FTO inhibitor composition and application thereof

Abstract

A double-stranded oligonucleotide that inhibits the expression of an FTO gene, said double-stranded oligonucleotide comprising a positive-sense strand and an antisense strand wherein the positive-sense strand comprises at least 15 contiguous nucleotides identical or substantially identical to SEQ ID NO: 449 (mRNA sequence of SEQ ID NO: 449), the basic complementation being that the mismatch of the positive-sense strand and the antisense strand in the double-stranded region does not exceed 3 nucleotides. The double-stranded oligonucleotide relieves the problem that in the prior art, nucleic acid drugs capable of inhibiting FTO gene expression and treating obesity or obesity-related diseases or Alzheimer's disease and other FTO gene expression-related diseases are lacked.

Description

FTO inhibitor composition and application thereof

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] The present disclosure claims priority to Chinese patent application No. 202310990258.X, filed with the State Intellectual Property Office of China on August 8, 2023, and entitled “FTO inhibitor composition and its application”, and Chinese patent application No. 202411030247.8, filed with the State Intellectual Property Office of China on July 30, 2024, and entitled “FTO inhibitor composition and its application”, the entire contents of which are incorporated by reference into the present disclosure. Technical Field

[0003] The present disclosure belongs to the technical field of nucleic acid drugs, and in particular relates to a double-stranded oligonucleotide and double-stranded RNAi for inhibiting FTO gene expression, a pharmaceutical composition comprising the double-stranded oligonucleotide and / or double-stranded RNAi, and applications thereof. Background Art

[0004] With economic development and improved living standards, obesity has become a major threat to human health. FTO (alpha-ketoglutarate-dependent dioxygenase) was the first candidate gene associated with obesity discovered by GWAS and is currently the most strongly associated susceptibility gene with obesity. FTO is a demethylase that, in response to oxygen, Fe(II), and vitamin C, catalyzes the demethylation of 3-methylthymine in single-stranded DNA in the cell nucleus, with a more pronounced demethylation of 3-methyluracil in single-stranded RNA. Studies have shown that FTO's demethylase activity plays an important role in regulating energy metabolism. Specifically, FTO mediates the expression of G6PC, a key gene for gluconeogenesis, in an m6A-dependent manner in liver tissue. Knockdown of FTO in liver tissue increases m6A levels in FOXO1 (forkhead box O1) messenger RNA, leading to downregulation of FOXO1 protein expression, which in turn reduces G6PC expression, resulting in weight loss and decreased blood glucose levels. Furthermore, unlike in liver tissue, FOXO1's inhibitory effect on transcription factors in adipose tissue can upregulate the expression of UCP1, a key factor in thermogenesis, thereby increasing thermogenesis in white adipose tissue. Inhibiting FTO activity in the presence of a high-sugar, high-fat diet can significantly reduce body fat content, increase thermogenesis, reduce weight, and improve various complications caused by obesity. These studies suggest that FTO could serve as a new liver-targeted drug target for inhibiting weight gain, promoting weight loss, lowering serum low-density lipoprotein cholesterol, LDL-C, or triglycerides, or for the treatment of obesity, obesity-related diseases, or Alzheimer's disease.

[0005] Inhibiting FTO gene expression through siRNA can prevent and treat diseases caused by abnormal body fat levels, particularly obesity, obesity-related diseases, and Alzheimer's disease. Currently, there are no commercially available RNAi therapeutics targeting FTO. Therefore, there is a need for siRNA therapeutics that can inhibit FTO gene expression and treat obesity, obesity-related diseases, or Alzheimer's disease.

[0006] Summary of the Invention

[0007] To address the clinical needs of various obesity-induced diseases, this disclosure has developed small nucleic acid drugs targeting FTO that can reduce body fat levels and thereby prevent or control obesity and other related diseases. This disclosure also provides double-stranded oligonucleotides that inhibit FTO gene expression, as well as methods and uses of these oligonucleotides for inhibiting or reducing FTO gene expression or treating FTO-mediated diseases or symptoms.

[0008] According to one aspect of the present disclosure, a double-stranded oligonucleotide (dsRNA) for inhibiting FTO gene expression is provided, wherein the double-stranded oligonucleotide comprises a sense strand and an antisense strand, wherein the antisense strand is complementary or substantially complementary to the sense strand; wherein the substantially complementary strand means that the mismatch between the sense strand and the antisense strand in the double-stranded region does not exceed 3 nucleotides.

[0009] The positive strand comprises at least 15 consecutive nucleotides that are identical or substantially identical to SEQ ID NO: 449, and the substantially identical means that there are no more than 3 nucleotide differences between the positive strand and at least 15 consecutive nucleotides in the SEQ ID NO: 449 sequence.

[0010] According to another aspect of the present disclosure, a double-stranded RNAi for inhibiting FTO gene expression is provided, the double-stranded RNAi comprising a sense strand and an antisense strand, wherein positions 2 to 19 in the nucleotide sequence of the antisense strand comprise a region complementary to the FTO RNA transcript, wherein the complementary region comprises a nucleotide sequence that differs by 0, 1, 2, or 3 nucleotides from any one of the antisense sequences listed in SEQ ID NO: 225 to SEQ ID NO: 448 in Table 1; and the nucleotide sequence of the sense strand comprises a nucleotide sequence that differs by 0, 1, 2, or 3 nucleotides from any one of the sequences listed in SEQ ID NO: 1 to SEQ ID NO: 224 in Table 1;

[0011] Furthermore, the sense strand and the antisense strand are complementary or substantially complementary to form a double-stranded region, and the double-stranded region is represented by the following formula (I):

[0012] SS:5'-(N)a'-(X)p'-(N)b'-(X)q'-(N)c'-(X)r'-(N)d'-3'

[0013] AS:3'-(N)a-(X)p-(N)b-(X)q-(N)c-5'(I),

[0014] Wherein, SS represents the sense strand, and AS represents the antisense strand;

[0015] All nucleotides of the sense strand and the antisense strand are modified nucleotides;

[0016] Each of the Ns independently represents a modified nucleotide: a 2'-O-methyl modified nucleotide, a 2'-fluoro modified nucleotide, or a 2'-deoxy modified nucleotide;

[0017] The X's are each independently a 2'-O-methoxyethyl modified nucleotide, a 2'-O-methyl modified nucleotide or a 2'-O(CH2) n OR substituted modified nucleotides, wherein n is 1 or 2, and R is selected from: substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 alkoxy; wherein the substituent is selected from halogen, C1-C6 alkyl, C1-C6 alkoxy, hydroxyl, and amino;

[0018] Said a, a', p, p', b, b', q, q', c, c', r', d' each independently represents the number of nucleotides, wherein: a' is selected from an integer of 3-8; p' is selected from an integer of 0-3; b' is selected from an integer of 4-13; q' is selected from an integer of 0-4; c' is selected from an integer of 3-9; r' is selected from an integer of 0-3; d' is selected from an integer of 0-9; a is selected from an integer of 4-7; p is selected from an integer of 0-1; b is selected from an integer of 4-8; q is selected from an integer of 0-4; c is selected from an integer of 6-10;

[0019] Furthermore, p', q', r', p, and q are not all 0 at the same time, and 0≤q'+r'≤4.

[0020] According to another aspect of the present disclosure, provided is a use of a double-stranded RNAi agent in the preparation of a medicament for alleviating, preventing and / or treating a disease or condition mediated by the FTO gene.

[0021] According to another aspect of the present disclosure, a pharmaceutical composition is provided, comprising the double-stranded RNAi agent of the present disclosure and pharmaceutically optional excipients.

[0022] According to another aspect of the present disclosure, a method for inhibiting FTO gene expression in cells in vitro is provided, the method comprising:

[0023] (a) contacting the cell with the double-stranded RNAi of the present disclosure or a pharmaceutical composition thereof; and

[0024] (b) maintaining the cells produced in step (a) for a period of time sufficient to obtain degradation of the mRNA transcript of the FTO gene, thereby inhibiting the expression of the FTO gene in the cells, wherein the expression of the FTO gene is inhibited by at least 70%.

[0025] According to another aspect of the present disclosure, provided is a use of the double-stranded RNAi or a pharmaceutical composition thereof in the preparation of a medicament for treating a subject suffering from an FTO-related disorder.

[0026] According to another aspect of the present disclosure, provided is a use of the double-stranded RNAi in preparing a medicament for treating a subject suffering from a disorder associated with FTO disorder.

[0027] According to another aspect of the present disclosure, a kit is provided, characterized in that the kit comprises the double-stranded RNAi agent or the pharmaceutical composition thereof according to the present disclosure.

[0028] According to another aspect of the present disclosure, a method for inhibiting FTO gene expression in a cell is provided, characterized in that the method comprises administering the double-stranded RNAi agent or the pharmaceutical composition thereof described in the present disclosure to a subject.

[0029] According to another aspect of the present disclosure, a method for alleviating, treating and / or preventing FTO-mediated diseases or conditions is provided, characterized in that the method comprises administering the double-stranded RNAi agent or pharmaceutical composition thereof described in the present disclosure to a subject.

[0030] It is well known to those skilled in the art that modified nucleotide groups can be introduced into the double-stranded RNAi described herein by using nucleoside monomers having corresponding modifications. Methods for preparing nucleoside monomers having corresponding modifications and methods for introducing modified nucleotide groups into double-stranded RNAi are also well known to those skilled in the art. All modified nucleoside monomers are commercially available or can be prepared using known methods.

[0031] Additional aspects and advantages of the present disclosure will be given in part in the description that follows and, in part, will be obvious from the description that follows, or will be learned through practice of the present disclosure. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] The above and / or additional aspects and advantages of the present disclosure will become apparent and readily understood from the description of the embodiments in conjunction with the following drawings, in which:

[0033] FIG1 shows the inhibitory activity of target genes in Balb / c-HDI mice after administration of double-stranded RNAi in Experiment 2.

[0034] FIG2 shows the inhibitory activity of target genes in C57BL / 6J mice after administration of double-stranded RNAi in Experiment 3.

[0035] FIG3 shows the expression of FTO protein in liver tissue of DIO mice after a single administration of RZM06005.

[0036] FIG4 shows the results of HE staining of liver tissues of DIO mice after a single administration of RZM06005.

[0037] FIG5 shows the results of Oil Red O staining of liver tissues of DIO mice after a single administration of RZM06005.

[0038] FIG6 shows the relative expression level of FTO mRNA in the liver tissue of Balbc-HDI mice after a single administration of RZ006037-RZ006043. DETAILED DESCRIPTION

[0039] The embodiments of the present disclosure are described in detail below. The embodiments described below are exemplary and are only used to explain the present disclosure, and should not be understood as limiting the present disclosure.

[0040] The following is a clear and complete description of the technical solutions in the embodiments of the present disclosure. Obviously, the embodiments described are only some of the embodiments of the present disclosure, not all of them. Based on the embodiments of the present disclosure, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of the present disclosure.

[0041] [definition]

[0042] In this document, above and below, unless otherwise specified, capital letters A (adenine), U (uracil), G (guanine), C (cytosine), and T (thymine, which also represents uracil in the sequence table) represent the base composition of nucleotides; lowercase letter m indicates that the nucleotide adjacent to the left of the letter m is a 2′-methoxy-modified nucleotide; lowercase letter f indicates that the nucleotide adjacent to the left of the letter f is a 2′-fluoro-modified nucleotide; (moe) indicates that the nucleotide adjacent to the left of the combination identifier is a 2′-O-methoxyethyl-modified nucleotide; lowercase letter s indicates that the two nucleotides adjacent to the left and right of the letter s are connected by phosphorothioate groups.

[0043] L96 represents a ligand moiety linked to Nu (double-stranded oligonucleotide) represented by the following formula.

[0044] As used herein, "fluorinated nucleotides" or "2'-fluorinated nucleotides" refer to nucleotides in which the hydroxyl group at the 2' position of the ribose group of the nucleotide is replaced by fluorine, and "non-fluorinated nucleotides" refer to nucleotides or nucleotide analogs in which the hydroxyl group at the 2' position of the ribose group of the nucleotide is replaced by a non-fluorinated group. The "methoxy-modified nucleotides" or "2'-O-methoxyethyl-modified nucleotides" refer to nucleotides in which the 2' hydroxyl group of the ribose group is replaced by a methoxy group or a methoxyethyl group. "Methoxy-modified nucleotides" are also referred to as 2'-OMe or 2'-O-methyl-modified nucleotides, which can be used interchangeably. "2'-O-methoxyethyl-modified nucleotides" are also referred to as 2'-MOE modifications. 2'-deoxynucleotides refer to nucleotides in which the 2' position of the ribose group is hydrogen.

[0045] As used herein, a double-stranded oligonucleotide is composed of two chains, wherein the chain that binds to the target sequence is called the antisense strand or guide strand, and the other chain is called the sense strand or passenger strand. The term "antisense strand" refers to a chain of a double-stranded oligonucleotide that includes a region that is completely or substantially complementary to the target sequence. The term "sense strand" refers to a chain of a double-stranded oligonucleotide that includes a region that is substantially complementary to the region of the antisense strand as defined herein. The term "complementary region" refers to a region on the antisense strand that is completely or substantially complementary to the target sequence. In the case where the complementary region is not completely complementary to the target sequence, the mismatch can be located in the interior or terminal regions of the molecule. As used herein, the term "complementary" refers to the ability of a first polynucleotide to hybridize with a second polynucleotide under certain conditions, such as stringent conditions. As used herein, double-stranded oligonucleotides and siRNA can be used interchangeably.

[0046] As used herein, the expressions "complementary" and "reverse complementary" are used interchangeably and have the meanings known to those skilled in the art, i.e., in a double-stranded nucleic acid molecule, the bases of one strand are paired with the bases of the other strand in a complementary manner.

