An rna i agent, composition and use for inhibiting expression of transthyretin (ttr)

Abstract

An RNAi agent and composition that inhibits expression of transthyretin (TTR) for use in the treatment and / or prevention of ATTR-associated diseases, including ATTR-PN (transthyretin amyloidosis polyneuropathy), ATTR-CM (transthyretin amyloidosis cardiomyopathy), senile systemic amyloidosis disease, leptomeningeal / central nervous system amyloidosis disease.

Description

RNAi reagent, composition and application for inhibiting thyroxine transporter (TTR) expression Technical Field

[0001] The present invention belongs to the field of biomedicine and relates to an RNAi reagent, composition and application for inhibiting the expression of thyroxine transporter (TTR), such as application in treating and preventing thyroxine transporter amyloidosis. Background Art

[0002] According to the central dogma, proteins are translated and synthesized using messenger RNA (mRNA) as a template. Scientists Andrew Fire and Craig Mello elucidated the RNA interference (RNAi) mechanism of Caenorhabditis elegans and were awarded the 2006 Nobel Prize in Physiology or Medicine for this groundbreaking discovery. RNAi interference can be triggered by exogenous double-stranded RNA (dsRNA) molecules. The dsRNA here is about 20-25bp and is called small or short interfering RNA (siRNA). siRNA selectively blocks or cuts complementary mRNA sequences to induce post-transcriptional gene silencing. Currently, Alnylam Pharmaceuticals has five siRNA drugs approved for marketing by the FDA and others, namely Patisiran, Givosiran, Lumasiran, Inclisiran, and Vutrisiran.

[0003] Transthyretin (TTR) (also known as prealbumin) is a serum protein primarily expressed in the liver, with other significant expression sites including the choroid plexus, retina (specifically, retinal pigment epithelial cells), and pancreas. It participates in the transport of thyroid hormone (T4) and retinol-binding protein (RBP). Because RBP binds to retinol (vitamin A), TTR also functions as a retinol transporter. TTR is a tetramer composed of four identical subunits (monomers), each containing 127 amino acids and rich in β-sheet structure. The potential for amyloidosis in transthyretin may be related to its extensive β-sheet structure; X-ray crystallographic studies have shown that mutations in certain amino acids can unbalance the tetrameric structure of the protein. Transthyretin tetramers dissociate into monomers, which misfold into amyloid and are deposited in tissues, organs, and extracellularly, ultimately leading to amyloidosis.

[0004] Amyloidosis is a general term for a group of amyloid diseases characterized by deposits of the protein amyloid. Amyloid diseases are classified based on their precursor protein. The name "amyloid" begins with "A" followed by the abbreviation of the precursor protein; for example, transthyretin amyloidosis is abbreviated as ATTR. ATTR is divided into two types: senile, wild-type transthyretin amyloidosis (ATTR wt), in which there is no mutation in the transthyretin gene sequence; and familial, mutant transthyretin amyloidosis (ATTR m), in which there is a pathogenic mutation in the transthyretin gene sequence. ATTR commonly causes lesions in the heart, peripheral nerves, autonomic nerves, eyes, and meninges. Renal lesions are uncommon, and the two diseases often coexist. Transthyretin (TTR) amyloidosis deposits in the myocardial interstitium, eventually leading to progressive heart failure (transthyretin amyloid cardiomyopathy, ATTR-CM). Transthyretin (TTR) amyloidosis deposits in peripheral nerves, primarily causing peripheral nerve damage (transthyretin amyloid polyneuropathy, ATTR-PN). ATTR wt is associated with cardiac amyloidosis in the elderly, also known as senile cardiac amyloidosis (SCA). ATTR wt is associated with systemic multi-organ amyloidosis in the elderly, also known as senile systemic amyloidosis (SSA). When ATTR m is associated with ATTR-PN, it is also called familial amyloidotic polyneuropathy (FAP); when ATTR m is associated with ATTR-CM, it is also called familial amyloidotic cardiomyopathy (FAC).

[0005] Current treatments for ATTR (transthyretin amyloidosis) primarily include thyroxine transporter stabilizers and gene silencing drugs. For example, diflunisal is a nonsteroidal anti-inflammatory drug approved in China only for the treatment of arthritis and pain. Currently, only clofazolin has been approved by the National Medical Products Administration for the treatment of ATTR-PN and ATTR-CM, and is included in medical insurance. Clofazolin stabilizes the tetrameric structure of transthyretin, inhibiting its dissociation into unstable monomers and reducing amyloid formation. Small interfering RNA (siRNA) drugs all work by inhibiting the expression of transthyretin messenger RNA (mRNA), reducing transthyretin production and ultimately reducing or eliminating the deposition of pathogenic transthyretin amyloid in the body. For example, International Patent Application Publication No. WO2013075035A1 by R.G. Caranzotasir et al., which is incorporated herein by reference in its entirety, is a patent family for Revusiran and Vutrisiran. Certain other TTR-specific RNA interference (RNAi) agents have been shown to inhibit TTR gene expression. The TTR RNAi agents disclosed herein have not been previously disclosed or known, and provide highly efficient inhibition of TTR gene expression.

[0006] Summary of the Invention

[0007] The present invention provides a novel TTR RNA interference (RNAi) reagent and composition that can selectively and effectively inhibit the expression of the TTR gene, which can be used to treat and / or prevent transthyretin amyloidosis.

[0008] In general, the invention features TTR gene-specific RNAi agents and compositions comprising the same, as well as methods of using the TTR RNAi agents and compositions comprising the same to inhibit TTR gene expression in vitro and / or in vivo. The TTR RNAi agents disclosed herein can selectively and effectively reduce or inhibit TTR gene expression, thereby reducing TTR protein levels in a subject (e.g., a human or animal subject).

[0009] The TTR RNAi agents can be used in methods for treating and / or preventing symptoms and diseases associated with ATTR, including, but not limited to, ATTR-PN (transthyretin amyloidosis polyneuropathy), ATTR-CM (transthyretin amyloidosis cardiomyopathy), senile systemic amyloidosis, and leptomeningeal / CNS (central nervous system) amyloidosis. The methods disclosed herein comprise administering one or more TTR RNAi agents to a subject (e.g., a human or animal subject) using methods known in the art, such as subcutaneous injection or intravenous administration.

[0010] The TTR RNAi reagent provided by the present invention comprises a sense strand (also known as a passenger strand or sense strand) and an antisense strand (also known as a guide strand), and the sense strand and the antisense strand may be partially, substantially, or completely reverse complementary to each other. The sense strand and the antisense strand of the RNAi reagent of the present invention may each be 16 to 30 nucleotides in length.

[0011] In some embodiments, the double-stranded RNAi agent for inhibiting the expression of the thyroxine transporter (TTR) gene provided by the present invention comprises a sense strand and an antisense strand that can form a double-stranded region, wherein the antisense strand is complementary to at least a portion of the mRNA encoding the thyroxine transporter (TTR). In some embodiments, the antisense strand comprises a sequence motif that is complementary to a portion of the mRNA sequence of the TTR gene. In some embodiments, the complementarity is partial, substantially, or completely reverse complementarity. In some embodiments, the sequence motif is complementary to the 118-140 fragment, the 411-433 fragment, the 542-566 fragment, and / or the 581-603 fragment of the TTR mRNA sequence as shown in SEQ ID NO: 884. In some preferred embodiments, the motif comprises at least 15, 16, 17, 18, 19, 20, 21, 22 or 23 consecutive nucleotides that differ from any of the following sequences by no more than 3, 2 or 1 nucleotides: SEQ ID NO: 90, 340, 424, 432, 444, 914 and 915.

[0012] In some more specific embodiments, the antisense strand comprises at least 15, 16, 17, 18, 19, 20, 21, 22, or 23 consecutive nucleotides that differ from any of the antisense strand nucleotide sequences listed in Table 1 by no more than 5, 4, 3, 2, or 1 nucleotides.

[0013] In some more specific embodiments, the sense strand comprises at least 15, 16, 17, 18, 19, 20, or 21 consecutive nucleotides that differ from any one of the sense strand nucleotide sequences in Table 1 by no more than 5, 4, 3, 2, or 1 nucleotides.

[0014] In some more specific embodiments, the antisense strand is complementary to a fragment 118-140, a fragment 411-433, a fragment 542-566, and / or a fragment 581-603 of the TTR mRNA sequence as set forth in SEQ ID NO:884, and / or the sense strand comprises at least 15, 16, 17, 18, 19, 20, or 21 consecutive nucleotides of a fragment 118-140, a fragment 411-433, a fragment 542-566, and / or a fragment 581-603 of the TTR mRNA sequence as set forth in SEQ ID NO:884.

[0015] In some specific embodiments, the double-stranded RNAi agent provided by the present invention is a paired siRNA sense chain and antisense chain combination selected from the group consisting of the sense and antisense chains listed in Table 2. In some preferred embodiments, the double-stranded RNAi agent provided by the present invention is a paired siRNA sense chain and antisense chain combination selected from the group consisting of the sense and antisense chains listed in Table 1.

[0016] In some preferred embodiments of the double-stranded RNAi reagent provided by the present invention, the sense strand / antisense strand respectively comprises SEQ ID NO: 89 / 90, SEQ ID NO: 339 / 340, SEQ ID NO: 417 / 418, SEQ ID NO: 419 / 420, SEQ ID NO: 421 / 422, SEQ ID NO: 423 / 424, SEQ ID NO: 425 / 426, SEQ ID NO: 427 / 428, SEQ ID NO: 429 / 430, SEQ ID NO: 431 / 432, SEQ ID NO: 431 / 914, SEQ ID NO: 431 / 915, SEQ ID NO: 433 / 434, SEQ ID NO: 435 / 436, SEQ ID NO: 437 / 438, SEQ ID NO: 439 / 440, SEQ ID NO: 441 / 442, SEQ ID NO: 443 / 444, SEQ ID NO: NO:445 / 446, SEQ ID NO:447 / 448, SEQ ID NO:449 / 450, SEQ ID NO:451 / 452, SEQ ID NO:453 / 454, SEQ ID NO:455 / 456, SEQ ID NO:457 / 458, SEQ ID NO:459 / 460, SEQ ID NO:461 / 462, SEQ ID NO:463 / 464, SEQ ID NO:465 / 466, SEQ ID NO:467 / 468 or SEQ ID NO:469 / 470, or respectively consist of the said sequences.

[0017] In some more specific embodiments, the antisense strand comprises at least 15, 16, 17, 18, 19, 20, 21, 22, or 23 consecutive nucleotides that differ by no more than 3, 2, or 1 nucleotides from any of the following sequences: SEQ ID NOs: 90, 340, 424, 432, 444, 914, and 915.

[0018] In some more specific embodiments, the sense strand comprises at least 15, 16, 17, 18, 19, 20, or 21 consecutive nucleotides that differ by no more than 3, 2, or 1 nucleotides from any of the following sequences: SEQ ID NOs: 89, 339, 423, 431, and 443.

[0019] In some more preferred embodiments of the double-stranded RNAi reagent provided by the present invention, the sense strand / antisense strand respectively comprises the nucleotide sequence shown in SEQ ID NO:89 / 90, SEQ ID NO:339 / 340, SEQ ID NO:423 / 424, SEQ ID NO:431 / 432, SEQ ID NO:431 / 914, SEQ ID NO:431 / 915, or SEQ ID NO:443 / 444, or respectively consists of the said sequence.

[0020] In yet another aspect, the present invention provides a double-stranded RNAi agent comprising at least one modified nucleotide.

[0021] In some embodiments, the RNAi agent of the present invention comprises a 4'-modified threose nucleic acid. In some embodiments, the 4'-modified threose nucleic acid has a structure of the following formula (A1), Wherein, Base represents a natural or modified nucleoside base, the natural nucleoside base is A, T, C, G or U, and R represents an alkyl group having 1-30 carbon atoms. In some preferred embodiments, the threose nucleic acid is located at the 5' end of the sense strand of the RNAi agent, and R represents an alkyl group having 10-30 carbon atoms, preferably a linear alkyl group having 12 carbon atoms; more preferably, the threose nucleic acid is a 4' modified threose nucleic acid having the following formula (A1'):

[0022] Wherein, Base is a natural nucleoside base A, T, C, G or U.

[0023] In some embodiments, the double-stranded RNAi agent for inhibiting thyroxine transporter (TTR) gene expression provided by the present invention comprises a sense strand and an antisense strand, wherein the sense strand and the antisense strand are reversely complementary by at least 18 nucleotides;

[0024] The sense strand comprises, consists of or essentially consists of the sequence represented by the following general formula (I):

[0025] 5'-n1-n2-n3-n4-n5-n6-n7-n8-n9-n 10 -n 11 -n 12 -n 13 -n 14 -n 15 -n 16 -n 17 -n 18 -n 19 -n 20 -n 21 -3' general formula (I);

[0026] The antisense strand comprises, consists of or essentially consists of the sequence represented by the following general formula (II):

[0027] 5'-N1-N2-N3-N4-N5-N6-N7-N8-N9-N 10 -N 11 -N 12 -N 13 -N 14 -N 15 -N 16 -N 17 -N 18 -N 19 -N 20 -N 21 -N 22 -N 23 -3' general formula (II);

[0028] Among them, n1~n 21 represents 21 consecutive nucleotides contained in the positive chain, N1 to N 23 represents 23 consecutive nucleotides contained in the antisense strand, and each n and N is independently a modified nucleotide, and the modifications include ribose group modification, backbone modification (such as phosphate group modification) and base modification.

[0029] In some embodiments, in the above-mentioned RNAi agent, the modified nucleotides are selected from: 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-O-methoxyethyl modified nucleotides, L-2'-fluoro modified nucleotides, 2'-deoxyribonucleotides, 2'-amino modified nucleotides, 2'-alkoxy modified nucleotides, 2',3'-bromonucleotide mimics, L-2'-O-methyl modified nucleotides, L-2'-deoxyribonucleotides, locked nucleic acids, threose nucleic acids, 4'-modified threose nucleic acids, inverted nucleotides, inverted 2'-O-methyl modified nucleotides, inverted 2'-deoxyribonucleotides, diol nucleotides, 5'-vinyl phosphate nucleotides; more preferably, at least one of the modified nucleotides is selected from the group consisting of: 4'-modified threose nucleic acids, 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-deoxyribonucleotides, the nucleotides are linked by phosphate groups or thiophosphate groups; and combinations thereof.

[0030] The 4'-modified threose nucleic acid may have a structure as defined above.

[0031] For the specific definitions and structures of these modifications, please refer to the "siRNA and modified siRNA" section of this article.

[0032] In some embodiments, in any of the above-mentioned RNAi reagents, the n1 and n 21 The nucleotide at the position is a threose nucleic acid, a 4'-modified threose nucleic acid, an inverted 2'-deoxyribonucleotide, an inverted nucleotide, an L-2'-O-methyl modified nucleotide, an L-2'-deoxyribonucleotide, or a 2'-O-methyl modified nucleotide.

[0033] In some embodiments, in any of the above-mentioned RNAi reagents, the n7, n9, n 10 and n 11 The position is a 2'-deoxyribonucleotide or a 2'-fluoro-modified nucleotide.

