Sirna inhibitor for inhibiting expression of angiotensinogen AGT gene and use thereof

Abstract

Provided are an siRNA for inhibiting expression of the angiotensinogen (AGT) gene, an siRNA conjugate, and a pharmaceutical composition thereof, as well as a method for reducing expression of the AGT gene using the siRNA, the siRNA conjugate, and the pharmaceutical composition thereof. The provided siRNA, siRNA conjugate, and pharmaceutical composition thereof can be used for preventing and / or treating diseases or disorders mediated by the AGT gene.

Description

A siRNA inhibitor that inhibits angiotensinogen AGT gene expression and its application Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to an siRNA inhibitor that inhibits the expression of the angiotensinogen AGT gene and its application. Background Technology

[0002] The renin-angiotensin-aldosterone system (RAAS) is a hormone cascade that plays a role in the dynamic homeostasis of arterial pressure, tissue perfusion, and extracellular volume. RAAS dysregulation is crucial in blood pressure regulation and fluid and electrolyte balance. Angiotensinogen (AGT, also known as SERPINA8, ANHU, or hFLT1) occupies a central upstream position in the RAAS system and is a precursor for bioactive peptides. AGT (aGT) is an α-2-globulin, constitutively produced by the liver and released into the bloodstream; its molecular weight is 59–64 kDa.

[0003] In the body's blood pressure regulation mechanism, renin secreted by the kidneys has a specific effect on AGT, hydrolyzing it to generate angiotensin I (Ang I). Angiotensin I, catalyzed by angiotensin-converting enzyme (ACE), can be further converted into angiotensin II (Ang II). Ang II is an active substance with potent vasoconstrictive function; it can raise blood pressure through a series of physiological processes, including promoting vasoconstriction and aldosterone release.

[0004] The dynamic changes in AGT levels are closely related to changes in plasma AngII levels, and this relationship directly affects blood pressure. Specifically, numerous experimental data show a significant positive correlation between elevated blood AGT concentrations and hypertension. For example, in AGT gene knockout mouse models, a significant decrease in blood pressure was observed; conversely, when AGT is overexpressed, blood pressure tends to increase. Furthermore, in-depth research has revealed that overactivated RAAS triggers a series of pathophysiological changes, including promoting cardiomyocyte proliferation and fibrosis, leading to myocardial hypertrophy. Simultaneously, AngII can induce growth, migration, superoxide ion production, adhesion molecule activation, and monocyte and macrophage activation in vascular smooth muscle cells. These processes promote the formation of atherosclerosis, gradually affecting kidney function and potentially leading to kidney failure in severe cases.

[0005] Existing traditional antihypertensive drugs, such as beta-blockers, angiotensin-converting enzyme inhibitors (ACEIs), angiotensin II receptor blockers (ARBs), and diuretics, have many drawbacks. Their blood pressure reduction is often erratic and fluctuates significantly, easily leading to stroke, and they have serious side effects, including electrolyte disturbances, bradycardia, dry cough, angioedema, and hyperkalemia. Furthermore, they require daily medication and are prone to drug resistance.

[0006] Therefore, there is an urgent need in this field to develop a drug that can efficiently and specifically inhibit AGT. Summary of the Invention

[0007] The purpose of this invention is to provide a drug that can efficiently and specifically inhibit AGT.

[0008] In a first aspect of the invention, a siRNA for inhibiting the expression of the angiotensinogen AGT gene is provided, the siRNA comprising a sense strand and an antisense strand, each nucleotide in the siRNA being independently modified or unmodified, wherein the antisense strand comprises at least 19 consecutive nucleotides differing from any antisense strand sequence shown in Table A or Table B by 0, 1, 2 or 3 nucleotides, and the sense strand and the antisense strand are complementary to each other by at least 15, 16, 17, 18, 19, 20 or 21 nucleotides.

[0009] In another preferred embodiment, the antisense strand is 19-23 bases complementary to the human AGT transcript (NM_000029.3).

[0010] In another preferred embodiment, the sequence of the human AGT transcript (NM_000029.3) is shown in SEQ ID NO:321.

[0011] In another preferred embodiment, the positive strand comprises at least 19 consecutive nucleotides that differ from any of the positive strand sequences shown in Table A or Table B by 0, 1, 2, or 3 nucleotides.

[0012] In another preferred embodiment, the positive strand has a 19-23 base pair complementary match with the human AGT transcript (NM_000029.3).

[0013] In another preferred embodiment, the lengths of the sense strand and the antisense strand are each independently 19-23 nucleotides.

[0014] In another preferred embodiment, the antisense strand has two nucleotide protrusions at the 3' end relative to the sense strand.

[0015] In another preferred embodiment, the antisense chain is a sequence selected from the following: SEQ ID NO:2m-1, where the value of m is any positive integer selected from 1 to 10.

[0016] In another preferred embodiment, the justice chain is a sequence selected from the following: SEQ ID NO:2n, where the value of n is any positive integer selected from 1 to 10.

[0017] In another preferred embodiment, the antisense strand comprises any of the antisense strands shown in Table A acting on the nucleotide sequence at position NM_000029.3, and the sense strand comprises any of the sense strands shown in Table A acting on the nucleic acid sequence at position NM_000029.3, wherein the sequence of NM_000029.3 is as shown in SEQ ID NO:321.

[0018] In another preferred embodiment, the siRNA is selected from any of the siRNA sequence codes shown in Table A.

[0019] In another preferred embodiment, the sense strand and antisense strand are selected from the sense strand and antisense strand shown by the siRNA sequence codes in Table A below.

[0020] In another preferred embodiment, the sense strand and antisense strand are selected from the sense strand and antisense strand indicated by the siRNA sequence codes in Table B below:

[0021] Table B

[0022] In another preferred embodiment, the justice chain and the antisense chain are shown as SEQ ID NO:1 and SEQ SEQ ID NO:2, respectively.

[0023] In another preferred embodiment, the justice chain and the antisense chain are shown as SEQ ID NO:3 and SEQ SEQ ID NO:4, respectively.

[0024] In another preferred embodiment, at least one nucleotide in the sense strand and the antisense strand is a modified nucleotide.

