Sirna for inhibiting expression of MAPT gene, conjugate thereof and use thereof

Abstract

The present invention relates to the field of biomedicine, and in particular to an siRNA for inhibiting expression of an MAPT gene, a conjugate thereof and a use thereof. The siRNA of the present invention comprises a sense strand and an antisense strand. The sense strand comprises nucleotide sequence I, and the antisense strand comprises nucleotide sequence II. Each nucleotide in the nucleotide sequence I and the nucleotide sequence II is a modified or unmodified nucleotide. The nucleotide sequence I and the nucleotide sequence II are at least partially reverse complementary to form a double-stranded region. The nucleotide sequence I is substantially identical to a first segment of nucleotide sequence. The first segment of nucleotide sequence is a segment of nucleotide sequence having a length of at least 15 nucleotides in an mRNA expressed by the MAPT gene. The siRNA and the conjugate thereof provided by the present invention can specifically induce the degradation of MAPT mRNA and thus inhibit the synthesis of MAPT, resulting in the reduction of Tau protein, thereby effectively preventing and / or treating neurodegenerative diseases.

Description

siRNA and conjugates thereof for inhibiting expression of MAPT gene and use thereof TECHNICAL FIELD

[0001] The present application belongs to the field of biological medicine, and relates to siRNA and conjugates for inhibiting expression of MAPT gene. BACKGROUND

[0002] In human body, Tau protein encoded by MAPT (Microtuble-Associated Protein Tau) gene plays an important role in microtubule assembly and stabilization in neurons. Abnormal expression and aggregation of Tau protein can cause damage and death of neurons, and is confirmed as an important pathological feature of various neurodegenerative diseases.

[0003] In cases of familial frontotemporal dementia (FTD), nearly 80 different missense mutations and intron mutations of MAPT gene are confirmed as direct causes of the disease. In addition, in Alzheimer's disease (AD), neurofibrillary tangles formed by abnormal aggregation of microtubule-associated protein Tau appear in neurons. Similar Tau protein deposition is also observed in other types of neurodegenerative diseases, such as progressive supranuclear palsy and corticobasal degeneration, and these depositions are usually not accompanied by extracellular deposits.

[0004] Alzheimer's disease (AD) is the most common neurodegenerative disease in the elderly and the most common type of dementia in clinical practice. At present, there are very few drugs for the clinical treatment of AD and the therapeutic effect is poor. In addition, macromolecular drugs targeting Tau protein have problems of blood-brain barrier permeability and cell membrane permeability, so small nucleic acid drugs based on targeting of MAPT gene become an important research direction for neurodegenerative diseases. SUMMARY

[0005] The purpose of the present application is to provide an siRNA molecule capable of inhibiting expression of MAPT gene, so as to provide a new treatment for neurodegenerative diseases.

[0006] In one aspect, the present application provides an siRNA, which comprises a sense strand and an antisense strand, the sense strand comprises a nucleotide sequence I, and the antisense strand comprises a nucleotide sequence II; each nucleotide in the nucleotide sequence I and the nucleotide sequence II is a modified or unmodified nucleotide; the nucleotide sequence I and the nucleotide sequence II are at least partially reverse complementary to form a double-stranded region; the nucleotide sequence I is substantially identical to a first nucleotide sequence, and the first nucleotide sequence is a nucleotide sequence of at least 15 nucleotides in length in mRNA expressed by MAPT gene.

[0007] In preferred embodiments, the first nucleotide sequence is a nucleotide sequence of 15-25 nucleotides in length, such as 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides, in the mRNA expressed by the MAPT gene.

[0008] In preferred embodiments, the first nucleotide sequence is a nucleotide sequence of at least 15 nucleotides in length, such as a nucleotide sequence of 15-25 nucleotides in length, in the high activity interval of the mRNA expressed by the MAPT gene. The high activity interval is position 182-204, 2191-2371, and 2750-2772, preferably position 182-204, 2191-2205, 2283-2305, 2284-2306, 2341-2363, 2349-2371, and 2750-2772, of the mRNA expressed by the MAPT gene, and the mRNA expressed by the MAPT gene is as shown in NCBI refseq ID NM_001377265.1, and specifically, the sequence of the mRNA expressed by the MAPT gene is as shown in SEQ ID NO: 1.

[0009] The high activity interval refers to the interval in which the siRNA and siRNA conjugate designed in the interval can effectively reduce the level of MAPT mRNA, and in FIG. 1, the intervals are divided according to whether the maximum inhibition rate of the siRNA and siRNA conjugate observed on MAPT mRNA falls within 40-60%, 60%-80%, or more than 80%.

[0010] In preferred embodiments, the nucleotide sequence I has at least 70%, at least 80%, at least 85%, at least 90%, at least 95% sequence identity to the first nucleotide sequence.

[0011] In some embodiments, the nucleotide sequence II in the siRNA described above is substantially, substantially complementary, or completely complementary to the first nucleotide sequence.

[0012] The sense strand and the antisense strand are the same or different in length, and the length of the sense strand is 16-23 nucleotides, and the length of the antisense strand is 19-26 nucleotides. In some embodiments, the length ratio of the siRNA sense strand and the antisense strand is 16 / 21, 19 / 21, 21 / 23, 19 / 24.

[0013] In specific embodiments, the nucleotide sequence I comprises at least 15 consecutive nucleotides, such as at least 15, 16, 17, 18, 19, 20, or 21 nucleotides, as shown in any one of SEQ ID NOs: 2-137.

[0014] In an embodiment, the nucleic acid sequence of the sense strand is set forth in Nucleotide Sequence I, which differs from any one of SEQ ID NOs: 2-137 by 1, 2, or 3 nucleotides.

[0015] In an embodiment, the nucleotide sequence I is set forth in any one of SEQ ID NOs: 2-137.

[0016] In an embodiment, the nucleotide sequence II comprises at least 15 contiguous nucleotides, such as at least 15, 16, 17, 18, 19, 20, 21, 22, or 23 nucleotides, set forth in any one of SEQ ID NOs: 138-273.

[0017] In an embodiment, the nucleic acid sequence of the antisense strand is set forth in Nucleotide Sequence II, which differs from any one of SEQ ID NOs: 138-273 by 1, 2, or 3 nucleotides.

[0018] In an embodiment, the nucleotide sequence II is set forth in any one of SEQ ID NOs: 138-273.

[0019] In an embodiment, the siRNA is an siRNA molecule set forth in Table 1, YGND21-1 to YGND21-136.

[0020] In an aspect, the siRNA provided herein comprises a modified nucleotide at each nucleotide position in the nucleotide sequence I and the nucleotide sequence II.

[0021] In an embodiment, the modified nucleotide is a fluorine-modified nucleotide or a non-fluorine-modified nucleotide.

[0022] In particular embodiments, the fluoro-modified nucleotide refers to a nucleotide having a hydroxyl group at the 2'-position of the ribosyl group of the nucleotide replaced by a fluorine to form a nucleotide having a structure shown in Formula (1) below. The non-fluoro-modified nucleotide refers to a nucleotide or a nucleotide analog having a hydroxyl group at the 2'-position of the ribosyl group of the nucleotide replaced by a non-fluoro group. In some embodiments, each non-fluoro-modified nucleotide is independently selected from one of a nucleotide or a nucleotide analog having a hydroxyl group at the 2'-position of the ribosyl group of the nucleotide replaced by a non-fluoro group. These nucleotides having a hydroxyl group at the 2'-position of the ribosyl group replaced by a non-fluoro group are well known to those skilled in the art, and can be selected from one of a 2'-alkoxy-modified nucleotide, a 2'-substituted alkoxy-modified nucleotide, a 2'-alkyl-modified nucleotide, a 2'-substituted alkyl-modified nucleotide, a 2'-amino-modified nucleotide, a 2'-substituted amino-modified nucleotide, and a 2'-deoxy nucleotide. In some embodiments, the 2'-alkoxy-modified nucleotide is a methoxy-modified nucleotide (2'-OMe), as shown in Formula (2) below. In some embodiments, the 2'-substituted alkoxy-modified nucleotide can be a 2'-O-methoxyethyl-modified nucleotide (2'-MOE), as shown in Formula (3) below. In some embodiments, the 2'-amino-modified nucleotide (2'-NH2) is as shown in Formula (4) below. In some embodiments, the 2'-deoxy nucleotide (DNA) is as shown in Formula (5) below.

[0023] A nucleotide analog refers to a group that can replace a nucleotide in a nucleic acid, but is structurally different from an adenine ribonucleoside, a guanine ribonucleotide, a cytosine ribonucleotide, a uracil ribonucleotide, or a thymine ribonucleotide. In some embodiments, the nucleotide analog can be an iso-nucleotide, a bridged nucleotide, or an acyclic nucleotide.

[0024] A bridged nucleotide refers to a constrained or inaccessible nucleotide. The bridged nucleotide can contain a five-membered ring, a six-membered ring, or a seven-membered ring with a fixed C3-endo sugar conformation. In some embodiments, the bridged nucleotide can be an LNA, an ENA, a cET BNA, etc.; wherein the LNA is as shown in Formula (6) below, the ENA is as shown in Formula (7) below, and the cET BNA is as shown in Formula (8) below.

[0025] An acyclic nucleotide is a class of nucleotides in which the sugar ring of the nucleotide is opened. In some embodiments, the acyclic nucleotide can be an unlocked nucleic acid (UNA) or a glycol nucleic acid (GNA); wherein the UNA is as shown in Formula (9) below, and the GNA is as shown in Formula (10) below.

[0026] In the above Formula (9) and Formula (10), R is selected from H, OH or alkoxy (O-alkyl).

[0027] An isonucleotide refers to a compound in which the position of a base on a ribose ring is changed. In some embodiments, an isonucleotide can be a compound in which a base is moved from the 1 '-position to the 2' or 3' position on a ribose ring. As shown in Formula (11) or Formula (12).

[0028] In the above Formula (11) and Formula (12), R is selected from H, OH, F or a non-fluorine group as described above.

[0029] In the above Formula (1) to Formula (12), Base represents a base, such as A, U, G, C or T.

[0030] In a specific embodiment, one or more of the nucleotides at positions 7, 9, 10, 11 of the nucleotide sequence I, in the 5'-end to 3'-end direction, is a fluorine-modified nucleotide; and one or more of the nucleotides at positions 2, 6, 8, 9, 14, 16 of the nucleotide sequence II, in the 5'-end to 3'-end direction, is a fluorine-modified nucleotide.

[0031] In a specific embodiment, some of the nucleotides of the nucleotide sequence III can be GNA-modified; more specifically, the nucleotide at position 7 of the nucleotide sequence III, in the 5'-end to 3'-end direction, is GNA-modified.

