein, effective doses and in vivo half-lives of the various iRNAs encompassed by the present disclosure can be estimated using conventional methods or based on in vivo testing in suitable animal models.

[0214] The pharmaceutical compositions of the disclosure can be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration can be topical (e.g., through a skin patch), pulmonary, e.g., by inhalation or insufflation of powders or aerosols, including by nebulizers; intratracheal; intranasal; epidermal and transdermal, oral or parenteral. Parenteral administration includes subcutaneous, intravenous, intraarterial, subdermal, intraperitoneal or intramuscular injection or infusion; subdermal, e.g., via implantation devices; or intracranial, e.g., intracerebral, intrathecal or intraventricular, administration.

[0215] The iRNAs for use in the compositions and methods of the disclosure can be formulated for delivery in a membranous molecular assembly, e.g., a liposome or a micelle. As used herein, the term "liposome" refers to a vesicle composed of amphiphilic lipids arranged in a bilayer (e.g., one bilayer or multiple bilayers) configuration. Liposomes include unilamellar or multilamellar vesicles which have a membrane that is formed from a lipophilic material and an aqueous interior. The aqueous portion contains the iRNA composition. The lipophilic material separates the aqueous interior from the aqueous exterior (although in some instances it can include the iRNA composition). Liposomes are useful for transferring and delivering active ingredients to a site of action. Because the liposome membrane is structurally similar to a biological membrane, when a liposome is administered to a tissue, the liposome bilayer fuses with the bilayer of a cell membrane. As fusion of the liposome with the cell proceeds, the internal aqueous contents, including the iRNA, are delivered into the cell, where the iRNA can specifically bind to a target RNA and can mediate RNAi. In some cases, the liposomes are also specifically targeted, e.g., to direct the iRNA to a particular cell type.

[0216] Liposomes comprising an RNAi agent can be prepared by a variety of methods. In one example, the lipid components of the liposome are dissolved in a detergent such that micelles are formed with the lipid components. For example, the lipid components can be amphipathic cationic lipids or lipid conjugates. The detergent can have a high critical micelle concentration and can be non-ionic. Exemplary detergents include cholate, CHAPS, octyl glucoside, deoxycholate, and lauroyl sarcosine. The RNAi agent formulation is then added to the micelles comprising the lipid components. The cationic groups on the lipids interact with the RNAi agent and condense around the RNAi agent to form liposomes. After condensation, the detergent is removed, e.g., by dialysis, to obtain a liposomal formulation of the RNAi agent.

[0217] An iRNA, e.g., a dsRNA of the disclosure, can be fully encapsulated in a lipid formulation, e.g., an LNP or other nucleic acid-lipid particle.

[0218] As used herein, the term "LNP" refers to a stable nucleic acid-lipid particle. LNPs contain a cationic lipid, a non-cationic lipid, and a lipid that prevents the particle from aggregating (e.g., a PEG-lipid conjugate). LNPs are extremely useful for synthetic applications because they exhibit extended circulation lifetimes following intravenous (i.v.) injection and accumulate at distal sites (e.g., at sites physically separate from the site of administration).

[0219] In one embodiment, the ratio of lipid to drug (mass / mass ratio) (e.g., the ratio of lipid to dsRNA) will be in a range from about 1 : 1 to about 50: 1, from about 1 : 1 to about 25: 1, from about 3: 1 to about 15: 1, from about 4: 1 to about 10: 1, from about 5: 1 to about 9: 1, or about 6: 1 to about 9: 1.

[0220] In some preferred embodiments, the lipid nanoparticle comprises a cationic lipid, a neutral lipid, a structural lipid, a polymer conjugated lipid.

[0221] In some preferred embodiments, the cationic lipid is a compound of structure (I), or an N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer thereof, wherein G1is C 1～6 alkylene; G2is C 2～8 alkylene; G3is C 1～3 alkylene; L1is C 6～15 linear alkyl; L2is C 12～25 branched alkyl. For example, YK-009 of structure (I-I) (see patent CN114044741B).

[0222]

[0223] In some preferred embodiments, the cationic lipid is a compound of structure (II), or an N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer thereof, wherein: G1is C 2～8 alkylene; G2is C 2～8 alkylene; L1is -C(O)O- or -OC(O)-; L2is -C(O)O- or -OC(O)-; R1is C 6～25 linear or branched alkyl; R2is C 6～25 linear or branched alkyl; G3is HO(CH2)2- or HO(CH2)3-; G4is HO(CH2)2- or HO(CH2)3-; L is (CH2)2- or -(CH2)3- or -(CH2)4-. For example, YK-401 of structure (II-I), YK-402 of structure (II-II) (see patent CN115784921B).

[0224]

[0225]

[0226] In some preferred embodiments, the cationic lipid is a compound of structure (III), or an N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer thereof, wherein: G1is C1～6 Alkylene; G2 is C 2～8 Alkylene; R1 is C 6～20 Straight-chain or branched alkyl; R2 is C 12～25 Branched alkyl groups; G3 is: HO(CH2)2N(CH3)(CH2)2-, HO(CH2)2N(CH2CH3)(CH2)2-, (HO(CH2)2)2N(CH2)2-, CH3O(CH2)2N(CH3)(CH2)2-, (CH3)2N(CH2)3SC(O)O(CH2)2-, (CH3)2N(CH2)3SC(O)-, CH3NH(CH2)2N(CH3)(CH2)2-, or CH3CH2NH(CH2)2-. For example, YK-201 with formula (III-I) structure, YK-202 with formula (III-II) structure (see patent CN115677518B).

[0227]

[0228] In some preferred embodiments, the cationic lipid is a compound of formula (IV), or its N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer, wherein G1 is C 1～8 Alkylene; G2 is C 2～8 Alkylene; R1 is C 6～25 Straight-chain or branched alkyl; R2 is C 12～25 Straight-chain or branched alkyl; G3 is: HO(CH2)2N(R3)CH2CH(OH)CH2-, where R3 is -CH3, -CH2CH3, or -CH2CH2OH. For example, YK-305 with formula (IV-I) and YK-310 with formula (IV-II) (see patent CN115745820B).

[0229]

[0230] In some preferred embodiments, the cationic lipid is a compound of formula (V), or its N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer, wherein G 1 and G 2 Each is independently unsubstituted C6-C 10 Alkylene; G 3 For unsubstituted C1-C 12 Alkylene; R 1 and R 2 Each independently is C6-C 24 Alkyl or C6-C 24 alkenyl; R 3 OR 5 N, -C (=O) OR4 -OC(=O)R 4 or -NR 5 C(=O)R 4 ; R 4 is a C1-C 12 alkyl group; and R 5 is H or a C1-C6alkyl group; for example, ALC0315 of structure (V-I) (see patent CN108368028B);

[0231]

[0232]

[0233] In some preferred embodiments, the cationic lipid is a compound of structure (VI), or an N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer thereof, wherein R4is selected from -(CH2) n Q and -(CH2) n CHQR; Q is selected from the group consisting of -OR, -OH, -O(CH2) n 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 a heterocycle; n is 1, 2, or 3; for example, SM102 of structure (VI-I) (see patent application CN110520409A).

[0234]

[0235] In some preferred embodiments, the cationic lipid is a compound of structure (VII), or an N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer thereof (see patent CN102625696B, DLIN-MC3-DMA),

[0236]

[0237] In some more preferred embodiments, the cationic lipid comprises YK-009, YK-401, YK-305, ALC0315, SM102, DLIN-MC3-DMA.

[0238] In some preferred embodiments, the molar ratio of the cationic lipid to the neutral lipid is 1 : 1 to 10: 1.

[0239] In some preferred embodiments, the molar ratio of the cationic lipid to the structural lipid is 1 : 1 to 5: 1.

[0240] 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).

[0241] 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).

[0242] In some more 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.

[0243] In some preferred embodiments, the neutral lipid comprises one or more of phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, ceramide, sterol, and derivatives thereof.

[0244] In some more preferred embodiments, the neutral lipid is selected from one or more of: 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-didodecanoyl-sn-glycero-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), dipalmitoyl phosphatidylglycerol (DPPG), palmitoyloleoyl phosphatidylethanolamine (POPE), distearoyl-phosphatidyl-ethanolamine (DSPE), dipalmitoyl phosphatidylethanolamine (DPPE), dimyristoyl phosphoethanolamine (DMPE), 1-stearoyl-2-oleoyl-stearoylethanolamine (SOPE), 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC), sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoyl phosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine (LPE), and mixtures thereof.

[0245] In some more preferred embodiments, the neutral lipid is DOPE and / or DSPC.

[0246] In some preferred embodiments, the structural lipid is selected from one or more of the following: cholesterol, non-sterols, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, ursolic acid, a-tocopherol, corticosteroids.

[0247] In some more preferred embodiments, the structural lipid is cholesterol.

[0248] In some preferred embodiments, the polymeric 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, PEG-modified dialkylglycerol.

[0249] In some more preferred embodiments, the polymeric conjugated lipid is selected from one or more of the following: distearoylphosphatidylethanolamine polyethylene glycol 2000 (DSPE-PEG2000), dimyristoylglycerol-3-methoxypolyethylene glycol 2000 (DMG-PEG2000), and methoxypolyethylene glycol bismyristyl acetyl amide (ALC-0159).

[0250] Pharmaceutical compositions of the present disclosure include, but are not limited to, solutions, emulsions, and liposome-containing formulations. These compositions can be generated from a variety of components that include, but are not limited to, preformed liquids, self-emulsifying solids, and self-emulsifying semisolids. Particularly preferred are formulations that target the liver when treating liver disorders, such as liver cancer.

[0251] Pharmaceutical formulations of the present disclosure, which can conveniently be presented in unit dosage form, can be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredients with a liquid carrier or a finely divided solid carrier or both, and then, if necessary, shaping the product.

[0252] Compositions of the present disclosure can be formulated in any of a number of possible dosage forms, such as, but not limited to, tablets, capsules, gelcaps, liquid syrups, soft gels, suppositories, and enemas. Compositions of the present disclosure can also be formulated as suspensions in aqueous, non-aqueous, or mixed media. Aqueous suspensions can further comprise a viscosifying agent to increase the viscosity of the suspension, such agents including, for example, sodium carboxymethylcellulose, sorbitol, and / or dextran. The suspension can also comprise a stabilizing agent.

[0253] Certain compositions of the present disclosure also incorporate a carrier compound into the formulation. As used herein, "carrier compound" or "carrier" can refer to a nucleic acid or its analog that is inert (i.e., not biologically active per se) but is considered a nucleic acid in vivo processes, e.g., by degrading or facilitating removal from circulation of biologically active nucleic acids, to reduce bioavailability of biologically active nucleic acids. Co-administration of a nucleic acid and a carrier compound, typically with the latter in excess, can result in a substantial reduction in the amount of nucleic acid recovered in the liver, kidney, or other extracirculatory reservoir, presumably due to 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 can reduce 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).

[0254] A "pharmaceutical carrier" or "excipient" in contrast to a carrier compound, is a pharmaceutically acceptable solvent, suspending agent or other vehicle with which non- nucleic acid pharmaceutical agents are administered to animals. Such excipients can be liquid or solid and are selected with the aim of providing an optimal medium for the agent being administered. Typical pharmaceutical carriers include, but are not limited to, binders (e.g., pregelatinized corn starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers (e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylate or dibasic calcium phosphate, etc.); lubricants (e.g., magnesium stearate, talc, silicon dioxide, colloidal silicon dioxide, stearic acid, metal stearates, hydrogenated vegetable oil, corn starch, polyethylene glycol, sodium benzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodium starch glycolate, etc.); and wetting agents (e.g., sodium lauryl sulfate, etc.).

[0255] Pharmaceutically acceptable organic or inorganic excipients, which do not deleteriously react with the nucleic acid, are also suitable for use in formulating compositions of the present disclosure for parenteral administration. Suitable pharmaceutically acceptable carriers include, but are not limited to, water, salt solutions, alcohol, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone, etc.

[0256] Formulations for topical administration of nucleic acids can include sterile or non-sterile aqueous solutions, non-aqueous solutions, or nucleic acid solutions in liquid or solid oil bases. These solutions also can include buffers, diluents, and other suitable additives. Pharmaceutically acceptable organic or inorganic excipients suitable for parenteral administration that do not cause toxic reactions with the nucleic acids can be used.

[0257] Suitable pharmaceutically acceptable excipients include, but are not limited to, water, saline, alcohol, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone, etc.

[0258] The present disclosure also provides methods for treating or preventing diseases and conditions that can be modulated by downregulating AGT gene expression. For example, treating hypertension, borderline hypertension, essential hypertension, secondary hypertension, hypertensive crisis, hypertensive emergency, isolated systolic and diastolic hypertension, pregnancy-related hypertension, diabetic hypertension, resistant hypertension, refractory hypertension, paroxysmal hypertension, renovascular hypertension, Goldblatt's hypertension, ocular hypertension, glaucoma, pulmonary hypertension, portal hypertension, systemic venous hypertension, systolic hypertension, labile hypertension; hypertensive heart disease, hypertensive nephropathy, atherosclerosis, arteriosclerosis, vasculopathy, diabetic nephropathy, diabetic retinopathy, chronic heart failure, cardiomyopathy, diabetic cardiomyopathy, glomerulosclerosis, coarctation of the aorta, aortic aneurysm, ventricular fibrosis, Cushing's syndrome and other glucocorticoid excess states (including chronic steroid treatment), pheochromocytoma, reninoma, secondary hyperaldosteronism and other mineralocorticoid excess states, sleep apnea, thyroid / parathyroid disease, heart failure, myocardial infarction, angina, stroke, diabetes, kidney disease, kidney failure, systemic sclerosis, intrauterine growth restriction (IUGR), and fetal growth restriction.

[0259] The RNAi agents of the present disclosure can be administered to a subject using any mode of administration known in the art, including, but not limited to, subcutaneous, intravenous, intramuscular, intraocular, intrabronchial, intrapleural, intraperitoneal, intraarterial, translymphatic, trans cerebrospinal, and any combination thereof. In preferred embodiments, the agents are administered subcutaneously.

[0260] In additional embodiments, the siRNA is administered in combination with an additional therapeutic agent. The siRNA and the additional therapeutic agent can be administered in combination in the same composition, e.g., parenterally, or the additional therapeutic agent can be administered as part of a separate composition or by another method described herein.

[0261] Examples of additional therapeutic agents include agents known to treat hypertension or agents known to treat cardiovascular and cerebrovascular diseases. For example, the additional therapeutic agent for treating hypertension is selected from the group consisting of angiotensin converting enzyme inhibitors (e.g., captopril, enalapril, benazepril, perindopril, etc.), angiotensin II receptor antagonists (e.g., losartan, losartan hydrochlorothiazide, valsartan, valsartan hydrochlorothiazide, telmisartan, telmisartan hydrochlorothiazide, olmesartan medoxomil, etc.), beta blockers (e.g., propranolol, bisoprolol, metoprolol tartrate, metoprolol succinate, etc.).

[0262] In one embodiment, the iRNA agent is administered to a patient and subsequently the additional therapeutic agent is administered to the patient (or vice versa). In another embodiment, the iRNA agent and the additional therapeutic agent are administered simultaneously.

[0263] The following examples are intended to illustrate the present disclosure, but not to limit the scope of the present disclosure. If not specifically stated, the technical means used in the examples are conventional means known to those skilled in the art, and the raw materials used are commercially available.

[0264] Nucleotide abbreviations used herein are as follows:

[0265] A = adenosine-3 '-phosphate

[0266] Am = 2'-methoxyadenosine-3 '-phosphate

[0267] Ams = 2'-methoxyadenosine-3 '-phosphorothioate

[0268] Af = 2'-fluoroadenosine-3 '-phosphate

[0269] Afs = 2'-fluoroadenosine-3 '-phosphorothioate

[0270] G = guanosine-3 '-phosphate

[0271] Gm = 2'-methoxyguanosine-3 '-phosphate

[0272] Gms = 2'-methoxyguanosine-3 '-phosphorothioate

[0273] Gf = 2'-fluoroguanosine-3 '-phosphate

[0274] Gfs = 2'-fluoroguanosine-3 '-phosphorothioate

[0275] C = cytidine-3 '-phosphate

[0276] Cm = 2'-methoxycytidine-3 '-phosphate

[0277] Cms = 2'-methoxycytidine-3 '-phosphorothioate

[0278] Cf = 2'-fluorocytidine-3'-phosphate

[0279] Cfs = 2'-fluorocytidine-3'-thiophosphate

[0280] U = uridine-3'-phosphate

[0281] Um = 2'-methoxyuridine-3'-phosphate

[0282] Ums = 2'-methoxyuridine-3'-thiophosphate

[0283] Uf = 2'-fluorouridine-3'-phosphate

[0284] Ufs = 2'-fluorouridine-3'-thiophosphate

[0285] AmsEVP = 5'-ethenyl-(E)-phosphonate-2'-methoxyadenosine-3'-thiophosphate

[0286] UmsEVP = 5'-ethenyl-(E)-phosphonate-2'-methoxyuridine-3'-thiophosphate

[0287] Agna = adenosine-diol nucleic acid

[0288] Cgna = cytidine-diol nucleic acid

[0289] Ggna = guanosine-diol nucleic acid

[0290] Tgna = thymidine-diol nucleic acid

[0291] Ugna = uridine-diol nucleic acid

[0292] The modifications correspond to the following English names herein:

[0293] 2'-methoxy: 2'-O-methyl

[0294] 2'-fluoro: 2'-fluoro

[0295] 3'-thiophosphate: 3'-thiophosphate

[0296] 5'-ethenyl-(E)-phosphonate: 5'-Ethenyl-(E)-phosphonate

[0297] 2'-deoxy: 2'-deoxyl

[0298] The compound numbering convention used herein is as follows:

[0299] Unmodified sequence is unmodified RNA sequence. For sequences with 21 / 23 bases in the sense / antisense strand, the number represents the position of the sequence on the AGT mRNA, such as compound 1579; for sequences with 20 / 22 bases in the sense / antisense strand, the letter "s" is added after the number representing the position to distinguish, such as compound 1578s.

[0300] Sequences with 2'-methoxy (2'-OMe) and 2'-fluoro (2'-F) alternating modification are added with "B" before the number of unmodified sequence and "-AL" after the number. For example, the 1578s sequence with alternating modification is numbered as B1578s-AL.

[0301] Compounds appearing in Example 4, sequences modified using DV25P-29P, DV32P-34P templates disclosed herein or sequences modified using prior art template DV22 are added with "C" before the number of unmodified sequence and the corresponding template name after the number. For example, the 1578s sequence modified using DV29P template is numbered as C1578s-DV29P.

[0302] Compounds appearing in Example 6, sequences modified using DV25P-29P / DV32P-34P templates disclosed herein or sequences modified using prior art template DV22 and possibly containing off-target prevention modification are added with "D" before the number of unmodified sequence and the corresponding template name and off-target prevention modification name in order after the number. For example, the unmodified sequence 1578s is modified using DV29P template and contains off-target prevention modification d7B, and is numbered as D578s-DV29Pd7B.

[0303] Compounds appearing in Example 7, sequences modified using DV25P-29P templates disclosed herein and possibly containing off-target prevention modification and connected with GalNAc at the 3' end of the sense strand to achieve liver-targeted delivery are numbered by adding G5 at the end of the numbering method of Example 6. For example, the D578s-DV29Pd7B sequence coupled with G5 is numbered as D578s-DV29Pd7BG5.

