CN111378658B

CN111378658B - SiRNA for inhibiting TIMP-1 gene expression, pharmaceutical composition containing same and application thereof

Info

Publication number: CN111378658B
Application number: CN201811627993.XA
Authority: CN
Inventors: 张鸿雁; 高山; 康代武
Original assignee: Suzhou Ruibo Biotechnology Co ltd
Current assignee: Suzhou Ruibo Biotechnology Co ltd
Priority date: 2018-12-28
Filing date: 2018-12-28
Publication date: 2024-03-15
Anticipated expiration: 2038-12-28
Also published as: CN111378658A

Abstract

An siRNA that inhibits expression of a TIMP-1 gene, comprising a sense strand and an antisense strand, said sense strand comprising nucleotide sequence I and said antisense strand comprising nucleotide sequence II, said nucleotide sequence I and said nucleotide sequence II each being 19 nucleotides in length, said nucleotide sequence II being at least partially complementary to a sequence of 19 consecutive nucleotides in length in the region 350-700 of TIMP-1 mRNA. The nucleotides at positions 7, 8 and 9 of the nucleotide sequence I are fluoro modified nucleotides, and the nucleotides at positions 2, 6, 14 and 16 of the nucleotide sequence II are fluoro modified nucleotides according to the direction from the 5 'end to the 3' end. The present disclosure also provides pharmaceutical compositions comprising the siRNA. The siRNA and the pharmaceutical composition containing the siRNA can treat or improve diseases related to TIMP-1 gene expression.

Description

SiRNA for inhibiting TIMP-1 gene expression, pharmaceutical composition containing same and application thereof

Technical Field

The present disclosure relates to a nucleic acid capable of inhibiting TIMP-1 gene expression and a pharmaceutical composition containing the same, and belongs to the field of nucleic acid pharmacy. The disclosure also relates to uses of these nucleic acids and pharmaceutical compositions.

Background

Liver fibrosis is a compensatory reaction in the course of tissue repair following various forms of chronic liver injury, and also a process of the progression of chronic liver disease to severe fatal diseases such as cirrhosis, liver cancer, etc., so anti-liver fibrosis is a serious issue in the treatment of chronic liver disease.

The current means for preventing and treating liver fibrosis are very limited, and mainly comprise two aspects: firstly, pathogenic factors such as virus resistance, alcohol abstinence and the like are removed aiming at the primary disease; second, treatments for liver fibrosis itself, such as by inhibiting inflammation or lipid peroxidation, or inhibiting proliferation activation of Hepatic Stellate Cells (HSCs), and promoting collagen degradation, etc. In clinical medicine, interferon is used to inhibit the activation of astrocytes and the expression of proliferation extracellular matrix, lamivudine is used to inhibit the replication of HBV DNA, and colchicine and silymarin are used to interfere with collagen secretion of cells. However, the medicines have an inaccurate treatment effect on liver fibrosis, have no obvious improvement on survival rate of patients suffering from liver fibrosis, and have obviously increased incidence of side effects.

Matrix metalloproteinase tissue inhibitor-1 (TIMP-1) is a protein that has been discovered in recent years to be produced by macrophages and connective tissue cells and to inhibit degradation of the extracellular matrix. From a molecular level, elevated TIMP-1 expression is one of the key events in the pathogenesis of fibrosis. Inhibiting the expression of this gene can enhance the degradation of extracellular matrix and reduce the synthesis and accumulation of collagen fibers in tissues, thereby potentially avoiding fibrosis. Early studies carried out liver-targeted delivery of siRNA against TIMP-1 using adenovirus as a vector, and the results showed that TIMP-1siRNA showed significant anti-fibrosis effects in both CCL4 and BDL fibrosis models (Am J pathol.2013,182 (5): 1607-16), suggesting that TIMP-1 may be a target gene for the treatment of fibrosis, for the treatment of fibrosis.

However, to date, the clinical application of siRNA drugs for treating diseases related to TIMP-1 gene expression has been slow, wherein the poor activity of siRNA itself and stability in blood is one of the reasons for the slow progress of such drugs. Therefore, there is an urgent need to develop an siRNA and an siRNA-containing pharmaceutical composition for treating diseases associated with TIMP-1 gene expression, which have potential clinical application values, have good stability and bioactivity.

Disclosure of Invention

In some embodiments, the present disclosure provides an siRNA capable of inhibiting expression of a TIMP-1 gene, the siRNA comprising a sense strand and an antisense strand, each nucleotide of the sense strand and the antisense strand being independently a fluoro-modified nucleotide or a non-fluoro-modified nucleotide, wherein the sense strand comprises nucleotide sequence I, the antisense strand comprises nucleotide sequence II, the nucleotide sequence I and the nucleotide sequence II are each 19 nucleotides in length, the nucleotide sequence I and the nucleotide sequence II are at least partially reverse-complementary to form a duplex region, the nucleotide sequence II is at least partially reverse-complementary to a first stretch of nucleotide sequences selected from the group consisting of sequences of 19 consecutive nucleotides in length in region 350-700 of a TIMP-1 mRNA; the nucleotides at positions 7, 8 and 9 of the nucleotide sequence I are fluoro modified nucleotides according to the direction from the 5 'end to the 3' end; the first nucleotide at the 5 '-end of the nucleotide sequence II is the first nucleotide at the 5' -end of the antisense strand, and the nucleotides at positions 2, 6, 14 and 16 of the nucleotide sequence II are fluoro-modified nucleotides.

In some embodiments, the present disclosure provides a pharmaceutical composition comprising an siRNA of the present disclosure and a pharmaceutically acceptable carrier.

In some embodiments, the present disclosure provides the use of an siRNA and/or a pharmaceutical composition of the present disclosure in the manufacture of a medicament for treating or ameliorating a disease associated with TIMP-1 gene expression.

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

Incorporated by reference

All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

Advantageous effects

The siRNA and the pharmaceutical composition containing the siRNA provided by the disclosure have good stability, higher gene inhibition activity, very low off-target effect and/or can obviously improve liver fibrosis level.

In some embodiments, the siRNA or the pharmaceutical composition comprising the siRNA provided by the present disclosure has higher stability and/or higher activity. In some embodiments, the siRNA or pharmaceutical compositions provided by the present disclosure exhibit a target gene expression inhibition of at least 20%,30%,40%,50%,60%,70%,80%, or 85%.

In some embodiments, the siRNA provided by the present disclosure or a pharmaceutical composition comprising the siRNA does not exhibit a significant off-target effect. 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.

In some embodiments, the sirnas provided by the present disclosure have good stability, maintaining consistent stability in vitro lysosome lysates, in human plasma, or in monkey plasma.

Therefore, the siRNA or the pharmaceutical composition provided by the disclosure can inhibit the expression of the TIMP-1 gene, effectively treat or improve the diseases related to the expression of the TIMP-1 gene, and has good application prospect.

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

Detailed Description

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

In the present disclosure, TIMP-1 gene and target gene all refer to mRNA sequences such as those shown in Genbank accession No. NM-003254.2.

Definition of the definition

In the above and below, capital C, G, U, A indicates the base composition of nucleotides unless otherwise specified; the lower case letter m indicates that the adjacent nucleotide to the left of the letter m is a methoxy modified nucleotide; the lower case letter f indicates that the adjacent nucleotide to the left of the letter f is a fluoro-modified nucleotide; the lower case letter s indicates that phosphorothioate linkages are between two nucleotides adjacent to the letter s; p1 indicates that one nucleotide adjacent to the right of P1 is a 5' -phosphonucleotide or a 5' -phosphoanalog modified nucleotide, the letter composition VP indicates that one nucleotide adjacent to the right of the letter composition VP is a vinyl phosphate modified nucleotide, the letter composition Ps indicates that one nucleotide adjacent to the right of the letter composition Ps is a phosphorothioate modified nucleotide, and the capital letter P indicates that one nucleotide adjacent to the right of the letter P is a 5' -phosphonucleotide.

In the above and below, the "fluoro-modified nucleotide" refers to a nucleotide in which the hydroxyl group at the 2 '-position of the ribosyl group of the nucleotide is substituted with fluorine, and the "non-fluoro-modified nucleotide" refers to a nucleotide or nucleotide analogue in which the hydroxyl group at the 2' -position of the ribosyl group of the nucleotide is substituted with a non-fluorine group. "nucleotide analog" refers to a group that is capable of replacing a nucleotide in a nucleic acid, but that differs in structure from adenine ribonucleotide, guanine ribonucleotide, cytosine ribonucleotide, uracil ribonucleotide, or thymine deoxyribonucleotide. Such as an iso-nucleotide, a bridged nucleotide (bridged nucleic acid, abbreviated as BNA) or an acyclic nucleotide. The "methoxy-modified nucleotide" refers to a nucleotide in which the 2' -hydroxyl group of the ribosyl group is replaced with a methoxy group.

In the present context, the terms "complementary" or "reverse complementary" are used interchangeably and have the meaning well known to those skilled in the art, i.e., in a double stranded nucleic acid molecule, the bases of one strand pair with the bases 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; "perfect complementarity" means that there is no base mismatch between 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.

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

As used herein, "treatment," "alleviating," or "improving" may be used interchangeably herein. These terms refer to methods 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.

As used herein, "prevent" and "prevent" are used interchangeably. These terms refer to methods of achieving a beneficial or desired result, including but not limited to prophylactic benefit. To obtain a "prophylactic benefit," the composition may be administered to a subject at risk of suffering from a particular disease, or to a subject reporting one or more pathological symptoms of the disease, even though a diagnosis of the disease may not have been made.

siRNA

The present disclosure provides an siRNA capable of inhibiting TIMP-1 gene expression.

The siRNA of the present disclosure 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.

The siRNA of the present disclosure comprises a sense strand and an antisense strand, each nucleotide of the sense strand and the antisense strand being independently a fluoro-modified nucleotide or a non-fluoro-modified 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 each 19 nucleotides in length, the nucleotide sequence I and the nucleotide sequence II are at least partially reverse-complementary to form a double-stranded region, the nucleotide sequence II is at least partially reverse-complementary to a first stretch of nucleotide sequence selected from the group consisting of sequences of 19 consecutive nucleotides in length in the region 350-700 of TIMP-1 mRNA; the nucleotides at positions 7, 8 and 9 of the nucleotide sequence I are fluoro modified nucleotides according to the direction from the 5 'end to the 3' end; the first nucleotide at the 5 '-end of the nucleotide sequence II is the first nucleotide at the 5' -end of the antisense strand, and the nucleotides at positions 2, 6, 14 and 16 of the nucleotide sequence II are fluoro-modified nucleotides.

In some embodiments, the first nucleotide sequence is selected from the group consisting of a sequence of 19 consecutive nucleotides in length at positions 370-388 of TIMP-1mRNA, defined as SEQ ID NO. 2, and the nucleotide sequence set forth in SEQ ID NO. 1 is a sequence that is fully reverse complement of SEQ ID NO. 2. The modified siRNA comprises a sense strand and an antisense strand, wherein each nucleotide in the sense strand and the antisense strand is independently a fluoro modified nucleotide or a non-fluoro modified nucleotide, the sense strand comprises a stretch of nucleotide sequence I, and the antisense strand comprises a stretch of 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 set forth in SEQ ID NO:1 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:2 and is NO more than 3 nucleotides different:

5'-CGUUAUGAGAUCAAGAUGZa ₁ -3'(SEQ ID NO:1)；

5'-Zb ₁ CAUCUUGAUCUCAUAACG-3'(SEQ ID NO:2)，

wherein Za ₁ Is A, zb ₁ Is a U-shaped structure, wherein the U is a U-shaped structure,

the nucleotide sequence I comprises a nucleotide sequence whose position corresponds to Za ₁ Nucleotide ZA of (A) ₁ The nucleotide sequence II comprises a nucleotide sequence whose position corresponds to Zb ₁ Nucleotide ZB of (A) ₁ The ZB ₁ Is the first nucleotide at the 5' end of the antisense strand.

In the above and in the following, "position correspondence" means that the same position in the nucleotide sequence is located from the same end of the nucleotide sequence. For example, nucleotide ZA at the 3' -end of nucleotide sequence I ₁ Is the 1 st nucleotide Za at the 3' -end corresponding to SEQ ID NO. 1 ₁ Is a nucleotide of (a).

In some embodiments, the first nucleotide sequence is selected from the group consisting of a sequence of 19 consecutive nucleotides in length at positions 497-515 of TIMP-1mRNA, defined as SEQ ID NO. 4, and the nucleotide sequence shown as SEQ ID NO. 3 is a sequence that is fully reverse complementary to SEQ ID NO. 4. The modified siRNA comprises a sense strand and an antisense strand, wherein each nucleotide in the sense strand and the antisense strand is independently a fluoro modified nucleotide or a non-fluoro modified nucleotide, the sense strand comprises a stretch of nucleotide sequence I, and the antisense strand comprises a stretch of nucleotide sequence II, which are at least partially reverse complementary to form a duplex region, wherein the nucleotide sequence I is equal in length to the nucleotide sequence set forth in SEQ ID NO:3 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:4 and is NO more than 3 nucleotides different:

5'-GCAGCGAGGAGUUUCUCAZa ₂ -3'(SEQ ID NO:3)；

5'-Zb ₂ UGAGAAACUCCUCGCUGC-3'(SEQ ID NO:4)，

Wherein Za ₂ Is U, zb ₂ In the case of the compound A,

the nucleotide sequence I comprises a nucleotide sequence whose position corresponds to Za ₂ Nucleotide ZA of (A) ₂ The nucleotide sequence II comprises a nucleotide sequence whose position corresponds to Zb ₂ Nucleotide ZB of (A) ₂ The ZB ₂ Is the first nucleotide at the 5' end of the antisense strand.

