CN117247940A

CN117247940A - siRNA, conjugates and pharmaceutical compositions for reducing PD-L1 expression

Info

Publication number: CN117247940A
Application number: CN202311267969.0A
Authority: CN
Inventors: 唐春雷; 薛凡; 姜虹羽; 范为正
Original assignee: Sanorri Biomedical Technology Wuxi Co ltd
Current assignee: Sanorri Biomedical Technology Wuxi Co ltd
Priority date: 2023-09-28
Filing date: 2023-09-28
Publication date: 2023-12-19

Abstract

The invention discloses siRNA, conjugate and pharmaceutical composition for reducing PD-L1 expression, belonging to the fields of molecular biology and biological medicine. RNA interference agents useful for inhibiting the expression of apoptosis-ligand 1 genes are disclosed. The inventionsiRNAs for apoptosis-ligand 1 (PD-L1) gene expression, and pharmaceutical compositions and siRNA conjugates containing the siRNAs are disclosed, as are methods of making and using the siRNAs, pharmaceutical compositions and siRNA conjugates. The average inhibition rate of the siRNA provided by the invention reaches 70-84%, and the inhibition rate IC ₅₀ Below 14nM, up to a minimum of 8.91nM.

Description

siRNA, conjugates and pharmaceutical compositions for reducing PD-L1 expression

Technical Field

The invention relates to siRNA, conjugate and pharmaceutical composition for reducing PD-L1 expression, belonging to the fields of molecular biology and biological medicine.

Background

Cell apoptosis-ligand 1 (Programmed cell death ligand 1, PD-L1), also known as surface antigen cluster 274 (cluster ofdifferentiation, CD 274) or B7 homolog (B7 homolog 1, B7-H1), is a protein in humans encoded by the CD274 gene. PD-L1 is a first type transmembrane protein with the size of 40kDa, and consists of an extracellular segment, a transmembrane anchoring region and an intracellular signal transduction region, and is mainly induced and expressed on the surfaces of activated T lymphocytes and B lymphocytes. It is believed to be associated with the suppression of the immune system in certain special situations (e.g., pregnancy, tissue transplantation, autoimmune diseases, and certain diseases such as hepatitis). The immune system normally responds to foreign antigens accumulated in the lymph nodes or spleen, triggering antigen-specific cytotoxic T cells (CD 8) ⁺ T cell) proliferation.

Ligands for apoptosis protein 1 (PD-1, also known as CD279, SLEB2 and/or hSLE 1) are B7-H1 (PD-L1) and B7-DC (PD-L2), which bind to inhibit T cell proliferation activation, down regulate T cell response, and are inhibitory receptors. PD-Ll is involved in evading chronic infections, e.g., chronic viral infections (including e.g., HIV, HBV, HCV and HTLV, etc.), chronic bacterial infections (including e.g., helicobacter pylori, etc.), and chronic parasitic infections (including e.g., schistosome). PD-L1 expression has been detected in a number of tissues and cell types, including T cells, B cells, macrophages, dendritic cells, and non-hematopoietic cells, including endothelial cells, hepatocytes, myocytes, and placenta. PD-L1 expression is also involved in the inhibition of anti-tumor immune activity. Tumors express antigens that can be recognized by host T cells, but immune clearance of tumors is rare. Part of the reason for this failure is immunosuppression of the tumor microenvironment. PD-L1 expression on many tumors is a component of this inhibitory environment and acts synergistically with other immunosuppressive signals. PD-Ll expression has been shown in situ in a variety of solid tumors including breast, lung, colon, ovary, melanoma, bladder, liver, saliva, stomach, glioma, thyroid, thymus epithelium, head and neck.

PD-1 and PD-L1 antibodies are already on the market at present, and block the inhibition of T cells by tumor cells. Cancer immunotherapy is a specific method of eliminating cancer cells by enhancing or modulating the host immune system. Immune checkpoint molecules regulate immune balance, neutralization of immune suppression checkpoints can lead to cancer elimination. Among these immune checkpoints, the blockade of PD-1 and its ligands 1 and 2 (PD-L1/2) is an intrinsic negative checkpoint, leading to one of the most successful immunotherapy by enhancing the immune response of T cells to tumor cells. Currently, the blocking of PD-1/PD-L1 can be achieved by three methods: antibody blocking, gene silencing and small molecule pathway inhibition. Commercial PD-L1 antibodies have shown great success, particularly for advanced cancers such as melanoma and non-small lung cancer. Among these three approaches, the gene silencing strategy has been less studied, but is now attracting more attention due to the method of inhibiting the PD-L1 pathway.