[0304] Table 49 Example Numbering Method

[0305]

[0306] In the following examples, the experimental data P value for comparison between groups is <0.05, and the difference is statistically significant.

[0307] Example

[0308] Example 1: Synthesis of small interfering oligonucleotides with alternating modification

[0309] According to the AGT mRNA sequence (NM_001384479.1), 99 siRNA unmodified sequences were designed. 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 thio-modified. For sequences with odd-numbered lengths of the sense strand, the odd-numbered positions of the sense strand were all 2'-F modified, and the even-numbered positions were all 2'-OMe modified. However, for sequences with even-numbered lengths of the sense strand, the odd-numbered positions of the sense strand were all 2'-OMe modified, and the even-numbered positions were all 2'-F modified. The odd-numbered positions of the antisense strand were all 2'-OMe modified, and the even-numbered positions were all 2'-F modified. In addition, the 5' end of the sense strand had two thio-modifications; the 5' and 3' ends of the antisense strand each had two thio-modifications. The alternately modified siRNA sequences are shown in Table 2.

[0310] 1. Synthesis of alternately modified sequence B1579-AL

[0311] The unmodified sequence of the small interfering ribonucleic acid with the sequence number B1579-AL in Table 1 is:

[0312] Sense strand: 5'-CCUUUUCUUCUAAUGAGUCGA-3' (SEQ ID NO: 4)

[0313] Antisense strand: 5'-UCGACUCAUUAGAAGAAAAGGUG-3' (SEQ ID NO: 17)

[0314] The odd-numbered positions of the sense strand and the even-numbered positions of the antisense strand were all 2'-F modified, and the other positions were all 2'-OMe modified. In addition, the 5' end of the sense strand had two thio-modifications; the 5' and 3' ends of the antisense strand each had two thio-modifications.

[0315] Instruments and reagents: Genesee 192P model DNA / RNA automatic synthesizer, with a general carrier of cross-linked polystyrene beads, model Primer support 5G Unylinker 350 (cytiva manufacturer).

[0316] Preparation method:

[0317] According to a monomer concentration of 0.15 M, the following nucleotide monomer solutions were prepared with acetonitrile: 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-A-F phosphoramidite monomer (Formula 5), DMT-C-F phosphoramidite monomer (Formula 6), DMT-G-F phosphoramidite monomer (Formula 7), and DMT-U-F phosphoramidite monomer (Formula 8).

[0318]

[0319] The following steps are used to prepare:

[0320] The solid support is loaded into the synthesizer at the designated position, and the corresponding fully protected product is obtained after several cycles of synthesis, which include (1) deprotection, (2) coupling, (3) oxidation / sulfurization, and (4) hydroxyl protection. The cycle process and the reagents used are described as follows:

[0321] (1) Deprotection

[0322] 3% dichloroacetic acid in toluene is used as the deprotection reagent to remove the DMT protecting group, and then acetonitrile is used for washing.

[0323] (2) Coupling

[0324] 0.25M 5-ethylthiotetrazole is used as the activator to couple the acetonitrile solution of each nucleotide monomer, and then acetonitrile is used for washing.

[0325] (3) Oxidation / sulfurization

[0326] Oxidation: 0.05M iodine in pyridine / water (90 / 10) is used as the oxidizing agent for oxidation, and then acetonitrile is used for washing.

[0327] Sulfurization: 3% hydrogenated xanthate in pyridine is used as the sulfurizing agent for sulfurization, and then acetonitrile is used for washing.

[0328] (4) Hydroxyl protection

[0329] 10% acetic anhydride in tetrahydrofuran (CAP A) tetrahydrofuran / pyridine / n-methylimidazole 74 / 10 / 16 (v / v / v) (CAP B) is used as the hydroxyl protection reagent for hydroxyl protection, and then acetonitrile is used for washing.

[0330] The above operations are repeated according to the set sequence cycle to obtain the fully protected product.

[0331] (5) 3% dichloroacetic acid in toluene is used as the deprotection reagent to remove the DMT protecting group of the last nucleotide, and then acetonitrile is used for washing.

[0332] (6) Ammonolysis and purification

[0333] The solid support is transferred to the reactor, and concentrated ammonia water (25-28%) is added. After ammonolysis at 60°C for 12h, the system is cooled to room temperature, and the mixture is transferred to a pressure filter tank, eluted with a mixed solution of purified water and ethanol, the filtrates are combined, passed through a chromatographic column, concentrated, and freeze-dried to obtain the product.

[0334] (7) Annealing

[0335] The purified sense and antisense strands were mixed in a 1:1 ratio, heated to 95°C for 3 min, and then slowly cooled to room temperature to form double-stranded siRNA.

[0336] B1579-AL purity 90.61%; measured molecular weight: 14437.86.

[0337] 2. Synthesis of other sequences

[0338] The other sequences listed in Table 1 were synthesized according to the above method, and a total of 99 siRNA sequences were synthesized.

[0339] Table 1: Alternating modification of small interfering ribonucleic acid sequences

[0340]

[0341]

[0342]

[0343]

[0344]

[0345]

[0346]

[0347]

[0348] Example 2: Inhibition of AGT gene by alternating modification sequences

[0349] After the 2'-OMe and 2'-F alternating modification siRNA sequences synthesized in Example 1 were transfected into HepG2 cells by lipid nanoparticles (LNP), the inhibition of AGT gene by each sequence was detected using qPCR technology.

[0350] 1. Experimental materials

[0351] Test samples: alternating modification small interfering ribonucleic acid sequences listed in Table 1 (synthesized in Example 1).

[0352] Cell type: HepG2 cell line

[0353] Drug vehicle: sterile enzyme-free water, gibco Opti-MEM.

[0354] 2. Experimental method

[0355] The inhibition of AGT mRNA expression in HepG2 cell line was detected by qRT-PCR.

[0356] 2.1 Cell culture

[0357] The subcultured HepG2 cell line was used, and the logarithmic growth cells were cultured in 10% fetal bovine serum DMEM culture medium (supplemented with 100x penicillin, 100x streptomycin 10 μL / mL) in a 37°C cell incubator with 5% CO2, and the medium was changed every day. The cells were subcultured by 0.25% trypsin digestion, centrifuged at 800 r / min for 3 min, the supernatant was discarded, and the cells were subcultured in fresh culture medium.

[0358] 2.2 Cell transfection

[0359] Transfection mixture preparation: mix Lipofectamine RNAiMAX and Opti-MEM at a ratio of 2:98, then vortex well.

[0360] Transfection reagent preparation: take 65 μL of siRNA solution diluted in Opti-MEM at a ratio of 1:1 (v / v) and add it to 65 μL of transfection mixture, vortex well, and let it stand at room temperature for 15 min to obtain lipid nanoparticles (LNP). Take 12.5 μL for encapsulation rate detection.

[0361] Blank control group transfection reagent: add 65 μL of prepared transfection mixture to 65 μL of Opti-MEM. Vortex well, and let it stand at room temperature for 15 min.

[0362] Add the prepared transfection reagent to a 24-well cell culture plate (100 μL per well), so that the final siRNA concentration per well is 0.07 nM. Add 500 μL of cell suspension (containing 1.5x10 5 After mixing with the cross method, place it in a 37°C, 5% CO2 cell incubator for 40 h.

[0363] 2.3 AGT mRNA detection

[0364] 1) RNA extraction

[0365] a. Remove the culture medium from the 12-well plate, wash the cells with 0.5 mL of 1x PBS per well, and remove the PBS. Add 0.5 mL of TRIzol reagent to the well, and blow the cells with a gun to fully lyse them. Transfer to a 1.5 mL RNase-free EP tube, and let it stand at room temperature for 5 min.

[0366] b. Add 0.1 mL of chloroform to each tube, and shake vigorously for 15 sec. Let it stand at room temperature for 5 min. Centrifuge at 4°C, 12000xg for 15 min, and take 200 μL of supernatant to a new EP tube.

[0367] c. Add equal volume of isopropanol, invert the tube several times to mix the contents, and incubate at -20℃ for 10 min. Centrifuge at 12000 x g for 15 min at 4℃. Discard the supernatant.

[0368] d. Add 0.5 mL of 75% ethanol, gently wash the RNA pellet, centrifuge at 12000 x g for 5 min at 4℃. Repeat the rinse once, centrifuge at 12000 x g for 1 min at 4℃. Remove the residual ethanol with a micropipette tip.

[0369] e. Air dry the residual ethanol at room temperature for 2-3 min, add 40 μL of RNase-free ddH2O, and dissolve.

[0370] 2) RNA concentration detection

[0371] Use nanodrop to detect the concentration of RNA. Use 2 μL of RNase-free ddH2O as a blank control, and detect 2 μL of RNA sample each time. Record the sample concentration.

[0372] 3) AGT mRNA reverse transcription

[0373] Mix the reverse transcription reagent, RNA solution, and water in a volume ratio of 2:5:3, and react in a PCR instrument at 37℃ for 15 min and at 85℃ for 5 s, and finally maintain at 4℃. Dilute the cDNA obtained at the end of the reaction 5 times with sterile enzyme-free water.

[0374] 4) AGT mRNA quantitative detection

[0375] In a 15 mL centrifuge tube, add qPCR reagents, upstream primer, and downstream primer in a volume ratio of 5:0.1:0.1 and mix well, labeled as solution A.

[0376] Label 1.5 mL EP tubes, add diluted cDNA and water in a volume ratio of 1:3.8 respectively and mix well, labeled as solution B.

[0377] Add 5.2 μL of solution A and 4.8 μL of solution B to each well of a 96-well PCR plate respectively. Cover with a sealing film, centrifuge at 3000 rpm for 1 min, and detect on the machine.

[0378] * This step is operated on ice to maintain low temperature conditions.

[0379] Place the plate in a qPCR instrument and run according to the following program:

[0380] Pre-denaturation: 95℃, 30 sec;

[0381] Cycling reaction: 95°C, 5 sec; 60°C, 34 sec; 40 cycles

[0382] Melting curve: 95°C, 15 sec; 60°C, 60 sec; 95°C, 15 sec.

[0383] The running time is about 2 h. Analyze the experimental results, calculate 2 -ΔΔCt .

[0384] 2.4 Data processing

[0385] The formula for calculating the expression rate (%) of AGT mRNA is:

[0386] Expression rate = (AGT mRNA expression amount / blank control group AGT mRNA expression amount) x 100%;

[0387] AGT gene expression inhibition rate = 100% - expression rate (%).

[0388] 3. Experimental results

[0389] The inhibition rate in this example is the average of 3 experiments, and the expression inhibition rate of AGT mRNA in HepG2 cells for each sequence is shown in Tables 2-5.

[0390] The experimental results show that some sequences with alternating modification of 2'-OMe and 2'-F, including B1789-AL sequence, have significant inhibitory effect on the expression of AGT mRNA in HepG2 cells, and the inhibition rate is more than 45%. Among them, B1576s-AL has an inhibition rate of more than 60%.

[0391] Other sequences have poor inhibitory effect on the expression of AGT mRNA, and the inhibition rate is less than 45%.

[0392] (1) Among the 99 designed sequences, 33 sequences have significant inhibitory effect on AGT gene, and the inhibition rate is more than 40%. Among them, the inhibition rate of some sequences is more than 60%.

[0393] Table 2 Alternating modification sequences with significant inhibition (inhibition rate higher than 40%) on AGT gene

[0394]

[0395]

[0396] As can be seen from Table 2, the alternating modification sequences in the above table (see Table 1 for specific sequences) have significant inhibitory effect on the expression of AGT mRNA, and the inhibition rate is more than 40%, among which the inhibition rate of the first 10 sequences is more than 60% Figure 1 ).

[0397] The sequence of the compounds in Table 2 is as follows in Table 3.

[0398] Table 3 siRNA sequences with significant inhibition of AGT gene

[0399]

[0400]

[0401]

[0402] (2) The inhibition rate of 23 sequences on AGT gene is between 25% and 40%. For example, the inhibition rate of B734-AL and B994-AL is 39.2% and 37.3% respectively.

[0403] Table 4 Alternating modification sequences with inhibition rate of AGT gene between 25% and 40%

[0404]

[0405]

[0406] The 22 sequences listed in Table 4 have poor inhibition effect on AGT gene, with inhibition rate less than 40%, between 25% and 40%. For example, the inhibition rate of B734-AL and B994-AL is 39.2% and 37.3% respectively. Figure 2

[0407] (3) The inhibition rate of 43 sequences on AGT gene is less than 25%. For example, the inhibition rate of B1017-AL is only 23.1%.

[0408] Table 5 Alternating modification sequences with inhibition rate of AGT gene less than 25%

[0409]

[0410]

[0411]

[0412] The 43 sequences listed in Table 5 have poor inhibition effect on AGT mRNA expression, with inhibition rate less than 25%. For example, the inhibition rate of B1017-AL is only 23.1% Figure 3 and Figure 4 .

[0413] (4) siRNA with similar sequences have very large activity difference. For example, the inhibition rate of B1365-AL is increased by 40.4% compared with B1367-AL, which is significantly improved. ​

[0414] The inhibition of AGT mRNA by some siRNAs with similar sequences is shown in Table 6.

[0415] Table 6 Comparison of the inhibition of AGT gene expression by siRNAs with similar sequences

[0416]

[0417] As shown in Table 6, the inhibition of AGT mRNA expression by some siRNAs with similar sequences differs greatly.

[0418] Unmodified sequence 1367 differs from unmodified sequence 1365 only in the last two bases. The sense strand: the 5' end of unmodified sequence 58 is GA, and the 3' end of unmodified sequence 59 is GU, and the other sequences are identical; the antisense strand: the 3' end of unmodified sequence 144 is AC, and the 5' end of unmodified sequence 145 is AC, and the other sequences are identical. However, the inhibition rate of alternating modification sequence B1365-AL is 40.4% higher than that of B1367-AL, which is significantly improved.

[0419] Unmodified sequence 1815 differs from unmodified sequence 1816 only in the last base. The sense strand: the 5' end of unmodified sequence 86 is U, and the 3' end of unmodified sequence 2 is U, and the other sequences are identical; the antisense strand: the 3' end of unmodified sequence 172 is G, and the 5' end of unmodified sequence 15 is A, and the other sequences are identical. However, the inhibition rate of alternating modification sequence B1816-AL is 30.5% higher than that of B1815-AL, which is significantly improved.

[0420] Unmodified sequence 729 differs from unmodified sequence 734s only in the last five bases. The sense strand: the 5' end of unmodified sequence 97 is AGAAC, and the 3' end of unmodified sequence 101 is GAAA, and the other sequences are identical; the antisense strand: the 3' end of unmodified sequence 183 is UCUGU, and the 5' end of unmodified sequence 187 is UUUC, and the other sequences are identical. However, the inhibition rate of alternating modification sequence B734s-AL is 40.0% higher than that of B729-AL, which is significantly improved.

[0421] Therefore, it is not easy to screen out sequences with significant inhibitory activity from a large number of oligonucleotide sequences designed for AGT mRNA sequences, and a lot of creative labor is required.

[0422] Summary:

[0423] (1) Of the 99 designed sequences, 33 sequences have significant inhibitory effect on AGT gene, with inhibition rate exceeding 40%. Among them, the inhibition rate of 10 sequences exceeds 60%.

[0424] (2) The inhibition rates of the 23 sequences on 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.

[0425] (3) The inhibition rate of the AGT gene by 43 sequences was less than 25%. For example, the inhibition rate of B1017-AL was only 23.1%.

[0426] (4) siRNAs with similar sequences can have very different activities. For example, the inhibition rate of B1365-AL is 40.4% higher than that of B1367-AL, which is a significant improvement. Therefore, it is not easy to screen out sequences with significant inhibitory activity from a very large number of oligonucleotide sequences designed for AGT mRNA sequences, and a lot of creative work is required.

[0427] Example 3: Repressive effect of unmodified sequences on the AGT gene

[0428] In this embodiment, some unmodified sequences corresponding to the modified sequences in Example 2 were synthesized and transfected into HepG2 cells via lipid nanoparticles (LNP). The inhibitory effect of each unmodified sequence on the AGT gene was detected by qPCR technology, and unmodified siRNA sequences with better inhibitory effects were screened out.

[0429] 1. Experimental Materials

[0430] Test sample:

[0431] The unmodified small interfering RNA sequences listed in Table 7.

[0432] 2. Synthesis of unmodified sequence 1579

[0433] The sequence of the small interfering RNA with sequence number 1579 in Table 7 is as follows:

[0434] Chain of Justice: 5'-CCUUUUCUUCUAAUGAGUCGA-3'(SEQ ID NO:4)

[0435] Antonym: 5'-UCGACUCAUUAGAAGAAAAGGUG-3' (SEQ ID NO:17)

[0436] Instruments and reagents: The Qingke 192P model DNA / RNA automated synthesizer, whose solid support is a universal carrier of cross-linked polystyrene beads, model Primer support 5G Unylinker 350 (Cytiva manufacturer).

[0437] Preparation method:

[0438] 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).

[0439]

[0440] The following procedure was used to prepare:

[0441] The solid support was loaded into the synthesizer at the designated position, and the corresponding fully protected product was obtained after several synthesis cycles, which included (1) deprotection, (2) coupling, (3) oxidation / sulfurization, and (4) hydroxyl protection. The cycle process and reagents used are described as follows:

[0442] (1) Deprotection

[0443] A 3% dichloroacetic acid in toluene solution was used as the deprotection reagent to remove the DMT protecting group, followed by acetonitrile wash.

[0444] (2) Coupling

[0445] A 0.25 M 5-ethylthiotetrazole solution was used as the activator for the acetonitrile solution of each nucleotide monomer, followed by acetonitrile rinse.

[0446] (3) Oxidation / Sulfurization

[0447] Oxidation: A 0.05 M iodine in pyridine / water (90 / 10) solution was used as the oxidizing agent, followed by acetonitrile rinse.

[0448] Sulfurization: A 3% hydroxylamine pyridine solution was used as the sulfurizing agent, followed by acetonitrile rinse.

[0449] (4) Hydroxyl Protection

[0450] A 10% acetic anhydride in tetrahydrofuran solution (CAP A) tetrahydrofuran / pyridine / azidemethylimidazole 74 / 10 / 16 (v / v / v) (CAP B) was used as the hydroxyl protection reagent, followed by acetonitrile rinse.

[0451] The above procedure was repeated in a set sequence cycle to obtain the fully protected product.

[0452] (5) A 3% dichloroacetic acid in toluene solution was used as the deprotection reagent to remove the DMT protecting group of the last nucleotide, followed by acetonitrile wash.

[0453] (6) Aminolysis and purification

[0454] The reacted solid support was transferred to a reactor, concentrated ammonia (25-28%) was added, and the aminolysis was carried out at 60°C for 12 h. The system was then cooled to room temperature, and the mixture was transferred to a filter tank, rinsed with a mixture of purified water and ethanol, and the filtrates were combined and passed through a chromatography column, concentrated, and lyophilized to obtain the 2'-O-TBDMS-protected product.

[0455] (7) Removal of TBDMS

[0456] DMSO and triethylamine hydrofluoride acid were added to the obtained product, and the reaction was carried out at 60°C for 2 h. Then, an aqueous ammonium acetate solution was added to the reaction solution, mixed well, and anhydrous ethanol was added. After mixing well, the mixture was crystallized at -20°C for 8-12 h. After centrifugation, the supernatant was discarded, and the precipitate was rinsed with anhydrous ethanol to obtain the unmodified single-stranded product.