[0047] As used herein, unless otherwise specified, "substantially reverse complementary" or "substantially complementary" means that there are no more than three base mismatches between the two nucleotide sequences involved; "substantially reverse complementary" means that there is no more than one base mismatch between the two nucleotide sequences; and "completely reverse complementary" means that there are no base mismatches between the two nucleotide sequences.

[0048] Exemplary FTO nucleotide sequences of the present disclosure are listed in Tables 1-3. The term "FTO" also refers to naturally occurring DNA sequence variations of the FTO gene. It also refers to single nucleotide polymorphisms in the FTO gene. Many sequence variations in the FTO gene have been identified and can be discovered, as is known in the art.

[0049] The terms "RNAi", "iRNA", "RNAi agent", "RNAi reagent", "RNA interference agent", and "RNA inhibitor" are used interchangeably herein to refer to molecules or agents comprising RNA and that can mediate targeted cleavage of RNA transcripts through the RNA-induced silencing complex (RISC) pathway. It is well known in the art that RNA directs sequence-specific degradation of mRNA through a process known as RNA interference (RNAi). In one embodiment, the RNAi agents of the present disclosure include single-stranded or double-stranded RNA that interacts with a target RNA sequence (e.g., FTO) to direct the cleavage of the target RNA. Therefore, in one aspect, the present disclosure relates to the term "siRNA" which can also be used to refer to the RNAi described above.

[0050] In some embodiments, the "RNAi" used in the compositions, uses, and methods of the present disclosure is double-stranded RNA, and the "RNAi agent" includes the double-stranded RNA, and the "RNAi agent" can refer to a "double-stranded RNAi agent", a "double-stranded RNA (dsRNA) molecule", a "dsRNA agent", a "siRNA agent", or a "dsRNA agent".

[0051] "dsRNA" or "siRNA" refers to a complex of ribonucleic acid molecules that has a double-stranded structure, comprising two antiparallel and substantially complementary nucleic acid strands, referred to as "sense" and "antisense" orientations relative to the target RNA (e.g., FTO gene).

[0052] In addition, as used in this specification, "RNAi reagents" or "RNAi agents" can include ribonucleotides with chemical modifications and / or ligands; RNAi reagents can include substantial modifications at multiple nucleotides. The term "modified nucleotides" refers to nucleotides that independently have modified sugar moieties, modified internucleotide linkages, and / or modified nucleobases. Thus, the term modified nucleotides encompasses substitutions, additions, or removals of, for example, functional groups or atoms of internucleotide linkages, sugars, or nucleobases. Modifications suitable for use in the agents of the present disclosure include all types of modifications disclosed herein or known in the art. As used in siRNA molecules, any such modification can be encompassed by "RNAi agent."

[0053] The term "nucleotide overhang" or "overhang" refers to at least one unpaired nucleotide that protrudes from the duplex structure of an iRNA (e.g., dsRNA). For example, a nucleotide overhang exists when the 3'-end of one strand of a dsRNA extends beyond the 5'-end of the other strand, or vice versa. The one or more overhangs can be on the sense strand, the antisense strand, or any combination thereof. Additionally, the one or more nucleotides of the overhang can be present on the 5' end, the 3' end, or both ends of the antisense or sense strand of the dsRNA.

[0054] The term "ligand" generally refers to any compound or molecule that can be covalently or otherwise chemically bound to a biologically active substance (such as an oligonucleotide). In certain embodiments, a ligand can interact directly or indirectly with another compound, such as a receptor. The receptor that interacts with the ligand can be present on the cell surface, or alternatively can be an intracellular and / or intercellular receptor. The interaction of the ligand with the receptor can result in a biochemical reaction, or can be simply a physical interaction or binding.

[0055] The term "administer" generally refers to introducing the disclosed pharmaceutical formulation into the body of a subject by any route of introduction or delivery. Any method known to those skilled in the art for contacting cells, organs, or tissues with the drug can be employed. The administration may include, but is not limited to, intravenous, intraarterial, intranasal, intraperitoneal, intramuscular, subcutaneous, or oral administration. The daily dose may be divided into one, two, or more doses in a suitable form for administration at one, two, or more times during a certain time period.

[0056] The term "contacting" generally refers to the contacting of two or more different types of substances in any order, in any manner, and for any duration. Contact can occur in vivo, ex vivo, or in vitro. In some embodiments, it can refer to direct contact of a RNAi agent or composition of the present disclosure with a cell or tissue. In other embodiments, the term refers to indirect contact of a RNAi agent or composition of the present disclosure with a cell or tissue.

[0057] The term "subject" generally refers to a human or non-human animal (including mammals) in need of diagnosis, prognosis, improvement, prevention and / or treatment of a disease, such as humans, non-human primates (apes, gibbons, gorillas, chimpanzees, orangutans, macaques), livestock (dogs and cats), farm animals (horses, cattle, goats, sheep, pigs) and experimental animals (mice, rats, rabbits, guinea pigs). Human subjects include fetuses, newborns, infants, adolescents and adult subjects. Subjects include animal disease models.

[0058] In this disclosure, the terms "include," "comprising," "having," "may," "containing," and variations thereof are generally intended to be open transitional phrases, terms, or words that do not exclude the possibility of additional actions or structures. The term "consisting of" generally indicates that no other components (or, similarly, features, integers, steps, etc.) can be present. Unless the context clearly dictates otherwise, nouns with an indefinite number also include plural referents.

[0059] In a first aspect of the present disclosure, a double-stranded oligonucleotide (dsRNA) for inhibiting FTO gene expression is provided, wherein the double-stranded oligonucleotide is capable of inhibiting FTO gene expression in mammals, including humans, monkeys, rats, or mice. The double-stranded oligonucleotide is capable of inhibiting FTO gene expression in cells in vitro and in vivo.

[0060] Specifically, the present disclosure provides a double-stranded oligonucleotide (dsRNA) for inhibiting FTO gene expression, the double-stranded oligonucleotide comprising a sense strand and an antisense strand, the antisense strand being complementary or substantially complementary to the sense strand; the substantially complementary strand means that the mismatch between the sense strand and the antisense strand in the double-stranded region is no more than 3 nucleotides. Wherein, the sense strand comprises at least 15 consecutive nucleotides identical or substantially identical to SEQ ID NO: 449, and the substantially identical strand means that there are no more than 3 nucleotide differences between the sense strand and at least 15 consecutive nucleotides in the SEQ ID NO: 449 sequence.

[0061] In some alternative embodiments, the sense strand comprises at least 15 consecutive nucleotides that differ from the nucleotide sequence of SEQ ID NO: 449 by no more than 2 nucleotides.

[0062] In some alternative embodiments, the sense strand comprises at least 15 consecutive nucleotides that differ from the nucleotide sequence of SEQ ID NO: 449 by no more than 1 nucleotide.

[0063] In some optional embodiments, the antisense strand is complementary or substantially complementary to at least 15, 16, 17, 18, 19, 20, 21, 22 or 23 consecutive nucleotides of the nucleotide sequence of SEQ ID NO: 449, wherein the substantially complementary strand has no more than 3 nucleotide mismatches in the complementary region.

[0064] In some optional embodiments, the double-stranded oligonucleotide includes a sense strand and an antisense strand, the sense strand comprising at least 15 consecutive nucleotides of any one of the sequences shown in SEQ ID NO:1 to SEQ ID NO:224 in Table 1, or a nucleotide sequence that differs from the at least 15 consecutive nucleotides by no more than 3 nucleotides; and / or, the antisense strand comprises at least 15 consecutive nucleotides of any one of the sequences shown in SEQ ID NO:225 to SEQ ID NO:448 in Table 1, or a nucleotide sequence that differs from the at least 15 consecutive nucleotides by no more than 3 nucleotides.

[0065] In some optional embodiments, the positive strand comprises a nucleotide sequence that differs by no more than 3 nucleotides from any one of the sequences shown in SEQ ID NO: 1 to SEQ ID NO: 224 in Table 1, and / or positions 2-19 of the antisense strand comprise a nucleotide sequence that differs by no more than 3 nucleotides from positions 2-19 of any one of the sequences shown in SEQ ID NO: 225 to SEQ ID NO: 448 in Table 1.

[0066] In some optional embodiments, the sense strand and the antisense strand are complementary or substantially complementary to form a double-stranded region; the substantially complementary means that the sense strand and the antisense strand have no more than 3 nucleotide mismatches in the double-stranded region.

[0067] In some optional embodiments, the sense strand comprises a nucleotide sequence that differs by no more than 1 or 2 nucleotides from any one of the sequences shown in SEQ ID NO: 1 to SEQ ID NO: 224 in Table 1, and / or the antisense strand comprises a nucleotide sequence that differs by no more than 1 or 2 nucleotides from any one of the sequences shown in SEQ ID NO: 225 to SEQ ID NO: 448 in Table 1.

[0068] In some optional embodiments, the sense strand comprises a nucleotide sequence that differs by no more than 1 nucleotide from any one of the sequences shown in SEQ ID NO: 1 to SEQ ID NO: 224 in Table 1, and / or the antisense strand comprises a nucleotide sequence that differs by no more than 1 nucleotide from any one of the sequences shown in SEQ ID NO: 225 to SEQ ID NO: 448 in Table 1.

[0069] In some optional embodiments, the sense strand comprises any one of the nucleotide sequences shown in SEQ ID NO: 1 to SEQ ID NO: 224 in Table 1, and / or the antisense strand comprises any one of the nucleotide sequences shown in SEQ ID NO: 225 to SEQ ID NO: 448 in Table 1.

[0070] In some optional embodiments, the sense strand or the antisense strand comprises any sense strand or antisense strand selected from the duplexes shown in the following numbers in Table 1: RN006007, RN006009, RN006017, RN006032, RN006033, RN006040, RN006052, RN006066, RN006076, RN006077, RN006082, RN006104, RN006107, RN006108, RN006109, RN 006120, RN006124, RN006132, RN006133, RN006137, RN006138, RN006149, RN006157, RN006172, RN006173, RN006 181, RN006182, RN006186, RN006188, RN006201, RN006218, RN006219, RN006220, RN006221, RN006222, RN006223;

[0071] In some specific embodiments, the sense strand or the antisense strand comprises a duplex nucleotide sequence selected from the group consisting of RN006222.

[0072] In some optional embodiments, the double-stranded oligonucleotide comprises one or more selected from the duplex group shown in the following numbers in Table 1: RN006007, RN006009, RN006017, RN006032, RN006033, RN006040, RN006052, RN006066, RN006076, RN006077, RN006082, RN006104, RN006107, RN006108, RN006109, RN0060 6120, RN006124, RN006132, RN006133, RN006137, RN006138, RN006149, RN006157, RN006172, RN006173, RN0061 81. RN006182, RN006186, RN006188, RN006201, RN006218, RN006219, RN006220, RN006221, RN006222, RN006223;

[0073] In some embodiments, the double-stranded oligonucleotide comprises a duplex nucleotide sequence selected from the group consisting of RN006222.

[0074] In some alternative embodiments, each nucleotide of the double-stranded oligonucleotide is an unmodified or modified nucleotide.

[0075] In some alternative embodiments, at least one nucleotide in the double-stranded oligonucleotide is modified.

[0076] In some alternative embodiments, all nucleotides in the sense strand and all nucleotides in the antisense strand of the double-stranded oligonucleotide are modified nucleotides.

[0077] In some alternative embodiments, the modified nucleotides are selected from 3'-terminal deoxy-thymine (dT) nucleotides, 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-deoxy-modified nucleotides, 2'-O-methoxyethyl modified nucleotides, 2'-O(CH2) n One or more nucleotides substituted with OR; wherein n is 1 or 2, and R is selected from: substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 alkoxy; wherein the substituent is selected from halogen, C1-C6 alkyl, C1-C6 alkoxy, hydroxyl, and amino.

[0078] In some optional embodiments, the modified nucleotide is selected from 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-O-methoxyethyl modified nucleotides, 2'-O(CH2) n OR one or more of the nucleotides substituted.

[0079] In some optional embodiments, the sense strand of the double-stranded oligonucleotide comprises any modified sense strand nucleotide sequence shown in Table 2, and / or the antisense strand comprises any modified antisense strand nucleotide sequence shown in Table 2.

[0080] In some optional embodiments, the length of the antisense strand and the sense strand is 17-25 nucleotides.

[0081] In some alternative embodiments, at least one of the antisense strand and the sense strand comprises a 3'-end overhang of 1-2 nucleotides.

[0082] In a second aspect of the present disclosure, the present disclosure provides a double-stranded RNAi for inhibiting FTO gene expression, the double-stranded RNAi comprising a sense strand and an antisense strand, wherein positions 2-19 in the antisense strand nucleotide sequence comprise a region complementary to the FTO RNA transcript, wherein the complementary region comprises a nucleotide sequence that differs by 0, 1, 2, or 3 nucleotides from any of the antisense sequences listed in SEQ ID NO: 225 to SEQ ID NO: 448 in Table 1; and, the nucleotide sequence of the sense strand comprises a nucleotide sequence that differs by 0, 1, 2, or 3 nucleotides from any of the sequences listed in SEQ ID NO: 1 to SEQ ID NO: 224 in Table 1; and, the sense strand and the antisense strand are complementary or substantially complementary to form a double-stranded region, wherein the substantially complementary region refers to a mismatch of no more than 3 nucleotides between the sense strand and the antisense strand in the double-stranded region, and the double-stranded region is represented by the following formula (I):

[0083] SS:5'-(N)a'-(X)p'-(N)b'-(X)q'-(N)c'-(X)r'-(N)d'-3'

[0084] AS:3'-(N)a-(X)p-(N)b-(X)q-(N)c-5'(I),

[0085] Wherein, SS represents the sense strand, and AS represents the antisense strand;

[0086] All nucleotides of the sense strand and the antisense strand are modified nucleotides;

[0087] Each of the Ns independently represents a modified nucleotide: a 2'-O-methyl modified nucleotide, a 2'-fluoro modified nucleotide, or a 2'-deoxy-modified nucleotide;

[0088] The Xs are each independently a 2'-O-methoxyethyl modified nucleotide, a 2'-O-methyl modified nucleotide or a 2'-O(CH2) n OR substituted modified nucleotides, wherein n is 1 or 2, and R is selected from: substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 alkoxy; wherein the substituent is selected from halogen, C1-C6 alkyl, C1-C6 alkoxy, hydroxyl, and amino;

[0089] Said a, a', p, p', b, b', q, q', c, c', r', d' each independently represents the number of nucleotides, wherein: a' is selected from an integer of 3-8; p' is selected from an integer of 0-3; b' is selected from an integer of 4-13; q' is selected from an integer of 0-4; c' is selected from an integer of 3-9; r' is selected from an integer of 0-3; d' is selected from an integer of 0-9; a is selected from an integer of 4-7; p is selected from an integer of 0-1; b is selected from an integer of 4-8; q is selected from an integer of 0-4; c is selected from an integer of 6-10;

[0090] Furthermore, p', q', r', p, and q are not all 0 at the same time, and 0≤q'+r'≤4.