PD-1, PD-L1 has become the new target point of treating tumor, and the anti-PD-1 antibody medicament that has been marketed abroad at present sometimes beautifies precious Nivolumab, keystuda of moxadong, etc. Still other drugs are in early clinical trial stages. However, such antibody drugs have a short duration of action, require long-term injections, and are expensive for patients. Thus, there is a need in the art for effective therapies directed against PD-L1. Diseases, such as infectious diseases, e.g., chronic intracellular infectious diseases, e.g., viral diseases, e.g., hepatitis infection, or bacterial infections, e.g., tuberculosis infection; and cancers, such as liver cancer, e.g., hepatocellular carcinoma. Despite the efforts in this area of research, current treatments do not fully meet patient needs and additional treatments suitable for most affected patient populations are highly desirable.

Thus, there is an unmet need for PD-L1 therapies, for example, agents that can selectively and effectively silence the PD-L1 gene using the RNAi machinery of the cell itself, which have high biological activity and in vivo stability, and which can effectively inhibit expression of the target PD-L1 gene.

Disclosure of Invention

The siRNA and the modified sequence thereof provided by the invention can specifically inhibit the expression of the apoptosis-ligand 1 (PD-L1) gene, and the pharmaceutical composition or the siRNA conjugate containing the siRNA can specifically target the liver, so that the expression of the apoptosis-ligand 1 gene can be inhibited, and the treatment of tumor diseases and/or symptoms can be realized.

In some embodiments, the invention provides a first siRNA capable of inhibiting expression of a PD-L1 gene, the siRNA comprising a sense strand and an antisense strand, each nucleotide in the siRNA being independently a modified or unmodified nucleotide, wherein the sense strand comprises a stretch of nucleotide sequence I and the antisense strand comprises a stretch of nucleotide sequence II, the nucleotide sequence I and the nucleotide sequence II being at least partially reverse complementary to form a double-stranded region, wherein the nucleotide sequence I is complementary to SEQ ID NO:1, and not more than 3 nucleotides, and the nucleotide sequence II is identical to the nucleotide sequence set forth in SEQ ID NO:2, and no more than 3 nucleotide differences:

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

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

In some embodiments, the invention provides a second siRNA capable of inhibiting expression of a PD-L1 gene, the siRNA comprising a sense strand and an antisense strand, each nucleotide in the siRNA being independently a modified or unmodified nucleotide, wherein the sense strand comprises a stretch of nucleotide sequence I and the antisense strand comprises a stretch of nucleotide sequence II, the nucleotide sequence I and the nucleotide sequence II being at least partially reverse complementary to form a double-stranded region, wherein the nucleotide sequence I is complementary to SEQ ID NO:3, and not more than 3 nucleotides, and said nucleotide sequence II is identical to SEQ ID NO:4, and no more than 3 nucleotide differences:

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

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

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

In some embodiments, the invention provides the use of the siRNA and/or pharmaceutical composition and/or siRNA conjugate in the manufacture of a medicament for treating a disease and/or disorder caused by PD-L1 gene expression.

In some embodiments, the invention provides a method of treating a disease and/or disorder caused by PD-L1 gene expression, the method comprising administering to a subject having a disease and/or disorder associated with metabolic disease an effective amount of an siRNA and/or pharmaceutical composition and/or siRNA conjugate of the invention.

In some embodiments, the invention provides a method of inhibiting PD-L1 gene expression, the method comprising contacting an effective amount of an siRNA and/or a pharmaceutical composition and/or an siRNA conjugate of the invention with the liver cell.

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

In some embodiments, the invention further comprises cells of the siRNA or the siRNA conjugate.

Advantageous effects

The siRNA, the pharmaceutical composition and the siRNA conjugate provided by the invention have good stability, higher PD-L1mRNA inhibition activity, lower off-target effect and/or can be used for remarkably treating diseases and/or symptoms related to PD-L1 abnormal expression.

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

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

Therefore, the siRNA, the pharmaceutical composition and the siRNA conjugate provided by the invention can inhibit the expression of PD-L1 genes, effectively treat related diseases and/or symptoms caused by PD-L1 abnormal expression, and have good application prospects.