In some embodiments, the first nucleotide sequence is selected from the group consisting of a sequence of 19 consecutive nucleotides in length at positions 642-660 of TIMP-1mRNA, defined as SEQ ID NO. 6, and the nucleotide sequence set forth in SEQ ID NO. 5 is a sequence that is fully reverse complementary to SEQ ID NO. 6. The modified siRNA comprises a sense strand and an antisense strand, wherein each nucleotide in the sense strand and the antisense strand is independently a fluoro modified nucleotide or a non-fluoro modified nucleotide, the sense strand comprises a stretch of nucleotide sequence I, and the antisense strand comprises a stretch of nucleotide sequence II, which are at least partially reverse complementary to form a duplex 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'-CACAGUGUUUCCCUGUUUZa ₃ -3'(SEQ ID NO:5)；

5'-Zb ₃ AAACAGGGAAACACUGUG-3'(SEQ ID NO:6)，

Wherein Za ₃ Is A, zb ₃ Is a U-shaped structure, wherein the U is a U-shaped structure,

the nucleotide sequence I comprises a nucleotide sequence whose position corresponds to Za ₃ Nucleotide ZA of (A) ₃ The nucleotide sequence II comprises a nucleotide sequence whose position corresponds to Zb ₃ Nucleotide ZB of (A) ₃ The ZB ₃ Is the first nucleotide at the 5' end of the antisense strand.

In some embodiments, nucleotide sequence II is substantially reverse complementary, or fully reverse complementary to the first nucleotide sequence.

In some embodiments, at least nucleotides 2-19 of the nucleotide sequence II are complementary to the first stretch of nucleotide sequence in a 5 'end to 3' end orientation.

In some embodiments, nucleotide 1 of the nucleotide sequence II is a or U in a 5 'to 3' end orientation.

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

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 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 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.

In some embodiments, the nucleotide difference between nucleotide sequence II and the nucleotide sequence set forth in SEQ ID NO. 2 comprises ZB ₁ At the position ofDifference, and ZB ₁ Selected from A, C or G. In some embodiments, the nucleotide difference is ZB ₁ Difference in position, ZB ₁ Selected from A, C or G. In some embodiments, ZA ₁ Is in combination with ZB ₁ Complementary nucleotides.

In some embodiments, the nucleotide difference between nucleotide sequence II and the nucleotide sequence set forth in SEQ ID NO. 4 comprises ZB ₂ Difference in position, and ZB ₂ Selected from U, C or G. In some embodiments, the nucleotide difference is ZB ₂ Difference in position, ZB ₂ Selected from U, C or G. In some embodiments, ZA ₂ Is in combination with ZB ₂ Complementary nucleotides.

In some embodiments, the nucleotide difference between nucleotide sequence II and the nucleotide sequence set forth in SEQ ID NO. 6 comprises ZB ₃ Difference in position, and ZB ₃ Selected from A, C or G. In some embodiments, the nucleotide difference is ZB ₃ Difference in position, ZB ₃ Selected from A, C or G. In some embodiments, ZA ₃ Is in combination with ZB ₃ Complementary nucleotides.

These nucleotide differences do not significantly reduce the target gene inhibition capacity of the siRNA, and these sirnas comprising nucleotide differences are also within the scope of the present disclosure.

In some embodiments, nucleotide sequence I is the nucleotide sequence set forth in SEQ ID NO. 7 and nucleotide sequence II is the nucleotide sequence set forth in SEQ ID NO. 8:

5'-CGUUAUGAGAUCAAGAUGZA ₁ -3'(SEQ ID NO:7)；

5'-ZB ₁ CAUCUUGAUCUCAUAACG-3'(SEQ ID NO:8)，

wherein the ZB ₁ Is the first nucleotide at the 5' -end of the antisense strand, ZA ₁ Selected from A, U, G or C, and ZB ₁ Is with ZA ₁ Complementary nucleotides; in some embodiments, ZA ₁ Is U, ZB ₁ Is A.

In some embodiments, nucleotide sequence I is the nucleotide sequence set forth in SEQ ID NO. 9 and nucleotide sequence II is the nucleotide sequence set forth in SEQ ID NO. 10:

5'-GCAGCGAGGAGUUUCUCAZA ₂ -3'(SEQ ID NO:9)；

5'-ZB ₂ UGAGAAACUCCUCGCUGC-3'(SEQ ID NO:10)，

wherein the ZB ₂ Is the first nucleotide at the 5' -end of the antisense strand, ZA ₂ Selected from A, U, G or C, and ZB ₂ Is with ZA ₂ Complementary nucleotides; in some embodiments, ZA ₂ Is U, ZB ₂ Is A.

In some embodiments, nucleotide sequence I is the nucleotide sequence set forth in SEQ ID NO. 11 and nucleotide sequence II is the nucleotide sequence set forth in SEQ ID NO. 12:

5'-CACAGUGUUUCCCUGUUUZA ₃ -3'(SEQ ID NO:11)；

5'-ZB ₃ AAACAGGGAAACACUGUG-3'(SEQ ID NO:12)，

Wherein the ZB ₃ Is the first nucleotide at the 5' -end of the antisense strand, ZA ₃ Selected from A, U, G or C, and ZB ₃ Is with ZA ₃ Complementary nucleotides; in some embodiments, ZA ₃ Is U, ZB ₃ Is A.

In some embodiments, the sense strand comprises only nucleotide sequence i and the antisense strand comprises only nucleotide sequence ii. In some embodiments, the sense strand is nucleotide sequence i and the antisense strand is nucleotide sequence ii. At this time, the length ratio of the sense strand to the antisense strand was 19/19.

In some embodiments, the sense strand further comprises a nucleotide sequence III, the antisense strand further comprises a nucleotide sequence IV, each nucleotide of the nucleotide sequence III and the nucleotide sequence IV is independently one of a non-fluoro modified nucleotide, the nucleotide sequence III and the nucleotide sequence IV are each 1-4 nucleotides in length, the nucleotide sequence III and the nucleotide sequence IV are equal in length and substantially reverse complementary or fully reverse complementary, the nucleotide sequence III is attached at the 5 'end of the nucleotide sequence I, the nucleotide sequence IV is attached at the 3' end of the nucleotide sequence II, the nucleotide sequence IV is substantially reverse complementary or fully reverse complementary to a second nucleotide sequence that is adjacent to the first nucleotide sequence in the target mRNA and is the same in length as the nucleotide sequence IV.

In some embodiments, the siRNA is one of the following sirnas:

1) The nucleotide sequence I is a sequence shown as SEQ ID NO. 7, the nucleotide sequence II is a sequence shown as SEQ ID NO. 8, at the moment, the siRNA also contains a nucleotide sequence III and a nucleotide sequence IV, the lengths of the nucleotide sequence III and the nucleotide sequence IV are 1 nucleotide, and the base of the nucleotide sequence III is G; at this time, the length ratio of the sense strand to the antisense strand was 20/20; alternatively, the length of the nucleotide sequence III and the length of the nucleotide sequence IV are 2 nucleotides, and the bases of the nucleotide sequence III are A and G in sequence according to the direction from the 5 'end to the 3' end; at this time, the length ratio of the sense strand to the antisense strand was 21/21; alternatively, the nucleotide sequences III and IV are 3 nucleotides in length, and the bases of the nucleotide sequence III are C, A and G in sequence according to the direction from the 5 'end to the 3' end; at this time, the length ratio of the sense strand to the antisense strand was 22/22; alternatively, the nucleotide sequences III and IV are 4 nucleotides in length, and the bases of the nucleotide sequence III are C, C, A and G in sequence according to the direction from the 5 'end to the 3' end; at this time, the length ratio of the sense strand to the antisense strand was 23/23.

2) The nucleotide sequence I is a sequence shown as SEQ ID NO. 9, the nucleotide sequence II is a sequence shown as SEQ ID NO. 10, at the moment, the siRNA also comprises a nucleotide sequence III and a nucleotide sequence IV, the lengths of the nucleotide sequence III and the nucleotide sequence IV are 1 nucleotide, and the base of the nucleotide sequence III is C; at this time, the length ratio of the sense strand to the antisense strand was 20/20; alternatively, the length of the nucleotide sequences III and IV is 2 nucleotides, and the bases of the nucleotide sequences III are C and C in sequence according to the direction from the 5 'end to the 3' end; at this time, the length ratio of the sense strand to the antisense strand was 21/21; alternatively, the nucleotide sequences III and IV are 3 nucleotides in length, and the bases of the nucleotide sequence III are A, C and C in sequence according to the direction from the 5 'end to the 3' end; at this time, the length ratio of the sense strand to the antisense strand was 22/22; alternatively, the nucleotide sequences III and IV are 4 nucleotides in length, and the bases of the nucleotide sequence III are A, A, C and C in sequence according to the direction from the 5 'end to the 3' end; at this time, the length ratio of the sense strand to the antisense strand was 23/23.

3) The nucleotide sequence I is a sequence shown as SEQ ID NO. 11, the nucleotide sequence II is a sequence shown as SEQ ID NO. 12, at the moment, the siRNA also contains a nucleotide sequence III and a nucleotide sequence IV, the lengths of the nucleotide sequence III and the nucleotide sequence IV are 1 nucleotide, and the base of the nucleotide sequence III is G; at this time, the length ratio of the sense strand to the antisense strand was 20/20; alternatively, the length of the nucleotide sequence III and the length of the nucleotide sequence IV are 2 nucleotides, and the bases of the nucleotide sequence III are U and G in sequence according to the direction from the 5 'end to the 3' end; at this time, the length ratio of the sense strand to the antisense strand was 21/21; alternatively, the nucleotide sequences III and IV are 3 nucleotides in length, and the bases of the nucleotide sequence III are A, U and G in sequence according to the direction from the 5 'end to the 3' end; at this time, the length ratio of the sense strand to the antisense strand was 22/22; alternatively, the nucleotide sequences III and IV are 4 nucleotides in length, and the bases of the nucleotide sequence III are A, A, U and G in sequence according to the direction from the 5 'end to the 3' end; at this time, the length ratio of the sense strand to the antisense strand was 23/23.

In some embodiments, nucleotide sequence III and nucleotide sequence IV are the same length and are fully reverse-complementary, thus, given the bases of nucleotide sequence III, the bases of nucleotide sequence IV are also determined.

In some embodiments, the siRNA further comprises a nucleotide sequence V, each nucleotide of the nucleotide sequence V being independently one of the non-fluoro modified nucleotides; the nucleotide sequence V is 1 to 3 nucleotides in length, and is attached to the 3 '-end of the antisense strand to constitute the 3' -overhang of the antisense strand. Thus, the length ratio of the sense strand and the antisense strand of the siRNA provided by the present disclosure can be 19/20, 19/21, 19/22, 20/21, 20/22, 20/23, 21/22, 21/23, 21/24, 22/23, 22/24, 22/25, 23/24, 23/25, or 23/26.

In some embodiments, each nucleotide in the nucleotide sequence V may be any nucleotide. In some embodiments, the nucleotide sequence V is 2 consecutive thymine deoxyribonucleotides (dTdT) or 2 consecutive uracil ribonucleotides (UU) in a 5 'to 3' end direction; alternatively, nucleotide sequence V is fully reverse-complementary to the third nucleotide sequence; the third sequence refers to a nucleotide sequence adjacent to the first or second nucleotide sequence and having a length equal to the nucleotide sequence V in the target mRNA. Thus, in some embodiments, the ratio of the length of the sense strand to the antisense strand of the siRNA of the present disclosure is 19/21 or 21/23, at which time the siRNA of the present disclosure has better mRNA silencing activity.

In some embodiments, the sense strand of the siRNA comprises a nucleotide sequence as set forth in SEQ ID NO. 13 and the antisense strand of the siRNA comprises a nucleotide sequence as set forth in SEQ ID NO. 14:

5'-CGUUAUGAGAUCAAGAUGZA ₁ -3'(SEQ ID NO:13)；

5'-ZB ₁ CAUCUUGAUCUCAUAACGCU-3'(SEQ ID NO:14)；

alternatively, the sense strand of the siRNA comprises the nucleotide sequence shown as SEQ ID NO. 15, and the antisense strand of the siRNA comprises the nucleotide sequence shown as SEQ ID NO. 16:

5'-AGCGUUAUGAGAUCAAGAUGZA ₁ -3'(SEQ ID NO:15)；

5'-ZB ₁ CAUCUUGAUCUCAUAACGCUGG-3'(SEQ ID NO:16)；

wherein the ZB ₁ Is the first nucleotide at the 5' -end of the antisense strand, ZA ₁ Selected from A, U, G or C, and ZB ₁ Is with ZA ₁ Complementary nucleotides.

In some embodiments, the sense strand of the siRNA comprises a nucleotide sequence as set forth in SEQ ID NO:17 and the antisense strand of the siRNA comprises a nucleotide sequence as set forth in SEQ ID NO: 18:

5'-GCAGCGAGGAGUUUCUCAZA ₂ -3'(SEQ ID NO:17)；

5'-ZB ₂ UGAGAAACUCCUCGCUGCGG-3'(SEQ ID NO:18)；

alternatively, the sense strand of the siRNA comprises the nucleotide sequence shown as SEQ ID NO. 19 and the antisense strand of the siRNA comprises the nucleotide sequence shown as SEQ ID NO. 20:

5'-CCGCAGCGAGGAGUUUCUCAZA ₂ -3'(SEQ ID NO:19)；

5'-ZB ₂ UGAGAAACUCCUCGCUGCGGUU-3'(SEQ ID NO:20)；

wherein the ZB ₂ Is the first nucleotide at the 5' -end of the antisense strand, ZA ₂ Selected from A, U, G or C, and ZB ₂ Is with ZA ₂ Complementary nucleotides.