[0034] In some embodiments, in any of the above-mentioned RNAi reagents, n2, n3, n4, n5, n6, n8, n9, 12 、n 13 、n 14 、n 15 、n 16 、n 17 、n 18 、n 19 and n 20 The nucleotides are 2'-O-methyl modified.

[0035] In some embodiments, in any of the above-mentioned RNAi agents, the 5' end of the sequence represented by general formula (I) contains at least one phosphorothioate group, and preferably one or more of n1 and n2, and n2 and n3 are connected by a phosphorothioate group.

[0036] The specific definition and structure of the phosphorothioate group can be found in the "siRNA and modified siRNA" section of this article.

[0037] In some embodiments, in any of the above-described RNAi agents, the N1 position is a 5'-vinyl phosphate-2'-O-methyl modified nucleotide, a 2'-O-methyl modified nucleotide, a threose nucleic acid, a 4'-modified threose nucleic acid, or an inverted nucleotide.

[0038] In some embodiments, in any of the above-mentioned RNAi agents, the N3 position is a 2'-O-methoxyethyl modified nucleotide or a 2'-O-methyl modified nucleotide.

[0039] In some embodiments, in any of the above-mentioned RNAi agents, the N5 position is a 2'-deoxyribonucleotide or a 2'-O-methyl modified nucleotide.

[0040] In some embodiments, in any of the above-mentioned RNAi reagents, the N6, N 11 The nucleotide position is a 2'-O-methyl modified nucleotide, a 2'-O-methoxyethyl modified nucleotide, or a 2'-fluoro modified nucleotide.

[0041] In some embodiments, in any of the above-described RNAi agents, the N7 position is a 2'-deoxyribonucleotide, a 2'-O-methyl modified nucleotide, a 2',3'-tetranucleotide mimic or a diol nucleotide.

[0042] In some embodiments, in any of the above-mentioned RNAi agents, the N 22 ~N 23 One or two of the positions are L-2'-O-methyl modified nucleotides, L-2'-deoxyribonucleotides, threose nucleic acids, 4'-modified threose nucleic acids, inverted 2'-deoxyribonucleotides, inverted nucleotides, inverted 2'-O-methyl modified nucleotides or 2'-O-methyl modified nucleotides.

[0043] In some embodiments, in any of the above-mentioned RNAi reagents, the N2, N 14 、N 16 The nucleotide position is a 2'-deoxyribonucleotide or a 2'-fluoro-modified nucleotide.

[0044] In some embodiments, in any of the above-mentioned RNAi reagents, the N4, N8, N9, N 10 、N 12 、N 13 、N 15 、N 17 、N 18 、N 19 、N 20 、N 21 It is a 2'-O-methyl modified nucleotide.

[0045] In some embodiments, in any of the RNAi agents described above, the 5' end and the 3' end of the sequence represented by the general formula (II) have at least one phosphorothioate group, preferably N1 and N2, N2 and N3, N 21 With N 22 、N 22 With N 23 One or more of the residues are linked by a phosphorothioate group.

[0046] In some embodiments, in any of the above-described RNAi agents, the sense strand consists of 21 nucleotides, and the antisense strand consists of 23 nucleotides.

[0047] In some embodiments, any of the above-described RNAi agents is obtained by independently modifying the nucleotides at each site of the sense strand and the antisense strand shown in Table 2.

[0048] In some embodiments, in any of the above-mentioned RNAi agents, the antisense strand comprises at least 15 consecutive nucleotides (preferably at least 16, 17, 18, 19 consecutive nucleotides, more preferably at least 20, 21, 22 or 23 consecutive nucleotides) of any modified antisense strand listed in Table 6; or differs from the modified antisense strand by no more than 3, 2 or 1 nucleotides in at least 15 consecutive nucleotides (preferably at least 16, 17, 18, 19 consecutive nucleotides, more preferably at least 20, 21, 22 or 23 consecutive nucleotides); or comprises at least 15 consecutive nucleotides that differ from the modified antisense strand by no more than 3, 2 or 1 nucleotides. Preferably, the antisense strand comprises, consists of or consists essentially of any modified antisense strand listed in Table 6.

[0049] In some embodiments, in any of the above-mentioned RNAi agents, the sense strand comprises at least 15 consecutive nucleotides (preferably at least 16, 17, 18, 19 consecutive nucleotides, more preferably at least 20 or 21 consecutive nucleotides) of any modified sense strand listed in Table 7; or differs from the modified sense strand in at least 15 consecutive nucleotides (preferably at least 16, 17, 18, 19 consecutive nucleotides, more preferably at least 20 or 21 consecutive nucleotides) by no more than 3, 2 or 1 nucleotides; or comprises at least 15 consecutive nucleotides that differ from the modified sense strand by no more than 3, 2 or 1 nucleotides. Preferably, the sense strand comprises, consists of or consists essentially of any modified sense strand listed in Table 7.

[0050] In some embodiments, in any of the above-described RNAi agents, the sense strand and the antisense strand are as shown in Table 7 and Table 6, respectively.

[0051] In some embodiments, in any of the above-described RNAi agents, the antisense strand comprises, consists of, or consists essentially of the following sequence: a sequence obtained by independently modifying the nucleotide at each position of a nucleotide sequence that differs from one of the following nucleotide sequences by 0, 1, 2, or 3 nucleotides:

[0052] 5'-AUCUAGAACUUUGACCAUCAGAG-3'(SEQ ID NO:90)

[0053] 5'-UAGGAGUAGGGGCUCAGCAGGGC-3'(SEQ ID NO:340)

[0054] 5'-UAGGUGAAAACACUGCUUUAGGG-3'(SEQ ID NO:424)

[0055] 5'-AUAUGAGGUGAAAACACUGCUGG-3'(SEQ ID NO:432)

[0056] 5'-AAUGUUUUAUUGUCUCUGCCUGG-3'(SEQ ID NO:444)

[0057] 5'-AUAUGAGGUGAAAACACUGCUUU-3'(SEQ ID NO:914)

[0058] 5'-AUAUGAGGUGAAAACACUGCUAU-3' (SEQ ID NO:915);

[0059] The difference is caused, for example, by substitution, insertion, deletion, or inversion of nucleotides in the listed nucleotide sequences.

[0060] In some embodiments, in any of the above-described RNAi agents, the sense strand comprises, consists of, or consists essentially of the following sequence: a sequence derived by independently modifying the nucleotide at each position of a nucleotide sequence that differs from one of the following nucleotide sequences by 0, 1, 2, or 3 nucleotides:

[0061] 5'-CUGAUGGUCAAAGUUCUAGAU-3'(SEQ ID NO:89)

[0062] 5'-CCUGCUGAGCCCCUACUCCUA-3'(SEQ ID NO:339)

[0063] 5'-CUAAAGCAGUGUUUUCACCUA-3'(SEQ ID NO:423)

[0064] 5'-AGCAGUGUUUUCACCUCAUAU-3'(SEQ ID NO:431)

[0065] 5'-AGGCAGAGACAAUAAAACAUU-3' (SEQ ID NO: 443);

[0066] The difference is caused, for example, by substitution, insertion, deletion, or inversion of nucleotides in the listed nucleotide sequences.

[0067] In some embodiments, any of the above-described RNAi agents comprises a duplex obtained by independently modifying the nucleotides at each position of each strand of any of the following pairs of duplexes:

[0068] Sense strand: 5'-CUGAUGGUCAAAGUUCUAGAU-3' (SEQ ID NO: 89) and

[0069] Antisense strand: 5′-AUCUAGAACUUUGACCAUCAGAG-3′ (SEQ ID NO: 90);

[0070] Sense strand: 5'-CCUGCUGAGCCCCUACUCCUA-3' (SEQ ID NO: 339) and

[0071] Antisense strand: 5′-UAGGAGUAGGGGCUCAGCAGGGC-3′ (SEQ ID NO: 340);

[0072] Sense strand: 5'-CUAAAGCAGUGUUUUCACCUA-3' (SEQ ID NO: 423) and

[0073] Antisense strand: 5'-UAGGUGAAAACACUGCUUUAGGG-3' (SEQ ID NO: 424)

[0074] Sense strand: 5'-AGCAGUGUUUUCACCUCAUAU-3' (SEQ ID NO: 431) and

[0075] Antisense strand: 5′-AUAUGAGGUGAAAACACUGCUGG-3′ (SEQ ID NO: 432);

[0076] Sense strand: 5'-AGCAGUGUUUUCACCUCAUAU-3' (SEQ ID NO: 431) and

[0077] Antisense strand: 5′-AUAUGAGGUGAAAACACUGCUUU-3′ (SEQ ID NO: 914);

[0078] Sense strand: 5'-AGCAGUGUUUUCACCUCAUAU-3' (SEQ ID NO: 431) and

[0079] Antisense strand: 5′-AUAUGAGGUGAAAACACUGCUAU-3′ (SEQ ID NO: 915);

[0080] Sense strand: 5'-AGGCAGAGACAAUAAAACAUU-3' (SEQ ID NO: 443) and

[0081] Antisense strand: 5'-AAUGUUUUAUUGUCUCUGCCUGG-3' (SEQ ID NO: 444).

[0082] In some embodiments, in any of the above-described RNAi agents, the antisense strand comprises, consists of, or consists essentially of a modified nucleotide sequence that differs from one of the following modified nucleotide sequences by 0, 1, 2, or 3 nucleotides:

[0083] 5'-UmsAfsGmGmUmGfAmAmAmAmCmAmCmUfGmCfUmUmUmAmGmsiGsiG-3' (SEQ ID NO: 597)

[0084] 5’-UmsAfsGmGmdTGmdAAmAmAmCmAmCmUfGmCfUmUmUmAmGmsGmsGm-3’(SEQ ID NO:604)

[0085] 5’-AmsUfsAmUmGmAfGmGmUmGmAmAmAmAfCmAfCmUmGmCmUmsGmsGm-3’(SEQ ID NO:606)

[0086] 5’-AmsUfsAmUmdGAmdGGmUmGmAmAmAmAfCmAfCmUmGmCmUmsGmsGm-3’(SEQ ID NO:620)

[0087] 5’-VpUmsUfsAmUmdGAmdGGmUmGmAmAmAmAfCmAfCmUmGmCmUmsGmsGm-3’(SEQ ID NO:621)

[0088] 5’-AmsAfsUmGmUmUfUmUmAmUmU(moe)GmUmCfUmCfUmGmCmCmUmsGmsGm-3’(SEQ ID NO:644)

[0089] 5’-AmsAfsUmGmUmUfdTUmAmUmUmGmUmCfUmCfUmGmCmCmUmsGmsGm-3’(SEQ ID NO:655)

[0090] 5’-AmsUfsAmUmdGAmdGGmUmGmAmAmAmAfCmAfCmUmGmCmUmsUmsUm-3’(SEQ ID NO:889)

[0091] 5’-AmsUfsAmUmdGAmdGGmUmGmAmAmAmAfCmAfCmUmGmCmUmsAmsUm-3’(SEQ ID NO:891)

[0092] The capital letters "G", "C", "A" and "U" represent ribonucleotides containing guanine, cytosine, adenine and uracil as bases, respectively; "T" represents a deoxyribonucleotide containing thymine as a base; the lowercase letter "m" represents that the nucleotide adjacent to the left of the letter "m" is a 2'-O-methyl modified nucleotide; the lowercase letter "f" represents that the nucleotide adjacent to the left of the letter "f" is a 2'-fluoro modified nucleotide; "moe" represents that the nucleotide adjacent to the left of the letter "moe" is a 2'-O-methoxyethyl modified nucleotide; "Vp" represents that the nucleotide adjacent to the right of the letter "Vp" is a 5'-vinyl phosphate nucleotide; the lowercase letter "i" represents that the nucleotide adjacent to the right of the letter "i" is an inverted nucleotide; the lowercase letter "d" represents that the nucleotide adjacent to the right of the letter "d" is a 2'-deoxyribonucleotide; the lowercase letter "s" represents that the connection between the two nucleotides adjacent to the letter "s" is a phosphorothioate connection; the meanings of these symbols in the sequence can also be found in Table 7 herein;

[0093] The difference is caused, for example, by substitution, insertion, deletion, or inversion of nucleotides in the listed nucleotide sequences.

[0094] In some embodiments, in any of the above-described RNAi agents, the sense strand comprises, consists of, or consists essentially of a modified nucleotide sequence that differs from one of the following modified nucleotide sequences by 0, 1, 2, or 3 nucleotides:

[0095] 5'-ChsUmsAmAmAmGmCfAmGfUfGfUmUmUmUmCmAmCmCmUmAm-3' (SEQ ID NO: 601)

[0096] 5'-iCsUmsAmAmAmGmCfAmGfUfGfUmUmUmUmCmAmCmCmUmAm-3' (SEQ ID NO: 603)

[0097] 5'-AmsGmsCmAmGmUmGfUmUfUfUfCmAmCmCmUmCmAmUmAmUm-3' (SEQ ID NO: 605)

[0098] 5'-AhsGmsCmAmGmUmGfUmUfUfUfCmAmCmCmUmCmAmUmAmUm-3' (SEQ ID NO: 617)

[0099] 5'-AmsGmsGmCmAmGmAfGmAfCfAfAmUmAmAmAmAmCmAmUmUm-3' (SEQ ID NO: 641)

[0100] 5'-AhsGmsGmCmAmGmAfGmAfCfAfAmUmAmAmAmAmCmAmUmUm-3' (SEQ ID NO: 653)

[0101] 5'-AhsGmsCmAmGmUmGfUmUfUfUfCmAmCmCmUmCmAmUmAmAm-3' (SEQ ID NO:744)

[0102] The capital letters "G", "C", "A" and "U" represent ribonucleotides containing guanine, cytosine, adenine and uracil as bases, respectively; the lowercase letter "m" indicates that the nucleotide adjacent to the left of the letter "m" is a 2'-O-methyl modified nucleotide; the lowercase letter "f" indicates that the nucleotide adjacent to the left of the letter "f" is a 2'-fluoro modified nucleotide; the lowercase letter "h" indicates that the nucleotide adjacent to the left of the letter "h" is a 4'-modified threose nucleic acid; "i" indicates that the nucleotide adjacent to the right of the letter "i" is an inverted nucleotide; the lowercase letter "s" indicates that the connection between the two nucleotides adjacent to the left and right of the letter "s" is a phosphorothioate connection; the meanings of these symbols in the sequence can also be found in Table 7 herein;

[0103] The difference is caused, for example, by substitution, insertion, deletion, or inversion of nucleotides in the listed nucleotide sequences.

[0104] In some embodiments, the RNAi agent of any of the above comprises any one of the following groups: SEQ ID NOs: 601 and 597, SEQ ID NOs: 601 and 604, SEQ ID NOs: 603 and 597, SEQ ID NOs: 603 and 604, SEQ ID NOs: 605 and 606, SEQ ID NOs: 605 and 620, SEQ ID NOs: 605 and 889, SEQ ID NOs: 617 and 606, SEQ ID NOs: 617 and 620, SEQ ID NOs: 744 and 621, SEQ ID NOs: 641 and 644, SEQ ID NOs: 641 and 655, SEQ ID NOs: 653 and 644, SEQ ID NOs: 653 and 655.

[0105] In some embodiments, in any of the above-described RNAi agents, the sense strand and / or antisense strand is linked to a targeting ligand to form a conjugate.