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

Detailed Description

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

In the invention, PD-L1 mRNA refers to mRNA having a sequence shown in Genbank accession No. NM-001267706.2. Further, unless otherwise indicated, the term "target gene" as used herein refers to a gene that is transcribed up to PD-L1 mRNA, and the term "target mRNA" refers to the PD-L1 mRNA described above.

Definition of the definition

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

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

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

As used herein, the term "PD-L1" interchangeably refers to the surface antigen cluster 274 (cluster of differentiation, CD 274) or the B7 homolog (B7 homolog 1, B7-H1), a protein in humans, encoded by the CD274 gene.

Other examples of PD-L1 mRNA sequences are readily available using public databases such as GenBank, uniProt and OMIM. The term "PD-L1" as used herein also refers to naturally occurring DNA sequence variations of the PD-L1 genome.

The term "diseases and/or conditions associated with aberrant expression of PD-L1" or "PD-L1-related diseases" as used herein is a disease or disorder caused by or associated with aberrant expression of PD-L1. The term "PD-L1-related disease" includes a disease, disorder or condition that benefits from reduced expression or replication of the PD-L1 gene. Non-limiting examples of PD-L1 related diseases include, for example, diabetes.

The term "inhibit", when referring to the expression of a given gene, means that the expression of the gene is reduced when the cell, cell population or tissue is treated with the siRNA, pharmaceutical composition and siRNA conjugate of the invention, as compared to a cell, cell population or tissue that has not been so treated.

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

As used herein, the phrase "inhibiting expression of PD-L1" or "inhibiting expression of a PD-L1 gene" includes inhibiting expression of any PD-L1 gene (e.g., a PD-L1 gene expressed by PD-L1 in PD-L1, a PD-L1 gene expressed by an expression construct in a cell), as well as variants or mutants of a PD-L1 gene encoding a PD-L1 protein. The term includes knockdown of any PD-L1 transcript encoding one or more PD-L1 proteins, as well as variants or mutants of the PD-L1 gene.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

In one aspect, the present invention provides the first to seventh siRNAs capable of inhibiting the expression of a PD-L1 gene. This will be described in detail in turn.

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

First siRNA

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

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

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

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

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

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

Second siRNA

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

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

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

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

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

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

As previously mentioned, the nucleotides in the sirnas disclosed herein are each independently modified or unmodified nucleotides. In some embodiments, each nucleotide in the siRNA of the invention is an unmodified nucleotide. In some embodiments, some or all of the nucleotides in the siRNA of the invention are modified nucleotides, and such modifications on the nucleotide groups do not result in a significant impairment or loss of function of the siRNA conjugates of the invention to inhibit PD-L1 gene expression.

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

In some embodiments, the presently disclosed siRNA contains at least 1 modified nucleotide. In the context of the present disclosure, the term "modified nucleotide" is used to refer to a nucleotide or nucleotide analogue formed by substitution of the hydroxyl group at the 2' -position of the ribosyl of the nucleotide with other groups, or a nucleotide having a modified base. The modified nucleotide does not result in a significant impairment or loss of function of the siRNA to inhibit gene expression. For example, modified nucleotides disclosed in J.K.Watts, G.F.Deleavey, and M.J.damha, chemically modified siRNA: tools and applications. Drug discovery Today,2008,13 (19-20): 842-55 may be selected.

In some embodiments, at least one nucleotide in the sense strand or the antisense strand of the siRNA provided herein is a modified nucleotide, and/or at least one phosphate group is a phosphate group having a modifying group. In other words, at least a portion of the phosphate groups and/or ribose groups in at least one single-stranded phosphate-sugar backbone in the sense strand and the antisense strand are phosphate groups and/or ribose groups having a modifying group.

In some embodiments, all of the nucleotides in the sense strand and/or the antisense strand are modified nucleotides. In some embodiments, each nucleotide in the sense strand and the antisense strand of the siRNA provided by the present disclosure is independently a fluoro-modified nucleotide or a non-fluoro-modified nucleotide.

In the context of the present disclosure, a "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 (1). "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 replaced 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 2' -methoxy (2 ' -OMe) -modified nucleotide, as shown in formula (2). In some embodiments, the 2' -substituted alkoxy modified nucleotide may be a 2' -methoxyethyl (2 ' - Μ o e) modified nucleotide as shown in formula (3). In some embodiments, 2 '-amino (2' -n-h) ₂ ) The modified nucleotide is shown as a formula (4). In some embodiments, the 2' -Deoxynucleotide (DNA) is represented by formula (5):

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, or an acyclic nucleotide.