In some embodiments, the sense strand of the siRNA comprises a nucleotide sequence as set forth in SEQ ID NO. 21 and the antisense strand of the siRNA comprises a nucleotide sequence as set forth in SEQ ID NO. 22:

5'-CACAGUGUUUCCCUGUUUZA ₃ -3'(SEQ ID NO:21)；

5'-ZB ₃ AAACAGGGAAACACUGUGCA-3'(SEQ ID NO:22)；

Alternatively, the sense strand of the siRNA comprises the nucleotide sequence shown as SEQ ID NO. 23 and the antisense strand of the siRNA comprises the nucleotide sequence shown as SEQ ID NO. 24:

5'-UGCACAGUGUUUCCCUGUUUZA ₃ -3'(SEQ ID NO:23)；

5'-ZB ₃ AAACAGGGAAACACUGUGCAUU-3'(SEQ ID NO:24)；

wherein the ZB ₃ Is the first nucleotide at the 5' -end of the antisense strand, ZA ₃ Selected from A, U, G or C, and ZB ₃ Is with ZA ₃ Complementary nucleotides.

In some embodiments, the siRNA of the present disclosure is sitmpa 1, sitmpa 2, sittimpb 1, sittimpb 2, sittimpc 1, or sittimpc 2:

siTIMPa1

sense strand: 5'-CGUUAUGAGAUCAAGAUGU-3' (SEQ ID NO: 25)

Antisense strand: 5'-ACAUCUUGAUCUCAUAACGCU-3' (SEQ ID NO: 26)

siTIMPa2

Sense strand: 5'-AGCGUUAUGAGAUCAAGAUGU-3' (SEQ ID NO: 27)

Antisense strand: 5'-ACAUCUUGAUCUCAUAACGCUGG-3' (SEQ ID NO: 28)

siTIMPb1

Sense strand: 5'-GCAGCGAGGAGUUUCUCAU-3' (SEQ ID NO: 29)

Antisense strand: 5'-AUGAGAAACUCCUCGCUGCGG-3' (SEQ ID NO: 30)

siTIMPb2

Sense strand: 5'-CCGCAGCGAGGAGUUUCUCAU-3' (SEQ ID NO: 31)

Antisense strand: 5'-AUGAGAAACUCCUCGCUGCGGUU-3' (SEQ ID NO: 32)

siTIMPc1

Sense strand: 5'-CACAGUGUUUCCCUGUUUU-3' (SEQ ID NO: 33)

Antisense strand: 5'-AAAACAGGGAAACACUGUGCA-3' (SEQ ID NO: 34)

siTIMPc2

Sense strand: 5'-UGCACAGUGUUUCCCUGUUUU-3' (SEQ ID NO: 35)

Antisense strand: 5'-AAAACAGGGAAACACUGUGCAUU-3' (SEQ ID NO: 36)

In some embodiments, the fluoro-modified nucleotides are located in nucleotide sequence I and nucleotide sequence II, the fluoro-modified nucleotides in nucleotide sequence I are no more than 5, and the nucleotides at positions 7, 8, 9 of nucleotide sequence I are fluoro-modified nucleotides in a 5 'end to 3' end direction; the number of the fluoro-modified nucleotides in the nucleotide sequence II is not more than 7, and the nucleotides at positions 2, 6, 14 and 16 of the nucleotide sequence II are fluoro-modified nucleotides.

In some embodiments, the fluoro-modified nucleotides and the non-fluoro-modified nucleotides are located in nucleotide sequence I and nucleotide sequence II, and the nucleotides at positions 7, 8, 9 of the nucleotide sequence I are fluoro-modified nucleotides, in a 5 'to 3' end direction, the nucleotides at the remaining positions in the sense strand being non-fluoro-modified nucleotides; the nucleotides at positions 2, 6, 14 and 16 of the nucleotide sequence II are fluoro modified nucleotides according to the direction from the 5 'end to the 3' end, and the nucleotides at the rest positions in the antisense strand are non-fluoro modified nucleotides.

In some embodiments, the fluoro-modified nucleotides and the non-fluoro-modified nucleotides are located in nucleotide sequence I and nucleotide sequence II, and the nucleotides at positions 5, 7, 8, 9 of the nucleotide sequence I are fluoro-modified nucleotides, in a 5 'end to 3' end direction, the nucleotides at the remaining positions in the sense strand being non-fluoro-modified nucleotides; the nucleotides at positions 2, 6, 14 and 16 of the nucleotide sequence II are fluoro modified nucleotides according to the direction from the 5 'end to the 3' end, and the nucleotides at the rest positions in the antisense strand are non-fluoro modified nucleotides.

In this context, the "fluoro-modified nucleotide" refers to a nucleotide in which the hydroxyl group at the 2' -position of the ribosyl group of the nucleotide is substituted with fluorine, which has a structure represented by the following formula (7). "non-fluoro modified nucleotide" refers to a nucleotide, or nucleotide analogue, in which the hydroxyl group at the 2' -position of the ribosyl of the nucleotide is substituted with a non-fluoro group. In some embodiments, each non-fluoro modified nucleotide is independently selected from one of the 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.

Nucleotides in which the hydroxyl group at the 2 '-position of the ribosyl group is substituted with a non-fluorine group are well known to those skilled in the art and may be selected from one of 2' -alkoxy-modified nucleotides, 2 '-substituted alkoxy-modified nucleotides, 2' -alkyl-modified nucleotides, 2 '-substituted alkyl-modified nucleotides, 2' -amino-modified nucleotides, 2 '-substituted amino-modified nucleotides, 2' -deoxynucleotides.

In some embodiments, the 2 '-alkoxy-modified nucleotide is a methoxy-modified nucleotide (2' -OMe), as shown in formula (8). In some embodiments, the 2' -substituted alkoxy-modified nucleotide may be, for example, a 2' -O-methoxyethyl-modified nucleotide (2 ' -MOE), as shown in formula (9). In some embodiments, 2 '-amino modified nucleotides (2' -NH) ₂ ) As shown in formula (10). In some embodiments, the 2' -Deoxynucleotide (DNA) is represented by formula (11):

nucleotide analogs refer to groups that are capable of replacing nucleotides in a nucleic acid, but that differ in structure from adenine ribonucleotide, guanine ribonucleotide, cytosine ribonucleotide, uracil ribonucleotide, or thymine deoxyribonucleotide. In some embodiments, the nucleotide analog may be an iso-nucleotide, a bridged nucleotide (bridged nucleic acid, abbreviated BNA), or an acyclic nucleotide.

BNA refers to a constrained or inaccessible nucleotide. BNA may contain a five-, six-, or seven-membered ring bridging structure with "fixed" C3' -endo-saccharides tucked. The bridge is typically incorporated at the 2'-, 4' -position of the ribose to provide a 2',4' -BNA nucleotide. In some embodiments, the BNA may be LNA, ENA, cret BNA, or the like, wherein LNA is shown as formula (12), ENA is shown as formula (13), cret BNA is shown as formula (14):

acyclic nucleotides are a class of nucleotides in which the sugar ring of a nucleotide is opened. In some embodiments, the acyclic nucleotide can be an Unlocking Nucleic Acid (UNA) or a Glycerol Nucleic Acid (GNA), wherein UNA is represented by formula (15), and GNA is represented by formula (16):

In the above formula (15) and formula (16), R represents a group selected from the group consisting of H, OH and alkoxy (O-alkyl).

An isopucleotide refers to a compound in which the position of a base on the ribose ring is changed in a nucleotide. In some embodiments, the isonucleotide may be a compound formed by a base moving from the 1' -position to the 2' -position or the 3' -position of the ribose ring, as shown in formula (17) or (18):

in the compounds of the above formulae (17) to (18), base represents a Base selected from A, U, G, C or T; r represents a group selected from the group consisting of H, OH, F and non-fluorine groups as described above.

In some embodiments, the nucleotide analog is selected from one of an iso-nucleotide, LNA, ENA, cET, UNA, and GNA. In some embodiments, each non-fluoro modified nucleotide is a methoxy modified nucleotide, which in the foregoing and below refers to a nucleotide formed by substitution of the 2' -hydroxy group of the ribosyl group with a methoxy group.

In the above and in the following, the meaning of "fluoro modified nucleotide", "2 '-fluoro modified nucleotide", "nucleotide with 2' -hydroxy group of ribose group replaced by fluoro" and "2 '-fluoro ribose group" are the same, and all refer to the compound having the structure shown in formula (7) formed by replacing 2' -hydroxy group of nucleotide by fluoro; the terms "methoxy-modified nucleotide", "2 '-methoxy-modified nucleotide", "nucleotide in which the 2' -hydroxy group of the ribose group is replaced by methoxy" and "2 '-methoxyribosyl" are the same, and refer to a compound having a structure shown in formula (8) formed by replacing the 2' -hydroxy group of the ribosyl group of a nucleotide by methoxy.

In some embodiments, the sirnas of the present disclosure can resist ribonuclease cleavage in blood, thereby increasing the blood stability of the sirnas, allowing the sirnas to have greater resistance to nuclease hydrolysis while maintaining higher target gene modulating activity.

In some embodiments, the sirnas of the present disclosure achieve a high balance of stability and efficiency of gene expression modulation, and some also have the advantage of being simpler and less costly.

In some embodiments, the siRNA of the present disclosure is an siRNA with modifications of:

the nucleotides at positions 7, 8 and 9 of the nucleotide sequence I in the sense strand of the siRNA are fluoro-modified nucleotides, the nucleotides at the rest of the sense strand of the siRNA are methoxy-modified nucleotides in the direction from the 5 'end to the 3' end, and the nucleotides at positions 2, 6, 14 and 16 of the nucleotide sequence II in the antisense strand of the siRNA are fluoro-modified nucleotides in the direction from the 5 'end to the 3' end, the nucleotides at the rest of the antisense strand of the siRNA are methoxy-modified nucleotides.

The nucleotides at positions 5, 7, 8 and 9 of the nucleotide sequence I in the sense strand of the siRNA are fluoro-modified nucleotides, the nucleotides at the rest of the sense strand of the siRNA are methoxy-modified nucleotides, and the nucleotides at positions 2, 6, 14 and 16 of the nucleotide sequence II in the antisense strand of the siRNA are fluoro-modified nucleotides, in the direction from the 5 'end to the 3' end.

In some embodiments, the sirnas of the present disclosure also contain other modified nucleotide groups that do not result in a significant impairment or loss of the function of the sirnas to modulate target gene expression.

Currently, there are a variety of ways available in the art for modifying siRNA, including, in addition to the ribose group modifications mentioned above, backbone modifications (e.g., phosphate group modifications) and base modifications, etc. (see, e.g., watts, J.K., G.F.Deleavey and M.J.damha, chemically modified siRNA: tools and applications. Drug discovery Today,2008.13 (19-20): p.842-55, the entire contents of which are incorporated herein by reference).

In some embodiments, at least 1 of the phosphate groups in the phosphate-sugar backbone of at least one single strand of the sense strand or the antisense strand of the siRNA provided by the present disclosure are phosphate groups having a modifying group. In some embodiments, the phosphate group having a modifying group is a phosphorothioate group formed by substitution of at least one oxygen atom of the phosphodiester bond in the phosphate group with a sulfur atom. In some embodiments, the phosphate group having a modifying group is a phosphorothioate group having a structure as shown in formula (1):

This modification stabilizes the double-stranded structure of the siRNA, maintaining high specificity and high affinity for base pairing.

In some embodiments, the present disclosure provides siRNA 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. In some embodiments, phosphorothioate linkages are present at all of the above positions except the 5' end of the sense strand. In some embodiments, phosphorothioate linkages are present at all of the above positions except the 3' end of the sense strand. In some embodiments, the phosphorothioate linkage is present in at least one of the following positions:

the 5' terminal end of the sense strand is between nucleotide 1 and nucleotide 2;

the 5' terminal end of the sense strand is between nucleotide 2 and nucleotide 3;

the 3' -terminal end of the sense strand is between nucleotide 1 and nucleotide 2;

the 3' -terminal end of the sense strand is between nucleotide 2 and nucleotide 3;

The 5' terminal end of the antisense strand is between nucleotide 1 and nucleotide 2;

the 5' terminal end of the antisense strand is between nucleotide 2 and nucleotide 3;

the 3' -terminal end of the antisense strand is between nucleotide 1 and nucleotide 2; and

the 3' -terminal end of the antisense strand is between nucleotide 2 and nucleotide 3.

In some embodiments, the 5' -terminal nucleotide of the siRNA antisense strand is a 5' -phosphonucleotide or a 5' -phosphoanalog modified nucleotide.

Commonly used such 5 '-phosphonucleotides or 5' -phosphoanalog modified nucleotides are well known to those skilled in the art. In some embodiments, the 5' -phosphate nucleotide has a structure represented by formula (2):

anastasia Khvorova and Jonathan K.Watts, the chemical evolution of oligonucleotide therapies of clinical units Nature Biotechnology,2017,35 (3): 238-48 discloses 5' -phosphoanalog modified nucleotides of formulas (3) to (6):

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

In some embodiments, the 5 '-phosphate analog modified nucleotide is a vinyl phosphate (5' - (E) -vinylphosphate, E-VP) -containing nucleotide of formula (3), or a phosphorothioate-containing nucleotide of formula (5).

In some embodiments, the sirnas provided by the present disclosure can be the sirnas shown in tables 1A-1C.

Table 1A siRNA sequences in some embodiments

Table 1B siRNA sequences in some embodiments

TABLE 1 siRNA sequences in some embodiments

The inventors of the present disclosure have unexpectedly found that the siRNA provided by the present disclosure not only has significantly enhanced plasma and lysosomal stability, but also retains very high gene suppression activity.

In the siRNA disclosed in the present disclosure, 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 a negative charge, and the siRNA can exist in a form of hydroxyl group or sulfhydryl group, and hydrogen ions in the hydroxyl group or sulfhydryl group can also be partially or completely replaced by cations. The cation may be any cation, such as a metal cation, ammonium ion NH4 ⁺ One of the organic ammonium cations. In some embodiments, 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 K ⁺ And/or Na ⁺ The tertiary amine forming cation may be triethylamine forming ammonium ion and/or N, N-diisopropylethylamine forming ammonium ion. Thus, the siRNA of the present disclosure may be present at least in part in the form of a salt. In one embodiment, the non-bridging oxygen or sulfur atoms in the phosphodiester or phosphorothioate linkages are at least partially bound to sodium ions, and the siRNA of the present disclosure is in the form of a sodium salt or partial sodium salt.