[0091] In some optional embodiments, the N is selected from 2′-O-methyl modified nucleotides, 2′-fluoro-modified nucleotides, and the nucleotide X is a 2′-O-methyl modified nucleotide or a 2′-O-methoxyethyl modified nucleotide;

[0092] In some alternative embodiments, the duplex includes at least one 2′-O-methoxyethyl modification or a 2′-O(CH2) n OR-modified nucleotides;

[0093] In some optional embodiments, the nucleotides of the sense strand and / or antisense strand of the double-stranded RNAi are all modified nucleotides, and, wherein, in the direction from the 5' end to the 3' end, at least three of the nucleotides at positions 7-10 of the nucleotide sequence in the sense strand are fluorine-modified nucleotides, and the nucleotides at the remaining positions are 2'-O-methyl-modified or 2'-O-methoxyethyl-modified nucleotides; and optionally, in the direction from the 5' end to the 3' end, at least four of the nucleotides at positions 2, 6, 9-12, 14 and 16 of the nucleotide sequence in the antisense strand are 2'-fluorine-modified nucleotides, and the nucleotides at the remaining positions are 2'-O-methyl-modified or 2'-O-methoxyethyl-modified nucleotides.

[0094] In some optional embodiments, the sense strand and / or the antisense strand contains at least one 2'-O-methoxyethyl modified nucleotide.

[0095] In some optional embodiments, in the double-stranded RNAi, in the direction from the 5' end to the 3' end, at least three of the nucleotides at positions 7 to 10 in the nucleotide sequence of the sense strand are fluorinated modified nucleotides, and the nucleotides at the remaining positions are 2'-O-methyl modified or 2'-O-methoxyethyl modified nucleotides;

[0096] In some optional embodiments, in the direction from the 5' end to the 3' end, at least four of the nucleotides at positions 2, 6, 9-12, 14 and 16 of the nucleotide sequence in the antisense strand are 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are 2'-O-methyl-modified or 2'-O-methoxyethyl-modified nucleotides.

[0097] In some optional embodiments, in the double-stranded RNAi, in the direction from the 5′ end to the 3′ end, the nucleotides at positions 7-10 of the nucleotide sequence in the sense strand are fluorinated nucleotides, and the nucleotides at the remaining positions are 2′-O-methyl modified or 2′-O-methoxyethyl modified nucleotides; and, the nucleotides at positions 2, 6, 14, and 16 of the nucleotide sequence in the antisense strand are 2′-fluorinated nucleotides, and any one nucleotide selected from positions 9 or 10 or 11 or 12 is a 2′-fluorinated nucleotide, and the nucleotides at the remaining positions are 2′-O-methyl modified or 2′-O-methoxyethyl modified nucleotides.

[0098] In some optional embodiments, in the double-stranded RNAi, in the direction from the 5′ end to the 3′ end, the nucleotides at positions 7 to 10 in the nucleotide sequence of the sense strand are fluorinated modified nucleotides, and the nucleotides at the remaining positions are 2′-O-methyl modified nucleotides;

[0099] Furthermore, positions 2, 6, 14, and 16 of the nucleotide sequence in the antisense strand are 2′-fluoro-modified nucleotides, and: any nucleotide selected from positions 9, 10, 11, or 12 is a 2′-fluoro-modified nucleotide, the nucleotide at position 15 is a 2′-O-methoxyethyl-modified nucleotide, and the remaining positions are 2′-O-methyl-modified nucleotides.

[0100] In some optional embodiments, the nucleotides of the sense strand and / or the antisense strand are all modified nucleotides; and the modifications of the sense strand and the antisense strand are selected from any one of the following (1)-(4):

[0101] (1) From the 5′ end to the 3′ end, the nucleotides at positions 7 to 10 in the nucleotide sequence of the sense strand are fluorinated nucleotides, and the nucleotides at the remaining positions are 2′-O-methyl modified nucleotides; the nucleotides at positions 2, 6, 9, 14, and 16 in the nucleotide sequence of the antisense strand are 2′-fluorinated nucleotides, and the nucleotides at the remaining positions are 2′-O-methyl modified nucleotides;

[0102] (2) From the 5′ end to the 3′ end, the nucleotides at positions 7 to 10 in the nucleotide sequence of the sense strand are fluorinated nucleotides, and the nucleotides at the remaining positions are 2′-O-methyl modified nucleotides; the nucleotides at positions 2, 6, 12, 14, and 16 in the nucleotide sequence of the antisense strand are 2′-fluorinated nucleotides, and the nucleotides at the remaining positions are 2′-O-methyl modified nucleotides;

[0103] (3) In the direction from the 5′ end to the 3′ end, the nucleotides at positions 7 to 10 in the nucleotide sequence of the sense strand are fluorinated nucleotides, and the nucleotides at the remaining positions are 2′-O-methyl modified nucleotides; the nucleotides at positions 2, 6, 9, 14, and 16 in the nucleotide sequence of the antisense strand are 2′-fluorinated nucleotides, the nucleotide at position 15 is a 2′-O-methoxyethyl modified nucleotide, and the remaining positions are 2′-O-methyl modified nucleotides;

[0104] (4) In the direction from the 5′ end to the 3′ end, the nucleotides at positions 7 to 10 in the nucleotide sequence of the sense strand are fluorinated nucleotides, and the nucleotides at the remaining positions are 2′-O-methyl modified nucleotides; the nucleotides at positions 2, 6, 12, 14, and 16 in the nucleotide sequence of the antisense strand are 2′-fluorinated nucleotides, the nucleotide at position 15 is a 2′-O-methoxyethyl modified nucleotide, and the nucleotides at the remaining positions are 2′-O-methyl modified nucleotides.

[0105] In some optional embodiments, the double-stranded RNAi further comprises a ligand; preferably, the ligand is conjugated to the 3' end of the sense strand of the double-stranded RNAi.

[0106] In some alternative embodiments, the ligand is one or more GalNAc (N-acetylgalactosamine) derivatives connected via a linker selected from a monovalent, divalent or trivalent linker.

[0107] In some optional embodiments, the ligand structure is as follows:

[0108] wherein represents the attachment site of the oligonucleotide molecule; m is 1, 2, 3 or 4; Z, p, q, R3, L and Y are defined as follows: Z is each independently selected from hydroxyl or sulfhydryl; p is each independently selected from 1, 2 or 3; q is each independently selected from 1, 2 or 3; each R3 is each independently selected from H, C1-C6 alkyl, C1-C6 haloalkyl or C1-C6 alkoxy; L is C1-C 20 The alkylene chain, or one or more C atoms in the chain, is substituted by any substituent selected from O, S, NH or -NH-C(O)-; Y is independently NH, O or S.

[0109] In some optional embodiments, the specific structure of the ligand is as follows:

[0110] In some alternative embodiments, the 3' end of the sense strand in the double-stranded RNAi is covalently coupled to a ligand.

[0111] In some alternative embodiments, the structure of the double-stranded RNAi of the present disclosure comprises a conjugate as shown in the following formula:

[0112] Here, Nu represents a double-stranded oligonucleotide molecule.

[0113] In some optional embodiments, the sense strand of the double-stranded oligonucleotide molecule comprises any modified sense strand nucleotide sequence shown in Table 2, and / or the antisense strand comprises any modified antisense strand nucleotide sequence shown in Table 2.

[0114] In some alternative embodiments, the ligand is attached to the 3' end of the sense strand.

[0115] Among them, Am, Cm, Gm and Um are 2'-O-methyladenosine-3'-phosphate, 2'-O-methylcytidine-3'-phosphate, 2'-O-methylguanosine phosphate and 2'-O-methyluridine 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; U(moe) is 2'-O-methoxyethyluridine-3'-phosphate, and s is phosphorothioate.

[0116] In some optional embodiments, the double-stranded RNAi disclosed herein may comprise a structure of Nu-(CR01008×3); wherein CR01008×3 is a ligand of the following structure:

[0117] In some alternative embodiments, the double-stranded RNAi of the present disclosure comprises a conjugate selected from the group consisting of conjugates numbered RZ006002, RZ006007, RZ006008, RZ006009, RZ006010, RZ006011, RZ006012, RZ006013, RZ006014, RZ006017, RZ006020, RZ006021, RZ006023, and RZ006029.

[0118] In some alternative embodiments, the sense strand and / or the antisense strand further comprises a 3' and / or 5' extension or overhang of 1-3 nucleotides in length.

[0119] In some alternative embodiments, the sense strand of the double-stranded region is 17-21 nucleotides long, and the antisense strand is 19-23 nucleotides long. Preferably, the double-stranded region is 19 to 23 nucleotide pairs long; or, the double-stranded region is 19 to 21 nucleotide pairs long.

[0120] In some alternative embodiments, the sense strand and / or the antisense strand of the RNAi agent independently comprises one or more phosphorothioate internucleoside linkages.

[0121] In some alternative embodiments, the sense strand comprises two consecutive phosphorothioate bonds between the terminal nucleotides at the 3' and 5' ends, or the antisense strand comprises two consecutive phosphorothioate bonds between the terminal nucleotides at the 3' and 5' ends.

[0122] In some alternative embodiments, at least one strand of the double-stranded region of the RNAi agent comprises a 3' overhang of 1-2 nucleotides.

[0123] In some alternative embodiments, the double-stranded RNAi comprises a sense strand and an antisense strand forming a double-stranded region, each strand being 14-25 nucleotides, and the double-stranded region is represented by the following formula II:

[0124] SS:5'-(N)a'-(X)p'-(N)b'-(X)q'-(N)c'-(X)r'-(N)d'-3'

[0125] AS:3'-(N)a-(X)p-(N)b-(X)q-(N)c-5'(II)

[0126] wherein SS represents the sense strand, AS represents the antisense strand, and the SS strand can be conjugated to a ligand; each of the Ns independently represents a 2'-O-methyl modified nucleotide, a 2'-fluoro modified nucleotide, or a 2'-deoxy-modified nucleotide; each of the Xs independently represents a 2'-O-methoxyethyl modified nucleotide;

[0127] wherein said a, a', p, p', b, b', q, q', c, c', r', d' each independently represents the number of nucleotides, wherein: a' is selected from an integer of 3-8; p' is selected from 0 or 1; b' is selected from an integer of 4-13; q' is selected from 0 or 1; c' is selected from an integer of 3-9; r' is selected from 0 or 1; d' is selected from an integer of 1-8; a is selected from an integer of 4-7; p is selected from 1; b is selected from an integer of 4-8; q is selected from 0 or 1; c is selected from an integer of 6-10; and p', q', r', p, q are not all 0, and 0≤q'+r'≤2;

[0128] Furthermore, there is at least one fluorinated-modified nucleotide in (N)a, and the 16th position of the antisense strand, counted from the 5'-end, is a fluorinated-modified nucleotide; there is at least one fluorinated-modified nucleotide in (N)b, and the 14th position of the antisense strand, counted from the 5'-end, is a fluorinated-modified nucleotide; there are at least two fluorinated-modified nucleotides in (N)c, and positions 2 and 6 of the antisense strand, counted from the 5'-end, are both fluorinated-modified nucleotides; and, counted from the 5'-end, the first four nucleotides of (N)b' contain at least two fluorinated-modified nucleotides.

[0129] In some optional embodiments, in the direction from the 5′ end to the 3′ end, the nucleotides at positions 7-10 of the nucleotide sequence in the sense chain of Formula II are fluorinated modified nucleotides, and the nucleotides at the remaining positions are 2′-O-methyl modified nucleotides; and, the nucleotides at positions 2, 6, 14, and 16 of the nucleotide sequence in the antisense chain are 2′-fluorinated modified nucleotides, and: any nucleotide selected from positions 9 or 10 or 11 or 12 is a 2′-fluorinated modified nucleotide, the nucleotide at position 15 is a 2′-O-methoxyethyl modified nucleotide, and the remaining positions are 2′-O-methyl modified nucleotides.

[0130] In some alternative embodiments, the double-stranded RNAi agent comprises a conjugate selected from the group consisting of the following numbers: RZ0060037-RZ006144.

[0131] In some alternative embodiments, the double-stranded RNAi is selected from the group consisting of the conjugates shown by the following numbers: RZ0060037-RZ006043.

[0132] In a third aspect of the present disclosure, the present disclosure provides a double-stranded RNAi for inhibiting FTO expression, the double-stranded RNAi comprising a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand comprises the nucleotide sequence shown in SEQ ID NO: 1 to SEQ ID NO: 224 in Table 1, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 225 to SEQ ID NO: 448 in Table 1. wherein each strand is no longer than 25 nucleotides, and both the sense strand and the antisense strand are modified nucleotides. the modified nucleotides comprise modified nucleotides selected from 2'-O-methyl modification and 2'-fluoro modification, and the sense strand comprises 1-3 phosphorothioate internucleotide bonds; wherein the modified nucleotides in the antisense strand comprise modified nucleotides selected from 2'-O-methyl modification, 2'-O-methoxyethyl modification and 2'-fluoro modification, wherein the antisense strand comprises 2-5 phosphorothioate internucleotide bonds; and wherein the sense strand is conjugated to a GalNAc ligand at the 3'-terminus.