[0032] In a specific embodiment, in the nucleotide sequences I and II, in addition to the fluorine-modified and GNA-modified nucleotides, the other nucleotides are methoxy-modified.

[0033] In a specific embodiment, at least one of the phosphates in the phosphate-sugar backbone of at least one of the single strands of the sense strand and the antisense strand of the siRNA provided by the present application is a phosphate with a modified group. In some embodiments, the phosphate with a modified group is a phosphorothioate group in which at least one of the oxygen atoms in the phosphodiester bond is replaced by a sulfur atom. In some embodiments, the phosphate with a modified group is a phosphorothioate group having a structure as shown in Formula (13):

[0034] In some embodiments, the siRNA provided herein, preferably, the phosphorothioate linkage is present at at least one of the following positions: between the 1st and 2nd nucleotides of the sense strand and / or the antisense strand; between the 2nd and 3rd nucleotides of the sense strand and / or the antisense strand; between the 19th and 20th nucleotides of the sense strand; between the 20th and 21st nucleotides of the sense strand; between the 21st and 22nd nucleotides of the antisense strand; between the 22nd and 23rd nucleotides of the antisense strand; or any combination thereof.

[0035] In some embodiments, the 5' terminal nucleotide of the antisense strand of the siRNA is a 5'-phosphate nucleotide or a 5'-phosphate analog modified nucleotide, such as shown in formula (14), formula (15) and formula (16):

[0036] In formula (14) to formula (16) above, Base represents a base, such as A, U, G, C or T.

[0037] In one aspect, the present application provides an siRNA conjugate, which comprises an siRNA as described above and a conjugate group conjugated to the siRNA.

[0038] In some embodiments, the conjugate group can be galactose or N-acetylgalactosamine, wherein the galactose or N-acetylgalactosamine molecule can be monovalent, divalent, trivalent, tetravalent.

[0039] In some embodiments, the conjugate group can be a normal alkyl group, such as n-hexadecyl (C16).

[0040] In some embodiments, the conjugation site of the siRNA and the conjugate group can be at the 3'-end or 5'-end of the sense strand of the siRNA, at the 3'-end of the antisense strand, or in the internal sequence of the siRNA.

[0041] In some specific embodiments, the siRNA conjugate provided herein, the conjugate group is L96 or C16, the structure of L96 is shown in formula (I) below, and the structure of the nucleoside conjugated with C16 is shown in formula (II) below:

[0042] In a specific embodiment, the siRNA conjugate is siRNA conjugate (YGND21-1M ~ YGND21-136M) as shown in Table 2, siRNA conjugate (YGND21-1M' ~ YGND21-136M') as shown in Table 3, and siRNA conjugate (YGND21-107M'G, YGND21-135M'G) as shown in Table 4.

[0043] In an aspect, the present application provides a composition comprising the aforementioned siRNA or siRNA conjugate. In a preferred embodiment, the composition is a pharmaceutical composition, further comprising a pharmaceutically acceptable carrier or excipient. The pharmaceutically acceptable carrier or excipient involved in the present application includes, but is not limited to, water for injection, sodium hydroxide, sodium dihydrogen phosphate monohydrate, sodium dihydrogen phosphate dihydrate, phosphoric acid, sodium chloride, potassium chloride, hydrochloric acid, anhydrous potassium dihydrogen phosphate, anhydrous disodium hydrogen phosphate, PEG2000, PEG6000, cholesterol, distearoylphosphatidylcholine, 1,2-dimyristin, dimethyl adipate.

[0044] In an aspect, the present application provides the use of the aforementioned siRNA, siRNA conjugate or pharmaceutical composition in the manufacture of a medicament for preventing and / or treating a neurodegenerative disease. Preferably, the neurodegenerative disease comprises Alzheimer's disease (AD), frontotemporal dementia (FTD), autism, epilepsy, progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), Pick's disease, primary age-related tauopathy (PART).

[0045] In an aspect, the present application provides a method for preventing and / or treating a neurodegenerative disease, comprising administering a prophylactically effective amount or a therapeutically effective amount of the siRNA, siRNA conjugate or pharmaceutical composition of the present application to a subject in need thereof.

[0046] In a specific embodiment, the neurodegenerative disease comprises Alzheimer's disease (AD), frontotemporal dementia (FTD), autism, epilepsy, progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), Pick's disease, primary age-related tauopathy (PART).

[0047] In an aspect, the present application provides the aforementioned siRNA, siRNA conjugate or pharmaceutical composition for use as a medicament.

[0048] In an aspect, the present application provides the aforementioned siRNA, siRNA conjugate or pharmaceutical composition for use in therapy.

[0049] The pharmaceutical composition of the present application can be used alone for treating the tau protein related neurodegenerative disease, and can also be used in combination with standard oral drugs, which provides experimental support for achieving diversified treatment plans.

[0050] The siRNA for inhibiting the expression of the MAPT gene of the present application is generally suitable in a dosage range of about 0.1 mg / kg to about 10.0 mg / kg, preferably about 0.3 mg / kg to about 3.0 mg / kg.

[0051] The administration routes of the drug of the present application include intracerebroventricular (ICV) injection, intrastriatal injection, intravenous administration, subcutaneous administration, intrathecal administration, intraspinal administration, intramuscular administration, airway administration (aerosol), rectal administration, transdermal administration, ocular administration, nasal administration, pulmonary administration, and topical administration (including oral administration and sublingual administration), etc.

[0052] The siRNA and conjugates thereof provided by the present application can specifically inhibit the synthesis of MAPT. In particular, the siRNA and conjugates thereof provided by the present application can specifically target the brain, complementarily pair with the brain MAPT mRNA sequence, induce the degradation of MAPT mRNA, thereby inhibiting the synthesis of MAPT in the brain, resulting in the reduction of tau protein. The siRNA and conjugates thereof provided by the present application have the advantages of strong inhibitory activity of MAPT gene and low cytotoxicity, can significantly reduce the expression of tau protein in animals, and can effectively prevent and / or treat neurodegenerative diseases. BRIEF DESCRIPTION OF DRAWINGS

[0053] Figure 1 is a schematic diagram of the binding site of the siRNA of the present application to the human MAPT gene;

[0054] Figure 2 is the result of the siRNA conjugate of the present application inhibiting the expression of MAPT in mice;

[0055] Figure 3 is the result of the siRNA conjugate of the present application inhibiting the expression of MAPT in the brain;

[0056] Figure 4 is the result of the siRNA conjugate of the present application inhibiting the expression of MAPT in the brain;

[0057] Figure 5 is the cytotoxicity result of the siRNA conjugate of the present application;

[0058] Figure 6 is the off-target analysis result of the siRNA conjugate of the present application. DETAILED DESCRIPTION

[0059] DEFINITIONS

[0060] In the present text, capital letters C, G, U, A represent nucleotides comprising cytosine, guanine, uracil, adenine as base, respectively, unless otherwise specified; lower case letter m indicates that the nucleotide adjacent to the left of the letter m is a methoxy-modified nucleotide; lower case letter f indicates that the nucleotide adjacent to the left of the letter f is a fluorinated-modified nucleotide; lower case letter s indicates that the two nucleotides adjacent to the left and right of the letter s are linked by phosphorothioate subunit; letter combination VP indicates that the nucleotide adjacent to the right of the letter combination VP is a 5'-(E)-vinylphosphonate (E-VP) modified nucleotide; L96 has the structure of Formula (I) linked to the 3'-end of the sense strand by a phosphonate linkage; hd indicates a C16 conjugate group linked to the siRNA, (Nhd) indicates that the nucleotide N in the siRNA is linked to C16, the structure of the C16 linked nucleoside is shown in Formula (II); GNA indicates that the nucleotide adjacent to the left of the letter combination (GNA) is a GNA modified nucleotide.

[0061] It is to be understood that the terms "nucleotide", "ribonucleotide" or "deoxyribonucleotide" herein can also refer to modified nucleotides (as described below).

[0062] In the present text, the terms "siRNA", "small interfering ribonucleic acid" or "dsRNA" are used interchangeably and refer to a double-stranded RNA molecule, sometimes also referred to as short interfering RNA. The siRNA typically comprises a sense strand and an antisense strand, the sense strand and the antisense strand having a length of 15 to 30 nucleotides, preferably 15 to 25 nucleotides. The antisense strand is complementary to a target nucleic acid sequence (e.g., a mature mRNA sequence), the degree of complementarity being, for example, at least 70% complementary, at least 80% complementary, at least 90% complementary, at least 95% complementary, at least 99% complementary, or completely complementary. The sense strand is complementary to the antisense strand to form a duplex or duplex region of, for example, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotide pairs in length.

[0063] In the present text, when the MAPT siRNA comprises a sense strand and an antisense strand that do not have unpaired nucleotides or analogs thereof at the termini, the termini are referred to as "blunt". When the MAPT siRNA comprises a sense strand and an antisense strand that have unpaired nucleotides or analogs thereof at the termini, the termini are referred to as "overhanging". The overhang can be 1, 2, or 3 nucleotides in length and can be a 5'-overhang and / or a 3'-overhang. In some embodiments, one terminus of the MAPT siRNA is blunt and the other terminus is overhanging. In other embodiments, both termini of the MAPT siRNA are blunt. In other embodiments, both the sense strand and the antisense strand have a 3'-overhang of 2 nucleotides in length.

[0064] In the present context, the term "antisense strand" refers to the strand of a MAPT siRNA comprising a region that is substantially complementary to a MAPT target sequence. The term "sense strand" refers to the strand of a MAPT siRNA comprising a region that is substantially complementary to a region of the antisense strand of a MAPT dsRNA.

[0065] In the present context, the term "complementary" or "complementarity" can be used interchangeably with "reverse complement" and refers to the ability of an oligonucleotide or nucleic acid to hybridize to another oligonucleotide or nucleic acid by base complementarity. In certain embodiments, the siRNA can comprise mismatches (i.e., the bases at the corresponding positions do not exist in a complementary pairing form) in the double-stranded nucleic acid and still retain its ability to bind to the target nucleic acid.

[0066] In the present context, the terms "complementary", "substantially complementary", "essentially complementary" or "fully complementary" can be used to refer to base matches between the sense strand and the antisense strand of a MAPT siRNA, base matches between the antisense strand of a MAPT siRNA and the sequence of a target MAPT mRNA, or base matches between a single-stranded antisense oligonucleotide and a target MAPT mRNA sequence. It is understood that the term "antisense strand of a MAPT dsRNA" can refer to the same sequence as a "MAPT antisense polynucleotide agent". Unless otherwise specified, "substantially complementary" means that there are no more than 3 base mismatches between the two nucleotide sequences involved; "essentially complementary" means that there are no more than 1 base mismatch between the two nucleotide sequences involved; "fully complementary" means that there are no base mismatches between the two nucleotide sequences involved.