The siRNAs of the present disclosure can be prepared by conventional methods, such as solid phase phosphoramidite synthesis, as is well known in the art, or can be prepared by commercial custom synthesis.

It is clear to those skilled in the art that modified nucleotide groups can be introduced into the siRNAs described in the present disclosure by using nucleoside monomers with 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.

Pharmaceutical composition

The present disclosure provides a pharmaceutical composition containing the siRNA as described above as an active ingredient and a pharmaceutically acceptable carrier.

The pharmaceutically acceptable carrier may be a carrier conventionally used in the field of siRNA administration, such as, but not limited to, magnetic nanoparticles (magnetic nanoparticles, e.g. based on Fe ₃ O ₄ Or Fe (Fe) ₂ O ₃ Carbon nanotubes), mesoporous silica (mesoporous silicon), calcium phosphate nanoparticles (calcium phosphate nanoparticles), polyethylenimine (PEI), polyamidedendrimers (polyamidoamine (PAMAM) dendrimer), polylysine (L-lysine), PLL, chitosan (chitosan), 1, 2-dioleoyl-3-trimethylammoniopropane (1, 2-dioleoyl-3-trimethylonium-propane, DOTAP), poly-D-or L-lactic acid/glycolic acid copolymers (poly (D) &L-lactic/glycolic acid) copolymer, PLGA), poly (aminoethylethylene phosphate) (poly (2-aminoethyl ethylene phosphate), PPEEA) and poly (N, N-dimethylaminoethyl methacrylate) (poly (2-dimethylaminoethyl methacrylate), PDMAEMA) and derivatives thereof.

In some embodiments, the amount of siRNA and pharmaceutically acceptable carrier in the pharmaceutical composition is not particularly limited, and in some embodiments, the weight ratio of siRNA to pharmaceutically acceptable carrier may be 1 (1-500), and in some embodiments, the weight ratio is 1 (1-50).

In some embodiments, the pharmaceutical composition may further comprise other pharmaceutically acceptable excipients, which may be one or more of various formulations or compounds conventionally employed in the art. For example, the pharmaceutically acceptable additional excipients may include at least one of a pH buffer, a protectant, and an osmolality adjusting agent.

The pH buffer solution can be a tris hydrochloride buffer solution with the pH value of 7.5-8.5 and/or a phosphate buffer solution with the pH value of 5.5-8.5, for example, the pH value of 5.5-8.5.

The protective agent may be at least one of inositol, sorbitol, sucrose, trehalose, mannose, maltose, lactose, and glucose. The protective agent may be present in an amount of 0.01 to 30% by weight, based on the total weight of the pharmaceutical composition.

The osmolality adjusting agent may be sodium chloride and/or potassium chloride. The osmolality adjusting agent is present in an amount such that the osmolality of the pharmaceutical composition is 200-700 milliosmoles per kilogram (mOsm/kg). The amount of osmolality adjusting agent can be readily determined by one skilled in the art based on the desired osmolality.

In some embodiments, the pharmaceutical composition may be a liquid formulation, such as an injection; or freeze-dried powder injection, and is mixed with liquid adjuvant to make into liquid preparation. The liquid formulation may be administered, but is not limited to, for subcutaneous, intramuscular or intravenous injection, and may be administered, but is not limited to, by spraying to the lungs, or by spraying through the lungs to other visceral tissues such as the liver. In some embodiments, the pharmaceutical composition is for intravenous administration.

In some embodiments, the pharmaceutical composition may be in the form of a liposomal formulation. In some embodiments, the pharmaceutically acceptable carrier used in the liposomal formulation comprises an amine-containing transfection compound (which may also be referred to hereinafter as an organic amine), a helper lipid, and/or a pegylated lipid. Wherein the organic amine, the helper lipid and the pegylated lipid may be selected from one or more of the amine-containing transfection compounds described in CN103380113a (which is incorporated herein by reference in its entirety) or pharmaceutically acceptable salts or derivatives thereof, the helper lipid and the pegylated lipid, respectively.

In some embodiments, the organic amine may be a compound as depicted in formula (201) described in CN103380113a or a pharmaceutically acceptable salt thereof:

wherein:

X ₁₀₁ and X ₁₀₂ Each independently is O, S, N-A or C-A, wherein A is hydrogen or se:Sub>A C1-C20 hydrocarbon chain;

Y ₁₀₁ and Z ₁₀₁ Each independently is c= O, C = S, S = O, CH-OH or SO ₂ ；

R ₁₀₁ 、R ₁₀₂ 、R ₁₀₃ 、R ₁₀₄ 、R ₁₀₅ 、R ₁₀₆ And R is ₁₀₇ Each independently is hydrogen, a cyclic or acyclic, substituted or unsubstituted, branched or straight chain aliphatic group, a cyclic or acyclic, substituted or unsubstituted, branched or straight chain heteroaliphatic group, a substituted or unsubstituted, branched or straight chain acyl group, a substituted or unsubstituted, branched or straight chain aryl group, a substituted or unsubstituted, branched or straight chain heteroaryl group;

x is an integer from 1 to 10;

n is an integer from 1 to 3, m is an integer from 0 to 20, and p is 0 or 1; wherein, if m=p=0, then R ₁₀₂ Is hydrogen;

and, if at least one of n or m is 2, then R ₁₀₃ And nitrogen in formula (201) forms a structure as shown in formula (202) or formula (203):

wherein g, e and f are each independently an integer of 1 to 6, "HCC" represents a hydrocarbon chain, and each of N represents a nitrogen atom in formula (201).

In some embodiments, R ₁₀₃ Is a polyamine. In other embodiments, R ₁₀₃ Is a ketal. In some embodiments, R in formula (201) ₁₀₁ And R is ₁₀₂ Independently any substituted or unsubstituted, branched or straight chain alkyl or alkenyl group having from 3 to about 20 carbon atoms, such as from 8 to about 18 carbon atoms, and from 0 to 4 double bonds, such as from 0 to 2 double bonds.

In some embodiments, if each of n and m independently has a value of 1 or 3, then R ₁₀₃ Any one of the following formulas (204) - (213) may be used:

wherein in the formulae (204) - (213), g, e and f are each independently an integer of 1 to 6, each "HCC" represents a hydrocarbon chain, and each shows R ₁₀₃ Possible points of attachment to the nitrogen atom in formula (201), wherein each H at any of the x positions may be replaced to effect attachment to the nitrogen atom in formula (201).

Wherein the compound of formula (201) may be prepared according to the description in CN103380113 a.

In some embodiments, the organic amine is an organic amine as shown in formula (214) and/or an organic amine as shown in formula (215):

the auxiliary lipid is cholesterol, cholesterol analogues and/or cholesterol derivatives;

the polyethylene glycol lipid is 1, 2-dipalmitoyl amide-sn-glycerin-3-phosphatidylethanolamine-N- [ methoxy (polyethylene glycol) ] -2000.

In some embodiments, the molar ratio between the organic amine, the helper lipid, and the pegylated lipid in the pharmaceutical composition is (19.7-80): (19.7-80): (0.3-50), for example, (50-70): (20-40): (3-20).

In some embodiments, the particles of the pharmaceutical composition formed from the siRNA of the present disclosure and the amine-containing transfection reagent described above have an average diameter of about 30nm to about 200nm, typically about 40nm to about 135nm, more typically the average diameter of the liposome particles is about 50nm to about 120nm, about 50nm to about 100nm, about 60nm to about 90nm, or about 70nm to about 90nm, e.g., the average diameter of the liposome particles is about 30, 40, 50, 60, 70, 75, 80, 85, 90, 100, 110, 120, 130, 140, 150, or 160nm.

In some embodiments, the weight ratio (weight/weight ratio) of siRNA to total lipid (e.g., organic amine, helper lipid, and/or pegylated lipid) in a pharmaceutical composition formed from an siRNA of the present disclosure and an amine-containing transfection reagent as described above is in the range of from about 1:1 to about 1:50, from about 1:1 to about 1:30, from about 1:3 to about 1:20, from about 1:4 to about 1:18, from about 1:5 to about 1:17, from about 1:5 to about 1:15, from about 1:5 to about 1:12, from about 1:6 to about 1:12, or from about 1:6 to about 1:10, e.g., the weight ratio of siRNA of the present disclosure to total lipid is about 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, or 1:18.

In some embodiments, the components of the pharmaceutical composition may be present independently at the point of sale and may be present in liquid formulations at the point of use. In some embodiments, the pharmaceutical compositions formed by the sirnas provided by the present disclosure and the pharmaceutically acceptable carriers described above can be prepared according to various known methods, except that the sirnas provided by the present disclosure are used instead of the existing sirnas; in some embodiments, it may be prepared as follows:

suspending organic amine, auxiliary lipid and polyethylene glycol lipid in alcohol according to the molar ratio, and uniformly mixing to obtain lipid solution; the amount of alcohol is such that the total mass concentration of the resulting lipid solution is 2-25mg/mL, for example, 8-18mg/mL. The alcohol is selected from pharmaceutically acceptable alcohols, such as alcohols that are liquid near room temperature, e.g., one or more of ethanol, propylene glycol, benzyl alcohol, glycerol, polyethylene glycol 200, polyethylene glycol 300, polyethylene glycol 400, e.g., may be ethanol.

The siRNA provided by the present disclosure is dissolved in a buffer salt solution to obtain an siRNA aqueous solution. The concentration of the buffer salt solution is 0.05-0.5M, for example, may be 0.1-0.2M, the pH of the buffer salt solution is adjusted to 4.0-5.5, for example, may be 5.0-5.2, and the amount of the buffer salt solution is such that the concentration of siRNA does not exceed 0.6mg/mL, for example, may be 0.2-0.4mg/mL. The buffer salt is selected from one or more of soluble acetate and soluble citrate, and can be sodium acetate and/or potassium acetate.

Mixing the lipid solution with siRNA water solution, and incubating the mixed product at 40-60deg.C for at least 2 min, such as 5-30 min, to obtain liposome preparation after incubation. The volume ratio of the lipid solution to the siRNA aqueous solution is 1: (2-5), for example, may be 1:4.

Concentrating or diluting the incubated liposome preparation, removing impurities, and sterilizing to obtain the pharmaceutical composition provided by the disclosure, wherein the physical and chemical parameters are that the pH value is 6.5-8, the encapsulation efficiency is not lower than 80%, the particle size is 40-200nm, the polydispersity index is not higher than 0.30, and the osmotic pressure is 250-400mOsm/kg; for example, the physical and chemical parameters can be pH 7.2-7.6, encapsulation efficiency not lower than 90%, particle size 60-100nm, polydispersity index not higher than 0.20, and osmotic pressure 300-400mOsm/kg.

Wherein concentration or dilution may be performed before, after, or simultaneously with removal of impurities. As a method for removing impurities, various methods are available, for example, a tangential flow system, a hollow fiber column, ultrafiltration at 100K Da, and Phosphate Buffer (PBS) of pH7.4 as an ultrafiltration exchange solution can be used. As a method of sterilization, various methods are available, and for example, filtration sterilization on a 0.22 μm filter can be used.

siRNA of the present disclosure, and application of pharmaceutical composition containing the same

In some embodiments, the present disclosure provides a method of treating or ameliorating a disease associated with TIMP-1 gene expression, the method comprising administering to a subject in need thereof an effective amount of an siRNA and/or a pharmaceutical composition of the present disclosure.

By administering the siRNA active ingredients of the present disclosure to a subject in need thereof, the goal of treating or ameliorating a disease associated with TIMP-1 gene expression can be achieved by a mechanism of RNA interference. Thus, the siRNA and/or pharmaceutical compositions of the present disclosure may be used for treating or ameliorating a disease associated with TIMP-1 gene expression, or for the preparation of a medicament for treating or ameliorating a disease associated with TIMP-1 gene expression.

The disease related to TIMP-1 gene expression refers to a disease caused by the overexpression of the TIMP-1 gene in cells, such as fibrosis disease and/or fibril formation disease. Specific examples of such diseases include, but are not limited to: liver fibrosis, kidney fibrosis, lung fibrosis, peritoneal fibrosis, vocal cord fibrosis, intestinal fibrosis, bone marrow fibrosis, heart fibrosis, fibrosis associated with cerebral infarction, abnormal scarring associated with all possible types of accidental or iatrogenic skin injury, scleroderma, glaucoma filtration failure, intestinal adhesion, cirrhosis or chronic liver injury.

The term "administration" as used herein refers to placement of an siRNA and/or pharmaceutical composition of the present disclosure into a subject by a method or route that results in, at least in part, positioning of the siRNA and/or pharmaceutical composition of the present disclosure at a desired site to produce a desired effect. Routes of administration suitable for the methods of the present disclosure include topical and systemic administration. In general, topical administration results in more siRNA active ingredient being delivered to a particular site than the entire body of the subject; whereas systemic administration results in delivery of the siRNA and/or pharmaceutical compositions of the present disclosure to substantially the entire body of the subject. It is contemplated that the present disclosure is directed to providing means for treating or ameliorating liver fibrosis, in some embodiments, a mode of administration capable of delivering a drug to the liver.

The administration to the subject may be by any suitable route known in the art, including but not limited to: oral or parenteral routes such as intravenous, intramuscular, subcutaneous, transdermal, airway (aerosol), pulmonary, nasal, rectal and topical (including buccal and sublingual). The frequency of administration may be 1 or more times daily, weekly, biweekly, tricyclically, monthly or yearly.

The dosages of siRNA or pharmaceutical compositions described in the present disclosure may be dosages conventional in the art, which may be determined according to various parameters, particularly the age, weight and sex of the subject. Toxicity and efficacy can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for LD ₅₀ (dose lethal to 50% of the population) and ED ₅₀ (in dose response refers to the dose that causes 50% of the maximum intensity of response, in mass response refers to the dose that causes 50% of the subjects to develop a positive response). The range of doses for human use can be derived based on data obtained from cell culture assays and animal studies.