[0457] (8) Annealing

[0458] The obtained unmodified sense and antisense strands were mixed at a ratio of 1:1, heated to 95°C and maintained for 3 min, and then slowly cooled to room temperature to form the unmodified double strand.

[0459] Unmodified sequence 1579: purity 97.4%; measured molecular weight: 13990.9.

[0460] 3. Synthesis of other sequences

[0461] The other sequences in Table 7 were synthesized according to the above method.

[0462] Table 7 Unmodified siRNA sequences

[0463]

[0464]

[0465]

[0466]

[0467] Cell type: HepG2 cells, a hepatoma cell line.

[0468] Drug solvent: sterile enzyme-free water, gibco Opti-MEM.

[0469] 3. Experimental method

[0470] The experimental procedure and siRNA concentration were as in Example 2.

[0471] 4. Experimental results

[0472] The inhibition rates of each unmodified sequence on AGT mRNA expression are shown in Table 8 and Table 9.

[0473] Unmodified sequences 1576s, 1578s, 1838, 1835, 1816, 1812, 1579, 1836, 1789, 1839, 1791, 1795 and 1585s have significant inhibitory effect on AGT mRNA expression in HepG2 cells, with inhibition rates all above 45%, among which 1576s, 1578s, 1838, 1835, 1812, 1579 and 1789 have inhibition rates exceeding 50%. See Table 29 for comparison of the inhibitory effect of unmodified sequences of the present disclosure and prior art sequences.

[0474] (1) 13 unmodified sequences have significant inhibitory effect on AGT gene, with inhibition rates all exceeding 45%, including 1576s, 1578s, 1838, 1835, 1816, 1812, 1579, 1836, 1789, 1839, 1791, 1795 and 1585s. Among them, 1576s, 1578s, 1838, 1835, 1812, 1579 and 1789 have inhibition exceeding 50%.

[0475] Table 8: Unmodified sequences having significant inhibitory effect on AGT gene (inhibition rate higher than 45%)

[0476] Sequence No. Inhibition rate (%) 1576s 59.1 1578s 55.2 1838 50.6 1835 51.6 1816 46.8 1812 56.8 1579 58.2 1836 46.5 1789 54.5 1839 45.7 1791 46.5 1795 49.6 1585s 48.9

[0477] Each unmodified sequence listed in Table 8, including 1576s, 1578s, 1838, 1835, 1816, 1812, 1579, 1836, 1789, 1839, 1791, 1795 and 1585s, has significant inhibitory effect on AGT mRNA expression, with inhibition rates all above 45%. Among them, 1576s, 1578s, 1838, 1835, 1812, 1579 and 1789 have inhibition exceeding 50%. Figure 5

[0478] (2) The inhibition rates of some unmodified sequences on AGT gene are all lower than 45%. For example, the inhibition rates of 731 and 1011 are only 1.3% and 2.1%.

[0479] Table 9: Unmodified sequences having inhibition rate on AGT gene lower than 45%

[0480]

[0481]

[0482]

[0483] ​The expression inhibition of AGT mRNA by each sequence in Table 9 is poor, and the inhibition rate is less than 45%. For example, the inhibition rates of 731 and 1011 are only 1.3% and 2.1%( Figure 5 ).

[0484] (3) The siRNAs with similar unmodified sequences have very different activities. For example, the inhibition rate of unmodified sequence 1812 is 32.3% higher than that of unmodified sequence 1810, which is significantly improved.

[0485] The inhibition of AGT mRNA by some siRNAs with similar sequences is compared in Table 10.

[0486] Table 10 Comparison of the expression inhibition rates of AGT gene by unmodified sequences with similar sequences

[0487]

[0488] As can be seen from Table 10, the inhibition of AGT mRNA by some siRNAs with similar sequences is very different.

[0489] Unmodified sequence 1608 is different from unmodified sequence 1612 only in the terminal 4 bases. The sense strand: the 5' terminal of unmodified sequence 76 is UGGA, and the 3' terminal of unmodified sequence 77 is UGGU, and the other sequences are completely identical; the antisense strand: the 3' terminal of unmodified sequence 162 is CAGC, and the 5' terminal of unmodified sequence 163 is ACCA, and the other sequences are completely identical. However, the inhibition rate of unmodified sequence 1608 is 29.7% higher than that of 1612, which is significantly improved.

[0490] Unmodified sequence 2025 is different from unmodified sequence 2030 only in the terminal 6 bases. The sense strand: the 5' terminal of unmodified sequence 92 is GUUAU, and the 3' terminal is A, and the 3' terminal of sequence 93 is GUAAUA, and the other sequences are completely identical; the antisense strand: the 3' terminal of unmodified sequence 178 is AACCA, and the 5' terminal is U, and the 5' terminal of unmodified sequence 179 is UAUUAC, and the other sequences are completely identical. However, the inhibition rate of unmodified sequence 2030 is 29.1% higher than that of 2025, which is significantly improved.

[0491] Unmodified sequence 731 is different from unmodified sequence 734 only in the terminal 3 bases. The sense strand: the 5' terminal of unmodified sequence 98 is AAC, and the 3' terminal of unmodified sequence 100 is AGA, and the other sequences are completely identical; the antisense strand: the 3' terminal of unmodified sequence 184 is UCU, and the 5' terminal of unmodified sequence 186 is UCU, and the other sequences are completely identical. However, the inhibition rate of unmodified sequence 734 is 41.7% higher than that of 731, which is significantly improved.

[0492] The only difference between unmodified sequence 1812 and unmodified sequence 1810 is the terminal 2 bases. The positive strand: the 3' terminal of unmodified sequence 3 is AA, the 5' terminal of unmodified sequence 83 is UC, and the other sequences are completely identical; the negative strand: the 5' terminal of unmodified sequence 16 is UU, the 3' terminal of unmodified sequence 169 is AC, and the other sequences are completely identical. The inhibition rate of unmodified sequence 1812 is increased by 32.3% compared with unmodified sequence 1810, which is significantly improved.

[0493] Therefore, it is not easy to screen out unmodified unmodified sequences with significant inhibitory activity from a very large number of oligonucleotide sequences designed for AGT mRNA sequences, and a lot of creative labor needs to be paid.

[0494] (4) The effect of different sequences after alternating modification on the activity is not consistent. Some are significantly improved, for example, unmodified sequence 731, the inhibition rate of alternating modification is increased by 25.1% compared with unmodified sequence; some are not obvious, for example, unmodified sequences 994, 1279 and 1591, the inhibition rates of alternating modification sequences and unmodified sequences are basically unchanged.

[0495] Table 11 Comparison of inhibition rates of alternating modification sequences and unmodified sequences on AGT gene

[0496]

[0497]

[0498]

[0499] As can be seen from Table 11, the effect of different sequences after alternating modification on the inhibition rate of AGT mRNA is not completely consistent. Some are significantly improved, for example, unmodified sequence 731, the inhibition rate of alternating modification is increased by 25.1% compared with unmodified sequence; some are not obvious, for example, unmodified sequences 994, 1279 and 1591, the inhibition rates of alternating modification sequences and unmodified sequences are basically unchanged.

[0500] Therefore, not all unmodified sequences can be significantly improved in activity after alternating modification, and the effect of alternating modification on the activity of different sequences is not consistent.

[0501] Summary:

[0502] (1) 13 sequences without modification have significant inhibitory effect on AGT gene, and the inhibition rate is more than 45%, including 1576s, 1579, 1812, 1578s, 1789, 1835, 1838, 1795, 1585s, 1816, 1791, 1836 and 1839. Among them, the inhibition rates of 1576s, 1578s, 1812, 1579, 1789, 1835 and 1838 are more than 50%.

[0503] (2) The inhibition rates of other sequences without modification on AGT gene are less than 45%. For example, the inhibition rates of 731 and 1011 are only 1.3% and 2.1%.

[0504] (3) The siRNAs with similar sequences have very different activities. For example, the inhibition rate of the sequence without modification 734 is increased by 41.7% compared with the sequence without modification 731, which is significantly improved.

[0505] (4) The effect of different sequences after alternating modification on activity is not consistent. Some have significantly improved inhibition rates, for example, the inhibition rate of the sequence without modification 731 is increased by 25.1% after alternating modification; some have no significant change, for example, the inhibition rates of the sequences without modification 994, 1279 and 1591 are basically unchanged.

[0506] Example 4: Inhibition effect of sequences with template modification on AGT gene

[0507] In this example, the sequences screened in Example 3, i.e., 13 sequences without modification 1576s, 1578s, 1838, 1835, 1816, 1812, 1579, 1836, 1789, 1839, 1791, 1795 and 1585s, are modified by using modified templates. Among them, DV25P, DV26P, DV27P, DV28P, DV29P, DV32P, DV33P and DV34P are new modified templates designed by the present disclosure, and DV22 is an Advanced ESC modified template (Foster, D. J., et al. (2018). "Advanced siRNA Designs Further Improve In Vivo Performance of GalNAc-siRNA Conjugates." Mol Ther 26(3): 708-717.).

[0508] Natural 5' end phosphorylation or simple direct 5' end phosphorylation can occur dephosphorylation in cells, direct 5' end phosphorylation oligonucleotide chain can be in the blood circulation 2 hours after the emergence of 90% dephosphorylation, and all disappear after 24 hours. 5' end phosphorylation design (5'-E-VP) uses E-vinyl phosphonate to replace the bridging oxygen, which has improved phosphorylation effect and stability. The 5' end of the antisense strand of the modified template DV25-29P, DV32-34P in the disclosure contains 5'-E-VP phosphorylation design.

[0509] 1. Experimental materials

[0510] Test samples:

[0511] Table 12 lists the examples 3 modified by different templates (DV25P, DV26P, DV27P, DV28P, DV29P, DV30P, DV32P, DV33P, DV34P and DV22) of sequences 1576s, 1578s, 1838, 1835, 1816, 1812, 1579, 1836, 1789, 1839, 1791, 1795 and 1585s, and the corresponding alternative modified sequences B1576s-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).

[0512] Sequence synthesis:

[0513] Synthesize siRNA sequences according to Example 1, and use monomers containing 5' end phosphonate groups, such as vinyl-(E)-phosphonate-A-OMe phosphoramidite monomers (Formula 9), vinyl-(E)-phosphonate-U-OMe phosphoramidite monomers (Formula 10), when synthesizing the base at the 5' end of the antisense strand (the last base), as shown in the structure:

[0514]

[0515] The modification principle of the modified template in the disclosure is as follows:

[0516] DV22, DV25-34P:

[0517] The antisense strand uses one of the modification methods shown in Table 46:

[0518] Table 46: Modification of antisense strand

[0519]

[0520]

[0521] One of the modifications in Table 47 is used for the sense strand:

[0522] Table 47 Modifications for the sense strand

[0523]

[0524]

[0525] In the above table, PS is a phosphorothioate backbone.

[0526] The siRNA modification template with the antisense strand modified with modification A and the sense strand modified with modification a is designated DV25P;

[0527] The siRNA modification template with the antisense strand modified with modification B and the sense strand modified with modification a is designated DV26P;

[0528] The siRNA modification template with the antisense strand modified with modification C and the sense strand modified with modification a is designated DV27P;

[0529] The siRNA modification template with the antisense strand modified with modification B and the sense strand modified with modification b is designated DV28P;

[0530] The siRNA modification template with the antisense strand modified with modification C and the sense strand modified with modification b is designated DV29P.

[0531] The siRNA modification template with the antisense strand modified with modification D and the sense strand modified with modification b is designated DV32P.

[0532] The siRNA modification template with the antisense strand modified with modification E and the sense strand modified with modification b is designated DV33P.

[0533] The siRNA modification template with the antisense strand modified with modification F and the sense strand modified with modification b is designated DV34P.

[0534] The siRNA modification template with the antisense strand modified with modification G and the sense strand modified with modification c is designated DV22.

[0535] For compounds having 20 / 22 bases in the sense / antisense strand, one of the modifications in Table 44 is used for the antisense strand:

[0536] Table 44 Modifications for the antisense strand

[0537]

[0538]

[0539] The sense strand is one of the modifications shown in Table 48:

[0540] Table 48 Modifications for sense strand

[0541]

[0542] 2'-OMe is 2'-methoxy; 2'-F is 2'-fluoro; PS is phosphorothioate backbone in the above table;

[0543] The siRNA modification template with antisense strand of modification A and sense strand of modification a is named as DV25P;

[0544] The siRNA modification template with antisense strand of modification B and sense strand of modification a is named as DV26P;

[0545] The siRNA modification template with antisense strand of modification C and sense strand of modification a is named as DV27P;

[0546] The siRNA modification template with antisense strand of modification B and sense strand of modification b is named as DV28P;

[0547] The siRNA modification template with antisense strand of modification C and sense strand of modification b is named as DV29P;

[0548] The siRNA modification template with antisense strand of modification D and sense strand of modification b is named as DV32P;

[0549] The siRNA modification template with antisense strand of modification E and sense strand of modification b is named as DV33P;

[0550] The siRNA modification template with antisense strand of modification F and sense strand of modification b is named as DV34P.

[0551] The modification sequences of each template are shown in Table 12, and the synthesis method of each sequence is the same as in Example 1.

[0552] Table 12 Sequences modified with different modification templates

[0553]

[0554]

[0555]

[0556]

[0557]

[0558]

[0559]

[0560]

[0561]

[0562]

[0563]

[0564] Cell type: HepG2 cell line cells

[0565] Drug solvent: sterile enzyme-free water, gibco Opti-MEM

[0566] 2. Experimental method

[0567] The inhibition of AGT gene mRNA expression in HepG2 cells by the test sample was detected by qRT-PCR.

[0568] 2.1 Cell culture

[0569] Take the subcultured HepG2 cell strain, and use 10% fetal bovine serum DMEM culture solution (supplemented with 100 μL / mL of each of ampicillin and streptomycin) to culture the logarithmically growing cells in a 37°C cell culture incubator containing 5% CO2, with daily medium replacement. Digest the subculture with 0.25% trypsin, centrifuge at 1000 r / min for 5 min, discard the supernatant, and add fresh culture medium for subculture.

[0570] 2.2 Cell transfection

[0571] Transfection mixture preparation: mix Lipofectamine RNAiMAX and Opti-MEM at a ratio of 2:98, and vortex to mix well.

[0572] Transfection reagent preparation: take 60 μL of siRNA solution diluted in Opti-MEM at a ratio of 1:1 (v / v) and add it to 60 μL of transfection mixture, vortex to mix well, and then let it stand at room temperature for 15 min to obtain lipid nanoparticles (LNP). Take 12.5 μL for encapsulation rate detection.

[0573] Blank control group transfection reagent: add 60 μL of prepared transfection mixture to 60 μL of Opti-MEM. Vortex to mix well, and let it stand at room temperature for 15 min.

[0574] The prepared transfection reagent was added into the 24-well cell culture plate (100 μL per well) to make the final siRNA concentration 0.02 nM per well. 500 μL of cell suspension (containing 1.5 x 10 5 After mixing with the cross method, it was placed in a 37°C, 5% CO2 cell incubator for 40 h.

[0575] 2.3 AGT mRNA detection

[0576] The procedure was the same as that in Example 2, "2.3 AGT mRNA detection".

[0577] 2.4 Data processing

[0578] The AGT mRNA expression rate (%) was calculated according to the following formula:

[0579] Expression rate = (AGT mRNA expression amount / blank control group AGT mRNA expression amount) x 100%;

[0580] AGT gene expression inhibition rate = 1 - expression rate (%).

[0581] 2.5 IC50 experiment

[0582] In this experiment, the siRNA concentration for EC50 experiment of each sequence was selected from 1.0 nM, 4-fold dilution, a total of 8 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), and the inhibition rate of each sequence at each concentration was determined, and a graph was drawn to calculate the IC50 concentration of each sequence.

[0583] 3. Experimental results

[0584] After 3 repeated experiments, the inhibition rates of each modified sequence on HepG2 cell AGT gene are shown in Tables 13-25.

[0585] The activity detection experiment results showed that 13 candidate sequences (unmodified sequences 1576s, 1578s, 1838, 1835, 1816, 1812, 1579, 1836, 1789, 1839, 1791, 1795 and 1585s) were modified by using the modified templates DV25P-29P and DV32P-34P designed according to the present disclosure, and had a significant inhibitory effect on AGT gene, with an inhibition rate of more than 40%. Among them, C1812-DV25P, C1579-DV33P and C1789-DV26P had an inhibition rate of 74.4%, 74.0% and 73.6%, respectively.

[0586] The inhibition rate of AGT gene expression of the template DV25P-29P modified sequence is significantly improved compared with the alternating modified sequence. For example, the inhibition rate of the unmodified sequence 1579 using the template DV33P modified sequence C1579-DV33 is improved by 12.9% compared with the alternating modified sequence, and the inhibition rate of the unmodified sequence 1812 using the template DV25P modified sequence C1812-DV25P is improved by 11.5% compared with the alternating modified sequence.

[0587] In addition, the activity difference is very large after the same siRNA sequence is modified by different modification templates. For example, the inhibition rate of the unmodified sequence 1579 using the modification template DV33P of the present disclosure is improved by 29.8% compared with the modification template DV28P of the present disclosure, and the inhibition rate is improved by 18.4% compared with the Advanced ESC template DV22.

[0588] The IC50 experiment shows that the IC50 value of the sequence modified by the modification template designed by the present disclosure is 0.0019-0.0615 nM. For example, the IC50 values of C1789-DV25P, C1812-DV25P and C1789-DV26P are 0.0019 nM, 0.0022 nM and 0.0026 nM respectively. These sequences can effectively inhibit the expression of AGT gene at low concentration.

[0589] (i) The inhibition of AGT gene by each unmodified sequence after modification by the template

[0590] (1) Unmodified sequence 1576s

[0591] Table 13 Inhibition rate of AGT gene by unmodified sequence 1576s after modification by the template

[0592]

[0593]

[0594] I. DV25P-29P template of the present disclosure

[0595] The inhibition rate of AGT gene by the unmodified sequence 1576s modified by the modification template DV25, DV26 and DV29 designed by the present disclosure is more than 70%, which is significantly improved compared with the 2' methoxy and 2' fluoro alternating modification. For example, the inhibition rates of C1576s-DV25P and C1576s-DV29P are improved by 8.7% and 14.7% respectively after modification by the templates DV25P and DV29P.

[0596] II. Advanced ESC template DV22 (fluorinated sites: antisense strand 2, 6, 14 and 16 sites, sense strand 7, 9, 10 and 11 sites) has been disclosed in prior art

[0597] The inhibition rate of C1576s-DV22 on AGT gene was 63.9% after the unmodified sequence 1576s was modified by the template DV22, which was 0.7% lower than the alternating modification sequence.

[0598] The inhibition rates of C1576s-DV25P and C1576s-DV29P were increased by 9.4% and 15.4% respectively compared with C1576s-DV22 after the unmodified sequence 1576s was modified by the modified templates DV25P and DV29P of the present disclosure, which was a significant increase.

[0599] Therefore, it can be seen that:

[0600] 1) The inhibition effect on AGT gene of the unmodified sequence 1576s modified by the modified templates DV25-29 of the present disclosure was significantly improved compared with the alternating modification sequence. For example, the inhibition rate of the unmodified sequence 1576s modified by the modified template DV26 was increased by 8.8% compared with the alternating modification sequence.