The bridged nucleotides (bridged nucleic acid, abbreviated BNA) may contain a five-ring-, six-or seven-membered ring bridging structure with "fixed" C3' -endo-sugar tucking. The bridge is typically incorporated at the 2'-, 4' -position of the ribose to provide a 2',4' -BNA nucleotide. In some embodiments, BNA may be LNA, ENA, cETBNA, etc., where LNA is shown in formula (6), ENA is shown in formula (7), ctbna is shown in formula (8).

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 (9), and GNA is represented by formula (10):

in the above formula (9) and formula (10), R _a Selected from H, OH or 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 (11) or (12):

in the compounds represented by the above formula (11) and formula (12), base represents a nucleobase, for example A, U, G, C or T; r is R _b Selected from H, OH,F or a non-fluorine group 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 hereinafter 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' -hydroxyl of ribose group substituted by fluoro" and "nucleotide with 2 '-fluoro ribose group" are the same, and all refer to a compound having a structure as shown in formula (1) formed by substituting 2' -hydroxyl of nucleotide by fluoro; "methoxy-modified nucleotide", "2 '-methoxy-modified nucleotide", "nucleotide in which the 2' -hydroxyl group of the ribose group is replaced by methoxy" and "nucleotide having a 2 '-methoxyribosyl" are the same in meaning, and refer to a compound having a structure shown in formula (2) in which the 2' -hydroxyl group of the ribosyl group of the nucleotide is replaced by methoxy.

In some embodiments, the sense strand or the antisense strand of the siRNA provided herein may comprise a base modification or substitution.

The siRNA with the modification can ensure that ribonuclease in blood is not easy to cut nucleic acid, thereby increasing the stability of the nucleic acid and ensuring that the nucleic acid has stronger performance of resisting nuclease hydrolysis. Meanwhile, the modified siRNA has higher activity of inhibiting target mRNA.

In some embodiments, at least a portion of the phosphate groups in the phosphate-sugar backbone of at least one single strand of the sense strand and the antisense strand of the siRNA provided herein 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 (13):

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

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

Commonly used nucleotides modified by such 5' -phosphonucleotides or 5' -phosphoanalogs are well known to those skilled in the art, e.g., the 5' -phosphonucleotides may have the structure of formula (14):

for another example, anastasia Khvorova and Jonathan K.Watts, the chemical evolution of oligonucleotide therapies of clinical U.S. Nature Biotechnology,2017,35 (3): 238-48 discloses the following 4 5' -phosphate analog modified nucleotides:

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

In some embodiments, the 5' -phosphate nucleotide is a nucleotide comprising a 5' -phosphate modification shown in formula (14), the 5' -phosphate analogue modified nucleotide is a nucleotide comprising a vinyl phosphate modification shown in formula (15), or is a phosphorothioate modified nucleotide shown in formula (17).

Abbreviations for the modified nucleotide monomers disclosed herein are shown in table 2.

TABLE 2 abbreviations for nucleotide monomers disclosed in the present invention

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In some embodiments, the siRNA provided by the invention is any one of the sirnas listed in table 3.

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

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

siRNA conjugates

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

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

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

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

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

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

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

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

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

wherein m is an integer of 1 to 3;

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

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

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

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

in the formula, the double helix structure represents siRNA.

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

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

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

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

In some embodiments, the siRNA conjugate has a structure as shown in formula (24), (25) or (26):

wherein,

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

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

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

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

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

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

each k2 is independently an integer from 1 to 20;

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

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

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

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

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

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

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

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

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In some embodiments, the P atom in formula S1 can be attached to any possible position in the siRNA sequence, e.g., the P atom in formula S1 can be attached to any one of the nucleotides of the sense strand or the antisense strand of the siRNA; in some embodiments, the P atom in formula S1 is attached to any one nucleotide of the sense strand of the siRNA. In some embodiments, the P atom in formula S1 is attached to the end of the sense strand or the antisense strand of the siRNA; in some embodiments, the P atom in formula S1 is attached to the 3' end of the sense strand of the siRNA. In the case of the above-described position of the sense strand linked to the siRNA, the siRNA conjugate shown in (24), (25) or (26) can release the separate antisense strand of siRNA upon unwinding to block the process of translation of the protein by PD-L1 mRNA and inhibit PD-L1 gene expression after entering the cell.