Upon administration of the siRNA and/or pharmaceutical compositions described in the present disclosure, for example, for male or female, C57BL/6J mice of 6-12 weeks of age, body weight 18-25g, in terms of siRNA: for pharmaceutical compositions of siRNA with a pharmaceutically acceptable carrier, the siRNA may be used in an amount of 0.001 to 50mg/kg body weight, in further embodiments 0.01 to 10mg/kg body weight, in still further embodiments 0.05 to 5mg/kg body weight, and in yet further embodiments 0.1 to 3mg/kg body weight.

In some embodiments, the present disclosure provides a method of inhibiting TIMP-1 gene expression in a cell, the method comprising contacting an effective amount of an siRNA and/or a pharmaceutical composition of the present disclosure with the cell, introducing the siRNA and/or the pharmaceutical composition of the present disclosure into the cell, and inhibiting TIMP-1 gene expression in the cell by a mechanism of RNA interference. The cells may be selected from hepatic stellate cells, renal stellate cells, or pulmonary stellate cells. In some embodiments, the cell is a hepatic stellate cell.

The methods provided by the present disclosure are used to inhibit expression of the TIMP-1 gene in a cell, the amount of the modified siRNA provided and/or the siRNA in the pharmaceutical composition is generally such that: it is sufficient to reduce expression of the target gene and results in an extracellular concentration of 1pM to 1. Mu.M, or 0.01nM to 100nM, or 0.05nM to 50nM, or to about 5nM at the surface of the target cell. The amount required to achieve this local concentration will vary depending on a variety of factors including the method of delivery, the site of delivery, the number of cell layers between the site of delivery and the target cell or tissue, whether the delivery is local or systemic, etc. The concentration at the delivery site may be significantly higher than the concentration at the surface of the target cell or tissue.

Kit for detecting a substance in a sample

The present disclosure provides a kit comprising an effective amount of at least one of the modified siRNA and the pharmaceutical composition of the present disclosure.

In some embodiments, the kits described herein can provide modified siRNA in one container. In some embodiments, the kits described herein can comprise a container that provides a pharmaceutically acceptable excipient. In some embodiments, other ingredients, such as stabilizers or preservatives, and the like, may also be included in the kit. In some embodiments, the kits described herein can comprise at least one additional therapeutic agent in a container other than the container in which the modified siRNA described herein is provided. In some embodiments, the kit can comprise instructions for mixing the modified siRNA with a pharmaceutically acceptable carrier and/or adjuvant or other ingredients, if any.

In the kits of the present disclosure, the modified siRNA and pharmaceutically acceptable carrier and/or adjuvant and the pharmaceutical composition and/or pharmaceutically acceptable adjuvant may be provided in any form, such as liquid form, dry form or lyophilized form. In some embodiments, the modified siRNA and pharmaceutically acceptable carrier and/or adjuvant, and the pharmaceutical composition and optional pharmaceutically acceptable adjuvant are substantially pure and/or sterile. In some embodiments, sterile water may be provided in a kit of the present disclosure.

The present disclosure will be further illustrated by the following examples, but the present disclosure is not limited thereby.

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)).

Hela cells were obtained from the university of Beijing institute of molecular medicine, nucleic acid technology laboratories, and cultured in DMEM complete medium (Hyclone) containing 20% fetal bovine serum (FBS, hyclone) and 0.2% by volume of Streptomyces griseus (Penicillin-Streptomycin, gibco, invitrogen) at 37℃in 5% CO ₂ Culture in an incubator with air at 95%.

Unless otherwise indicated, the reagent ratios provided below are all calculated as volume ratios (v/v).

PREPARATION EXAMPLE 1 Synthesis of siRNA sequences

In siRNA synthesis, unless otherwise specified, the nucleoside monomer (nucleoside monomer) refers to a modified nucleoside phosphoramidite monomer (modified RNA phosphoramidites) used in phosphoramidite solid phase synthesis depending on the kind and order of nucleotides in the siRNA to be prepared. Phosphoramidite solid phase synthesis is a method well known to those skilled in the art for use in RNA synthesis. The nucleoside monomers used are commercially available unless otherwise specified.

(1-a) solid phase phosphoramidite method

The siRNA sequences listed in Table 2 were obtained by the solid phase phosphoramidite method.

For the sense strand, a universal solid support (UnyLinker TMloaded)HL Solid Supports, kinovate Life Sciences company) and ligating nucleoside monomers one by one from the 3'-5' direction in the nucleotide arrangement order. Each nucleoside monomer attached includes four steps of deprotection, coupling, capping, oxidation or vulcanization. Wherein, when two nucleotides are connected by phosphate, the connection of the latter nucleoside monomer comprises deprotection, coupling, capping and oxidation. When phosphorothioate is adopted to connect two nucleotides, the protection is included when the latter nucleoside monomer is connected, Coupling, capping and vulcanizing.

The synthesis conditions were given as follows:

the nucleoside monomer was provided as a 0.1M acetonitrile solution, the deprotection conditions were the same for each step, i.e., the temperature was 25 ℃, the reaction time was 70 seconds, the deprotection reagent was a dichloromethane solution of dichloroacetic acid (3% v/v), and the molar ratio of dichloroacetic acid to 4,4' -dimethoxytrityl protecting group on the solid support was 5:1.

The coupling reaction conditions of each step are the same, the temperature is 25 ℃, the mole ratio of the nucleic acid sequence connected on the solid carrier to the nucleoside monomer is 1:10, the mole ratio of the nucleic acid sequence connected on the solid carrier to the coupling reagent is 1:65, the reaction time is 600 seconds, and the coupling reagent is a 0.5M acetonitrile solution of 5-ethylthio-1H-tetrazole.

The capping conditions were the same for each step, including a temperature of 25℃and a reaction time of 15 seconds. The capping reagent solution is a mixed solution of CapA and CapB with a mol ratio of 1:1, wherein CapA is a pyridine/acetonitrile mixed solution of 20 volume percent N-methylimidazole, and the volume ratio of pyridine to acetonitrile is 3:5; capB is a 20% by volume acetic anhydride in acetonitrile. The molar ratio of the capping reagent to the nucleic acid sequence attached to the solid support is acetic anhydride: N-methylimidazole: nucleic acid sequence attached to the solid support=1:1:1.

The oxidation reaction conditions are the same in each step, the temperature is 25 ℃, the reaction time is 15 seconds, and the oxidizing agent is iodine water with the concentration of 0.05M. The molar ratio of iodine to nucleic acid sequence attached to the solid support in the coupling step was 30:1. The reaction was carried out in a mixed solvent of tetrahydrofuran, water, pyridine=3:1:1.

The conditions for each step of sulfiding reaction were the same, including a temperature of 25 ℃, a reaction time of 300 seconds, and the sulfiding reagent was hydrogenation Huang Yuansu. The molar ratio of sulfiding reagent to nucleic acid sequence attached to the solid support in the coupling step was 120:1. The reaction was carried out in a mixed solvent of acetonitrile: pyridine=1:1.

The cleavage and deprotection conditions were as follows: the synthesized nucleotide sequence with the carrier attached was added to ammonia water at a concentration of 25wt% at an amount of 0.5 ml/. Mu.mol, reacted at 55℃for 16 hours, the liquid was removed, and concentrated to dryness in vacuo.

Purifying and desalting: purification of nucleic acids was accomplished by gradient elution with NaCl using a preparative ion chromatography purification column (Source 15Q). Specifically, the method comprises the following steps: eluent A:20mM sodium phosphate (pH 8.1), solvent water/acetonitrile=9:1 (volume ratio); eluent B:1.5M sodium chloride, 20mM sodium phosphate (pH 8.1), solvent water/acetonitrile=9:1 (volume ratio); elution gradient: eluent a, eluent b=100:0-50:50 gradient elution. Collecting and combining product eluents, desalting by using a reversed phase chromatographic purification column, wherein specific conditions comprise desalting by using a sephadex column, eluting with deionized water by using sephadex G25 as a filler.

And (3) detection: purity was checked using ion exchange chromatography (IEX-HPLC) and molecular weight was analyzed using liquid chromatography-mass spectrometry (LC-MS). The measured value matches the theoretical value, indicating that the sense strand S was synthesized.

For the antisense strand, the synthesis conditions are the same as for the sense strand, except that the corresponding nucleoside monomers are used in the nucleotide order of the antisense strand.

(1-b) introduction of 5' -VP modification

For siRNA 2, the first nucleotide at the 5' -end of its antisense strand has a 5' -VP modification, which is introduced by using a vinyl phosphate modified 2' -methoxy modified uracil nucleoside monomer (VP-Um) in the above solid phase phosphoramidite synthesis procedure, which is synthesized as follows:

synthesis of (1-b-1) VP-U-2

VP-U-2 molecules were synthesized according to the following procedure:

2 '-methoxy-modified uridine (2' -OMe-U,51.30g,91.6 mmol), tert-butyldiphenylchlorosilane (TBDPSCl, 50.35g,183.2 mmol), imidazole (12.47 g,183.2 mmol) were dissolved in 450ml of N, N-Dimethylformamide (DMF) and stirred at room temperature for 20h. DMF was distilled off, dissolved in 600ml of dichloromethane, washed with 300ml of saturated sodium bicarbonate, the aqueous phase was extracted 3 times with Dichloromethane (DCM), 300ml each time, the organic phases were combined, washed with 5% oxalic acid to pH <5, and the solvent was evaporated to dryness to give crude VP-U-1 which was used directly in the subsequent synthesis of VP-U-2.

After the VP-U-1 crude product is dissolved by using 100ml of dichloromethane, the mixture is stirred for 10 minutes by adding ice bath, 450ml of 2% p-toluenesulfonic acid solution (the solvent is a mixed solvent of methanol and dichloromethane with the volume ratio of 3:7) which is refrigerated in a refrigerator at the temperature of 4 ℃ in advance is added, and the reaction is carried out for 10 minutes. The reaction was quenched by addition of 200ml of saturated sodium bicarbonate and the organic phase was washed with saturated aqueous sodium bicarbonate to ph=8. The aqueous phases were combined, extracted 2 times with 200ml of dichloromethane each time, the organic phases were combined, washed once with 200ml of saturated brine and the solvent evaporated to dryness. Purifying by 200-300 mesh normal phase silica gel column, loading petroleum ether into column, gradient eluting with petroleum ether, ethyl acetate, dichloromethane, methanol=1:1:0.05-1:1:1:0.25, collecting product eluent, evaporating solvent under reduced pressure, and foaming and drying by vacuum oil pump to obtain pure VP-U-2 40.00g. ¹ H NMR(400MHz,DMSO-d6)δ7.96(d,J＝7.8Hz,1H),7.64(dtd,J＝5.1,4.0,2.2Hz,4H),7.41–7.30(m,6H),6.79(d,J＝4.7Hz,1H),5.73(d,J＝7.6Hz,1H),4.94(t,J＝7.0Hz,1H),4.12(td,J＝4.6,3.9Hz,1H),4.05(dd,J＝4.8,4.0Hz,1H),3.96(t,J＝4.7Hz,1H),3.68(ddd,J＝11.8,7.0,4.6Hz,1H),3.57–3.46(m,1H),3.39(s,3H),1.05(s,8H).MS m/z：C ₂₆ H ₃₃ N ₂ O ₆ Si，[M+H] ⁺ Theory: 497.21, found: 497.45.

(1-b-2) Synthesis of VP-U-4:

VP-U-2 (19.84 g,40.0 mmol), dicyclohexylcarbodiimide (DCC, 16.48g,80.0 mmol), pyridine (4.20 g,53.2 mmol), trifluoroacetic acid (6.61 g,53.2 mmol) were mixed and dissolved in 200ml of dimethyl sulfoxide (DMSO), and the mixture was stirred at room temperature for 20 hours to obtain a reaction solution for use. Taking another partTetraethyl methylenediphosphate (21.44 g,74.4 mmol) was dissolved in 120ml of THF, cooled in an ice bath, t-BuOK (11.36 g,101.2 mmol) was added at ice bath temperature, reacted for 10min at ice bath temperature, then allowed to react at room temperature for 0.5h, then added to the above reaction solution, after about 1h addition, reacted for 1h at ice bath temperature, and then allowed to react at room temperature for 18h. The reaction was quenched with water and the aqueous phase was extracted 3 times with 200ml of dichloromethane. The organic phases were combined, washed once with 200ml of saturated brine and the solvent was evaporated to dryness. Purifying with 200-300 mesh normal phase silica gel column, loading petroleum ether into column, gradient eluting with petroleum ether:ethyl acetate=1:1-1:4, collecting product eluent, evaporating solvent under reduced pressure, and vacuum oil pump foaming and drying to obtain pure VP-U-4 14.00g. ¹ H NMR(400MHz,DMSO-d6)δ7.96(d,J＝7.8Hz,1H),7.64(dtd,J＝5.1,4.0,2.2Hz,4H),7.41–7.30(m,6H),6.82–6.71(m,2H),5.90(ddd,J＝25.9,15.0,1.0Hz,1H),5.73(d,J＝7.6Hz,1H),4.36–4.21(m,3H),4.18(t,J＝4.9Hz,1H),4.05(ddq,J＝9.7,8.5,6.9Hz,2H),3.87(t,J＝4.8Hz,1H),3.39(s,3H),1.32(td,J＝6.9,0.7Hz,6H),1.05(s,8H).MS m/z：C ₃₁ H ₄₂ N ₂ O ₈ PSi，[M+H] ⁺ Theory: 629.24, found: 629.51.