[0067] When the complementary region is not fully complementary to the target sequence, the mismatches can be present in the interior or at the ends of the siRNA molecule. It is known in the art that mismatches are acceptable for the efficacy of the siRNA. In some embodiments, the mismatches are located in the terminal regions, e.g., within 5, 4, 3, 2, or 1 nucleotides of the 5'-end and / or 3'-end. In other embodiments, the MAPT siRNA does not comprise mismatches.

[0068] In the present context, "inhibiting the expression of MAPT" or "inhibiting the expression of a MAPT gene" includes inhibiting the expression of any MAPT gene and variants thereof, including but not limited to the mouse MAPT gene, the rat MAPT gene, the monkey MAPT gene, the human MAPT gene. The MAPT gene can be, for example, a wild-type MAPT gene, a mutant MAPT gene, or a transgenic MAPT gene.

[0069] In the present context, the term "inhibit" can be used interchangeably with "silence", "reduce", "down-regulate" and the like, and includes any level of inhibition. Thus, "inhibiting expression of MAPT" or "inhibiting MAPT gene expression" includes at least partial inhibition of expression of the MAPT gene, such as at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% inhibition of expression of the MAPT gene.

[0070] In the present context, "target nucleic acid" refers to any nucleic acid molecule whose expression or activity can be modulated by an siRNA. In some embodiments, the target nucleic acid is DNA or RNA. In some specific embodiments, the target nucleic acid is mRNA, non-coding RNA, pre-mRNA, pri-microRNA, mature microRNA, etc. In some specific embodiments, the target nucleic acid is a MAPT mRNA.

[0071] In the present context, and particularly in the description of the preparation of the siRNA, pharmaceutical composition or siRNA conjugate of the application, the nucleoside monomers, unless otherwise specified, refer to modified or unmodified nucleoside phosphoramidites monomers (unmodified or modified RNA phosphoramidites, sometimes also referred to as Nucleoside phosphoramidites) used in the phosphoramidite solid phase synthesis according to the kind and order of nucleotides in the siRNA or siRNA conjugate to be prepared. The phosphoramidite solid phase synthesis is a method well known to the person skilled in the art for the synthesis of siRNA. All nucleoside monomers used in the present application are commercially available.

[0072] In Tables 1, 2, 3 and 4, the 5'-terminal nucleotide of the sense strand or of the modified sense strand to which the conjugation group is attached is represented on the left side of the 5'-terminal nucleotide if not marked with VP, meaning that the 5'-terminal nucleotide is not attached to a 5'-phosphate group or a 5'-phosphate derivative, the structure of which is represented by formula (III):

[0073] wherein Base represents a base, such as A, U, G, C or T; and R is a hydroxyl group or is substituted by various groups known to the person skilled in the art, for example, R can be 2'-fluoro (2'-F), 2'-alkoxy, 2'-substituted alkoxy, 2'-alkyl, 2'-substituted alkyl, 2'-amino, 2'-substituted amino, 2'-deoxynucleotide.

[0074] In Tables 1, 2, 3, and 4, if the 5'-terminal nucleotide of the antisense strand or the modified antisense strand is not labeled with VP, it means that the 5'-terminal nucleotide is not linked with a 5'-phosphate group or a 5'-phosphate derivative group, and its structure is also shown in Formula (III).

[0075] In Tables 1, 2, 3, and 4, the 3' position of the 3'-terminal nucleotide of the sense strand, the 3'-terminal nucleotide of the antisense strand, and the 3'-terminal nucleotide of the modified antisense strand is hydroxyl.

[0076] In the present disclosure, "siRNA" can also refer to "base sequence", "siRNA motif", or "motif", the meaning of which includes the order of nucleotide arrangement of the siRNA duplex. "Motif" refers to each nucleotide in the siRNA is an unmodified nucleotide, as shown in Table 1. In some embodiments, the 5'-terminal nucleotide of the antisense strand of the motif is linked with a 5'-phosphate group or a 5'-phosphate derivative group. In other embodiments, the 5'-terminal nucleotide of the antisense strand of the motif is not linked with a 5'-phosphate group or a 5'-phosphate derivative group.

[0077] In the present disclosure, the term "nucleotide analogue" refers to a group that is structurally different from adenine ribonucleotide, guanine ribonucleotide, cytosine ribonucleotide, uracil ribonucleotide, or thymine deoxyribonucleotide, but can replace nucleotides in nucleic acids. Specifically, nucleotide analogues include, but are not limited to, iso-nucleotides, bridged nucleotides (BNAs), or acyclic nucleotides.

[0078] In the present disclosure, "modified nucleotide" refers to modification of at least one nucleotide in the siRNA molecule to improve the stability or efficacy of the siRNA. In the present disclosure, siRNA containing modified nucleotides is also referred to as "siRNA modifier". In the present disclosure, different modifications can be made to the motif of the siRNA, thereby obtaining the corresponding siRNA modifier. In some embodiments, the siRNA modifier is obtained by alternating modification of the motif. In other embodiments, the siRNA modifier is obtained by modifying the motif using a specific modification template. In yet other embodiments, the siRNA modifier is obtained by modifying the motif using the off-target protection modification method in the present disclosure. In yet other embodiments, the siRNA modifier is obtained by modifying the motif using multiple different modification methods simultaneously.

[0079] In the present disclosure, the term "alternating modification" refers to modification of each nucleotide of the motif according to the order of nucleotide arrangement of the motif using modification methods such as 2'-methoxy (2'-OMe) modification, 2'-fluoro (2'-F) modification, etc.

[0080] In the present context, "siRNA conjugate" refers to an siRNA or siRNA modifier linked to a conjugate group, resulting in an siRNA conjugate or a conjugate of an siRNA modifier.

[0081] In the present context, a "conjugate group" is a GalNAc derivative or a n-alkyl group (e.g. n-hexadecyl) linked to an oligonucleotide. In some embodiments, the conjugate group comprises a targeting group (also known as a ligand). In other embodiments, the conjugate group can further comprise a linker. Specifically, a GalNAc derivative linked to an oligonucleotide via a linker (e.g. a divalent, trivalent or tetravalent branched linker arm), or a GalNAc derivative linked to an oligonucleotide via a monovalent linker arm.

[0082] In the present context, when referring to any nucleotide position of any strand of an siRNA motif, an siRNA modifier, an siRNA conjugate, an siRNA duplex, etc., the 5' to 3' direction is meant, unless otherwise specified.

[0083] In the present context, the term "sequence identity" means that a nucleic acid sequence comprises a sequence having at least about 70% or more sequence identity, preferably at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity, compared to a reference sequence. The percentage of sequence identity is determined by comparing the best alignment of two sequences over a comparison window. The present invention includes nucleotide sequences that are essentially identical to the sequences in Tables 1-4. In some embodiments, the nucleotide sequence is identical to the sequences in Tables 1-4, or has at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity.

[0084] Modifications

[0085] In some embodiments, the MAPT siRNAs of the application are unmodified and do not include chemical modifications known in the art and described herein. In other embodiments, the MAPT siRNAs of the application can include ribonucleotides having chemical modifications to enhance stability or other beneficial properties. The modifications can include all types of modifications known in the art. In some embodiments, all of the nucleotides of the MAPT siRNA are modified. In other embodiments, substantially all of the nucleotides in the MAPT siRNA are modified. In yet other embodiments, there are no more than 5, 4, 3, 2, or 1 unmodified nucleotides in a strand of the MAPT siRNA.

[0086] The MAPT siRNAs of the application can be synthesized or modified by methods recognized in the art, such as those described in "Current protocols in Nucleic Acid Chemistry," incorporated herein by reference. Modifications include, for example, terminal modifications, such as 5'-terminal modifications (phosphorylation, conjugation, inverted linkage, etc.), 3'-terminal modifications (conjugation, DNA nucleotides, inverted linkage, etc.); base modifications, such as replacement of bases with stabilizing bases, destabilizing bases, or bases that base pair with extended partner pools, abasic (abasic nucleotides), or conjugated bases; sugar modifications (e.g., at the 2' position or 4' position) or replacement of sugars; or backbone modifications, including modification or replacement of phosphodiester linkages. Particular examples of MAPT siRNAs, siRNA conjugates useful in the application include, but are not limited to, RNAs comprising modified backbones or no natural internucleoside linkages. In addition to these, RNAs with modified backbones include those with no phosphorus atom in the backbone. In some embodiments, the modified RNA has a phosphorus atom in its internucleoside backbone.

[0087] Modified RNA backbones can include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl-phosphotriesters, methyl and other alkyl phosphonates including 3'-alkylene phosphonates and chiral phosphonates, phosphinates, amino-phosphinates, including 3'-amino amino-phosphinates and aminoalkyl-phosphinates, thio-phosphinates, thioalkyl-phosphonates, thioalkyl-phosphotriesters, and boranophosphonates having normal 3'-5' linkages, 2'-5' linked analogs of these, and those with inverted polarity wherein the adjacent nucleoside units are linked 3'-5' to 5'-3', or 2'-5' to 5'-2'. Various salts, mixed salts and free acid forms are also included. In some embodiments, the RNAi agents of the application are in the free acid form. In other embodiments, the RNAi agents of the application are in the salt form, such as the sodium salt form.

[0088] The phosphorus-free modified RNA backbone has a backbone formed from: short-chain alkyl or cycloalkyl nucleoside inter-linking bonds, mixed heteroatom and alkyl or cycloalkyl nucleoside inter-linking bonds, and one or more short-chain heteroatom or heterocyclic nucleoside inter-linking bonds. These include those with morpholine bonds (partially formed from the sugar moiety of the nucleoside); siloxane backbones; sulfide, sulfoxide, and sulfone backbones; methacetyl and thiomethacetyl backbones; methylenemethacetyl and thiomethacetyl backbones; olefin-containing backbones; aminosulfonate backbones; methyleneimino and methylenehydrazine backbones; sulfonate and sulfonamide backbones; amide backbones; and other moieties mixed with N, O, S, and CH2 components. Methods for preparing phosphorus-free modified RNA backbones are conventional in the art.

[0089] In some embodiments, the MAPT siRNA or siRNA conjugate, the siRNA sense strand, and the siRNA antisense strand of the present invention contain RNA mimics, wherein the sugar and nucleoside internucleotide bonds (i.e., the backbone) of the nucleotide units are replaced by novel groups. Base units are maintained to hybridize with suitable MAPT nucleic acid target compounds. One such oligomeric compound (an RNA mimic that has been shown to have excellent hybridization properties) is called peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of the RNA is replaced by a backbone containing an amide, particularly an aminoethylglycine backbone. Nucleobases are retained and bind directly or indirectly to the nitrogen atoms of the amide moiety in the backbone. The method for preparing the RNA mimics is conventional in the art.