[0601] 2) The activity difference was very large after the same siRNA sequence was modified by different modified templates. For example, the inhibition rate of the unmodified sequence 1576s modified by the modified templates DV25P and DV29P of the present disclosure was increased by 20.5% at most compared with the modified template DV28P of the present disclosure, which was a significant increase; and the inhibition rate was increased by 15.4% at most compared with the modified sequence of the disclosed Advanced ESC template DV22, which was a significant increase.

[0602] 3) The activity difference of siRNA sequences modified by different templates was as large as 20.5%. Therefore, it is uncertain which template modification can be used for the siRNA sequence to have high activity.

[0603] (2) Unmodified sequence 1578s

[0604] Table 14 Inhibition rate of unmodified sequence 1579 modified by templates on AGT gene

[0605]

[0606] I. DV25P-29P templates of the present disclosure

[0607] The inhibition rate of AGT gene of unmodified sequence 1578s modified by modification templates DV25P, DV27P and DV29P designed by the present disclosure is all more than 70%, which is significantly improved compared with the alternating modification of 2' methoxy and 2' fluoro.

[0608] II. The Advanced ESC template DV22 (fluorinated sites: antisense strand 2, 6, 14 and 16 sites, sense strand 7, 9, 10 and 11 sites) disclosed in the prior art

[0609] The inhibition rate of AGT gene of C1578s-DV22 of unmodified sequence 1578s modified by template DV22 is 61.1%, which is reduced by 2.8% compared with the alternating modification sequence.

[0610] The inhibition rates of C1578s-DV25P and C1578s-DV29P of sequence modified by modification templates DV25P and DV29P of the present disclosure are increased by 14.7% and 16.0% respectively compared with C1578s-DV22, which is significantly improved.

[0611] Therefore, it can be seen that:

[0612] 1) The inhibition effect of AGT gene of unmodified sequence 1578s modified by modification templates DV25P-29P of the present disclosure is significantly improved compared with the alternating modification sequence. For example, the inhibition rate of unmodified sequence 1578s modified by DV27P is increased by 9.2% compared with the alternating modification sequence.

[0613] 2) The activity difference is very large after the same siRNA sequence is modified by different modification templates. For example, the inhibition rate of unmodified sequence 1578s modified by modification templates DV25P and DV29P of the present disclosure is increased by 20.2% at most compared with the modification sequence of modification template DV28P of the present disclosure, which is significantly improved; and the inhibition rate is increased by 16.0% at most compared with the modification sequence of Advanced ESC template DV22 disclosed in the prior art, which is significantly improved.

[0614] 3) The activity difference of siRNA sequence modified by different templates is as large as 20.2%. Therefore, it is uncertain which template modification can be used for siRNA sequence to have high activity.

[0615] (3) Unmodified sequence 1838

[0616] Table 15 Inhibition rate of AGT gene of unmodified sequence 1579 modified by templates

[0617]

[0618] I. The DV25P-29P templates of the present disclosure

[0619] The inhibition rate of the unmodified sequence 1838 modified by the modified templates DV27P and DV29P designed by the present disclosure on the AGT gene is more than 70%, which is significantly improved compared with the alternating modification of 2' methoxy and 2' fluoro. For example, after modification by templates DV27P and DV29P, the inhibition rates of C1838-DV27P and C1838-DV29P are increased by 8.7% and 12.0%, respectively.

[0620] II. The Advanced ESC template DV22 (fluorinated sites: antisense strand 2, 6, 14 and 16, and sense strand 7, 9, 10 and 11) disclosed in the prior art

[0621] After the unmodified sequence 1838 is modified by the template DV22, the inhibition rate of C1838-DV22 on the AGT gene is 62.7%, which is reduced by 0.9% compared with the alternating modification sequence.

[0622] After the sequence is modified by the modified templates DV27P and DV29P of the present disclosure, the inhibition rates of C1838-DV27P and C1838-DV29P are increased by 9.6% and 12.9% compared with C1838-DV22, respectively, which is significantly improved.

[0623] Therefore, it can be seen that:

[0624] 1) The unmodified sequence 1838 modified by the modified templates DV25P-29P of the present disclosure has a significantly improved inhibition effect on the AGT gene compared with the alternating modification sequence. For example, the unmodified sequence 1838 modified by the DV27P modification sequence has an inhibition rate increased by 8.7% compared with the alternating modification sequence.

[0625] 2) The activity of the same siRNA sequence modified by different modification templates is very different. For example, the unmodified sequence 1838 modified by the modified templates DV27P and DV29P of the present disclosure has an inhibition rate increased by 15.3% at most compared with the modified sequence by the modified template DV28P of the present disclosure, which is significantly improved; and has an inhibition rate increased by 12.9% at most compared with the modified sequence by the disclosed Advanced ESC template DV22, which is significantly improved.

[0626] 3) The activity of siRNA sequences modified by different templates is very different, with a difference of 15.3% at most. Therefore, it is uncertain which template modification can be used for the siRNA sequence to have high activity.

[0627] (4) Unmodified sequence 1579

[0628] Table 16 Inhibition rate of AGT gene by unmodified sequence 1579 and modified sequence using modification templates of the present disclosure

[0629]

[0630] I. DV25P-29P templates of the present disclosure

[0631] The inhibition rate of AGT gene by C1579-DV25P and C1579-DV29P modified using modification templates DV25P and DV29P of the present disclosure is more than 70%, which is significantly improved compared with the alternating modification of 2'-methoxy and 2'-fluoride. For example, the inhibition rate of C1579-DV25P and C1579-DV29P is increased by 11.1% and 10.6% respectively after modification using templates DV25P and DV29P. Figure 7

[0632] II. Other modification templates of the present disclosure

[0633] The inhibition rate of AGT gene by C1579-DV32P, C1579-DV33P and C1579-DV34P modified using templates DV32P, DV33P and DV34P is 68.9%, 74.0 and 63.9% respectively, which is increased by 7.8%, 12.9% and 2.8% respectively compared with the corresponding alternating modification sequence.

[0634] The inhibition rate of C1579-DV25P and C1579-DV29P modified using modification templates DV25P and DV29P of the present disclosure is increased by 3.3% and 2.8% respectively compared with C1579-DV32P, decreased by 1.8% and 2.3% respectively compared with C1579-DV33P, and increased by 8.3% and 7.8% respectively compared with C1579-DV34P.

[0635] III. Advanced ESC template DV22 (fluoride sites: 2, 6, 14 and 16 sites of antisense strand, 7, 9, 10 and 11 sites of sense strand) disclosed in prior art

[0636] The inhibition rate of AGT gene by C1579-DV22 modified using template DV22 is 55.6%, which is decreased by 5.5% compared with the alternating modification sequence.

[0637] The inhibition rate of C1579-DV25P and C1579-DV29P modified using modification templates DV25P and DV29P of the present disclosure is increased by 16.6% and 16.1% respectively compared with C1579-DV22, which is significantly improved.

[0638] Therefore, it can be seen that:​

[0639] 1) The unmodified sequence 1579 adopts the modified template DV25-29 of the present disclosure, and the inhibition effect on the AGT gene is significantly improved compared with the alternating modified sequence. For example, the unmodified sequence 1579 adopts the DV26 modified sequence, and the inhibition rate is increased by 9.7% compared with the alternating modified sequence.

[0640] 2) The activity difference is very large after the same siRNA sequence is modified by different modified templates. For example, the unmodified sequence 1579 adopts the modified template DV25P and DV29P of the present disclosure, and the inhibition rate is increased by 28.0% at most compared with the modified template DV28P of the present disclosure, which is significantly improved; and the inhibition rate is increased by 16.6% at most compared with the modified template DV22 of the Advanced ESC template disclosed, which is significantly improved.

[0641] 3) The activity difference of siRNA sequences modified by different templates is large, and the difference is 28.0% at most. Therefore, it is uncertain which template modification can be used for the siRNA sequence to have high activity.

[0642] (5) Unmodified sequence 1585s

[0643] Table 17 Inhibition rate of unmodified sequence 1585s after modification by template

[0644]

[0645] I. DV26P-29P template of the present disclosure

[0646] The unmodified sequence 1585s adopts the templates DV26P, DV27P and DV29P designed by the present disclosure for modification, and the inhibition rate on the AGT gene is more than 50%, which is significantly improved compared with the alternating modification of 2' methoxy and 2' fluoro. For example, after modification by the templates DV26P and DV27P, the inhibition rates of C1585s-DV26P and C1585s-DV27P are increased by 3.3% and 5.3% respectively.

[0647] II. Other modified templates of the present disclosure

[0648] After the unmodified sequence 1585s is modified by the templates DV32P and DV34P, the inhibition rates of C1585s-DV32P and C1585s-DV34P on the AGT gene are 61.8% and 54.1% respectively, which are increased by 2.5% and -5.2% respectively compared with the corresponding alternating modified sequences.

[0649] The inhibition rates of C1585s-DV26P and C1585s-DV27P modified by the modification templates DV26P and DV27P of the present disclosure are increased by 0.8% and 2.8% respectively compared with C1585s-DV32P, and increased by 8.5% and 10.5% respectively compared with C1585s-DV34P.

[0650] III. The Advanced ESC template DV22 (fluorinated sites: antisense strand 2, 6, 14 and 16 sites, and sense strand 7, 9, 10 and 11 sites) disclosed in the prior art

[0651] The inhibition rate of C1585s-DV22 on AGT gene is 55.2% after the unmodified sequence 1585s is modified by the template DV22, which is decreased by 4.1% compared with the alternating modification sequence.

[0652] The inhibition rates of C1585s-DV26P and C1585s-DV27P modified by the modification templates DV26P and DV27P of the present disclosure are increased by 7.4% and 9.4% respectively compared with C1585s-DV22, which is significantly improved.

[0653] Therefore, it can be seen that:

[0654] 1) The inhibition effect on AGT gene of the unmodified sequence 1585s modified by the modification templates DV26P-29P of the present disclosure is significantly improved compared with the alternating modification sequence. For example, the inhibition rate of the unmodified sequence 1585s modified by the modification template DV27P is increased by 5.3% compared with the alternating modification sequence.

[0655] 2) The activity difference is very large after the same siRNA sequence is modified by different modification templates. For example, the inhibition rate of the unmodified sequence 51 modified by the modification template DV27P of the present disclosure is increased by 15.1% compared with the modification template DV28P of the present disclosure, which is significantly improved; and the inhibition rate is increased by 9.4% at most compared with the modification template DV22 of the Advanced ESC disclosed in the prior art, which is significantly improved.

[0656] 3) The activity difference of siRNA sequences modified by different templates is as large as 15.1%. Therefore, it is uncertain which template modification can be used for the siRNA sequence to have high activity.

[0657] (6) Unmodified sequence 1789

[0658] Table 181 Inhibition rates of unmodified sequence 1789 modified by templates on AGT gene

[0659]

[0660] I. The DV25P-29P templates of the present disclosure are used for modification

[0661] The inhibition rates of the unmodified sequence 1789 modified by the DV25P-29P templates of the present disclosure on the AGT gene are all more than 60%, and the inhibition rates are all improved compared with the 2'-methoxy and 2'-fluoro alternative modification. For example, the inhibition rates of C1789-DV25P and C1789-DV26P are increased by 5.5% and 10.1% respectively after the modification by the templates DV25P and DV26P.

[0662] II. Other modified templates of the present disclosure

[0663] The inhibition rates of the unmodified sequence 1789 modified by the DV32P-34P templates on the AGT gene are 61.8%, 57.9 and 56.9% respectively for C1789-DV32P, C1789-DV33P and C1789-DV34P, which are decreased by 1.7%, 5.6 and 6.6% respectively compared with the corresponding alternative modified sequences.

[0664] The inhibition rates of the unmodified sequence 1789 modified by the DV25P and DV26P templates of the present disclosure are increased by 7.2% and 11.8% respectively for C1789-DV25P and C1789-DV26P compared with C1789-DV32P, by 11.1% and 15.7% respectively compared with C1789-DV33P, and by 12.1% and 16.7% respectively compared with C1789-DV34P.

[0665] III. The Advanced ESC template DV22 (fluorinated sites: 2, 6, 14 and 16 sites of the antisense strand, and 7, 9, 10 and 11 sites of the sense strand) disclosed in the prior art

[0666] The inhibition rate of the unmodified sequence 1789 modified by the DV22 template on the AGT gene is 60.3%, which is decreased by 3.2% compared with the alternative modified sequence.

[0667] The inhibition rates of the unmodified sequence 1789 modified by the DV25P and DV26P templates of the present disclosure are increased by 8.7% and 13.3% respectively for C1789-DV25P and C1789-DV26P compared with C1789-DV22, which is a significant improvement.

[0668] Therefore, it can be seen that:

[0669] 1) The unmodified sequence 1789 modified by the DV25P-29P templates of the present disclosure has a significant improvement in the inhibition of the AGT gene compared with the alternative modified sequence. For example, the inhibition rate of the unmodified sequence 81 modified by the DV26P template is increased by 10.1% compared with the alternative modified sequence.

[0670] 2) The same siRNA sequence is modified by different modification templates, and the activity difference is very large. For example, the unmodified sequence 1789 is modified by the modification template DV25P and DV26P of the present disclosure, and the inhibition rate is increased by 16.7% at most compared with the modification sequence of the modification template DV32P-34P of the present disclosure, which is significantly improved; and the inhibition rate is increased by 13.3% at most compared with the modification sequence of the Advanced ESC template DV22 disclosed, which is significantly improved.

[0671] 3) The activity difference of siRNA sequences modified by different templates is large, and the difference is up to 16.7%. Therefore, it is uncertain which template modification can be used for the siRNA sequence to have high activity.

[0672] (7) Unmodified sequence 1791

[0673] Table 19 Inhibition rate of unmodified sequence 1791 modified by template on AGT gene

[0674]

[0675] I. DV26P-29P template of the present disclosure

[0676] The inhibition rate of the unmodified sequence 1791 modified by the modification template DV26-29P designed by the present disclosure on the AGT gene is more than 55%, which is significantly improved compared with the 2' methoxy and 2' fluoro alternative modification. For example, after modified by the templates DV26P and DV28P, the inhibition rates of C1791-DV26P and C1791-DV28P are increased by 8.5% and 2.8%, respectively.

[0677] II. Other modification templates of the present disclosure

[0678] After the unmodified sequence 1791 is modified by the templates DV32P and DV34P, the inhibition rates of C1791-DV32P and C1791-DV34P on the AGT gene are 65.9% and 66.7%, respectively, which are increased by 4.1% and 4.9%, respectively, compared with the 2' methoxy and 2' fluoro alternative modification.

[0679] After the unmodified sequence 1791 is modified by the modification template DV26P of the present disclosure, the inhibition rate of C1791-DV26P is increased by 4.4% and 3.6% compared with C1791-DV32P and C1791-DV34P, respectively.

[0680] Therefore, it can be known that:

[0681] 1) The unmodified sequence 1791 was modified by the modification template DV26-29P of the present disclosure, and the inhibition effect on the AGT gene was significantly improved compared with the alternating modification sequence. For example, the unmodified sequence 1791 was modified by the modification template DV26P, and the inhibition rate was increased by 8.5% compared with the alternating modification sequence.

[0682] 2) The activity of the same siRNA sequence modified by different modification templates is very different. For example, the unmodified sequence 1791 was modified by the modification template DV26P of the present disclosure, and the inhibition rate was increased by 10.9% compared with the modification template DV29P of the present disclosure, which was significantly improved.

[0683] 3) The activity of siRNA sequences modified by different templates is very different, and the difference can be up to 10.9%. Therefore, it is uncertain which template modification can be used for the siRNA sequence to have high activity.

[0684] (8) Unmodified sequence 1795

[0685] Table 20 Inhibition rate of unmodified sequence 1795 modified by templates on AGT gene

[0686]

[0687]

[0688] I. DV25-29P templates of the present disclosure

[0689] The unmodified sequence 1795 was modified by the modification templates DV26-29P designed by the present disclosure, and the inhibition rate on the AGT gene was more than 50%, which was improved compared with the alternating modification of 2' methoxy and 2' fluoro. For example, after modification by templates DV27P and DV29P, the inhibition rates of C1795-DV27P and C1795-DV29P were increased by 5.8% and 4.1%, respectively.

[0690] II. Other modification templates of the present disclosure

[0691] After the unmodified sequence 1795 was modified by templates DV32P and DV34P, the inhibition rates of C1795-DV32P and C1795-DV34P on the AGT gene were 55.8% and 55.0%, respectively, which were decreased by 1.7% and 2.5% compared with the corresponding alternating modification sequences.

[0692] The inhibition rates of C1795-DV27P and C1795-DV29P modified by the modification templates DV27P and DV29P of the present disclosure were increased by 7.5% and 5.8% compared with C1795-DV32P, and increased by 8.3% and 6.6% compared with C1795-DV34P.

[0693] Therefore, it can be seen that:

[0694] 1) The unmodified sequence 1795 adopts the modification template DV25P-29P of the present disclosure, and the inhibition effect on the AGT gene is significantly improved compared with the alternating modification sequence. For example, the unmodified sequence 84 adopts the modification template DV27P, and the inhibition rate is increased by 5.8% compared with the alternating modification sequence.

[0695] 2) After the same siRNA sequence is modified by different modification templates, the activity difference is very large. For example, the unmodified sequence 1795 adopts the modification template DV27P of the present disclosure, and the inhibition rate is increased by 11.4% compared with the modification template DV28P of the present disclosure, which is significantly improved.

[0696] 3) The activity difference of siRNA sequences modified by different templates is huge, and the maximum difference can be 11.4%. Therefore, it is uncertain which template modification can have high activity for siRNA sequences.

[0697] (9) Unmodified sequence 1812

[0698] Table 21 Inhibition rate of unmodified sequence 1812 after modification by template on AGT gene

[0699]

[0700] I. Modification by DV25P-29P template of the present disclosure

[0701] The unmodified sequence 1812 adopts the template DV25P-29P designed by the present disclosure for modification, and the inhibition rate on the AGT gene is more than 55%, which is improved compared with the 2' methoxy and 2' fluoro alternating modification. For example, after modification by the template DV25P, the inhibition rate of C1812-DV25P is increased by 11.5%.

[0702] II. Other modification templates of the present disclosure

[0703] After the unmodified sequence 1812 is modified by the template DV32P-34P, the inhibition rates of C1812-DV32P, C1812-DV33P and C1812-DV34P on the AGT gene are 63.6%, 54.2% and 60.7% respectively, which are increased by 0.7%, -8.7% and -2.2% respectively compared with the corresponding alternating modification sequences.

[0704] The inhibition rate of C1812-DV25P is 10.8% higher than that of C1812-DV32P, 20.2% higher than that of C1812-DV33P, and 13.7% higher than that of C1812-DV34P, which is significantly improved.

[0705] III. The Advanced ESC template DV22 (fluorinated sites: antisense strand 2, 6, 14 and 16, and sense strand 7, 9, 10 and 11) disclosed in the prior art

[0706] After the unmodified sequence 1812 is modified by the template DV22, the inhibition rate of C812-DV22 on the AGT gene is 63.6%, which is 0.7% higher than that of the alternating modification sequence.

[0707] The inhibition rate of C1812-DV25P is 10.8% higher than that of C1812-DV22, which is significantly improved.

[0708] Therefore, it can be seen that:

[0709] 1) The unmodified sequence 1812 is modified by the modification template DV25-29P of the present disclosure, and the inhibition effect on the AGT gene is significantly improved compared with the alternating modification sequence. For example, the unmodified sequence 1812 is modified by the DV25P modification sequence, and the inhibition rate is 11.5% higher than that of the alternating modification sequence.

