CN117187242A

CN117187242A - siRNA and conjugate thereof

Info

Publication number: CN117187242A
Application number: CN202311088727.5A
Authority: CN
Inventors: 唐春雷; 范为正; 袁昕; 姜虹羽
Original assignee: Sanorri Biomedical Technology Wuxi Co ltd
Current assignee: Sanorri Biomedical Technology Wuxi Co ltd
Priority date: 2023-08-28
Filing date: 2023-08-28
Publication date: 2023-12-08

Abstract

The invention discloses siRNA and a conjugate thereof, and belongs to the field of biological medicine. The siRNA and the conjugate thereof and the pharmaceutical composition containing the siRNA and the conjugate can specifically target the liver, thereby inhibiting the expression of the angiotensinogen gene, having lower off-target effect and good stability and realizing the treatment of diseases and/or symptoms related to the expression of the angiotensinogen.

Description

siRNA and conjugate thereof

Technical Field

The invention discloses siRNA and a conjugate thereof, siRNA for regulating the expression of Angiotensinogen (AGT) genes, a pharmaceutical composition containing the siRNA and the siRNA conjugate, and also discloses a preparation method and application of the siRNA, the pharmaceutical composition and the siRNA conjugate, belonging to the field of biological medicine.

Background

Angiotensinogen (AGT) is a member of the serpin family and is a component of the renin-angiotensin-aldosterone system (RAAS). It is produced mainly in the liver and released into the blood circulation where renin converts it into angiotensin I. Angiotensin I is then converted to angiotensin II by Angiotensin Converting Enzyme (ACE). Angiotensin II is a peptide hormone that causes vasoconstriction, which in turn can increase blood pressure. Angiotensin II also stimulates secretion of the hormone aldosterone in the adrenal cortex. Aldosterone causes the kidneys to increase reabsorption of sodium and water, resulting in an increase in the volume of fluid in the body, which in turn may increase blood pressure. Excessive stimulation or activity of the RAAS pathway can lead to high blood pressure. Chronic hypertension is known as hypertension. The high blood pressure of hypertensive patients requires the heart to make more effort to circulate blood through the blood vessels.

Hypertension is the most common, controllable disease in developed countries, affecting 20-50% of the adult population. The World Health Organization (WHO) has identified hypertension as a major cause of cardiovascular morbidity. Hypertension is a major risk factor for a variety of diseases, disorders and conditions, such as shortened life expectancy, chronic kidney disease, stroke, myocardial infarction, heart failure, aneurysms (e.g., aortic aneurysms), peripheral arterial disease, cardiac injury (e.g., heart dilatation or hypertrophy), and other cardiovascular-related diseases, disorders and/or conditions. Furthermore, hypertension has been shown to be an important risk factor for cardiovascular morbidity and mortality, accounting for or constituting 62% of all strokes and 49% of all heart disease cases. In 2017, changes in the guidelines for diagnosis, prevention and treatment of hypertension have occurred, providing a goal for even lower blood pressure to further reduce the risk of developing hypertension-related diseases and disorders.

Antihypertensive drugs, renal sympathetic denervation, baroreceptor activation therapy, dietary changes, and lifestyle changes can reduce hypertension and reduce diseases, disorders, and/or conditions associated with hypertension (Paulis et al, nat Rev Cardiol 2012, 9:276-285). However, current approved therapies for treating hypertension have limitations in that all significant subgroups of hypertensive patients fail to achieve adequate blood pressure control. For example, drugs targeting portions of the renin-angiotensin system (RAS) pathway such as ACE inhibitors and Angiotensin Receptor Blockers (ARBs) limit their ability to inhibit the RAAS pathway (Nobakh et al, nat RevNephrol,2011, 7:356-359). In addition, although the number of antihypertensive drugs for treating hypertension is large, more than two thirds of subjects cannot be controlled with one antihypertensive drug and two or more antihypertensive drugs selected from different drug classes are required. This further reduces the number of subjects with controlled blood pressure, as compliance decreases and side effects increase with increasing dosing. If all three antihypertensive drugs cannot control blood pressure to a normal range, the blood pressure is called refractory hypertension, which is quite common in clinic.

Thus, there is a need to find alternative therapies to inhibit the RAAS pathway and treat hypertension. Antisense technology is becoming an effective means of reducing the expression of certain gene products. However, early antisense oligonucleotides targeting AGT provided limited benefit or were only applicable to AGT in animals (WO 2014018930 A1). The compounds and compositions herein provide novel, highly potent and tolerable compounds that inhibit human AGT, and are suitable for use in human subjects. In addition, the compounds disclosed herein are predicted to alleviate tolerability problems of traditional RAS blockers in patients at risk of hyperkalemia and/or kidney disease by using a conjugate strategy that delivers antisense compounds to the liver and limits their renal distribution and activity.

Disclosure of Invention

The siRNA and the modified sequence thereof provided by the invention can specifically inhibit the expression of the Angiotensinogen (AGT) gene, and the pharmaceutical composition or the siRNA conjugate containing the siRNA can specifically target the liver, so that the expression of the Angiotensinogen (AGT) gene can be inhibited and regulated, and the treatment of diseases and/or symptoms related to hypertension is realized.

In some embodiments, the invention provides a first siRNA capable of inhibiting expression of an Angiotensinogen (AGT) gene, the siRNA comprising a sense strand and an antisense strand, each nucleotide in the siRNA being independently a modified or unmodified nucleotide, wherein the sense strand comprises a stretch of nucleotide sequence I and the antisense strand comprises a stretch of nucleotide sequence II, the nucleotide sequence I and the nucleotide sequence II being at least partially reverse complementary to form a duplex region, wherein the nucleotide sequence I is complementary to SEQ ID NO:1, and not more than 3 nucleotides, and the nucleotide sequence II is identical to the nucleotide sequence set forth in SEQ ID NO:2, and no more than 3 nucleotide differences:

5’-ACUUCUUGGGCUUCUGUAUUU-3’(SEQ ID NO:1)；

5’-AUACGGAAGCCCAAGAAGUUU-3’(SEQ ID NO:2)。

In some embodiments, the invention provides a second siRNA capable of inhibiting expression of an Angiotensinogen (AGT) gene, the siRNA comprising a sense strand and an antisense strand, each nucleotide in the siRNA being independently a modified or unmodified nucleotide, wherein the sense strand comprises a stretch of nucleotide sequence I and the antisense strand comprises a stretch of nucleotide sequence II, the nucleotide sequence I and the nucleotide sequence II being at least partially reverse complementary to form a double-stranded region, wherein the nucleotide sequence I is complementary to SEQ ID NO:3, and not more than 3 nucleotides, and said nucleotide sequence II is identical to SEQ ID NO:4, and no more than 3 nucleotide differences:

5’-CGUUGCUGCUGAGAAGAUUUU-3’(SEQ ID NO:3)；

5’-AAUCUUCUCAGCAGCAACGUU-3’(SEQ ID NO:4)。

in some embodiments, the invention provides a third siRNA capable of inhibiting expression of an Angiotensinogen (AGT) gene, the siRNA comprising a sense strand and an antisense strand, each nucleotide in the siRNA being independently a modified or unmodified nucleotide, wherein the sense strand comprises a stretch of nucleotide sequence I and the antisense strand comprises a stretch of nucleotide sequence II, the nucleotide sequence I and the nucleotide sequence II being at least partially reverse complementary to form a double-stranded region, wherein the nucleotide sequence I is complementary to SEQ ID NO:5, and not more than 3 nucleotides, and the nucleotide sequence II is identical to the nucleotide sequence set forth in SEQ ID NO:6, and no more than 3 nucleotide differences:

5’-GGUGCUGAACAGCAUUUUUUU-3’(SEQ ID NO:5)；

5’-AAAAAUGCUGUUCAGCACCUU-3’(SEQ ID NO:6)。

In some embodiments, the invention provides a fourth siRNA capable of inhibiting expression of an Angiotensinogen (AGT) gene, the siRNA comprising a sense strand and an antisense strand, each nucleotide in the siRNA being independently a modified or unmodified nucleotide, wherein the sense strand comprises a stretch of nucleotide sequence I and the antisense strand comprises a stretch of nucleotide sequence II, the nucleotide sequence I and the nucleotide sequence II being at least partially reverse complementary to form a duplex region, wherein the nucleotide sequence I is complementary to SEQ ID NO:7, and the nucleotide sequence II is identical in length to the nucleotide sequence set forth in SEQ ID NO:8, and no more than 3 nucleotide differences:

5’-AGUCUACCCAACAGCUUAAUU-3’(SEQ ID NO:7)；

5’-UUAAGCUGUUGGGUAGACUUU-3’(SEQ ID NO:8)。

in some embodiments, the invention provides a pharmaceutical composition comprising an siRNA of the invention and a pharmaceutically acceptable carrier.

In some embodiments, the invention provides the use of said siRNA and/or pharmaceutical composition and/or siRNA conjugate in the manufacture of a medicament for the treatment of a disease and/or disorder caused by the expression of the Angiotensinogen (AGT) gene.

In some embodiments, the invention provides a method of treating a disease and/or disorder caused by the expression of the Angiotensinogen (AGT) gene, the method comprising administering to a subject suffering from a hypertension-associated disease and/or disorder an effective amount of an siRNA and/or a pharmaceutical composition and/or an siRNA conjugate of the invention.

In some embodiments, the invention provides a method of inhibiting expression of an Angiotensinogen (AGT) gene, the method comprising contacting an effective amount of an siRNA and/or a pharmaceutical composition and/or an siRNA conjugate of the invention with said liver cells.

In some embodiments, the invention provides a kit comprising an siRNA and/or a pharmaceutical composition and/or an siRNA conjugate of the invention.

Advantageous effects

The siRNA, the pharmaceutical composition and the siRNA conjugate provided by the invention have the advantages of remarkably enhanced stability of plasma and lysosome, higher inhibition activity of angiotensinogen mRNA, lower off-target effect and/or remarkably treating diseases and/or symptoms related to hypertension.

In some embodiments, the siRNA, pharmaceutical compositions, or siRNA conjugates provided herein exhibit excellent target gene inhibition activity in vitro experiments. In some embodiments, the siRNA, pharmaceutical compositions, or siRNA conjugates provided herein exhibit a target gene expression inhibition rate of at least 50%, 60%, 70%, 80%, 90%, or 95% in hepatocytes.

The siRNA, pharmaceutical composition or siRNA conjugate provided by the present invention did not show significant off-target effects. The off-target effect may be, for example, inhibition of normal gene expression of non-target genes. It is believed that the off-target effect is not significant if the binding/inhibition of off-target gene expression is less than 50%, 40%, 30%, 20% or 10% compared to the effect at the target gene.

Therefore, the siRNA, the pharmaceutical composition and the siRNA conjugate provided by the invention can inhibit the expression of the Angiotensinogen (AGT) gene, effectively treat diseases and/or symptoms related to hypertension, and have good application prospect.

Additional features and advantages of the invention will be set forth in the detailed description which follows.

Detailed Description

The following describes specific embodiments of the present invention in detail. It should be understood that the detailed description and specific examples, while indicating and illustrating the invention, are not intended to limit the invention.

In the present invention, AGT mRNA means mRNA having a sequence shown in Genbank accession No. NM-000029.3. Further, unless otherwise indicated, the term "target gene" as used herein refers to a gene that is transcribed up to AGT mRNA, and the term "target mRNA" refers to the AGT mRNA described above.