[0133] In some alternative embodiments, the double-stranded RNAi of the present disclosure comprises a conjugate selected from the group consisting of conjugates shown as RZ0060037-RZ006144.

[0134] In some alternative embodiments, the double-stranded RNAi of the present disclosure comprises a conjugate selected from the group consisting of conjugates numbered RZ0060037-RZ006043.

[0135] In a fourth aspect of the present disclosure, the present disclosure provides use of a double-stranded RNAi agent in the preparation of a medicament for alleviating, preventing and / or treating a disease or condition mediated by the FTO gene.

[0136] In some optional embodiments, the disease or condition comprises obesity or obesity-related diseases (particularly obesity-related diabetes, hyperglycemia, diabetic nephropathy, hyperlipidemia, coronary heart disease, atherosclerosis, hypertension, cardiovascular or cerebrovascular disease) or Alzheimer's disease.

[0137] In a fifth aspect of the present disclosure, the present disclosure provides a pharmaceutical composition comprising the double-stranded RNAi agent of the present disclosure and pharmaceutically optional excipients.

[0138] In some alternative embodiments, the double-stranded RNAi described herein can be administered in an unbuffered solution or a buffer, wherein the unbuffered solution is saline or water, and the buffer comprises acetate, citrate, prolamin, carbonate, or phosphate, or any combination thereof; alternatively, wherein the buffer is phosphate-buffered saline (PBS).

[0139] In a sixth aspect of the present disclosure, the present disclosure provides a method for inhibiting FTO gene expression in cells in vitro, the method comprising:

[0140] (a) contacting the cell with the double-stranded RNAi of the present disclosure or a pharmaceutical composition thereof; and

[0141] (b) maintaining the cells produced in step (a) for a period of time sufficient to obtain degradation of the mRNA transcript of the FTO gene, thereby inhibiting the expression of the FTO gene in the cells, wherein the expression of the FTO gene is inhibited by at least 70%.

[0142] In a seventh aspect of the present disclosure, the present disclosure provides use of the double-stranded RNAi or a pharmaceutical composition thereof in the preparation of a medicament for treating a subject suffering from an FTO-related disorder.

[0143] In an eighth aspect of the present disclosure, the present disclosure provides use of the double-stranded RNAi in preparing a medicament for treating a subject suffering from a disorder related to FTO disorder, wherein the medicament is subcutaneously administered to the subject, optionally, wherein the subject is a human or other mammal.

[0144] In some alternative embodiments, the double-stranded RNAi is administered at a dose of 0.01 mg / kg to 10 mg / kg or 0.5 mg / kg to 50 mg / kg.

[0145] In some alternative embodiments, the double-stranded RNAi is administered subcutaneously or intravenously.

[0146] In a ninth aspect of the present disclosure, the present disclosure provides a kit, characterized in that the kit comprises the double-stranded RNAi agent or the pharmaceutical composition thereof according to the present disclosure.

[0147] In a tenth aspect of the present disclosure, the present disclosure provides a method for inhibiting FTO gene expression in a cell, characterized in that the method comprises administering the double-stranded RNAi agent or the pharmaceutical composition thereof described in the present disclosure to a subject.

[0148] In some alternative embodiments, the expression of FTO is inhibited by at least 50%, 60%, 70%, 80%, 90% or 95%. In some alternative embodiments, the inhibition of FTO expression reduces the level of FTO protein in the subject by at least 50%, 60%, 70%, 80%, 90% or 95%.

[0149] In the eleventh aspect of the present disclosure, the present disclosure provides a method for alleviating, treating and / or preventing FTO-mediated diseases or conditions, characterized in that the method comprises administering the double-stranded RNAi agent or pharmaceutical composition thereof described in the present disclosure to a subject.

[0150] In some alternative embodiments, the FTO-mediated disease or disorder includes a disease associated with the mRNA level of FTO gene expression.

[0151] In some optional embodiments, the FTO-related disease includes obesity or obesity-related diseases (particularly obesity-related diabetes, hyperglycemia, diabetic nephropathy, hyperlipidemia, coronary heart disease, atherosclerosis, hypertension, cardiovascular or cerebrovascular disease) or Alzheimer's disease.

[0152] According to the experimental data of the Examples, the double-stranded RNAi agent or its pharmaceutical composition provided by the present disclosure can effectively regulate the expression level of the FTO gene, and thus can effectively treat and / or prevent diseases or symptoms associated with dysregulated FTO gene expression.

[0153] In some optional embodiments, the pharmaceutical composition can be administered by any suitable means, such as in the form of an injection or infusion solution (e.g., subcutaneously, intramuscularly, or intravenously), and the administration of the double-stranded oligonucleotide or the pharmaceutical composition includes, but is not limited to, single administration or multiple administration. The dosage range is 0.1 mg / kg to 100 mg / kg, 0.5 mg / kg to 50 mg / kg, such as 3 mg / kg, 10 mg / kg, or 33 mg / kg.

[0154] The mRNA sequence encoding FTO involved in the present disclosure (SEQ ID NO: 449, NM_001363897.1) is as follows:

[0155] The dsRNA sequences used in the present disclosure were all commissioned to Kunshan Aotai Biotechnology Co., Ltd. for synthesis; the PCR primers used in the present disclosure were all commissioned to Sangon Biotechnology (Shanghai) Co., Ltd. for synthesis; the human liver cancer cell line HepG2 used in the present disclosure was purchased from Wuhan Punosai Life Science Co., Ltd.; the experimental animal C57BL / 6J mice used in the present disclosure were purchased from Zhejiang Weitonglihua Experimental Animal Technology Co., Ltd.; the experimental animal C57BL / 6JGpt DIO mice used in the present disclosure were purchased from Jiangsu Jicui Pharmaceutical Kang Biotechnology Co., Ltd. Changzhou Branch. The mouse serum samples in the present disclosure were tested by Suzhou Xishan Biotechnology Co., Ltd. using a fully automatic biochemical detector. The mouse tissue samples in the present disclosure were observed by Wuhan Saiweier Biotechnology Co., Ltd. using hematoxylin-eosin staining (HE) and Oil Red O staining to observe the degree of liver fat deposition.

[0156] In the context of this disclosure, unless otherwise stated, the real-time PCR detection data of the activity experiments involved in this disclosure are all calculated using the ΔΔCt method to perform relative quantitative calculation of the target gene mRNA in each test group. The calculation method is summarized as follows:

[0157] ΔCt(test group) = Ct(test group target gene) - Ct(test group reference gene)

[0158] ΔCt(control group) = Ct(control group target gene) - Ct(control group internal reference gene)

[0159] ΔΔCt(test group)=ΔCt(test group)-ΔCt(control group average)

[0160] ΔΔCt(control group) = ΔCt(control group) - ΔCt(control group average)

[0161] The mRNA expression level of the target gene in the test group was normalized with the control group as the benchmark, and the remaining expression level of the target gene mRNA in the control group was defined as 100%.

[0162] Relative residual expression level of target gene mRNA in the test group = 2 -ΔΔCt (Test group) × 100%

[0163] The target gene mRNA inhibition rate in the test group = 100% - the relative expression level of the target gene mRNA in the test group.

[0164] In the context of this disclosure, unless otherwise stated, in vivo activity experimental data are presented as The experimental data were plotted and analyzed using GraphPad prism 8.0 software.

[0165] In the context of the present disclosure, unless otherwise stated, the reagent ratios provided below are calculated on a volume basis (v / v).

[0166] The sequences of the unmodified double-stranded oligonucleotides (dsRNA) described in the present disclosure are shown in Table 1.

[0167] Table 1 Sequence list of unmodified double-stranded oligonucleotides

[0168] Table 2 Modified double-stranded oligonucleotide sequence list

[0169] Preparation of ligands

[0170] Preparation Example 1 Preparation of CR01008 ligand

[0171] (1.1) Synthesis of compound CR01008

[0172] The synthetic route of compound CR01008 is as follows:

[0173] (1.1.1) Synthesis of Compound 2

[0174] Compound 1 (trans-4-(Boc-amino)cyclohexylcarboxaldehyde, 10.0 g, 1.0 eq) and formaldehyde solution (8.9 g, 37% by mass, 2.4 eq) were dissolved in 33 ml of methanol, and 13 ml of a 45.3% by mass KOH aqueous solution was added dropwise. After the addition was complete, the mixture was stirred at 25 ° C for 30 minutes, heated to 60 ° C and refluxed at 60 ° C for 2 hours. After the reaction was completed, the reaction solution was cooled to room temperature and evaporated to dryness under reduced pressure to obtain a crude white solid. A small amount of water was added to the crude product to slurry, and filtered to obtain compound 2 (9 g, yield 78.9%) as a white solid. MS-ESI (m / z) = 260 [M + H] +.

[0175] (1.1.2) Synthesis of compound 3

[0176] Compound 2 (9 g, 1 eq) prepared in step (1.1.1) was dissolved in 70 mL of 1,4-dioxane. A 4 M solution of hydrogen chloride in 1,4-dioxane (45 mL) was added, and the mixture was stirred at 25°C for 1 hour. After completion of the reaction, the reaction solution was evaporated to dryness under reduced pressure to obtain compound 3 (6.8 g, 100% yield) as a white solid.

[0177] (1.1.3) Synthesis of compound 5

[0178] Compound 3 (1.8 g, 2.0 eq), compound 4 (5-[[(2R,3R,4R,5R,6R)-3-acetylamino-4,5-diacetoxy-6-(acetoxymethyl)-2-tetrahydropyranyl]oxy]pentanoic acid, 2.1 g, 1.0 eq), and DIEA (N,N-diisopropylethylamine, 3.5 g, 6.0 eq) prepared in step (1.1.2) were dissolved in 15 ml of DMF. HBTU (1.9 g, 1.1 eq) was added, and the mixture was stirred at 25°C under a N2 atmosphere for 3 hours. After completion of the reaction, the reaction solution was evaporated to dryness under reduced pressure and reverse-phase purified (22% acetonitrile in water) to obtain compound 5 (1.78 g, 64.4% yield) as a white solid. MS-ESI (m / z) = 589 [M+H] + .

[0179] (1.1.4) Synthesis of Compound 6

[0180] Compound 5 (1.54 g, 1.0 eq) prepared in step (1.1.3) was dissolved in 15 ml of pyridine. The reaction system was cooled to 0°C using an ice-water bath and DMTrCl (4,4'-dimethoxytriphenylmethane chloride, 1.32 g, 1.5 eq) was added at 0°C. The reaction was allowed to react at 25°C for 3 hours. 15 ml of methanol was added to the reaction solution to quench the reaction. After completion of the reaction, the reaction solution was evaporated to dryness under reduced pressure and reverse-phase purified (60% acetonitrile in water) to obtain compound 6 (1 g, 42.7% yield) as a yellow solid. MS-ESI (m / z) = 891 [M+H] + .

[0181] (1.1.5) Synthesis of compound CR01008

[0182] Compound 6 (1.08 g, 1.0 eq) prepared in step (1.1.4) was dissolved in 20 ml of anhydrous dichloromethane. DCI (115 mg, 0.8 eq) and compound 7 (bis(diisopropylamino)(2-cyanoethoxy)phosphine, 732 mg, 2.1 eq) were added, respectively. The atmosphere was purged with nitrogen three times, and the mixture was stirred at 25°C for 2 hours. After completion of the reaction, 20 ml of saturated aqueous sodium bicarbonate solution was added to the reaction solution, and the mixture was extracted three times with 20 ml of dichloromethane (3×20 ml). The organic phases were combined and evaporated to dryness under reduced pressure. After reverse purification (72 vol% acetonitrile in water), the mixture was dried under vacuum for 12 hours to obtain compound CR01008 (1 g, 76.0% yield) as a white powder. MS-ESI (m / z) = 1091 [M+Na] + .

[0183] 1H NMR(400MHz, DMSO-d6)δ1.05(d,J=6.7Hz,6H).1.14(d,J=6.7Hz,6H),1.37-1.17(m,5H),1.60-1.40(m,6H),1.68-1.62(m,1H),1.80(s,3H), 1.80(s,3H),1.92(s,3H),2.02(s,5H),2.13(s,3H),2.71(t,J=5.9Hz,2H),2.79(d,J=8.4Hz,1H),2.87(d,J=8.4Hz,1H),3.36(s,1H),3.58- 3.39(m,3H),3.69-3.60(m,2H),3.75(s,7H),3.90(dt,J=11.2,8.8Hz,1H),4.05(s,3H),4.51(d,J=8.4Hz,1H),4.99(dd,J=11.3,3.4Hz,1H) ,5.24(d,J=3.4Hz,1H),5.78(s,1H),6.93-6.87(m,4H),7.35-7.21(m,7H),7.44-7.37(m,2H),7.66(d,J=7.8Hz,1H),7.84(d,J=9.2Hz,1H).

[0184] (1.2) Synthesis of compound CR01008Z

[0185] Compound CR01008Z was obtained by linking compound 6, which was used to synthesize compound CR01008, to a solid phase support CPG.

[0186] The synthetic route of compound CR01008Z is as follows:

[0187] (1.2.1) Synthesis of compound 9

[0188] Compound 6 (500 mg) prepared in step (1.1.4) was dissolved in 10 ml of dichloromethane. Compound 8 (succinic anhydride, 112 mg), DMAP (6.8 mg), and TEA (226.2 mg) were added. The atmosphere was purged with nitrogen three times and stirred at 25°C for 16 hours. Flash purification was performed to obtain compound 9 (300 mg, 53.6% yield). MS-ESI (m / z) = 1013 [M + Na] + .