[0710] 2) The activity of the same siRNA sequence modified by different modification templates is very different. For example, the unmodified sequence 1812 is modified by the modification template DV25P of the present disclosure, and the inhibition rate is 20.2% higher than that of the modification template DV33P of the present disclosure, which is significantly improved; and the inhibition rate is at most 10.8% higher than that of the modification sequence of the Advanced ESC template DV22 disclosed in the prior art, which is significantly improved.

[0711] 3) The activity of siRNA sequences modified by different templates is very different, and the difference is at most 20.2%. Therefore, it is uncertain which template modification can be used for the siRNA sequence to have high activity.

[0712] (10) Unmodified sequence 1816

[0713] Table 22 Inhibition rate of unmodified sequence 1816 modified by template on AGT gene

[0714]

[0715] I. Modification by the DV25P-29P template of the present disclosure

[0716] The inhibition rate of AGT gene of the unmodified sequence 1816 modified by the templates DV25P, DV26P, DV27P and DV29P designed by the present disclosure is all more than 55%, which is higher than that of the 2'-methoxy and 2'-fluoro alternative modification. For example, the inhibition rate of C1812-DV25P is increased by 7.6% after modified by the template DV29P.

[0717] II. Other modified templates of the present disclosure

[0718] The inhibition rate of AGT gene of the unmodified sequence 1816 modified by the templates DV32P-34P is 58.6%, 48.4% and 38.5% respectively for C1816-DV32P, C1816-DV33P and C1816-DV34P, which is decreased by 2.3%, 12.5% and 22.4% respectively compared with the corresponding alternative modified sequence.

[0719] The inhibition rate of C1816-DV25P modified by the modified template DV29P of the present disclosure is increased by 9.9% compared with C1816-DV32P, 20.1% compared with C1816-DV33P and 30.0% compared with C1816-DV34P, which is significantly increased.

[0720] III. The prior art has disclosed an Advanced ESC template DV22 (fluorinated sites: 2, 6, 14 and 16 sites of the antisense strand, and 7, 9, 10 and 11 sites of the sense strand)

[0721] The inhibition rate of AGT gene of the unmodified sequence 1816 modified by the template DV22 is 66.7% for C1816-DV22, which is increased by 5.8% compared with the alternative modified sequence.

[0722] The inhibition rate of C1816-DV29P modified by the modified template DV29P of the present disclosure is increased by 1.8% compared with C1816-DV22.

[0723] Therefore, it can be known that:

[0724] 1) The inhibition effect of AGT gene of the unmodified sequence 1816 modified by the modified templates DV25P-29P of the present disclosure is significantly increased compared with the alternative modified sequence. For example, the inhibition rate of the unmodified sequence 1816 modified by the template DV29P is increased by 7.6% compared with the alternative modified sequence.

[0725] 2) The same siRNA sequence has very different activity after modification with different modification templates. For example, the unmodified sequence 1816 modified with the modification template DV29P of the present disclosure has an inhibition rate that is 30.0% higher than that of the sequence modified with the modification template DV34P of the present disclosure, and is significantly improved; and is 1.8% higher than that of the sequence modified with the Advanced ESC template DV22 that has been disclosed.

[0726] 3) The siRNA sequences modified with different templates have a huge difference in activity, with a maximum difference of 30.0%. Therefore, it is uncertain which template modification can be used for the siRNA sequence to have high activity.

[0727] (11) Unmodified sequence 1835

[0728] Table 23 Inhibition rate of unmodified sequence 1835 modified with templates on AGT gene

[0729]

[0730] I. Modified with DV25P-29P templates of the present disclosure

[0731] The unmodified sequence 1835 modified with the DV25P-29P templates designed by the present disclosure has an inhibition rate on AGT gene that exceeds 50%, and except for DV27P, the inhibition rate on AGT gene is improved compared with the 2' methoxy and 2' fluoro alternative modification. For example, after modification with the template DV26P, the inhibition rate of C1835-DV26P is improved by 9.5%.

[0732] II. Other modification templates of the present disclosure

[0733] After modification with the templates DV32P-34P, the inhibition rates of C1835-DV32P, C1835-DV33P and C1835-DV34P on AGT gene are 53.3%, 64.9% and 71.8% respectively, which are improved by -9.0%, 2.6% and 9.5% respectively compared with the corresponding alternative modification sequences.

[0734] The inhibition rate of C1835-DV26P modified with the modification template DV26P of the present disclosure is 18.5% higher than that of C1835-DV32P, 10.7% higher than that of C1835-DV33P, and 0.0% higher than that of C1835-DV34P.

[0735] III. Advanced ESC template DV22 (fluorinated sites: antisense strand 2, 6, 14 and 16, and sense strand 7, 9, 10 and 11) that has been disclosed in the prior art

[0736] The inhibition rate of C1835-DV22 on AGT gene was 58.2% after the unmodified sequence 1835 was modified by using the template DV22, which was 4.1% lower than that of the alternative modified sequence.

[0737] The inhibition rate of C1835-DV26P was 13.6% higher than that of C1835-DV22 after the unmodified sequence 1835 was modified by using the modified template DV25P of the present disclosure, which was significantly improved.

[0738] Therefore, it can be seen that:

[0739] 1) The inhibition effect on AGT gene of the unmodified sequence 1835 by using the modified template DV25-29P of the present disclosure was significantly improved compared with the alternative modified sequence. For example, the inhibition rate of the unmodified sequence 1835 by using the modified template DV26P was 9.5% higher than that of the alternative modified sequence.

[0740] 2) The activity difference was very large after the same siRNA sequence was modified by using different modified templates. For example, the inhibition rate of the unmodified sequence 1835 by using the modified template DV26P of the present disclosure was 20.6% higher than that of the modified template DV27P of the present disclosure, which was significantly improved; and the inhibition rate was at most 13.6% higher than that of the modified sequence DV22 of the Advanced ESC template, which was significantly improved.

[0741] 3) The activity difference of siRNA sequences modified by different templates was as large as 20.6%. Therefore, it is uncertain which template modification can be used for the siRNA sequence to have high activity.

[0742] (12) Unmodified sequence 1836

[0743] Table 24 Inhibition rate of unmodified sequence 1836 on AGT gene after modification by using templates

[0744]

[0745]

[0746] I. Modified by using the DV26-29P template of the present disclosure

[0747] The inhibition rate of the unmodified sequence 1836 on AGT gene after modification by using the templates DV26P-29P designed by the present disclosure was all more than 55%, which was improved compared with the 2' methoxy and 2' fluoro alternative modification. For example, the inhibition rate of C1836-DV26P and C1836-DV28P was improved by 4.0% after modification by using the templates DV26P and DV28P.

[0748] II. Other modified templates of the present disclosure

[0749] The inhibition rates of C1836-DV32P and C1836-DV34P on AGT gene were 49.2% and 63.0% respectively, which were 2.9% and 10.6% lower than the corresponding alternative modified sequences.

[0750] The inhibition rates of C1836-DV26P and C1836-DV28P were 14.6% and 0.8% higher than C1836-DV32P and C1836-DV34P respectively.

[0751] Therefore, it can be seen that:

[0752] 1) The unmodified sequence 1836 is modified by the modified template DV26-29P of the present disclosure, and the inhibition effect on AGT gene is significantly improved compared with the alternative modified sequence. For example, the unmodified sequence 1836 is modified by DV26P and DV28P, and the inhibition rate is 4.0% higher than the alternative modified sequence.

[0753] 2) The activity of the same siRNA sequence modified by different modified templates is very different. For example, the unmodified sequence 1836 is modified by the modified template DV26P and DV28P of the present disclosure, and the inhibition rate is 14.6% higher than the modified template DV32P of the present disclosure, which is significantly improved.

[0754] 3) The activity of siRNA sequences modified by different templates is very different, and the difference can be up to 14.6%. Therefore, it is uncertain which template modification can have high activity for siRNA sequences.

[0755] (13) Unmodified sequence 1839

[0756] Table 25 Inhibition rate of unmodified sequence 1839 modified by template on AGT gene

[0757]

[0758]

[0759] I. Modified by DV26-29P template of the present disclosure

[0760] The unmodified sequence 1839 is modified by the template DV26-29P designed by the present disclosure, and the inhibition rate on AGT gene is more than 50%, which is improved compared with the 2' methoxy and 2' fluoro alternative modification. For example, after modified by template DV29P, the inhibition rate of C1839-DV29P is improved by 5.1%.

[0761] II. Other modified templates of the present disclosure

[0762] After the unmodified sequence 1839 was modified by the templates DV32P and DV34P, the inhibition rates of C1839-DV32P and C1839-DV34P on the AGT gene were 63.7% and 55.9%, respectively, which were increased by 4.6% and -3.2% compared with the corresponding alternate modified sequences.

[0763] After the unmodified sequence 1839 was modified by the modified template DV29P of the present disclosure, the inhibition rate of C1839-DV29P was increased by 0.5% and 8.3% compared with C1839-DV32P and C1839-DV34P, respectively.

[0764] Therefore, it can be seen that:

[0765] 1) The unmodified sequence 1839 was modified by the modified template DV26-29P of the present disclosure, and the inhibition effect on the AGT gene was significantly improved compared with the alternate modified sequence. For example, the unmodified sequence 1839 was modified by the DV29P modified sequence, and the inhibition rate was increased by 5.1% compared with the alternate modified sequence.

[0766] 2) After the same siRNA sequence was modified by different modified templates, the activity difference was very large. For example, the unmodified sequence 1839 was modified by the modified template DV29P of the present disclosure, and the inhibition rate was increased by 15.6% compared with the modified sequence modified by the modified template DV26P of the present disclosure, which was significantly improved.

[0767] 3) The activity difference of siRNA sequences modified by different templates was huge, and the difference could be up to 15.6%. Therefore, it is uncertain which template modification can have high activity for siRNA sequences.

[0768] Summary:

[0769] (1) The unmodified sequences 1576s, 1578s, 1838, 1835, 1816, 1812, 1579, 1836, 1789, 1839, 1791, 1795 and 1585s were modified by the modified templates DV25P-29P designed by the present disclosure, and had a significant inhibition effect on the expression of the AGT gene, and the inhibition rate was more than 40%. Among them, the inhibition rates of C1812-DV25P, C1579-DV33P and C1789-DV26P were 74.4%, 74.0% and 73.6%, respectively.

[0770] (2) The above-mentioned 12 sequences use the modification template DV25-29P of the present disclosure to modify the sequence, and compared with the alternating modification sequence, the inhibition rate of AGT gene expression is significantly improved. For example, the unmodified sequence 1812 uses the template DV25P modification sequence C1812-DV25P, and the inhibition rate is increased by 11.5% compared with the alternating modification sequence. The unmodified sequence 1791 uses the template DV26P modification sequence C1791-DV26P, and the inhibition rate is increased by 8.5% compared with the alternating modification sequence.

[0771] (3) The same sequence uses the modification template DV25-29P of the present disclosure to modify, and compared with the modification template disclosed by the prior art, the inhibition rate of AGT gene expression is significantly improved. For example, the unmodified sequence 1579 uses the modification template DV25P of the present disclosure to modify the sequence, and compared with the modification sequence using the disclosed Advanced ESC template DV22, the inhibition rate is increased by 16.6%.

[0772] (4) The same sequence uses the modification template DV25-29P of the present disclosure to modify, and compared with the other modification templates DV32-34P of the present disclosure, the inhibition rate of AGT gene expression is significantly improved. For example, the unmodified sequence 1812 uses the modification template DV25P of the present disclosure to modify the sequence, and compared with the modification sequence using the modification template DV33P of the present disclosure, the inhibition rate is increased by 20.2%.

[0773] (5) The activities of different template modification sequences are greatly different. For example, the unmodified sequence 1816 uses DV29P modification, and compared with the DV34 modification, the inhibition rate is increased by 30.0%. The unmodified sequence 1579 uses DV33P modification, and compared with the DV28P modification, the inhibition rate is increased by 29.8%. Therefore, it is uncertain that which modification template is used to modify the siRNA sequence to have high activity.

[0774] (ii) IC50 experiment

[0775] IC50 experiment was carried out on 24 template modified sequences including C1816-DV29P, C1816-DV25P, C1812-DV25P, C1579-DV32P, C1789-DV26P, C1789-DV25P, C1839-DV29P, C1795-DV27P, C1585s-DV27P, C1835-DV26P, C1835-DV25P, C1579-DV25P, C1836-DV26P, C1791-DV26P, C1585s-DV29P, C1604-DV29P, C1606-DV29P, C1607-DV29P, C1835-DV29P, C1838-DV29P, C1579-DV29P, C1576s-DV29P, C1578s-DV29P and C1579s-DV29P to determine the IC50 concentration of each sequence. The experimental results are shown in Table 26.

[0776] Table 26 IC50 experimental results of each modified sequence

[0777]

[0778]

[0779] As can be seen from Table 26, the IC50 values of the 24 sequences are between 1.884 pM and 61.498 pM. Among them, the IC50 values of C1789-DV25P, C1812-DV25P and C1789-DV26P are 1.884 pM, 2.160 pM and 2.643 pM respectively. It is shown that at low concentration, these sequences can effectively inhibit the expression of AGT gene.

[0780] Example 5: Comparison of AGT gene inhibition effect with prior art disclosed sequences

[0781] In this example, the AGT gene inhibition efficiency of the unmodified sequences 1579, 1789, 1812, 1576s, 1578s, 1585s, 1816, 1835, 1836, 1838, 1839 and 1791 disclosed in this application, the unmodified sequences similar to the prior art disclosed sequences, the sequences using the alternating modification sequence disclosed in this application and the sequences using DV25P-29P template modification, were compared.

[0782] 1. Experimental materials

[0783] Test samples:

[0784] (1) Prior art disclosed sequences, see Table 27.

[0785] Table 27 Prior art disclosed sequences

[0786]

[0787] (2) siRNA sequence with 2'-OMe and 2'-F alternately modified and terminal thio-modified in Example 2; (3) siRNA sequence modified by using modification template DV25-29P, see Table 28 for specific sequence.

[0788] Table 28: Each sequence modified by using modification template DV25P-29P

[0789]

[0790]

[0791]

[0792]

[0793]

[0794]

[0795] Cell type: HepG2 cells, provided by Selleck (H1-1701).

[0796] HepG2 cells were cultured in DMEM medium (ATCC-30-2003) containing 10% fetal bovine serum (FBS, ExCell Bio-FSP500), 1% penicillin-streptomycin (HyClone-SV30010).

[0797] Drug vehicle: sterile enzyme-free water, gibco Opti-MEM.

[0798] 2. Experimental method

[0799] qRT-PCR was used to detect the inhibition of HepG2 cell line AGT gene mRNA expression by the test sample.

[0800] 2.1 Cell culture

[0801] The subcultured HepG2 cell strain was used, and the logarithmically growing cells were cultured in 10% fetal bovine serum DMEM culture medium (supplemented with 100 μL / mL of penicillin and streptomycin each) and placed in a cell culture incubator at 37°C with 5% CO2, with daily medium replacement. The cells were subcultured by 0.25% trypsin digestion, centrifuged at 1000 r / min for 5 min, the supernatant was discarded, and the cells were subcultured in fresh culture medium.

[0802] 2.2 Cell transfection

[0803] Transfection mixture preparation: mix Lipofectamine RNAiMAX and Opti-MEM at a ratio of 2:98, and vortex well.

[0804] Transfection reagent preparation: take 60 μL of siRNA solution diluted in Opti-MEM and add 60 μL of transfection mixture at a ratio of 1:1 (v / v), vortex well, and stand at room temperature for 15 min to obtain lipid nanoparticles (LNP). Take 12.5 μL for encapsulation rate detection.

[0805] Blank control group transfection reagent: add 60 μL of prepared transfection mixture to 60 μL of Opti-MEM. Vortex well, and stand at room temperature for 15 min.

[0806] Add the prepared transfection reagent to a 24-well cell culture plate (100 μL per well), so that the final siRNA concentration per well is 0.02 nM. Add 500 μL of cell suspension (containing 1.5 x 10 5 After mixing with the cross method, place in a 37°C, 5% CO2 cell incubator for 40 h.

[0807] 2.3 AGT mRNA detection

[0808] 1) RNA extraction

[0809] The steps are the same as "1) RNA extraction" in "2.3 AGT mRNA detection" in Example 2.

[0810] 2) RNA concentration detection

[0811] The steps are the same as "2) RNA concentration detection" in "2.3 AGT mRNA detection" in Example 2.

[0812] 3) AGT mRNA quantitative detection

[0813] In a 15 mL centrifuge tube, add the remaining ingredients except for primers and templates, and label as A.

[0814] Label 1.5 mL EP tubes, and add 77 μL of A and 420 ng of total RNA per tube, and mix well for standby.

[0815] Mix the upstream and downstream primers of the internal reference gene GAPDH and the target gene AGT for standby.

[0816] Add 11 μL of B and 1 μL of C to each well of the PCR plate. Cover with sealing film, centrifuge at 3000 rpm for 1 min, and load onto the machine.

[0817] * This step is operated on ice to maintain low temperature conditions.

[0818] Put the plate into the qPCR instrument and run according to the following procedure:

[0819] Reverse transcription: 55°C, 15 min;

[0820] Pre-denaturation: 95°C, 30 sec;

[0821] Cycle reaction: 95°C, 10 sec; 60°C, 35 sec; 40 cycles;

[0822] Melting curve: 95°C, 15 sec; 60°C, 60 sec; 95°C, 15 sec.

[0823] The running time is about 2 h. Analyze the experimental results and calculate 2-ΔΔCt.

[0824] 2.4 Data processing

[0825] The AGT mRNA expression rate (%) is calculated according to the following formula:

[0826] Expression rate = (experimental group AGT mRNA expression amount / blank control group AGT mRNA expression amount) x 100%;

[0827] AGT gene expression inhibition rate = 1 - expression rate (%).

[0828] 3. Experimental results

[0829] The specific experimental results are shown in Table 29.

[0830] The experimental results show that the alternating modification sequence and the sequence modified by the specific modification template in the present disclosure have significantly improved AGT inhibition activity compared with the similar unmodified sequence disclosed in the prior art. For example, the sequence 812P in the prior art is compared with the sequence 1812 in the present disclosure, the 3' end of the positive strand is 1 base A less; the 5' end of the negative strand is 1 base U less, and the others are completely the same. However, the inhibition rate of the unmodified sequence 1812 in the present disclosure is increased by 14.3% compared with 812P, the inhibition rate of the alternating modification sequence is 62.9%, which is increased by 20.4% compared with 812P, and after modification by the modification template DV25 in the present disclosure, the inhibition rate reaches 74.4%, which is increased by 31.9% compared with 31P.

[0831] (1) Compared with the similar unmodified sequence disclosed in the prior art, the unmodified sequence, the alternating modification sequence and the sequence modified by the specific modification template in the present disclosure have significantly improved AGT gene inhibition rate, which can be increased by 91.0% at most.

[0832] Compared with the sequence similar to the prior art, the inhibition rate of the AGT gene of the unmodified sequence, the alternately modified sequence and the sequence modified by the specific modification template in the present disclosure is significantly improved. For example, the inhibition rate of the unmodified sequence 812P is 42.5%, the inhibition rate of the unmodified sequence 1812 similar to the sequence in the present disclosure is 56.8%, which is increased by 14.3% compared with 812P, the inhibition rate of the alternately modified sequence is 62.9%, which is increased by 20.4% compared with 812P, and after being modified by the modification template DV25 in the present disclosure, the inhibition rate reaches 74.4%, which is increased by 31.9% compared with 812P.