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

The siRNA conjugate of the invention has remarkably improved stability in blood plasma, low off-target effect and higher PD-L1mRNA silencing activity. In some embodiments, the siRNA of the invention may be any of the sirnas shown in table 1 or table 3. siRNA conjugates containing these siRNAs exhibit higher PD-L1mRNA silencing activity.

In the siRNA or siRNA conjugate according to the present invention, each adjacent nucleotide is formed by phosphodiester bond or phosphorothioateThe non-bridging oxygen or sulfur atoms in the ester linkages, phosphodiester linkages or phosphorothioate linkages carry a negative charge and may be present in the form of a hydroxyl or sulfhydryl group, the hydrogen ion in which may also be partially or fully replaced by a cation. The cation may be any cation, such as a metal cation, ammonium ion NH4 ⁺ One of organic ammonium cations. In one embodiment, the cation is selected from one or more of an alkali metal ion, a tertiary amine-forming ammonium cation, and a quaternary ammonium cation for improved solubility. The alkali metal ion may be and/or Na+, and the cation formed by the tertiary amine may be an ammonium ion formed by triethylamine and/or an ammonium ion formed by N, N-diisopropylethylamine. Thus, the siRNA or siRNA conjugate of the invention may be at least partially present in salt form. In one mode, the non-bridging oxygen or sulfur atoms in the phosphodiester or phosphorothioate linkages are at least partially bound to sodium ions and the siRNA or siRNA conjugates of the invention are in the form of sodium salts or partial sodium salts.

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

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

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

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

Wherein:

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Pharmaceutical composition

The invention also includes pharmaceutical compositions and formulations comprising the siRNA conjugates of the invention. In some embodiments, provided herein are pharmaceutical compositions comprising an siRNA conjugate as described herein and a pharmaceutically acceptable carrier. Pharmaceutical compositions comprising the siRNA conjugates are useful for treating diseases or disorders associated with the expression or activity of PD-L1 gene. Such pharmaceutical compositions are formulated based on the mode of delivery. One example is a composition formulated for systemic administration by parenteral delivery, such as by Subcutaneous (SC), intramuscular (IM), or Intravenous (IV) delivery. In certain embodiments, the invention provides compositions formulated for organ-specific (e.g., liver) intra-arterial, intratumoral, intradermal, intravitreal injection, topical ocular, ophthalmic (eye drops), nebulization, topical or other topical ocular route, suppository or oral administration. In a preferred embodiment, the composition is administered subcutaneously.

The pharmaceutical compositions of the invention may be administered in a dose sufficient to inhibit PD-L1 gene expression. In some embodiments, the siRNA conjugate is administered at the following doses: about 0.5mg/kg to 50mg/kg per dose, or 0.3mg/kg to 20mg/kg, or 3mg/kg to 10mg/kg, or preferably 3mg/kg to 10mg/kg per dose. For example, the siRNA conjugates may be administered at a dose of about 0.5mg/kg, 1mg/kg, 1.5mg/kg, 2mg/kg, 3mg/kg, 10mg/kg, 20mg/kg, 30mg/kg, 40mg/kg, or 50mg/kg per single dose.

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

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

The pharmaceutical composition may be administered indefinitely, e.g. in a subject with one or more signs or symptoms of aberrant expression of PD-L1. Such as chronic viral infections (including e.g. HIV, HBV, HCV and HTLV, etc.), chronic bacterial infections (including e.g. helicobacter pylori, etc.), and chronic parasitic infections (including e.g. schistosome), or cancers (including e.g. breast cancer, lung cancer, colorectal cancer, ovarian cancer, melanoma, bladder cancer, liver cancer, stomach cancer, glioma, thyroid cancer, head and neck cancer). In some embodiments, treatment with the siRNA conjugate is performed for a discrete or defined period of time and provides a functional cure.

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

A. Excipient

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

B. Other components

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

In some embodiments, the pharmaceutical compositions characterized in the present invention comprise (a) one or more siRNA conjugate compounds and (b) one or more agents that function by a non-RNAi mechanism and are useful in the treatment of PD-L1 related disorders. Examples of such agents include, but are not limited to, anti-inflammatory agents, anti-steatosis agents, antiviral agents, and anti-fibrotic agents.