[0025] In another preferred embodiment, the modified nucleotide is selected from: 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-deoxynucleotides, 2'-deoxy-2'-fluoro modified nucleotides, 2'-methoxyethyl modified nucleotides, 2'-amino modified nucleotides, 2'-alkyl modified nucleotides, 2'-alkoxy modified nucleotides, phosphate thioester modified nucleotides, debased nucleotides, and locked nucleotides.

[0026] In another preferred embodiment, the modified nucleotide is selected from: 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-deoxynucleotides, phosphate thioester modified nucleotides, or combinations thereof.

[0027] In another preferred embodiment, the 5' end of the sense strand comprises one or two phosphate-thioester modified nucleotides; and / or the 5' end and 3' end of the antisense strand each independently comprise one or two phosphate-thioester modified nucleotides.

[0028] In another preferred embodiment, the antisense strand comprises any one of the antisense strand nucleotide sequences shown in Table C, and the sense strand comprises any one of the sense strand nucleic acid sequences shown in Table C.

[0029] In another preferred embodiment, the sense strand and antisense strand are selected from the sense strand and antisense strand shown by the siRNA sequence codes in Table C:

[0030] Table C

[0031] Wherein, mG represents 2'-O-methylguanosine, mA represents 2'-O-methyladenosine, mU represents 2'-O-methyluridine, and mC represents 2'-O-methylcytidine; 2FG represents 2'-fluoroguanosine, 2FA represents 2'-fluoroadenosine, 2FU represents 2'-fluorouridine, and 2FC represents 2'-fluorocytidine; S represents a thiophosphate group; dA represents 2'-deoxyadenosine, dC represents 2'-deoxycytidine, dG represents 2'-deoxyguanosine, and dT represents 2'-deoxythymidine.

[0032] In another preferred embodiment, the siRNA is selected from any of the siRNA sequence codes shown in Table C.

[0033] In another preferred embodiment, the siRNA is selected from the siRNAs shown by the following siRNA sequence codes in Table C: 20-11, 20-20, 20-26, 43-12, 43-15, 43-16, 43-22, 43-25, 43-32, 43-34, 43-35, 43-42, 43-45, 43-48, 43-52, 43-53 or 95-18.

[0034] In another preferred embodiment, the siRNA is selected from the siRNAs shown by the following siRNA sequence codes in Table C: 20-20, 43-25, or 43-52.

[0035] In another preferred embodiment, the justice chain and the antisense chain are as shown in SEQ ID NO: 28 and SEQ ID NO: 116, respectively.

[0036] In another preferred embodiment, the justice chain and the antisense chain are shown as SEQ ID NO: 41 and SEQ ID NO: 147, respectively.

[0037] In another preferred embodiment, the justice chain and the antisense chain are shown as SEQ ID NO: 46 and SEQ ID NO: 167, respectively.

[0038] In another preferred embodiment, the justice chain and the antisense chain are shown as SEQ ID NO: 25 and SEQ ID NO: 107, respectively.

[0039] In another preferred embodiment, the justice chain and the antisense chain are as shown in SEQ ID NO: 30 and SEQ ID NO: 122, respectively.

[0040] In another preferred embodiment, the justice chain and the antisense chain are shown as SEQ ID NO: 36 and SEQ ID NO: 134, respectively.

[0041] In another preferred embodiment, the justice chain and the antisense chain are as shown in SEQ ID NO: 37 and SEQ ID NO: 137, respectively.

[0042] In another preferred embodiment, the justice chain and the antisense chain are as shown in SEQ ID NO: 38 and SEQ ID NO: 138, respectively.

[0043] In another preferred embodiment, the justice chain and the antisense chain are as shown in SEQ ID NO: 40 and SEQ ID NO: 144, respectively.

[0044] In another preferred embodiment, the justice chain and the antisense chain are shown as SEQ ID NO: 43 and SEQ ID NO: 154, respectively.

[0045] In another preferred embodiment, the justice chain and the antisense chain are as shown in SEQ ID NO: 43 and SEQ ID NO: 156, respectively.

[0046] In another preferred embodiment, the justice chain and the antisense chain are shown as SEQ ID NO: 44 and SEQ ID NO: 157, respectively.

[0047] In another preferred embodiment, the justice chain and the antisense chain are shown as SEQ ID NO: 45 and SEQ ID NO: 164, respectively.

[0048] In another preferred embodiment, the justice chain and the antisense chain are as shown in SEQ ID NO: 47 and SEQ ID NO: 170, respectively.

[0049] In another preferred embodiment, the justice chain and the antisense chain are shown as SEQ ID NO: 48 and SEQ ID NO: 174, respectively.

[0050] In another preferred embodiment, the justice chain and the antisense chain are shown as SEQ ID NO: 48 and SEQ ID NO: 175, respectively.

[0051] In a second aspect of the invention, an siRNA conjugate is provided, the siRNA conjugate comprising the siRNA described in the first aspect of the invention and a targeting group.

[0052] In another preferred embodiment, the conjugate further comprises a linker, and the siRNA, the linker, and the targeting group are covalently or non-covalently linked.

[0053] In another preferred embodiment, the targeting group contains a group derived from a carbohydrate, including monosaccharides, disaccharides, trisaccharides, or polysaccharides.

[0054] In another preferred embodiment, the targeting group is selected from the following monosaccharides: allose, maltose, arabinose, cladinose, brown sugar, erythrose, fructose, D-fucoitol, L-fucoitol, fucose glycosamine, fucose, fucose, galactosamine, D-galactosamine alcohol, N-acetyllactose (GalNAc), galactose, glucosamine, N-acetyl-glucosamine, glucosamine alcohol, glucose, glucose-6-phosphate, glucosylglyceraldehyde, L-glycerol-D-mannose-heptose, glycerol, glycerol, glucose, iodosose, lythose, mannose glycosamine, mannose, mannose-6-phosphate, allulose, quinovose, quinovosamine, rhamnitol, rhamnose, ribose, ribulose, heptose, sorbose, tagatose, tartaric acid, threose, xylose, and xylose.

[0055] In another preferred embodiment, the targeting group contains a group derived from N-acetylgalactosamine (GalNAc) or a derivative thereof.