[0189] (1.2.2) Synthesis of compound CR01008Z

[0190] Compound 9 (50 mg), aminoCPG (1.25 g, 80 μmol / g, 0.1 mmol), HBTU (27 mg), and DIEA (12 mg) prepared in step (1.2.1) were added to a 20 ml sample vial and shaken for 16 hours. After the reaction, the reaction solution was filtered to obtain a filter cake, which was washed once with 10 ml of acetonitrile (1×10 ml) and then dried in vacuo. The dried filter cake, DMAP (3 mg), Cap1 (10 ml, 200 V), and Cap2 (1 ml, 20 V) were added to a 20 ml sample vial and shaken for 6 hours. After the reaction, the reaction solution was filtered to obtain a filter cake, which was washed once with 10 ml of acetonitrile (1×10 ml) and then dried in vacuo to obtain compound CR01008Z (1.03 g, loading 20-30 μmol / g).

[0191] Cap1 and Cap2 are capping reagents, Cap1 is a 20% by volume N-methylimidazole mixed solution in pyridine / acetonitrile, with a volume ratio of pyridine to acetonitrile of 3:5; Cap2 is a 20% by volume acetic anhydride solution in acetonitrile.

[0192] Preparation Example 2 Preparation of (CR01008) × 3 ligand

[0193] By the phosphoramidite nucleic acid solid phase synthesis method, the solid phase support compound (CR01008Z) described in Preparation Example 1 was used as the starting cycle to connect the compounds (CR01008) that were not connected to the solid phase support one by one.

[0194] Each compound connection involves four steps: deprotection, coupling, capping, oxidation or sulfurization. The synthesis conditions are given below:

[0195] The compound CR01008 not bound to a solid support was separately prepared into a 0.1 M solution using acetonitrile.

[0196] The deprotection reaction conditions for each step were identical: 25°C, 70 seconds, a 3% vol. dichloroacetic acid solution in dichloromethane as the deprotection reagent, and a 5:1 molar ratio of dichloroacetic acid to the 4,4'-dimethoxytrityl protecting group on the solid support.

[0197] The conditions for each coupling reaction were the same. The coupling reaction conditions were: a temperature of 25°C, a molar ratio of the compound attached to the solid support to the compound not attached to the solid support of 1:10, a molar ratio of the compound attached to the solid support to the coupling reagent of 1:65, a reaction time of 600 seconds, a 0.5 M solution of 5-ethylthio-1H-tetrazole in acetonitrile as the coupling reagent, and a 0.2 M solution of hydrogenated xanthan gum in acetonitrile / pyridine (1:1 volume ratio of acetonitrile to pyridine) as the thiolation reagent.

[0198] The capping reaction conditions were identical for each step. The capping reaction conditions were: 25°C; 2 minutes; a 1:1 molar ratio of Cap1 and Cap2; Cap1: a 20% by volume N-methylimidazole solution in pyridine / acetonitrile (with a 3:5 volume ratio of pyridine to acetonitrile); and Cap2: a 20% by volume solution of acetic anhydride in acetonitrile. The molar ratio of the N-methylimidazole in Cap1 to the acetic anhydride in Cap2 to the compound attached to the solid support was 1:1:1.

[0199] The conditions for each oxidation or sulfidation reaction were identical. The oxidation reaction conditions were: temperature, 25°C; reaction time, 3 seconds; oxidizing agent concentration, 0.05 M iodine solution; a molar ratio of iodine to the nucleic acid sequence attached to the solid support during the coupling reaction, 30:1; and the oxidation reaction was performed in a water / pyridine mixture (water:pyridine volume ratio, 1:9). The sulfidation reaction conditions were: temperature, 25°C; reaction time, 360 seconds; sulfidation agent concentration, 0.2 M hydrogenated xanthan gum in pyridine solution; a molar ratio of sulfidation agent to the compound attached to the solid support during the coupling reaction, 4:1; and the sulfidation reaction was performed in a water / pyridine mixture (water:pyridine volume ratio, 1:9).

[0200] Three clusters of CR01008 (denoted as (CR01008)×3 or CR01008×3) were obtained through the above method.

[0201] The structural formula of the three-cluster CR01008 is as follows:

[0202] Preparation of double-stranded RNAi

[0203] Example 1 Preparation of Double-stranded RNAi with L96 as Ligand

[0204] Compound L96-PS was purchased from Ailiying Pharmaceutical Group (Tianjin) Co., Ltd. with a loading of 120±12 μmol / g (detection method: UV / HPLC). The structural formula of compound L96-PS is as follows:

[0205] Wherein, PS represents polystyrene resin solid phase carrier.

[0206] The desired single-stranded RNAi agent sequence was introduced into an ABI394 synthesizer and synthesized using Universal CPG / PS as a carrier according to the specified synthesis protocol. Each base was introduced through a four-step reaction process: DMTr removal, coupling, oxidation, and capping. 2'-OMe, 2'-F, and 2'-MOE-modified nucleoside phosphoramidite monomers were purchased from Shanghai Zhaowei Technology Development Co., Ltd. 5-Ethylmercapto-1H-tetrazole (ETT) was used as an activator (0.6 M in acetonitrile), a 0.2 M solution of xanthan gum in a 1:1 volume ratio of acetonitrile and pyridine (Suzhou Kelema) was used as a thiolation reagent, and a 0.05 M solution of iodine in a 9:1 volume ratio of pyridine and water (Suzhou Kelema) was used as an oxidant. After synthesis, the carrier was removed and air-dried. Deprotection was performed using concentrated ammonia (50°C, 16 h). After the reaction, the reaction was cooled in a -20°C refrigerator for 10 min. After centrifugation, the supernatant was transferred to another centrifuge tube, concentrated to dryness, and then purified using RP-HPLC, with mobile phase A consisting of 0.1 M TEAA and mobile phase B consisting of acetonitrile. The purified fractions were analyzed by HPLC, MS, and UV. Qualified fractions were collected and lyophilized to obtain single-stranded RNAi agents. The resulting single-stranded RNAi agents were then annealed to obtain double-stranded RNAi agents.

[0207] The prepared double-stranded RNAi information is shown in the following table:

[0208] Table 3 Double-stranded RNAi sequences with L96 as ligand

[0209] In this sequence, the base composition and modification meanings are as follows: capital letters A, U, G, C, and T represent the base composition of the nucleotides; the lowercase letter m indicates that the nucleotide adjacent to the left of the letter m is a 2'-methoxy-modified nucleotide; the lowercase letter f indicates that the nucleotide adjacent to the left of the letter f is a 2'-fluoro-modified nucleotide; the lowercase letter s indicates that the two nucleotides adjacent to the left and right of the letter s are connected by a phosphorothioate bond; L96 is an N-acetylgalactosamine derivative.

[0210] Example 2 Preparation of double-stranded RNAi with (CR01008) × 3 as ligand

[0211] The (CR01008)×3 ligand was prepared by the method shown in Preparation Example 2.

[0212] According to the method described in Example 1, a double-stranded RNAi with a ligand of (CR01008)×3 was prepared. The information is shown in the following table:

[0213] Table 4: Double-stranded RNAi sequences of (CR01008)×3 ligands

[0214] Biological detection experiments

[0215] Unless otherwise stated, the reagents and consumables (Table 5) and instruments and equipment (Table 6) used in this disclosure were all commercially available products from the following manufacturers.

[0216] Table 5 Main reagents and consumables

[0217] Table 6 Main instruments and equipment

[0218] In the following examples, FTO mRNA refers to the mRNA having the sequence shown in Genbank Accession No. NM_001363897.1. Further, unless otherwise specified, in the following examples, "target gene" refers to the gene that transcribes the above-mentioned FTO mRNA, and "target mRNA" refers to the above-mentioned FTO mRNA.

[0219] Experiment 1 Evaluation of in vitro activity of modified double-stranded oligonucleotides

[0220] This example uses the target gene inhibitory activity assessment method in the human liver cancer cell line HepG2 to evaluate the inhibitory activity of RX006001 to RX006224 on the target gene FTO in cells at the same FTO target site, with RX000001 as a negative control;

[0221] Test sample preparation:

[0222] After centrifugation of each modified double-stranded oligonucleotide test sample, add an appropriate amount of PBS according to the specifications of each tube to dissolve it and prepare a 20 μM stock solution. The stock solution is further diluted with PBS in a gradient manner to make 0.1 μM and 0.01 μM working solutions. Dosage tests are performed with final duplex concentrations of 1 nM and 0.1 nM.

[0223] 96-well transfection and detection:

[0224] HepG2 cells grown to near confluence were digested with trypsin, and the cells were washed to prepare a cell suspension. 100 μL of cell suspension was added to each well of a 96-well plate, with 12,000 cells per well. The cells were cultured in a 37°C, 5% CO2 incubator. After the cells adhered for 24 hours, the DMEM medium in the 96-well plate was aspirated and 80 μL of Opti-MEM was added to each well. TMThe 96-well plate was then placed in an incubator for further incubation. 1 μL of the 0.1 μM and 0.01 μM working solutions were dispersed in 9 μL of Opti-MEM to form a siRNA mixture. 0.3 μL of RNAiMAX was dispersed in 9.7 μL of Opti-MEM and mixed with each siRNA mixture to form a transfection complex. The transfection complex was incubated at room temperature for 10 minutes, and then the transfection complex was added to the 96-well plate at a rate of 20 μL / well. After 4 hours of incubation, 100 μL of DMEM medium supplemented with 20% FBS was added to each well, and the 96-well plate was placed in an incubator for an additional 24 hours.

[0225] The 96-well plate was removed and total RNA was extracted using a fully automatic nucleic acid extractor (purchased from Zhejiang Hanwei Technology Co., Ltd.) and a nucleic acid extraction kit (purchased from Zhejiang Hanwei Technology Co., Ltd., GO-MNTR-100) according to the standard operating procedures for total RNA extraction.

[0226] Use reverse transcription kit (Thermo Fisher Scientific, RevertAid First Strand cDNA Synthesis Kit, K1622) and select Oligo (dT) 18 Reverse transcription primers, according to the method of reverse transcription kit specification sheet, configure 20 μ L reverse transcription system and complete reverse transcription reaction.Then use real-time fluorescence quantitative PCR kit (Thermo Fisher Scientific company, TaqMan Fast Advanced Master Mix, 4444557) to detect the expression of target gene mRNA in HepG2 cells on fluorescence quantitative PCR instrument (Bio-Rad company CFX Opus 384).In this real-time fluorescence quantitative PCR method, using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene as internal reference gene, use primers for target gene and primers for GAPDH internal reference gene to detect target gene and GAPDH internal reference gene respectively.The sequence of detection primers is shown in Table 7.

[0227] Table 7 Sequences of detection primers

[0228] In the real-time fluorescence quantitative PCR method, the expression level and inhibition rate of the target gene mRNA in each test group were relatively quantitatively calculated using the ΔΔCt method according to the technical method described in the embodiment.

[0229] Table 8 Inhibitory activity of target genes in HepG2 cells after administration of modified double-stranded oligonucleotides

[0230] Experiment 2 Evaluation of the in vivo activity of RNAi agents with L96 ligand in mice using the high-pressure hydrodynamic injection (HDI) model

[0231] In this example, the Balb / c mouse hydrodynamic injection model was used to evaluate the inhibitory activity of the same FTO target double-stranded RNAi sense strand 3' end conjugated with L96 ligand on the target gene FTO, with RZ000002 as a negative control.

[0232] Plasmid construction: pcDNA-CMV-RG006 plasmid was constructed by Sangon Biotech (Shanghai) Co., Ltd. The full-length FTO (NM_001363897.1) was inserted into the multiple cloning site of the pcDNA-CMV vector by whole gene synthesis.

[0233] Mouse model construction:

[0234] The Balb / c mouse hydrodynamic injection model is established by rapidly injecting a pcDNA-CMV-RG006 plasmid solution through the tail vein at high pressure. On day 3 of the experiment, mice were injected with 10 μg of pcDNA-CMV-RG006 via the tail vein hydrodynamic injection over 5 seconds, at an injection volume of 8% of their body weight. The plasmid DNA for injection was diluted with normal saline, prepared immediately prior to injection, and stored at 4°C.

[0235] Animal grouping, drug administration and tissue sample collection:

[0236] 6-8 week old Balb / c mice were randomly divided into groups (all female) according to body weight, with 5 mice in each group. Each test group was given a predetermined dose of drug conjugate and a PBS control group was added. All mice were dosed according to body weight, and a single dose was administered by subcutaneous injection in the abdomen. Each drug conjugate was administered in the form of a 0.3 mg / mL (calculated as double-stranded RNAi) PBS solution, and the administration volume was 10 mL / kg mouse body weight, that is, the dosage of each drug conjugate was 3 mg / kg mouse body weight (calculated as double-stranded RNAi). The PBS control group was given the same volume of PBS solution (not containing drug conjugate). The day of administration was recorded as day 0 (recorded as D0), and plasmid injection was performed on the 3rd day after administration (recorded as D3). On the 4th day (recorded as D4), 5 mice of all groups were killed. The mice were subjected to gross anatomy and the liver tissue of each mouse was collected. The liver tissue was cut into about 2 mm 3 Small pieces were stored with RNA later.

[0237] For each mouse, an appropriate amount of liver tissue sample was taken from RNA later, and the liver tissue sample was disrupted in a Tissuelyser II fully automatic tissue homogenizer for 60 seconds. Then, total RNA was extracted using a fully automatic nucleic acid extractor (purchased from Zhejiang Hanwei Technology Co., Ltd.) and a nucleic acid extraction kit (purchased from Zhejiang Hanwei Technology Co., Ltd., GO-MNTR-100) according to the standard operating procedures for total RNA extraction.