[0090] In some embodiments, the MAPT siRNA or siRNA conjugate, the siRNA sense strand, and the siRNA antisense strand of the present invention comprise RNA with a phosphate thioester backbone and an oligonucleotide with a heteroatom backbone, and are prepared using methods conventional in the art. In some embodiments, the MAPT siRNA or siRNA conjugate, the siRNA sense strand, and the siRNA antisense strand of the present invention have a morpholine backbone structure.

[0091] The modified RNA may also contain one or more substituted sugar moieties. The MAPT siRNA or siRNA conjugate, the siRNA sense strand, and the siRNA antisense strand of the present invention may contain one of the following at the 2' position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S-, or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl, and alkynyl groups may be substituted or unsubstituted C1 to C2 groups. 10 Alkyl or C2 to C 10 Alkenyl and ynyl groups. Exemplary modifications include: O[(CH2)] n O] m CH3, O(CH2) n OCH3, O(CH2) nNH2, O(CH2) n CH3, O(CH2) n ONH2 and O(CH2) n ON[(CH2) n CH3)]2, wherein n and m are 1 to about 10. In other embodiments, the MAPT siRNA or siRNA conjugate, the siRNA sense strand, and the siRNA antisense strand of the present invention include one of the following at the 2' position: C1 to C 10 Lower alkyl groups, substituted lower alkyl groups, alkylaryl groups, aryl alkyl groups, O-alkylaryl or O-aryl alkyl groups, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocyclic alkyl groups, heterocyclic alkylaryl groups, aminoalkylamino groups, polyalkylamino groups; substituted silyl groups, RNA cleavage groups, reporter groups, intercalating agents; groups used to improve the pharmacokinetic properties of MAPT siRNA; or groups used to improve the pharmacodynamic properties of MAPT siRNA or siRNA conjugates, the sense strand of siRNA, the antisense strand of siRNA, and other substituents with similar properties. In some embodiments, the modification includes 2'-methoxyethoxy (2'-O-CH2CH2OCH3, also known as 2'-O-(2-methoxyethyl) or 2'-MOE, i.e., alkoxy-alkoxy. Another exemplary modification is 2'-dimethylaminoethoxyethoxy, i.e., the O(CH2)2ON(CH3)2 group, also known as 2'-DMAOE, and 2'-dimethylaminoethoxyethoxy (also known as 2'-O-dimethylaminoethoxyethyl or 2'-DMAEOE), i.e., 2'-O-CH2-O-CH2-N(CH2)2.

[0092] Other modifications include 2'-methoxy (2'-OCH3), 2'-aminopropoxy (2'-OCH2CH2CH2NH2), and 2'-fluorine (2'-F). Similar modifications may also be made at other positions of the MAPT siRNA or siRNA conjugate, the siRNA sense strand, and the siRNA antisense strand of the present invention, particularly at the 3' position of the 3'-terminal nucleotide, or at the 5' position of the sugar in the 2'-5' linked MAPT siRNA or siRNA conjugate, the siRNA sense strand, and the siRNA antisense strand, as well as at the 5' position of the 5'-terminal nucleotide. The MAPT siRNA or siRNA conjugate, the siRNA sense strand, and the siRNA antisense strand may also have a sugar mimic, such as replacing the cyclobutyl moiety of furanylpentose. The methods for preparing modified RNAs, such as those described, are conventional methods in the art.

[0093] In some implementations, the MAPT siRNA or siRNA conjugate, the siRNA sense strand, and the siRNA antisense strand may include nucleobase (usually referred to simply as "base") modifications or substitutions. As used herein, "unmodified" or "natural" nucleobases include purine bases adenine (A) and guanine (G), and pyrimidine bases thymine (T), cytosine (C), and uracil (U). Modified nucleobases include other synthetic and natural nucleobases, such as deoxythymidine (dT), 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymidine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyluracil and cytosine, 6-azouracil. Pyridine, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil; 8-halogen, 8-amino, 8-thio, 8-thioalkyl, 8-hydroxy and other 8-substituted adenine and guanine; 5-halogen, specifically 5-bromo, 5-trifluoromethyl and other 5-substituted uracil and cytosine; 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-azaguanine and 7-azaadenine, and 3-deadenine and 3-deadenine. Other and editable materials include those disclosed in U.S. Patent No. 3,687,808, and those disclosed in *Modified Nucleosides in Biochemistry, Biotechnology and Medicine, Herdewijn, P.Ed. Wiley-VCH, 2008; *The Concise Encyclopedia Of Polymer Science and Engineering, pages 858-859; Kroschwitz, JL, Ed. John Wiley & Sons, 1990; English et al., Angewandte Chemie, International Edition, 1991, 30, 613; Sanghvi, Y S., Chapter 15, dsRNA Research and MAPTlications, pages 289-302; Crooke, STand Lebleu, B., Ed., CRC Press, 1993. The methods for preparing MAPT siRNA or siRNA conjugates, siRNA sense strands, and siRNA antisense strands comprising nucleobase modifications and / or substitutions are conventional methods in the art.

[0094] In some embodiments, the MAPT siRNA or siRNA conjugate, the siRNA sense strand, and the siRNA antisense strand of the present invention can be modified to include one or more open-loop nucleic acids (UNAs, also known as unlocked nucleic acids) and / or glycol nucleic acids (GNAs). GNAs contain an acyclic, three-carbon propylene glycol (1,2-propanediol) backbone that replaces the (deoxy)ribose sugars of DNA and RNA, forming the simplest chemically stable nucleic acid structure. It is known in the art that nucleic acids modified with GNAs exhibit better thermal stability and can mitigate off-target effects.

[0095] In some embodiments, the 3'-terminus of the MAPT siRNA or siRNA conjugate of the present invention, the siRNA sense strand, and the siRNA antisense strand comprises at least one modified nucleotide, wherein the at least one modified nucleotide includes: ribitol, reverse nucleotide, abase-free nucleotide, reverse 2'-OMe nucleotide, reverse abase-free nucleotide, and reverse 2'-deoxynucleotide. It is known in the art that comprising abase-free or reverse abase-free nucleotide at the oligonucleotide terminus can enhance stability.

[0096] In some embodiments, the MAPT siRNA or siRNA conjugate of the present invention comprises a 5' phosphate ester or a 5' phosphate ester mimic, such as a 5' terminal phosphate ester or phosphate ester mimic on the antisense strand. Suitable phosphate ester mimics are known in the art, such as those disclosed in U.S. Patent Publication No. 2012 / 0157511.

[0097] deliver

[0098] In some embodiments, the present invention includes chemically linking MAPT siRNA to one or more ligands, moieties, or conjugates that enhance the in vivo delivery activity, cellular distribution, or uptake of MAPT siRNA. Such moieties include, but are not limited to, lipid moieties, such as cholesterol moieties.

[0099] In some embodiments, the ligand alters the distribution, targeting, or lifetime of the RNAi agent it incorporates. In some embodiments, the ligand does not participate in double-strand pairing in the nucleic acid molecule. The ligand may comprise naturally occurring substances such as proteins (e.g., human serum albumin, low-density lipoprotein, or globulin); carbohydrates (e.g., dextran, amylopectin, chitin, chitosan, inulin, cyclodextrin, N-acetylglucosamine, N-acetylgalactosamine, or hyaluronic acid); or lipids. The ligand may also be a recombinant or synthetic molecule, such as a synthetic polymer, for example, synthetic polyamino acids, including examples of polylysine, poly-L-aspartic acid, poly-L-glutamic acid, styrene-maleic anhydride copolymer, poly(L-lactide-co-glycolic acid) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer, polyethylene glycol, polyvinyl alcohol, polyurethane, poly(2-ethylacrylic acid), N-isopropylacrylamide polymer, or polyphosphonazine. Examples of polyamines include polyethyleneimine, polylysine, spermine, spermidine, pseudopeptide-polyamine, pseudopeptide-polyamine, dendritic polyamine, arginine, amidine, protamine, cationic lipids, cationic porphyrins, quaternary salts of polyamines, or α-helical peptides.

[0100] The ligand may also contain a targeting group, such as a cell or tissue target, like a lectin, glycoprotein, lipid, or protein, such as an antibody that binds to a specific cell type (e.g., brain cells). The targeting group can be thyroid-stimulating hormone, melanocyte-stimulating hormone, lectin, glycoprotein, surfactant protein A, mucin carbohydrate, polygalactose, polygalactose, N-acetyl-galactosamine, N-acetyl-glucosamine, polymannose, polyfucose, glycosylated polyamino acids, polygalactose, transferrin, bisphosphonates, polyglutamate, polyaspartate, lipids, cholesterol, steroids, bile acids, folic acid, vitamin B12, vitamin A, biotin, or RGD peptide or RGD peptide mimics. In some embodiments, the ligand is a polygalactose, such as GalNAC. In other embodiments, the ligand is a positive-chain alkyl group.

[0101] In some embodiments, in vivo delivery may also be achieved via β-glucan delivery systems, such as those described in U.S. Patent Nos. 5,032,401 and 5,607,677, and U.S. Publication No. 2005 / 0281781, the entire contents of which are incorporated herein by reference. In other embodiments, the MAPT siRNA of the present invention is introduced into cells from in vitro using methods known in the art, such as electroporation and lipid transfection. In still other embodiments, the MAPT siRNA is delivered without a target agent. These RNAs can be delivered as “naked” RNA molecules. As a non-limiting example, the MAPT siRNA of the present invention can be administered to a subject in a pharmaceutical composition containing an RNAi reagent but not a target agent (e.g., a GalNAc targeting compound) to treat neurodegenerative diseases.

[0102] The MAPT siRNA of the present invention can be administered to subjects in a quantity and manner that effectively reduces the level and activity of MAPT peptides in cells and / or subjects. In some embodiments, one or more MAPT siRNAs are administered to cells and / or subjects to treat neurodegenerative diseases. In some embodiments, the method of the present invention includes administering one or more MAPT siRNAs to a subject in need to alleviate the subject's neurodegenerative disease. The MAPT siRNA of the present invention can be administered to reduce MAPT expression and / or activity in one or more cells in vitro, ex vivo, and in vivo.