[0106] In some embodiments, in the conjugate of the above-mentioned RNAi agent, the targeting ligand comprises one or more (e.g., 1, 2, 3, or 4) N-acetylgalactosamine (GalNAc) and / or its derivatives, for example, N-acetylgalactosamine is covalently conjugated to the sense strand and / or antisense strand in a monovalent state, a divalent state, a trivalent state, or a tetravalent state;

[0107] The structural formula of N-acetylgalactosamine (GalNAc) is shown in formula (BI):

[0108] In some embodiments, in any of the above-described RNAi agents, in the conjugate, the targeting ligand is L96(GalNAc3), and its structural formula is shown in Formula (B-II):

[0109] In some embodiments, in any of the above-described RNAi agents, in the conjugate, the targeting ligand is conjugated to the sense strand.

[0110] In some embodiments, in any of the above-described RNAi agents, in the conjugate, the targeting ligand is conjugated to the 3' end or the 5' end of the sense strand.

[0111] In some embodiments, in any of the above-described RNAi agents, the structural formula of the conjugate is as shown in Formula (B-III):

[0112] in represents the double-stranded oligonucleotide of the RNAi, 3' represents the 3' end of the sense strand, and X is O or S.

[0113] In some embodiments, in any of the above-described RNAi agents, the conjugate is as shown in Table 5.

[0114] In some embodiments, in any of the above-mentioned RNAi agents, the conjugate is BPR-30221218, BPR-30221221, BPR-30221223, BPR-30221228, BPR-30221686, BPR-30221617, BPR-30221618, BPR-30221620, BPR-30221672, BPR-30221676, BPR-30221678, BPR-30222203, BPR-30222212, or BPR-30222218 of the present invention.

[0115] The present invention provides a cell comprising the RNAi agent of the present invention.

[0116] The TTR RNAi agents disclosed herein can be incorporated into compositions. Thus, the present invention also provides a composition comprising one or more TTR RNAi agents disclosed herein and at least one pharmaceutically acceptable diluent, carrier, and / or excipient. In some embodiments, the compositions disclosed herein comprising one or more pharmaceutically acceptable diluents, carriers, and / or excipients of the disclosed TTR RNAi agents are pharmaceutical compositions.

[0117] The pharmaceutically acceptable diluent, carrier or excipient may be any suitable diluent, carrier and / or excipient conventionally used in the art.

[0118] In some embodiments, in the above composition, the diluent is PBS buffer, physiological saline or water.

[0119] In some embodiments, any of the above-described compositions may further comprise one or more additional therapeutic agents, such as any therapeutic agent effective for treating and / or preventing diseases and / or conditions mediated at least in part by TTR gene expression.

[0120] The present invention also provides a method for preparing any of the above-described compositions, comprising mixing one or more TTR RNAi agents disclosed herein with at least one pharmaceutically acceptable diluent, carrier, and / or excipient. In some embodiments, the method comprises mixing the TTR RNAi agents disclosed herein with a diluent. In some embodiments, the method comprises mixing the TTR RNAi agents disclosed herein with water, saline, or PBS buffer.

[0121] The present invention also provides a method for inhibiting the expression of a TTR gene in a cell in vivo or in vitro, comprising introducing an effective amount of any of the above-described RNAi agents or any of the above-described compositions into the cell.

[0122] In some embodiments, in any of the methods described above, the cell is in a subject.

[0123] In some embodiments, in any of the methods described above, the subject is a human.

[0124] In some embodiments, in any of the methods described above, the expression of the TTR gene is inhibited by at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, 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%, at least about 99%, or about 100%.

[0125] The present invention also provides use of any of the above-described RNAi agents or any of the above-described compositions for preparing a medicament for treating and / or preventing a disease, disorder or symptom mediated at least in part by TTR gene expression.

[0126] In some embodiments, in the above uses, the diseases include ATTR-PN (transthyretin amyloidosis polyneuropathy), ATTR-CM (transthyretin amyloidosis cardiomyopathy), senile systemic amyloidosis, and leptomeningeal / central nervous system amyloidosis.

[0127] The present invention also provides a method for treating or preventing a disease, disorder or symptom mediated at least in part by TTR gene expression, comprising administering a therapeutically effective amount of any of the aforementioned RNAi agents or any of the aforementioned compositions to a patient in need thereof.

[0128] In some embodiments, in the above methods, the patient is a human.

[0129] In some embodiments, in any of the above methods, the disease includes ATTR-PN (transthyretin amyloidosis polyneuropathy), ATTR-CM (transthyretin amyloidosis cardiomyopathy), senile systemic amyloidosis, and leptomeningeal / central nervous system amyloidosis.

[0130] In some embodiments, in any of the above methods, the RNAi agent or the composition is administered to the patient by subcutaneous, intravenous, intramuscular, intrabronchial, intrapleural, intraperitoneal, intraarterial, lymphatic and / or cerebrospinal administration.

[0131] In some embodiments, in any of the methods described above, the RNAi agent or the composition is delivered to the liver, choroid plexus, retina, and / or pancreas of the patient.

[0132] In some embodiments, in any of the above methods, the RNAi agent is administered at a dose of about 1-300 mg / kg body weight.

[0133] In some embodiments, in any of the above methods, the RNAi agent is administered at a dosage of about 1-250 mg / kg body weight, about 1-200 mg / kg body weight, about 1-150 mg / kg body weight, about 1-100 mg / kg body weight, about 1-90 mg / kg body weight, about 1-80 mg / kg body weight, about 1-70 mg / kg body weight, about 1-60 mg / kg body weight, about 1-50 mg / kg body weight, about 1-40 mg / kg body weight, about 1-30 mg / kg body weight, about 1-20 mg / kg body weight, about 1-10 mg / kg body weight, about 1-9 mg / kg body weight, about 1-8 mg / kg body weight, about 1-7 mg / kg body weight, about 1-6 mg / kg body weight, about 1-5 mg / kg body weight, about 1-4 mg / kg body weight, about 1-3 mg / kg body weight, or about 1-2 mg / kg body weight.

[0134] In some embodiments, in any of the above methods, the RNAi agent is administered at a dose of about 2-300 mg / kg body weight, about 3-300 mg / kg body weight, about 4-300 mg / kg body weight, 5-300 mg / kg body weight, about 6-300 mg / kg body weight, about 7-300 mg / kg body weight, about 8-300 mg / kg body weight, about 9-300 mg / kg body weight, about 10-300 mg / kg body weight, about 20-300 mg / kg body weight, about 30-300 mg / kg body weight, about 40-300 mg / kg body weight, about 50-300 mg / kg body weight, about 60-300 mg / kg body weight, about 70-300 mg / kg body weight, about 80-300 mg / kg body weight, about 90-300 mg / kg body weight, about 100-300 mg / kg body weight, about 150-300 mg / kg body weight, about 200-300 mg / kg body weight, about 250-300 mg / kg body weight.

[0135] In some embodiments, in any of the above methods, the RNAi agent is administered at a dosage of about 1 mg / kg body weight, about 2 mg / kg body weight, about 3 mg / kg body weight, about 4 mg / kg body weight, about 5 mg / kg body weight, about 6 mg / kg body weight, about 7 mg / kg body weight, about 8 mg / kg body weight, about 9 mg / kg body weight, about 10 mg / kg body weight, about 20 mg / kg body weight, about 30 mg / kg body weight, about 40 mg / kg body weight, about 50 mg / kg body weight, about 60 mg / kg body weight, about 70 mg / kg body weight, about 80 mg / kg body weight, about 90 mg / kg body weight, about 100 mg / kg body weight, about 150 mg / kg body weight, about 200 mg / kg body weight, about 250 mg / kg body weight, about 300 mg / kg body weight, or any range or value therebetween.

[0136] In some embodiments, in any of the methods described above, the RNAi agent is administered once or more every day, every week, every two weeks, every three weeks, every 1 month, every 2 months, every 3 months, every 4 months, every 5 months, every 6 months, every 7 months, every 8 months, every 9 months, every 10 months, every 11 months, or every 12 months, for example, every day, every week, every two weeks, every three weeks, every 1 month, every 2 months, every 3 months, every 4 months, every 5 months, every 6 months, every 7 months, every 8 months, every 9 months, every 10 months, every 11 months, or every 12 months. 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 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, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111

[0137] In some embodiments, in any of the methods described above, the total number of times the RNAi agent or composition is administered can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50. For example, the RNAi agent or composition can be administered about 1, 2, 3, or 4 times.

[0138] The present invention also provides a kit comprising any of the above-mentioned RNAi reagents or any of the above-mentioned compositions and optionally instructions for use. Beneficial effects

[0139] The novel TTR RNA interference (RNAi) reagents and compositions provided by the present invention can selectively and effectively inhibit the expression of the TTR gene, which can be used to treat and prevent thyroxine translocase amyloidosis diseases, including but not limited to ATTR-PN (thyroxine translocase amyloidosis polyneuropathy), ATTR-CM (thyroxine translocase amyloidosis cardiomyopathy), senile systemic amyloidosis diseases, and leptomeningeal / CNS (central nervous system) amyloidosis diseases. In addition, the novel TTR RNA interference (RNAi) reagents and compositions provided by the present invention can also effectively inhibit the expression of the TTR gene in the evaluation of free uptake activity of primary hepatocytes, and can also effectively inhibit the expression of the TTR gene in vivo in the evaluation of the efficacy of humanized TTR mice and cynomolgus monkeys. The modification scheme provided in this application is applicable to all sequences provided in Table 2, and is not limited to the sequences specifically verified in the examples (such as Table 5). Other unverified RNAi reagents containing modified sequences have the same or similar technical effects as the RNAi reagent sequences verified in the examples.

[0140] The present invention evaluated the transfection activity of 235 pairs of siRNAs shown in Table 2 in vitro on liver cancer cells HepG2 and Hep3B, and found that the TTR inhibition rates of siRNA in HepG2 were approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, and 90%, with the highest inhibition rate being 98.60%; the TTR inhibition rates of siRNA in Hep3B were approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, and 90%, with the highest inhibition rate being 95.85%, as shown in Table 3.

[0141] The present invention also evaluated the free uptake activity of GalNAc-siRNA in primary hepatocytes. In the free uptake test in human primary hepatocytes, the highest mean inhibition rate of TTR gene expression was 94.45% at a dose of 20 nM; 92.05% at a dose of 10 nM; and 88.55% at a dose of 5 nM, as shown in Table 8. In the free uptake test in monkey primary hepatocytes, the highest mean inhibition rate of TTR gene expression was 92.61% at a dose of 20 nM; and 84.26% at a dose of 10 nM, as shown in Table 9.

[0142] GalNAc-siRNA was also evaluated for efficacy in humanized TTR mice, with the results shown in Figure 1. BPR-30213022, BPR-30221223, BPR-30221686, BPR-30221617, BPR-30221618, BPR-30222201, and BPR-30222218 all knocked down serum TTR protein levels in mice on Day 7 after administration, with the lowest knockdown rate being 61% and the highest being 96%. The highest knockdown rate of serum TTR protein in mice was 90% on Day 42 after administration.

[0143] The efficacy of GalNAc-siRNA in crab-eating monkeys was also evaluated. The experimental results showed that BPR-30221223, BPR-30221228, BPR-30221686, BPR-30221617, BPR-30221618, BPR-30221620, BRP-30222201, BPR-3022218, etc., were able to effectively knock down the serum TTR protein content in crab-eating monkeys after administration.

[0144] The GalNAc-siRNA of the present invention can effectively inhibit the expression of the TTR gene and can be used to treat and / or prevent symptoms and diseases associated with ATTR, including but not limited to ATTR-PN (transthyretin amyloidosis polyneuropathy), ATTR-CM (transthyretin amyloidosis cardiomyopathy), senile systemic amyloidosis, and leptomeningeal / CNS (central nervous system) amyloidosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0145] FIG1 is a graph showing the change in hTTR protein content in the serum of humanized mice at different time points relative to the inhibition rate before administration, after subcutaneous injection of a single dose of 3 mg / kg of the pharmaceutical composition comprising the GalNAc-siRNA conjugate in Example 6 into TTR gene humanized mice.

[0146] FIG2 is a graph showing the mean change in the inhibition rate of serum TTR protein in each experimental group at different time points, relative to that before administration, after subcutaneous administration of a single dose of 1 mg / kg of the pharmaceutical composition comprising the GalNAc-siRNA conjugate in Example 6 to humanized B6-hTTR mice.

[0147] FIG3 is a graph showing the mean change in the inhibition rate of serum TTR protein in each group of mice at different time points, equivalent to that before administration, after a single dose of the pharmaceutical composition comprising the GalNAc-siRNA conjugate of Example 7 was subcutaneously administered to cynomolgus monkeys up to day 42 of administration. DETAILED DESCRIPTION

[0148] The present invention will be further described below with reference to specific examples. It should be understood that the following examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. Unless otherwise specified, the techniques used in the examples are conventional in the art, or in accordance with the experimental methods recommended by the kit and instrument manufacturers. The reagents and biological materials used in the examples are all commercially available unless otherwise specified.

[0149] As used herein, the terms "deoxyribonucleotide" and "DNA" refer to a nucleotide or polynucleotide comprising at least one sugar moiety having an H at its 2' position instead of an OH.

[0150] As used herein, "transthyretin" (TTR) is well known in the art. TTR is also known as prealbumin, HsT2651, PALB, and TBPA. TTR binds to thyroxine (T4) and retinol-binding protein (RBP), functioning as a transporter for thyroxine (T4) and retinol, and also acts as a protease. The liver secretes TTR into the blood, and the choroid plexus secretes TTR into the cerebrospinal fluid. TTR is also expressed in the pancreas and retinal pigment epithelial cells. The greatest clinical relevance of TTR is that both normal and mutant TTR proteins can form amyloid fibrils and aggregate outside cells, causing amyloidosis. The sequence of human TTR mRNA can be found in the National Center for Biotechnology Information (NCBI) RefSeq accession number NM_000371.4 (SEQ ID NO: NM_000371.4). The sequence of mouse TTR mRNA can be found in RefSeq Accession No. NM_013697.2, and the sequence of rat TTR mRNA can be found in RefSeq Accession No. NM_012681.1. Additional examples of TTR mRNA sequences are readily available using publicly available databases such as GenBank, UniProt, and OMIM.

[0151] As used herein, a "target sequence" refers to a continuous portion of the nucleotide sequence of an mRNA molecule formed during transcription of a TTR gene, including mRNA that is a product of RNA processing as a product of the initial transcription. In one example, the target portion of the sequence is at least long enough to serve as a substrate for RNAi-guided cleavage at or near the nucleotide sequence portion of the mRNA molecule formed during transcription of the TTR gene. In one example, the target sequence is within the protein-coding region of the TTR gene. In another example, the target sequence is within the 3'UTR of the TTR gene.

[0152] The term "interfering RNA" or "RNAi" or "interfering RNA sequence" includes single-stranded RNA (e.g., mature miRNA, ssRNAi oligonucleotide) or double-stranded RNA (i.e., duplex RNA, such as siRNA, dsRNA, shRNA, aiRNA or miRNA) that, when the interfering RNA and the target gene or sequence are in the same cell, can reduce or inhibit the expression of the target gene (e.g., by mediating degradation and inhibiting translation of mRNA complementary to the interfering RNA sequence). Interfering RNA therefore refers to a single-stranded RNA that is complementary to the target sequence or a double-stranded RNA formed by two complementary strands or by a single self-complementary strand.