[0238] For each mouse, 1 μg of total RNA was collected and reverse transcription kit (Thermo Fisher Scientific, RevertAid First Strand cDNA Synthesis Kit, K1622) was used to select Oligo (dT) 18 Reverse transcription primers were prepared according to the method described in the reverse transcription kit instructions to configure 20 μL reverse transcription systems and complete the reverse transcription reaction. After the reaction was completed, 60 μL RNase-Free water was added to the reverse transcription system to obtain a cDNA solution. The expression of the target gene mRNA in the animal was then detected using a real-time fluorescence quantitative PCR kit (Thermo Fisher Scientific, TaqMan Fast Advanced Master Mix, 4444557) and a fluorescence quantitative PCR instrument (Bio-Rad, CFX Opus 384). In this real-time fluorescence quantitative PCR method, the Nero gene on the plasmid backbone was used as an internal reference gene, and the target gene and the Nero internal reference gene were detected using primers for the target gene and primers for the Nero internal reference gene, respectively. The sequences of the detection primers are shown in Table 9.

[0239] Table 9 Sequences of detection primers

[0240] According to the instructions of the real-time fluorescence quantitative PCR kit, 10 μL of real-time PCR reaction system was prepared for each PCR detection well. Each reaction system contained 4 μL of the cDNA solution obtained by the reverse transcription reaction, 5 μL of TaqMan TMFast Advanced Master Mix (2×), 0.15 μL 10 μM upstream primer, 0.15 μL 10 μM downstream primer, 0.15 μL 10 μM probe primer, 0.55 μL RNase-Free H2O. The configured reaction system was placed on a real-time fluorescence quantitative PCR instrument (Bio-Rad, CFX Opus 384), and Real-time PCR amplification was performed using a two-step method. The amplification program was 50°C for 2 min, followed by pre-denaturation at 95°C for 20 s, denaturation at 95°C for 3 s, annealing and extension at 60°C for 30 s, and the denaturation, annealing and extension process was repeated for 40 cycles. In this real-time fluorescence quantitative PCR method, the expression level and inhibition rate of the target gene mRNA in each test group were relatively quantitatively calculated using the ΔΔCt method according to the technical method described in the embodiment.

[0241] Table 10 Inhibitory activity of target genes in Balb / c-HDI mice after administration of double-stranded RNAi

[0242] As shown in Figure 1 and Table 10, at a dose of 3 mg / kg, RZ006002, RZ006007, RZ006008, RZ006009, RZ006010, RZ006011, RZ006012, RZ006013, RZ006014, RZ006017, RZ006020, RZ006021, RZ006023, and RZ006029 could significantly inhibit the expression of FTO mRNA in mouse liver tissue, with an inhibition rate greater than 80%.

[0243] Experiment 3 Evaluation of double-stranded RNAi activity in C57BL / 6J mice

[0244] This example evaluates the mRNA inhibition activity of double-stranded RNAi RZM06002-RZM06005 with L96 as the ligand in the liver tissue of C57BL / 6J mice.

[0245] Animal grouping, drug administration and tissue sample collection:

[0246] 6-8 week old C57BL / 6J male mice (Zhejiang Weitong Lihua Experimental Animal Technology Co., Ltd.) were randomly divided into groups according to body weight, with 5 mice in each group. Each test group was given a predetermined dose of drug conjugate and a PBS control group was added. The drug dose for all mice was calculated based on body weight, and the administration volume was 10 mL / kg of mouse body weight. Single administration was performed by subcutaneous injection in the abdomen, and each drug conjugate was administered in the form of 0.3 mg / mL and 0.9 mg / mL (calculated as double-stranded RNAi) PBS solution, i.e., the dosage of each drug conjugate was 3 mg / kg and 9 mg / kg of mouse body weight (calculated as double-stranded RNAi). The PBS control group was given the same volume of PBS solution (without drug conjugate).

[0247] The day of administration was recorded as day 0 (denoted as D0), and all groups of mice were killed on the 7th day after administration (denoted as D7). The mice were subjected to gross dissection and the liver tissue of each mouse was collected. The liver tissue was cut into small pieces of about 2 mm3 and preserved with RNA later for FTO mRNA expression level determination. In the real-time fluorescence quantitative PCR method, the GAPDH gene was used as the internal reference gene, and primers for the target gene and the primers for the GAPDH internal reference gene were used for detection. The sequences of the detection primers are shown in Table 11.

[0248] Table 11 Sequences of detection primers

[0249] In the real-time fluorescence quantitative PCR method, the expression level and inhibition rate of the target gene mRNA in each test group were relatively quantitatively calculated using the ΔΔCt method according to the technical method described in the embodiment.

[0250] Table 12 Inhibitory activity of target genes in liver tissue of C57BL / 6J mice after administration of double-stranded RNAi

[0251] As shown in Figure 2 and Table 12, RZM06004 and RZM06005 can significantly reduce the mRNA expression level in the liver tissue of C57BL / 6J mice. The inhibitory effect is greater than 80% at a single dose of 9 mg / kg and greater than 70% at a single dose of 3 mg / kg.

[0252] Experiment 4 Evaluation of the in vivo activity of RNAi agents with L96 as ligand in C57BL / 6JGpt DIO mice

[0253] In this example, C57BL / 6JGpt DIO mice were used to evaluate the in vivo target gene FTO protein reduction and fat deposition improvement effects of different doses of RZM06005.

[0254] Animal grouping, drug administration and tissue sample collection:

[0255] C57BL / 6JGpt mice were fed with a 45% high-fat diet to obtain 42-week-old C57BL / 6JGpt DIO mice. DIO mice (Changzhou Branch of Jiangsu Jicui Pharmaceutical Kang Biotechnology Co., Ltd.) were randomly divided into groups according to body weight, with 6 mice in each group. The drug dose for all mice was calculated based on body weight, and a single dose was used. The drug conjugate RZM06005 was administered in the form of 0.6 mg / mL and 0.9 mg / mL (calculated as siRNA) of PBS solution, and the administration volume was 10 mL / kg mouse body weight, that is, the dosage of the drug conjugate RZM06005 was 6 mg / kg and 9 mg / kg mouse body weight (calculated as siRNA), respectively. The PBS control group was given the same volume of PBS solution (without drug conjugate). The day of administration was designated as day 0 (D0). On the 14th day after administration (D14), liver tissues were collected from all groups of mice. Some tissues were quick-frozen in liquid nitrogen and transferred to -80°C for storage for FTO protein detection. Some liver tissues were fixed with 10% neutral formalin and sent to Wuhan Sevier Biotechnology Co., Ltd. for hematoxylin-eosin staining (HE) and Oil Red O staining to observe the degree of liver fat deposition.

[0256] Liver tissue protein sample processing and determination:

[0257] Liver tissue was added to RIPA lysis buffer I (Shanghai Sangon Biotech Co., Ltd., C500005) and protein was extracted using a tissue grinder (TF-800, Shanghai Jingxin Industrial Development Co., Ltd.). The absorbance of each sample at 562 nm was measured using a multi-function microplate reader (SYNERGY H1, BioTek) according to the instructions in the BCA reagent (Thermo, 23225). Protein concentration was calculated from a standard curve and diluted to a quantified protein concentration of 1 μg / mL using SDS-PAGE protein loading buffer (5×) (Biyuntian, P0015). The samples were denatured at 95°C for 10 minutes.

[0258] SDS-PAGE gel electrophoresis:

[0259] A 10% separating gel was prepared based on the target protein size. After the separating gel solidified, a 5% stacking gel was added. After loading the protein, electrophoresis was performed at 70V for 30 minutes and then at 120V for 30 minutes. Transfer buffer was poured into a transfer box. Filter paper, PVDF membrane (Miniprint), and nylon pad were placed in the box containing transfer buffer and allowed to soak for 30 minutes. A plywood was assembled in the order of negative plate, sponge, filter paper, gel, PVDF membrane, filter paper, sponge, and positive plate. The plywood was placed in a transfer tank and transferred at a constant current of 300 mA for 2 hours at 4°C or on ice. After transfer, the membrane was rinsed in TBST solution, then 5% skim milk powder was added and blocked on a shaker at room temperature for 2 hours. Anti-FTO antibody (ab94482) and Anti-GAPDH antibody (ab181602) were diluted 1:500 in primary antibody diluent (Biyuntian, P0023A) and incubated overnight at 4°C. Goat anti-rabbit antibody (HRP) (ab97051) was diluted 1:2000 in TBST solution and incubated on a shaker at room temperature for 1 hour. After washing the PVDF membrane, ELC chemiluminescence reagent (Biyuntian, P0018AM) was added and the membrane was imaged using an automated gel image analysis system (Tianneng).

[0260] Experimental results:

[0261] Figure 3 shows immunoblotting results in mouse liver tissue. A single subcutaneous injection of different doses of the RZM06005 conjugate significantly reduced FTO protein expression in the livers of C57BL / 6JGpt DIO mice. H&E histopathological sections (Figure 4) and Oil Red O staining (Figure 5) revealed significant fat deposition in the livers of C57BL / 6JGpt DIO mice. A single subcutaneous injection of different doses of the RZM06005 conjugate significantly reduced fat deposition in the livers of C57BL / 6JGpt DIO mice, with a single dose of 9 mg / kg significantly reducing fat deposition in the livers of mice.

[0262] Experiment 5: Evaluation of the in vivo activity of RNAi agents with CR01008 × 3 ligands in mice using the high-pressure hydrodynamic injection (HDI) model

[0263] In this example, the hydrodynamic injection model of Balb / c mice was used to evaluate the inhibitory activity of the same FTO target double-stranded RNAi sense strand 3' end conjugated with (CR01008) × 3 ligands on the target gene FTO.

[0264] Plasmid construction: pcDNA-CMV-RG006 plasmid was constructed by Sangon Biotech (Shanghai) Co., Ltd. The full-length FTO (NM_001363897.1) was inserted into the multiple cloning site of the pcDNA-CMV vector by whole gene synthesis.

[0265] Mouse model construction:

[0266] The Balb / c mouse hydrodynamic injection model is established by rapidly injecting a pcDNA-CMV-RG006 plasmid solution through the tail vein at high pressure. On day 3 of the experiment, mice were injected with 10 μg of pcDNA-CMV-RG006 via the tail vein hydrodynamic injection over 5 seconds, at an injection volume of 8% of their body weight. The plasmid DNA for injection was diluted with normal saline, prepared immediately prior to injection, and stored at 4°C.

[0267] Animal grouping, drug administration and tissue sample collection:

[0268] 6-8 week old Balb / c mice were randomly divided into groups (all female) by body weight, with 5 mice in each group. Each test group was given a predetermined dose of drug conjugate and a PBS control group was added. All mice were given a dose according to body weight, and a single dose was administered by subcutaneous injection in the abdomen. Each drug conjugate was administered in the form of a 0.3 mg / mL (calculated as double-stranded RNAi) PBS solution, and the administration volume was 10 mL / kg mouse body weight, that is, the dosage of each drug conjugate was 3 mg / kg mouse body weight (calculated as double-stranded RNAi). The PBS control group was given the same volume of PBS solution (not containing drug conjugate). The administration day was recorded as day 0 (recorded as D0), and plasmid injection was performed on the 3rd day after administration (recorded as D3), and 5 mice of all groups were killed on the 4th day (recorded as D4). The mice were subjected to gross anatomy and the liver tissue of each mouse was collected and cut into about 2 mm 3 Small pieces were stored with RNA later.

[0269] For each mouse, an appropriate amount of liver tissue sample was taken from RNA later, and the liver tissue sample was disrupted in a Tissuelyser II fully automatic tissue homogenizer for 60 seconds. Then, total RNA was extracted using a fully automatic nucleic acid extractor (purchased from Zhejiang Hanwei Technology Co., Ltd.) and a nucleic acid extraction kit (purchased from Zhejiang Hanwei Technology Co., Ltd., GO-MNTR-100) according to the standard operating procedures for total RNA extraction.

[0270] For each mouse, 1 μg of total RNA was collected and reverse transcription kit (Thermo Fisher Scientific, RevertAid First Strand cDNA Synthesis Kit, K1622) was used to select Oligo (dT) 18Reverse transcription primers were prepared according to the method described in the reverse transcription kit instructions to configure 20 μL reverse transcription systems and complete the reverse transcription reaction. After the reaction was completed, 60 μL RNase-Free water was added to the reverse transcription system to obtain a cDNA solution. The expression of the target gene mRNA in the animal was then detected using a real-time fluorescence quantitative PCR kit (Thermo Fisher Scientific, TaqMan Fast Advanced Master Mix, 4444557) and a fluorescence quantitative PCR instrument (Bio-Rad, CFX Opus 384). In this real-time fluorescence quantitative PCR method, the Nero gene on the plasmid backbone was used as an internal reference gene, and the target gene and the Nero internal reference gene were detected using primers for the target gene and primers for the Nero internal reference gene, respectively. The sequences of the detection primers are shown in Table 13.

[0271] Table 13 Sequences of detection primers

[0272] According to the instructions of the real-time fluorescence quantitative PCR kit, 10 μL of real-time PCR reaction system was prepared for each PCR detection well. Each reaction system contained 4 μL of the cDNA solution obtained by the reverse transcription reaction, 5 μL of TaqMan TM Fast Advanced Master Mix (2×), 0.15 μL 10 μM upstream primer, 0.15 μL 10 μM downstream primer, 0.15 μL 10 μM probe primer, 0.55 μL RNase-Free H2O. The configured reaction system was placed on a real-time fluorescence quantitative PCR instrument (Bio-Rad, CFX Opus 384), and Real-time PCR amplification was performed using a two-step method. The amplification program was 50°C for 2 min, followed by pre-denaturation at 95°C for 20 s, denaturation at 95°C for 3 s, annealing and extension at 60°C for 30 s, and the denaturation, annealing and extension process was repeated for 40 cycles. In this real-time fluorescence quantitative PCR method, the expression level and inhibition rate of the target gene mRNA in each test group were relatively quantitatively calculated using the ΔΔCt method according to the technical method described in the embodiment.