In addition, other substances commonly used to protect the liver, such as silymarin, may also be used in combination with the siRNA conjugates described herein. Other agents useful in the treatment of liver disease include telbivudine, entecavir and protease inhibitors such as telaprevir and other drugs disclosed in, for example, US2005/0148548, US2004/0167116, US2003/0144217 and US 2004/0127088.

Toxicity and therapeutic efficacy of such compounds may be determined by standard pharmaceutical procedures in cell culture or experimental animals (e.g., for determining LD ₅₀ (50% of the population lethal dose) and ED ₅₀ (a therapeutically effective dose in 50% of the population)). The dose ratio between toxicity and therapeutic efficacy is the therapeutic index, and it can be expressed as LD ₅₀ /ED ₅₀ Ratio. In some embodiments, compounds exhibiting high therapeutic indices are preferred.

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

Examples

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

Example 1 design of siRNA

A set of siRNAs targeting the human PD-L1 gene (human NCBI refseqID NM-001267706.2;NCBI Gene ID:29126) was designed on-line using oligo alk. Human NM-001267706 REFSEQ mRNA, second version has 3292 bases in length. Meanwhile, in order to avoid toxicity caused by any sequence, sequences similar to those of human genes need to be excluded.

A detailed list of unmodified PD-L1 sense and antisense strand nucleotide sequences is shown in Table 1. A detailed list of modified PD-L1 sense and antisense strand nucleotide sequences is shown in Table 3.

EXAMPLE 2 preparation of siRNA or siRNA conjugates

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

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

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

Annealing of the oligoribonucleotides results in siRNA conjugates: the RNA oligomer to be annealed is treated with sterile RNaseF reeH ₂ O (no RNA hydrolase) was formulated at 200. Mu.m. The annealing reaction system is set as follows, the mixed solution with the total volume of 100 mu L is placed in a water bath kettle with the temperature of 95 ℃ for 10 min (the required quantity of more than or equal to 100nmol is required to be high temperature for 20 min), the mixed solution is rapidly placed in a water bath with the temperature of 60 ℃ for natural cooling, and the solution after annealing is not placed in the high temperature for storage. Complementary strands are mixed by combining equimolar RNA solutions.

Table 4 shows PD-L1 siRNA conjugates synthesized using the methods described above.

TABLE 4 modified PD-L1 siRNA conjugate nucleotide sequences

Example 3 in vitro Activity assay of siRNA

Quantitative detection of PD-L1 mRNA content in RKO cells by qPCR, as EC of the compound ₅₀ The value is used as an index to evaluate the inhibitory activity of the siRNA conjugate on PD-L1.

Experimental materials and reagents:

cell line: RKO cells.

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

Reagent: pancreatin (Invitrogen-25300062); DMSO (Sigma-D2650-1 OOML); transfection reagent Lipofectamine RNAiMAX (Invitrogen-13778-150); MEM Medium (HyClone-SH 30024.01); ULtraPure Distilled Water (DNAse, RNAse, free) (Invitrogen-10977-015); opti-MEM I (1X) (Gibco-31985-070);

Phosphate Buffered Saline(PBS)(Gibco)；PrimeScript ^TM RT reagentKitwith gDNAEraser(takara-RR047A)；ChamQ Universal SYBR qPCR Master Mix(vyzme-Q711-02)

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

the experimental steps are as follows:

siRNA or siRNA conjugate was transfected into RKO cells as follows: RKO cells were taken, washed with PBS, digested with trypsin, adjusted to appropriate density, 24h later, siRNA was transferred into RKO cells using Lipofectamine RNAiMax, inoculated into 48 well plates at a density of 100,000 cells per well, and 500. Mu.L per well of culture medium. Cells were exposed to 5% CO ₂ Culturing in incubator at 37 deg.c for 48 hr.

The siRNA tested was tested at 2 concentration points, 2 duplicate wells. In control, 4 concentration points, 2 duplicate wells, were tested.

24 hours after transfection, cells were collected, RNA was extracted, and total intracellular PD-L1-RNA was detected by RT-PCR.

The procedure for the detection of PD-L1 RNA is briefly described as follows: total RNA in cells was extracted by the trizol method, and was reverse transcribed into cDNA by adding random primers, referring to the reverse transcription kit (takara) instructions, and then the PD-L1 cDNA in the sample was detected by qPCR. Meanwhile, GAPDH primers and probes specifically detect GAPDH cDNA.