[0153] The term "siRNA" as used herein refers to a small inhibitory RNA duplex that induces the RNA interference (RNAi) pathway, mediating targeted cleavage of RNA transcripts through the RNA-induced silencing complex (RISC) pathway, and guiding sequence-specific degradation of mRNA. siRNAs can vary in length (usually between 18-30 base pairs) and have varying degrees of complementarity with the target mRNA.

[0154] Typically, siRNA comprises a sense strand (SS) and an antisense strand (AS). Most of the nucleotides of each chain of siRNA are ribonucleotides, and one or both of the two chains may also include one or more non-ribonucleotides, for example, deoxyribonucleotides and / or modified nucleotides. In addition, as used herein, "siRNA" may include ribonucleotides with chemical modifications; siRNA may include substantial modifications at multiple nucleotides. As used herein, the term "modified nucleotides" refers to nucleotides independently having modified sugar moieties, modified internucleotide bonds, and / or modified core bases. Therefore, the term "modified nucleotides" encompasses substitutions, additions, or removals (such as functional groups or atoms) on internucleotide bonds, sugar moieties, or bases. Modifications suitable for use in the medicament of the present invention include all modification types disclosed herein or known in the art.

[0155] The term "antisense strand" or "guide strand" refers to a strand that is substantially complementary to a target sequence (e.g., TTR mRNA). As used herein, the term "region of complementarity" refers to the region where the antisense strand is substantially complementary to the target sequence, and may have mismatches in the interior or terminal regions of the molecule.

[0156] The term "sense strand" or "passenger strand" or "sense strand" refers to the RNAi strand that includes a region that is substantially complementary to the antisense strand as defined herein.

[0157] The term "nucleotide overhang" refers to at least one unpaired nucleotide that protrudes from the duplex structure of RNAi (e.g., dsRNA). For example, a nucleotide overhang is present when the 3' end of one strand of a dsRNA extends beyond the 5' end of the other strand, or vice versa. A dsRNA can include an overhang having at least one nucleotide; the overhang can comprise at least one nucleotide, at least two nucleotides, at least three nucleotides, at least four nucleotides, at least five nucleotides or more. The nucleotide overhang can comprise or consist of nucleotide / nucleoside analogs (including deoxynucleotides / nucleosides). The one or more overhangs can be on the sense strand, the antisense strand, or a combination thereof. Additionally, the one or more nucleotides of the overhang can be present at the 5' end, the 3' end, or both ends of the antisense strand of the dsRNA.

[0158] The capital letters "G", "C", "A" and "U" represent ribonucleotides containing guanine, cytosine, adenine and uracil as bases, respectively; "T" represents deoxyribonucleotides containing thymine as a base.

[0159] The term "complementary" refers to the ability of polynucleotides to pair with each other to form base pairs. Base pairs are typically formed by hydrogen bonds between nucleotide units in antiparallel polynucleotide chains. Complementary polynucleotide chains can base pair in a Watson-Crick manner (e.g., A to T, A to U, C to G), or in any other manner that allows the formation of a duplex, such as the Hoogsteen base pairing method or the wobble base pairing method.

[0160] The term "conjugate" or "coupled compound" refers to a new compound formed by covalently linking (coupling) two or more compounds through a bivalent or multivalent compound with a linking function. The conjugate can be represented by GalNAc-siRNA, where the GalNAc can be L96 (i.e., the GalNAc3 ligand), serving as a liver-targeted delivery vehicle. L96 can be coupled to the 3' end of the sense strand of the siRNA to form a conjugate, or it can be coupled to the 5' end of the sense strand of the siRNA to form a conjugate.

[0161] As used herein, the phrase "inhibiting TTR gene expression" encompasses inhibiting the expression of any TTR gene (such as a mouse TTR gene, a rat TTR gene, a monkey TTR gene, or a human TTR gene) as well as variants or mutants of a TTR gene encoding a TTR protein.

[0162] Inhibiting TTR gene expression comprises any level of TTR gene inhibition, e.g., at least partial inhibition of TTR gene expression, such as at least about 20% inhibition. In certain embodiments, the inhibition is at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, 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%.

[0163] TTR gene expression can be assessed based on the level of any variable related to TTR gene expression, such as TTR mRNA levels or TTR protein levels. Inhibition can be assessed by a decrease in the absolute or relative level of one or more of these variables compared to a control level. The control level can be any type of control level used in the art, such as a pre-dose baseline level or a level determined from a similar untreated or control-treated subject, cell, or sample (e.g., a buffer-only control or an inert agent control).

[0164] The subject includes any human or non-human animal. The term "non-human animal" includes all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, etc. As used herein, the term "cyno" or "Monkey" refers to cynomolgus monkeys.

[0165] The terms "therapeutically effective amount," "therapeutically effective dose," and "effective amount" refer to an amount of the siRNA, GalNAc-siRNA, or pharmaceutical composition of the present invention, when administered alone or in combination with other therapeutic agents to a cell, tissue, or subject, that is effective to prevent or ameliorate the symptoms of one or more diseases or conditions or the progression of the disease or condition. The effective amount for a particular subject may vary depending on a variety of factors, such as the disease to be treated, the patient's overall health, the method, route, and dosage of administration, and the severity of side effects. The effective amount may be the maximum dose or dosing regimen that avoids significant side effects or toxic effects. When administered to an individual, the therapeutically effective amount refers to the individual ingredient. When administered in combination, the therapeutically effective amount refers to the combined amount of the active ingredients that produces the therapeutic effect, regardless of whether they are administered in combination, sequentially, or simultaneously. A therapeutically effective amount will alleviate symptoms, typically by at least 10%, usually by at least 20%, preferably by at least about 30%, more preferably by at least 40%, and most preferably by at least 50%.

[0166] The term "treat" refers to a beneficial or desired result, including, for example, a reduction in transthyretin amyloid deposits. The term "treat" also includes, but is not limited to, alleviation or improvement of one or more symptoms of ATTR-PN, ATTR-CM, SSA, and leptomeningeal / CNS amyloidosis.

[0167] "Treat," "treat," or "ameliorate" are used interchangeably herein. These terms refer to an approach for obtaining beneficial or desired results, including but not limited to therapeutic benefit.

[0168] siRNA and modified siRNA

[0169] Provided herein are siRNAs for inhibiting TTR gene expression. Each siRNA comprises a sense strand and an antisense strand. The sequence information for BPR-302045, BPR-302170, BPR-302212, BPR-302216, and BPR-302222, as well as their specific locations within TTR mRNA (NM_000371.4), are shown in Table 1. The sense strand (SS) of the siRNA consists of 21 nucleotides, and the antisense strand (AS) consists of 23 nucleotides. The 3' end of the antisense strand (AS) has a two-base overhang. The antisense strand and the sense strand have at least 18 consecutive nucleotides in reverse complementation.

[0170] Table 1

[0171] In order to increase the specificity, stability and effectiveness of double-stranded RNA, each nucleotide of the sense strand (SS) and antisense strand (AS) of siRNA has been independently modified, including ribose group modification, backbone modification (such as phosphate group modification) and base modification.

[0172] The modified nucleotide is selected from the group consisting of: 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-O-methoxyethyl modified nucleotides, L-2'-fluoro modified nucleotides, 2'-deoxyribonucleotides, 2'-amino modified nucleotides, 2'-alkoxy modified nucleotides, 2',3'-bromonucleotide mimics, L-2'-O-methyl modified nucleotides, L-2'-deoxyribonucleotides, locked nucleic acids, threose nucleic acids, 4'-modified threose nucleic acids, inverted nucleotides, inverted 2'-O-methyl modified nucleotides, inverted 2'-deoxyribonucleotides, diol nucleotides, 5'-vinyl phosphate nucleotides;

[0173] Among them, the 2'-O-methyl modified nucleotide refers to a nucleotide in which the hydroxyl group at the 2' position of the ribose group of the nucleotide is replaced by a methoxy group, and its structure is shown in A1;

[0174] 2'-Fluoro-modified nucleotides refer to nucleotides in which the hydroxyl group at the 2' position of the ribose group of the nucleotide is replaced by fluorine, and its structure is shown in A2;

[0175] A 2'-O-methoxyethyl modified nucleotide refers to a nucleotide in which the hydrogen of the hydroxyl group at the 2' position of the ribose group of the nucleotide is replaced by a methoxyethyl group, and its structure is shown in A3;

[0176] L-2'-fluoro-modified nucleotides refer to nucleotides in which the chirality of the 1', 2', 3', and 4' positions of the ribose group of the nucleotide is completely opposite to that of natural nucleotides, and the hydroxyl group at the 2' position is replaced by fluorine. Its structure is shown in A4;

[0177] 2'-deoxyribonucleotide (DNA) refers to a nucleotide in which the hydroxyl group at the 2' position of the ribose group of the nucleotide is replaced by hydrogen, and its structure is shown in A5;

[0178] A 2'-amino modified nucleotide refers to a nucleotide in which the hydroxyl group at the 2' position of the ribose group of the nucleotide is replaced by an amino group, and its structure is shown in A6;

[0179] A 2'-alkoxy modified nucleotide refers to a nucleotide in which the hydrogen of the hydroxyl group at the 2' position of the ribose group of the nucleotide is replaced by an alkyl group, and its structure is shown in A7;

[0180] A 2',3'-broken nucleotide mimetic is a nucleotide formed by breaking the carbon-carbon bonds at the 2' and 3' positions of the ribose group of the nucleotide, and its structure is shown in A8;

[0181] L-2'-O-methyl modified nucleotides refer to nucleotides in which the chirality of the 1', 2', 3' and 4' positions of the ribose group of the nucleotide is completely opposite to that of natural nucleotides, and the hydroxyl group at the 2' position is replaced by a methoxy group. Its structure is shown in A9;

[0182] L-2'-deoxyribonucleotides are nucleotides in which the chirality of the 1', 3', and 4' positions of the ribose group is completely opposite to that of natural nucleotides, and the hydroxyl group at the 2' position is replaced by hydrogen. Its structure is shown in A10.

[0183] Locked nucleic acid (LNA) refers to a nucleotide in which the 2'-O position and the 4'-C position of the ribose group of the nucleotide form an oxymethylene bridge, and its structure is shown in A11;

[0184] Threonose nucleic acid (TNA) refers to a nucleotide with a threose structure, and its structure is shown in A12;

[0185] The structure of the 4'-modified threose nucleic acid is shown in A13, wherein R represents a C10-C30 alkyl group; preferably, R represents a C10-C30 straight-chain alkyl group;

[0186] An inverted nucleotide refers to a nucleotide in which the 3' position of the ribose group is replaced by phosphate and is connected to the 3' end of a non-inverted nucleotide, and a nucleotide in which the 5' position is a hydroxyl group and is connected to the 5' end of a non-inverted nucleotide, and its structure is shown in A14; in this article, unless otherwise specified, "inverted nucleotide" does not include any substitution of other position groups (such as the hydroxyl group at the 2' position).

[0187] A reverse 2'-O-methyl modified nucleotide refers to a nucleotide in which the 3' position of the ribose group of the nucleotide is replaced by a phosphate, the 5' position is a hydroxyl group, and the 2' position hydroxyl group is replaced by a methoxy group. Its structure is shown in A15;

[0188] Reverse 2'-deoxynucleotides are nucleotides in which the 3' position of the ribose group of the nucleotide is replaced by phosphate, the 5' position is a hydroxyl group, and the 2' position of the hydroxyl group is replaced by hydrogen. Its structure is shown in A16;

[0189] Diol nucleotides refer to nucleotide analogs with a diol structure, the structure of which is shown in A17, where R represents H, OH, or alkoxy;

[0190] 5'-vinyl phosphate nucleotides are nucleotides in which the 5'-position of the ribose group of the nucleotide is substituted with vinyl phosphate and the hydroxyl group at the 2'-position is substituted with hydrogen. The structure thereof is shown in A18.

[0191] At least one of the phosphate groups in the phosphate-sugar backbone of at least one single strand of the sense strand and the antisense strand is a phosphate group with a modified group. The phosphate group with a modified group is a phosphorothioate group formed by replacing at least one oxygen atom in the phosphodiester bond of the phosphate group with a sulfur atom. For example, a non-bridging oxygen atom in the phosphodiester bond is replaced with a phosphorothioate diester bond, i.e., the connection between the two nucleotides is a phosphorothioate linkage. This modification can stabilize the structure of the siRNA and maintain high base pairing specificity and affinity.

[0192] The structural formula of the phosphorothioate group mentioned in this article is shown in A19:

[0193] Details of the modified sense strand are shown in Table 7, and details of the modified antisense strand are shown in Table 6.

[0194] Those skilled in the art will appreciate that the siRNAs described herein can be obtained by conventional siRNA preparation methods in the art (e.g., solid phase synthesis and liquid phase synthesis methods). Solid phase synthesis is commercially available on a custom basis. Modified nucleotides can be introduced into the siRNAs described herein using correspondingly modified nucleotide monomers. Methods for preparing correspondingly modified nucleotide monomers and methods for introducing modified nucleotides into siRNAs are also well known to those skilled in the art.

[0195] GalNAc-siRNA conjugates

[0196] The conjugate comprises the above-mentioned siRNA disclosed herein and a conjugated group connected to the siRNA. Generally speaking, the conjugated group comprises at least one pharmaceutically acceptable targeting group (i.e., a targeting ligand) and optionally a linker, and the siRNA, the linker, and the targeting ligand are connected in sequence.

[0197] In some embodiments, the conjugate group can be attached to the end of the siRNA strand, such as the 5' end and / or 3' end of the sense strand, and / or the 5' end and / or 3' end of the antisense strand. When the conjugate group is attached to the end of the siRNA strand, the conjugate group is typically attached to a phosphate group of a nucleotide.

[0198] The present invention uses N-acetylgalactosamine (GalNAc) as a delivery vector for siRNA drugs to specifically deliver drugs to hepatocytes to reduce or inhibit the expression of the TTR gene, thereby reducing the TTR protein level in a subject (e.g., a human or animal subject), thereby achieving the purpose of preventing and / or treating symptoms and diseases associated with ATTR, including but not limited to ATTR-PN (transthyretin amyloidosis polyneuropathy), ATTR-CM (transthyretin amyloidosis cardiomyopathy), senile systemic amyloidosis, and leptomeningeal / CNS (central nervous system) amyloidosis.

[0199] In some embodiments, the 3' end of the sense strand of the siRNA disclosed herein is conjugated to three N-acetylgalactosamine (GalNAc) molecules (i.e., L96) to obtain a GalNAc-siRNA conjugate, the structural formula of which is shown in Formula (B-III):

[0200] in represents the double-stranded oligonucleotide of the RNAi, 3' represents the 3' end of the sense strand, and X is O or S.

[0201] In some embodiments, the specific information of the conjugate is shown in Table 5.

[0202] The above conjugates can be synthesized by methods that have been described in detail in the prior art.

[0203] Pharmaceutical composition

[0204] The present invention provides pharmaceutical compositions comprising the disclosed specifically modified siRNAs and GalNAc-siRNAs as active ingredients and a pharmaceutically acceptable diluent, carrier, and / or excipient (e.g., PBS buffer, saline, water). The purpose of the pharmaceutical compositions is to facilitate administration to an organism, facilitate absorption of the active ingredient, and thereby exert its biological activity.