[0273] Table 14 Inhibitory activity of target genes in Balb / c-HDI mice after administration of double-stranded RNAi

[0274] As shown in Figure 6 and Table 14, at a dose of 3 mg / kg, RZ006037, RZ006038, RZ006039, RZ006040, RZ006041, and RZ006043 could significantly inhibit the expression of FTO mRNA in mouse liver tissue, with an inhibition rate greater than 90%.

[0275] Experiment 6 Evaluation of the activity of RNAi agents with (CR01008) × 3 as ligands in cynomolgus monkeys

[0276] This embodiment adopts non The inhibitory activity of the same FTO target double-stranded RNAi sense strand 3' end conjugated with (CR01008) × 3 ligands on the target gene FTO was evaluated in normal cynomolgus monkeys.

[0277] Animal grouping, drug administration and tissue sample collection:

[0278] 2-6 years old non Normal cynomolgus monkeys (all male) were randomly divided into 8 groups according to body weight, with 3 animals in each group. Each test group was given a predetermined dose of drug conjugate and a 0.9% sodium chloride injection control group was added. All animals were dosed according to body weight and administered a single dose by subcutaneous injection on the back. The day of administration was designated as day 0 (denoted as D0). Each drug conjugate was administered as a 6 mg / mL (calculated as double-stranded RNAi) solution of 0.9% sodium chloride injection. The administration volume was 1 mL / kg cynomolgus monkey body weight, that is, the administration dose of each drug conjugate was 6 mg / kg cynomolgus monkey body weight (calculated as double-stranded RNAi). The control group was given the same volume of 0.9% sodium chloride injection (without drug conjugate). On the third day before administration (denoted as D-3), the day of administration, day 0 (denoted as D0), day 14 (denoted as D14), day 28 (denoted as D28), day 42 (denoted as D42) and day 56 (denoted as D56), the animals were anesthetized and liver puncture samples were performed with the assistance of an ultrasound machine. The liver puncture samples were preserved in RNA later.

[0279] Liver tissue RNA sample extraction and determination:

[0280] The liver puncture sample was disrupted in a Tissuelyser II fully automatic tissue homogenizer for 60 seconds, and then total RNA was extracted using a fully automatic nucleic acid extractor (purchased from Zhejiang Hanwei Technology Co., Ltd.) and a nucleic acid extraction kit (purchased from Zhejiang Hanwei Technology Co., Ltd., GO-MNTR-100) according to the standard operating procedures for total RNA extraction.

[0281] For each animal, 1 μg of total RNA was collected and reverse transcription was performed using a reverse transcription kit (Thermo Fisher Scientific, RevertAid First Strand cDNA Synthesis Kit, K1622) and Oligo(dT) was selected. 18 Reverse transcription primers were prepared according to the method described in the reverse transcription kit instructions to configure 20 μL of reverse transcription system and complete the reverse transcription reaction. After the reaction was completed, 60 μL of RNase-Free water was added to the reverse transcription system to obtain a cDNA solution. Real-time fluorescence quantitative PCR kit (Thermo Fisher Scientific, TaqMan Fast Advanced Master Mix, 4444557) was then used to detect the expression of target gene mRNA in the animal on a fluorescence quantitative PCR instrument (Bio-Rad, CFX Opus 384). In this real-time fluorescence quantitative PCR method, the cynomolgus monkey GAPDH gene was used as an internal reference gene, and primers for the target gene and the GAPDH internal reference gene were used to detect the target gene and the GAPDH internal reference gene, respectively. The sequences of the detection primers are shown in Table 15.

[0282] Table 15 Sequences of detection primers

[0283] According to the instructions of the real-time fluorescence quantitative PCR kit, 10 μL of real-time PCR reaction system was prepared for each PCR detection well. Each reaction system contained 4 μL of the cDNA solution obtained by the reverse transcription reaction, 5 μL of TaqMan TM Fast Advanced Master Mix (2×), 0.15 μL 10 μM upstream primer, 0.15 μL 10 μM downstream primer, 0.15 μL 10 μM probe primer, 0.55 μL RNase-Free H2O. The prepared reaction system was placed on a real-time fluorescence quantitative PCR instrument (Bio-Rad, CFX Opus 384), and Real-time PCR amplification was performed using a two-step method. The amplification program was 50°C for 2 minutes, followed by pre-denaturation at 95°C for 20 seconds, denaturation at 95°C for 3 seconds, annealing and extension at 60°C for 30 seconds, and the denaturation, annealing and extension process was repeated for 40 cycles. In this real-time fluorescence quantitative PCR method, the samples at each time point were calculated using the ΔΔCt method according to the technical method described in the embodiment, and then the mRNA expression level of the target gene in the test group was normalized based on the pre-drug D-3 mRNA expression level, and the remaining expression level of the target gene mRNA before administration was defined as 100%. Experimental results:

[0284] Table 16 Inhibitory activity of target genes in cynomolgus monkeys after administration of double-stranded RNAi

[0285] The results in Table 16 show that after a single subcutaneous injection of 6 mg / kg, the FTO mRNA level in each experimental group was significantly suppressed.

[0286] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present disclosure, rather than to limit them. Although the present disclosure has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present disclosure. Industrial Applicability

[0287] The present disclosure provides a double-stranded oligonucleotide for inhibiting FTO gene expression, which alleviates the problem in the prior art of the lack of nucleic acid drugs that can inhibit FTO gene expression and treat obesity, obesity-related diseases, or Alzheimer's disease and other diseases related to FTO gene expression.

Claims

1. A double-stranded oligonucleotide (dsRNA) for inhibiting FTO gene expression, characterized in that: The double-stranded oligonucleotide includes a sense strand and an antisense strand, wherein the antisense strand is complementary or substantially complementary to the sense strand; the substantially complementary means that the mismatch between the sense strand and the antisense strand in the double-stranded region does not exceed 3 nucleotides; wherein the sense strand contains at least 15 consecutive nucleotides that are identical or substantially identical to SEQ ID NO:449, and the substantially identical means that there are no more than 3 nucleotide differences between the sense strand and at least 15 consecutive nucleotides in the SEQ ID NO:449 sequence.

2. The double-stranded oligonucleotide according to claim 1, characterized in that The sense strand comprises at least 15 consecutive nucleotides that differ from the nucleotide sequence of SEQ ID NO: 449 by no more than 2 nucleotides; Optionally, the sense strand comprises at least 15 consecutive nucleotides that differ from the nucleotide sequence of SEQ ID NO:449 by no more than 1 nucleotide.

3. The double-stranded oligonucleotide according to claim 1, characterized in that The antisense strand is complementary or substantially complementary to at least 15, 16, 17, 18, 19, 20, 21, 22 or 23 consecutive nucleotides of the nucleotide sequence of SEQ ID NO: 449, wherein the substantially complementary strand has no more than 3 nucleotide mismatches in the complementary region.

4. The double-stranded oligonucleotide according to claim 1, characterized in that The sense strand comprises at least 15 consecutive nucleotides of any sequence shown in SEQ ID NO: 1 to SEQ ID NO: 224 in Table 1, or a nucleotide sequence that differs from the at least 15 consecutive nucleotides by no more than 3 nucleotides; and / or, the antisense strand comprises at least 15 consecutive nucleotides of any sequence shown in SEQ ID NO: 225 to SEQ ID NO: 448 in Table 1, or a nucleotide sequence that differs from the at least 15 consecutive nucleotides by no more than 3 nucleotides; Optionally, the sense strand comprises a nucleotide sequence that differs by no more than 3 nucleotides from any of the sequences shown in SEQ ID NO:1 to SEQ ID NO:224 in Table 1, and / or positions 2-19 of the antisense strand comprise a nucleotide sequence that differs by no more than 3 nucleotides from positions 2-19 of any of the sequences shown in SEQ ID NO:225 to SEQ ID NO:448 in Table 1.

5. The double-stranded oligonucleotide according to claim 1, characterized in that The sense strand comprises a nucleotide sequence that differs from any of the sequences shown in SEQ ID NO: 1 to SEQ ID NO: 224 in Table 1 by no more than 1 or 2 nucleotides, and / or the antisense strand comprises a nucleotide sequence that differs from any of the sequences shown in SEQ ID NO: 225 to SEQ ID NO: 448 in Table 1 by no more than 1 or 2 nucleotides; Optionally, the sense strand comprises a nucleotide sequence that differs by no more than 1 nucleotide from any of the sequences shown in SEQ ID NO: 1 to SEQ ID NO: 224 in Table 1, and / or the antisense strand comprises a nucleotide sequence that differs by no more than 1 nucleotide from any of the sequences shown in SEQ ID NO: 225 to SEQ ID NO: 448 in Table 1; Optionally, the sense strand comprises any one of the nucleotide sequences shown in SEQ ID NO: 1 to SEQ ID NO: 224 in Table 1, and / or the antisense strand comprises any one of the nucleotide sequences shown in SEQ ID NO: 225 to SEQ ID NO: 448 in Table 1.

6. The double-stranded oligonucleotide according to any one of claims 1 to 5, characterized in that The sense strand or the antisense strand comprises any sense strand or antisense strand selected from the duplexes shown in the following numbers: RN006007, RN006009, RN006017, RN006032, RN006033, RN006040, RN006052, RN006066, RN006076, RN006077, RN006082, RN006104, RN006107, RN006108, RN006109, RN006120, RN006124, RN006132, RN006133, RN006137, RN006138, RN006149, RN006157, RN006172, RN006173, RN006181, RN006182, RN006186, RN006188, RN006201, RN006218, RN006219, RN006220, RN006221, RN006222, RN006223.

7. The double-stranded oligonucleotide according to any one of claims 1 to 5, characterized in that The double-stranded oligonucleotide comprises one or more selected from the duplex group shown in the following numbers: RN006007, RN006009, RN006017, RN006032, RN006033, RN006040, RN006052, RN006066, RN006076, RN006077, RN006082, RN006104, RN006107, RN006108, RN006109, RN006120, RN 006124, RN006132, RN006133, RN006137, RN006138, RN006149, RN006157, RN006172, RN006173, RN006181, R N006182, RN006186, RN006188, RN006201, RN006218, RN006219, RN006220, RN006221, RN006222, RN006223.

8. The double-stranded oligonucleotide according to any one of claims 1 to 7, characterized in that All nucleotides of the sense strand and all nucleotides of the antisense strand are modified nucleotides; Optionally, the antisense strand and the sense strand have a length of 17-25 nucleotides; Optionally, at least one of the antisense strand and the sense strand comprises a 3'-terminal overhang of 1-2 nucleotides.

9. The double-stranded oligonucleotide according to claim 8, characterized in that The modified nucleotides are selected from 3'-terminal deoxy-thymine (dT) nucleotides, 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-deoxy-modified nucleotides, 2'-O-methoxyethyl modified nucleotides, 2'-O(CH2) n One or more of the nucleotides substituted by OR; wherein n is 1 or 2, and R is selected from substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 alkoxy; wherein the substituent is selected from halogen, C1-C6 alkyl, C1-C6 alkoxy, hydroxyl, and amino; Optionally, the modified nucleotide is selected from 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-O-methoxyethyl modified nucleotides, 2'-O(CH2) n One or more of the nucleotides substituted by OR; Optionally, the sense strand of the double-stranded oligonucleotide comprises any modified sense strand nucleotide sequence shown in Table 2, and / or the antisense strand comprises any modified antisense strand nucleotide sequence shown in Table 2.

10. A double-stranded RNAi for inhibiting FTO gene expression, characterized in that: The double-stranded RNAi comprises a sense strand and an antisense strand, wherein positions 2 to 19 in the antisense strand nucleotide sequence comprise a region complementary to an FTO RNA transcript, wherein the complementary region comprises a nucleotide sequence that differs by 0, 1, 2 or 3 nucleotides from any antisense sequence listed in SEQ ID NO: 225 to SEQ ID NO: 448 in Table 1; and, the nucleotide sequence of the sense strand comprises a nucleotide sequence that differs by 0, 1, 2 or 3 nucleotides from any sequence listed in SEQ ID NO: 1 to SEQ ID NO: 224 in Table 1; Furthermore, the sense strand and the antisense strand are complementary or substantially complementary to form a double-stranded region, wherein the substantially complementary means that the mismatch between the sense strand and the antisense strand in the double-stranded region does not exceed 3 nucleotides, and the double-stranded region is shown in the following formula (I): SS:5'-(N)a'-(X)p'-(N)b'-(X)q'-(N)c'-(X)r'-(N)d'-3' AS:3'-(N)a-(X)p-(N)b-(X)q-(N)c-5' (I), Wherein, SS represents the sense strand, and AS represents the antisense strand; All nucleotides of the sense strand and the antisense strand are modified nucleotides; The Ns each independently represent the following modified nucleotides: 2'-O-methyl modified nucleotides, 2'-fluorine modified nucleotides, 2'-deoxy-modified nucleotides; The Xs are each independently a 2'-O-methoxyethyl modified nucleotide, a 2'-O-methyl modified nucleotide or a 2'-O(CH2) n OR substituted modified nucleotides; wherein n is 1 or 2, and R is selected from substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 alkoxy; wherein the substituent is selected from halogen, C1-C6 alkyl, C1-C6 alkoxy, hydroxyl, and amino; Said a, a', p, p', b, b', q, q', c, c', r', d' each independently represents the number of nucleotides, wherein a' is selected from an integer of 3-8; p' is selected from an integer of 0-3; b' is selected from an integer of 4-13; q' is selected from an integer of 0-4; c' is selected from an integer of 3-9; r' is selected from an integer of 0-3; d' is selected from an integer of 0-9; a is selected from an integer of 4-7; p is selected from an integer of 0-1; b is selected from an integer of 4-8; q is selected from an integer of 0-4; c is selected from an integer of 6-10; Furthermore, p', q', r', p, and q are not all 0 at the same time, and 0≤q'+r'≤4.