[0056] In a third aspect of the invention, a pharmaceutical composition is provided, the pharmaceutical composition comprising:

[0057] (a) the siRNA described in the first aspect of the present invention, and / or the conjugate described in the second aspect of the present invention; and

[0058] (b) Pharmaceutically acceptable carriers.

[0059] In another preferred embodiment, the pharmaceutical composition is used to inhibit the expression of the AGT gene.

[0060] In another preferred embodiment, the inhibition rate of the expression can be calculated using the following formula:

[0061] In another preferred embodiment, the value of Y is 50% to 90%.

[0062] In a fourth aspect of the invention, the use of the siRNA described in the first aspect of the invention, the conjugate described in the second aspect of the invention, and the pharmaceutical composition described in the third aspect of the invention in the preparation of a medicament for the prevention and / or treatment of AGT-mediated diseases or conditions, for inhibiting the expression of the AGT gene, is provided.

[0063] In another preferred embodiment, the AGT-mediated disease or condition is a disease or condition of AGT overexpression.

[0064] In another preferred embodiment, the disease or condition includes lipid metabolism disorder.

[0065] In another preferred embodiment, the disease or condition includes: hypertension, atherosclerosis, hypercholesterolemia, hypertriglyceridemia, myocardial infarction, and cardiovascular disease.

[0066] In another preferred embodiment, the formulation includes a laboratory formulation.

[0067] In another preferred embodiment, the composition comprises a pharmaceutical composition.

[0068] In another preferred embodiment, the inhibition rate of the expression can be calculated using the following formula:

[0069] In another preferred embodiment, the value of Y is 50% to 90%.

[0070] In a fifth aspect of the present invention, a method for inhibiting AGT expression in cells in vitro is provided, the method comprising the following steps:

[0071] (z1) Co-culture cells with an effective amount of the siRNA described in the first aspect of the present invention, the conjugate described in the second aspect of the present invention, or the pharmaceutical composition described in the third aspect of the present invention.

[0072] In another preferred embodiment, the method is non-diagnostic and non-therapeutic.

[0073] In another preferred embodiment, the siRNA described in the first aspect of the invention, the conjugate described in the second aspect of the invention, or the pharmaceutical composition described in the third aspect of the invention enters the cell via endocytosis.

[0074] In a sixth aspect of the invention, a method for treating AGT-mediated diseases or conditions is provided, comprising administering a therapeutically effective amount of the siRNA described in the first aspect of the invention, the conjugate described in the second aspect of the invention, or the pharmaceutical composition described in the third aspect of the invention to a subject.

[0075] In another preferred embodiment, the AGT-mediated disease or condition is a disease or condition of AGT overexpression.

[0076] In another preferred embodiment, the dosage is 1–20 mg / kg.

[0077] In another preferred embodiment, the disease or condition includes lipid metabolism disorder.

[0078] In another preferred embodiment, the disease or condition includes: hypertension, atherosclerosis, hypercholesterolemia, hypertriglyceridemia, myocardial infarction, and cardiovascular disease.

[0079] In another preferred embodiment, the subject is a human or a non-human mammal.

[0080] In another preferred embodiment, the subject is a human being.

[0081] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0082] Figure 1 shows the cytotoxicity test results of modified siRNA on Hep3B cells in one embodiment of the present invention. Detailed Implementation

[0083] Through extensive and in-depth research, the inventors have developed, for the first time, a class of siRNA drugs that inhibit AGT protein expression. These drugs can potently reduce the production of Ang I and Ang II at the source; they also exhibit sustained and stable blood pressure reduction with high specificity. Compared to traditional drugs, they significantly reduce interference with normal physiological processes, lowering side effects and complications. They are long-acting, solving the problem of frequent medication and eliminating the need for daily dosing. Based on this, the present invention was completed.

[0084] The siRNA provided by this invention includes a sense strand and an antisense strand. The antisense strand induces the formation of an RNA-induced silencing complex (RISC) and binds to the human AGT transcript (NM_000029.3) through base complementarity pairing, thereby cleaving the AGT gene transcript and reducing AGT expression.

[0085] the term

[0086] As used herein, the term "AGT" refers to angiotensinogens from humans, rodents, and primates, including the full-length unprocessed precursor form, the mature form (signal peptide cleavage), and the protease-processed AGT protein. The human AGT mRNA transcript is numbered NM_000029.3, and the predicted sequence number of the cynomolgus monkey AGT mRNA is (AB170313.1).

[0087] The terms "interfering RNA" or "RNAi" or "interfering RNA sequence" include single-stranded RNA (e.g., mature miRNA, ssRNAi oligonucleotide, ssDNAi oligonucleotide) or double-stranded RNA (i.e., double-stranded RNA such as siRNA, dsRNA, shRNA, aiRNA, or precursor miRNA) that, when the interfering RNA is in the same cell as the target gene or sequence, can reduce or inhibit the expression of the target gene or sequence (e.g., by mediating degradation and inhibiting the translation of mRNA complementary to the interfering RNA sequence). Interfering RNA therefore refers to a single-stranded RNA complementary to the target mRNA sequence or a double-stranded RNA formed by two complementary strands or a single self-complementary strand.

[0088] Interfering RNA includes “small interfering RNA” or “siRNA”, each strand of which contains about 15 to about 60 nucleotides (e.g., about 15-60, 15-50, 15-40, 15-30, 15-25, 17-25, 19-25, 17-23, 17-21, 19-23, or 19-21 nucleotides, or 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides). The ranges and lengths listed above, as well as intermediate values, are also conceivable to be part of this invention. In a preferred embodiment, the siRNA is chemically synthesized. The siRNA of this invention is capable of silencing the expression of target sequences in vitro and / or in vivo. In other embodiments, the siRNA contains at least one modified nucleotide, for example, the siRNA contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more modified nucleotides in the double-stranded region.

[0089] As used herein, the terms “dsRNA,” “double-stranded ribonucleic acid,” or “precursor RNAi molecule” are intended to include any precursor molecule processed in vivo by a nuclease to produce active siRNA.