The PCR reaction procedure was: 95℃for 2 minutes, then enter a cyclic mode, 95℃for 10 seconds, followed by 60℃for 30 seconds for a total of 40 cycles. The PD-L1 RNA content in the samples is calculated according to the Ct value of each sample.

The PCR primers were as follows:

Homo-PDLI-F1(192bp):5’-TGCCTTGGTGTAGCACTGAC-3’；

Homo-PDLI-R1(192bp):5’-CCCCGATGAACCCCTAAACC-3’。

the expression level of the target gene PD-L1 mRNA was calculated by a DeltaCt relative quantification method for each sample. The relative expression level of the target gene is expressed by using 2-delta CT, and the calculation method is as follows:

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

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

Δct=ct (gene of interest) -Ct (gapdh);

ΔΔΔCt =Δct (detection of sample) - Δct (Mock) Relative mRNA expression to Mock =2 ^-ΔΔCt ；

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

The inhibition rate was calculated as follows: (1- (2- ^ΔΔCt ))*100％。

Table 5 shows the inhibitory activity of the siRNA of the present invention on PD-L1.

TABLE 5 inhibition of PD-L1 RNA by siRNA of the invention

Table 6 shows the inhibitory activity (IC) of the siRNA conjugates of the invention on PD-L1 ₅₀ )。

TABLE 6 inhibitory Activity of siRNA conjugates of the invention on PD-L1 (IC ₅₀ )

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

Claims

1. An siRNA targeting programmed death-ligand 1, the siRNA comprising a sense strand and an antisense strand, each nucleotide in the siRNA being independently a modified or unmodified nucleotide, wherein the sense strand comprises a stretch of nucleotide sequence I and the antisense strand comprises a stretch of nucleotide sequence II, the nucleotide sequence I and the nucleotide sequence II being at least partially complementary in antiphase to form a duplex region, the nucleotide sequence I and the nucleotide sequence II being selected from the group of sequences set forth in (I) or (II):

(i) The sequence I and SEQ ID NO:1, and not more than 3 nucleotides, and the nucleotide sequence II is identical to the nucleotide sequence set forth in SEQ ID NO:2, and no more than 3 nucleotide differences:

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

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

(ii) The sequence I and SEQ ID NO:3, and not more than 3 nucleotides, and said nucleotide sequence II is identical to SEQ ID NO:4, and no more than 3 nucleotides different in length;

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

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

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

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

3. The siRNA of claim 1 or 2, wherein at least one nucleotide of the sense strand or the antisense strand is a modified nucleotide and/or at least one phosphate group is a phosphate group having a modification group.

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

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

6. The siRNA according to claim 5, wherein the nucleotide formed by substituting the hydroxyl group at the 2 '-position of the ribosyl of the nucleotide with a non-fluorine group is selected from one of a 2' -alkoxy-modified nucleotide, a 2 '-substituted alkoxy-modified nucleotide, a 2' -alkyl-modified nucleotide, a 2 '-substituted alkyl-modified nucleotide, a 2' -amino-modified nucleotide, a 2 '-substituted amino-modified nucleotide, and a 2' -deoxynucleotide; the nucleotide analogue is selected from one of an iso nucleotide, LNA (6), ENA (7), cET (8), UNA (9) and GNA (10):

wherein R in the above formula (9) and formula (10) _a Selected from H, OH or alkoxy (O-alkyl).

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

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

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

10. an siRNA conjugate targeting apoptosis-ligand 1, said siRNA conjugate comprising the siRNA of any one of claims 1-9 and a conjugate group conjugated to the siRNA.

11. The siRNA conjugate of claim 10, wherein the conjugate group comprises a pharmaceutically acceptable targeting group and a linker, and the siRNA, linker and targeting group are sequentially covalently or non-covalently linked.

12. The siRNA conjugate of claim 11, wherein the linker has a structure as shown in formula (19):

wherein m is an integer of 1 to 3;

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

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

13. The siRNA conjugate of any one of claims 10-12, wherein the siRNA conjugate has a structure as shown in formula (23):

wherein the double helix structure represents the siRNA.

14. The siRNA conjugate of any of claims 10 to 13, wherein said linker is attached to the 3 'end of the sense strand or the 5' end of the sense strand of said siRNA.