[0205] The pharmaceutically acceptable diluents, carriers and / or excipients used in the present invention include any suitable pharmaceutically acceptable diluents, carriers and / or excipients known in the art.

[0206] application

[0207] The present invention provides disclosed specifically modified siRNAs, GalNAc-siRNAs, and pharmaceutical compositions for use in treating and / or preventing diseases or disorders mediated by TTR gene expression (eg, related to transthyretin amyloidosis).

[0208] The present invention provides siRNA, GalNAc-siRNA, and pharmaceutical compositions for inhibiting TTR gene expression in cells in vivo or in vitro, which promote degradation of TTR gene mRNA through RNAi, thereby reducing TTR gene expression in cells. In some embodiments, TTR gene expression is reduced or inhibited by at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, 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%, at least about 99%, or about 100%.

[0209] In some embodiments of the treatment and prevention provided herein, the subject is a mammal, such as a primate, rodent, or human. Administration of the GalNAc-siRNA or pharmaceutical composition of the present invention results in a decrease in serum TTR protein in the subject. Vutrisiran and Patisiran have been shown to effectively treat and prevent diseases or conditions mediated by TTR gene expression (e.g., transthyretin amyloidosis-related) by reducing serum TTR protein levels, including ATTR-PN (transthyretin amyloidosis polyneuropathy), ATTR-CM (transthyretin amyloidosis cardiomyopathy), and senile systemic amyloidosis.

[0210] Administration to a patient can be by any suitable route known in the art, including but not limited to, subcutaneous, intravenous, intramuscular, intrabronchial, intrapleural, intraperitoneal, intraarterial, lymphatic, and / or cerebrospinal.

[0211] The dosage of the RNAi agents and compositions disclosed herein can be determined based on the patient's weight, age, sex, severity of the disease, etc. Based on the amount of siRNA contained therein, the dosage of the RNAi agents and compositions disclosed herein is about 1-300 mg / kg body weight.

[0212] The frequency of administration can be daily, weekly, every two weeks, every three weeks, every 1 month, every 2 months, every 3 months, every 4 months, every 5 months, every 6 months, every 7 months, every 8 months, every 9 months, every 10 months, every 11 months or yearly, once or more.

[0213] The administration cycle is one or more times daily, weekly, biweekly, three-weekly, monthly, two-monthly, three-monthly, four-monthly, five-monthly, six-monthly, seven-monthly, eight-monthly, nine-monthly, ten-monthly, eleven-monthly, or annually. The total number of times the RNAi agent or composition is administered is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 times. For example, the RNAi agent or composition can be administered about 1, 2, 3, or 4 times.

[0214] In some embodiments, after introduction into cells expressing the thyroxine transporter (TTR) gene, the RNAi reagents and compositions disclosed herein can inhibit the expression of thyroxine transporter (TTR) mRNA. In some embodiments, the cells are hepatocellular carcinoma cells or primary hepatocytes. In some embodiments, the cells are human hepatocellular carcinoma cells or primary hepatocytes. In some embodiments, the human hepatocellular carcinoma cells are HepG2. In some embodiments, the human hepatocellular carcinoma cells are Hep3B. In some embodiments, the human primary hepatocytes are PHH. In some embodiments, the monkey primary hepatocytes are PCH.

[0215] Various drug delivery systems are known and can be used for the RNAi agents and compositions of the present disclosure, such as encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the compound, receptor-mediated endocytosis, constructing nucleic acids as part of retroviruses or other vectors. In some embodiments, the receptor-mediated endocytosis is endocytosis mediated by the binding of GalNAc to the asialoglycoprotein receptor (ASGPR) on the cell surface. In some embodiments, the drug delivery system is endocytosis mediated by the binding of GalNAc3 to the cell surface receptor ASGPR.

[0216] In some embodiments, the RNAi agents and compositions of the present invention can be packaged in a kit. The RNAi agent and pharmaceutically acceptable diluent, carrier or excipient in the kit are provided in liquid form or dry form. In some embodiments, the kit includes instructions for mixing the RNAi agent with a pharmaceutically acceptable diluent, carrier or excipient or other ingredients.

[0217] In some embodiments, BPR-30213022, BPR-30221223, BPR-30221686, BPR-30221617, BPR-30221618, BPR-30222201, and BPR-30222218 all knocked down serum TTR protein levels in mice on Day 7 after administration, with the highest knockdown rate reaching 96%. The highest knockdown rate for serum TTR protein in mice reached 90% on Day 42 after administration. See Figure 1 for details.

[0218] In some embodiments, the activity of GalNAc-siRNA is evaluated in cynomolgus monkeys. BPR-30221223, BPR-30221228, BPR-30221686, BPR-30221617, BPR-30221618, BPR-30221620, BPR-30222201, and BPR-3022218 of the present invention can effectively knock down the serum TTR protein level in cynomolgus monkeys after administration.

[0219] The present invention confirms that the RNAi reagents and compositions disclosed in the present invention reduce the production of thyroxine transporter by inhibiting the expression of thyroxine transporter messenger RNA (mRNA), thereby ultimately reducing or eliminating the deposition of pathogenic thyroxine transporter amyloid in the body.

[0220] Description of the abbreviations of materials used in this invention:

[0221] Unless otherwise specified, the following in vivo / in vitro experimental data were analyzed using GraphPad Prism statistical analysis software. The independent sample T test (T-test) was used for comparison between two groups of samples, and the one-way ANOVA test was used for comparison of multiple groups, followed by Dunnett's post hoc test.

[0222] Example 1: Preparation of hTTR siRNA

[0223] 1. Human thyroxine transporter (hTTR) mRNA sequence

[0224] NM_000371.4 (Gene ID: 7276) from the NCBI RefSeq library, as shown in SEQ ID NO: 884.

[0225] Based on the hTTR mRNA, 235 pairs of hTTR siRNAs were designed, as shown in Table 2.

[0226] Table 2 hTTR siRNA

[0227] In Table 2, SS is the sense strand and AS is the antisense strand; the capital letters "G", "C", "A" and "U" in the AS and SS chains represent ribonucleotides containing guanine, cytosine, adenine and uracil as bases, respectively.

[0228] 2. Preparation of modified siRNA

[0229] Table 2 hTTR siRNA was synthesized using an oligonucleotide solid-phase synthesis process. Specifically, the SS strand was modified as follows: positions 1, 2, 3, 4, 5, 6, 8, 12, 13, 14, 15, 16, 17, 18, 19, 20, and 21 were 2'-O-methyl modified nucleotides, and positions 7, 9, 10, and 11 were 2'-fluoro modified nucleotides. For the AS strand, positions 1, 3, 4, 5, 7, 8, 9, 10, 11, 12, 13, 15, 17, 18, 19, 20, 21, 22, and 23 were 2'-O-methyl modified nucleotides, and positions 2, 6, 14, and 16 were 2'-fluoro modified nucleotides, counting from the 5' end to the 3' end.

[0230] Using a universal solid phase synthesis carrier (UnyLinker TM Using loaded HL Solid Supports (Kinovate Life Sciences) as supports and phosphoramidite nucleoside monomers as starting materials, the nucleoside monomers were linked one by one in the 3'-5' direction according to the oligonucleotide arrangement order via phosphoramidite solid-phase synthesis. Aminolysis was then used to obtain a crude oligonucleotide. After purification, ultrafiltration, and lyophilization, the crude product yielded the two single-stranded oligonucleotides that comprised the siRNA. Finally, the two single-stranded oligonucleotides were annealed to form a double-stranded siRNA with reverse-complementary base pairing.

[0231] The solid-phase synthesis process consists of four steps (a, b, c, d), forming one cycle. This cycle is repeated, with the desired nucleoside phosphoramidite monomers of different types added sequentially according to the oligonucleotide sequence. The 5'-DMTr group at the end of the oligonucleotide is removed, and the cyanoethyl-protected phosphate is removed using a 20% (v / v) diethylamine solution in acetonitrile. The solid-phase reaction concludes, yielding a single-stranded oligonucleotide attached to a solid support.

[0232] a. Deprotection of dimethoxytrityl groups: DMTr (dimethoxytrityl) protecting groups on the vector / nucleotide are removed using dichloroacetic acid at room temperature (20-25°C) to obtain active hydroxyl groups that can undergo coupling reactions. The deprotection reagent is either a 3% (v / v) solution of dichloroacetic acid in toluene or a 3% (v / v) solution of dichloroacetic acid in dichloromethane.

[0233] b. Coupling Reaction: Nucleotide phosphoramidite monomers and an activator are simultaneously introduced into the solid-phase synthesis. The phosphoramidite groups are activated and undergo a coupling condensation reaction with the active hydroxyl groups to form a phosphite triester. The activator is a 0.6 M solution of 5-ethylthio-1H-tetrazole (ETT) in acetonitrile.

[0234] c. Oxidation reaction: Under the action of iodine water as an oxidant, the phosphite triester generated in the previous coupling condensation reaction is converted into a stable phosphate triester. The oxidant is a 0.04M iodine / water / pyridine solution with a v (water): v (pyridine) ratio of 1:9.

[0235] d. Capping reaction: The active hydroxyl groups that were not completely reacted during the coupling reaction were capped to prevent them from participating in subsequent reactions. The capping reagents were CapA (acetic anhydride / acetonitrile) and CapB (NMI:Py:acetonitrile = 2:3:5).

[0236] The solid support containing the oligonucleotide is aminolyzed using an aminolysis reagent (25% to 28% concentrated ammonia solution). This cleaves the oligonucleotide from the support and simultaneously removes the various protecting groups on the nucleoside bases. The resulting solution is concentrated to produce the crude oligonucleotide. The crude product is then sent for analysis, and the target molecular weight is determined using high-resolution liquid chromatography-mass spectrometry (LC-MS).

[0237] The crude oligonucleotides were purified by anion exchange chromatography and purified by gel column (HiTrap TM Desalting was performed. The desalted sense and antisense strands were mixed in an equimolar ratio, heated to 65°C, held for 30 minutes, and then naturally cooled to room temperature. The two single strands formed a double-stranded structure, i.e., siRNA, through hydrogen bonding. Molecular weight determination was performed using ion-pair reversed-phase chromatography (IPRP-HPLC) and high-resolution liquid chromatography-mass spectrometry (LC-MS) to confirm that the modified siRNAs with the motifs shown in Table 2 were prepared.

[0238] Example 2: Detection of hTTR siRNA activity in liver cancer cells

[0239] 1. Cell culture and transfection

[0240] HepG2 (ATCC, HB-8065) ​​and Hep3B (ATCC, HB-8064) liver cancer cell lines were cultured in EMEM medium (ATCC, 30-2003) at 37°C and 5% CO2 until near confluence, and then the cells were released from the plate by trypsinization. 5 μL of siRNA and 5 μL of mix (4.9 μL of Opti-MEM plus 0.1 μL of Lipofectamine RNAiMax (Invitrogen, catalog number 13778-150)) were added to each 96-well plate and incubated at room temperature for 15-20 minutes. Then, 90 μL of complete growth medium without antibiotics but containing 4,000 liver cancer cells was added to each well of the 96-well plate for reverse transfection; a negative control group was also set up for transfection without siRNA compound (i.e., only the solvent PBS).

[0241] 2. RT-qPCR

[0242] After incubation at 37°C and 5% CO₂ for 48 hours, the transfected cells were lysed, RNA was extracted, and RT-qPCR was performed. RNA extraction was performed using the Fast Pure Universal Plant Total RNA Isolation Kit (VAZYME, Catalog No. RC411-01), and reverse transcription was performed using the HiScriptIII 1st Strand cDNA Synthesis Kit (+gDNA wiper) (VAZYME, Catalog No. R312-01), all according to the manufacturer's instructions. PCR mixture (10 μl 2XSYBR Mix, 0.4 μl Primer mix (0.2 μl 10 μM forward primer and 0.2 μl 10 μM reverse primer)) was added to a 96-well plate (Applied Biosystems, Catalog No. 4326659). Then, 2 μl of the corresponding cDNA was added to each well and centrifuged to mix. qPCR was used to detect the expression levels of the target gene TTR and the internal reference gene GAPDH mRNA. The primer sequences are shown below:

[0243] TTR forward primer: 5′-CCCCTACTCCTATTCCACC-3′ (SEQ ID NO: 885);

[0244] TTR reverse primer: 5′-GAAATCCCATCCCTCGTC-3′ (SEQ ID NO: 886);

[0245] GAPDH forward primer: 5′-CCCTACTCCTATTCCACCAC-3′ (SEQ ID NO: 887);

[0246] GAPDH reverse primer: 5'-GAAATCCCATCCCTCGTC-3' (SEQ ID NO: 888).

[0247] The relative expression of TTR mRNA was calculated using 2 -ΔΔCT The calculation formula is as follows:

[0248] TTR mRNA relative expression = 2 -ΔΔCT

[0249] TTR mRNA relative inhibition rate (%) = (1-TTR mRNA relative expression level in the test group / TTR mRNA relative expression level in the negative control group) × 100%

[0250] 3. In vitro liver cancer cell activity screening results

[0251] The TTR inhibition rates of single-concentration siRNA in HepG2 cells varied from approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, and 90%, with a maximum inhibition rate of 98.60%. The TTR inhibition rates of single-concentration siRNA in Hep3B cells varied from approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, and 90%, with a maximum inhibition rate of 95.85%. The results are shown in Table 3.

[0252] Table 3

[0253] ND means the sample was not tested.

[0254] Table 4_Hep3B multi-concentration screening

[0255] Example 3: Preparation of 4'-modified threonucleosides

[0256] 1. Synthesis of Compound 11

[0257] 1) Synthesis of compound 2:

[0258] Dry compound 1 (20.0 g, 105.15 mmol, 1.0 eq.) was dissolved in DCM (200 mL). Imidazole (17.9 g, 262.88 mmol, 2.5 eq.) was added to the reaction system, and the reaction mixture was cooled to 0°C and stirred for 30 minutes. Tert-butyldiphenylsilyl chloride (31.79 g, 115.67 mmol, 1.1 eq.) was then slowly added dropwise to the reaction system. After the addition, the ice bath was removed and the reaction mixture was gradually allowed to return to room temperature. The reaction mixture was allowed to react overnight at room temperature. TLC confirmed the complete reaction of compound 1. Water was added to the reaction system, and the mixture was extracted twice with ethyl acetate. The organic phases were combined and washed with water and saturated brine. The organic phases were dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain the crude product. The crude product was purified by column chromatography (PE / EA = 100 / 15) to afford compound 2 (35.24 g, 82.29 mmol, 78.3% yield). ESI-MS: m / z 451.2[M+Na] +

[0259] 2) Synthesis of compound 3:

[0260] Compound 2 (38.2 g, 89.13 mmol, 1.0 eq.) was dissolved in DMF (300 mL) and stirred until completely dissolved. The reaction system was cooled to approximately 0°C. NaH (5.35 g, 133.70 mmol, 1.5 eq.) was slowly added portionwise to the reaction. After addition, the reaction system was stirred at 0°C for 30 minutes. Benzyl bromide (22.87 g, 133.70 mmol, 1.5 eq.) was slowly added dropwise to the reaction system, maintaining the reaction temperature at approximately 0°C. After addition, the reaction system was allowed to return to room temperature and allowed to react overnight. TLC confirmed the complete reaction of compound 2. The reaction system was slowly poured into a saturated aqueous ammonium chloride solution at approximately 0°C. The mixture was extracted twice with ethyl acetate, and the organic phases were combined. The organic phases were washed with water and saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain crude compound 3 (68.2 g), which was used directly in the next step. ESI-MS: m / z 519.3[M+H] +

[0261] 3) Synthesis of compound 4:

[0262] Crude compound 3 (68.2 g) was dissolved in THF (400 mL) and stirred until completely dissolved. TBAF (1 M in THF, 100 mL, 100 mmol, 1.12 eq.) was added to the reaction mixture, and stirred at room temperature overnight until reaction of compound 3 was complete. The reaction solution was concentrated under reduced pressure to remove the solvent, and the resulting crude product was purified by column chromatography (PE / EA = 10 / 3) to afford compound 4 (22.07 g, 78.73 mmol, 88.3% yield over two steps).