Definition of the definition

The following terms and phrases used herein are intended to have the following meanings unless otherwise indicated. A particular term or phrase, unless otherwise specifically defined, should not be construed as being ambiguous or otherwise clear, but rather should be construed in a generic sense. When trade names are presented herein, it is intended to refer to their corresponding commercial products or active ingredients thereof.

The term "linked" as used herein, when referring to a link between two molecules, refers to the linking of the two molecules by covalent bonds or the association of the two molecules via non-covalent bonds (e.g., hydrogen or ionic bonds).

An "oligonucleotide" as used herein is a nucleotide sequence containing 10-50 nucleotides or nucleotide base pairs. In some embodiments of the invention, the oligonucleotide has a nucleobase sequence that is at least partially complementary to a coding sequence in a target gene expressed in a cell. The nucleotide may optionally be modified. In some embodiments of the invention, the oligonucleotide is capable of inhibiting or blocking expression of a gene in vitro or in vivo after delivery of the oligonucleotide to a cell expressing the gene.

The term "angiotensinogen", which is used interchangeably with the term "AGT" according to the present invention, refers to a class of peptide substances having potent vasoconstrictor and stimulation of aldosterone secretion by the adrenal cortex, involved in the regulation of blood pressure and body fluids.

Other examples of AGT mRNA sequences are readily available using public databases such as GenBank, uniProt and OMIM. The method can be used for the following steps: international angiotensinogen data store (Intemational Repository for Hepatitis B Virus Strain Data) was accessed on the// www.hpa-bioinformation. Org. Uk/HepSEQ/main. Php.

As used herein, the term "AGT" also refers to naturally occurring DNA sequence variants of the AGT genome (e.g., genotypes a-J and variants thereof).

The term "hypertension related diseases and/or disorders" or "AGT related diseases" as used herein is a disease or disorder caused by or associated with the excessive replication of AGT. The term "AGT-related disease" includes diseases, disorders or conditions that benefit from reduced expression or replication of the AGT gene. Non-limiting examples of AGT related diseases include, for example, hypertension, borderline hypertension, primary hypertension, secondary hypertension, hypertension risk, hypertension emergency status, isolated systolic and diastolic hypertension, pregnancy-related hypertension, diabetic hypertension, intractable hypertension, refractory hypertension, paroxysmal hypertension, renal vascular hypertension, godbla's hypertension, ocular hypertension, glaucoma, pulmonary arterial hypertension, portal hypertension, systemic venous hypertension, systolic hypertension, unstable hypertension

The term "inhibit", as used herein, when expressing a given gene, means that the gene expression is reduced when the cell, cell population or tissue is treated with an siRNA, pharmaceutical composition or siRNA conjugate as described herein, as compared to a cell, cell population or tissue that has not been treated.

The term "inhibit" is used interchangeably with "reduce," "silence," "down-regulate," "inhibit," and other similar terms, and includes any level of inhibition. Preferably, inhibition comprises a statistically significant inhibition or a clinically significant inhibition.

As used herein, the phrase "inhibiting the expression of AGT" or "inhibiting the expression of an AGT gene" includes inhibiting the expression of any AGT gene (e.g., an AGT gene expressed by AGT in AGT, an AGT gene expressed by an expression construct in a cell), as well as variants or mutants of an AGT gene encoding an AGT protein. The term includes the absence of any AGT transcription encoding one or more AGT proteins as well as the knockdown of variants and mutants of the AGT gene.

Each nucleotide in the sense strand and the antisense strand is independently a modified or unmodified nucleotide. In the context of the present invention, unless otherwise indicated, "conjugated" means that two or more chemical moieties each having a specific function are linked to each other by covalent linkage; accordingly, "conjugate" refers to a compound formed by covalent linkage between the chemical moieties. Further, "siRNA conjugate" means a compound formed by covalently attaching one or more chemical moieties having specific functions to an siRNA. Hereinafter, the siRNA conjugate of the present invention is also sometimes simply referred to as "conjugate". siRNA conjugates are to be understood as the generic term of siRNA conjugates, either the first or the second siRNA conjugate, or the siRNA sense strand conjugate or the siRNA antisense strand conjugate, depending on the context.

In some embodiments, the conjugate group may be attached to the phosphate group, the 2 '-hydroxyl group, the 5' -hydroxyl group, or the base of the nucleotide. In some embodiments, the conjugate group may also be attached to the 3' -hydroxyl group, in which case the nucleotides are linked using a 2' -5' phosphodiester linkage. When a conjugate group is attached to the end of the siRNA strand, the conjugate group is typically attached to the phosphate group of the nucleotide; when a conjugate group is attached to the internal sequence of the siRNA, the conjugate group is typically attached to a ribose sugar ring or base. Various connection means can be referred to as: muthiah Manoharan et al, siraconjugates carrying sequentially assembled trivalentN-acetylgalactosamine linked through nucleosides elicit robust gene silencing in vivo in hepatoducts, ACS chemical biology,2015,10 (5): 1181-7.

In some embodiments, the siRNA and the conjugate group may be linked by acid labile, or reducible, chemical bonds that degrade in the acidic environment of the intracellular inclusion bodies, thereby allowing the siRNA to be in a free state. For non-degradable conjugation, the conjugation group can be attached to the sense strand of the siRNA, thereby minimizing the effect of conjugation on siRNA activity.

In the above and below, unless otherwise specified, "G", "C", "a", "T" and "U" each represent a nucleotide containing guanine, cytosine, adenine, thymine and uracil as bases. However, it is understood that the term "ribonucleotide" or "nucleotide" may also refer to modified nucleotides, nucleotide analogs (surrogate replacement moiety), as described in further detail below.

Wherein a, c, g and u are 2 '-O-methyladenosine-3' -phosphate, 2 '-O-methylcytidine-3' -phosphate, 2 '-O-methylguanosine-3' -phosphate and 2 '-O-methyluridine-3' -phosphate, respectively;

af. Cf, gf and Uf are 2 '-fluoroadenosine-3' -phosphate, 2 '-fluorocytidine-3' -phosphate, 2 '-fluoroguanosine-3' -phosphate and 2 '-fluorouridine-3' -phosphate, respectively;

dA. dC, dG and dT are 2 '-deoxyadenosine-3' -phosphate, 2 '-deoxycytidine-3' -phosphate, 2 '-deoxyguanosine-3' -phosphate and 2 '-deoxythymidine-3' -phosphate, respectively;

(Agn) is an adenosine-diol nucleic acid (GNA); and s is a phosphorothioate linkage.

In the context of the present invention, the expressions "complementary" or "reverse complementary" are used interchangeably and have the meaning known to the person skilled in the art, i.e. in a double stranded nucleic acid molecule the bases of one strand are each paired with a base on the other strand in a complementary manner. In DNA, the purine base adenine (a) is always paired with the pyrimidine base thymine (T) (or uracil (U) in RNA); the purine base guanine (C) is always paired with the pyrimidine base cytosine (G). Each base pair includes a purine and a pyrimidine. When adenine on one strand always pairs with thymine (or uracil) on the other strand, and guanine always pairs with cytosine, the two strands are considered complementary to each other, and the sequence of the strand can be deduced from the sequence of its complementary strand. Accordingly, "mismatch" means in the art that bases at corresponding positions do not exist in complementary pairs in a double-stranded nucleic acid.

In the above and in the following, unless otherwise specified, "substantially reverse complementary" means that there are no more than 3 base mismatches between the two nucleotide sequences involved; "substantially reverse complementary" means that there is no more than 1 base mismatch between two nucleotide sequences; "complete reverse complement" means that there is no base mismatch between the two nucleotide sequences. In the above and below, the "nucleotide difference" between one nucleotide sequence and another nucleotide sequence means that the base type of the nucleotide at the same position is changed as compared with the former, for example, when one nucleotide base is A in the latter, when the corresponding nucleotide base at the same position in the former is U, C, G or T, it is determined that there is a nucleotide difference between the two nucleotide sequences at the position. In some embodiments, a nucleotide difference is also considered to occur at an original position when the nucleotide is replaced with an abasic nucleotide or its equivalent.

In the above and in the following, particularly in describing the preparation method of the siRNA, the siRNA-containing pharmaceutical composition or the siRNA conjugate of the invention, unless otherwise specified, the nucleoside monomer (nucleoside monomer) means a modified or unmodified nucleoside phosphoramidite monomer (unmodified ormodifiedRNAphosphoramidites, sometimes also referred to as RNAphosphoramidites Nucleoside phosphoramidites) used in phosphoramidite solid phase synthesis according to the kind and order of nucleotides in the siRNA or siRNA conjugate to be prepared. Phosphoramidite solid phase synthesis is a method well known to those skilled in the art for use in RNA synthesis. Nucleoside monomers useful in the present invention are commercially available.

As used herein, "optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not. For example, "optionally substituted" alkyl "includes" alkyl "and" substituted alkyl "as defined below, as will be understood by those of skill in the art, for any group comprising one or more substituents, such groups are not intended to introduce any substitution or pattern that is sterically impractical, synthetically infeasible, and/or inherently unstable.

The term "subject" as used herein refers to any animal, such as a mammal or a pouched animal. Subjects of the invention include, but are not limited to, humans, non-human primates (e.g., rhesus monkeys or other types of macaques), mice, pigs, horses, cows, rats, or any kind of poultry.

As used herein, "treatment" refers to a method of achieving a beneficial or desired result, including but not limited to therapeutic benefit. By "therapeutic benefit" is meant eradication or amelioration of the underlying disorder being treated. In addition, therapeutic benefit is obtained by eradicating or ameliorating one or more of the physiological symptoms associated with the underlying disorder such that an improvement is observed in the subject, although the subject may still be afflicted with the underlying disorder.

In one aspect, the present invention provides the first to ninth siRNAs capable of inhibiting the expression of AGT gene. This will be described in detail in turn.

The siRNA of the present invention contains a nucleotide group as a basic structural unit, which is well known to those skilled in the art, and the nucleotide group contains a phosphate group, a ribose group and a base, and is not described herein.

First siRNA

According to the present invention, the siRNA may be a first siRNA.

The first siRNA comprises a sense strand and an antisense strand, each nucleotide in the first siRNA is independently a modified or unmodified nucleotide, wherein the sense strand comprises a nucleotide sequence I, the antisense strand comprises a nucleotide sequence II, the nucleotide sequence I and the nucleotide sequence II are at least partially reverse complementary to form a double-stranded region, wherein the nucleotide sequence I is equal in length to the nucleotide sequence shown in SEQ ID NO:1 and is not more than 3 nucleotides different, and the nucleotide sequence II is equal in length to the nucleotide sequence shown in SEQ ID NO:2 and is not more than 3 nucleotides different:

5’-ACUUCUUGGGCUUCUGUAUUU-3’(SEQ ID NO:1)；

5’-AUACGGAAGCCCAAGAAGUUU-3’(SEQ ID NO:2)。

in some embodiments, the sense strand comprises only nucleotide sequence I and the antisense strand comprises only nucleotide sequence II. In some embodiments, the nucleotide sequence I differs from the nucleotide sequence set forth in SEQ ID NO. 1 by NO more than 1 nucleotide, and/or the nucleotide sequence II differs from the nucleotide sequence set forth in SEQ ID NO. 2 by NO more than 1 nucleotide.

In some embodiments, the nucleotide sequence I and the nucleotide sequence II are substantially reverse complementary, or fully reverse complementary; by substantially reverse complement is meant that there are no more than 3 base mismatches between the two nucleotide sequences; by substantially reverse complement is meant that there is no more than 1 base mismatch between the two nucleotide sequences; complete reverse complementarity refers to the absence of a base mismatch between two nucleotide sequences.