[0090] As used herein, "dsRNA" refers to a double helix structure consisting of two antiparallel, complementary nucleic acid strands, forming a "sense strand" and an "antisense strand" relative to the target gene mRNA, depending on whether they are complementary.

[0091] The term "siRNA" refers to a molecule containing siRNA as defined herein, and that the molecule mediates targeted cleavage of RNA transcripts via an RNA-induced silencing complex (RISC) pathway. iRNA guides the sequence-specific degradation of mRNA via a process known as RNA interference (RNAi). iRNA regulates (e.g., inhibits) the expression of AGT in cells (e.g., cells within a subject, such as a mammalian subject).

[0092] Typically, the majority of nucleotides in each strand of siRNA are ribonucleotides; however, as detailed herein, each or both of the two strands may also include one or more non-ribonucleotides, such as deoxyribonucleotides and / or modified nucleotides. Additionally, as used herein, "siRNA" may include chemically modified ribonucleotides; siRNA may include substantial modifications at multiple nucleotide sites. As used herein, the term "modified nucleotide" refers to a nucleotide that independently has a modified sugar moiety, a modified internucleotide link, and / or a modified nucleobase. Thus, the term modified nucleotide encompasses substitution, addition, or removal of, for example, a functional group or atom, from the internucleotide link, sugar moiety, or nucleobase. Modifications suitable for use in this invention include all types of modifications disclosed herein or known in the art.

[0093] As used herein, “G”, “C”, “A”, and “U” represent nucleotides containing guanine, cytosine, adenine, and uracil as bases, respectively. “Ribosonucleotide” or “nucleotide” can also indicate modified nucleotides. “dT” represents 2’-deoxythymidine.

[0094] As used in this article, mG represents 2'-O-methylguanosine monophosphate, mA represents 2'-O-methyladenosine monophosphate, mU represents 2'-O-methyluridine monophosphate, and mC represents 2'-O-methylcytidine monophosphate; 2FG represents 2'-fluoroguanosine monophosphate, 2FA represents 2'-fluoroadenosine monophosphate, 2FU represents 2'-fluorouridine monophosphate, and 2FC represents 2'-fluorocytidine monophosphate; the letter S indicates that the two nucleotides adjacent to the letter S are linked by thiophosphate groups; dA represents 2'-deoxyadenosine monophosphate, dC represents 2'-deoxycytidine monophosphate, dG represents 2'-deoxyguanosine monophosphate, dT represents 2'-deoxythymidine monophosphate, and GalNac represents L96.

[0095] The siRNA of this invention comprises a single-stranded RNA that interacts with the AGT mRNA sequence, thereby guiding the cleavage of the mRNA. Upon entering the cell, the double-stranded siRNA binds to AGO2, unwinds the double helix, and then enters the RISC, further guiding the antisense strand to recognize and complementarily pair with the AGT mRNA. Ultimately, endonucleases such as AGO2 within the RISC cleave the AGT mRNA, reducing AGT mRNA levels.

[0096] As used herein, the term "nucleotide overhang" refers to an unpaired nucleotide that protrudes from the double helix structure of siRNA. For example, when the 3′ end of the antisense strand of siRNA extends beyond the 5′ end of the sense strand, this extended portion of nucleotide is called a nucleotide overhang.

[0097] As used herein, the term "antisense strand" or "guide strand" refers to a double-stranded siRNA containing a nucleotide sequence complementary to the target sequence (AGT mRNA). This complementary nucleotide sequence is defined as the antisense strand. The antisense strand and the target mRNA sequence may not be perfectly matched; mismatches can occur in the terminal regions.

[0098] As used herein, the term "complementarity" refers to the ability of one nucleotide sequence to hybridize with another nucleotide sequence under certain conditions and form a double-stranded structure. Complete base pairing across the entire length of the two strands of siRNA is called "complete complementarity," while the presence of 1-3 base mispairings is termed "basic complementarity." The terms "complementarity," "complete complementarity," and "basic complementarity" can be used within double-stranded siRNAs or in the context of base pairing between the antisense strand of the siRNA and the target gene mRNA.

[0099] As used herein, the term “inhibition” is used interchangeably with similar descriptive terms such as “reduction,” “silencing,” and “downregulation” to indicate any level of inhibition. “Inhibition of AGT expression” includes inhibition of the expression of any AGT gene mRNA and its variants mRNA, as well as the expression of AGT and its variant proteins.

[0100] As used in this article, "delivering siRNA into cells" refers to the in vitro delivery of siRNA into target cells via lipid transfection, where siRNA encapsulated in liposomes is incubated with the cells. In in vivo experiments, siRNA needs to be coupled to the ligand GalNAc. GalNAc binds to the desialylate glycoprotein receptor on the surface of liver cells, thereby specifically delivering siRNA into liver cells.

[0101] As used in this article, "lipid metabolism disorder" refers to disorders or diseases or conditions related to lipid metabolism, such as hyperlipidemia, caused by disturbances in lipid metabolism and imbalances in metabolic balance. The main manifestation is an abnormally high level of certain lipid metabolites and lipoproteins in the blood. "Lipid metabolism disorder" includes (1) hereditary diseases: familial hypertriglyceridemia; and (2) acquired disorders, such as dietary or drug-related abnormalities. In a general sense, "lipid metabolism disorder" is considered to include various types of diseases such as atherosclerosis, dyslipidemia, hypertriglyceridemia, acute pancreatitis related to hypertriglyceridemia, chylomicron syndrome, familial chylomicronemia, hyperlipidemia (including familial complex hyperlipidemia), and hypercholesterolemia. In cardiovascular diseases, coronary artery disease (ischemic heart disease) and inflammation related to coronary artery disease are also considered to be related to "lipid metabolism disorder."

[0102] The term "conjugate" (sometimes also referred to as a conjugate, coupling compound, or coupling agent, and sometimes as a conjugate in the literature) used in this invention corresponds to the English word "conjugate" or "conjugates." A conjugate is a new compound formed by the covalent connection (coupling) of two or more compound molecules through bivalent or multivalent compound molecules with a linking function. Conjugates can also be formed directly from two molecules through coupling or condensation. A common example is the antibody-drug conjugate (ADC), also known as an antibody-drug conjugate. In some embodiments, the product resulting from the coupling of siRNA molecules to a target group via a linker (or linker) is also a conjugate or siRNA conjugate. In some embodiments, the product resulting from the coupling of siRNA to a target group is an siRNA conjugate.