15. The siRNA conjugate of claim 10, wherein the conjugate has a structure represented by formula (24), (25) or (26):

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

wherein E is ₁ Is OH or SH, nu is the siRNA of any one of claims 1-9;

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

M ₁ represents a targeting group;

indicating the site of covalent attachment of the group.

16. The siRNA conjugate of claim 15, wherein each L ₁ Independently selected from the group consisting of ₁ -A ₁₄ Groups and any combination of groups:

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

each k2 is independently an integer from 1 to 20;

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

17. The siRNA conjugate of claim 16, wherein L ₁ Is a linked combination of at least 2 of the groups A1, A4, A8, A10, A11.

18. The siRNA conjugate of any of claims 15 to 17, wherein L ₁ Is 3 to 20 atoms in length.

19. The siRNA conjugate of any of claims 16 to 18, wherein k1 is an integer from 3 to 5, k2 is an integer from 3 to 5, R _c Is one of methyl, ethyl and isopropyl, R _d Is A15 or A16, R _e Is one of methyl, ethyl, isopropyl and butyl.

20. The siRNA conjugate of any of claims 10-19, wherein each of said targeting groups is independently selected from one of D-galactose, L-galactose, a-D-glucopyranose, β -D-glucopyranose, a-D-glucofuranose, β -D-glucofuranose, a-D-fructopyranose, a-D-galactopyranose, β -D-galactopyranose, a-D-galactofuranose, β -D-galactofuranose, glucosamine, sialic acid, galactosamine, N-acetylgalactosamine, N-trifluoroacetylgalactosamine, N-propionylgalactosamine, N-butyryl galactosamine, N-isobutyryl galactosamine.

21. The siRNA conjugate of claim 20, wherein at least one or each of said targeting groups is galactose or N-acetylgalactosamine.

22. The siRNA conjugate of any of claims 15 to 21, wherein R ₁ Containing both a linking site attached to an N atom on a nitrogen-containing backbone and R ₂ A linking site to which the P atom is linked.

23. The siRNA conjugate of any of claims 15 to 22, wherein R ₁ The above-mentioned site bonded to N atom on the nitrogen-containing skeleton forms an amide bond with N, the above-mentioned site bonded to R ₂ The P atom on the substrate is linked to P to form a phosphate bond or a phosphorothioate bond.

24. The siRNA conjugate of any of claims 15 to 23, wherein a P atom in formula (S1) is attached to the end of the sense strand or the antisense strand of the siRNA.

25. The siRNA conjugate of any of claims 15-24, wherein the P atom in formula (S1) is linked to the 2' position, the 3' position or the 5' position of a nucleotide in the siRNA via a phosphodiester linkage.

26. The siRNA of any one of claims 1-9, or the siRNA conjugate of any one of claims 10-25, wherein the siRNA or siRNA conjugate inhibits expression of apoptosis-ligand 1 in a cell.

27. A pharmaceutical composition comprising the siRNA of any one of claims 1-9, and/or the siRNA conjugate of any one of claims 10-25 and a pharmaceutically acceptable excipient.

28. Use of the siRNA of any one of claims 1-9, the siRNA conjugate of any one of claims 10-25 and/or the pharmaceutical composition of claim 27 in the manufacture of a medicament for treating a disease and/or disorder associated with abnormal expression of a apoptosis-ligand 1 gene.

29. The use of claim 28, wherein the disease and/or disorder associated with aberrant expression of the apoptosis-ligand 1 gene is a chronic viral infection, a chronic bacterial infection, and a chronic parasitic infection or cancer; alternatively, the chronic viral infection comprises HIV, HBV, HCV or HTLV, the chronic bacterial infection comprises helicobacter pylori, the chronic parasitic infection comprises schistosome, and the cancer comprises breast cancer, lung cancer, colorectal cancer, ovarian cancer, melanoma, bladder cancer, liver cancer, gastric cancer, glioma, thyroid cancer or head and neck tumor.

30. A cell comprising the siRNA of any one of claims 1-9 or the siRNA conjugate of any one of claims 10-25.

31. A method of apoptosis-ligand 1 gene expression in a cell, the method comprising contacting an effective amount of the siRNA of any one of claims 1-9, the siRNA conjugate of any one of claims 10-25 and/or the pharmaceutical composition of claim 27 with the cell, thereby inhibiting expression of the apoptosis-ligand 1 gene in the cell.