[0833] Table 29 Inhibition rate of AGT gene of prior art and sequence similar to the present disclosure

[0834]

[0835]

[0836]

[0837] I. Sequence structure comparison

[0838] The sequence disclosed in the prior art in Table 30 is very close to the sequence in the present disclosure, and the difference is small. The specific comparison is shown in the following table:

[0839] Table 30 Comparison of sequence disclosed in prior art and sequence disclosed in the present disclosure

[0840]

[0841]

[0842] As can be seen from Table 30:

[0843] The prior art disclosed sequence 579P and the sequence 1579 in the present disclosure are different in that the 5' end of the positive strand has one more base A, and the 3' end has one base U; the 5' end of the antisense strand has one base A, and the 3' end has one more base G.

[0844] The prior art disclosed sequence 789P and the sequence 1789 in the present disclosure are different in that the 5' end of the positive strand has one less base G, and the 3' end has one base A; the 5' end of the antisense strand has one base U, and the 3' end has one less base A.

[0845] The prior art disclosed sequence 812P and the sequence 1812 in the present disclosure are different in that the 3' end of the positive strand has one less base A; the 5' end of the antisense strand has one less base U.

[0846] The prior art disclosed sequence 576P and the sequence 1576s in the present disclosure are different in that the 3' end of the positive strand has two bases GU; the 5' end of the antisense strand has two bases AC.

[0847] The prior art disclosed sequence 578P has 1 base C more at the 5' end of the sense strand and 1 base G more at the 3' end of the antisense strand than the sequence 1578s of the present disclosure.

[0848] The prior art disclosed sequence 585P has 1 base C less at the 5' end of the sense strand and 3 bases GAA less at the 3' end of the antisense strand than the sequence 1585s of the present disclosure.

[0849] The prior art disclosed sequence 816P has 1 base U less at the 5' end of the sense strand and 1 base U different at the 5' end and 1 base A less at the 3' end of the antisense strand than the sequence 1816 of the present disclosure.

[0850] The prior art disclosed sequence 835P has 2 bases UU less at the 5' end of the sense strand and 2 bases UU less at the 3' end of the antisense strand than the sequence 1835 of the present disclosure.

[0851] The prior art disclosed sequence 836P has 2 bases UG less at the 5' end of the sense strand and 2 bases AU less at the 3' end of the antisense strand than the sequence 1836 of the present disclosure.

[0852] The prior art disclosed sequence 839P has the same sense strand and 2 bases UG less at the 5' end of the antisense strand than the sequence 1839 of the present disclosure. Compared with the sequence 1838 of the present disclosure, the sense strand has 1 base G less at the 5' end and 2 bases CA different at the 3' end; the antisense strand has 1 base U less at the 5' end and 1 base A less at the 3' end.

[0853] The prior art disclosed sequence 791P has 1 base U different at the 3' end of the sense strand and 2 bases UA less at the 5' end and 2 bases UU different at the 3' end of the antisense strand than the sequence 1791 of the present disclosure.

[0854] II. Activity comparison

[0855] 1) The unmodified sequence of the present disclosure has significantly improved activity compared with the similar unmodified sequence disclosed in the prior art. For example, the unmodified sequence 1576s of the present disclosure has an inhibition rate of 20.9% higher than the similar sequence 576P (inhibition rate of 47.1%) in the prior art. Figure 6 ).

[0856] The unmodified sequence 1579 of the present disclosure has an inhibition rate of 58.2% on AGT gene, which is 11.2% higher than the similar sequence 579P (inhibition rate of 47%) in the prior art, and is significantly improved.

[0857] The unmodified sequence 1789 of the present disclosure has an inhibition rate of 54.5% on AGT gene, which is 2.6% higher than the similar sequence 789P (inhibition rate of 51.9%) in the prior art.

[0858] The AGT gene inhibition rate of the unmodified sequence 1812 in the present disclosure is 56.8%, which is increased by 14.3% compared with the similar sequence 812P (inhibition rate of 42.5%) disclosed in the prior art, and is significantly improved.

[0859] The AGT gene inhibition rate of the unmodified sequence 1576s in the present disclosure is 59.1%, which is increased by 20.9% compared with the similar sequence 576P (inhibition rate of 38.2%) disclosed in the prior art, and is significantly improved.

[0860] The AGT gene inhibition rate of the unmodified sequence 1578s in the present disclosure is 55.2%, which is increased by 21.6% compared with the similar sequence 578P (inhibition rate of 33.6%) disclosed in the prior art, and is significantly improved.

[0861] The AGT gene inhibition rate of the unmodified sequence 1585s in the present disclosure is 48.9%, which is increased by 1.57% compared with the similar sequence 585P (inhibition rate of 47.33%) disclosed in the prior art.

[0862] The AGT gene inhibition rate of the unmodified sequence 1816 in the present disclosure is 46.8%, which is increased by -17.7% compared with the similar sequence 816P (inhibition rate of 64.5%) disclosed in the prior art, and is significantly improved.

[0863] The AGT gene inhibition rate of the unmodified sequence 1835 in the present disclosure is 51.6%, which is increased by 37.7% compared with the similar sequence 835P (inhibition rate of 13.9%) disclosed in the prior art, and is significantly improved.

[0864] The AGT gene inhibition rate of the unmodified sequence 1836 in the present disclosure is 46.5%, which is increased by 4.6% compared with the similar sequence 836P (inhibition rate of 41.9%) disclosed in the prior art.

[0865] The AGT gene inhibition rate of the unmodified sequence 1839 in the present disclosure is 45.7%, which is increased by 61.1% compared with the similar sequence 839P (inhibition rate of -15.4%) disclosed in the prior art, and is significantly improved.

[0866] The AGT gene inhibition rate of the unmodified sequence 1838 in the present disclosure is 50.6%, which is increased by 66.0% compared with the similar sequence 839P (inhibition rate of -15.4%) disclosed in the prior art, and is significantly improved.

[0867] The AGT gene inhibition rate of the unmodified sequence 1791 in the present disclosure is 46.5%, which is decreased by 4.3% compared with the similar sequence 791P (inhibition rate of 50.8%) disclosed in the prior art.

[0868] 2) The activity of the alternately modified sequence in the present disclosure is significantly improved compared with the prior art disclosed unmodified sequence. For example, the inhibition rate of the alternately modified sequence B1579-AL is increased by 14.1% compared with sequence 579P.

[0869] After the unmodified sequence 1579 in the present disclosure is alternately modified, the inhibition rate of B1579-AL on AGT gene is 61.1%, which is increased by 14.1% compared with the prior art disclosed unmodified sequence 579P, and is significantly improved.

[0870] After the unmodified sequence 1789 in the present disclosure is alternately modified, the inhibition rate of B1789-AL on AGT gene is 63.5%, which is increased by 11.6% compared with the prior art disclosed unmodified sequence 789P, and is significantly improved.

[0871] After the unmodified sequence 1812 in the present disclosure is alternately modified, the inhibition rate of B1812-AL on AGT gene is 62.9%, which is increased by 20.4% compared with the prior art disclosed unmodified sequence 812P, and is significantly improved.

[0872] After the unmodified sequence 1576s in the present disclosure is alternately modified, the inhibition rate of B1576s-AL on AGT gene is 64.6%, which is increased by 26.4% compared with the prior art disclosed unmodified sequence 576P, and is significantly improved.

[0873] After the unmodified sequence 1578s in the present disclosure is alternately modified, the inhibition rate of B1578s-AL on AGT gene is 63.9%, which is increased by 30.3% compared with the prior art disclosed unmodified sequence 578P, and is significantly improved.

[0874] After the unmodified sequence 1585s in the present disclosure is alternately modified, the inhibition rate of B1585s-AL on AGT gene is 59.3%, which is increased by 11.97% compared with the prior art disclosed unmodified sequence 585P, and is significantly improved.

[0875] After the unmodified sequence 1816 in the present disclosure is alternately modified, the inhibition rate of B1816-AL on AGT gene is 60.9%, which is decreased by 3.6% compared with the prior art disclosed unmodified sequence 816P.

[0876] After the unmodified sequence 1835 in the present disclosure is alternately modified, the inhibition rate of B1835-AL on AGT gene is 62.3%, which is increased by 48.4% compared with the prior art disclosed unmodified sequence 835P, and is significantly improved.

[0877] After the unmodified sequence 1836 in the present disclosure is alternately modified, the inhibition rate of B1836-AL on AGT gene is 59.8%, which is increased by 17.9% compared with the prior art disclosed unmodified sequence 836P, and is significantly improved.

[0878] After the alternating modification of the unmodified sequence 1839 of the present disclosure, the inhibition rate of B1839-AL on AGT gene is 59.1%, which is increased by 74.5% compared with the unmodified sequence 839P disclosed in the prior art, and is significantly improved.

[0879] After the alternating modification of the unmodified sequence 1838 of the present disclosure, the inhibition rate of B1838-AL on AGT gene is 63.6%, which is increased by 79% compared with the unmodified sequence 839P disclosed in the prior art, and is significantly improved.

[0880] After the alternating modification of the unmodified sequence 1791 of the present disclosure, the inhibition rate of B1791-AL on AGT gene is 61.8%, which is increased by 11% compared with the unmodified sequence 791P disclosed in the prior art, and is significantly improved.

[0881] 3) The activity of the sequence modified by the modification template of the present disclosure is significantly improved compared with the unmodified sequence disclosed in the prior art. For example, compared with sequence 579P, the inhibition rate of the modified sequence C1579-DV26P of the present disclosure is increased by 25.2%.

[0882] After the modification of the unmodified sequence 1579 of the present disclosure by the modification template DV25P-29P, the inhibition rate can be increased by more than 20% compared with the unmodified sequence 579P disclosed in the prior art. For example, C1579-DV26P is increased by 25.2%, which is significantly improved.

[0883] After the modification of the unmodified sequence 1789 of the present disclosure by the modification template DV25P-29P, the inhibition rate can be increased by more than 20% compared with the unmodified sequence 789P disclosed in the prior art. For example, C1789-DV26P is increased by 21.7%, which is significantly improved.

[0884] After the modification of the unmodified sequence 1812 of the present disclosure by the modification template DV25P-29P, the inhibition rate can be increased by more than 30% compared with the unmodified sequence 812P disclosed in the prior art. For example, C1812-DV25P is increased by 31.9%, which is significantly improved.

[0885] After the modification of the unmodified sequence 1576s of the present disclosure by the modification template DV25P-29P, the inhibition rate can be increased by more than 40% compared with the unmodified sequence 576P disclosed in the prior art. For example, C1576s-DV29P is increased by 41.1%, which is significantly improved.

[0886] After the modification of the unmodified sequence 1578s of the present disclosure by the modification template DV25P-29P, the inhibition rate can be increased by more than 40% compared with the unmodified sequence 578P disclosed in the prior art. For example, C1578s-DV29P is increased by 43.5%, which is significantly improved.

[0887] The unmodified sequence 1585s of the present disclosure is modified by the modification template DV25P-29P, and compared with the unmodified sequence 585P disclosed in the prior art, the inhibition rate can be increased by more than 10%. For example, C1585s-DV27P is increased by 17.27%, which is significantly improved.

[0888] The unmodified sequence 1816 of the present disclosure is modified by the modification template DV25P-29P, and compared with the unmodified sequence 816P disclosed in the prior art, the inhibition rate is not significantly improved.

[0889] The unmodified sequence 1835 of the present disclosure is modified by the modification template DV25P-29P, and compared with the unmodified sequence 835P disclosed in the prior art, the inhibition rate can be increased by more than 50%. For example, C1835-DV26P is increased by 57.9%, which is significantly improved.

[0890] The unmodified sequence 1836 of the present disclosure is modified by the modification template DV25P-29P, and compared with the unmodified sequence 836P disclosed in the prior art, the inhibition rate can be increased by more than 20%. For example, C1836-DV26P is increased by 21.9%, which is significantly improved.

[0891] The unmodified sequence 1839 of the present disclosure is modified by the modification template DV25P-29P, and compared with the unmodified sequence 839P disclosed in the prior art, the inhibition rate can be increased by more than 70%. For example, C1839-DV29P is increased by 79.6%, which is significantly improved.

[0892] The unmodified sequence 1838 of the present disclosure is modified by the modification template DV25P-29P, and compared with the unmodified sequence 839P disclosed in the prior art, the inhibition rate can be increased by more than 90%. For example, C1838-DV29P is increased by 91%, which is significantly improved.

[0893] The unmodified sequence 1791 of the present disclosure is modified by the modification template DV25P-29P, and compared with the unmodified sequence 791P disclosed in the prior art, the inhibition rate can be increased by more than 10%. For example, C1791-DV26P is increased by 19.5%, which is significantly improved.

[0894] Therefore, compared with the unmodified sequence similar to the prior art, the unmodified sequence of the present disclosure, and the modified sequence using the alternating modification and template modification of the present disclosure, the inhibition activity on AGT can be significantly enhanced, and the inhibition rate can be increased by 91% at most.

[0895] Summary:

[0896] The inhibition effect of the unmodified sequence, the alternately modified sequence and the sequence modified by the specific modification template in the present disclosure is significantly improved compared with the sequence similar to the prior art disclosure. For example, the inhibition rate of the unmodified sequence 1839 in the present disclosure is increased by 61.1% compared with the sequence 839P similar to the sequence in the prior art disclosure. The inhibition rate of the alternately modified sequence B1835-AL of the sequence 1835 in the present disclosure is increased by 48.4% compared with the unmodified sequence 835P similar to the sequence 1835 in the prior art disclosure. The inhibition rate of the sequence C1812-DV25P modified by the modification template DV25P in the present disclosure is increased by 31.9% compared with the unmodified sequence 812P similar to the sequence 1812 in the prior art disclosure.

[0897] Example 6: Off-target effect experiment of modified sequence

[0898] In the actual application of siRNA, there are many cases of non-target mRNA expression being inhibited only partially complementary to the guide strand (antisense strand). The research of Alnylam Corporation shows that the hepatotoxicity of n-acetylgalactosamine (GalNAc) conjugated siRNA is mainly due to the off-target effect causing gene inhibition on the wrong target through the recognition mechanism similar to microRNA (miRNA).

[0899] In order to study whether the sequence in the present disclosure will produce off-target effect, the present example studies the inhibition efficiency of the potential off-target genes of the sequences with higher activity in Example 4 in HepG2 cells, and compares with the drug Zilebesiran which has entered the clinical phase II. For 13 sequences such as 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, the potential off-target genes with higher sequence similarity are searched by blast. No gene with higher similarity is searched for C1585s-DV27P, so no detection is performed. The remaining 12 sequences have obvious inhibition effect on AGT at the concentration of 16pM to 10nM; the inhibition effect of the sequences except C1578s-DV29P and C1835-DV26P on the potential off-target genes is not significant (IC 50 The difference is more than 200 times).

[0900] 1. Experimental materials

[0901] 1) Test sample:

[0902] The 12 sequences with higher activity in Example 4 and the positive control sequence APC-ZL (Table 31). APC-ZL is a Zilebesiran sequence without GalNAc delivery carrier.

[0903] Table 31 Template modification of each sequence

[0904]

[0905]

[0906] Cell type: HepG2 cells, provided by Selleck (H1-1701).

[0907] HepG2 cells were cultured in DMEM medium (ATCC-30-2003) containing 10% fetal bovine serum (FBS, ExCell Bio-FSP500), 1% penicillin-streptomycin (HyClone-SV30010).

[0908] Drug vehicle: sterile enzyme-free water, gibco Opti-MEM.

[0909] 2. Experimental method

[0910] qRT-PCR was used to detect the inhibition of the mRNA expression of the AGT gene of the HepG2 cell line by the test sample.

[0911] 2.1 Cell culture

[0912] Take the subcultured HepG2 cell strain, and use 10% fetal bovine serum DMEM culture medium (supplemented with 100 μL / mL of penicillin and streptomycin) to culture the logarithmic growth cells in a 37°C cell culture incubator containing 5% CO2, and change the liquid every day. Digest the subculture with 0.25% trypsin, centrifuge at 1000 r / min for 5 min, discard the supernatant, and add fresh culture medium for subculture.

[0913] 2.2 Cell transfection

[0914] Transfection mixture preparation: mix Lipofectamine RNAiMAX and Opti-MEM at a ratio of 2:98, and vortex to mix well.

[0915] Transfection reagent preparation: take 60 μL of siRNA solution diluted with Opti-MEM at a ratio of 1:1 (v / v) and add it to 60 μL of transfection mixture, vortex to mix well, and then stand at room temperature for 15 min to obtain lipid nanoparticles (LNP). Take 12.5 μL for encapsulation rate detection.

[0916] Blank control group transfection reagent: add prepared transfection mixture 60 μL in 60 μL Opti-MEM. Mix well by vortex, and stand at room temperature for 15 min.

[0917] Add prepared transfection reagent to 24-well cell culture plate (100 μL per well), so that the final siRNA concentration per well is 0.016 nM / 0.08 nM / 0.4 nM / 2 nM / 10 nM. Add 500 μL cell suspension (containing 1.5 x 10 5 After mixing by cross method, place in 37 °C, 5% CO2 cell incubator, and culture for 40 h.

[0918] 2.3 RNA extraction and reverse transcription

[0919] After transfection for 24 h, remove culture medium and collect cells for RNA extraction. Extract total RNA according to kit instructions using RNeasy Plus Mini Kit (QIAGEN-74182). Then synthesize cDNA according to instructions using FastKing RT Kit (With gDNase) (TIANGEN-KR116-02).

[0920] 2.4 RT-qPCR

[0921] See mRNA quantitative detection part in section 2.3 of Example 2.

[0922] 2.5 Data analysis

[0923] According to the Ct value of each sample, calculate the RNA expression level of the target gene in the sample, and calculate by ΔΔCt relative quantification method. The relative expression of the target gene is represented by 2-ΔΔCT.

[0924] The calculation formula is as follows:

[0925] ΔCT = average Ct value of target gene - average Ct value of internal reference gene;

[0926] ΔΔCT = ΔCT (drug group) - ΔCT (RNAiMAX control group);

[0927] Relative expression of target gene mRNA = 2-ΔΔCT

[0928] Inhibition rate = (1 - sample relative expression / RNAiMAX control average expression) x 100%

[0929] 3. Experimental results

[0930] 3.1 Each sequence has obvious inhibitory effect on AGT gene. For example, the inhibition efficiency of C1576s-DV29P on AGT at 10 nM reached 83.8%.​

[0931] The inhibition efficiency of each sequence on AGT at different concentrations is shown in Table 32 below. Each sequence has a significant inhibitory effect on AGT. For example, the inhibition efficiency of C1576s-DV29P on AGT at 10 nM reached 83.8%.

[0932] Table 32 Inhibition rate of sequences on AGT at different concentrations

[0933]

[0934]

[0935] 3.2 C1578s-DV29P and C1835-DV26P have certain off-target effects, and other sequences have no obvious off-target effects.

[0936] The inhibition efficiency of each sequence on AGT and potential off-target genes at each concentration, IC 50 is shown in Table 33 below.

[0937] Table 33 Inhibition rate of sequences on potential off-target genes at different concentrations and IC 50

[0938]

[0939]

[0940]

[0941] From the above table, we can know:

[0942] (1) Some sequences have a low inhibition efficiency on potential off-target genes. For example, C1789-DV25P has an inhibition rate of only 2.7% on MRPL42 at 10 nM, and the inhibitory effect is not obvious.