[0263] 4) Synthesis of compound 5:

[0264] Compound 4 (22.07 g, 78.73 mmol, 1.0 eq.) was dissolved in DMF (200 mL) and stirred thoroughly. The reaction mixture was cooled to 0°C and stirred for 30 minutes. Bromododecane (23.55 g, 94.48 mmol, 1.2 eq.) was slowly added dropwise to the reaction system. After addition, the reaction mixture was allowed to cool to room temperature and allowed to react overnight. TLC confirmed the complete reaction of compound 4. The reaction mixture was slowly poured into ice water to quench the mixture. The mixture was extracted twice with ethyl acetate, and the organic phases were combined. The organic phases were washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain the crude compound. The crude product was purified by column chromatography (PE / EA = 100 / 9) to afford compound 5 (32.0 g, 71.37 mmol, 90.6% yield).

[0265] 5) Synthesis of compound 6:

[0266] Compound 5 (35.32 g, 78.73 mmol, 1.0 eq.) was added to a 500 mL round-bottom three-necked flask. Acetic acid (120 mL) and acetic anhydride (40.19 g, 393.65 mmol, 5.0 eq.) were added to the reaction system and stirred until completely dissolved. The reaction mixture was cooled to 0°C and stirred at this temperature for 30 minutes. Concentrated sulfuric acid (5 mL) was slowly added dropwise to the reaction system, maintaining the temperature between 0 and 5°C. After addition, the reaction system was allowed to return to room temperature and stirred for 8 hours until compound 5 was completely reacted. The reaction system was cooled to approximately -5°C and neutralized with aqueous ammonia to a pH of approximately 7. Water was added to the reaction system, and the reaction mixture was extracted twice with ethyl acetate. The organic phases were combined. The organic phases were washed with water and saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to yield crude product 6. The crude product 6 was purified by column chromatography (PE / EA=10 / 2) to give compound 6 (19.0 g, 38.59 mmol, yield 49%).

[0267] 6) Synthesis of compound 7:

[0268] Compound 6 (5.0 g, 10.15 mmol, 1.00 eq.) and uracil (2.28 g, 20.30 mmol, 2.0 eq.) were added to a 500 mL three-necked round-bottom flask. Ultra-dry acetonitrile (60 mL) was added and stirred to dissolve. BSA (6.19 g, 30.45 mmol, 3.0 eq.) was then added. The reaction system was heated to 80°C in an oil bath and stirred at this temperature for 1 hour. The entire reaction was carried out under nitrogen. After completion of the reaction, the reaction system was stirred in a 0°C ice-water bath for 30 minutes. TMSOTf (2.26 g, 10.15 mmol, 1.0 eq.) was slowly added dropwise to the reaction system. After the addition, the reaction system was slowly heated to 80°C in an oil bath and allowed to react overnight at this temperature. TLC and LCMS analysis confirmed the complete reaction of compound 6. The reaction was removed from the oil bath and cooled to room temperature. Saturated aqueous sodium bicarbonate was added to the reaction system to quench the reaction. The system was extracted with ethyl acetate, and the organic phases were combined. The organic phases were washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain crude product 7. Column chromatography (PE / EA = 5 / 2) yielded compound 7 (4.71 g, 8.65 mmol, 85.2% yield).

[0269] 7) Synthesis of Compound 8:

[0270] Compound 7 (4.71 g, 8.65 mmol) was added to a 250 mL three-necked flask. Ultra-dry dichloromethane (50 mL) was added to the reaction flask. The reaction system was cooled to -20°C and stirred at this temperature for 30 minutes. Boron trichloride (1 M in toluene, 26 mL, 25.95 mmol, 3.0 eq.) was slowly added dropwise to the reaction system, and the reaction was continued for 5 hours until compound 7 was completely reacted. The reaction was quenched with triethylamine and methanol at -20°C. The reaction mixture was returned to room temperature and water was added. The mixture was extracted with ethyl acetate, and the organic phases were combined. The organic phases were washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain crude product 8. The crude product was purified by column chromatography (PE / EA = 2 / 3) to yield compound 8 (2.0 g, 4.4 mmol, 50.9% yield). ESI-MS: m / z 455.3 [M+H] +

[0271] 8) Synthesis of compound 9:

[0272] Compound 8 (2.0 g, 4.40 mmol, 1.0 eq.) was added to a 250 ml round-bottom flask. Ultra-dry DCE (30 mL) was added to the reaction flask and stirred until completely dissolved. DMTrCl (7.45 g, 22.00 mmol, 5.0 eq.), silver nitrate (747 mg, 4.40 mmol, 1.0 eq.), and 2,4,6-trimethylpyridine (5.33 g, 44 mmol, 10.0 eq.) were added to the reaction at room temperature and stirred until uniform. The reaction was heated to 80°C in an oil bath and allowed to react overnight. TLC and LCMS confirmed the complete reaction of compound 8. The reaction was returned to room temperature and quenched by the addition of methanol. The reaction was diluted with ethyl acetate and filtered through celite to obtain the filtrate. The filter cake was washed twice with ethyl acetate. The combined filtrates were concentrated under reduced pressure to yield crude compound 9. The crude product was purified by column chromatography (PE / EA=100 / 35) to give compound 9 (3.2 g, 4.23 mmol, yield 96%). ESI-MS: m / z 757.4 [M+H] +

[0273] 9) Synthesis of compound 10:

[0274] Compound 9 (3.2 g, 4.23 mmol, 1.0 eq.) was added to a 100 mL round-bottom flask. 7 M aqueous ammonia solution (50 mL) was then added to the reaction at room temperature. The reaction was stirred at room temperature for 3 hours until the reaction of compound 9 was complete. After completion of the reaction, the reaction system was concentrated under reduced pressure at 40°C to obtain crude product 10. This crude product was purified by column chromatography (PE / EA = 1 / 1) to afford compound 10 (2.9 g, 4.06 mmol, 96% yield).

[0275] 10) Synthesis of compound 11:

[0276] Dry compound 10 (2.9 g, 4.06 mmol, 1.0 eq.) was added to a 100 mL round-bottom flask, and ultra-dry dichloromethane (30 mL) was added and stirred until completely dissolved. DIPEA (1.05 g, 8.12 mmol, 2.0 eq.) and DMAP (99 mg, 0.81 mmol, 0.2 eq.) were added to the reaction mixture and stirred at room temperature for 15 minutes. The reaction system was purged with nitrogen and carried out under a nitrogen atmosphere. CEP-Cl (1.44 g, 6.09 mmol, 1.5 eq.) was added dropwise to the reaction mixture at room temperature and allowed to react for 30-60 minutes until compound 10 was completely reacted. The reaction system was quenched by the addition of a saturated aqueous sodium bicarbonate solution and extracted twice with dichloromethane. The organic phases were combined, washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to yield crude product 11. The crude product was purified by column chromatography (PE / EA=1 / 1) to give compound 11 (3.16 g, 3.46 mmol, yield 85.2%). 1 H NMR(400MHz, DMSO-d6)δ11.35(dd,J=14.6,2.2Hz,1H),7.93(d,J=8.1Hz,1H),7.47–7.36(m,2H),7. 33–7.23(m,7H),6.89–6.83(m,4H),5.71–5.51(m,2H),4.34(dt,J=8.8,4.7Hz,1H),4.11–3.90(m,1H ),3.84–3.76(m,1H),3.75(s,6H),3.69–3.36(m,6H),2.71(t,J=6.0Hz,1H),2.60–2.39(m,1H),1.6 2–1.52(m,2H),1.42–1.20(m,20H),1.10(t,J=6.6Hz,6H),1.02(d,J=6.7Hz,6H),0.89–0.81(m,3H). 31 P NMR(162MHz,DMSO-d6)δ151.30,148.65.,ESI-MS:m / z 915.6[M+H] +

[0277] 2. Synthesis of Compound 16

[0278] 1) Synthesis of compound 12:

[0279] Compound 10 (2.6 g, 3.64 mmol, 1.0 eq.) was added to a 100 mL round-bottom flask. Ultra-dry DMF (30 mL) was added and stirred to dissolve. Imidazole (991 mg, 14.56 mmol, 4.0 eq.) was added at room temperature and stirred for 10 minutes. TBSCl (1.10 g, 7.28 mmol, 2.0 eq.) was then slowly added portionwise to the reaction system. The reaction was allowed to proceed overnight at room temperature under nitrogen. TLC and LCMS confirmed the complete reaction of compound 10. Water was added to quench the reaction. The mixture was extracted twice with ethyl acetate, and the organic phases were combined. The organic phases were washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain the crude product. The crude product was purified by column chromatography (PE / EA = 2 / 1) to afford compound 12 (2.75 g, 3.32 mmol, 91.2% yield). ESI-MS: m / z 829.5[M+H] +

[0280] 2) Synthesis of compound 13:

[0281] Compound 12 (2.75 g, 3.32 mmol, 1.0 eq.) was added to a 250 mL round-bottom flask. Ultra-dry acetonitrile (30 mL) was added and stirred until completely dissolved. Triethylamine (672 mg, 6.64 mmol, 2.0 eq.) and DMAP (811 mg, 6.64 mmol, 2.0 eq.) were added to the reaction system and stirred until uniform. The reaction mixture was cooled to 0-5°C and TPSCl (2.01 g, 6.64 mmol, 2.0 eq.) was slowly added portionwise. After the addition, the ice bath was removed and the mixture was allowed to return to room temperature. The reaction was stirred at room temperature overnight. TLC confirmed the complete reaction of compound 12. Aqueous ammonia (20 mL) was added to the reaction mixture at room temperature and stirred for approximately 12 hours until the intermediate was completely reacted. Saturated brine was added to the reaction mixture, and the mixture was extracted twice with ethyl acetate. The organic phases were combined. The organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give crude product 13 (8.3 g, calculated based on 100% yield). ESI-MS: m / z 828.6 [M+H] +

[0282] 3) Synthesis of compound 14:

[0283] Crude compound 13 (8.3 g, 1.0 eq.) was added to a 100 mL round-bottom flask, and pyridine (50 mL) was added and stirred until the crude compound 13 was completely dissolved. The reaction system was cooled to 0°C, and BzCl (933 mg, 6.64 mmol, 2.0 eq.) was slowly added dropwise to the reaction system. The mixture was stirred at 0°C for 1 hour until the reaction of compound 13 was complete. Nitrogen was used throughout the reaction. The reaction system was returned to room temperature and quenched by adding methanol and water. The mixture was extracted twice with ethyl acetate, and the organic phases were combined. The organic phases were washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain the crude compound. The crude product was purified by column chromatography (PE / EA = 1 / 1) to obtain compound 14 (2.35 g, 2.52 mmol, 76% total yield for two steps). ESI-MS: m / z 931.5 [M+H] +

[0284] 4) Synthesis of Compound 15:

[0285] Compound 14 (2.35 g, 2.52 mmol, 1.0 eq.) was added to a 100 mL round-bottom flask, and THF (30 mL) was added and stirred until completely dissolved. Triethylamine trihydrofluoride (5.0 mL) was neutralized with triethylamine (17 mL) until alkaline and then added to the above reaction system. The reaction was placed in a 40°C oil bath and stirred overnight under nitrogen. TLC and LCMS confirmed the complete reaction of compound 14. Water was added to the reaction mixture, and the mixture was extracted twice with ethyl acetate. The organic phases were combined. The organic phases were washed with water, saturated sodium bicarbonate aqueous solution, and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain the crude product. The crude product was purified by column chromatography (PE / EA = 1 / 1) to obtain compound 15 (1.57 g, 1.92 mmol, 76.2% yield). ESI-MS: m / z 818.5 [M+H] +

[0286] 5) Synthesis of compound 16:

[0287] Dried compound 15 (1.57 g, 1.92 mmol) was added to a 100 mL round-bottom flask. Ultra-dry dichloromethane (20 mL) was added and stirred to dissolve. DIPEA (496 mg, 3.84 mmol, 2.0 eq.) and DMAP (47 mg, 0.38 mmol, 0.2 eq.) were added to the reaction mixture, and the reaction was purged with nitrogen. The reaction mixture was cooled to approximately 0°C, and CEP-Cl (682 mg, 2.93 mmol, 1.5 eq.) was slowly added dropwise. The reaction mixture was allowed to react at this temperature under nitrogen for 1 hour until compound 15 was completely reacted. After completion of the reaction, saturated sodium bicarbonate aqueous solution was added to the reaction mixture to quench the reaction. The mixture was extracted twice with dichloromethane, and the organic phases were combined. The organic phases were washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain the crude product. The crude product was purified by column chromatography (PE / EA=1 / 1) to give compound 16 (1.7 g, 1.67 mmol, yield 87%). 1 H NMR (400MHz, DMSO-d6) δ11.27(d,J=17.5Hz,1H),8.46(dd,J=30.3,7.5Hz,1H),8.01(d,J=7.6Hz,2H ),7.62(t,J=7.3Hz,1H),7.55–7.44(m,2H),7.39–7.13(m,9H),6.87–6.82(m,4H),5.71(d,J=25.0H z,1H),4.23–3.90(m,3H),3.78–3.38(m,13H),2.74(dd,J=10.7,5.6Hz,1H),2.61–2.32(m,1H),1.6 1–1.50(m,2H),1.38–1.19(m,20H),1.12–1.04(m,9H),0.96(d,J=6.7Hz,3H),0.83(t,J=6.6Hz,3H), 31 P NMR(162MHz,DMSO-d6)δ149.87,149.34,ESI-MS:m / z 1018.6[M+H] +

[0288] 3. Synthesis of Compound 21

[0289] 1) Synthesis of compound 17:

[0290] Compound 6 (27 g, 54.81 mmol, 1.00 eq.) was added to a 500 mL three-necked round-bottom flask. Ultra-dry acetonitrile (250 mL) was added and stirred to dissolve. BSA (33.45 g, 164.43 mmol, 3.0 eq.) and ABz (26.22 g, 109.62 mmol, 2.0 eq.) were then added. The reaction system was heated to 80°C in an oil bath and stirred at this temperature for 1 hour. The entire reaction was carried out under nitrogen. After completion of the reaction, the reaction system was stirred in a 0°C ice-water bath for 30 minutes. TMSOTf (12.18 g, 54.81 mmol, 1.0 eq.) was slowly added dropwise to the reaction system. After the addition, the reaction system was slowly heated to 80°C in an oil bath and allowed to react overnight at this temperature. TLC and LCMS analysis confirmed the complete reaction of compound 6. The reaction was removed from the oil bath and cooled to room temperature. Saturated aqueous sodium bicarbonate was added to the reaction system to quench the reaction. The system was extracted with ethyl acetate, and the organic phases were combined. The organic phases were washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain crude product 17. Column chromatography (PE / EA = 1 / 1) yielded compound 17 (25 g, 37.23 mmol, 67.9% yield).