(1-b-3) Synthesis of VP-U-5:

VP-U-4 (14.00 g,22.29 mmol) was dissolved in 100ml tetrahydrofuran, and triethylamine trihydrofluoric acid (17.96 g,111.45 mmol) was added thereto, followed by stirring at room temperature for 20 hours to complete the reaction. The solvent was evaporated directly to dryness, dissolved with dichloromethane and evaporated to dryness 2 times, using 50ml of dichloromethane each time, to give the crude product. Purifying with 200-300 mesh normal phase silica gel column, loading petroleum ether into column, gradient eluting with petroleum ether, ethyl acetate, dichloromethane, methanol=1:1:1:0.05-1:1:1:0.25, collecting product eluate, evaporating solvent under reduced pressure, and foaming and drying with vacuum oil pump to obtain pure VP-U-5 with total weight of 6.70g. ¹ H NMR(400MHz,DMSO-d6)δ7.96(d,J＝7.8Hz,1H),6.77(dd,J＝15.0,6.2Hz,1H),5.99–5.82(m,2H),5.73(d,J＝7.6Hz,1H),5.27(d,J＝5.1Hz,1H),5.10(dd,J＝5.3,4.7Hz,1H),4.29(ddq,J＝9.8,8.6,7.0Hz,2H),4.17(ddd,J＝6.2,5.2,1.0Hz,1H),4.12–3.98(m,3H),3.39(s,2H),1.32(td,J＝6.9,0.6Hz,6H).MS m/z：C ₁₅ H ₂₄ N ₂ O ₈ P，[M+H] ⁺ Theory: 391.13, found: 391.38.

(1-b-4) Synthesis of VP-U-6:

VP-U-5 (399mg, 1.0 mmol), pyridine trifluoroacetate (0.232 g,1.2 mmol), N-methylimidazole (0.099 g,1.2 mmol) and bis (diisopropylamino) (2-cyanoethoxy) phosphine (0.452 g,1.5 mmol) were added to 10ml of anhydrous dichloromethane under argon atmosphere, and the mixture was stirred at room temperature for 5 hours. Evaporating the solvent to dryness, purifying by column chromatography (200-300 mesh normal phase silica gel, dichloromethane: acetonitrile (containing 0.5 wt% triethylamine) =3:1-1:3 gradient elution), collecting product eluent, concentrating to remove the solvent to obtain the target product VP-U-6 of 508mg. ³¹ P NMR(161MHz,DMSO-d6)δ150.34,150.29,17.07,15.50.MS m/z：C ₂₄ H ₄₁ N ₄ O ₉ P ₂ ，[M+H] ⁺ Theory: 591.23, found: 591.55. VP-U-6 is shown to be the target product VP-Um, and is used as a nucleoside monomer to participate in RNA chain synthesis.

(1-c) introduction of 5' -P modification

In the preparation of antisense strand according to solid-phase phosphoramidite method, 5' -P modification is carried out on the first nucleotide of 5' -end of antisense strand of siRNA 1, 3, 4, 5, 9, and CPR-I monomer (Suzhou Ji Ma, cat# 13-2601-XX) is connected to 5' -end of antisense strand by four steps of deprotection, coupling, capping and oxidation after the last nucleoside monomer of antisense strand is connected, thus forming 5' -phosphate modification (5 ' -P modification).

The conditions and reagents used for the deprotection, coupling, capping, and oxidation reactions are the same as those for the solid phase phosphoramidite method described above.

After the introduction of the 5' -P modification described above, the cleavage-deprotection step AS described above is further performed to obtain the antisense strand AS.

(1-d) annealing to form double-stranded siRNA

The equimolar sense strand and antisense strand were dissolved separately using DEPC water (available from Amresco, cat. No. E174) to prepare the desired concentrations, which were heated to 70-95℃and then cooled at room temperature to form a double-stranded structure via hydrogen bonds.

For the obtained double-stranded siRNA, the molecular weight thereof was detected by LC-MS, confirming that the product structure was consistent with the target siRNA, indicating that the double-stranded siRNA having the target sequence was obtained.

TABLE 2 siRNA sequences

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dT represents thymine deoxyribonucleotide.

Experimental example 1 detection of the inhibition efficiency of siRNA on TIMP-1mRNA expression level in HeLa cells.

The siRNA obtained in preparation 1 was transfected into HeLa cells using Lipofectamine 2000 at a final siRNA concentration of 50nM, respectively. 3 duplicate wells were transfected with each siRNA. Cells not treated with any siRNA served as a blank.

The amount of TIMP-1mRNA expressed in HeLa cells transfected with each siRNA was detected by real-Time fluorescent quantitative PCR (Quantitative Real-Time PCR). The method comprises the following specific steps: after 24 hours of culturing the transfected cells, total RNA was extracted from the cells using Trizol (Thermo Fisher Co.) according to standard procedures for total RNA extraction; mu.g of total RNA was taken and reverse transcribed to obtain cDNA using a reverse transcription kit (Promega Corp., cat. No. A3500) according to the procedure described in the specification. TIMP-1mRNA expression level was measured by using a 2X Ultra SYBR Mixture (with ROX) (Beijing Kao is century Biotech Co., ltd., product No. CW 0956) kit and using cDNA as a template according to the procedure of the specification. Among them, PCR primers for amplifying TIMP-1 and GAPDH as an internal reference gene are shown in Table 3.

TABLE 3 primer information

TIMP-1mRNA expression level was calculated as follows: TIMP-1mRNA expression level= (test set TIMP-1mRNA expression level/test set GAPDH mRNA expression level)/(control set TIMP-1mRNA expression level/control set GAPDH mRNA expression level) ×100%.

mRNA inhibition ratio= (1-TIMP-1 mRNA expression amount) ×100%. Wherein each test group is Hela cells treated with each siRNA respectively, and the control group is Hela cells not treated with siRNA. The results are shown in Table 4.

TABLE 4 in vitro Activity assay of siRNA

siRNA	Numbering device	mRNA inhibition Rate (%)
			siRNA N1	siTIMP1	86
siRNA 1	siTIMPa1M1SP	83
			siRNA 2	siTIMPa1UM1SVP	85
siRNA 3	siTIMPa1M2SP	75
			siRNA N2	siTIMP2	86
siRNA 4	siTIMPb1M1SP	85
			siRNA 5	siTIMPb1M1S	82
siRNA 6	siTIMPb1M3S	60
			siRNA N3	siTIMP3	85
siRNA 7	siTIMPc1M1SP	82
			siRNA 8	siTIMPc1M1	80
siRNA 9	siTIMPc1M2	70
			siRNA 10	siTIMPc1M3	62

mRNA inhibition was the average of the inhibition of TIMP-1mRNA by siRNA in 3 wells.

As can be seen from table 4, the modified siRNA provided by the present disclosure has higher inhibitory activity in Hela cell line.

Experimental example 2 stability assay of siRNA in lysosomes

Test sample preparation by lysosome lysate treatment: 6 μl of each siRNA (20 μM) of preparation 1 was mixed with 27.2 μl sodium citrate aqueous solution (pH5.0), 4.08 μl deionized water and 2.72 μl murine lysosome lysate (Rat Liver Tritosomes, xenotech company, cat# R0610.LT, lot # 1610069) respectively, to give a final acid phosphatase concentration of 0.2 mU/. Mu.L. Incubate at 37 ℃. Mu.l of each of the mixtures was taken out at 0, 2, 6 and 24 hours, denatured by adding to 15. Mu.l of 9M urea solution, followed by adding to 4. Mu.l of 6 Xload buffer (Soy Corp., cat. 20160830), and immediately freezing to-80℃to terminate the reaction. And 0 hours represents the moment when the sample to be tested is immediately taken out after being uniformly mixed with the lysosome lysate.

Reference sample preparation without lysosomal lysate treatment: 1.5. Mu.l of each siRNA (20. Mu.M) was mixed with 7.5. Mu.L of aqueous sodium citrate (pH 5.0) and 1. Mu.L of deionized water, respectively, and denatured by adding 30. Mu.L of 9M urea solution, followed by adding 8. Mu.L of 6 Xloading buffer, mixing, and immediately freezing to terminate the reaction in a-80℃refrigerator. Marked M.

Preparing 16 wt% non-denatured polyacrylamide gel, taking 20 μl of each of the test sample and the reference sample, loading to gel, electrophoresis under 20mA constant current condition for 10min, and continuing electrophoresis under 40mA constant current condition for 30min. After the electrophoresis was completed, the gel was placed on a shaking table and stained with Gelred dye (BioTium Co., ltd., cat. 13G 1203) for 10min. And (3) observing and photographing gel imaging, and carrying out gray analysis on the electrophoresis strip. Table 5 shows the results of semi-quantitative determination of the stability of the test sirnas listed in table 2 in vitro lysosomal lysates. The results are expressed as the Ratio (RL) of the longest fragment remaining after incubation of the siRNA with Tritosomes to the longest fragment of the siRNA without Tritosome treatment.

TABLE 5 Tritosome stability semi-quantitative results for siRNA

As can be seen from table 5, the modified siRNA provided by the present disclosure was able to be stably present in murine lysosomes for at least 24 hours.

While some embodiments of the present disclosure have been described in detail above, the present disclosure is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solutions of the present disclosure within the scope of the technical concept of the present disclosure, and all the simple modifications belong to the protection scope of the present disclosure.

It should be noted that, in the case where the specific features described in the above embodiments are not contradictory, they may be combined in any suitable manner, and in order to avoid unnecessary repetition, the present disclosure does not describe the various possible combinations.

Moreover, any combination between the various embodiments of the present disclosure is possible as long as it does not depart from the spirit of the present disclosure, which should also be construed as the disclosure of the present disclosure.

Sequence listing

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<213> Artificial sequence (Artificial Sequence)

<400> 55

cguuaugaga ucaagaugu 19

<210> 56

<211> 21

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<400> 56

acaucuugau cucauaacgc u 21

<210> 57

<211> 21

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<400> 57

agcguuauga gaucaagaug u 21

<210> 58

<211> 23

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<400> 58

acaucuugau cucauaacgc ugg 23

<210> 59

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<400> 59

gcagcgagga guuucucau 19

<210> 60

<211> 21

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<400> 60

augagaaacu ccucgcugcg g 21

<210> 61

<211> 21

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<400> 61

ccgcagcgag gaguuucuca u 21

<210> 62

<211> 23

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<400> 62

augagaaacu ccucgcugcg guu 23

<210> 63

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<400> 63

gcagcgagga guuucucau 19

<210> 64

<211> 21

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<400> 64

augagaaacu ccucgcugcg g 21

<210> 65

<211> 21

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<400> 65

ccgcagcgag gaguuucuca u 21

<210> 66

<211> 23

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<400> 66

augagaaacu ccucgcugcg guu 23

<210> 67

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<400> 67

gcagcgagga guuucucau 19

<210> 68

<211> 21

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<400> 68

augagaaacu ccucgcugcg g 21

<210> 69

<211> 21

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<400> 69

ccgcagcgag gaguuucuca u 21

<210> 70

<211> 23

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<400> 70

augagaaacu ccucgcugcg guu 23

<210> 71

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<400> 71

gcagcgagga guuucucau 19

<210> 72

<211> 21

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<400> 72

augagaaacu ccucgcugcg g 21

<210> 73

<211> 21

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<400> 73

ccgcagcgag gaguuucuca u 21

<210> 74

<211> 23

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<400> 74

augagaaacu ccucgcugcg guu 23

<210> 75

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<400> 75

cacaguguuu cccuguuuu 19

<210> 76

<211> 21

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<400> 76

aaaacaggga aacacugugc a 21

<210> 77

<211> 21

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<400> 77

ugcacagugu uucccuguuu u 21

<210> 78

<211> 23

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<400> 78

aaaacaggga aacacugugc auu 23

<210> 79

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<400> 79

cacaguguuu cccuguuuu 19

<210> 80

<211> 21

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<400> 80

aaaacaggga aacacugugc a 21

<210> 81

<211> 21

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<400> 81

ugcacagugu uucccuguuu u 21

<210> 82

<211> 23

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<400> 82

aaaacaggga aacacugugc auu 23

<210> 83

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<400> 83

cacaguguuu cccuguuuu 19

<210> 84

<211> 21

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<400> 84

aaaacaggga aacacugugc a 21

<210> 85

<211> 21

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<400> 85

ugcacagugu uucccuguuu u 21

<210> 86

<211> 23

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<400> 86

aaaacaggga aacacugugc auu 23

<210> 87

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<400> 87

cacaguguuu cccuguuuu 19

<210> 88

<211> 21

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<400> 88

aaaacaggga aacacugugc a 21

<210> 89

<211> 21

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<400> 89

ugcacagugu uucccuguuu u 21

<210> 90

<211> 23

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<400> 90

aaaacaggga aacacugugc auu 23

<210> 91

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<400> 91

cguuaugaga ucaagaugu 19

<210> 92

<211> 21

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<400> 92

acaucuugau cucauaacgc u 21

<210> 93

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<400> 93

cguuaugaga ucaagauga 19

<210> 94

<211> 21

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<400> 94

ucaucuugau cucauaacgc u 21

<210> 95

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<400> 95

cguuaugaga ucaagaugu 19

<210> 96

<211> 21

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<400> 96

acaucuugau cucauaacgc u 21

<210> 97

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<400> 97

gcagcgagga guuucucau 19

<210> 98

<211> 21

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<400> 98

augagaaacu ccucgcugcg g 21

<210> 99

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<400> 99

gcagcgagga guuucucau 19

<210> 100

<211> 21

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<400> 100

augagaaacu ccucgcugcg g 21

<210> 101

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<400> 101

gcagcgagga guuucucau 19

<210> 102

<211> 21

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<400> 102

augagaaacu ccucgcugcg g 21

<210> 103

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<400> 103

cacaguguuu cccuguuuu 19

<210> 104

<211> 21

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<400> 104

aaaacaggga aacacugugc a 21

<210> 105

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<400> 105

cacaguguuu cccuguuuu 19

<210> 106

<211> 21

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<400> 106

aaaacaggga aacacugugc a 21

<210> 107

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<400> 107

cacaguguuu cccuguuuu 19

<210> 108

<211> 21

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<400> 108

aaaacaggga aacacugugc a 21

<210> 109

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<400> 109

cacaguguuu cccuguuuu 19

<210> 110

<211> 21

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<400> 110

aaaacaggga aacacugugc a 21

<210> 111

<211> 21

<212> DNA/RNA

<213> Artificial sequence (Artificial Sequence)