[0943] (2) Some sequences have a certain inhibitory effect on potential off-target genes at high concentrations, but the IC 50 for inhibiting potential off-target genes is much higher than the IC 50 for inhibiting AGT. For example, C1579-DV25P has an inhibition rate of 42.3% on GLTP at 10 nM, but the IC 50 for GLTP is greater than 10 nM, while the IC 50 for AGT is 0.011 nM, and the ratio is much greater than 200, which can be considered as no obvious off-target effect.

[0944] (3) C1578s-DV29P has an inhibition rate of 66.9% on ERRFI1 at 10 nM, and the IC 50 is 0.021 nM. The IC50 0.004 nM, IC 50 The ratio is only 5.3; C1835-DV26P has an inhibition rate of 74.6% on CCNA2 at 10 nM, and the IC 50 1.766 nM, and the IC 50 less than 0.016 nM, and the IC 50 The minimum value of the ratio is 110.4. The above two sequences can be considered to have certain off-target effects, which need to be reduced by off-target prevention modification.

[0945] Summary:

[0946] (1) Each sequence has obvious inhibitory effect on AGT. For example, C1576s-DV29P has an inhibition efficiency of 83.8% on AGT at 10 nM.

[0947] (2) C1578s-DV29P and C1835-DV26P have certain off-target effects, and other sequences have no obvious off-target effects.

[0948] Example 7: Inhibition of AGT and off-target genes by sequences with specific off-target prevention design

[0949] In the actual application of siRNA, there are many cases of non-target mRNA expression being inhibited with only partial complementarity to the guide strand (antisense strand). Research by Arima Corporation shows that the hepatotoxicity of n-acetylgalactosamine (GalNAc) conjugated siRNA is mainly due to the gene inhibition of the wrong target by the recognition mechanism similar to microRNA (miRNA) caused by off-target effects.

[0950] To solve this problem, Arima Corporation used a glycol nucleic acid (GNA) modification at position 7 of the antisense strand of siRNA in the latest fifth-generation template design to disrupt the seed region of the antisense strand, thereby significantly reducing off-target effects and alleviating hepatotoxicity. Such modification can affect the way siRNA binds to non-design targets through seed region recognition, inhibiting off-target effects.

[0951] In addition, in a study in 2008, researchers found that replacing all 8 base pairs of the siRNA double strand located at the 5' end of the antisense strand with DNA can significantly reduce the off-target effects of siRNA without affecting the activity of siRNA. Studies on mRNA cleavage sites show that DNA replacement does not affect the RISC complex to cleave mRNA between the 10th and 11th nucleotides of the antisense strand, but affects the cleavage of mRNA at the secondary site. The mechanism may be that DNA replacement inhibits the non-specific cleavage of RNA enzyme on double-stranded RNA.

[0952] In summary, in order to reduce the off-target effect of the sequence, the DV25-29P is used to modify the unmodified sequences 1578s and 1835 in this embodiment, the 7th nucleotide of the antisense strand or the 6th and 7th nucleotides are replaced with DNA, and the nucleotides of the complementary position of the sense strand are also replaced with DNA to reduce the off-target effect of the sequence, and the two off-target prevention modifications are compared with the off-target prevention design of replacing the 7th nucleotide of the antisense strand with (S)-GNA.

[0953] The experimental results show that the sequences with off-target prevention design have significant inhibitory effect on the target gene AGT at a concentration of 10 nM to 16 pM, and the inhibitory effect of some sequences on the off-target gene is significantly reduced. It is shown that the use of off-target prevention design does not affect the inhibitory effect of the alternating modification and template modification sequences of the present disclosure on the AGT gene, but can inhibit the off-target effect of some sequences.

[0954] 1. Experimental materials

[0955] 1) Test samples:

[0956] The unmodified sequences 1578s, 1835 and 1838 are template modified sequences, and the sequences with template modification and off-target prevention design (Table 34).

[0957] 2) Sequence synthesis:

[0958] I. Template modified sequence

[0959] The synthesis method refers to part of the synthesis of the alternating modification sequence in Example 1.

[0960] II. siRNA sequence with DNA off-target prevention design

[0961] The siRNA sequence is synthesized according to Example 1, and DNA monomer 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) are used when synthesizing the 6th or 7th nucleotide at the 5' end of the antisense strand, and the structures are as follows:

[0962]

[0963] III. siRNA sequence with GNA off-target prevention design

[0964] The siRNA sequence is synthesized according to Example 1, and GNA monomer is used to synthesize the sequence when synthesizing the 7th base at the 5' end of the antisense strand, and the structure of the GNA monomer is as follows:

[0965]

[0966] Table 34 Template modification, off-target design for each sequence

[0967]

[0968]

[0969] Cell type: HepG2 cells, provided by Selleck (H1-1701).

[0970] HepG2 cells were cultured in DMEM medium (ATCC-30-2003) containing 10% fetal bovine serum (FBS, ExCell Bio-FSP500), 1% penicillin-streptomycin (HyClone-SV30010).

[0971] Drug vehicle: sterile enzyme-free water, gibco Opti-MEM.

[0972] 2. Experimental method

[0973] qRT-PCR was used to detect the inhibition of mRNA expression of AGT and potential off-target genes in HepG2 cell lines by the test samples.

[0974] 2.1 Cell culture

[0975] Take the subcultured HepG2 cell strain, and use 10% fetal bovine serum DMEM culture medium (supplemented with 100 μL / mL of penicillin and streptomycin) to culture the logarithmic growth cells in a 37°C cell incubator containing 5% CO2, and change the liquid every day. Digest the subculture with 0.25% trypsin, centrifuge at 1000 r / min for 5 min, discard the supernatant, and add fresh culture medium for subculture.

[0976] 2.2 Cell transfection

[0977] Transfection mixture preparation: mix Lipofectamine RNAiMAX and Opti-MEM at a ratio of 2:98, and vortex to mix well.

[0978] Transfection reagent preparation: take 60 μL of siRNA solution diluted in Opti-MEM at a ratio of 1:1 (v / v) and add it to 60 μL of transfection mixture, vortex to mix well, and then let it stand at room temperature for 15 min to obtain lipid nanoparticles (LNP). Take 12.5 μL for encapsulation rate detection.

[0979] Blank control group transfection reagent: add 60 μL of prepared transfection mixture to 60 μL of Opti-MEM. Vortex to mix well, and let it stand at room temperature for 15 min.

[0980] The prepared transfection reagent was added into 24-well cell culture plates (100 μL per well) to make the final siRNA concentration 16 pM / 80 pM / 400 pM / 2 nM / 10 nM per well. 500 μL of cell suspension (containing 1.5 x 10 5 After mixing with cross method, it was put into 37℃, 5% CO2 cell incubator for 40 h.

[0981] 2.3 RNA extraction and reverse transcription

[0982] After transfection for 24 h, the culture medium was removed and the cells were collected for RNA extraction. Total RNA was extracted according to the kit instructions using RNeasy Plus Mini Kit (QIAGEN-74182). Then cDNA was synthesized according to the instructions using FastKing RT Kit (With gDNase) (TIANGEN-KR116-02).

[0983] 2.4 RT-qPCR

[0984] The target cDNA was detected by qPCR, and GAPDH cDNA was detected as an internal control. 8 μL of prepared qPCR reaction solution and 2 μL of sample cDNA were added in 384-well plates. The TaqMan qPCR reaction program was as follows: heating at 95℃ for 10 min, then entering the cycle mode, heating at 95℃ for 15 s, then at 60℃ for 1 min, for a total of 40 cycles. The SYBR qPCR reaction program was as follows: heating at 50℃ for 2 min, heating at 95℃ for 10 min, then entering the cycle mode, heating at 95℃ for 15 s, then at 60℃ for 1 min, for a total of 40 cycles; finally, the melting curve was heated at 95℃ for 15 s, at 60℃ for 1 min, and at 95℃ for 15 s.

[0985] 2.5 Data analysis

[0986] The RNA expression level of the target gene in each sample was calculated according to the Ct value, and the relative quantification method was used for calculation. The relative expression of the target gene was represented by 2-ΔΔCT.

[0987] The calculation formula is as follows:

[0988] ΔCT = average Ct value of target gene - average Ct value of internal reference gene;

[0989] ΔΔCT = ΔCT (drug group) - ΔCT (RNAiMAX control group);

[0990] Relative expression of target gene mRNA = 2-ΔΔCT

[0991] ​Inhibition rate = (1 - sample relative expression amount / RNAiMAX control average expression amount) x 100%

[0992] 3. Experimental results

[0993] (1) Different modified sequences have significant inhibitory effect on AGT gene. For example, the inhibition rate of template modified sequence D578s-DV29P reaches 85.7%, and the inhibition rate of template modified and off-target prevention modified sequence D578s-DV29Pd7B reaches 81.3%.

[0994] The inhibition rates of all tested samples on AGT gene are shown in Table 35.

[0995] All template modified sequences except D838-DV29P+ have significant inhibitory effect on AGT at a concentration of 10 nM. For example, the inhibition rate of template modified sequence D578s-DV29P reaches 85.7%.

[0996] Table 35 Inhibition rates of template modified and off-target prevention modified sequences on AGT gene

[0997]

[0998] After template modification and off-target prevention design, the modified sequences have significant inhibitory activity on AGT gene expression. For example, the inhibition rate of sequence D578s-DV29Pd7B reaches 81.3% after template modification of unmodified sequence 1578s using template DV29P and off-target prevention design.

[0999] (2) After modification using any one or more of the various modification methods of the present disclosure, off-target prevention design is performed, which has significant off-target prevention effect on some off-target genes and reduces the inhibitory effect on off-target genes.

[1000] As shown in Table 36, the experimental results show that:

[1001] The template modified sequences of the present disclosure can reduce the inhibitory effect on off-target genes, i.e., have off-target prevention effect, after off-target prevention design. The template modified sequences of the present disclosure have off-target prevention effect after off-target prevention design, and the inhibition rate on off-target genes can be reduced by at most 72.535%.

[1002] Table 36 Inhibition rates of template modified sequences and their off-target prevention modifications on off-target genes

[1003]

[1004]

[1005] 1) Only template modification design

[1006] Part of the sequence modified by template modification has certain off-target effect. For example, the modified sequence D578s-DV29P of the unmodified sequence 1578s using DV29P modification template has an inhibition efficiency of 66.894% on the potential off-target gene ERRFI1; the modified sequence D835-DV26P of the unmodified sequence 1835 using DV26P template has an inhibition efficiency of 74.619% on the potential off-target gene CCNA2.

[1007] 2) The off-target prevention design is used at the same time (indicated by "d67B" or "d7B" in the sequence number)

[1008] Part of the sequence using template modification and off-target prevention modification at the same time has a significant decrease in the inhibition rate on the off-target gene, and has a significant off-target prevention effect. For example, the inhibition rate of D578s-DV25Pd7B on ERRFI1 is reduced by 72.535%, which is significantly reduced; the inhibition rate of D835-DV26Pd7B on CCNA2 is reduced by 53.666%, which is significantly reduced.

[1009] Summary:

[1010] 1. Different modified sequences have significant inhibition effect on AGT gene. For example, the inhibition rate of the template modified sequence D578s-DV29P reaches 85.7%, and the inhibition rate of the template modified and off-target prevention modified sequence D578s-DV29Pd7B is as high as 81.3%.

[1011] 2. After using any one or more of the modification methods in the present disclosure, the off-target prevention design is used, which has a significant off-target prevention effect on some off-target genes and reduces the inhibition effect on the off-target genes.

[1012] 1) Part of the sequence modified by template modification has certain off-target effect. For example, the modified sequence D578s-DV29P of the unmodified sequence 1578s using DV29P modification template has an inhibition efficiency of 66.894% on the potential off-target gene ERRFI1; the modified sequence D835-DV26P of the unmodified sequence 1835 using DV26P template has an inhibition efficiency of 74.619% on the potential off-target gene CCNA2.

[1013] 2) The template modified sequence of the present disclosure uses off-target prevention design, which can reduce the inhibition effect on some off-target genes, that is, has off-target prevention effect, and the inhibition rate on the off-target gene can be reduced by 72.535% at most.

[1014] Example 8: Inhibition effect of the sequence modified by the modified template of the present disclosure on AGT in human primary hepatocytes

[1015] This example investigated the inhibitory efficiency of the highly active sequences from Example 4 against potential off-target genes in human primary hepatocytes and compared it with that of Zilebesiran, a drug that has entered Phase II clinical trials. For five sequences—C1576s-DV29P, C1578s-DV29P, C1579-DV25P, C1812-DV25P, and C1585s-DV27P—each was modified using its own template or simultaneously using both a template and an off-target prevention modifier, and GalNAc was conjugated to achieve free uptake by hepatocytes. Human primary hepatocytes were treated with the sequences in Table 37 at concentrations ranging from 0.0064 nM to 100 nM in a free uptake manner. Dose-response curves were fitted, and the IC50 value for each sequence was calculated. 50 The results show the IC of partial sequences. 50 Superior to Zilebesiran, such as IC D576s-DV29PG5 50 The value was 4.698 nM, which was better than that of Zilebesiran (7.071 nM).

[1016] 1. Experimental Materials

[1017] 1) Test sample:

[1018] The sequences in Table 37 are those using template modification and those using both template modification and off-target protection design. These sequences are coupled to GalNAc ligand G5 at the 3' end of the positive strand:

[1019]

[1020] The method for conjugating the oligonucleotide to ligand G5 is the same as the method for preparing conjugates 4, 5, 6, and 7 in Example 3 of Chinese Patent CN116854754A, that is, conjugating YK-GAL-304, YK-GAL-305, YK-GAL-306, and YK-GAL-307 to the oligonucleotide. The synthesis methods of YK-GAL-304, YK-GAL-305, YK-GAL-306, and YK-GAL-307 are the same as those in Example 1 of CN116854754A.

[1021] Oligonucleotides and ligands form conjugates as shown below:

[1022]

[1023] The specific sequences are shown in Table 7. In 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.

[1024] Table 37 Template Modifications for Each Sequence

[1025]

[1026] Cell type: Human primary hepatocytes, provided by Livzon Biotech Company (LV-PHH001).

[1027] Drug vehicle: Sterile enzyme-free water, human primary hepatocyte maintenance medium (LV-WEM001).

[1028] 2. Experimental methods

[1029] The inhibition of human primary hepatocyte AGT gene mRNA expression by the test sample was detected by qRT-PCR.

[1030] 2.1 Resuscitation and culture of human primary hepatocytes

[1031] The culture medium was preheated at 37°C for more than 30 min. 300 μL of PBS was added to each well of the collagen-coated 24-well plate, and after shaking several times, the PBS was aspirated. The cell cryopreservation tube was taken out of the liquid nitrogen tank and placed in a 37°C water bath, and gently shaken until only a small amount of ice crystals remained in the tube. The cell suspension was poured into the resuscitation culture medium at one time. The inner wall of the cryopreservation tube was rinsed with 1 mL of resuscitation culture medium for 2-3 times. The resuscitation culture medium cell suspension was inverted and mixed well. Centrifugation was performed at room temperature for 5 min at 50g, with the acceleration setting at 5 and the deceleration setting at 3. The cells were diluted to 3 x 10 5 6 / mL using plating medium, and inoculated into the 24-well plate at 0.5 mL per well, and placed in the incubator for 24 h.

[1032] 2.2 Free uptake of human primary hepatocytes

[1033] The test product was diluted to 100-0.0064 nM using human primary hepatocyte maintenance medium. The cultured human primary hepatocytes were taken out, and the plating medium was aspirated. 500 μL of diluted test product was added to each well, and placed back into the 37°C, 5% CO2 cell incubator for 40 h.

[1034] 2.3 RNA extraction and reverse transcription

[1035] After transfection for 24 h, the culture medium was removed and the cells were collected for RNA extraction. Total RNA was extracted using the RNeasy Plus Mini 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.

[1036] 2.4 RT-qPCR

[1037] See the mRNA quantification detection section in section 2.3 of Example 2.

[1038] ​2.5 Data analysis

[1039] The RNA expression level of the target gene in each sample was calculated according to the Ct value of each sample, and was calculated by the ΔΔCt relative quantification method. The relative expression of the target gene was represented by 2-ΔΔCT.

[1040] The calculation formula is as follows:

[1041] ΔCT = average Ct value of target gene - average Ct value of internal reference gene;

[1042] ΔΔCT = ΔCT (drug group) - ΔCT (RNAiMAX control group);

[1043] Relative expression of target gene mRNA = 2-ΔΔCT

[1044] Inhibition rate = (1 - sample relative expression / RNAiMAX control average expression) x 100%

[1045] 3. Experimental results

[1046] The inhibition efficiency of each sequence on AGT at different concentrations is shown in Table 38. Each sequence has obvious inhibitory effect on AGT. For example, the inhibition efficiency of D576s-DV29PG5 on AGT at 100 nM reached 83.1%. In addition, the IC 50 is better than that of Zilebesiran, for example, the IC 50 of D576s-DV29PG5 is 4.698 nM, which is better than 7.071 nM of Zilebesiran.

[1047] Table 38 Inhibition rate of sequences on AGT at different concentrations and IC 50

[1048]

[1049]

[1050] Example 9: Inhibition of AGT in mouse serum by sequences modified by the template modification of the present disclosure

[1051] Some sequences were selected as examples in this embodiment, including unmodified sequences 1576s, 1578s, 1579, 1585s, 1789, 1791, 1795, 1812, 1816, 1835, 1838 and 1839, and these sequences were modified, for example, only using template modification, and using template modification and off-target prevention design at the same time. The positive control drug uses the siRNA drug Zilebesiran which is currently in the clinical phase II. Using transgenic mice expressing human AGT gene, the inhibitory effect of each sequence on serum AGT at different time points was detected by ELISA.

[1052] 1. Experimental materials

[1053] Test drugs:

[1054] Each sequence in Table 39 is a sequence using template modification and a sequence using template modification and off-target prevention design at the same time, and the 3' end of the sense strand of each sequence is coupled with the GalNAc ligand G5:

[1055]

[1056] The coupling method of the oligonucleotide and the ligand G5 is the same as the preparation method of conjugate 4, conjugate 5, conjugate 6 and conjugate 7 in Example 3 of Chinese Patent CN116854754A, that is, YK-GAL-304, YK-GAL-305, YK-GAL-306 and YK-GAL-307 are coupled with the oligonucleotide. The synthesis method of YK-GAL-304, YK-GAL-305, YK-GAL-306 and YK-GAL-307 is the same as Example 1 of CN116854754A.

[1057] The oligonucleotide and the ligand form the conjugate as shown below:

[1058]

[1059] The specific sequences of each sequence are shown in Table 39, and G5 in the sequence number indicates that the sequence is coupled with the GalNAc ligand G5, and GL indicates that the sequence is coupled with the GalNAc ligand L96.

[1060] The structure of G5 is as follows:

[1061]

[1062] [[(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]

[1063] [[(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]

[1064] The GL structure is as follows:

[1065]

[1066] [[(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]methyl hydrogenphosphate]

[1067] [[[(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-triazanonacosyl-1-yl]-4-hydroxy-2-pyrrolidinyl]methyl hydrogen phosphate]

[1068] Table 39 Sequences used in animal experiments

[1069]

[1070]

[1071]

[1072] Test drug preparation:

[1073] Drug solvent: physiological saline

[1074] Preparation conditions: sterile environment

[1075] Identification method: the prepared administration preparation is identified by a label, and the outer package is marked with a special topic number, name, concentration, quantity, preparation date, preparer, and storage condition.