Second siRNA

According to the present invention, the siRNA may be a second siRNA.

The second siRNA comprises a sense strand and an antisense strand, each nucleotide in the second siRNA is independently a modified or unmodified nucleotide, wherein the sense strand comprises a nucleotide sequence I and the antisense strand comprises a nucleotide sequence II, which are at least partially reverse complementary to form a double-stranded region, wherein the nucleotide sequence I is equal in length to the nucleotide sequence shown in SEQ ID NO:3 and is not more than 3 nucleotides different, and the nucleotide sequence II is equal in length to the nucleotide sequence shown in SEQ ID NO:4 and is not more than 3 nucleotides different:

5’-CGUUGCUGCUGAGAAGAUUUU-3’(SEQ ID NO:3)；

5’-AAUCUUCUCAGCAGCAACGUU-3’(SEQ ID NO:4)。

In some embodiments, the sense strand comprises only nucleotide sequence I and the antisense strand comprises only nucleotide sequence II. In some embodiments, the nucleotide sequence I differs from the nucleotide sequence set forth in SEQ ID NO. 3 by NO more than 1 nucleotide and/or the nucleotide sequence II differs from the nucleotide sequence set forth in SEQ ID NO. 4 by NO more than 1 nucleotide.

In some embodiments, the nucleotide sequence I and the nucleotide sequence II are substantially reverse complementary, or fully reverse complementary.

Third siRNA

According to the present invention, the siRNA may be a third siRNA.

The third siRNA comprises a sense strand and an antisense strand, each nucleotide in the third siRNA being independently a modified or unmodified nucleotide, wherein the sense strand comprises a stretch of nucleotide sequence I and the antisense strand comprises a stretch of nucleotide sequence II, the nucleotide sequence I and the nucleotide sequence II being at least partially reverse complementary to form a double-stranded region, wherein the nucleotide sequence I is equal in length to the nucleotide sequence set forth in SEQ ID NO:5 and is NO more than 3 nucleotides different, and the nucleotide sequence II is equal in length to the nucleotide sequence set forth in SEQ ID NO:6 and is NO more than 3 nucleotides different:

5’-GGUGCUGAACAGCAUUUUUUU-3’(SEQ ID NO:5)；

5’-AAAAAUGCUGUUCAGCACCUU-3’(SEQ ID NO:6)。

In some embodiments, the sense strand comprises only nucleotide sequence I and the antisense strand comprises only nucleotide sequence II. In some embodiments, the nucleotide sequence I differs from the nucleotide sequence set forth in SEQ ID NO. 5 by NO more than 1 nucleotide and/or the nucleotide sequence II differs from the nucleotide sequence set forth in SEQ ID NO. 6 by NO more than 1 nucleotide.

Fourth siRNA

According to the present invention, the siRNA may be a fourth siRNA.

The fourth siRNA comprises a sense strand and an antisense strand, each nucleotide in the fourth siRNA is independently a modified or unmodified nucleotide, wherein the sense strand comprises a nucleotide sequence I and the antisense strand comprises a nucleotide sequence II, which are at least partially reverse complementary to form a double-stranded region, wherein the nucleotide sequence I is equal in length to the nucleotide sequence shown in SEQ ID NO:7 and is not more than 3 nucleotides different, and the nucleotide sequence II is equal in length to the nucleotide sequence shown in SEQ ID NO:8 and is not more than 3 nucleotides different:

5’-AGUCUACCCAACAGCUUAAUU-3’(SEQ ID NO:7)；

5’-UUAAGCUGUUGGGUAGACUUU-3’(SEQ ID NO:8)。

In some embodiments, the sense strand comprises only nucleotide sequence I and the antisense strand comprises only nucleotide sequence II. In some embodiments, the nucleotide sequence I differs from the nucleotide sequence set forth in SEQ ID NO. 7 by NO more than 1 nucleotide, and/or the nucleotide sequence II differs from the nucleotide sequence set forth in SEQ ID NO. 8 by NO more than 1 nucleotide.

As previously mentioned, the nucleotides in the sirnas disclosed herein are each independently modified or unmodified nucleotides. In some embodiments, each nucleotide in the siRNA of the invention is an unmodified nucleotide. In some embodiments, some or all of the nucleotides in the siRNA of the invention are modified nucleotides, and such modifications on the nucleotide groups do not result in a significant impairment or loss of the function of the siRNA conjugates of the invention to inhibit AGT gene expression.

In some embodiments, the siRNA of the invention may be any of the unmodified sirnas listed in table 1.

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The siRNA provided by the invention not only has obviously enhanced stability of plasma and lysosomes, but also has very high target mRNA inhibition activity.

The siRNA provided by the present invention may be obtained by methods of siRNA preparation conventional in the art (e.g., solid phase synthesis). Among them, solid phase synthesis is already commercially available as a custom service. Methods of preparing nucleoside monomers having corresponding modifications and methods of introducing modified nucleotide groups into siRNA can also be known to those of skill in the art by introducing modified nucleotide groups into siRNA described herein using nucleoside monomers having corresponding modifications.

siRNA conjugates

The present invention provides an siRNA conjugate comprising the above siRNA and a conjugate group conjugated to the siRNA.

In general, the conjugate group comprises at least one pharmaceutically acceptable targeting group and optionally a linker (linker), and the siRNA, the linker and the targeting group are sequentially linked. In some embodiments, the targeting group is 2-4. The siRNA molecule may be non-covalently or covalently conjugated to the conjugate group, e.g., may be covalently conjugated to the conjugate group. The conjugation site of the siRNA to the conjugation group may be at the 3 'or 5' end of the sense strand of the siRNA, or may be in the internal sequence of the siRNA. In some embodiments, the conjugation site of the siRNA to the conjugation group is at the 3 'end or the 5' end of the sense strand of the siRNA.

In some embodiments, the conjugate group may be attached to the phosphate group, the 2' -hydroxyl group, or the base of the nucleotide. In some embodiments, the conjugate group may also be attached to the 3' -hydroxyl group, in which case the nucleotides are linked using a 2' -5' phosphodiester linkage. When a conjugate group is attached to the end of the siRNA strand, the conjugate group is typically attached to the phosphate group of the nucleotide; when a conjugate group is attached to the internal sequence of the siRNA, the conjugate group is typically attached to a ribose sugar ring or base. Various connection means can be referred to as: muthiah Manoharan et al, siRNA conjugates carrying sequentially assembled trivalent N-acetylgalactosamine linked through nucleosides elicit robust gene silencing in vivo in hepatocytocytocytosis, ACS Chemical biology,2015,10 (5): 1181-7.

In some embodiments, the siRNA and the conjugate group may be linked by acid labile or reducible chemical bonds that degrade in the acidic environment of the intracellular inclusion bodies, thereby allowing the siRNA to be in a free state. For non-degradable conjugation, the conjugation group can be attached to the sense strand of the siRNA, thereby minimizing the effect of conjugation on siRNA activity.

In some embodiments, the pharmaceutically acceptable targeting group can be a ligand conventionally used in the art of siRNA administration, such as the various ligands described in W02009082607A2, the entire disclosure of which is incorporated herein by reference.

In some embodiments, the targeting group comprises an asialoglycoprotein receptor ligand. In some embodiments, the asialoglycoprotein receptor ligand comprises or consists of one or more galactose derivatives. As used herein, the term "galactose derivative" includes galactose and lactose derivatives having an affinity for the asialoglycoprotein receptor equal to or greater than that of galactose. Galactose derivatives include, but are not limited to: galactose, galactosamine, N-formylgalactosamine, N-acetylgalactosamine, N-propionyl-galactosamine, N-N-butyryl-galactosamine and N-isobutyryl galactosamine (see, e.g., iobst, S.T. and Drickamer, K.J.B.C.1996, vol 271, page 6686). Galactose derivatives and galactose derivative clusters that can be used for targeting oligonucleotides and other molecules to the liver in vivo are known in the art (see, e.g., baenziger and Fiete,1980, cell,22,611-620;Connolly et al, 1982, j. Biol. Chem.,257, 939-945). Galactose derivatives have been used to target molecules to hepatocytes in vivo through their binding to asialoglycoprotein receptors (ASGPr) expressed on the surface of hepatocytes. Binding of ASGPr ligands to ASGPr(s) facilitates cell-specific targeting of hepatocytes and entry of endocytic molecules into hepatocytes. The ASGPr ligand may be a monomer (e.g., having a single galactose derivative) or a multimer (e.g., having multiple galactose derivatives). Galactose derivatives or galactose derivative clusters can be attached to the 3 'or 5' end of the siRNA using methods known in the art.

In some embodiments, the pharmaceutically acceptable targeting group in the siRNA conjugate may be galactose or N-acetylgalactosamine, wherein the galactose or N-acetylgalactosamine molecule may be monovalent, divalent, trivalent, tetravalent. It should be understood that the monovalent, divalent, trivalent, tetravalent means that after the siRNA molecule forms an siRNA conjugate with a conjugate group containing galactose or N-acetylgalactosamine molecules as a targeting group, the molar ratio of siRNA molecule to galactose or N-acetylgalactosamine molecules in the siRNA conjugate is 1:1, 1:2, 1:3, or 1:4, respectively. In some embodiments, the pharmaceutically acceptable targeting group is N-acetylgalactosamine. In some embodiments, when the siRNA of the invention is conjugated to a conjugate group comprising N-acetylgalactosamine, the N-acetylgalactosamine molecule is trivalent or tetravalent. In some embodiments, the N-acetylgalactosamine molecule is trivalent when the siRNA of the invention is conjugated to a conjugate group comprising N-acetylgalactosamine.

The targeting group can be attached to the siRNA molecule via a suitable linker, which can be selected by one skilled in the art depending on the particular type of targeting group. The types of these linkers, targeting groups, and the manner of attachment to the siRNA can be found in the disclosure of W02015006740A2, which is incorporated herein by reference in its entirety.

In some embodiments, when the targeting group is N-acetylgalactosamine, a suitable linker may be of the structure shown in formula (19):

wherein m is an integer of 1 to 3;

L ^A is a chain-like moiety comprising an amide bond having a structure represented by formula (20), each of the L ^A At both ends thereof with one of said targeting group and said L ^C Part is connected by ether linkage:

L ^B is a N-acyl pyrrolidine-containing chain moiety having a structure represented by the formula (21), the chain moiety having a carbonyl group at one end thereof and being bonded to the L ^C Part is linked by an amide bond, has an oxygen atom at the other end and is linked to the siRNA by a phosphate bond:

L ^C is a 2-4 valent linking group based on hydroxymethyl aminomethane, dimethylol aminomethane or trimethylol aminomethane, said L ^C Via an oxygen atom with each of said L ^A Part is linked by an ether linkage and is bound to the L via a nitrogen atom ^B The moieties are linked by amide linkages.

In some embodiments, the linker is- (L) ^A ) ₃ Trimethylolaminomethane-L ^B -an siRNA conjugate formed by linking an N-acetylgalactosamine molecule and an siRNA molecule, having the structure shown in formula (22) below:

in the formula, the double helix structure represents siRNA.

Also, the conjugation site of the siRNA to the conjugation group may be at the 3 'end or 5' end of the sense strand of the siRNA, or may be in the internal sequence of the siRNA.

In some embodiments, the 3 '-end of the sense strand of the siRNA of the present invention is linked to the 3' -end of the sense strand via a linker- (L) ^A ) ₃ Trimethylolaminomethane-L ^B Covalent conjugation with three N-acetylgalactosamine (GalNAc) molecules, resulting in siRNA conjugates with a molar ratio of siRNA molecules to GalNAc molecules of 1:3, which may also be referred to as (GalNAc) hereinafter ₃ -siRNA having the structure shown in formula (23):

wherein the duplex structure represents the siRNA and the linker is attached to the 3' end of the sense strand of the siRNA.