<400> 111

cguuaugaga ucaagaugut t 21

<210> 112

<211> 21

<212> DNA/RNA

<213> Artificial sequence (Artificial Sequence)

<400> 112

acaucuugau cucauaacgt t 21

<210> 113

<211> 21

<212> DNA/RNA

<213> Artificial sequence (Artificial Sequence)

<400> 113

gcagcgagga guuucucaut t 21

<210> 114

<211> 21

<212> DNA/RNA

<213> Artificial sequence (Artificial Sequence)

<400> 114

augagaaacu ccucgcugct t 21

<210> 115

<211> 21

<212> DNA/RNA

<213> Artificial sequence (Artificial Sequence)

<400> 115

cacaguguuu cccuguuuut t 21

<210> 116

<211> 21

<212> DNA/RNA

<213> Artificial sequence (Artificial Sequence)

<400> 116

aaaacaggga aacacugugt t 21

<210> 117

<211> 21

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 117

tgttgttgct gtggctgata g 21

<210> 118

<211> 21

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 118

gttgtgggac ctgtggaagt a 21

<210> 119

<211> 19

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 119

ggtcggagtc aacggattt 19

<210> 120

<211> 19

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 120

ccagcatcgc cccacttga 19

Claims

1. An siRNA capable of inhibiting expression of a TIMP-1 gene, said siRNA comprising a sense strand and an antisense strand, each nucleotide of said sense strand and antisense strand being independently a fluoro-modified nucleotide or a non-fluoro-modified nucleotide, wherein said sense strand comprises nucleotide sequence I, said antisense strand comprises nucleotide sequence II, said nucleotide sequence I and said nucleotide sequence II are each 19 nucleotides in length, at least positions 2-19 of said nucleotide sequence II being reverse complementary to a first stretch of nucleotide sequence selected from the group consisting of sequences of 19 consecutive nucleotides in length in the region 350-700 of TIMP-1 mRNA; the nucleotides at positions 7, 8 and 9 of the nucleotide sequence I are fluoro-modified nucleotides according to the direction from the 5' end to the 3' end, the nucleotides at the rest positions in the sense strand are non-fluoro-modified nucleotides, the first nucleotide at the 5' end of the nucleotide sequence II is the first nucleotide at the 5' end of the antisense strand, the nucleotides at positions 2, 6, 14 and 16 of the nucleotide sequence II are fluoro-modified nucleotides, the nucleotides at the rest positions in the antisense strand are non-fluoro-modified nucleotides, each non-fluoro-modified nucleotide is a methoxy-modified nucleotide, and the methoxy-modified nucleotide refers to a nucleotide formed by methoxy substitution of the 2' -hydroxyl of the ribosyl;

Wherein,

the nucleotide sequence I is equal to the nucleotide sequence shown in SEQ ID NO. 1 in length and is not more than 1 nucleotide difference, and the nucleotide sequence II is equal to the nucleotide sequence shown in SEQ ID NO. 2 in length and is not more than 1 nucleotide difference:

5'-CGUUAUGAGAUCAAGAUGZa ₁ -3'(SEQ ID NO:1)；

5'-Zb ₁ CAUCUUGAUCUCAUAACG-3'(SEQ ID NO:2)，

the nucleotide sequence I comprises a nucleotide sequence whose position corresponds to Za ₁ Nucleotide ZA of (A) ₁ The nucleotide sequence II comprises a nucleotide sequence whose position corresponds to Zb ₁ Nucleotide ZB of (A) ₁ The ZB ₁ Is the first nucleotide at the 5' -end of the antisense strand, and the nucleotide difference between the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO. 2 comprises ZB ₁ Difference in position, and ZB ₁ Selected from A, C or G, ZA ₁ Is in combination with ZB ₁ Complementary nucleotides; or alternatively

The nucleotide sequence I is equal to the nucleotide sequence shown in SEQ ID NO. 3 in length and is not more than 1 nucleotide difference, and the nucleotide sequence II is equal to the nucleotide sequence shown in SEQ ID NO. 4 in length and is not more than 1 nucleotide difference:

5'-GCAGCGAGGAGUUUCUCAZa ₂ -3'(SEQ ID NO:3)；

5'-Zb ₂ UGAGAAACUCCUCGCUGC-3'(SEQ ID NO:4)，

wherein Za ₂ Is U, zb ₂ In the case of the compound A,

the nucleotide sequence I comprises a nucleotide sequence whose position corresponds to Za ₂ Nucleotide ZA of (A) ₂ The nucleotide sequence II comprises a nucleotide sequence whose position corresponds to Zb ₂ Nucleotide ZB of (A) ₂ The ZB ₂ Is the first nucleotide at the 5' -end of the antisense strand, and the nucleotide difference between the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO. 4 comprises ZB ₂ Difference in position, and ZB ₂ Selected from U, C or G, ZA ₂ Is in combination with ZB ₂ Complementary nucleotides; or alternatively

The nucleotide sequence I is equal to the nucleotide sequence shown in SEQ ID NO. 5 in length and is not more than 1 nucleotide difference, and the nucleotide sequence II is equal to the nucleotide sequence shown in SEQ ID NO. 6 in length and is not more than 1 nucleotide difference:

5'-CACAGUGUUUCCCUGUUUZa ₃ -3'(SEQ ID NO:5)；

5'-Zb ₃ AAACAGGGAAACACUGUG-3'(SEQ ID NO:6)，

the nucleotide sequence I comprises a nucleotide sequence whose position corresponds to Za ₃ Nucleotide ZA of (A) ₃ The nucleotide sequence II comprises a nucleotide sequence whose position corresponds to Zb ₃ Nucleotide ZB of (A) ₃ The ZB ₃ Is the first nucleotide at the 5' -end of the antisense strand, and the nucleotide difference between the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO. 6 comprises ZB ₃ Difference in position, and ZB ₃ Selected from A, C or G, ZA ₃ Is in combination with ZB ₃ Complementary nucleotides;

the position corresponds to the same position in the nucleotide sequence from the same end of the nucleotide sequence.

2. The siRNA according to claim 1, wherein said nucleotide sequence I is the sequence shown as SEQ ID NO. 7 and said nucleotide sequence II is the sequence shown as SEQ ID NO. 8; or alternatively

The nucleotide sequence I is a sequence shown as SEQ ID NO. 9, and the nucleotide sequence II is a sequence shown as SEQ ID NO. 10; or alternatively

The nucleotide sequence I is a sequence shown as SEQ ID NO. 11, and the nucleotide sequence II is a sequence shown as SEQ ID NO. 12;

5'-CGUUAUGAGAUCAAGAUGZA ₁ -3'(SEQ ID NO:7)

5'-ZB ₁ CAUCUUGAUCUCAUAACG-3'(SEQ ID NO:8)

5'-GCAGCGAGGAGUUUCUCAZA ₂ -3'(SEQ ID NO:9)

5'-ZB ₂ UGAGAAACUCCUCGCUGC-3'(SEQ ID NO:10)

5'-CACAGUGUUUCCCUGUUUZA ₃ -3'(SEQ ID NO:11)

5'-ZB ₃ AAACAGGGAAACACUGUG-3'(SEQ ID NO:12)

wherein,

ZB ₁ is the first nucleotide at the 5' -end of the antisense strand, ZA ₁ Selected from A, U, G or C, and ZB ₁ Is with ZA ₁ Complementary nucleotides;

ZB ₂ is the first nucleotide at the 5' -end of the antisense strand, ZA ₂ Selected from A, U, G or C, and ZB ₂ Is with ZA ₂ Complementary nucleotides;

ZB ₃ is the first nucleotide at the 5' -end of the antisense strand, ZA ₃ Selected from A, U, G or C, and ZB ₃ Is with ZA ₃ Complementary nucleotides.

3. The siRNA of claim 2, wherein ZA ₁ Is U, ZB ₁ Is A; ZA (zam) ₂ Is U, ZB ₂ Is A; ZA (zam) ₃ Is U, ZB ₃ Is A.

4. The siRNA of claim 1, wherein the sense strand further comprises a nucleotide sequence III, wherein the antisense strand further comprises a nucleotide sequence IV, wherein each of the nucleotides of nucleotide sequence III and of nucleotide sequence IV is independently one of non-fluoro modified nucleotides, wherein each of the nucleotides of nucleotide sequence III and of nucleotide sequence IV is 1-4 nucleotides in length, wherein the nucleotide sequence III and of nucleotide sequence IV are equal in length and substantially reverse complementary or fully reverse complementary, wherein the nucleotide sequence III is attached at the 5 'end of the nucleotide sequence I, and wherein the nucleotide sequence IV is attached at the 3' end of the nucleotide sequence II, wherein the nucleotide sequence IV is substantially reverse complementary or fully reverse complementary to a second nucleotide sequence adjacent to the first nucleotide sequence in the target mRNA and of the same length as the nucleotide sequence IV, wherein the substantially reverse complementary means that there are no more than 1 base between the two nucleotide sequences, and wherein the mismatch between the two nucleotide sequences is not present.

5. The siRNA of claim 4, wherein the siRNA is one of the following sirnas:

1) The nucleotide sequence I is a sequence shown as SEQ ID NO. 7, the nucleotide sequence II is a sequence shown as SEQ ID NO. 8, the lengths of the nucleotide sequences III and IV are 1 nucleotide, and the base of the nucleotide sequence III is G; alternatively, the length of the nucleotide sequence III and the length of the nucleotide sequence IV are 2 nucleotides, and the bases of the nucleotide sequence III are A and G in sequence according to the direction from the 5 'end to the 3' end; alternatively, the nucleotide sequences III and IV are 3 nucleotides in length, and the bases of the nucleotide sequence III are C, A and G in sequence according to the direction from the 5 'end to the 3' end; alternatively, the nucleotide sequences III and IV are 4 nucleotides in length, and the bases of the nucleotide sequence III are C, C, A and G in sequence according to the direction from the 5 'end to the 3' end;

2) The nucleotide sequence I is a sequence shown as SEQ ID NO. 9, the nucleotide sequence II is a sequence shown as SEQ ID NO. 10, the lengths of the nucleotide sequences III and IV are 1 nucleotide, and the base of the nucleotide sequence III is C; alternatively, the length of the nucleotide sequences III and IV is 2 nucleotides, and the bases of the nucleotide sequences III are C and C in sequence according to the direction from the 5 'end to the 3' end; alternatively, the nucleotide sequences III and IV are 3 nucleotides in length, and the bases of the nucleotide sequence III are A, C and C in sequence according to the direction from the 5 'end to the 3' end; alternatively, the nucleotide sequences III and IV are 4 nucleotides in length, and the bases of the nucleotide sequence III are A, A, C and C in sequence according to the direction from the 5 'end to the 3' end;

3) The nucleotide sequence I is a sequence shown as SEQ ID NO. 11, the nucleotide sequence II is a sequence shown as SEQ ID NO. 12, the lengths of the nucleotide sequences III and IV are 1 nucleotide, and the base of the nucleotide sequence III is G; alternatively, the length of the nucleotide sequence III and the length of the nucleotide sequence IV are 2 nucleotides, and the bases of the nucleotide sequence III are U and G in sequence according to the direction from the 5 'end to the 3' end; alternatively, the nucleotide sequences III and IV are 3 nucleotides in length, and the bases of the nucleotide sequence III are A, U and G in sequence according to the direction from the 5 'end to the 3' end; alternatively, the nucleotide sequences III and IV are 4 nucleotides in length, and the bases of the nucleotide sequence III are A, A, U and G in sequence according to the direction from the 5 'end to the 3' end.

6. The siRNA of claim 4, wherein the siRNA further comprises a nucleotide sequence V, each nucleotide of the nucleotide sequence V being independently a non-fluoro modified nucleotide; the length of the nucleotide sequence V is 1 to 3 nucleotides, and the nucleotide sequence V is connected to the 3 '-end of the antisense strand to form the 3' -protruding end of the antisense strand.

7. The siRNA of claim 6, wherein the nucleotide sequence V is 2 nucleotides in length; the nucleotide sequence V is 2 consecutive thymidylate nucleotides, 2 consecutive uracil ribonucleotides, or is completely reverse-complementary to the third nucleotide sequence in the 5 '-terminal to 3' -terminal direction; the third nucleotide sequence refers to a nucleotide sequence adjacent to the second nucleotide sequence in the target mRNA and having a length equal to the nucleotide sequence V.

8. The siRNA of claim 1, wherein the siRNA further comprises a nucleotide sequence V, each nucleotide of the nucleotide sequence V being independently a non-fluoro modified nucleotide; the length of the nucleotide sequence V is 1 to 3 nucleotides, and the nucleotide sequence V is connected to the 3 '-end of the antisense strand to form the 3' -protruding end of the antisense strand.

9. The siRNA of claim 8, wherein the nucleotide sequence V is 2 nucleotides in length; the nucleotide sequence V is 2 consecutive thymidylate nucleotides, 2 consecutive uracil ribonucleotides, or is completely reverse-complementary to the third nucleotide sequence in the 5 '-terminal to 3' -terminal direction; the third nucleotide sequence refers to a nucleotide sequence adjacent to the first nucleotide sequence and having a length equal to the nucleotide sequence V in the target mRNA.