[0103] In some embodiments, the MAPT siRNA of the present invention can be administered alone or in combination with one or more other MAPT siRNAs. In some embodiments, a variety of independently selected MAPT siRNAs are administered to the subject. In some embodiments, the MAPT siRNA is administered to the subject in combination with one or more other treatment regimens for treating neurodegenerative diseases. Other treatment regimens may be administered before, simultaneously with, or after the administration of the MAPT siRNA of the present invention. Non-limiting examples of non-MAPT siRNA therapeutics include: cholinesterase inhibitors; excitatory amino acid receptor antagonists such as memantine or memantine combined with donepezil, rivastigmine; medications for psychotropic symptoms such as risperidone, quetiapine, aripiprazole, olanzapine; monoclonal antibodies targeting Aβ, including lencanemab, adunatumab, and donepemab; antiepileptic and antimigraine medications such as lamotrigine, levetiracetam, lacosamide, clobazine, valproic acid, tiapride, verapamil, and other medications such as mannocycline, minocycline; immunosuppressive drugs such as mycophenolate mofetil, cyclophosphamide, azathioprine, intravenous immunoglobulin, rituximab, etc. Other therapeutic agents, such as diuretics, beta-blockers, angiotensin-converting enzyme (ACE) inhibitors, angiotensin II receptor antagonists, vasodilators, calcium channel blockers, aldosterone antagonists, α2-agonists, renin inhibitors, α-blockers, peripherally acting adrenergic agents, selective D1 receptor partial agonists, non-selective α-adrenergic antagonists, synthetic steroidal anti-mineralocorticoid agents, or any combination thereof. Administration of the MAPT siRNA of the present invention to cells or subjects, along with one or more other therapeutic agents or active ingredients, can act synergistically to treat neurodegenerative diseases, thereby increasing the effectiveness of treatment for neurodegenerative diseases.

[0104] The treatment methods of the present invention include administering the MAPT siRNA of the present invention before and / or when a neurodegenerative disease is present, for example, at all times before and after the early, middle, and late stages of the disease or condition.

[0105] Pharmaceutical Composition

[0106] The present invention also includes pharmaceutical compositions comprising the MAPT siRNA of the present invention, its modifications or conjugates, and pharmaceutically acceptable carriers or excipients.

[0107] The term "pharmaceutically acceptable carrier or excipient" refers to a carrier or excipient used for the administration of a therapeutic agent. Such carriers or excipients include, but are not limited to, water for injection, sodium hydroxide, sodium dihydrogen phosphate monohydrate, sodium dihydrogen phosphate dihydrate, sodium chloride, potassium chloride, phosphoric acid, hydrochloric acid, anhydrous potassium dihydrogen phosphate, anhydrous disodium hydrogen phosphate, PEG2000, PEG6000, cholesterol, distearate, glyceryl 1,2-dimyristate, dimethyl adipate, and combinations thereof. This term explicitly excludes cell culture media. For orally administered drugs, pharmaceutically acceptable carriers include, but are not limited to, pharmaceutically acceptable excipients such as inert diluents, disintegrants, binders, lubricants, sweeteners, flavorings, colorants, and preservatives. Suitable inert diluents include sodium carbonate and calcium carbonate, sodium phosphate and calcium phosphate, and lactose, while corn starch and alginate are suitable disintegrants. Binders may include starch and gelatin, while lubricants (if present) are typically magnesium stearate, stearic acid, or talc. If necessary, tablets can be coated with materials such as glyceryl monostearate or glyceryl distearate to delay absorption in the gastrointestinal tract.

[0108] The pharmaceutical compositions of the present invention can be formulated based on the delivery mode. One example is formulation for parenteral delivery, such as subcutaneous, intramuscular, or intravenous delivery for systemic administration. Another example is formulation for intracerebral delivery. The pharmaceutical compositions of the present invention can be administered in amounts sufficient to inhibit the expression of the MAPT gene.

[0109] Dosage

[0110] The MAPT siRNA or siRNA conjugate of the present invention is delivered in a pharmaceutical composition at a dose sufficient to inhibit MAPT gene expression. In some embodiments, the dose of MAPT siRNA or siRNA conjugate is 0.01-200.0 mg per kilogram of recipient body weight per day, for example 1-50 mg / kg body weight, 5-40 mg / kg body weight, 10-30 mg / kg body weight, 1-20 mg / kg body weight, 1-10 mg / kg body weight, or 4-15 mg / kg body weight per day. For example, each single dose of MAPT siRNA or siRNA conjugate may be administered in an amount ranging from about 0.01 mg / kg, 0.05 mg / kg, 0.1 mg / kg, 0.2 mg / kg, 0.3 mg / kg, 0.4 mg / kg, 0.5 mg / kg, 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, 10 mg / kg, 15 mg / kg, 20 mg / kg, 30 mg / kg, 40 mg / kg, 50 mg / kg body weight or any value within the range thereof.

[0111] Several factors can be considered when determining the delivery dose and timing of the MAPT siRNA or siRNA conjugate of the present invention. The absolute amount of MAPT siRNA or siRNA conjugate delivered will depend on a variety of factors, including co-treatment, dose number, and individual subject parameters, including age, physical condition, body size, and weight. These are factors well known to those skilled in the art and can be resolved through routine experiments. In some embodiments, a maximum dose may be used, i.e., the highest safe dose based on reasonable medical judgment.

[0112] In some embodiments, the method of the present invention may include administering to a subject doses of MAPT siRNA or siRNA conjugates of 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, 10 mg / kg, or more. In some cases, a dose of the pharmaceutical compound (e.g., containing MAPT siRNA or siRNA conjugates) may be administered to the subject at least daily, every other day, weekly, every other week, monthly, etc., and may be administered once daily or more than once daily, for example, 2, 3, 4, 5, or more times within a 24-hour cycle. The pharmaceutical composition of the present invention may be administered once daily; or the MAPT siRNA or siRNA conjugate may be administered at appropriate intervals throughout the day in two, three, or more sub-dose doses, or even delivered using continuous infusion or by controlled-release formulations. In some embodiments of the method of the present invention, the pharmaceutical composition of the present invention may be administered to the subject once daily or more, once weekly or more, once monthly or more, or once annually or more.

[0113] In some aspects, the methods of the present invention include administering a pharmaceutical compound alone; in combination with one or more other MAPT siRNAs or siRNA conjugates; and / or in combination with other pharmaceutical therapies or treatment activities or protocols administered to a subject suffering from a neurodegenerative disease. The pharmaceutical compound may be administered in the form of a pharmaceutical composition. The pharmaceutical composition used in the methods of the present invention may be sterile and contain a quantity of MAPT siRNA or siRNA conjugate that reduces the activity of the MAPT peptide to a level sufficient to produce the desired response in a weight or volume unit suitable for administration to the subject. The dose of the pharmaceutical composition containing MAPT siRNA or siRNA conjugate administered to the subject may be selected according to various parameters to reduce MAPT protein activity, particularly depending on the route of administration used and the condition of the subject. Other factors include the required duration of treatment. If the subject's response at the initial dose is insufficient, a higher dose may be used (or the dose may be effectively increased via a different, more localized delivery route) within the limits of patient tolerance.

[0114] Uses and treatments

[0115] The MAPT siRNA, its modifications or conjugates, and pharmaceutical compositions of the present invention can be used to reduce the expression of the MAPT gene in cells or subjects and can be used to treat neurodegenerative diseases. Accordingly, the MAPT siRNA, its modifications or conjugates, and pharmaceutical compositions of the present invention can be used to prepare medicaments for the prevention and / or treatment of neurodegenerative diseases. The present invention also provides a method for the prevention and / or treatment of degenerative diseases, comprising administering a preventively effective amount or a therapeutically effective amount of MAPT siRNA, its modifications or conjugates, and pharmaceutical compositions to a subject in need.

[0116] In this document, "patient" or "subject" is intended to include humans or non-human animals, preferably mammals such as monkeys. More preferably, the subject or patient is a human.

[0117] In this article, "preventive effective dose" refers to the amount of siRNA agent sufficient to prevent or improve the disease or one or more symptoms of the disease when administered to a subject who has not yet experienced or exhibited symptoms of a neurodegenerative disease but may be susceptible to it. Improving the disease includes slowing its progression or reducing the severity of subsequent disease development. This "preventive effective dose" can vary depending on the siRNA agent, how it is administered, the risk level for the disease, and medical history, age, weight, family history, genetic makeup, type of prior or concomitant treatment (if any), and other individual characteristics of the patient to be treated.

[0118] In this article, "therapeutic effective dose" is intended to include the amount of siRNA agent that is sufficient to achieve treatment of a neurodegenerative disease (e.g., by attenuating, improving, or maintaining the existing disease or symptoms of one or more diseases) when administered to a patient. This "therapeutic effective dose" can vary depending on the siRNA agent, how it is administered, the disease and its severity, and medical history, age, weight, family history, genetic makeup, stage of the pathological process mediated by MAPT expression, type of prior or concomitant treatment (if any), and other individual characteristics of the patient to be treated.

[0119] In this document, "preventative effective amount" or "therapeutic effective amount" also includes the amount of siRNA agent that produces a desired local or systemic effect at a reasonable benefit / risk ratio suitable for any treatment. The siRNA agent used in the method of the present invention may be administered in an amount sufficient to produce a reasonable benefit / risk ratio suitable for such treatment.

[0120] It is understood that the amount of MAPT siRNA or siRNA conjugate or pharmaceutical composition administered to the subject can be modified, at least in part, based on the results of such determinations of the subject's disease and / or symptom state and / or physiological characteristics. The therapeutic dose can be changed, for example, by altering the composition of the MAPT siRNA or siRNA conjugate administered, by changing the route of administration, by changing the time of administration, etc., to increase or decrease the amount of MAPT siRNA or siRNA conjugate. The effective amount of MAPT siRNA or siRNA conjugate will vary depending on the specific condition being treated, the age and physical condition of the subject, the severity of the condition, the duration of treatment, the nature of any co-treatments, the specific route of administration, and other factors within the knowledge and expertise of the healthcare practitioner. For example, the effective amount may depend on the amount of MAPT peptide effective for treating neurodegenerative diseases and / or the desired level of MAPT gene expression. Those skilled in the art can determine the effective amount of a particular MAPT siRNA or siRNA conjugate used in the methods of the present invention empirically without excessive experimentation. Based on the teachings provided herein, effective preventative or therapeutic treatment regimens can be devised to effectively treat specific subjects by selecting from the various MAPT siRNAs or siRNA conjugates of the present invention and weighing factors such as potency, relative bioavailability, patient weight, severity of adverse side effects, and preferred route of administration. As used in embodiments of the present invention, the effective amount of the MAPT siRNA or siRNA conjugate of the present invention can be the amount that produces the desired biological effect in the cells upon contact with them.