In some embodiments, the linker is attached to the 5' end of the sense strand of the siRNA.

In some embodiments, the siRNA conjugates have a structure as shown in formula (24), (25) or (26)

Wherein,

R ₂ a group having a structure represented by the formula (S1):

wherein E is ₁ OH or SH, nu is the siRNA of the invention;

R ₁ is a linear or cyclic alkylene group of 1 to 20 carbon atoms in length, wherein one or more carbon atoms are optionally replaced by any one or more selected from the group consisting of: c (O), NH, O, S, CH = N, S (O) ₂ 、C ₂ -C ₁₀ Alkenylene, C ₂ -C ₁₀ Alkynylene, C ₆ -C ₁₀ Arylene group, C ₃ -C ₁₈ Heterocyclylene and C ₅ -C ₁₀ Heteroarylene; and wherein R is ₁ Optionally having substituents of any one or more of the group consisting of: c (C) ₁ -C ₁₀ Alkyl, C ₆ -C ₁₀ Aryl, C ₅ -C ₁₀ Heteroaryl, C ₁ -C ₁₀ Haloalkyl, -OC ₁ -C ₁₀ Alkyl, -OC ₁ -C ₁₀ Alkylphenyl radicals C ₁ -C ₁₀ alkyl-OH, -OC ₁ -C ₁₀ Haloalkyl, -SC ₁ -C ₁₀ Alkyl, -SC ₁ -C ₁₀ Alkylphenyl radicals C ₁ -C ₁₀ alkyl-SH, -SC ₁ -C ₁₀ Haloalkyl, halogen substituent, -OH, -SH, -NH ₂ 、-C ₁ -C ₁₀ alkyl-NH ₂ 、-N(C ₁ -C ₁₀ ) Alkyl (C) ₁ -C ₁₀ Alkyl), -NH (C) ₁ -C ₁₀ Alkyl), -N (C) ₁ -C ₁₀ Alkyl) (C) ₁ -C ₁₀ Alkylphenyl), -NH (C) ₁ -C ₁₀ ) Alkylphenyl), cyano, -CO ₂ H、C(O)O(C ₁ -C ₁₀ ) Alkyl), -CON (C) ₁ -C ₁₀ ) Alkyl (C) ₁ -C ₁₀ Alkyl), -CONH (C) ₁ -C ₁₀ ) Alkyl, -CONH ₂ 、-NH C(O)(C ₁ -C ₁₀ ) Alkyl), -NHC (O) (phenyl), -N (C) ₁ -C ₁₀ ) Alkyl, -N (C) ₁ -C ₁₀ Alkyl) C (O) (C ₁ -C ₁₀ ) Alkyl), -N (C) ₁ -C ₁₀ ) Alkyl) C (O) (phenyl), -C (O) C ₁ -C ₁₀ Alkyl, -C (O) C ₁ -C ₁₀ Alkylphenyl, -C (O) C ₁ -C ₁₀ Haloalkyl, -OC (O) C ₁ -C ₁₀ Alkyl, -SO ₂ (C ₁ -C ₁₀ ) Alkyl, -SO ₂ (phenyl) -SO ₂ (C ₁ -C ₁₀ ) Haloalkyl) -SO ₂ NH ₂ ,-SO ₂ NH(C ₁ -C ₁₀ Alkyl), -SO ₂ NH (phenyl) -NHSO ₂ (C ₁ -C ₁₀ ) Alkyl), -NHSO ₂ (phenyl) and-NHSO ₂ (C ₁ -C) haloalkyl);

each L ₁ Independently is a linear alkylene group of 1 to 40 carbon atoms in length, wherein one or more carbon atoms are optionally replaced by any one or more selected from the group consisting of: c (O), NH, O, S, CH = N, S (O) ₂ 、C ₂ -C ₁₀ Alkenylene, C ₂ -C ₁₀ Alkynylene, C ₆ -C ₁₀ Arylene group, C ₃ -C ₁₈ Heterocyclylene and C ₅ -C ₁₀ Heteroarylene; and wherein R is ₁ Optionally having substituents of any one or more of the group consisting of: c (C) ₁ -C ₁₀ Alkyl, C ₆ -C ₁₀ Aryl, C ₅ -C ₁₀ Heteroaryl, C ₁ -C ₁₀ Haloalkyl, -OC ₁ -C ₁₀ Alkyl, -OC ₁ -C ₁₀ Alkylphenyl radicals C ₁ -C ₁₀ alkyl-OH, -OC ₁ -C ₁₀ Haloalkyl, -SC ₁ -C ₁₀ Alkyl, -SC ₁ -C ₁₀ Alkylphenyl radicals C ₁ -C ₁₀ alkyl-SH, -SC ₁ -C ₁₀ Haloalkyl, halogen substituent, -OH, -SH, -NH ₂ 、-C ₁ -C ₁₀ alkyl-NH ₂ 、-N(C ₁ -C ₁₀ ) Alkyl (C) ₁ -C ₁₀ Alkyl), -NH (C) ₁ -C ₁₀ Alkyl), -N (C) ₁ -C ₁₀ Alkyl) (C) ₁ -C ₁₀ Alkylphenyl), -NH (C) ₁ -C ₁₀ ) Alkylphenyl), cyano, -CO ₂ H、C(O)O(C ₁ -C ₁₀ ) Alkyl), -CON (C) ₁ -C ₁₀ ) Alkyl (C) ₁ -C ₁₀ Alkyl), -CONH (C) ₁ -C ₁₀ ) Alkyl, -CONH ₂ 、-NH C(O)(C ₁ -C ₁₀ ) Alkyl), -NHC (O) (phenyl), -N (C) ₁ -C ₁₀ ) Alkyl, -N (C) ₁ -C ₁₀ Alkyl) C (O) (C ₁ -C ₁₀ ) Alkyl), -N (C) ₁ -C ₁₀ ) Alkyl) C (O) (phenyl), -C (O) C ₁ -C ₁₀ Alkyl, -C (O) C ₁ -C ₁₀ Alkylphenyl, -C (O) C ₁ -C ₁₀ Haloalkyl, -OC (O) C ₁ -C ₁₀ Alkyl group、-SO ₂ (C ₁ -C ₁₀ ) Alkyl, -SO ₂ (phenyl) -SO ₂ (C ₁ -C ₁₀ ) Haloalkyl) -SO ₂ NH ₂ ,-SO ₂ NH(C ₁ -C ₁₀ Alkyl), -SO ₂ NH (phenyl) -NHSO ₂ (C ₁ -C ₁₀ ) Alkyl), -NHSO ₂ (phenyl) and-NHSO ₂ (C ₁ -C ₁₀ ) A haloalkyl group);

in some embodiments, L ₁ May be selected from the group consisting of A1-a14 groups or any linked combination thereof, wherein the structures and definitions of A1-a14 are as follows:

Wherein each k1 is independently an integer from 1 to 20;

each k2 is independently an integer from 1 to 20;

each R _c Independently C ₁ -C ₁₀ An alkyl group;

each R _d Selected from the group consisting of a15-a19 and any combination thereof:

each R _e Independently C ₁ -C ₁₀ An alkyl group;indicating the site of covalent attachment of the group.

The skilled artisan will appreciate that L, although for convenience ₁ Is defined as a linear alkylene group, but it may not be a linear group or be named differently, such as an amine or alkenyl group resulting from the substitution and/or substitution described above. For the purposes of the present disclosure, L ₁ Is the number of atoms in the chain connecting the two points of attachment. For this purpose, the ring obtained by substitution of the carbon atoms of the linear alkylene groupSuch as heterocyclylene or heteroarylene) is an atom.

M ₁ Represents a targeting group, the definition and optional scope of which are the same as the targeting groups described above. In some embodiments, each M ₁ Independently selected from one of the ligands having an affinity for asialoglycoprotein receptors on the surface of mammalian liver cells.

R ₂ A group of the structure represented by the formula (S1), wherein E ₁ OH or SH.

R ₁ Is selected to achieve a linkage to S1 from the N atom on the nitrogen-containing backbone. R is R ₁ Any linking group capable of linking the S1 group to the N atom on the nitrogen-containing backbone in a suitable manner. In some embodiments, in the case of preparing the siRNA conjugates represented by formulas (24), (25) or (26) by a process of solid phase synthesis, R ₁ The group needs to contain both a linking site to the N atom on the nitrogen-containing skeleton and R ₂ A junction site to which the P atom of (C) is attached. In some embodiments, R ₁ Wherein the site bonded to the N atom on the nitrogen-containing skeleton forms an amide bond with the N atom, the site bonded to R ₂ The P atom-attached site forms a phosphate bond with the P atom.

In some embodiments, the siRNA conjugates have a structure as shown by the formula (Z1-Nu), (Z2-Nu), (Z3-Nu), (Z4-Nu), (Z5-Nu), (Z6-Nu), (Z7-Nu), (Z8-Nu), (Z9-Nu), (Z10-Nu), (Z11-Nu), (Z12-Nu), (Z13-Nu), (Z14-Nu), (Z15-Nu), (Z16-Nu), (Z17-Nu), (Z18-Nu), (Z19-Nu), (Z20-Nu), (Z21-Nu), (Z22-Nu), (Z23-Nu), (Z24-Nu), (Z25-Nu), (Z26-Nu), (Z27-Nu), (Z28-Nu), (Z29-Nu), (Z30-Nu), (Z31-Nu), (Z32-Nu), wherein Z1-Z32 is a conjugated group.

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In some embodiments, the P atom in formula S1 can be attached to any possible position in the siRNA sequence, e.g., the P atom in formula S1 can be attached to any one of the nucleotides of the sense strand or the antisense strand of the siRNA; in some embodiments, the P atom in formula S1 is attached to any one nucleotide of the sense strand of the siRNA. In some embodiments, the P atom in formula S1 is attached to the end of the sense strand or the antisense strand of the siRNA; in some embodiments, the P atom in formula S1 is attached to the 3' end of the sense strand of the siRNA. In the case of the above-described position of the sense strand linked to the siRNA, the siRNA conjugate shown in (24), (25) or (26) can release the separate antisense strand of siRNA upon unwinding to block the process of translation of protein by AGT mRNA and inhibit the expression of AGT gene after entering the cell.

In some embodiments, the P atom in formula S1 can be attached to any possible position on the nucleotide in the siRNA, for example, the 5' position of the nucleotide, the 2' position of the nucleotide, the 3' position of the nucleotide, or the base of the nucleotide. In some embodiments, the P atom in formula S1 can be linked to the 2', 3', or 5' position of a nucleotide in the siRNA by formation of a phosphodiester linkage. In some embodiments, the P atom in formula S1 is attached to an oxygen atom formed after dehydrogenation of the 3' hydroxyl group of the 3' terminal nucleotide of the siRNA sense strand (in this case, the P atom in S1 can also be considered as a P atom in a phosphate group contained in the siRNA), or the P atom in formula S1 is attached to the nucleotide by replacing hydrogen in the 2' -hydroxyl group of one nucleotide in the siRNA sense strand, or the P atom in formula S1 is attached to the nucleotide by replacing hydrogen in the 5' hydroxyl group of the 5' terminal nucleotide of the siRNA sense strand.