10. The siRNA of claim 1, wherein the siRNA is one of the following sirnas:

the sense strand of the siRNA comprises a nucleotide sequence shown as SEQ ID NO. 13, and the antisense strand comprises a nucleotide sequence shown as SEQ ID NO. 14:

5'-CGUUAUGAGAUCAAGAUGZA ₁ -3'(SEQ ID NO:13)；

5'-ZB ₁ CAUCUUGAUCUCAUAACGCU-3'(SEQ ID NO:14)；

alternatively, the sense strand of the siRNA comprises the nucleotide sequence shown as SEQ ID NO. 15, and the antisense strand comprises the nucleotide sequence shown as SEQ ID NO. 16:

5'-AGCGUUAUGAGAUCAAGAUGZA ₁ -3'(SEQ ID NO:15)；

5'-ZB ₁ CAUCUUGAUCUCAUAACGCUGG-3'(SEQ ID NO:16)；

Wherein the ZB ₁ Is the first nucleotide at the 5' -end of the antisense strand, ZA ₁ Selected from A, U, G or C, and ZB ₁ Is with ZA ₁ Complementary nucleotides; or alternatively

The sense strand of the siRNA contains a nucleotide sequence shown as SEQ ID NO. 17, and the antisense strand of the siRNA contains a nucleotide sequence shown as SEQ ID NO. 18:

5'-GCAGCGAGGAGUUUCUCAZA ₂ -3'(SEQ ID NO:17)；

5'-ZB ₂ UGAGAAACUCCUCGCUGCGG-3'(SEQ ID NO:18)；

5'-CCGCAGCGAGGAGUUUCUCAZA ₂ -3'(SEQ ID NO:19)；

5'-ZB ₂ UGAGAAACUCCUCGCUGCGGUU-3'(SEQ ID NO:20)；

wherein the ZB ₂ Is the first nucleotide at the 5' -end of the antisense strand, ZA ₂ Selected from A, U, G or C, and ZB ₂ Is with ZA ₂ Complementary nucleotides; or alternatively

The sense strand of the siRNA contains a nucleotide sequence shown as SEQ ID NO. 21, and the antisense strand of the siRNA contains a nucleotide sequence shown as SEQ ID NO. 22:

5'-CACAGUGUUUCCCUGUUUZA ₃ -3'(SEQ ID NO:21)；

5'-ZB ₃ AAACAGGGAAACACUGUGCA-3'(SEQ ID NO:22)；

5'-UGCACAGUGUUUCCCUGUUUZA ₃ -3'(SEQ ID NO:23)；

5'-ZB ₃ AAACAGGGAAACACUGUGCAUU-3'(SEQ ID NO:24)；

11. The siRNA of claim 10, wherein the siRNA is one of the following sirnas:

siTIMPa1

Sense strand: 5'-CGUUAUGAGAUCAAGAUGU-3' (SEQ ID NO: 25)

Antisense strand: 5'-ACAUCUUGAUCUCAUAACGCU-3' (SEQ ID NO: 26)

siTIMPa2

Sense strand: 5'-AGCGUUAUGAGAUCAAGAUGU-3' (SEQ ID NO: 27)

Antisense strand: 5'-ACAUCUUGAUCUCAUAACGCUGG-3' (SEQ ID NO: 28)

siTIMPb1

Sense strand: 5'-GCAGCGAGGAGUUUCUCAU-3' (SEQ ID NO: 29)

Antisense strand: 5'-AUGAGAAACUCCUCGCUGCGG-3' (SEQ ID NO: 30)

siTIMPb2

Sense strand: 5'-CCGCAGCGAGGAGUUUCUCAU-3' (SEQ ID NO: 31)

Antisense strand: 5'-AUGAGAAACUCCUCGCUGCGGUU-3' (SEQ ID NO: 32)

siTIMPc1

Sense strand: 5'-CACAGUGUUUCCCUGUUUU-3' (SEQ ID NO: 33)

Antisense strand: 5'-AAAACAGGGAAACACUGUGCA-3' (SEQ ID NO: 34)

siTIMPc2

Sense strand: 5'-UGCACAGUGUUUCCCUGUUUU-3' (SEQ ID NO: 35)

Antisense strand: 5'-AAAACAGGGAAACACUGUGCAUU-3' (SEQ ID NO: 36).

12. The siRNA of claim 1, wherein at least 1 of the phosphate groups in the phosphate-sugar backbone of at least one of the sense strand and the antisense strand is a phosphate group having a modification group, and/or the 5' -terminal nucleotide of the antisense strand is a 5' -phosphate nucleotide or a 5' -phosphate analogue modified nucleotide.

13. The siRNA of claim 12, 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 in the phosphate group with a sulfur atom, the phosphorothioate group linkage being present at least one of the group consisting of:

14. The siRNA of claim 12, wherein the 5 '-phosphonucleotide or 5' -phosphoanalogue modified nucleotide is a nucleotide represented by one of formula (2) -formula (6):

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

15. The siRNA of claim 1, wherein the siRNA is any one of sitmpa 1M1, siT1MPa2M1, sitmpa 1M1S, siTIMPa M1S, siTIMPa M1P1, sitmpa 2M1P1, sitmpa 1M1SP1, and sitmpa 2M1SP 1;

siTIMPa1M1：

S：

CmGmUmUmAmUmGfAfGfAmUmCmAmAmGmAmUmGmUm

(SEQ ID NO:43)

AS：

AmCfAmUmCmUfUmGmAmUmCmUmCmAfUmAfAmCmGmCmUm

(SEQ ID NO:44)

siTIMPa2M1：

S：

AmGmCmGmUmUmAmUmGfAfGfAmUmCmAmAmGmAmUmGmUm

(SEQ ID NO:45)

AS：

AmCfAmUmCmUfUmGmAmUmCmUmCmAfUmAfAmCmGmCmUmGmGm

(SEQ ID NO:46)

siTIMPa1M1S：

S：

CmsGmsUmUmAmUmGfAfGfAmUmCmAmAmGmAmUmGmUm

(SEQ ID NO:47)

AS：

AmsCfsAmUmCmUfUmGmAmUmCmUmCmAfUmAfAmCmGmsCmsUm

(SEQ ID NO:48)

siTIMPa2M1S：

S：

AmsGmsCmGmUmUmAmUmGfAfGfAmUmCmAmAmGmAmUmGmUm

(SEQ ID NO:49)

AS：

AmsCfsAmUmCmUfUmGmAmUmCmUmCmAfUmAfAmCmGmCmUmsGmsGm

(SEQ ID NO:50)

siTIMPa1M1P1：

S：

CmGmUmUmAmUmGfAfGfAmUmCmAmAmGmAmUmGmUm

(SEQ ID NO:51)

AS：

P1-AmCfAmUmCmUfUmGmAmUmCmUmCmAfUmAfAmCmGmCmUm

(SEQ ID NO:52)

siTIMPa2M1P1：

S：

AmGmCmGmUmUmAmUmGfAfGfAmUmCmAmAmGmAmUmGmUm

(SEQ ID NO:53)

AS：

P1-AmCfAmUmCmUfUmGmAmUmCmUmCmAfUmAfAmCmGmCmUmGmGm

(SEQ ID NO:54)

siTIMPa1M1SP1：

S：

CmsGmsUmUmAmUmGfAfGfAmUmCmAmAmGmAmUmGmUm

(SEQ ID NO:55)

AS：

P1-AmsCfsAmUmCmUfUmGmAmUmCmUmCmAfUmAfAmCmGmsCmsUm

(SEQ ID NO:56)

siTIMPa2M1SP1：

S：

AmsGmsCmGmUmUmAmUmGfAfGfAmUmCmAmAmGmAmUmGmUm

(SEQ ID NO:57)

AS：

P1-AmsCfsAmUmCmUfUmGmAmUmCmUmCmAfUmAfAmCmGmCmUmsGmsGm(SEQ ID NO:58)

Alternatively, the siRNA is any one of sittimpb 1M1, sittimpb 2M1, sittimpb 1M1S, siTIMPb M1S, siTIMPb M1P1, sittimpb 2M1P1, sittimpb 1M1SP1, and sittimpb 2M1SP 1:

siTIMPb1M1：

S：

GmCmAmGmCmGmAfGfGfAmGmUmUmUmCmUmCmAmUm

(SEQ ID NO:59)

AS：

AmUfGmAmGmAfAmAmCmUmCmCmUmCfGmCfUmGmCmGmGm

(SEQ ID NO:60)

siTIMPb2M1：

S：

CmCmGmCmAmGmCmGmAfGfGfAmGmUmUmUmCmUmCmAmUm

(SEQ ID NO:61)

AS：

AmUfGmAmGmAfAmAmCmUmCmCmUmCfGmCfUmGmCmGmGmUmUm

(SEQ ID NO:62)

siTIMPb1M1 S：

S：

GmsCmsAmGmCmGmAfGfGfAmGmUmUmUmCmUmCmAmUm

(SEQ ID NO:63)

AS：

AmsUfsGmAmGmAfAmAmCmUmCmCmUmCfGmCfUmGmCmsGmsGm(SEQ ID NO:64)

siTIMPb2M1 S：

S：

CmsCmsGmCmAmGmCmGmAfGfGfAmGmUmUmUmCmUmCmAmUm(SEQ ID NO:65)

AS：

AmsUfsGmAmGmAfAmAmCmUmCmCmUmCfGmCfUmGmCmGmGmsUmsUm(SEQ ID NO:66)

siTIMPb1M1P1：

S：

GmCmAmGmCmGmAfGfGfAmGmUmUmUmCmUmCmAmUm

(SEQ ID NO:67)

AS：

P1-AmUfGmAmGmAfAmAmCmUmCmCmUmCfGmCfUmGmCmGmGm(SEQ ID NO:68)

siTIMPb2M1P1：

S：

CmCmGmCmAmGmCmGmAfGfGfAmGmUmUmUmCmUmCmAmUm(SEQ ID NO:69)

AS：

P1-AmUfGmAmGmAfAmAmCmUmCmCmUmCfGmCfUmGmCmGmGmUmUm(SEQ ID NO:70)

siTIMPb1M1 SP1：

S：

GmsCmsAmGmCmGmAfGfGfAmGmUmUmUmCmUmCmAmUm

(SEQ ID NO:71)

AS：

P1-AmsUfsGmAmGmAfAmAmCmUmCmCmUmCfGmCfUmGmCmsGmsGm(SEQ ID NO:72)

siTIMPb2M1 SP1：

S：

CmsCmsGmCmAmGmCmGmAfGfGfAmGmUmUmUmCmUmCmAmUm(SEQ ID NO:73)

AS：

P1-AmsUfsGmAmGmAfAmAmCmUmCmCmUmCfGmCfUmGmCmGmGmsUmsUm(SEQ ID NO:74)

alternatively, the siRNA is any one of sittimpc 1M1, sittimpc 2M1, sittimpc 1M1S, siTIMPc M1S, siTIMPc M1P1, sittimpc 2M1P1, sittimpc 1M1SP1 and sittimpc 2M1SP 1:

siTIMPc1M1：

S：

CmAmCmAmGmUmGfUfUfUmCmCmCmUmGmUmUmUmUm

(SEQ ID NO:75)

AS：

AmAfAmAmCmAfGmGmGmAmAmAmCmAfCmUfGmUmGmCmAm

(SEQ ID NO:76)

siTIMPc2M1：

S：

UmGmCmAmCmAmGmUmGfUfUfUmCmCmCmUmGmUmUmUmUm

(SEQ ID NO:77)

AS：

AmAfAmAmCmAfGmGmGmAmAmAmCmAfCmUfGmUmGmCmAmUmUm

(SEQ ID NO:78)

siTIMPc1M1S：

S：

CmsAmsCmAmGmUmGfUfUfUmCmCmCmUmGmUmUmUmUm

(SEQ ID NO:79)

AS：

AmsAfsAmAmCmAfGmGmGmAmAmAmCmAfCmUfGmUmGmsCmsAm

(SEQ ID NO:80)

siTIMPc2M1S：

S：

UmsGmsCmAmCmAmGmUmGfUfUfUmCmCmCmUmGmUmUmUmUm

(SEQ ID NO:81)

AS：

AmsAfsAmAmCmAfGmGmGmAmAmAmCmAfCmUfGmUmGmCmAmsUmsUm

(SEQ ID NO:82)

siTIMPc1M1P1：

S：

CmAmCmAmGmUmGfUfUfUmCmCmCmUmGmUmUmUmUm

(SEQ ID NO:83)

AS：

P1-AmAfAmAmCmAfGmGmGmAmAmAmCmAfCmUfGmUmGmCmAm

(SEQ ID NO:84)

siTIMPc2M1P1：

S：

UmGmCmAmCmAmGmUmGfUfUfUmCmCmCmUmGmUmUmUmUm

(SEQ ID NO:85)

AS：

P1-AmAfAmAmCmAfGmGmGmAmAmAmCmAfCmUfGmUmGmCmAmUmUm

(SEQ ID NO:86)

siTIMPc1M1SP1：

S：

CmsAmsCmAmGmUmGfUfUfUmCmCmCmUmGmUmUmUmUm

(SEQ ID NO:87)

AS：

P1-AmsAfsAmAmCmAfGmGmGmAmAmAmCmAfCmUfGmUmGmsCmsAm

(SEQ ID NO:88)

siTIMPc2M1SP1：

S：

UmsGmsCmAmCmAmGmUmGfUfUfUmCmCmCmUmGmUmUmUmUm

(SEQ ID NO:89)

AS：

P1-AmsAfsAmAmCmAfGmGmGmAmAmAmCmAfCmUfGmUmGmCmAmsUmsUm

(SEQ ID NO:90)

wherein S refers to the sense strand and AS refers to the antisense strand; capital C, G, U, A indicates the base composition of the nucleotide; the lower case letter m indicates that the adjacent nucleotide to the left of the letter m is a methoxy modified nucleotide; the lower case letter f indicates that the adjacent nucleotide to the left of the letter f is a fluoro-modified nucleotide; the lower case letter s indicates that phosphorothioate linkages are between two nucleotides adjacent to the letter s; p1 represents that one nucleotide adjacent to the right of P1 is a 5 '-phosphonucleotide or a 5' -phosphoanalog modified nucleotide.

16. A pharmaceutical composition comprising the siRNA of any one of claims 1-15 and a pharmaceutically acceptable carrier.

17. The pharmaceutical composition of claim 16, wherein the weight ratio of said siRNA to said pharmaceutically acceptable carrier is 1 (1-500).

18. The pharmaceutical composition of claim 17, wherein the weight ratio of siRNA to pharmaceutically acceptable carrier is 1 (1-50).

19. A kit comprising the siRNA of any one of claims 1-15 and/or the pharmaceutical composition of any one of claims 16-18.