11. The double-stranded RNAi according to claim 10, characterized in that a' is selected from an integer of 3-8; p' is selected from 0 or 1; b' is selected from an integer of 4-13; q' is selected from 0 or 1; c' is selected from an integer of 3-9; r' is selected from 0 or 1; d' is selected from an integer of 1-8; a is selected from an integer of 4-7; p is selected from 1; b is selected from an integer of 4-8; q is selected from 0 or 1; c is selected from an integer of 6-10; Optionally, the N is selected from 2'-O-methyl modified nucleotides, 2'-fluoro-modified nucleotides, and the X is selected from 2'-O-methoxyethyl modified nucleotides; Optionally, the duplex includes at least one 2'-O-methoxyethyl modification or 2'-O(CH2) n OR modified nucleotides.

12. The double-stranded RNAi according to any one of claims 10 to 11, characterized in that The nucleotides of the double-stranded RNAi sense strand and / or antisense strand are all modified nucleotides; Wherein, in the direction from the 5' end to the 3' end, at least three of the nucleotides at positions 7 to 10 in the nucleotide sequence of the sense strand are fluorinated modified nucleotides, and the nucleotides at the remaining positions are 2'-O-methyl modified or 2'-O-methoxyethyl modified nucleotides; Optionally, in the direction from the 5' end to the 3' end, at least four of the nucleotides at positions 2, 6, 9-12, 14 and 16 of the nucleotide sequence in the antisense strand are 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are 2'-O-methyl-modified or 2'-O-methoxyethyl-modified nucleotides; Optionally, the sense strand and / or the antisense strand contains at least one 2'-O-methoxyethyl modified nucleotide.

13. The double-stranded RNAi according to claim 12, characterized in that In the direction from the 5' end to the 3' end, the nucleotides at positions 7 to 10 in the nucleotide sequence of the sense strand are fluorinated modified nucleotides, and the nucleotides at the remaining positions are 2'-O-methyl modified or 2'-O-methoxyethyl modified nucleotides; Furthermore, the nucleotides at positions 2, 6, 14, and 16 of the nucleotide sequence in the antisense strand are 2'-fluoro-modified nucleotides, and any one nucleotide selected from positions 9, 10, 11, or 12 is a 2'-fluoro-modified nucleotide, and the nucleotides at the remaining positions are 2'-O-methyl-modified or 2'-O-methoxyethyl-modified nucleotides.

14. The double-stranded RNAi according to claim 12 or 13, characterized in that In the direction from the 5' end to the 3' end, the nucleotides at positions 7 to 10 in the nucleotide sequence of the sense strand are fluorinated modified nucleotides, and the nucleotides at the remaining positions are 2'-O-methyl modified nucleotides; Furthermore, the nucleotide sequence at positions 2, 6, 14, and 16 in the antisense strand is a 2'-fluoro-modified nucleotide, and any nucleotide selected from positions 9, 10, 11, or 12 is a 2'-fluoro-modified nucleotide, the nucleotide at position 15 is a 2'-O-methoxyethyl-modified nucleotide, and the remaining positions are 2'-O-methyl-modified nucleotides.

15. The double-stranded RNAi according to any one of claims 10 to 11, characterized in that The nucleotides of the sense strand and / or the antisense strand are all modified nucleotides; and the modifications of the sense strand and the antisense strand are selected from any one of the following (1)-(4): (1) From the 5' end to the 3' end, the nucleotides at positions 7 to 10 in the nucleotide sequence of the sense strand are fluorinated nucleotides, and the nucleotides at the remaining positions are 2'-O-methyl modified nucleotides; the nucleotides at positions 2, 6, 9, 14, and 16 in the nucleotide sequence of the antisense strand are 2'-fluorinated nucleotides, and the nucleotides at the remaining positions are 2'-O-methyl modified nucleotides; (2) From the 5' end to the 3' end, the nucleotides at positions 7 to 10 of the nucleotide sequence in the sense strand are fluorinated nucleotides, and the nucleotides at the remaining positions are 2'-O-methyl modified nucleotides; the nucleotides at positions 2, 6, 12, 14, and 16 of the nucleotide sequence in the antisense strand are 2'-fluorinated nucleotides, and the remaining positions are 2'-O-methyl modified nucleotides; (3) In the direction from the 5' end to the 3' end, the nucleotides at positions 7 to 10 in the nucleotide sequence of the sense strand are fluorinated modified nucleotides, and the nucleotides at the remaining positions are 2'-O-methyl modified nucleotides; the nucleotides at positions 2, 6, 9, 14, and 16 in the nucleotide sequence of the antisense strand are 2'-fluorinated modified nucleotides, the nucleotide at position 15 is a 2'-O-methoxyethyl modified nucleotide, and the remaining positions are 2'-O-methyl modified nucleotides; (4) In the direction from the 5' end to the 3' end, the nucleotides at positions 7 to 10 of the nucleotide sequence in the sense strand are fluorinated nucleotides, and the nucleotides at the remaining positions are 2'-O-methyl modified nucleotides; the nucleotides at positions 2, 6, 12, 14, and 16 of the nucleotide sequence in the antisense strand are 2'-fluorinated nucleotides, the nucleotide at position 15 is a 2'-O-methoxyethyl modified nucleotide, and the remaining positions are 2'-O-methyl modified nucleotides.

16. The double-stranded RNAi according to any one of claims 10 to 15, characterized in that The double-stranded RNAi further comprises a ligand; Optionally, the ligand is conjugated to the 3' end of the sense strand of the double-stranded RNAi; Optionally, the ligand is one or more GalNAc (N-acetylgalactosamine) derivatives attached via a linker.

17. The double-stranded RNAi according to claim 16, characterized in that The ligand structure is as follows: Wherein, * represents the connection site of the oligonucleotide molecule; m is 1, 2, 3 or 4; Z, p, q, R3, L and Y are defined as follows: Z is each independently selected from hydroxyl or thiol; p is each independently selected from 1, 2 or 3; q is each independently selected from 1, 2 or 3; each R3 is each independently selected from H, C1-C6 alkyl, C1-C6 haloalkyl or C1-C6 alkoxy; L is a C1-C20 alkylene chain, or one or more C atoms on the chain are replaced by any group selected from O, S, NH or -NH-C(O)-; Y is each independently NH, O or S.

18. The double-stranded RNAi according to claim 16 or 17, characterized in that The ligand structure is as follows:

19. The double-stranded RNAi according to any one of claims 10 to 18, characterized in that The double-stranded RNAi is shown in the following formula: Wherein, Nu represents a double-stranded oligonucleotide molecule; Optionally, the sense strand of the double-stranded oligonucleotide comprises any modified sense strand nucleotide sequence shown in Table 2, and / or, the antisense strand comprises any modified antisense strand nucleotide sequence shown in Table 2; Optionally, the ligand is attached to the 3' end of the sense strand.

20. The double-stranded RNAi according to claim 19, characterized in that The 3' end of the sense strand in the double-stranded RNAi is covalently coupled to a ligand having the following structure:

21. The double-stranded RNAi according to claim 20, characterized in that The double-stranded RNAi is selected from the group consisting of duplexes numbered RZ006002, RZ006007, RZ006008, RZ006009, RZ006010, RZ006011, RZ006012, RZ006013, RZ006014, RZ006017, RZ006020, RZ006021, RZ006023, and RZ006029; wherein Am, Cm, Gm and Um are 2'-O-methyladenosine-3'-phosphate, 2'-O-methylcytidine-3'-phosphate, 2'-O-methylguanosine phosphate and 2'-O-methyluridine 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; U(moe) is 2'-O-methoxyethyluridine-3'-phosphate, and s is phosphorothioate; Among them, CR01008×3 is a ligand with the following structure:

22. The double-stranded RNAi according to any one of claims 10 to 21, characterized in that The sense strand and / or the antisense strand further comprises a 3' and / or 5' extension or overhang of 1-3 nucleotides in length; Optionally, the sense strand is 17-21 nucleotides long, and the antisense strand is 19-23 nucleotides long; Optionally, the sense strand and / or the antisense strand of the RNAi agent independently comprises one or more phosphorothioate internucleoside linkages; Optionally, the sense strand comprises two consecutive phosphorothioate bonds between the terminal nucleotides at the 3' and 5' ends, or the antisense strand comprises two consecutive phosphorothioate bonds between the terminal nucleotides at the 3' and 5' ends; Optionally, the double-stranded region is 19 to 23 nucleotide pairs in length; or, the double-stranded region is 19 to 21 nucleotide pairs in length; Optionally, at least one strand of the double-stranded region of the RNAi agent comprises a 3' overhang of 1-2 nucleotides.

23. The double-stranded RNAi according to claim 10, wherein the double-stranded RNAi comprises a sense strand and an antisense strand forming a double-stranded region, each strand being 14 to 25 nucleotides in length, and the double-stranded region is represented by the following formula II: SS:5'-(N)a'-(X)p'-(N)b'-(X)q'-(N)c'-(X)r'-(N)d'-3' AS:3'-(N)a-(X)p-(N)b-(X)q-(N)c-5'(II) in, SS represents the sense strand, AS represents the antisense strand; the Ns each independently represent the following modified nucleotides: 2'-O-methyl modified nucleotides, 2'-fluorine modified nucleotides, 2'-deoxy-modified nucleotides; the Xs each independently represent 2'-O-methoxyethyl modified nucleotides; wherein said a, a', p, p', b, b', q, q', c, c', r', d' each independently represents the number of nucleotides, wherein a' is selected from an integer of 3-8; p' is selected from an integer of 0 or 1; b' is selected from an integer of 4-13; q' is selected from an integer of 0 or 1; c' is selected from an integer of 3-9; r' is selected from an integer of 0 or 1; d' is selected from an integer of 1-8; a is selected from an integer of 4-7; p is selected from 1; b is selected from an integer of 4-8; q is selected from 0 or 1; c is selected from an integer of 6-10; and p', q', r', p, q are not all 0 at the same time, and 0≤q'+r'≤2; Furthermore, (N)a contains at least one fluorinated modified nucleotide, and the 16th position of the antisense strand is a fluorinated modified nucleotide starting from the 5'-end; (N)b contains at least one fluorinated modified nucleotide, and the 14th position of the antisense strand is a fluorinated modified nucleotide starting from the 5'-end; (N)c contains at least two fluorinated modified nucleotides, and the 2nd and 6th positions of the antisense strand are both fluorinated modified nucleotides starting from the 5'-end; and the first four nucleotides of (N)b' contain at least two fluorinated modified nucleotides starting from the 5'-end; Optionally, in the direction from the 5' end to the 3' end, the nucleotides at positions 7 to 10 of the nucleotide sequence in the sense strand are fluorinated modified nucleotides, and the nucleotides at the remaining positions are 2'-O-methyl modified nucleotides; and, the nucleotides at positions 2, 6, 14, and 16 of the nucleotide sequence in the antisense strand are 2'-fluorinated modified nucleotides, and any one nucleotide selected from positions 9 or 10 or 11 or 12 is a 2'-fluorinated modified nucleotide, the nucleotide at position 15 is a 2'-O-methoxyethyl modified nucleotide, and the remaining positions are 2'-O-methyl modified nucleotides; Wherein, the sense chain is conjugated to a ligand.

24. The double-stranded RNAi according to claim 23, characterized in that The ligand structure is as follows:

25. The double-stranded RNAi according to any one of claims 10-20 and 22-24, characterized in that The double-stranded RNAi is selected from the group consisting of duplexes numbered RZ0060037-RZ006144; wherein CR01008×3 is selected from the ligands of the following structures: Optionally, the double-stranded RNAi comprises a duplex selected from the group consisting of duplexes shown in numbers RZ0060037-RZ006043.

26. Use of the double-stranded oligonucleotide according to any one of claims 1 to 9, and / or the double-stranded RNAi according to any one of claims 10 to 25 in the preparation of a medicament for alleviating, preventing and / or treating a disease or condition mediated by the FTO gene; Optionally, the disease or disorder comprises obesity or obesity-related diseases or Alzheimer's disease, and the obesity-related diseases comprise diabetes, hyperglycemia, diabetic nephropathy, hyperlipidemia, coronary heart disease, atherosclerosis, hypertension, cardiovascular, cerebrovascular diseases.

27. A pharmaceutical composition, characterized in that The method comprises the double-stranded oligonucleotide according to any one of claims 1 to 9, and / or the double-stranded RNAi according to any one of claims 10 to 25, and further comprises pharmaceutically optional excipients.

28. A kit, characterized in that The kit comprises the double-stranded oligonucleotide according to any one of claims 1 to 9, and / or the double-stranded RNAi according to any one of claims 10 to 25, and / or the pharmaceutical composition according to claim 27.

29. A method for inhibiting the expression of FTO gene in cells, characterized in that: The method comprises administering to a subject a double-stranded oligonucleotide according to any one of claims 1 to 9, and / or a double-stranded RNAi according to any one of claims 10 to 25, and / or a pharmaceutical composition according to claim 27; Optionally, expression of the FTO gene is inhibited by at least 70%, 80%, 90% or 95%; Optionally, inhibiting the expression of the FTO gene reduces the level of FTO protein in the subject by at least 50%, 60%, 70%, 80%, 90% or 95%.

30. A method for alleviating, treating and / or preventing a disease or condition mediated by FTO, characterized in that: The method comprises administering to a subject a double-stranded oligonucleotide according to any one of claims 1 to 9, and / or a double-stranded RNAi according to any one of claims 10 to 25, and / or a pharmaceutical composition according to claim 27; Optionally, the FTO-mediated disease or disorder includes a disease associated with the mRNA level of FTO gene expression; Optionally, the disease or disorder comprises obesity or obesity-related diseases or Alzheimer's disease, and the obesity-related diseases comprise diabetes, hyperglycemia, diabetic nephropathy, hyperlipidemia, coronary heart disease, atherosclerosis, hypertension, cardiovascular, cerebrovascular diseases.

Images