The siRNA conjugate of the invention has remarkably improved stability in blood plasma, low off-target effect and higher AGT mRNA silencing activity. In some embodiments, the siRNA of the invention may be any one of the sirnas shown in table 1 or table 3. siRNA conjugates containing these sirnas exhibited higher AGT mRNA silencing activity.

In the siRNA or siRNA conjugate, each adjacent nucleotide is connected by a phosphodiester bond or a phosphorothioate bond, the non-bridging oxygen atom or sulfur atom in the phosphodiester bond or the phosphorothioate bond carries negative charge, the siRNA or siRNA conjugate can exist in a hydroxyl or sulfhydryl form, and hydrogen ions in the hydroxyl or sulfhydryl can be partially or completely replaced by cations. The cation may be any cation, such as a metal cation, ammonium ion NH4 ⁺ One of organic ammonium cations. In one embodiment, the cation is selected from one or more of an alkali metal ion, a tertiary amine-forming ammonium cation, and a quaternary ammonium cation for improved solubility. The alkali metal ion may be and/or Na+, and the cation formed by the tertiary amine may be an ammonium ion formed by triethylamine and/or an ammonium ion formed by N, N-diisopropylethylamine. Thus, the siRNA or siRNA conjugate of the invention may be at least partially present in salt form. In one mode, the non-bridging oxygen or sulfur atom of the phosphodiester or phosphorothioate linkage is at least partially bound to sodium ionsIn addition, the siRNA or siRNA conjugates of the present invention exist in the form of sodium salt or partial sodium salt.

It is known to those skilled in the art that modified nucleotide groups can be introduced into the siRNA of the present invention by using nucleoside monomers having corresponding modifications. Methods of preparing nucleoside monomers with corresponding modifications and methods of introducing modified nucleotide groups into siRNA are also well known to those of skill in the art. All modified nucleoside monomers are commercially available or can be prepared using known methods.

Preparation of siRNA conjugates represented by the formulas (24), (25) or (26)

The siRNA conjugates of formulas (24), (25) or (26) may be prepared using any reasonable synthetic route.

In some embodiments, the siRNA conjugates represented by formulas (24), (25) or (26) can be prepared by a method comprising sequentially ligating nucleoside monomers in a 3 'to 5' direction under conditions of phosphoramidite solid phase synthesis according to the nucleotide species and sequence of the sense strand and the antisense strand of the siRNA, respectively, the ligating of each nucleoside monomer comprising a deprotection, coupling, capping, oxidation or sulfidation four-step reaction; separating a sense strand and an antisense strand of the siRNA, and annealing, wherein the siRNA is the siRNA of the invention; and, the method further comprises contacting the compound represented by formula (27), (28) or (29) with a nucleoside monomer or a nucleotide sequence attached to a solid support in the presence of a coupling reagent under coupling reaction conditions, so that the compound represented by formula (27), (28) or (29) is attached to the nucleotide sequence by coupling reaction. Hereinafter, the compound represented by the formula (27), (28) or (29) is also referred to as a conjugate molecule.

Wherein:

R ₃ is a group capable of binding to siRNA represented by Nu in the compound represented by formula (24), (25) or (26). In some embodiments, R3 is a group capable of binding to an siRNA represented by Nu via a covalent bond. In some implementationsIn embodiments, R ₃ A group that is any functional group capable of being conjugated to siRNA represented by Nu through a phosphodiester bond by reaction;

each T ₁ Independently is a group formed by substitution of all active hydroxyl groups in M1 with YCOO-groups, wherein each Y is independently selected from one of methyl, trifluoromethyl, difluoromethyl, monofluoromethyl, trichloromethyl, dichloromethyl, monochloromethyl, ethyl, n-propyl, isopropyl, phenyl, halophenyl and alkylphenyl; in some embodiments, Y is methyl. L (L) ₁ The definition and optional scope of (a) is as described above.

R ₃ Is selected to achieve attachment to the N atom on the nitrogen-containing backbone and to provide a suitable reaction site for synthesizing siRNA conjugates represented by formulas (24), (25) or (26). In some embodiments, R ₃ Includes R ₁ Linking group or protected R ₁ A linking group, and a functional group that can react with the siRNA to form a structure shown as S1.

In some embodiments, R ₃ Comprising the 1 st functional group which can form a phosphite with a group on a siRNA or nucleoside monomer represented by Nu, the 2 nd functional group which can react with a hydroxyl group or an amino group to form a covalent bond, or a solid support linked by the covalent bond. In some embodiments, the 1 st functional group is a phosphoramidite, a hydroxyl group, or a protected hydroxyl group. In some embodiments, the 2 nd functional group is a phosphoramidite, a carboxyl group, or a carboxylate. In some embodiments, the 2 nd functional group is a solid support attached to the rest of the molecule via a covalent bond formed by a hydroxyl or amino group. In some embodiments, the solid support is linked via a phosphate bond, a carboxylate bond, or an amide bond. In some embodiments, the solid support is a resin.

In some embodiments, the 1 st functional group contains a hydroxyl group, -ORm, or a group of formula (C3); the 2 nd functional group contains a structure represented by formula (C1), (C2), (C3), (C1 ') or (C3'):

wherein q1 is an integer of 1 to 4, X is O or NH, M ⁺ Is a cation, R _m Is a hydroxyl protecting group, SPS represents a solid support,indicating the site at which the group attaches to the covalent moiety.

In some embodiments, the 1 st functional group contains a phosphoramidite group, as shown in formula (C3), which can undergo a coupling reaction with a hydroxyl group at any position on the nucleotide, such as a 2' -hydroxyl group, a 3' -hydroxyl group, or a 5' -hydroxyl group, to form a phosphite, and oxidized or sulfided to form a phosphodiacetyl bond or phosphorothioate bond shown in formula S1, to conjugate the conjugated molecule to the siRNA. At this time, even if the 2 nd functional group is not present, the compound represented by formula (27), (28) or (29) can be conjugated to a nucleotide without affecting the obtaining of the siRNA conjugate represented by formula (24), (25) or (26). In this case, after obtaining the sense strand or antisense strand of the siRNA via a phosphoramidite solid phase synthesis or the like, the compound represented by formula (27), (28) or (29) is reacted with a hydroxyl group on a terminal nucleotide in the nucleotide sequence, and a phosphodiester linkage or phosphorothioate linkage is formed in a subsequent oxidation or vulcanization process, and the compound represented by formula (27), (28) or (29) is conjugated to the siRNA.

In some embodiments, the 1 st functional group contains a protected hydroxyl group. In some embodiments, the 2 nd functional group comprises a group that is reactive with the solid support, the reaction providing a conjugated molecule comprising the solid support. In some embodiments, the 2 nd functional group contains a carboxyl group, carboxylate, or phosphoramidite, as shown in formula (C1), (C2), or (C3), and when the 2 nd functional group contains a carboxyl group or carboxylate, the compound of formula (27), (28), or (29) undergoes an esterification reaction or amidation reaction with a solid support, such as a hydroxyl group or an amino group on a resin, to form a conjugate molecule comprising a solid support linked via a carboxylic acid ester linkage. When the 2 nd functional group comprises a phosphoramidite functional group, the compound of formula (27), (28) or (29) is coupled to a general solid support, such as a hydroxyl group on a resin, and oxidized to form a conjugated molecule comprising a solid support linked via a phosphodiester linkage. Subsequently, the above-mentioned product after the solid phase carrier is attached is used as an initial, and nucleoside monomers are sequentially attached according to a phosphoramidite solid phase synthesis method, so as to obtain the sense strand or antisense strand of the siRNA with the attached conjugate group. During the solid phase synthesis of phosphoramidite, the 1 st functional group is deprotected and then coupled to the phosphoramidite group on the nucleoside monomer under coupling reaction conditions.

In some embodiments, the 1 st functional group contains a hydroxyl group or a protected hydroxyl group; the 2 nd functional group contains a solid phase carrier connected by a carboxylic ester bond or a solid phase carrier connected by an amide bond or a solid phase carrier connected by a phosphoric ester bond, as shown in formula (C1 ') or (C3'). At this time, nucleoside monomers were sequentially linked by phosphoramidite solid phase synthesis starting from the compounds represented by the formulas (27), (28) and (29) instead of the solid phase carrier, to obtain the sense strand or antisense strand of the siRNA to which the conjugate group was linked.

In some embodiments, each T ₁ Independently M ₁ . In some embodiments, each S ₁ M is independently ₁ At least one active hydroxyl group of the polymer is protected by a hydroxyl protecting group. In some embodiments, the protected hydroxyl group may be represented by the formula YCOO-, wherein each Y is independently selected from one of methyl, trifluoromethyl, difluoromethyl, monofluoromethyl, trichloromethyl, dichloromethyl, monochloromethyl, ethyl, n-propyl, isopropyl, phenyl, halophenyl, and alkylphenyl; in some embodiments, Y is methyl.

In some embodiments, R _m Is one or more of MMTr (4-methoxytrityl), DMTr (4, 4 '-dimethoxytrityl) and TMTr (4, 4' -trimethoxytrityl). In some embodiments, R _m May be DMTr, 4'-dimethoxytrityl (4, 4' -dimethoxytrityl).

Accordingly, unless otherwise indicated, in the following description relating to the preparation of conjugates and/or conjugate molecules, when reference is made to "deprotection," "coupling," "capping," "oxidation," "sulfidation," etc. reactions, it is to be understood that the reaction conditions and reagents involved in solid phase synthesis of phosphoramidite nucleic acids, which are well known in the art, are equally applicable to these reactions. Exemplary reaction conditions and reagents will be described in detail later.

As described above, the preparation method of the siRNA conjugate represented by formula (24), (25) or (26) further comprises the steps of: the other strand of the siRNA is synthesized (e.g., when the steps described above synthesize the sense strand of the siRNA to which the conjugate molecule is attached, also include synthesizing the antisense strand of the siRNA according to a solid phase synthesis method, and vice versa), separating the sense strand and the antisense strand, and annealing. Specifically, in the separation step, the solid phase carrier linked to the nucleotide sequence and/or the conjugate molecule is cleaved, while the necessary protecting groups are removed (at this time, each S1 group in the compound represented by formula (27), (28) or (29) is converted into a corresponding M1 targeting group), and the siRNA sense strand (or antisense strand) and the corresponding antisense strand (or sense strand) linked to the conjugate molecule are obtained, and the sense strand and the antisense strand are annealed to form a double-stranded RNA structure, thereby obtaining the siRNA conjugate represented by formula (24), (25) or (26).

In some embodiments, the method of preparing the siRNA conjugate of formula (24), (25) or (26) comprises the steps of: contacting a compound shown in a formula (27), (28) or (29) with a first nucleoside monomer at the 3' end of a sense strand or an antisense strand under coupling reaction conditions and in the presence of a coupling reagent, connecting the first nucleotide in the sequence to the compound shown in the formula (27), (28) or (29), and sequentially connecting the nucleoside monomers in the 3' to 5' direction under the condition of phosphoramidite solid phase synthesis according to the expected sense strand or antisense strand nucleotide species and sequence to synthesize the sense strand or antisense strand of the siRNA; wherein the compound shown in the formula (27), (28) or (29) is a compound in which R2 contains a 1 st functional group and a 2 nd functional group, the 1 st functional group contains a protected hydroxyl group, and the 2 nd functional group has a structure shown as a formula (C1 ') or (C3'), and the compound shown in the formula (27), (28) or (29) is deprotected before being connected with the first nucleoside monomer; the connection of each nucleoside monomer comprises four steps of deprotection, coupling, capping, oxidation or vulcanization reaction; obtaining a sense strand or an antisense strand of the nucleic acid to which the conjugate group is attached; under the condition of the solid-phase synthesis of the bony amide, sequentially connecting nucleoside monomers according to the nucleotide types and sequences of the antisense strand or the sense strand and the direction from 3 'to 5', and synthesizing the antisense strand or the sense strand of the nucleic acid; the connection of each nucleoside monomer comprises four steps of deprotection, coupling, capping, oxidation or vulcanization reaction; removing protecting group, cutting with solid phase carrier, separating and purifying to obtain sense strand and antisense strand, and annealing.