[0103] The term "coupling" as used in this invention refers to a chemical process in which two or more compound molecules undergo a reaction to form new chemical bonds and new molecules. In certain contexts, "coupling" may be used interchangeably with or instead of "linking".

[0104] As used herein, “inhibit AGT gene expression” includes any level of AGT gene inhibition, such as at least partial inhibition of AGT gene expression, like at least about 20% inhibition. In some embodiments, inhibition is 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%.

[0105] AGT mRNA or AGT protein levels can be assessed based on the levels of any variable associated with AGT gene expression. Inhibition can be assessed by a reduction in the absolute or relative level of one or more of these variables compared to a control level. This control level can be any type of control level used in the art, such as baseline levels before administration or levels determined from similar untreated or controlled (e.g., buffer-only or inert control) subjects, cells, or samples.

[0106] As used herein, "therapeutic effective amount," "therapeutic effective dose," and "effective amount" refer to the amount of the siRNA of the present invention, when administered alone or in combination with other therapeutic agents to cells, tissues, or subjects, that effectively prevents or improves the symptoms of one or more diseases or conditions, or the development of such diseases or conditions. Therapeutic effective dose also refers to the amount of antibody or its antigen-binding fragment sufficient to cause symptom improvement, such as the amount used to treat, cure, prevent, or improve an associated medical condition, or to accelerate the treatment, cure, prevention, or improvement of such conditions. When administered to an individual as a single active ingredient, the therapeutic effective dose refers only to that ingredient. When administered in combination, the therapeutic effective dose refers to the combined amount of active ingredients that cause a therapeutic effect, whether administered in combination, sequentially, or simultaneously. The effective amount of the therapeutic agent will result in an increase of at least 10%, typically at least 20%, preferably at least about 30%, more preferably at least 40%, and most preferably at least 50% in diagnostic criteria or parameters.

[0107] The siRNA of the present invention

[0108] This document provides siRNAs for inhibiting AGT gene expression. Each siRNA comprises a sense strand and an antisense strand. The antisense strand is substantially complementary to the AGT gene mRNA sequence, with the complementary region being 19-23 nucleotides in length. The sense and antisense strands are each 19-23 nucleotides in length. The sense and antisense strands may be of the same length or different lengths. In some embodiments, the sense and antisense strands are each independently 19-23 nucleotides in length. In some embodiments, the sense strand is 19-21 nucleotides in length, and the antisense strand is 19-23 nucleotides in length. In some embodiments, the sense strand is about 19 nucleotides in length, and the antisense strand is about 20 nucleotides in length. In some embodiments, the sense strand is about 19 nucleotides in length, and the antisense strand is about 21 nucleotides in length. After delivery of the siRNA of this invention into cells, the siRNA inhibits AGT gene expression by at least about 50%. AGT gene expression levels are determined using PCR and Western blotting techniques. The siRNA can be synthesized using an automated Oligo synthesizer. The siRNA of the present invention is prepared by the following steps: (1) preparing the sense strand and antisense strand of the double-stranded RNA molecule separately; (2) annealing the sense strand and antisense strand in a 1:1 molar ratio to assemble the double-stranded siRNA composition. Both the sense strand and antisense strand of the double-stranded RNA molecule are prepared by solid-phase organic synthesis. The siRNA of the present invention comprises two nucleotide sequences, one sense sequence and one antisense sequence. The sense strand is selected from the sequences provided in Tables A to C, and the corresponding antisense strand is selected from the sequences in Tables A to C. The two sequences are complementary, wherein the antisense strand is substantially complementary to the AGT mRNA sequence.

[0109] The siRNA provided by this invention may contain one or more mismatches relative to the target gene mRNA sequence, generally no more than three mismatches. For a 21-nucleotide siRNA, the mismatches are typically not located in the central 2-14 nucleotide region. If the antisense strand of the siRNA contains a mismatch of the target gene mRNA sequence, the mismatch region is preferably limited to the last 5 nucleotides from the 5′ or 3′ end of the complementary region. The methods described herein or methods known in the art can be used to determine whether an siRNA containing a mismatch of the target sequence effectively inhibits AGT gene expression.

[0110] In a preferred embodiment, the sense and antisense strands of the siRNA of the present invention are selected from the sense and antisense strands shown by the siRNA sequence codes in Table A:

[0111] Table A

[0112] Wherein, NM_000029.3 refers to the human AGT transcript number with a sequence as shown in SEQ ID NO:321.

[0113] In another preferred embodiment, the justice chain and antisense chain are shown in Table B:

[0114] Table B

[0115] The modified siRNA of this invention

[0116] This invention also provides siRNA chemically modified with RNA, thereby enhancing the stability of siRNA. The nucleic acid characterized by this invention can be synthesized and modified using methods established in the art. Modifications include: (1) terminal modification, 5′ terminal modification (phosphorylation) or 3′ terminal modification (covalent binding of conjugates, etc.); morpholino modification at the 5′ end of the positive strand, etc.; (2) base modification, replacing bases at specific sites, generally by replacing with stable bases, stable bases, or removing bases (non-base nucleotides); (3) ribose modification: introducing a methoxy group at the 2′ position of the ribose, or introducing a fluoride group after deoxygenation, etc.; (4) backbone modification, including 3′ terminal modification or the introduction of thiophosphate at the 3′ end. In one embodiment, the siRNA composition has a GalNac ligand covalently bound to the 3′ end of the positive strand, thereby enhancing the distribution of the siRNA in hepatocytes and its targeting ability in liver tissue.

[0117] In another preferred embodiment, the modified nucleotide is selected from: 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-deoxynucleotides, 2'-deoxy-2'-fluoro modified nucleotides, 2'-methoxyethyl modified nucleotides, 2'-amino modified nucleotides, 2'-alkyl modified nucleotides, 2'-alkoxy modified nucleotides, phosphate thioester modified nucleotides, debased nucleotides, and locked nucleotides.