[0291] 2) Synthesis of compound 18:

[0292] Compound 17 (3.0 g, 4.47 mmol, 1.0 eq.) was added to a 250 mL three-necked flask. Extra-dry dichloromethane (30 mL) was added to the reaction flask. The reaction system was cooled to -10°C and stirred at this temperature for 30 minutes. A 1.0 mol / L boron trichloride solution in dichloromethane (13.4 mL, 3.0 eq.) was slowly added dropwise to the reaction system, and the reaction was continued for 5 hours until compound 17 was fully reacted. The reaction was quenched with triethylamine and methanol at -20°C. The reaction mixture was cooled to room temperature, water was added, and the mixture was extracted with ethyl acetate. The organic phases were combined. The organic phases were washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to yield crude product 18. The crude product was purified by column chromatography (PE / EA = 1 / 2) to yield compound 18 (1.0 g, 1.72 mmol, 38.5% yield).

[0293] 3) Synthesis of compound 19:

[0294] Compound 18 (1.0 g, 1.72 mmol, 1.0 eq.) was added to a 100 mL round-bottom flask. Ultra-dry DCE (15 mL) was added to the reaction flask and stirred until completely dissolved. DMTrCl (2.9 g, 8.6 mmol, 5.0 eq.), silver nitrate (0.29 g, 1.72 mmol, 1.0 eq.), and 2,4,6-trimethylpyridine (2.08 g, 17.2 mmol, 10.0 eq.) were added to the reaction at room temperature and stirred until uniform. The reaction was heated to 80°C in an oil bath and allowed to react overnight. TLC and LCMS confirmed the complete reaction of compound 18. The reaction was returned to room temperature and quenched by the addition of methanol. The reaction was diluted with ethyl acetate and filtered through celite to obtain the filtrate. The filter cake was washed twice with ethyl acetate. The combined filtrates were concentrated under reduced pressure to obtain crude compound 19. The crude product was purified by column chromatography (PE / EA=2 / 3) to give compound 19 (1.2 g, 1.36 mmol, yield 72%). ESI-MS: m / z 884.5 [M+H] +

[0295] 4) Synthesis of Compound 20:

[0296] Compound 19 (1.2 g, 1.36 mmol, 1.0 eq.) was added to a 100 mL round-bottom flask. THF (10 mL) was added and the reaction was stirred until completely dissolved. A 5.4 mol / L sodium methoxide solution (0.76 mL, 4.08 mmol, 3.0 eq.) was added to the reaction mixture and stirred at room temperature for 3 hours until compound 19 was completely reacted. After completion of the reaction, the reaction system was concentrated under reduced pressure at 40°C to obtain crude product 20. Column chromatography of the crude product afforded compound 20 (0.82 g, 0.97 mmol, 73% yield). ESI-MS: m / z 842.4 [M+H] +

[0297] 5) Synthesis of Compound 21:

[0298] Dried compound 20 (0.82 g, 0.97 mmol, 1.0 eq.) was added to a 50 mL round-bottom flask, and ultra-dry dichloromethane (10 mL) was added and stirred until completely dissolved. DIPEA (0.25 g, 1.94 mmol, 2.0 eq.) and DMAP (23.8 mg, 0.194 mmol, 0.2 eq.) were added to the reaction mixture and stirred at room temperature for 15 minutes. The reaction system was purged with nitrogen and carried out under a nitrogen atmosphere. CEP-Cl (0.35 g, 1.46 mmol, 1.5 eq.) was added dropwise to the reaction mixture at room temperature and allowed to react for 30-60 minutes until compound 20 was completely reacted. The reaction system was quenched by the addition of a saturated aqueous sodium bicarbonate solution and extracted twice with dichloromethane. The organic phases were combined, washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to yield crude product 21. The crude product was purified by column chromatography to obtain compound 21 (0.74 g, 0.71 mmol, 73.2% yield). 1 H NMR (400MHz, DMSO-d6) δ11.21(d,J=4.0Hz,1H),8.71(d,J=5.9Hz,1H),8.60(d,J=29.9Hz ,1H),8.04(d,J=7.6Hz,2H),7.65(t,J=7.3Hz,1H),7.57(t,J=7.6Hz,2H),7.35(t,J=8.6H z,2H),7.21–7.12(m,7H),6.83–6.71(m,4H),6.02(dd,J=36.3,3.4Hz,1H),4.78–4.31(m ,2H),3.81–3.36(m,15H),2.67–2.38(m,2H),1.62–1.48(m,2H),1.42–1.21(m,18H),1.12 –0.99(m,9H),0.93–0.78(m,6H).ESI-MS:m / z 1042.6[M+H] +

[0299] 4. Synthesis of Compound 26

[0300] 1) Synthesis of compound 22:

[0301] Compound 6 (20 g, 40.60 mmol, 1.00 eq.) was added to a 500 mL three-necked round-bottom flask. Ultra-dry acetonitrile (200 mL) was added and stirred to dissolve. BSA (24.78 g, 121.8 mmol, 3.0 eq.) and GiBu (17.96 g, 81.2 mmol, 2.0 eq.) were then added. The reaction system was heated to 80°C in an oil bath and stirred at this temperature for 1 hour. The entire reaction was carried out under nitrogen. After completion of the reaction, the reaction system was stirred in a 0°C ice-water bath for 30 minutes. TMSOTf (9.02 g, 40.6 mmol, 1.0 eq.) was slowly added dropwise to the reaction system. After the addition, the reaction system was slowly heated to 80°C in an oil bath and allowed to react overnight at this temperature. TLC and LCMS analysis confirmed the complete reaction of compound 6. The reaction was removed from the oil bath and cooled to room temperature. Saturated aqueous sodium bicarbonate solution was added to the reaction system to quench the reaction. The system was extracted with ethyl acetate, and the organic phases were combined. The organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain crude product 22. The crude product was purified by column chromatography (PE / EA = 1 / 1) to obtain compound 22 (18 g, 27.53 mmol, 67.8% yield). ESI-MS: m / z 654.5 [M+H] +

[0302] 2) Synthesis of compound 23:

[0303] Compound 22 (5.0 g, 7.64 mmol, 1.0 eq.) was added to a 250 mL three-necked flask. Extra-dry dichloromethane (50 mL) was added to the reaction flask. The reaction system was cooled to -20°C and stirred at this temperature for 30 minutes. A 1.0 mol / L boron trichloride solution in dichloromethane (22.92 mL, 3.0 eq.) was slowly added dropwise to the reaction system, and the reaction was continued for 5 hours until compound 22 was fully reacted. The reaction was quenched with triethylamine and methanol at -20°C. The reaction mixture was returned to room temperature and water was added. The mixture was extracted with ethyl acetate, and the organic phases were combined. The organic phases were washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to yield crude product 23. The crude product was purified by column chromatography (PE / EA = 1 / 2) to yield compound 23 (2.6 g, 4.61 mmol, 60.3% yield). ESI-MS: m / z 564.3 [M+H] +

[0304] 3) Synthesis of compound 24:

[0305] Compound 23 (2.6 g, 4.61 mmol, 1.0 eq.) was added to a 250 mL round-bottom flask. Ultra-dry DCE (35 mL) was added to the reaction flask and stirred until completely dissolved. DMTrCl (7.8 g, 23.05 mmol, 5.0 eq.), silver nitrate (0.8 g, 4.61 mmol, 1.0 eq.), and 2,4,6-trimethylpyridine (5.59 g, 46.1 mmol, 10.0 eq.) were added to the reaction at room temperature and stirred until uniform. The reaction was heated to 80°C in an oil bath and allowed to react overnight. TLC and LCMS confirmed the complete reaction of compound 23. The reaction was returned to room temperature and quenched by the addition of methanol. The reaction was diluted with ethyl acetate and filtered through celite to obtain the filtrate. The filter cake was washed twice with ethyl acetate. The combined filtrates were concentrated under reduced pressure to yield crude compound 24. The crude product was purified by column chromatography (PE / EA=2 / 3) to give compound 24 (3.18 g, 3.67 mmol, yield 79.6%). ESI-MS: m / z 866.6 [M+H] +

[0306] 4) Synthesis of Compound 25:

[0307] Compound 24 (3.18 g, 3.67 mmol, 1.0 eq.) was added to a 100 mL round-bottom flask. THF (30 mL) was added to the reaction and stirred until completely dissolved. 5.4 mol / L sodium methoxide solution (2.04 mL, 11.01 mmol, 3.0 eq.) was added to the reaction and stirred at room temperature for 3 hours until compound 24 was completely reacted. After the reaction was complete, the reaction system was concentrated under reduced pressure at 40°C to obtain crude product 25. The crude product was purified by column chromatography to obtain compound 25 (2.48 g, 3.01 mmol, 82.0% yield). ESI-MS: m / z 824.5 [M+H]+

[0308] 5) Synthesis of Compound 26:

[0309] Dry compound 25 (2.0 g, 2.43 mmol, 1.0 eq.) was added to a 50 mL round-bottom flask, and ultra-dry dichloromethane (30 mL) was added and stirred until completely dissolved. DIPEA (0.63 g, 4.86 mmol, 2.0 eq.) and DMAP (59.4 mg, 0.486 mmol, 0.2 eq.) were added to the reaction mixture and stirred at room temperature for 15 minutes. The reaction system was purged with nitrogen and carried out under a nitrogen atmosphere. CEP-Cl (0.86 g, 3.65 mmol, 1.5 eq.) was added dropwise at room temperature and allowed to react at room temperature for 30-60 minutes until compound 25 was completely reacted. The reaction system was quenched by the addition of saturated aqueous sodium bicarbonate solution and extracted twice with dichloromethane. The organic phases were combined, washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to yield crude product 26. The crude product was purified by column chromatography to give compound 26 (2.08 g, 2.06 mmol, 83.5% yield). 1 H NMR (400MHz, DMSO-d6) δ12.08(s,1H),11.48(s,1H),8.18(d,J=29.6Hz,1H),7.52–7.11(m,9H),6.93– 6.84(m,4H),5.72(dd,J=38.6,4.8Hz,1H),5.21–5.04(m,1H),4.53(t,J=5.5Hz,1H),3.81–3.69(m,8H ),3.61–3.34(m,3H),3.30–3.23(m,2H),3.11(d,J=8.4Hz,1H),2.79–2.64(m,1H),2.59–2.48(m,2H), 2.46–2.35(m,1H),1.51(d,J=6.6Hz,2H),1.38–1.18(m,18H),1.13–0.98(m,13H),0.88–0.74(m,8H). 31 P NMR(162MHz,DMSO-d6)δ151.94,148.86,ESI-MS:m / z 1024.6[M+H] +

[0310] 5. Synthesis of Compound 27

[0311] Nucleoside compound 27 is a 2'-phosphoramidite group, 3'-ODMTr, Base=U, R=C 14 H 29 The preparation route refers to compound 11, except that the intermediate compound 4 is used to synthesize compound 5 from raw material C. 12 H 25Br(bromododecane) is changed to C 14 H 29 Br, the remaining steps are similar to compound 11, and the structure and NMR data of the prepared nucleoside compound 27 are shown below:

[0312] 1 H NMR(400MHz, DMSO-d6)δ11.35(dd,J=14.6,2.2Hz,1H),7.93(d,J=8.1Hz,1H),7.47–7.36(m,2H),7. 33–7.23(m,7H),6.89–6.83(m,4H),5.71–5.51(m,2H),4.34(dt,J=8.8,4.7Hz,1H),4.11–3.90(m,1H ),3.84–3.76(m,1H),3.75(s,6H),3.69–3.36(m,6H),2.71(t,J=6.0Hz,1H),2.60–2.39(m,1H),1.6 2–1.52(m,2H),1.42–1.20(m,24H),1.10(t,J=6.6Hz,6H),1.02(d,J=6.7Hz,6H),0.89–0.81(m,3H). 31 P NMR(162MHz,DMSO-d6)δ151.32,148.63.,ESI-MS:m / z 943.3[M+H] +

[0313] 6. Synthesis of Compound 28

[0314] Nucleoside compound 28 is a 2'-phosphoramidite group, 3'-ODMTr, Nu=U, R=C 16 H 33 The preparation route refers to compound 11, except that the intermediate compound 4 is used to synthesize compound 5 from raw material C. 12 H 25 Br changed to C 16 H 33 Br, the remaining steps are similar to compound 11, and the structure and NMR data of the prepared nucleoside compound 28 are shown below:

[0315] 1H NMR(400MHz, DMSO-d6)δ11.35(dd,J=14.6,2.2Hz,1H),7.93(d,J=8.1Hz,1H),7.47–7.36(m,2H),7. 33–7.23(m,7H),6.89–6.83(m,4H),5.71–5.51(m,2H),4.34(dt,J=8.8,4.7Hz,1H),4.11–3.90(m,1H ),3.84–3.76(m,1H),3.75(s,6H),3.69–3.36(m,6H),2.71(t,J=6.0Hz,1H),2.60–2.39(m,1H),1.6 2–1.52(m,2H),1.42–1.20(m,28H),1.10(t,J=6.6Hz,6H),1.02(d,J=6.7Hz,6H),0.89–0.81(m,3H). 31 P NMR(162MHz,DMSO-d6)δ151.35,148.66.,ESI-MS:m / z 971.5[M+H] +

[0316] 7. Synthesis of Compound 29

[0317] Nucleoside compound 29 is a 2'-phosphoramidite group, 3'-ODMTr, Nu=C, R=C 14 H 29 The preparation route refers to compound 16, except that R=C in the starting compound 10. 12 H 25 Change to C 14 H 29 The remaining steps are similar to those of compound 16. The structure and NMR data of the prepared nucleoside compound 29 are shown below:

[0318] 1H NMR (400MHz, DMSO-d6) δ11.27(d,J=17.5Hz,1H),8.46(dd,J=30.3,7.5Hz,1H),8.01(d,J= 7.6Hz,2H),7.62(t,J=7.3Hz,1H),7.55–7.44(m,2H),7.39–7.13(m,9H),6.87–6.82(m,4H) ,5.71(d,J=25.0Hz,1H),4.23–3.90(m,3H),3.78–3.38(m,13H),2.74(dd,J=10.7,5.6Hz, 1H),2.61–2.32(m,1H),1.61–1.50(m,2H),1.38–1.19(m,24H),1.12–1.04(m,9H),0.96(d, J=6.7Hz,3H),0.83(t,J=6.6Hz,3H), 31 P NMR(162MHz,DMSO-d6)δ149.82,149.30,ESI-MS:m / z 1045.6[M+H] +