[0121] In this document, the term "prevention" or "taking preventative measures," when used to refer to a disease, condition, or illness from which reduced MAPT gene expression would benefit, means a reduced likelihood of developing symptoms associated with such disease, condition, or illness, such as those associated with diseases or illnesses associated with excessive MAPT expression, such as neurodegenerative diseases. A reduced likelihood of developing a neurodegenerative disease is considered effective prevention if, for example, an individual has one or more risk factors for a neurodegenerative disease but has not developed the disease or has only developed a less severe neurodegenerative disease, and relative to a population with the same risk factors who have not received the treatments described herein, they have failed to develop the relevant disease, condition, or illness, or the development of symptoms associated with such disease, condition, or illness is reduced (e.g., a reduction of at least about 10% on a scale indicating clinical prevalence of the disease or illness), or the onset of symptoms is delayed (e.g., delayed by days, weeks, months, or years).

[0122] In some aspects of the invention, the MAPT siRNA or siRNA conjugate of the invention may be administered to the subject at one or more times before or after the diagnosis of a neurodegenerative disease. In some aspects of the invention, the subject is at risk of having or developing a neurodegenerative disease. A subject at risk of developing a neurodegenerative disease is a subject with an increased likelihood of developing a neurodegenerative disease compared to a control risk level. In some embodiments of the invention, the risk level is statistically significant compared to a control risk level. Subjects at risk may include, for example, those who are or will be subjects with pre-existing diseases and / or genetic abnormalities that make them more susceptible to neurodegenerative diseases than control subjects without pre-existing diseases or genetic abnormalities; subjects with a family and / or personal history of neurodegenerative diseases; and subjects who have previously received treatment for neurodegenerative diseases. It should be understood that pre-existing diseases and / or genetic abnormalities that make a subject more susceptible to neurodegenerative diseases may be diseases or genetic abnormalities that, when present, have previously been identified as being associated with a higher likelihood of developing a neurodegenerative disease.

[0123] It should be understood that MAPT siRNA or siRNA conjugates can be administered to subjects based on their individual medical conditions. For example, healthcare provided to a subject can assess MAPT levels measured in samples obtained from the subject and determine that reducing the subject's MAPT levels by administering the MAPT siRNA or siRNA conjugate of the present invention is desirable. In a non-limiting example, a biological sample, such as a blood or serum sample, can be obtained from the subject, and the subject's MAPT levels can be determined in the sample. MAPT siRNA or siRNA conjugates are administered to the subject, and a blood or serum sample is obtained from the subject after administration, and the MAPT level is determined using this sample. This result is compared to the result determined in a sample prior to administration. A decrease in the subject's MAPT level in the subsequent sample compared to the pre-administration level indicates the efficacy of the administered MAPT siRNA or siRNA conjugate in reducing the subject's MAPT levels.

[0124] Therefore, some embodiments of the present invention include assessing changes in one or more physiological characteristics induced by prior treatment of the subject to adjust the amount of MAPT siRNA or siRNA conjugate of the present invention subsequently administered to the subject. Some embodiments of the method of the present invention include 1, 2, 3, 4, 5, 6 or more measurements of physiological characteristics of neurodegenerative diseases; assessing and / or monitoring the efficacy of the administered MAPT siRNA or siRNA conjugate of the present invention; and optionally using the results of the measurements to adjust one or more of the following: the dose, dosing regimen, and / or dosing frequency of the MAPT siRNA or siRNA conjugate of the present invention in the treatment of the neurodegenerative disease in the subject. In some embodiments of the method of the present invention, the desired result of administering an effective amount of the MAPT siRNA or siRNA conjugate of the present invention to the subject is a decrease in MAPT levels and a reduction in the manifestation of neurodegenerative diseases.

[0125] As used herein, the terms “treatment,” “therapeutic,” or “therapeutic” when used in relation to neurodegenerative diseases can refer to preventative treatment, reducing the likelihood of a subject developing a neurodegenerative disease, and can also refer to treatment undertaken after a subject has developed a neurodegenerative disease in order to eliminate or reduce the level of the neurodegenerative disease, prevent the neurodegenerative disease from becoming more severe, and / or slow the progression of the neurodegenerative disease in a subject compared to a subject in the absence of a therapy that reduces MAPT peptide activity in the subject.

[0126] Application method

[0127] The administration routes of the MAPT siRNA or siRNA conjugate or pharmaceutical composition of the present invention can employ any route of administration known in the art. The choice of a particular route of administration will depend at least in part on the specific condition being treated and the dose required for therapeutic efficacy. In some embodiments, the MAPT siRNA or siRNA conjugate or pharmaceutical composition can be administered orally, enterically, mucosally, subcutaneously, intracerebrally, and / or parenterally. The term "parentereal" includes subcutaneous, intravenous, intrathecal, intramuscular, intraperitoneal, and intrasternal injection or infusion techniques. Other routes include, but are not limited to, nasal (e.g., via a gastrointestinal tube), percutaneously, vaginally, rectally, sublingually, and inhalation. Routes of administration of the present invention may include intrathecal, intracardiac, or intracranial. In some embodiments, the MAPT siRNA or siRNA conjugate may be placed in a sustained-release matrix and administered by placing the matrix into the subject.

[0128] The MAPT siRNA or siRNA conjugate or pharmaceutical composition of the present invention can be administered in formulation form or in a pharmaceutically acceptable solution, which typically contains pharmaceutically acceptable concentrations of salts, buffers, preservatives, compatible carriers, adjuvants, and optionally other therapeutic ingredients. In some embodiments, the MAPT siRNA or siRNA conjugate or pharmaceutical composition can be formulated with another therapeutic agent for simultaneous administration.

[0129] medicine box

[0130] The present invention provides a medicine box comprising a suitable container for a pharmaceutical formulation containing at least one of the MAPT siRNA, siRNA conjugates, and pharmaceutical compositions of the present invention.

[0131] The kit of the present invention comprises one or more dsRNAs and instructions for use, such as instructions for administering a preventative or therapeutically effective amount of dsRNA. The dsRNA may be in a vial or a pre-filled syringe. The kit may optionally further include a device for administering the dsRNA (e.g., an injection device, such as a pre-filled syringe) or a device for measuring MAPT inhibition (e.g., a device for measuring inhibition of MAPT mRNA, MAPT protein, and / or MAPT activity). Such a device for measuring MAPT inhibition may include a device for obtaining a sample (e.g., a plasma sample) from a subject. The kit of the present invention may optionally further include a device for determining a therapeutically or preventatively effective amount.

[0132] In some embodiments, the individual components of the drug formulation may be provided in a single container, such as a vial or a pre-filled syringe. In some embodiments, the components of the drug formulation may be provided separately in two or more containers; for example, one container may be for dsRNA, and at least another for a carrier compound. The kit may be packaged in many different configurations, such as one or more containers in a single box. Different components may be combined, for example, according to the instructions provided with the kit. In some embodiments, the kit may also include a delivery device.

[0133] Example 1: siRNA Synthesis

[0134] 1.1 siRNA Design

[0135] A set of siRNAs targeting the human MAPT gene (human MAPT gene: NCBI ref seq ID NM_001377265.1; NCBI Gene ID: 4137) was designed online using oligowalk. The human NM_001377265.1 REF SEQ mRNA has a length of 6815 bases. Sequences similar to the human gene were excluded to avoid any toxicity.

[0136] Human NM_001377265.1 REF SEQ mRNA (NCBI refseq ID NM_001377265.1):

[0137] 1.2 siRNA sequence synthesis

[0138] siRNA was synthesized according to a standard oligonucleotide solid-phase synthesis protocol, including a negative control siRNA (siCtrl).

[0139] Oligonucleotide solid-phase synthesis protocol: Commercially available 5'-DMT-2'-TBDMS-rU phosphoramide monomers, 5'-DMT-2'-TBDMS-rA(Bz) phosphoramide monomers, 5'-DMT-2'-TBDMS-rC(Ac) phosphoramide monomers, and 5'-DMT-2'-TBDMS-rG(iBu) phosphoramide monomers were used. RNA was synthesized on a 500 nmol scale. A phosphoramide solution was prepared at a concentration of 50 mM, and 0.3 M benzylthiotetrazole (BTT) acetonitrile solution was used as an activator. During synthesis, a 0.1 M oxidizing agent (pyridine:THF:water = 20:78:2) was used to convert trivalent phosphorus to pentavalent phosphorus to stabilize the phosphate backbone. After synthesis, the sequence was ammonolyzed from the solid support and precipitated. The 2'-2'-O-tert-butyldimethylsilyl protecting group was removed with triethylamine trihydrofluoric acid.

[0140] For the synthesized RNA sequence, ammonolysis was performed at 55°C for 40 minutes using an ammonia:methylamine ratio of 1:1. After ammonolysis, the solid support was removed, and the supernatant was dried. A protecting group removal agent of triethylamine trihydrofluoric acid:triethylamine:NMP = 6:4:3 was added, and the reaction was carried out at 60°C for 2 hours. Then, n-butanol was added at a ratio of 1:5, and the mixture was allowed to stand at -20°C for 30 minutes. The precipitate was collected by centrifugation. The precipitate was dissolved in RNase-free water and purified by reversed-phase chromatography (0.1M triethylamineacetic acid (TEAA) and acetonitrile). The purified sample was desalted by ultrafiltration with PBS and then annealed to obtain siRNA. The obtained siRNA was verified, and the results showed that the target siRNA was successfully prepared.

[0141] 1.3 siRNA sequence modification and conjugate synthesis

[0142] Modified siRNAs are synthesized according to oligonucleotide solid-phase synthesis schemes. The modified nucleotide groups can be introduced into the siRNAs disclosed herein using nucleoside monomers with corresponding modifications. Methods for preparing nucleoside monomers with corresponding modifications are well known to those skilled in the art. L96 is conjugated to siRNA to synthesize siRNA conjugates, referring to the synthesis methods disclosed in WO2014025805A1 or WO2017015109A1. The synthetic method disclosed in Osborn MF, Khvorova A. Improving siRNA Delivery In Vivo Through Lipid Conjugation. Nucleic Acid Ther. 2018 Jun; 28(3):128-136. doi:10.1089 / nat.2018.0725.Epub 2018 May 10.PMID:29746209; PMCID:PMC5994667 involves conjugating C16 (hexadecyl) to siRNA to synthesize siRNA conjugates. The conjugating group L96 is shown in formula (I), and the nucleoside structure conjugated by C16 is shown in formula (II), with C16 attached to the 2'-O position of the nucleoside or nucleotide.