[0118] The GalNAc covalently bound siRNA conjugate provided by this invention can be prepared conventionally using solid-phase synthesis techniques, or it can be commissioned to a commercial company with such synthesis technology for preparation.

[0119] In a preferred embodiment, the sense and antisense strands of the modified siRNA of the present invention are selected from the sense and antisense strands shown by the siRNA sequence codes in Table C:

[0120] Table C

[0121] Pharmaceutical Composition

[0122] This invention also includes pharmaceutical compositions and formulations. These comprise the siRNA, siRNA conjugates, and clinically applicable formulations of the present invention, thereby forming pharmaceutical compositions. The siRNA of the present invention is formulated using various buffer solutions. Buffer solutions may include acetate, citrate, carbonate, or phosphate. In one embodiment, the buffer solution is phosphate-buffered saline (PBS). The pharmaceutical composition of this siRNA can be used to treat diseases or disorders related to AGT gene expression or activity, including but not limited to hypertension, lipid metabolism disorders (e.g., hypertriglyceridemia), etc. The pharmaceutical compositions provided by the present invention can be administered intravenously (IV) or subcutaneously. The effect of a single dose of the pharmaceutical composition of the present invention on AGT levels can be sustained for a long time, therefore the pharmaceutical composition can be administered once daily, or at intervals of 3, 4, or 5 days, no more than 1, 2, or 3 days, or 4 weeks.

[0123] The pharmaceutical compositions of the present invention comprise an active ingredient within a safe and effective range and a pharmacologically acceptable excipient or carrier. "Safe and effective range" refers to an amount of active ingredient sufficient to significantly improve the condition without causing serious side effects. Typically, the pharmaceutical composition contains 1-2000 mg of active ingredient per dose, more preferably 10-200 mg of active ingredient per dose. Preferably, "one dose" refers to one injection.

[0124] "Pharmaceutically acceptable carriers" refer to one or more compatible solid or liquid fillers or gelling substances that are suitable for human use and must have sufficient purity and sufficiently low toxicity. "Compatibility" here refers to the ability of the components in the composition to interact with and incorporate with the active ingredient without significantly reducing the efficacy of the active ingredient. Examples of pharmaceutically acceptable carriers include cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose, etc.), gelatin, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, olive oil, etc.), polyols (such as propylene glycol, mannitol, sorbitol, etc.), and emulsifiers (such as...). Wetting agents (such as sodium dodecyl sulfate), stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

[0125] The administration method of the pharmaceutical composition described in this invention is consistent with the administration method (or delivery method) described in this invention.

[0126] The dosage form and administration method of the pharmaceutical composition described in this invention are consistent, such as (but not limited to): tablets, capsules, powders, pills, granules, syrups, solutions, suspensions, emulsions, sprays, aerosols, powder sprays, volatile liquids, injections, powder injections, external solutions, lotions, pouring solutions, liniments, poultices, plasters, ointments, rubber plasters, ointments, hard plasters, pastes, eye drops, nasal drops, ophthalmic ointments, mouthwashes, sublingual tablets, suppositories, etc.

[0127] The method of the present invention

[0128] This invention also provides methods for inhibiting AGT expression in cells using siRNA, siRNA conjugates, or pharmaceutical compositions containing the same. These methods include: delivering the siRNA of this invention into cells and maintaining the cells for a period of time, thereby effectively degrading AGT mRNA and inhibiting AGT gene expression in the cells. The reduction in gene expression can be assessed by any method known in the art, including qRT-PCR to determine AGT mRNA expression levels and Western blotting to determine AGT protein levels.

[0129] This invention also provides methods for inhibiting AGT gene expression in animals. These methods include: targeting and delivering a GalNAc-conjugate siRNA to liver cells via intravenous or subcutaneous injection, and continuing to feed the animals for a period of time, thereby effectively reducing AGT expression levels. The reduction in gene expression can be assessed by any method known in the art, including qRT-PCR to determine AGT mRNA expression levels; and Western blotting to determine AGT protein levels.

[0130] This invention further provides methods for treating subjects in need using siRNA or pharmaceutical compositions thereof. These treatment methods of the invention include administering the siRNA, siRNA conjugate, or pharmaceutical composition of the invention to a subject suffering from diseases including, but not limited to, hypertension and atherosclerotic cardiovascular disease. The above methods can be used in combination with other drugs and / or other treatment methods. Examples include cholesterol synthesis inhibitors (statins), bile acid sequestrants; cholesterol acyltransferase inhibitors; and farnesol X receptor antagonists. Other therapeutic agents include those that increase high-density lipoprotein, such as cholesterol ester transfer protein inhibitors.

[0131] The main advantages of this invention are:

[0132] 1. The siRNA of the present invention can effectively inhibit the expression of the AGT gene, thereby effectively treating diseases or symptoms caused by AGT overexpression.

[0133] 2. The siRNA of the present invention is a class of siRNA drugs with a long-acting dosing interval, providing a more convenient treatment method for cardiovascular-related diseases, including the preventive treatment of hypertension.

[0134] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0135] Example 1: Design and synthesis of siRNA:

[0136] Based on the analysis of the AGT gene NM_000029.3 (sequence shown in SEQ ID NO: 321), the AGT gene sequence is as follows:

[0137] 131 pairs of siRNAs were designed as shown in Table 1. The sequences were sent to Beijing Qingke Biotechnology Co., Ltd. (hereinafter referred to as Qingke), where 0.2-1 μmol of oligonucleotides were synthesized using a solid-phase oligonucleotide synthesis protocol. In this protocol, G represents guanylic acid, A represents adenosine acid, U represents uridine acid, C represents cytidine acid, and dT represents 2'-deoxythymidine.