In some embodiments, the method of preparing the siRNA conjugate of formula (24), (25) or (26) comprises the steps of: sequentially connecting nucleoside monomers according to the nucleotide types and sequences of a sense strand or an antisense strand in the double-stranded siRNA and the direction from 3 'to 5' to synthesize the sense strand and the antisense strand, wherein the connection of each nucleoside monomer comprises four steps of deprotection, coupling, capping, oxidation or sulfuration reaction to obtain the sense strand connected to a solid carrier and the antisense strand connected to the solid carrier; contacting a compound represented by the formula (27), (28) or (29) with a sense strand attached to a solid support or an antisense strand attached to a solid support under coupling reaction conditions and in the presence of a coupling reagent to attach the compound represented by the formula (27), (28) or (29) to the sense strand or the antisense strand, wherein the compound represented by the formula (27), (28) or (29) is R ₃ The compound contains a 1 st functional group, wherein the 1 st functional group is a phosphoramidite group and is shown in the formula (27), (28) or (29); removing protecting groups, cutting with a solid phase carrier, separating and purifying to obtain a sense strand or an antisense strand of the siRNA, and annealing, wherein the sense strand or the antisense strand of the siRNA is connected with a conjugation group.

In some embodiments, the P atom in formula S1 is attached to the 3' end of the sense strand in the siRNA, and the method of preparing the siRNA conjugate of formula (24), (25) or (26) comprises:

(1) Removing the compound shown in the formula (27), (28) OR (29) (wherein the compound shown in the formula (27), (28) OR (29) is R3 containing the 1 st functional group and the 2 nd functional group, and the 1 st functional group contains the protected hydroxyl OR) _m A hydroxyl protecting group R in a compound having a structure as shown in formula (C1 ') or (C3') as the 2 nd functional group _m The method comprises the steps of carrying out a first treatment on the surface of the At the position ofContacting the deprotected product with a nucleoside monomer under coupling reaction conditions and in the presence of a coupling reagent to obtain a nucleoside monomer linked to a solid support via a conjugate molecule;

(2) Synthesizing the sense strand of the siRNA by a phosphoramidite solid phase synthesis method according to the 3'-5' direction starting from the nucleoside monomer attached to the solid phase carrier through the conjugate molecule;

(3) Synthesizing antisense strand of siRNA through phosphoramidite solid phase synthesis method;

(4) The sense strand and the antisense strand of the siRNA are separated and annealed to obtain the siRNA conjugate represented by formula (24), (25) or (26).

After obtaining the conjugate, in some embodiments, the synthesized siRNA conjugate of formula (24), (25) or (26) may also be characterized by means of molecular weight detection, etc., using a method such as liquid chromatography, etc., to determine that the synthesized siRNA conjugate is the target designed siRNA conjugate of formula (24), (25) or (26), and that the sequence of the synthesized siRNA is the sequence of the desired siRNA.

In some embodiments, the solid support is a solid support known in the art to be useful in solid phase synthesis of nucleic acids.

Pharmaceutical composition

The invention also includes pharmaceutical compositions and formulations comprising the siRNA conjugates of the invention. In some embodiments, provided herein are pharmaceutical compositions comprising an siRNA conjugate as described herein and a pharmaceutically acceptable carrier. Pharmaceutical compositions comprising the siRNA conjugates are useful for treating diseases or disorders associated with the expression or activity of AGT genes. Such pharmaceutical compositions are formulated based on the mode of delivery. One example is a composition formulated for systemic administration by parenteral delivery, such as by Subcutaneous (SC), intramuscular (IM), or Intravenous (IV) delivery. In certain embodiments, the invention provides compositions formulated for organ-specific (e.g., liver) intra-arterial, intratumoral, intradermal, intravitreal injection, topical ocular, ophthalmic (eye drops), nebulization, topical or other topical ocular route, suppository or oral administration. In a preferred embodiment, the composition is administered subcutaneously.

The pharmaceutical composition of the invention may be administered in a dose sufficient to inhibit AGT gene expression. In some embodiments, the siRNA conjugate is administered at the following doses: about 0.5mg/kg to 50mg/kg per dose, or 0.3mg/kg to 20mg/kg, or 3mg/kg to 10mg/kg, or preferably 3mg/kg to 10mg/kg per dose. For example, the siRNA conjugates may be administered at a dose of about 0.5mg/kg, 1mg/kg, 1.5mg/kg, 2mg/kg, 3mg/kg, 10mg/kg, 20mg/kg, 30mg/kg, 40mg/kg, or 50mg/kg per single dose.

The composition may also be prepared and packaged in a fixed dose for the subject independent of body weight. Exemplary dosage levels may be calculated by multiplying each kilogram of body weight by the average subject's body weight. For example, average adult weight is generally considered to be about 70 kg.

Repeated dose regimens may include periodic administration of a therapeutic amount of the siRNA conjugate, e.g., once a month, once every other month, or once every third month. In a preferred embodiment, the siRNA conjugate is administered at a frequency of no more than once a month. Following the initial treatment regimen, the treatment may be administered less frequently.

The skilled artisan will appreciate that certain factors may influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health or age of the subject, and other diseases present. Furthermore, treating a subject with a therapeutically effective amount of a composition may include monotherapy or a series of therapies. As described elsewhere herein, the effective dose and in vivo half-life of the individual siRNA conjugates encompassed by the invention can be estimated using conventional methods or based on in vivo testing using an appropriate animal model.

A. Excipient

A "pharmaceutical carrier" or "pharmaceutical excipient" is a pharmaceutically acceptable solvent, suspending agent, or any other pharmaceutically inert vehicle for delivering one or more nucleic acids to an animal. Such agents are well known in the art.

B. Other components

The compositions of the present invention may additionally comprise other auxiliary components conventionally present in pharmaceutical compositions at levels of use established in the art. Thus, for example, the composition may comprise additional, compatible pharmaceutically active substances, such as antipruritics, astringents, local anesthetics or anti-inflammatory agents, or may comprise additional substances such as preservatives, antioxidants and stabilizers useful in physically formulating the compositions of the present invention in various dosage forms. However, such materials should not unduly interfere with the biological activity of the components of the compositions of the present invention when added. The formulation may be sterilized and, if desired, mixed with adjuvants which do not adversely interact with the nucleic acids of the formulation, such as preservatives, stabilizers, wetting agents, emulsifiers, salts or buffers which affect osmotic pressure, and the like.

In some embodiments, the pharmaceutical compositions characterized in the present invention comprise (a) one or more siRNA conjugate compounds and (b) one or more agents that function by a non-RNAi mechanism and are useful in the treatment of hypertension-related disorders.

As noted above, in addition to their administration, the siRNA conjugates characterized herein may be administered in combination with other known agents effective in treating hypertension. Regardless, the administering physician can adjust the amount and timing of siRNA conjugate administration based on the results observed using standard efficacy measurements known in the art or described herein.

Examples

Unless otherwise specified, reagents and media used in the following examples are commercially available, and the procedures for nucleic acid electrophoresis, real-time PCR, and the like used are carried out by the method described in Molecular Cloning (Cold Spring Harbor Laboratory Press (1989)). The conjugate molecules of formula (27), (28) or (29) used were purchased from Nanjing Rai pharmaceutical technologies Co.

Example 1 design of siRNA

A set of siRNAs targeting the human AGT gene (human NCBI refseqID NM-000029.3;NCBI Gene ID:183) was designed on-line using oligo alk. Human NM-000029 REFSEQ mRNA, third edition has a length of 2587 bases. Meanwhile, in order to avoid toxicity caused by any sequence, sequences similar to those of human genes need to be excluded.

A detailed list of the nucleotide sequences of the sense and antisense strands of unmodified AGT is shown in Table 3. A detailed list of modified AGT sense and antisense strand nucleotide sequences is shown in Table 4.

EXAMPLE 2 preparation of siRNA or siRNA conjugates

And (3) synthesis: sense and antisense strand sequences were synthesized according to phosphoramidite solid phase synthesis techniques, on a 1. Mu. Mol scale using solid phase carrier mediated phosphoramidite chemistry on a Mermade 192 synthesizer (BioAutomation). The solid support is a controlled pore glass loaded with custom GalNAc ligand molecules (CPG,) Or a universal solid support. Auxiliary synthesis reagents, such as 2'-F and 2' -O-methyl RNA phosphoramidite, are commercially available reagents. The corresponding phosphoramidites were used to introduce 2' -F, 2' -O-methyl, GNA (diol nucleic acid), 5' -phosphate and abasic modifications. Synthesis of 3' GalNAc conjugated single strands was performed on GalNAc modified CPG supports. CPG universal solid phase carriers are used for synthesis of antisense single strands, or synthesis of 5' GalNAc conjugated single strands. The coupling time for all phosphoramidites (dissolved in anhydrous acetonitrile, 100 mM) was 5 minutes using 5-ethylthio-1H-tetrazole (ETT) as activator (0.6M in acetonitrile). Phosphorothioate linkages were generated using a solution of 50mm 3- ((dimethylamino-methylene) amino) -3H-1,2, 4-dithiazole-3-thione (DDTT) in anhydrous acetonitrile/pyridine (vv=1/1) for a reaction time of 3 minutes. All sequences were synthesized after the final removal of DMT groups.

Cleavage and deprotection of bound oligomers on CPG: after termination of the solid phase synthesis, the protecting group was removed by treatment with an acetonitrile solution containing 20% diethylamine for 30 minutes without cleavage of the oligonucleotide from the CPG. Subsequently, the dried CPG was treated with concentrated ammonia at 40℃for 18 hours. After centrifugation, the supernatant was transferred to a new tube and CPG was washed with ammonia. The combined solutions were concentrated to give a solid mixture.

Purifying: purification was by anion exchange HPLC using NanoQ. Buffer a was 10mM sodium perchlorate solution, 20mM Tris,1mM EDTA,pH7.4 and contained 20% acetonitrile, buffer B,500mM sodium perchlorate, 20mM Tris,1mM EDTA,pH7.4 and contained 20% acetonitrile. The target product was isolated and desalted using a reverse phase C18 column.

Annealing of the oligoribonucleotides results in siRNA conjugates: the RNA oligomer to be annealed is treated with sterile RNase Free H ₂ O (no RNA hydrolase) was formulated as a 200. Mu.m solution. The annealing reaction system was set up as follows, the above solution (duplex concentration 10 nmol) with total volume of 100. Mu.L was placed in a 95℃water bath for 10 minutes (. Gtoreq.100 nmol demand requires high temperature 20 minutes). Fwdarw.rapidly placed in a 60℃water bath for natural cooling. Fwdarw.the annealed solution was stored at 4 ℃. Complementary strands are mixed by combining equimolar RNA solutions.

Table 4 shows AGT siRNA conjugates synthesized using the methods described above.