[0143] The structure of the conjugation group L96 is shown below:

[0144] The structure of the nucleoside conjugated with the C16 conjugation group is shown below:

[0145] Annealing of oligonucleotides to produce siRNA conjugates: The RNA oligomers to be annealed were prepared into a 200 μM solution using sterile RNase-free H2O (RNA hydrolase-free). The annealing reaction system was set up as follows: 100 μL of the above solution (10 nmol doublet concentration) was placed in a 95°C water bath for 10 minutes (≥100 nmol requires 20 minutes at high temperature) → immediately cooled in a 60°C water bath → the annealed solution was stored at 4°C. Equimolar amounts of RNA solution were combined to mix complementary strands. The siRNA molecule was confirmed to be correctly constructed. The siRNA solution was prepared into a dry powder for later use.

[0146] The synthesized siRNA molecule sequences are shown in Table 1 below, and the binding sites with the human MAPT gene are shown in Figure 1:

[0147] Table 1. siRNAs targeting MAPT

[0148] The sequences of the synthesized siRNA conjugates are shown in Table 2 below:

[0149] Table 2. siRNA conjugates targeting MAPT

[0150] Table 3. siRNA conjugates targeting MAPT

[0151] Table 4. siRNA conjugates targeting MAPT

[0152] In this context, lowercase letter m indicates that the nucleotide adjacent to the left of letter m is methoxy-modified; lowercase letter f indicates that the nucleotide adjacent to the left of letter f is fluorinated; lowercase letter s indicates that the two nucleotides adjacent to the left and right of letter s are linked by thiophosphate subunits; VP indicates that the nucleotide adjacent to the right of the letter combination VP is 5'-(E)-vinylphosphonate (E-VP) modified; GNA indicates that the nucleotide adjacent to the left of the letter combination (GNA) is GNA modified; L96 indicates the L96 conjugate group linked to siRNA; hd indicates the C16 conjugate group linked to siRNA; and (Nhd) indicates that the nucleotide N in siRNA is linked to C16, specifically, the 2'-O in the nucleotide N is linked to C16.

[0153] Example 2. In vitro activity screening of siRNA HCT116 cell line

[0154] 2.1 Experimental Procedure

[0155] 2.1.1 Cell Culture

[0156] HCT116 cells (BNCC, BNCC287750) were cultured in DMEM complete medium (Eallbio, with 10% FBS added) at 37°C and 5% CO2. When the confluence reached 80%-90%, the cells were digested with trypsin, counted, and transfected.

[0157] 2.1.2 Preparation of siRNA dilution buffer

[0158] (1) The dry powder of the siRNA to be tested was centrifuged at low temperature and high speed, and then dissolved in ultrapure distilled water to prepare a 100 μM siRNA stock solution.

[0159] (2) Prepare 200 nM siRNA dilution solution Y.

[0160] a) Take 50 μl of the 100 μM siRNA stock solution obtained in step (1) above, add 50 μl of ultrapure distilled water to obtain a siRNA dilution solution with a final concentration of 50 μM.

[0161] b) Take 2 μl of the 50 μM siRNA dilution solution obtained in step a) and add 18 μl of ultrapure distilled water to obtain siRNA stock solution X with a final concentration of 5 μM.

[0162] c) Take 2 μl of the prepared siRNA stock solution X and add 48 μl of Opti-medium (Gibco, 31985070) to obtain 200 nM siRNA dilution solution Y.

[0163] 2.1.3 HCT116 cell transfection

[0164] Pick 0.6 μl of transfection reagent was added to 10 μl of Opti-medium to obtain... Transfection reagent dilution solution; The transfection reagent diluent and the 200nM siRNA diluent Y prepared in step 2.1.2 were mixed at a volume ratio of 1:1 to prepare a transfection mixture. After standing for 5 minutes, 10 μl of the transfection mixture was added to a 96-well plate, along with 90 μl of HCT116 cells cultured in step 2.1.1 (final volume 100 μl / well, cell number 20,000 / well; taking siRNA diluent Y as 200nM as an example, the concentration of siRNA in this system is 10nM). The plate was then cultured for 24 hours after transfection.

[0165] 2.1.4 RNA Extraction

[0166] Total RNA was extracted from HCT116 cells obtained in step 2.1.3 according to the FlysisAmp Cells-to-CT 1-Step SYBR Green Kit product instructions.

[0167] 2.1.5 Quantitative Real-Time PCR

[0168] The extracted total RNA was analyzed by reverse transcription and real-time PCR using the FlysisAmp Cells-to-CT 1-Step SYBR Green Kit.

[0169] 2.1.6 Results Analysis

[0170] (1) Use the software of the 7500 real-time fluorescence quantitative PCR instrument (Thermo Fisher) to automatically calculate the Ct value;

[0171] (2) Calculate the relative expression level of the gene using the following formula:

[0172] ΔCt1=Ct(MAPT group)–Ct(MAPT group's ACTIN)

[0173] ΔCt2=Ct(siCtrl group)–Ct(siCtrl group's ACTIN)

[0174] ΔCt = ΔCt1 (MAPT group) - ΔCt2 (siCtrl group), where the siCtrl group is the negative control group;

[0175] mRNA expression relative to the siCtrl group = 2 -ΔΔCt

[0176] Inhibition rate (%) = (1 - mRNA expression relative to siCtrl group) × 100%.

[0177] 2.2 Experimental Results

[0178] The inhibitory effects of the siRNA of this invention are shown in Table 5 below:

[0179] Table 5. Results of in vitro screening of HCT116 cell lines using siRNA

[0180] As can be seen from Table 5, some of the siRNAs of the present invention can significantly inhibit the expression of the MAPT gene in HCT116 cells at 10 nM.

[0181] Example 3. siRNA IC50 assay

[0182] This embodiment examines the dose-response relationship between drug dosage and biological effect by calculating the half-maximal inhibitory concentration (IC50) of each siRNA, thereby quantitatively reflecting the ability of the drug to cause changes in this indicator.

[0183] 3.1 The IC50 of siRNA inhibiting MAPT gene expression was determined using a method similar to that in Example 2, wherein the concentrations of transfected siRNA were 25 nM, 5 nM, 1 nM, 0.2 nM, 0.04 nM, 0.008 nM, and 0.0016 nM, respectively.

[0184] 3.2 Results Analysis

[0185] (1) Use the software of the 7500 real-time fluorescence quantitative PCR instrument (Thermo Fisher) to automatically calculate the Ct value;

[0186] (2) Calculate the relative expression level of the gene using the following formula:

[0187] ΔCt1=Ct(MAPT group)–Ct(MAPT group's ACTIN)

[0188] ΔCt2=Ct(siCtrl group)–Ct(siCtrl group's ACTIN)

[0189] ΔCt = ΔCt1 (MAPT group) - ΔCt2 (siCtrl group), where the siCtrl group is the negative control group;

[0190] mRNA expression relative to the siCtrl group = 2 -ΔΔCt

[0191] Inhibition rate (%) = (1 - mRNA expression relative to siCtrl group) × 100%.

[0192] Using the log value of siRNA concentration as the X-axis and the percentage inhibition rate as the Y-axis, analysis software was used to fit the dose-response curve to obtain the IC50 value of each siRNA.

[0193] The fitting formula is: Y = 100 / (1 + 10^((LogIC50 - X) × HillSlope))

[0194] Where HillSlope represents the slope of the percentage inhibition rate curve.

[0195] 3.3 Experimental Results

[0196] The IC50 assay results of the siRNA of this invention are shown in Table 6 below:

[0197] Table 6. IC50 assay results of siRNA in HCT116 cell line

[0198] As can be seen from Table 6, some of the siRNAs of the present invention can significantly inhibit MAPT gene expression, with an IC50 as low as 26.26 pM.

[0199] Based on the in vitro screening results of Examples 2 and 3, the highly efficient activity regions for inhibiting MAPT mRNA were determined to be positions 182-204, 2191-2371, and 2750-2772, calculated according to NCBI refseqID NM_001377265.1.

[0200] Example 4. In vivo activity screening of siRNA conjugates

[0201] Based on the in vitro screening results of Examples 2 and 3 above, some siRNA sequences with good in vitro activity were selected, and their conjugates were used to verify their in vivo activity.

[0202] 4.1 Experimental Procedure

[0203] Six- to eight-week-old male mice (C57BL / 6) were purchased from Spiford (Beijing) Biotechnology Co., Ltd. Each mouse was intravenously injected with 1×10⁻⁶ ppm. 11 A recombinant adeno-associated virus 8 (AAV8) vector with one genome copy was packaged from an AAV8 capsid protein expression plasmid and a transfer plasmid carrying an AAV2 ITR. The transfer plasmid carried the human MAPT sequence (NM_001377265.1) from positions 319 to 1758, controlled by the human thyroxine-binding globulin promoter (AAV8-TBG-hMAPT). The AAV8-hMAPT transgenic mouse model was established 14 days after injection. Mice were then subcutaneously administered the conjugate at a dose of 3 mg / kg per mouse. Mice were sacrificed on the day of administration, day 7 (D7), and day 14 (D14), with six mice in each group. Liver tissue was collected for mRNA expression detection. Total RNA was extracted using the Trizol method. mRNA was reverse transcribed using the HiScript III RT SuperMix for qPCR (+gDNAwiper) kit (catalog number: R323-01, Vazyme). Real-time quantitative PCR was performed using the ChamQ Universal SYBR qPCR Master Mix kit (catalog number: Q711-03, Vazyme). In vivo activity screening results are shown in Table 7 and Figures 2 and 3.

[0204] Table 7. In vivo screening results of siRNA conjugates

[0205] 4.2 Experimental Results

[0206] The chemically modified siRNA sequences showed good activity in mice. YGND21-3M, YGND21-41M, YGND21-42M, YGND21-53M, YGND21-56M, and YGND21-75M all significantly reduced MAPT expression levels in mice. Among them, YGND21-53M had an inhibition rate as high as 71.03% and YGND21-56M had an inhibition rate as high as 67.6% on day 14.

[0207] Example 5. Brain activity results of siRNA conjugates

[0208] 5.1 Experimental Procedure

[0209] Six- to eight-week-old male mice (C57BL / 6) were purchased from Spiford (Beijing) Biotechnology Co., Ltd., weighing approximately 20g. Each mouse was intravenously injected with 1×10 11 A recombinant adeno-associated virus (AAV-PHP.eB) vector (purchased from Yunzhou Biotechnology (Guangzhou) Co., Ltd.) with 1 copy of the genome was used. The recombinant AAV-PHP.eB vector plasmid carried the human MAPT sequence (NM_001377265.1). The transgenic mouse model was established 14 days after injection. The mice were then divided into 8 treatment groups and 1 control group, with 3 mice in each group. On day 0, 120 μg of siRNA conjugate was injected intracerebroventricularly (ICV) into each mouse. On day 14, central nervous system tissue and spinal cord tissue, including the cerebral cortex, hippocampus, and thoracic spinal cord, were collected, flash-frozen in liquid nitrogen, and mRNA was detected by RT-qPCR. The brain activity results of different siRNA conjugates are shown in Figure 4.