[0138] Table 1. Sense and antisense strand sequences of unmodified siRNA

[0139] Example 2. In vitro activity assay of unmodified siRNA

[0140] Hep3B cells (purchased from Pronosei Biotechnology Co., Ltd.) were cultured in DMEM high-glucose medium (Adamas) supplemented with 10% fetal bovine serum (FBS, ExCell) and 1% NeAA (Gibco) at 5% CO2 and 37°C. After the cells nearly completely covered the culture flask, they were digested with trypsin (Gibco) and resuspended. The resuspended cell density was adjusted to 3 × 10⁶ cells / year. 5 Cells were seeded into 6-well plates and cultured until 80%-90% confluence. The medium in each well was then replaced with 2 ml of DMEM / F-12 (Adamas) medium supplemented with 10% FBS. The siRNA, transfection reagent (Polyplus), and OptiPRO SFM (Gibco) were mixed and suspended in the 6-well plates for transfection. The plates were then incubated overnight at 37°C with 5% CO2. The final concentration of siRNA used for initial screening was 5 nM, and the IC50 was [not specified]. 50 During the assay, siRNA was initially diluted to a concentration of 5 nM, followed by seven 3-fold dilutions to a final concentration of 0.07 nM. In addition to the experimental group, a control group (CK) without siRNA and a positive control group (PC group, Zilebesiran) were also included.

[0141] Approximately 24 hours post-transfection, total RNA was extracted using an RNA kit (Qingke). cDNA synthesis was performed using a reverse transcription kit (Titan). 1 μg RNA, 2 μL gDNA Remover, and 5 μL 5×RT Mix were added, and Nuclease-free ddH2O was added to bring the volume to 20 μL. The mixture was briefly centrifuged to the bottom of the tube. cDNA synthesis was then performed using a PCR instrument (ThermoFisher), incubated at 42°C for 5 min, followed by incubation at 95°C for 30 s, and subsequently stored at -20°C.

[0142] Quantitative PCR was performed using the SYBR Green assay (Qingke). The human HMBS gene was used as an internal reference gene. The upstream primer sequence for HMBS was 5'-TACTGGCACACTGCAGCCTC-3' (SEQ ID NO. 322), and the downstream primer sequence was 5'-CACGATCCCGAGACTCTGCT-3' (SEQ ID NO. 323). The upstream primer sequence for the human AGT gene was 5'-GGGAAGAAGCTGCCGTTGT-3' (SEQ ID NO. 324), and the downstream primer sequence was 5'-CGAGGTGGAAGGGGTGTATG-3' (SEQ ID NO. 325). Using 2- △△Ct The relative mRNA level of AGT was detected. Quantitative PCR was performed using a QuantStudio3 (ABI) instrument. The reaction conditions were: (1) pre-denaturation 95℃, 30 seconds; (2) denaturation 95℃, 10 seconds, annealing extension 60℃, 30 seconds. Step (2) was repeated for 40 cycles. (3) Melting curves: 95℃, 15 seconds; 60℃, 60 seconds; 95℃, 15 seconds. The results were normalized using controls to obtain the relative mRNA expression level and knockdown efficiency.

[0143] Excel software was used for graphing during screening. If IC50 calculation was required, Graphpad Prism was used for four-parameter fitting. The initial screening results of unmodified siRNA are shown in Table 2; the IC50 results of unmodified siRNA are shown in Table 3.

[0144] Table 2. In vitro activity assay of unmodified siRNA (5 nM concentration)

[0145] Table 3. IC50 determination of unmodified siRNA

[0146] Example 3: Design and preparation of modified siRNA

[0147] According to the experimental results obtained in Tables 2 and 3 above, multiple pairs of siRNAs inhibited the expression of AGT in Hep3B cells to varying degrees. Among them, siRNAs 20, 43, 69, 70, 74, 83, 88, 89, 95 and 125 showed relatively high inhibitory activity against AGT, comparable to the activity of the positive reference (Zilebesiran, i.e., the PC group).

[0148] To improve the stability and activity of siRNA in vivo, sequences 20, 43, 69, 70, 74, 83, 88, 89, 95, and 125 were modified. Each sequence underwent multiple modifications. Specifically, mG represents 2'-O-methylguanylic acid, mA represents 2'-O-methyladenosine acid, mU represents 2'-O-methyluridine acid, and mC represents 2'-O-methylcytidine acid; 2FG represents 2'-fluoroguanylic acid, 2FA represents 2'-fluoroadenosine acid, 2FU represents 2'-fluorouridine acid, and 2FC represents 2'-fluorocytidine acid; S represents a thiophosphate group; dA represents 2'-deoxyadenosine acid, dC represents 2'-deoxycytidine acid, dG represents 2'-deoxyguanylic acid, dT represents 2'-deoxythymidine acid, and GalNac represents GalNac-L96. For specific sequence modifications, please refer to Table 4. The sequences were sent to Beijing Qingke, where 0.2–1 μmol of oligonucleotides were synthesized using a solid-phase oligonucleotide synthesis protocol.

[0149] Table 4. Sensitive and antisense strand sequences of modified siRNA

[0150] Example 4. In vitro activity assay of modified siRNA

[0151] For specific methods, please refer to Example 2. The sample screening concentration was adjusted to two concentrations, such as PC-H representing the high concentration (1 nM) of the positive drug Zilebesiran and PC-L representing the low concentration (0.1 nM) of the positive drug. The experimental results are shown in Tables 5 and 6.

[0152] Table 5. In vitro activity assay of modified siRNA

[0153] Table 6. IC50 assay of modified siRNA

[0154] The results in Tables 5 and 6 show that the activity of some modified sequences was significantly affected, with inhibition rates at both high and low concentrations lower than those of the positive control drug. Further IC50 determination was performed on the modified siRNAs with superior activity compared to the positive control drug, and the IC50 of the modified siRNAs numbered 20-20, 43-12, 43-15, 43-16, 43-22, 43-24, 3-52, and 43-53 was superior to that of the positive control drug Zilebesiran.

[0155] Example 5: Detection of siRNA-modified cytotoxicity

[0156] Hep3B cells were fed at a rate of 2 × 10 4 Cells were seeded into 96-well plates and incubated at 37°C in a 5% CO2 incubator.

[0157] Transfection was performed 24 hours after plating. siRNA, transfection reagent, and OptiPRO SFM were mixed and added to a 96-well plate for transfection. The initial concentration of the modified siRNA was 50 nM, diluted 3-fold ten times to a final concentration of 0.003 nM. CK cells served as a Hep3B control and were not transfected.