TABLE 4 modified AGT siRNA conjugate nucleotide sequences

Example 3 in vitro Activity assay of siRNA

Quantitative determination of AGT mRNA content in HepG2 cells by qPCR as IC of the Compound ₅₀ The value is used as an index to evaluate the inhibition activity of the siRNA conjugate on AGT;

experimental materials and reagents:

cell line: hepG2 cell (provided by stem cell bank of China academy of sciences)

HepG2 cell culture medium (DMEM, invitrogen-11330032;10% serum, invitrogen-10099141;100units/mL penicillin and 100. Mu.g/mL streptomycin, hyclone-SV30010;1% non-essential amino acids, invitrogen-11140050;2mM L-glutamine, invitrogen-25030081;1mM sodium pyruvate, gibco-11360-070; 500. Mu.g/mL Geneticin, invitrogen-10131027).

Reagent: pancreatin (Invitrogen-25300062); DMSO (Sigma-D2650-100 ML); transfection reagent Lipofectamine RNAiMAX (Invitrogen-13778-150); MEM Medium (HyClone-SH 30024.01); ULtraPure DistilledWater (DNAse, RNAse, free) (Invitrogen-10977-015);Opti-MEM I(1X)(Gibco-31985-070)；Phosphate Buffered Saline(PBS)(Gibco)；PrimeScript ^TM RT reagent Kit with gDNA Eraser(takara-RR047A)；ChamQ Universal SYBR qPCR Master Mix(vyzme-Q711-02)。

consumable and instrument: 48 well cell culture plates (timing-3599); CO ₂ Incubator (HERA-CELL-240);Microplate(Axygen-PCR-96-FLT-C)；qPCR equipment(QIANGE)。

the experimental steps are as follows:

the siRNA or siRNA conjugate was transfected into HepG2 cells as follows: hepG2 cells were taken, washed with PBS, digested with trypsin, adjusted to the appropriate density, and after 24h, siRNA was transferred into AGT cells using transfection reagent Lipofectamine RNAiMax and inoculated into 48 well plates at a density of 10,000 cells per well, with 500. Mu.L per well of HepG2 cell culture medium. Cells were exposed to 5% CO ₂ Culturing in incubator at 37 deg.c for 48 hr. 48 hours after transfection, cells were collected, RNA was extracted, and total AGT-RNA in the cells was detected by RT-PCR.

The siRNA tested was tested at 2 concentration points, 3 duplicate wells. Control was set to nm_000029.3, 4 concentration points were tested, 2 wells.

The procedure for detection of AGT RNA is briefly described as follows: total RNA in cells was extracted by the trizol method, reverse transcribed into cDNA by adding random primers, and then qPCR was performed to detect AGT cDNA in the sample, referring to the reverse transcription kit (takara) instructions. Meanwhile, GAPDH primers and probes specifically detect GAPDH cDNA.

The PCR reaction procedure was: 95℃for 2 minutes, then enter a cyclic mode, 95℃for 10 seconds, followed by 60℃for 30 seconds for a total of 40 cycles. The AGT RNA content in the samples was calculated from the Ct value of each sample.

The PCR primers were as follows:

Human AGT-Forward 5-ACTTCACAGAACTGGATGTTGCTGC-3；

Human AGT-Reverse 5-AACAGACACTGAGGTGCTGTTGTCCAC-3。

Human GAPDH-Forward 5-GGAGCGAGATCCCTCCAAAAT-3；

Human GAPDH-Reverse 5-GGCTGTTGTCATACTTCTCATGG-3。

the expression level of the AGT mRNA of the gene of interest was calculated by a relative quantification method of DeltaDeltaCt for each sample. The relative expression level of the target gene is expressed by using 2-delta CT, and the calculation formula is as follows:

a) The Ct value is automatically calculated according to the default settings of the Quant Studio 7 software. The Ct value is exported as an Excel file.

b) The relative expression amount of the gene was calculated using the following formula:

delta ct=ct (gene of interest) -Ct (gapdh)

ΔΔΔCt =Δct (detection of sample) relative to DeltaCt (Mock)

mRNA expression of Mock = 2 ^-ΔΔCt

Wherein Mock represents a negative control to which equal concentrations of Lipofectamine RNAiMax were added but no siRNA.

The inhibition rate was calculated as follows: (1- (2 ^-ΔΔCt ) 100%. Table 5 shows the inhibitory activity of the siRNA of the present invention on AGT.

TABLE 5 inhibition of AGT RNA by siRNA of the invention

Transfected into HepG2 cells using the same method as described above, the inhibitory activity (IC) of the siRNA conjugates of the present invention on AGT was measured ₅₀ ) Data fitting using GraphPad to derive IC ₅₀ Numerical values. Table 6 shows the inhibitory activity (IC) of siRNA conjugates of the invention on AGT ₅₀ )。

TABLE 6 inhibition Activity of siRNA conjugates of the invention on AGT (IC ₅₀ )

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From Table 6, it can be seen that the siRNA conjugates provided by the present invention have higher inhibitory activity in HepG2 cells.

While the invention has been described with reference to the preferred embodiments, it is not limited thereto, and various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

Claims

1. An siRNA comprising a sense strand and an antisense strand, each nucleotide in the siRNA being independently a modified or unmodified nucleotide, wherein the sense strand comprises nucleotide sequence I and the antisense strand comprises nucleotide sequence II, which are at least partially complementary in opposite phase to form a double-stranded region, the nucleotide sequence I and the nucleotide sequence II being selected from the group of sequences shown in (I) - (iv):

(i) The nucleotide sequence I and SEQ ID NO:1, and optionally having NO more than 3 nucleotide differences, and the nucleotide sequence II is identical to the nucleotide sequence set forth in SEQ ID NO:2, and optionally having no more than 3 nucleotide differences: 5'-ACUUCUUGGGCUUCUGUAUUU-3' (SEQ ID NO: 1);

5’-AUACGGAAGCCCAAGAAGUUU-3’(SEQ ID NO:2)；

(ii) The nucleotide sequence I and SEQ ID NO:3, and optionally having NO more than 3 nucleotide differences, and the nucleotide sequence II is identical to the nucleotide sequence set forth in SEQ ID NO:4, and optionally having no more than 3 nucleotide differences: 5'-CGUUGCUGCUGAGAAGAUUUU-3' (SEQ ID NO: 3);

5’-AAUCUUCUCAGCAGCAACGUU-3’(SEQ ID NO:4)；

(iii) The nucleotide sequence I and SEQ ID NO:5, and optionally having NO more than 3 nucleotide differences, and the nucleotide sequence II is identical to the nucleotide sequence set forth in SEQ ID NO:6, and optionally having no more than 3 nucleotide differences:

5’-GGUGCUGAACAGCAUUUUUUU-3’(SEQ ID NO:5)；

5’-AAAAAUGCUGUUCAGCACCUU-3’(SEQ ID NO:6)；

(iv) The nucleotide sequence I and SEQ ID NO:7, and optionally with NO more than 3 nucleotide differences, and the nucleotide sequence II is identical to the nucleotide sequence set forth in SEQ ID NO:8, and optionally having no more than 3 nucleotide differences:

5’-AGUCUACCCAACAGCUUAAUU-3’(SEQ ID NO:7)；

5’-UUAAGCUGUUGGGUAGACUUU-3’(SEQ ID NO:8)。

2. The siRNA of claim 1, wherein said nucleotide sequence I is identical to SEQ ID NO:1, and/or said nucleotide sequence II differs from the nucleotide sequence set forth in SEQ ID NO:2 optionally having no more than 1 nucleotide difference between the nucleotide sequences shown in 2;

alternatively, the nucleotide sequence I is identical to SEQ ID NO:3, and/or said nucleotide sequence II differs from the nucleotide sequence set forth in SEQ ID NO:4 optionally having no more than 1 nucleotide difference between the nucleotide sequences shown in fig;

alternatively, the nucleotide sequence I is identical to SEQ ID NO:5, and/or said nucleotide sequence II differs from the nucleotide sequence set forth in SEQ ID NO:6 optionally having no more than 1 nucleotide difference between the nucleotide sequences shown in fig. 6;

alternatively, the nucleotide sequence I is identical to SEQ ID NO:7, and/or said nucleotide sequence II differs from the nucleotide sequence set forth in SEQ ID NO:8 optionally having no more than 1 nucleotide difference between the nucleotide sequences shown in figure 8.

3. siRNA according to claim 1 or 2, wherein at least one nucleotide of said sense strand or said antisense strand is a modified nucleotide, preferably all nucleotides of said sense strand and/or said antisense strand are modified nucleotides, and/or at least one phosphate group is a phosphate group with a modification group.

4. The siRNA of any of claims 1-3, wherein each nucleotide in the sense strand and the antisense strand is independently a fluoro-modified nucleotide or a non-fluoro-modified nucleotide.

5. The siRNA according to claim 4 wherein each non-fluoro modified nucleotide is independently selected from one of nucleotides or nucleotide analogs formed by substitution of the hydroxyl group at the 2' position of the ribosyl of the nucleotide with a non-fluoro group.

6. The siRNA according to claim 5, wherein,

the nucleotide formed by substituting the hydroxyl at the 2 '-position of the ribosyl of the nucleotide with a non-fluorine group is selected from one of a 2' -alkoxy modified nucleotide, a 2 '-substituted alkoxy modified nucleotide, a 2' -alkyl modified nucleotide, a 2 '-substituted alkyl modified nucleotide, a 2' -amino modified nucleotide, a 2 '-substituted amino modified nucleotide and a 2' -deoxynucleotide;

the nucleotide analogue is selected from an iso nucleotide, a bridged nucleotide or an acyclic nucleotide;

in particular, an isonucleotide is a compound in which a base is displaced from the 1' -position to the 2' -position or the 3' -position of the ribose ring;

the bridged nucleotide is one selected from LNA shown in formula (6), ENA shown in formula (7) and cET shown in formula (8),

The acyclic nucleotide is one selected from the group consisting of UNA represented by formula (9) and GNA represented by formula (10),

wherein in the above formulae (6) to (10), base represents a nucleobase，R _a Selected from H, OH or C ₁ -C ₁₀ Alkoxy (O-alkyl).

7. The siRNA of any of claims 4-6, wherein each non-fluoro modified nucleotide is a methoxy modified nucleotide, preferably said methoxy modified nucleotide is a nucleotide formed by substitution of the 2' -hydroxy group of the ribosyl group with methoxy.

8. The siRNA according to claim 3, wherein the phosphate group having a modifying group is a phosphorothioate group formed by substitution of at least one oxygen atom of a phosphodiester bond of a phosphate group with a sulfur atom.

9. The siRNA of claim 3 or 8, wherein the phosphate group having a modifying group is a phosphorothioate group having a structure as shown in formula (13):

10. the siRNA according to claim 8 or 9, wherein,

the phosphorothioate linkage is present at least one of the group consisting of: between the first and second nucleotides at either end of the sense strand or the antisense strand; between the second and third nucleotides at either end of the sense strand or the antisense strand; or any combination of the above;

Alternatively, phosphorothioate linkages may be present at all of the above positions except at the 5' end of the sense strand,

alternatively, phosphorothioate linkages are present at all of the above positions except the 3' end of the sense strand.

11. The siRNA of claim 1, wherein the 5' terminal nucleotide of the antisense strand is a 5' -phosphonucleotide or a 5' -phosphoanalog modified nucleotide;

in particular, the nucleotide 5 '-phosphate is a nucleotide containing a modification of 5' -phosphate represented by the formula (14),

the 5' -phosphate analogue modified nucleotide is a nucleotide containing a vinyl phosphate modification as shown in formula (15) or a phosphorothioate modification as shown in formula (17),

wherein R is selected from the group consisting of H, OH, methoxy and fluorine; base represents a nucleobase selected from A, U, C, G or T.