[1076] Storage conditions: use fresh, and store the remaining sample at -80°C.

[1077] Information of experimental animals:

[1078] Species / strain: hAGT transgenic mice

[1079] Grade: SPF

[1080] Gender: male

[1081] Number: 150

[1082] Age: 6-8 weeks

[1083] Weight: 18-28 g

[1084] Source: Jiangsu Jicui Yekang Biotechnology Co., Ltd.

[1085] Production license number: SCXK(Su)2018-0008

[1086] Ethical review of experimental animals (IACUC):

[1087] The experimental animals were housed at Beijing Baoreshe Technology Co., Ltd. after receiving, with license number: SYXK(Jing)2022-0025. This project has been reviewed by the Experimental Animal Ethics Committee of Beijing Baoreshe Technology Co., Ltd. The test process is strictly in accordance with the requirements of IACUC, and the animal welfare is guaranteed.

[1088] Feeding and management:

[1089] Feeding conditions: The experimental animals were housed at Beijing Baoreshe Technology Co., Ltd. after receiving, with license number: SYXK(Jing)2022-0025. They were housed in cages with dimensions of length x width x height = 29.0 cm x 18.5 cm x 13.0 cm; the temperature range was set to 20-26℃, the humidity range was set to 40%-70%, the air exchange frequency was not less than 15 times of fresh air per hour, and 12 hours of artificial lighting was provided.

[1090] The environmental conditions were controlled by a combined air conditioning unit according to the standards of the People's Republic of China National Standard GB14925-2010.

[1091] The animals were allowed to eat and drink freely. The water bottles and the water used by the animals should be replaced at least twice a week, and the used water bottles should be sterilized by a pulsating vacuum sterilizer before reuse.

[1092] The animal cages and bedding were replaced at least once a week, and all animal cages and bedding were sterilized by a pulsating vacuum sterilizer before entering the barrier environment; the animal cage racks were cleaned, disinfected and wiped at least once a week.

[1093] The animal feeding observation room was cleaned and disinfected every day, including flat shelves, floors, tables, etc.

[1094] The disinfectants used in the barrier environment include: 6.67% new jieer solution, 0.5% 84 disinfectant, 75% disinfectant, 0.08% hundred poison killer, and the four disinfectants are used alternately, but not mixed.

[1095] Experimental animal feed: SPF mouse maintenance feed: produced by Sibeifeng (Beijing) Biotechnology Co., Ltd., with animal feed production license number SCXK(Jing)2019-0010, issued by Beijing Municipal Science and Technology Committee.

[1096] Feed detection: Each batch of feed has a quality certificate, and the company conducts microbial detection once every quarter, and the feed supply unit provides a recent third-party feed detection report every half year. The nutritional component detection of feed refers to the national standard GB14924.3-2010 of the People's Republic of China, and the pollutant index detection refers to the national standard GB14924.2-2001 of the People's Republic of China.

[1097] Experimental animal drinking water: drinking water: sterile water prepared by a filtration system, directly filled with a drinking water bottle.

[1098] Drinking water testing: microbiological testing is performed by the company every quarter, and water quality testing is performed by a third-party testing agency every year. Drinking water testing refers to the People's Republic of China National Standard GB5749-2006.

[1099] Animal bedding: corn cob bedding: Spero (Beijing) Biotechnology Co., Ltd., Animal Bedding Production License No. SCXK(Jing)2019-0004, issued by Beijing Municipal Science and Technology Committee.

[1100] Bedding testing: microbiological testing is performed by the company every quarter, and bedding testing reports are provided by the bedding supplier at least once every six months. Bedding testing refers to the People's Republic of China National Standard GB14924.2-2001.

[1101] 2. Experimental method

[1102] Dose design and grouping

[1103] Test date definition: the day when the test animals are given the solvent or the test drug is defined as day 0 (day 0).

[1104] Grouping and dosing: after 3 days of acclimation of test animals, randomly divide into negative control group and test drug group, 6 in each group, according to serum AGT protein content. Through subcutaneous single injection, the dose is 3mg / kg, the volume is 5mL / kg, the concentration is 0.6mg / mL, and the day of administration is recorded as day 0.

[1105] Animal identification uses ear tag number for individual identification. The feeding cage is identified by hanging cage card. Laboratory identification plate is hung at the entrance of the laboratory. The grouping information is shown in Table 40 below:

[1106] Table 40 Grouping information

[1107] Group Drug Dose (mg / kg) Administration mode / frequency Period (days) Number of animals Negative control group Vehicle / / 56 6 Test drug group siRNA 3 s.c. / once 56 6

[1108] Detection index

[1109] (1) General observation

[1110] From 1 week before administration to the end of the experiment, observe once a day.

[1111] Observation content: observe the death or near-death, mental state, behavior activity, feces shape, feed and water supply, etc. of animals beside the cage.

[1112] Test animals: all animals in the negative control group and the test drug group.

[1113] (2) Expression of AGT protein in serum

[1114] Detection time: day-3 before administration (day-3), day 7 after administration for 1 week (1w), day 14 after administration for 2 weeks (2w), day 21 after administration for 3 weeks (3w), day 28 after administration for 4 weeks (4w), day 35 after administration for 5 weeks (5w).

[1115] AGT protein level detection method: ELISA kit was used for detection; serum samples were avoided from repeated freeze-thawing.

[1116] Detection animals: all animals in the negative control group and the test drug group.

[1117] (4) Data processing and statistical analysis

[1118] The test data is represented by mean ± standard deviation (Mean ± SD), and the data analysis is performed by using GraphPad Prism 8.3 analysis software. Statistical analysis is performed by using two-way ANOVA and post-hoc test, LSD test is used for variance homogeneity, Dunnett T3 is used for variance heterogeneity, and P<0.05 indicates statistical significance.

[1119] 3. Experimental results

[1120] The specific experimental results are shown in Table 41.

[1121] It can be seen that these sequences can continuously and significantly inhibit AGT protein in serum. For example, D576s-DV29PG5 reached 84.09%, 82.11% and 85.25% inhibition rates at 7th day, 14th day and 28th day, respectively.

[1122] (1) The sequences of the present disclosure, such as unmodified sequences 1579, 1789, 1812, 1576s, 1578s and 1585s, have significant inhibitory effect on the expression of AGT protein in serum by using different modifications. For example, D576s-DV29PG5 reached 84.09%, 82.11% and 85.25% inhibition rates at 7th day, 14th day and 21st day, respectively. Figure 8 ).

[1123] Table 41 Inhibition rate of different sequences on AGT protein in serum

[1124]

[1125]

[1126] As can be seen from Table 41, the conjugates formed by conjugating each sequence with the GalNAc compound have a significant inhibitory effect on AGT protein expression in serum. For example, D576s-DV29PG5 reached 84.09%, 82.11% and 85.25% inhibition rates on day 7, day 14 and day 21, respectively. Figure 9

[1127] It is shown that the unmodified sequences designed in the present disclosure, such as 1579, 1789, 1812, 1576s, 1578s and 1585s, conjugated with the GalNAc compound, can be efficiently delivered to the liver of animals and significantly inhibit AGT gene expression.

[1128] Summary:

[1129] The unmodified sequences designed in the present disclosure, such as 1579, 1789, 1812, 1576s, 1578s and 1585s, conjugated with the GalNAc compound, can be efficiently delivered to the liver of animals and significantly inhibit AGT gene expression.

[1130] For example, D576s-DV29PG5 reached 84.09%, 82.11% and 85.25% inhibition rates on day 7, day 14 and day 21, respectively.

[1131] Conclusion:

[1132] The present disclosure designs a series of siRNA based on AGT mRNA sequence, which are alternately modified and modified using a specific set of modification templates, and some of the sequences are designed to prevent off-targeting. The results show that:

[1133] (1) 13 unmodified sequences have a significant inhibitory effect on AGT gene, with an inhibition rate of more than 45%, including 1576s, 1579, 1812, 1578s, 1789, 1835, 1838, 1795, 1585s, 1816, 1791, 1836 and 1839.

[1134] (2) The highest inhibition rate of the modified sequence can reach more than 70%. And the modified sequence conjugated with the GalNAc compound can be efficiently delivered to the liver of animals and significantly inhibit AGT gene expression.

[1135] Specifically as follows:

[1136] 1. The alternately modified sequences of the present disclosure have a significant inhibitory effect on AGT gene

[1137] ​(1) Among the 99 designed sequences, 33 sequences have significant inhibitory effect on AGT gene, and the inhibition rate is more than 40%. Among them, the inhibition rate of 10 sequences is more than 60%.

[1138] (2) The inhibition rate of 23 sequences on AGT gene is 25%-40%. For example, the inhibition rate of B734-AL and B994-AL is 39.2% and 37.3% respectively.

[1139] (3) The inhibition rate of 43 sequences on AGT gene is less than 25%. For example, the inhibition rate of B1017-AL is only 23.1%.

[1140] (4) The activity difference of siRNA with similar sequences is very large. For example, the inhibition rate of B1365-AL is increased by 40.4% compared with B1367-AL, which is significantly improved, and the two only differ in the terminal 2 bases; the inhibition rate of B1816-AL is increased by 30.5% compared with B1815-AL, which is significantly improved, and the two only differ in the terminal 1 base. Therefore, it is not easy to screen out sequences with significant inhibitory activity from a large number of oligonucleotide sequences designed according to AGT mRNA sequence, and a lot of creative labor needs to be paid.

[1141] 2. The present disclosure has significant inhibitory effect on AGT gene

[1142] (1) 13 unmodified sequences have significant inhibitory effect on AGT gene, and the inhibition rate is more than 45%, including 1576s, 1579, 1812, 1578s, 1789, 1835, 1838, 1795, 1585s, 1816, 1791, 1836 and 1839. Among them, 1576s, 1578s, 1812, 1579, 1789, 1835 and 1838 are inhibited by more than 50%.

[1143] (2) The inhibition rate of other unmodified sequences on AGT gene is less than 45%. For example, the inhibition rate of 731 and 1011 is only 1.3% and 2.1%.

[1144] (3) The activity difference of siRNA with similar sequences is very large. For example, the inhibition rate of unmodified sequence 734 is increased by 41.7% compared with unmodified sequence 731, which is significantly improved.

[1145] (4) After the modification of different sequences, the effect on activity is not consistent. Some have significant improvement in inhibition rate, for example, the inhibition rate of unmodified sequence 731 is increased by 25.1% after alternating modification; some are not obvious, for example, the inhibition rate of unmodified sequences 994, 1279 and 1591 does not change significantly after alternating modification.

[1146] 3. The sequence modified by the modification template of the present disclosure has a significant inhibitory effect on the AGT gene

[1147] (1) The unmodified sequences 1576s, 1578s, 1838, 1835, 1816, 1812, 1579, 1836, 1789, 1839, 1791, 1795 and 1585s screened have a significant inhibitory effect on the expression of AGT gene after being modified by the modification template DV25P-29P designed by the present disclosure, and the inhibition rate is more than 40%. Among them, C1812-DV25P, C1579-DV33P and C1789-DV26P have inhibition rates of 74.4%, 74.0% and 73.6%, respectively.

[1148] (2) The 12 sequences above are modified by the modification template DV25-29P of the present disclosure, and the inhibition rate of the expression of AGT gene is significantly improved compared with the alternate modification sequence. For example, the inhibition rate of the unmodified sequence 1812 modified by the template DV25P C1812-DV25P is increased by 11.5% compared with the alternate modification sequence, and the inhibition rate of the unmodified sequence 1791 modified by the template DV26P C1791-DV26P is increased by 8.5% compared with the alternate modification sequence.

[1149] (3) The same sequence is modified by the modification template DV25-29P of the present disclosure, and the inhibition rate of the expression of AGT gene is significantly improved compared with the modification template disclosed by the prior art. For example, the unmodified sequence 1579 is modified by the modification template DV25P of the present disclosure, and the inhibition rate is increased by 16.6% compared with the modification sequence modified by the disclosed Advanced ESC template DV22.

[1150] (4) The same sequence is modified by the modification template DV25-29P of the present disclosure, and the inhibition rate of the expression of AGT gene is significantly improved compared with the modification template DV32-34P of the present disclosure. For example, the unmodified sequence 1812 is modified by the modification template DV25P of the present disclosure, and the inhibition rate is increased by 20.2% compared with the modification sequence modified by the modification template DV33P of the present disclosure.

[1151] (5) The activity of different modification template modification sequences is very different. For example, the unmodified sequence 1816 is modified by DV29P, and the inhibition rate is increased by 30.0% compared with the modification by DV34; the unmodified sequence 1579 is modified by DV33P, and the inhibition rate is increased by 29.8% compared with the modification by DV28P. Therefore, it is uncertain which modification template can be used to modify the siRNA sequence to have high activity.

[1152] (6) The IC50 values of the above 24 sequences are between 1.884 pM and 61.498 pM. Among them, the IC50 values of C1789-DV25P, C1812-DV25P and C1789-DV26P are 1.884 pM, 2.160 pM and 2.643 pM respectively. It shows that these sequences can effectively inhibit AGT gene expression at low concentration.

[1153] 4. The sequences modified by the alternating modification and the modification template of the present application have significantly improved AGT inhibition activity compared with the unmodified sequences with little difference from the sequences disclosed in the prior art.

[1154] Compared with the unmodified sequences with little difference from the sequences disclosed in the prior art, the unmodified sequences, the alternating modified sequences and the sequences modified by the specific modification template of the present application have significantly improved AGT gene inhibition rate, which can be increased by 91% at most.

[1155] I. The activity of the unmodified sequences of the present application is significantly improved compared with the similar unmodified sequences disclosed in the prior art. For example, the inhibition rate of the unmodified sequence 1835 of the present application is increased by 37.7% compared with the similar sequence 835P.

[1156] II. The activity of the alternating modified sequences of the present application is significantly improved compared with the similar unmodified sequences disclosed in the prior art. For example, the inhibition rate of the alternating modified sequence B1812-AL of the present application is increased by 20.4% compared with the sequence 812P.

[1157] III. The activity of the sequences modified by the modification template of the present application is significantly improved compared with the similar unmodified sequences disclosed in the prior art. For example, the inhibition rate of the alternating modified sequence C1576s-DV29P of the present application is increased by 41.1% compared with the sequence 576P.

[1158] 5. The alternating modified sequences and the template modified sequences of the present application have significant inhibition effect on AGT gene by using specific off-target prevention design, and the inhibition effect on off-target genes is significantly reduced.

[1159] (1) Different modified sequences have significant inhibition effect on AGT gene. For example, the inhibition rate of the template modified sequence D576s-DV29P reaches 83.8%, and the inhibition rate of the template modified and off-target prevention modified sequence D576s-DV29Pd7B is as high as 85.4%.

[1160] (2) After being modified by any one or more of the various modification methods of the present application, and then being designed for off-target prevention, the sequences have significant off-target prevention effect on some off-target genes, and the inhibition effect on off-target genes is reduced, i.e. the inhibition rate on off-target genes is reduced by 72.535% at most.

[1161] 6. The sequence modified by the template modification of the present disclosure or significantly inhibits the expression of AGT in mouse serum.

[1162] The unmodified sequences designed in the present disclosure, such as 1579, 1789, 1812, 1576s, 1578s and 1585s, are modified by the template modification of the present disclosure, and are conjugated with GalNAc compound, which can be delivered to the liver of animals efficiently and can significantly inhibit the expression of AGT protein in serum for a long time.

[1163] For example, the inhibition rate of D576s-DV29PG5 reached 84.09%, 82.11% and 85.25% at 7th day, 14th day and 21st day, respectively.

[1164] Sequence list of the present disclosure

[1165]

[1166]

[1167]

[1168]

[1169]

[1170]

[1171]

[1172]

[1173]

[1174]

[1175]

[1176]

[1177]

[1178]

[1179]

[1180]

[1181]

[1182]

Claims

1. A double stranded RNAi agent comprising an oligonucleotide duplex consisting of the following sense and antisense strands: (1) the sequence of the sense strand is set forth in SEQ ID NO: 423; the sequence of the antisense strand is set forth in SEQ ID NO: 488; (2) the sequence of the sense strand is set forth in SEQ ID NO: 209; the sequence of the antisense strand is set forth in SEQ ID NO:

222.

2. A conjugate for reducing expression of AGT comprising the double stranded RNAi agent of claim 1, and a ligand conjugated thereto.

3. The conjugate of claim 2, wherein the ligand is conjugated to the 3 '-end or 5 '-end of the sense strand of the oligonucleotide.

4. The conjugate of claim 2 or 3, wherein the ligand is a GalNAc derivative attached with a divalent or trivalent branched linker.

5. The conjugate of claim 2 or 3, wherein the ligand is: ， wherein X is a hydroxyl protecting group selected from acetyl, benzoyl and isobutyryl; Y is an amine protecting group selected from formyl, acetyl, propionyl, n-butyryl and isobutyryl; n is an integer between 0-20; q, r and s are independently integers between 1-7.

6. The conjugate of claim 5, wherein the ligand is: 。 7. The conjugate of claim 2 or 3, wherein the ligand is: ， wherein X is oxygen, nitrogen or sulfur; Y is alkyl or aryl; R1 is oxygen or sulfur; R2is 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 -, wherein a is an integer from 1 to 15, b is an integer from 1 to 7, c is an integer from 1 to 7, and d is an integer from 1 to 5; B is -(CH2) e wherein e is an integer from 0 to 7; L is -CONH- or -NHCO-; X1is -(CH2) f - or -(CH2CH2O) f CH2- and f is an integer from 1 to 5; X2 is -(CH2) g - and g is an integer from 1 to 6; Y1 is 0 or 1; Y2 is 0, 1 or 2; Y3 is 1, 2 or 3; m is an integer from 0-4; n is an integer from 0-4.

8. The conjugate of claim 2 or 3, wherein the ligand is G4, G5, G6 or G7, having the following structures: ， ， ， 。 9. The conjugate of claim 2 or 3, having the following structure: ， ， ， or 。 10. The conjugate of claim 2 or 3, wherein the double stranded RNAi agent comprises an oligonucleotide duplex consisting of the following sense and antisense strands: the sequence of the sense strand is set forth in SEQ ID NO: 423; and the sequence of the antisense strand is set forth in SEQ ID NO: 488; wherein the oligonucleotide duplex is conjugated to ligand G5, having the following structure: 。 11. The conjugate of claim 2 or 3, wherein the double stranded RNAi agent comprises an oligonucleotide duplex consisting of the following sense and antisense strands: the sequence of the sense strand is set forth in SEQ ID NO: 554; and the sequence of the antisense strand is set forth in SEQ ID NO: 488; wherein the oligonucleotide duplex is conjugated to ligand G5, having the following structure: 。 12. A nucleic acid protein composition comprising the double stranded region of the double stranded RNAi agent of claim 1, and a nuclease; or, the nucleic acid protein composition comprises the antisense strand of the double stranded region of the double stranded RNAi agent of claim 1, and a nuclease. ​ 13. The nucleic acid protein composition of claim 12, wherein the nuclease is an AGO protein.

14. A pharmaceutical composition comprising the double stranded RNAi agent of claim 1, the conjugate of any one of claims 2-11, or the nucleic acid protein composition of claim 12 or 13, and a pharmaceutically acceptable carrier.

15. Use of the double stranded RNAi agent of claim 1, the conjugate of any one of claims 2-11, the nucleic acid protein composition of claim 12 or 13, or the pharmaceutical composition of claim 14 in the manufacture of a medicament for treating hypertension.

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