[0210] 5.2 Explanation of Experimental Results

[0211] The siRNA conjugates exhibited good activity in the mouse brain. YGND21-53M', YGND21-75M', YGND21-107M', and YGND21-135M' all significantly reduced MAPT expression levels in the mouse brain. On day 14, YGND21-135M' showed the highest inhibitory efficiency, with inhibition rates as high as 87.76% in the cerebral cortex, 93.38% in the hippocampus, and 88.99% in the thoracic spinal cord.

[0212] Example 6. Results of brain activity of siRNA conjugates

[0213] 6.1 Experimental Procedure

[0214] Four-week-old male FAD3T transgenic mice, a model of Alzheimer's disease, were purchased from Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd. They were divided into four treatment groups and one control group, with three mice in each group. On day 0, each mouse was injected with 120 μg of siRNA conjugate via ICV. On day 14, central nervous system tissues, including the cerebral cortex and hippocampus, were collected, flash-frozen in liquid nitrogen, and mRNA was extracted and detected by RT-qPCR. The brain activity results of different siRNA conjugates are shown in Figure 5.

[0215] 6.2 Explanation of Experimental Results

[0216] The siRNA conjugates showed good activity in the mouse brain. YGND21-107M', YGND21-41M', YGND21-107M'G, and YGND21-135M'G all significantly reduced MAPT expression levels in the mouse brain. YGND21-107M' showed the highest inhibitory efficiency on day 14.

[0217] Example 7. Cytotoxicity Results of siRNA Conjugates

[0218] 7.1 Experimental Procedure

[0219] Following experimental step 3.1 in Example 3, BE(2)-C cell culture and transfection were performed, with siRNA concentrations of 5 nM and 50 nM in the system. After 72 hours of culture, the cytotoxicity of each siRNA conjugate was measured by determining the cell viability / cytotoxicity ratio in each sample. Cell viability was measured by determining intracellular ATP content using a CellTiter-Glo (Promega, catalog number G7570) according to the manufacturer's protocol. Cytotoxicity in the supernatant was measured using a ToxiLight™ (Lonza, catalog number LT07-217) according to the manufacturer's protocol.

[0220] 7.2 Experimental Results

[0221] The cytotoxicity results of the siRNA conjugates are shown in Figure 6.

[0222] The results showed that some of the siRNA conjugates disclosed in this paper have low cytotoxicity and good cell compatibility.

[0223] Example 8. Off-target analysis of siRNA conjugates

[0224] 8.1 Experimental Procedure

[0225] Following experimental step 2.1 in Example 2, BE(2)-C cell culture and transfection were completed. After 24 hours of culture, total RNA was extracted from BE(2)-C cells according to the instructions of the FlysisAmp Cells-to-CT 1-Step SYBR Green Kit. The RNA was sent to a third-party company for transcriptome analysis to identify differentially expressed genes. In the differentially expressed gene analysis, genes with padj≤0.05 and |log2FoldChange|>0.5145 were defined as differentially expressed genes. Here, padj≤0.05 indicates the significance level after multiple validation correction, ensuring that the identification results of differentially expressed genes have a confidence level of more than 95%; while |log2FoldChange|>0.5145 corresponds to a gene expression level that is at least upregulated by 1.43-fold (2^0.5145) or downregulated to 70% of the control group (2^-0.5145), thereby excluding minor fluctuations and focusing on genes with significant changes in expression levels. The specific results are shown in Table 8.

[0226] 8.2 Experimental Results

[0227] Transcriptome results showed that some of the siRNA conjugates disclosed herein caused less intracellular gene variation, exhibited high safety, and could effectively prevent off-target effects.

[0228] Table 8. Transcriptome results of siRNA conjugates

Claims

1. An siRNA for inhibiting the expression of a MAPT gene, the siRNA comprising a sense strand and an antisense strand, the sense strand comprising a nucleotide sequence I, and the antisense strand comprising a nucleotide sequence II; each nucleotide in the nucleotide sequence I and the nucleotide sequence II is a modified or unmodified nucleotide; the nucleotide sequence I and the nucleotide sequence II are at least partially reverse-complementary to form a double-stranded region; the nucleotide sequence I is substantially identical to a first nucleotide sequence of at least 15 nucleotides in length in a mRNA expressed by the MAPT gene, preferably the first nucleotide sequence is a nucleotide sequence of 15-25 nucleotides in length in the mRNA expressed by the MAPT gene, such as 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides; Preferably, the first nucleotide sequence is a nucleotide sequence of at least 15 nucleotides in length in a highly active region of the mRNA expressed by the MAPT gene, preferably a nucleotide sequence of 15-25 nucleotides in length, the highly active region is position 182-204, 2191-2371, and 2750-2772, preferably position 182-204, 2191-2205, 2283-2305, 2284-2306, 2341-2363, 2349-2371, and 2750-2772, of the mRNA expressed by the MAPT gene; the mRNA expressed by the MAPT gene is as shown in NCBI ref seq ID NM_001377265.1; in particular, the sequence of the mRNA expressed by the MAPT gene is as shown in SEQ ID NO:

1.

2. The siRNA of claim 1, wherein, The nucleotide sequence I has at least 70%, at least 80%, at least 85%, at least 90%, at least 95% sequence identity to the first nucleotide sequence.

3. The siRNA of claim 1 or 2, wherein, The nucleotide sequence I comprises at least 15 contiguous nucleotides, such as at least 15, 16, 17, 18, 19, 20, or 21 nucleotides, of any one of SEQ ID NOs: 2-137; or the nucleic acid sequence of the sense strand is as shown in the nucleotide sequence I, which differs from any one of SEQ ID NOs: 2-137 by 1, 2, or 3 nucleotides; preferably, the nucleotide sequence I is as shown in SEQ ID NOs: 2-137; The nucleotide sequence II comprises at least 15 contiguous nucleotides, such as at least 15, 16, 17, 18, 19, 20, 21, 22, or 23 nucleotides, of any one of SEQ ID NOs: 138-273; or the nucleic acid sequence of the antisense strand is as shown in the nucleotide sequence II, which differs from any one of SEQ ID NOs: 138-273 by 1, 2, or 3 nucleotides; preferably, the nucleotide sequence II is as shown in SEQ ID NOs: 138-273.

4. The siRNA according to any one of claims 1 to 3, wherein the siRNA is selected from the siRNA molecules shown in Table 1.

5. The siRNA according to any one of claims 1 to 4, wherein, Each of the nucleotides in the nucleotide sequence I and the nucleotide sequence II is a modified nucleotide, which is a fluorine-modified nucleotide or a non-fluorine-modified nucleotide; preferably, the fluorine-modified nucleotide refers to a nucleotide in which the hydroxyl group at the 2'-position of the ribosyl group of the nucleotide is replaced by fluorine, which has the structure shown in the following formula (1); the non-fluorine-modified nucleotide refers to a nucleotide or a nucleotide analogue in which the hydroxyl group at the 2'-position of the ribosyl group of the nucleotide is replaced by a non-fluorine group, which has the structure shown in any one of the following formulae (2) to (12): In formula (1) to formula (12), Base represents a base. In formula (9) and formula (10), R is selected from H, OH or alkoxy (O-alkyl). In formula (11) and formula (12), R is selected from H, OH, F or the non-fluorine group.

6. The siRNA of claim 5, wherein, one or more of the nucleotides at positions 7, 9, 10, 11 of the nucleotide sequence I are fluorine-modified nucleotides in the direction from the 5'-end to the 3'-end; and one or more of the nucleotides at positions 2, 6, 8, 9, 14, 16 of the nucleotide sequence II are fluorine-modified nucleotides in the direction from the 5'-end to the 3'-end; or one or more of the nucleotides at positions 7, 9, 10, 11 of the nucleotide sequence I are fluorine-modified nucleotides in the direction from the 5'-end to the 3'-end; and one or more of the nucleotides at positions 2, 6, 8, 9, 14, 16 of the nucleotide sequence II are fluorine-modified nucleotides in the direction from the 5'-end to the 3'-end; or 7. The siRNA of claim 5 or 6, wherein, at least one of the phosphates in the phospho-sugar backbone of at least one of the sense strand and the antisense strand of the siRNA is a phosphate having a modification group, the phosphate having a modification group being a phosphorothioate group in which at least one of the oxygen atoms in the phosphodiester bond of the phosphate is replaced by a sulfur atom; preferably, the phosphate having a modification group is a phosphorothioate group having a structure as shown in formula (13): one or more of the nucleotides at positions 7, 9, 10, 11 of the nucleotide sequence I are fluorine-modified nucleotides in the direction from the 5'-end to the 3'-end; and one or more of the nucleotides at positions 2, 6, 8, 9, 14, 16 of the nucleotide sequence II are fluorine-modified nucleotides in the direction from the 5'-end to the 3'-end; or More preferably, the 5'-terminal nucleotide of the siRNA antisense strand is a 5'-phosphate nucleotide or a 5'-phosphate analog modified nucleotide, such as shown in Formula (14), Formula (15), and Formula (16): In formula (14) to formula (16), Base represents a base.

8. An siRNA conjugate comprising the siRNA of any one of claims 1 to 7 and a conjugate group conjugated to the siRNA, wherein, The conjugate group is L96, which has the structure shown in formula (I) below; or the conjugate group is C16, and the nucleoside conjugated with C16 has the structure shown in formula (II) below: Preferably, the siRNA conjugate is selected from the siRNA conjugates shown in Table 2, Table 3 or Table 4.

9. A composition comprising the siRNA according to any one of claims 1 to 7 or the siRNA conjugate according to claim 8; preferably, the composition is a pharmaceutical composition further comprising a pharmaceutically acceptable carrier or excipient; further preferably, the carrier or excipient comprises, but is not limited to, water for injection, sodium hydroxide, sodium phosphate monobasic monohydrate, sodium phosphate monobasic dihydrate, phosphoric acid, sodium chloride, potassium chloride, hydrochloric acid, anhydrous potassium phosphate monobasic, anhydrous sodium phosphate dibasic, PEG2000, PEG6000, cholesterol, distearoylphosphatidylcholine, 1,2-dimyristylglycerol, dimethyl adipate.

10. Use of the siRNA according to any one of claims 1 to 7 or the siRNA conjugate according to claim 8 or the pharmaceutical composition according to claim 9 in the manufacture of a medicament for preventing and / or treating a neurodegenerative disease. Preferably, the neurodegenerative disease comprises Alzheimer's disease (AD), frontotemporal dementia (FTD), autism, epilepsy, progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), Pick's disease, primary age-related tauopathy (PART).

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