[0158] 24 hours after transfection, add 10 μL of CCK-8 assay solution (CCK-8 Cell Proliferation-Virus Assay Kit DOjinDO) to each well of a 96-well plate. Incubate at 37°C for 30 minutes, then shake well. Set the molecular device to 450 nm and measure the OD value. Calculate Cell Viability (%) using the formula: Cell Viability (%) = (Exp / CK) × 100%.

[0159] As shown in Figure 1, after transfection of cells with each modified siRNA (e.g., siRNAs numbered 20-20, 43-25, and 43-52), the cell viability was all above 83%, with minimal impact on cell viability. No cytotoxicity was observed after transfection.

[0160] Example 6: Transgenic Mouse Experiment

[0161] Using the transgenic B6-Rosa26-hAGT mouse as the experimental system, different test products were administered via single subcutaneous injection. The inhibitory effect of the test products on serum hAGT was investigated by measuring serum hAGT protein levels at different time points, and compared with the positive control Zilebesiran.

[0162] Forty-eight 6-8 week old male AGT humanized transgenic mice (B6-Rosa26-hAGT, Jiangsu Jicui Yaokang Biotechnology Co., Ltd.) were randomly divided into eight groups of six mice each based on their hAGT levels. A placebo control group and a positive control group (Zilebesiran) were included. Different doses of the test substance were administered via single subcutaneous injection. Serum hAGT concentrations were collected weekly after administration, and protein expression rate was calculated (protein expression rate (%) = (concentration at detection point - concentration at zero time point) / concentration at zero time point × 100%, where zero time point is the concentration on the day of administration, i.e., D0). The results are shown in Table 7.

[0163] Table 7. Changes in hAGT protein levels in mice after a single dose of hAGT.

[0164] Note: D0 to D56 represent days 0 to 56 after administration, respectively. hAGT protein change rate (%) = hAGT protein expression level at the detection point - protein expression level at D0. For example, in the table, the hAGT protein change rate at D7 in the placebo group is "-9.6%", which means that compared with day D0, the placebo inhibited 9.6% of hAGT protein expression on day 7 after administration.

[0165] The results showed that, compared with the placebo group, the modified siRNA groups numbered 20-20, 43-25, and 43-52 were able to inhibit hAGT protein expression in a dose-dependent manner. Furthermore, the inhibition level of each modified siRNA group on day 56 after administration was greater than that of the positive control Zilebesiran group on day 49. Specifically, on day 56 after administration, the inhibition rate of the positive control Zilebesiran was only 5.1%, while the inhibition rate of the siRNAs 43-25 and 43-52 (at a dose of 10 mg / kg) of this invention was over 55%, approximately 11 times that of the positive control. This indicates that the siRNAs provided by this invention maintain the inhibition of angiotensinogen AGT gene expression for a longer period than the positive control. The siRNAs provided by this invention exhibit a sustained and stable blood pressure-lowering effect, high specificity, reduced side effects and complications, and long-lasting efficacy.

[0166] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A siRNA for inhibiting the expression of the angiotensinogen AGT gene, said siRNA comprising a sense strand and an antisense strand, wherein each nucleotide in said siRNA is independently a modified or unmodified nucleotide. in, The antisense strand comprises at least 19 consecutive nucleotides that differ from any of the antisense strand sequences shown in Table A or Table B by 0, 1, 2, or 3 nucleotides. The sense strand and the antisense strand are complementary to each other by at least 15, 16, 17, 18, 19, 20 or 21 nucleotides.

2. The siRNA as described in claim 1, characterized in that, The sense and antisense strands are selected from the sense and antisense strands indicated by the following siRNA sequence designations: Wherein, NM_000029.3 refers to the human AGT transcript number with a sequence as shown in SEQ ID NO:

321.

3. The siRNA as described in claim 1, characterized in that, The sense and antisense strands are selected from the sense and antisense strands indicated by the following siRNA sequence designations:

4. The siRNA as described in claim 1, characterized in that, At least one nucleotide in the sense strand and the antisense strand is a modified nucleotide, wherein the modified nucleotide is selected from: 2'-O-methyl modified nucleotide, 2'-fluoro modified nucleotide, 2'-deoxynucleotide, 2'-deoxy-2'-fluoro modified nucleotide, 2'-methoxyethyl modified nucleotide, 2'-amino modified nucleotide, 2'-alkyl modified nucleotide, 2'-alkoxy modified nucleotide, phosphate thioester modified nucleotide, debased nucleotide, and locked nucleotide.

5. The siRNA as described in claim 4, characterized in that, The sense and antisense strands are selected from the sense and antisense strands indicated by the following siRNA sequence designations: Wherein, mG represents 2'-O-methylguanosine, mA represents 2'-O-methyladenosine, mU represents 2'-O-methyluridine, and mC represents 2'-O-methylcytidine; 2FG represents 2'-fluoroguanosine, 2FA represents 2'-fluoroadenosine, 2FU represents 2'-fluorouridine, and 2FC represents 2'-fluorocytidine; S represents a thiophosphate group; dA represents 2'-deoxyadenosine, dC represents 2'-deoxycytidine, dG represents 2'-deoxyguanosine, and dT represents 2'-deoxythymidine.

6. An siRNA conjugate, characterized in that, The siRNA conjugate comprises the siRNA of claim 1 and a targeting group.

7. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises: (a) the siRNA of claim 1, and / or the conjugate of claim 6; and (b) Pharmaceutically acceptable carriers.

8. Use of the siRNA of claim 1, the conjugate of claim 6, or the pharmaceutical composition of claim 7 in the preparation of a medicament for the prevention and / or treatment of AGT-mediated diseases or conditions, for the purpose of inhibiting the expression of the AGT gene.

9. The use as described in claim 1, characterized in that, The diseases or conditions mentioned include: hypertension, atherosclerosis, hypercholesterolemia, hypertriglyceridemia, myocardial infarction, and cardiovascular diseases.

10. A method for inhibiting AGT expression in cells in vitro, characterized in that, The method includes the following steps: (z1) Co-culturing cells with an effective amount of the siRNA of claim 1, the conjugate of claim 6, or the pharmaceutical composition of claim 7, wherein the method is non-diagnostic and non-therapeutic.

Images