12. The siRNA of any one of claims 1-11, selected from the following table:

13. an siRNA conjugate comprising the siRNA of any one of claims 1-12 and a conjugate group conjugated to the siRNA.

14. The siRNA conjugate of claim 13, wherein the conjugate group comprises a pharmaceutically acceptable targeting group and a linker, and the siRNA, the linker and the targeting group are sequentially covalently or non-covalently linked.

15. The siRNA conjugate of claim 14, wherein the linker has a structure as shown in formula (19):

wherein m is an integer of 1 to 3;

16. The siRNA conjugate of any of claims 13 to 15, wherein said connector is attached to the 3 'end of the sense strand or the 5' end of the sense strand of said siRNA.

17. The siRNA conjugate of claim 13, wherein the conjugate has a structure represented by formula (24), (25) or (26):

Wherein R is ₂ A group having a structure represented by the formula (S1):

wherein E is ₁ OH or SH;

nu is the siRNA of any one of claims 1-12;

R ₁ is a linear or cyclic alkylene group of 1 to 20 carbon atoms in length, wherein one or more carbon atoms are optionally replaced by any one or more selected from the group consisting of: c (O), NH, O, S, CH = N, S (O) ₂ 、C ₂ -C ₁₀ Alkenylene, C ₂ -C ₁₀ Alkynylene, C ₆ -C ₁₀ Arylene group, C ₃ -C ₁₈ Heterocyclylene and C ₅ -C ₁₀ Heteroarylene; and wherein R is ₁ Optionally having substituents of any one or more of the group consisting of: c (C) ₁ -C ₁₀ Alkyl, C ₆ -C ₁₀ Aryl, C ₅ -C ₁₀ Heteroaryl, C ₁ -C ₁₀ Haloalkyl, -OC ₁ -C ₁₀ Alkyl, -OC ₁ -C ₁₀ Alkylphenyl radicals C ₁ -C ₁₀ alkyl-OH, -OC ₁ -C ₁₀ Haloalkyl, -SC ₁ -C ₁₀ Alkyl, -SC ₁ -C ₁₀ Alkylphenyl radicals C ₁ -C ₁₀ alkyl-SH, -SC ₁ -C ₁₀ Haloalkyl, halogen substituent, -OH, -SH, -NH ₂ 、-C ₁ -C ₁₀ alkyl-NH ₂ 、-N(C ₁ -C ₁₀ Alkyl) (C) ₁ -C ₁₀ Alkyl), -NH (C) ₁ -C ₁₀ Alkyl), -N (C) ₁ -C ₁₀ Alkyl) (C) ₁ -C ₁₀ Alkylphenyl), -NH (C) ₁ -C ₁₀ Alkylphenyl), cyano, -CO ₂ H、C(O)O(C ₁ -C ₁₀ Alkyl), -CON (C) ₁ -C ₁₀ Alkyl) (C) ₁ -C ₁₀ Alkyl), -CONH (C) ₁ -C ₁₀ Alkyl), -CONH ₂ 、-NHC(O)(C ₁ -C ₁₀ Alkyl), -NHC (O) (phenyl), -N (C) ₁ -C ₁₀ Alkyl), -N (C) ₁ -C ₁₀ Alkyl) C (O) (C ₁ -C ₁₀ Alkyl), -N (C) ₁ -C ₁₀ Alkyl) C (O) (phenyl), -C (O) C ₁ -C ₁₀ Alkyl, -C (O) C ₁ -C ₁₀ Alkylphenyl, -C (O) C ₁ -C ₁₀ Haloalkyl, -OC (O) C ₁ -C ₁₀ Alkyl, -SO ₂ (C ₁ -C ₁₀ Alkyl), -SO ₂ (phenyl) -SO ₂ (C ₁ -C ₁₀ Haloalkyl) -SO ₂ NH ₂ 、-SO ₂ NH(C ₁ -C ₁₀ Alkyl), -SO ₂ NH (phenyl) -NHSO ₂ (C ₁ -C ₁₀ Alkyl), -NHSO ₂ (phenyl) and-NHSO ₂ (C ₁ -C ₁₀ A haloalkyl group);

each L ₁ Independently is a linear alkylene group of 1 to 40 carbon atoms in length, wherein one or more carbon atoms are optionally replaced by any one or more selected from the group consisting of: c (O), NH, O, S, CH = N, S (O) ₂ 、C ₂ -C ₁₀ Alkenylene, C ₂ -C ₁₀ Alkynylene, C ₆ -C ₁₀ Arylene group, C ₃ -C ₁₈ Heterocyclylene and C ₅ -C ₁₀ Heteroarylene; and wherein R is ₁ Optionally having substituents of any one or more of the group consisting of: c (C) ₁ -C ₁₀ Alkyl, C ₆ -C ₁₀ Aryl, C ₅ -C ₁₀ Heteroaryl, C ₁ -C ₁₀ Haloalkyl, -OC ₁ -C ₁₀ Alkyl, -OC ₁ -C ₁₀ Alkylphenyl radicals C ₁ -C ₁₀ alkyl-OH, -OC ₁ -C ₁₀ Haloalkyl, -SC ₁ -C ₁₀ Alkyl, -SC ₁ -C ₁₀ Alkylphenyl radicals C ₁ -C ₁₀ alkyl-SH, -SC ₁ -C ₁₀ Haloalkyl, halogen substituent, -OH, -SH, -NH ₂ 、-C ₁ -C ₁₀ alkyl-NH ₂ 、-N(C ₁ -C ₁₀ Alkyl) (C) ₁ -C ₁₀ Alkyl), -NH (C) ₁ -C ₁₀ Alkyl), -N (C) ₁ -C ₁₀ Alkyl) (C) ₁ -C ₁₀ Alkylphenyl), -NH (C) ₁ -C ₁₀ Alkylphenyl), cyano, -CO ₂ H、C(O)O(C ₁ -C ₁₀ Alkyl), -CON (C) ₁ -C ₁₀ Alkyl) (C) ₁ -C ₁₀ Alkyl), -CONH (C) ₁ -C ₁₀ Alkyl), -CONH ₂ 、-NHC(O)(C ₁ -C ₁₀ Alkyl), -NHC (O) (phenyl), -N (C) ₁ -C ₁₀ Alkyl), -N (C) ₁ -C ₁₀ Alkyl) C (O) (C ₁ -C ₁₀ Alkyl), -N (C) ₁ -C ₁₀ Alkyl) C (O) (phenyl), -C (O) C ₁ -C ₁₀ Alkyl, -C (O) C ₁ -C ₁₀ Alkylphenyl, -C (O) C ₁ -C ₁₀ Haloalkyl, -OC (O) C ₁ -C ₁₀ Alkyl, -SO ₂ (C ₁ -C ₁₀ Alkyl), -SO ₂ (phenyl) -SO ₂ (C ₁ -C ₁₀ Haloalkyl) -SO ₂ NH ₂ 、-SO ₂ NH(C ₁ -C ₁₀ Alkyl), -SO ₂ NH (phenyl) -NHSO ₂ (C ₁ -C ₁₀ Alkyl), -NHSO ₂ (phenyl) and-NHSO ₂ (C ₁ -C ₁₀ A haloalkyl group);

M ₁ represents a targeting group;

indicating the site of covalent attachment of the group.

18. The siRNA conjugate of claim 17, wherein each L ₁ Independently selected from the group consisting of ₁ -A ₁₄ And any combination of:

wherein each k1 is independently an integer from 1 to 20;

each k2 is independently an integer from 1 to 20;

each R _c Independently C ₁ -C ₁₀ An alkyl group;

19. The siRNA conjugate of claim 18, wherein L ₁ Is a linked combination of at least 2 of the groups A1, A4, A8, A10, A11.

20. The siRNA conjugate of any of claims 17 to 19, wherein L ₁ Is 3 to 20 atoms in length.

21. The siRNA conjugate of any of claims 18 to 20, wherein k1 is an integer from 3 to 5, k2 is an integer from 3 to 5, R _c Is one of methyl, ethyl and isopropyl, R _d Is A15 or A16, R _e Is one of methyl, ethyl, isopropyl and butyl.

22. The siRNA conjugate of any of claims 14 to 21, wherein each of said targeting groups is independently one selected from the group consisting of D-galactose, L-galactose, a-D-glucopyranose, β -D-glucopyranose, a-D-glucofuranose, β -D-glucofuranose, a-D-fructofuranose, a-D-galactopyranose, β -D-galactopyranose, a-D-galactofuranose, β -D-galactofuranose, glucosamine, sialic acid, galactosamine, N-acetylgalactosamine, N-trifluoroacetylgalactosamine, N-propionylgalactosamine, N-butyryl galactosamine, N-isobutyryl galactosamine.

23. The siRNA conjugate of any of claims 17 to 22, wherein R ₁ Containing both a linking site attached to an N atom on a nitrogen-containing backbone and R ₂ A linking site to which the P atom is linked.

24. The siRNA conjugate of any of claims 17 to 23, wherein R ₁ The above-mentioned site bonded to N atom on the nitrogen-containing skeleton forms an amide bond with N, the above-mentioned site bonded to R ₂ The P atom on the substrate is linked to P to form a phosphate bond or a phosphorothioate bond.

25. The siRNA conjugate of claims 17-24, wherein the P atom in formula (S1) is attached to the end of the sense strand or the antisense strand of the siRNA.

26. The siRNA conjugate of any of claims 17-25, wherein the P atom in formula (S1) is linked to the 2' position, the 3' position or the 5' position of a nucleotide in the siRNA via a phosphodiester linkage.

27. The siRNA conjugate according to claim 13, wherein the siRNA conjugate has a structure represented by the formula (Z1-Nu), (Z2-Nu), (Z3-Nu), (Z4-Nu), (Z5-Nu), (Z6-Nu), (Z7-Nu), (Z8-Nu), (Z9-Nu), (Z10-Nu), (Z11-Nu), (Z12-Nu), (Z13-Nu), (Z14-Nu), (Z15-Nu), (Z16-Nu), (Z17-Nu), (Z18-Nu), (Z19-Nu), (Z20-Nu), (Z21-Nu), (Z22-Nu), (Z23-Nu), (Z24-Nu), (Z25-Nu), (Z26-Nu), (Z27-Nu), (Z28-Nu), (Z29-Nu), (Z30-Nu), (Z31-Nu) or (Z32-Nu),

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wherein Nu is the siRNA of any one of claims 1-12.

28. The siRNA conjugate of claim 13, wherein the siRNA conjugate has a structure as shown in formula (23):

wherein the double helix structure represents the siRNA of any one of claims 1-12.

29. Use of the siRNA of any one of claims 1-12 and/or the siRNA conjugate of any one of claims 13-28 in the manufacture of a medicament for inhibiting expression of a renin gene in a cell.

30. A pharmaceutical composition comprising the siRNA of any one of claims 1-12 and/or the siRNA conjugate of any one of claims 13-28 and optionally a pharmaceutically acceptable carrier.

31. Use of the siRNA of any one of claims 1-12, the siRNA conjugate of any one of claims 13-28 and/or the pharmaceutical composition of claim 30 in the manufacture of a medicament for treating a disease and/or disorder associated with expression of a renin gene.

32. The use of claim 31, wherein the disease and/or disorder associated with expression of the angiotensinogen gene is a disease and/or disorder associated with hypertension.

33. A method of inhibiting expression of an angiotensinogen gene in a cell, comprising contacting the cell with an effective amount of the siRNA of any one of claims 1-12, the siRNA conjugate of any one of claims 13-28, and/or the pharmaceutical composition of claim 30, thereby inhibiting expression of the angiotensinogen gene in the cell.