[0319] 8. Synthesis of Compound 30

[0320] Nucleoside compound 30 is a 2'-phosphoramidite group, 3'-ODMTr, Nu=C, R=C 16 H 33 The preparation route refers to compound 16, except that R=C in the starting compound 10. 12 H 33 Change to C 16 H 29 The remaining steps are similar to those of compound 16. The structure and NMR data of the prepared nucleoside compound 30 are shown below:

[0321] 1H NMR (400MHz, DMSO-d6) δ11.27(d,J=17.5Hz,1H),8.46(dd,J=30.3,7.5Hz,1H),8.01(d,J=7.6Hz,2H ),7.62(t,J=7.3Hz,1H),7.55–7.44(m,2H),7.39–7.13(m,9H),6.87–6.82(m,4H),5.71(d,J=25.0H z,1H),4.23–3.90(m,3H),3.78–3.38(m,13H),2.74(dd,J=10.7,5.6Hz,1H),2.61–2.32(m,1H),1.6 1–1.50(m,2H),1.38–1.19(m,28H),1.12–1.04(m,9H),0.96(d,J=6.7Hz,3H),0.83(t,J=6.6Hz,3H), 31 P NMR(162MHz,DMSO-d6)δ149.79,149.31,ESI-MS:m / z 1073.6[M+H] +

[0322] 9. Synthesis of Compound 31

[0323] Nucleoside compound 31 is a 2'-phosphoramidite group, 3'-ODMTr, Nu=A, R=C 14 H 33 The preparation route refers to compound 21, except that R=C in the starting compound 6 12 H 33 Change to C 14 H 29 The remaining steps are similar to those of compound 21. The structure and NMR data of the prepared nucleoside compound 31 are shown below:

[0324] 1H NMR (400MHz, DMSO-d6) δ11.26(d,J=4.0Hz,1H),8.74(d,J=5.9Hz,1H),8.56(d,J=29.9Hz,1H),8. 24(d,J=7.6Hz,2H),7.65(t,J=7.3Hz,1H),7.53(t,J=7.6Hz,2H),7.36(t,J=8.6Hz,2H),7.24–7.1 2(m,7H),6.80–6.61(m,4H),6.03(dd,J=36.3,3.4Hz,1H),4.74–4.32(m,2H),3.82–3.39(m,15H) ,2.67–2.35(m,2H),1.61–1.49(m,2H),1.41–1.20(m,22H),1.03–0.96(m,9H),0.90–0.76(m,6H). 31 P NMR(162MHz,DMSO-d6)δ149.66,148.93.ESI-MS:m / z 1070.5[M+H] +

[0325] 10. Synthesis of Compound 32

[0326] Nucleoside compound 32 is a 2'-phosphoramidite group, 3'-ODMTr, Nu=A, R=C 16 H 33 The preparation route refers to compound 21, except that R=C in the starting compound 6 12 H 33 Change to C 16 H 29 The remaining steps are similar to those of compound 21. The structure and NMR data of the prepared nucleoside compound 32 are shown below:

[0327] 1H NMR (400MHz, DMSO-d6) δ11.19(d,J=4.0Hz,1H),8.71(d,J=5.9Hz,1H),8.49(d,J=29.9Hz,1H),8. 27(d,J=7.6Hz,2H),7.35(t,J=7.3Hz,1H),7.55(t,J=7.6Hz,2H),7.37(t,J=8.6Hz,2H),7.25–7.1 7(m,7H),6.81–6.60(m,4H),6.05(dd,J=36.3,3.4Hz,1H),4.75–4.33(m,2H),3.89–3.37(m,15H) ,2.69–2.32(m,2H),1.62–1.45(m,2H),1.43–1.22(m,26H),1.13–0.79(m,9H),0.91–0.77(m,6H). 31 P NMR(162MHz,DMSO-d6)δ149.29,148.76.ESI-MS:m / z1098.4[M+H] +

[0328] 11. Synthesis of Compound 33

[0329] Nucleoside compound 33 is a 2'-phosphoramidite group, 3'-ODMTr, Nu=G, R=C 14 H 33 The preparation route refers to compound 21, except that R=C in the starting compound 6 12 H 33 Change to C 14 H 29 The remaining steps are similar to those of compound 21. The structure and NMR data of the prepared nucleoside compound 33 are shown below:

[0330] 1 H NMR (400MHz, DMSO-d6) δ12.08(s,1H),11.48(s,1H),8.18(d,J=29.6Hz,1H),7.52–

[0331] 7.11(m,9H),6.93–6.84(m,4H),5.72(dd,J=38.6,4.8Hz,1H),5.21–5.04(m,1H) ,4.53(t,J=5.5Hz,1H),3.81–3.69(m,8H),3.61–3.34(m,3H),3.30–3.23(m,2H), 3.11(d,J=8.4Hz,1H),2.79–2.64(m,1H),2.59–2.48(m,2H),2.46–2.35(m,1H),1 .51(d,J=6.6Hz,2H),1.38–1.18(m,22H),1.13–0.98(m,13H),0.88–0.74(m,8H). 31 P NMR(162MHz,DMSO-d6)δ151.92,148.83,ESI-MS:m / z 1038.3[M+H] +

[0332] 12. Synthesis of Compound 34

[0333] Nucleoside compound 34 is a 2'-phosphoramidite group, 3'-ODMTr, Nu=G, R=C 16 H 33 The preparation route refers to compound 21, except that R=C in the starting compound 6 12 H 33 Change to C 16 H 29 The remaining steps are similar to those of compound 21. The structure and NMR data of the prepared nucleoside compound 34 are shown below:

[0334] 1H NMR (400MHz, DMSO-d6) δ12.08(s,1H),11.48(s,1H),8.18(d,J=29.6Hz,1H),7.52– 7.11(m,9H),6.93–6.84(m,4H),5.72(dd,J=38.6,4.8Hz,1H),5.21–5.04(m,1H) ,4.53(t,J=5.5Hz,1H),3.81–3.69(m,8H),3.61–3.34(m,3H),3.30–3.23(m,2H), 3.11(d,J=8.4Hz,1H),2.79–2.64(m,1H),2.59–2.48(m,2H),2.46–2.35(m,1H),1 .51(d,J=6.6Hz,2H),1.38–1.18(m,22H),1.13–0.98(m,13H),0.88–0.74(m,8H). 31 P NMR(162MHz,DMSO-d6)δ151.96,148.87,ESI-MS:m / z 1066.5[M+H] +

[0335] Example 4: Preparation of GalNAc-siRNA conjugate

[0336] 1. The synthesis process of GalNAc-siRNA conjugate is as follows:

[0337] According to the above process steps, L96 (GalNAc3) and an amino carrier (polystyrene resin or CPG powder) are coupled and capped to prepare a solid-phase synthetic carrier pre-loaded with L96. The carrier loading capacity is measured through analysis and testing.

[0338] 2. Load the solid-phase synthesis support onto a solid-phase reaction synthesis column and install a nucleic acid synthesizer. Using a standard oligonucleotide solid-phase synthesis method, synthesize the L96-coupled oligonucleotide sequence (modified SS chain) by cycling the four steps of dedimethoxytritylation, coupling, oxidation, and capping reactions as described in Example 1. If the target product has a phosphorothioate modification, a thiolation reaction is used instead of an oxidation reaction. Specifically, the oxidation reaction in step c. of Example 1 is replaced with a thiolation reaction: the phosphite triester generated in the previous coupling condensation step is converted to a stable phosphorothioate triester using the thiolation reagent PADS / ADTT. To synthesize the desired sequence, remove the cyanoethyl protecting group with 20% diethylamine, dry the solid-phase support with argon gas, and deprotect the support by ammoniation with concentrated ammonia at 55°C for 5 to 16 hours. After deprotection, remove the support by filtration. The ammonium hydrolysis solution is concentrated to remove the ammonia, and the remaining concentrated sample is analyzed by LC-MS to confirm that the sample molecular weight is consistent with the theoretical molecular weight.

[0339] 3. The L96-coupled oligonucleotide sample (L96 is located at the 3' end of the modified SS chain) was purified on an AKTA Pure 150 using an ion exchange method and desalted on a gel column to obtain a single-stranded sample that meets the requirements.

[0340] 4. Use the universal solid-phase synthesis carrier Nitto Phase HLUny Linker to synthesize the antisense chain (modified AS chain) according to the standard solid-phase synthesis method.

[0341] 5. Add the modified SS chain and the modified AS chain into the annealing container in an equal molar ratio, heat to 55°C, maintain for about 30 minutes, and cool naturally to room temperature.

[0342] 6. The annealed double-stranded samples are divided into freeze-drying containers according to the required amount and freeze-dried to obtain GalNAc-siRNA conjugates.

[0343] The structure of the GalNAc-siRNA conjugate is shown in formula (B-III). In each GalNAc-siRNA, the 3' end of the sense strand is coupled with L96:

[0344] in represents the double-stranded oligonucleotide of the RNAi, 3' represents the 3' end of the sense strand, and X is O or S.

[0345] The sense and antisense strands contained in the GalNAc-siRNA conjugates are shown in Table 5. Each conjugate was analyzed by LC-MS to confirm identity, quantified by UV (260 nm), and determined by HPLC analysis for purity.

[0346] Table 5 Sense and antisense chains contained in the conjugates

[0347] Table 6

[0348] Table 7

[0349] In Table 6 and Table 7, the meanings of the symbols in the sequence are shown in Table 8.

[0350] Table 8

[0351] Example 5: Detection of free uptake activity of conjugates in primary hepatocytes

[0352] On day 0, cryopreserved primary human hepatocytes (PHH) or primary cynomogus hepatocytes (PCH) were thawed and the cells were adjusted to 6×10 5Cells were plated at 400 μL of siRNA per well and 90 μL was inoculated into a 96-well plate. The culture medium was Invitro GROCP Medium (BioIVT, catalog number Z990005) containing 10% FBS and 1% P / S. Simultaneously, 10 μL of the tested GalNAc-siRNA was added to the plate. A control well without GalNAc-siRNA (PBS negative control group) was also set up. Each sample was tested at 3 or 2 concentrations, with 3 replicate wells for each concentration. After 48 hours of culture, cells were collected and RNA was extracted. RT-qPCR was used to detect the mRNA expression level of the TTR gene in the sample (probe ID: Hs00174914), and the mRNA expression level of the internal reference gene GAPDH (probe ID: Hs02786624) was also detected. Intracellular RNA was extracted using the RNeasy kit (QIAGEN-74182) according to the instructions, and reverse transcribed into cDNA using the Fast King RT Kit (With gDNase) (TIANGEN-KR116), followed by qPCR quantification.

[0353] The relative expression of TTR mRNA was calculated using 2 -ΔΔCT The calculation formula is as follows:

[0354] TTR mRNA relative expression = 2 -ΔΔCT

[0355] TTR mRNA relative inhibition rate (%) = (1-TTR mRNA relative expression level in the test group / TTR mRNA relative expression level in the negative control group) × 100%

[0356] The results of the free uptake activity test of the conjugates in primary hepatocytes are shown in Tables 9, 10 and 11.

[0357] Table 9 Results of free uptake activity of conjugates in human primary hepatocytes

[0358] The results in Table 9 show that in the free uptake test of human primary hepatocytes, at a dose of 20 nM, the average maximum inhibition rate of TTR gene was 94.45%; at a dose of 10 nM, the average maximum inhibition rate of TTR gene was 92.05%; at a dose of 5 nM, the average maximum inhibition rate of TTR gene was 88.55%.

[0359] Table 10 Free uptake activity results of conjugates in monkey primary hepatocytes

[0360] The results in Table 10 show that in the free uptake test of monkey primary hepatocytes, at a dose of 20 nM, the average maximum inhibition rate of TTR gene was 92.61%; at a dose of 10 nM, the average maximum inhibition rate of TTR gene was 84.26%.

[0361] Table 11 Results of free uptake activity of conjugates in monkey primary hepatocytes

[0362] Note: AD-649282 and AD-649286 are derived from patent WO2023014677A1

[0363] The results in Table 11 show that in the free uptake test of monkey primary hepatocytes, at different screening concentrations, the conjugates screened by the present invention have equivalent or better TTR gene inhibition effects than the positive control.

[0364] Example 6: Drug Efficacy Evaluation in Humanized Mice

[0365] In this example, a single subcutaneous injection of a TTR-humanized mouse, B6-hTTR (Jiangsu Jicui Yaokang Biotechnology Co., Ltd.), was performed to investigate the ability of the pharmaceutical composition to inhibit TTR gene expression in vivo. At least one week in advance, 6-8-week-old male mice were placed in a fertilization room for adaptive feeding. During the adaptive feeding period, blood samples (pre-dose blood samples) and serum TTR protein levels were collected. After completion of the adaptive feeding period, mice were randomly divided into groups based on body weight and pre-dose serum TTR protein levels, with 3-5 animals per group. An AD-65492 (see Table 3 of CN108138182A) positive control group, a PBS negative control group, a BPR-30213022 experimental group, a BPR-30221223 experimental group, a BPR-30221686 experimental group, a BPR-30221617 experimental group, a BPR-30221618 experimental group, a BPR-30222201 experimental group, and a BPR-30222218 experimental group were set up. Based on the weight of the mice, a single subcutaneous injection dose of 3 mg / kg was administered in a volume of 10 μL / g. The day of administration was designated as Day 0. In addition, experimental groups for BPR-30221672, BPR-30221676, BPR-30221678, and BPR-30221686 were established. Based on mouse body weight, a single subcutaneous dose of 1 mg / kg was administered in a volume of 10 μL / g, with dosing occurring on Day 0. Blood samples were collected at various time points after dosing, including Day 7, Day 14, Day 21, Day 28, and Day 35 / Day 42. Approximately 100 μL of blood was collected and centrifuged to obtain serum. Serum TTR protein was quantitatively assayed using an ELISA kit (Abcam ab231920), following the same procedures as described in the kit's instructions. The relative inhibition rate of serum TTR protein at each time point after dosing was calculated for each animal using the formula: 100% * (TTR serum protein level at each time point after dosing / TTR serum protein level before dosing in the corresponding individual - 1). The results are shown in Figures 1 and 2.

[0366] Figure 1 shows that BPR-30213022, BPR-30221223, BPR-30221686, BPR-30221617, BPR-30221618, BPR-30222201, and BPR-30222218 all knocked down serum TTR protein levels in mice on Day 7 after administration, with the highest knockdown rate reaching 96%. On Day 42 after administration, the highest knockdown rate for serum TTR protein in mice reached 90%. Some conjugates exhibited superior activity to AD-65492, as described in patent CN108138182A.

[0367] Figure 2 shows that at 28 days and 35 days after administration, the TTR protein inhibition rate of BPR-30221676 was weaker than that of BPR-30221672, BPR-30221678 and BPR-30221686, and the TTR protein inhibition rate of BPR-30221672 was weaker than that of BPR-30221678 and BPR-30221686.

[0368] Example 7: Evaluation of drug efficacy in cynomolgus monkeys

[0369] In this example, a single subcutaneous dose of the pharmaceutical composition was administered to non-human primates to investigate its ability to inhibit TTR gene expression in vivo. Wild male cynomolgus macaques were pre-adapted to the acclimation setting. During this period, blood was collected (as a pre-dose blood sample) and serum TTR protein levels were measured. After completion of the acclimation period, the macaques were randomly divided into groups based on body weight and pre-dose serum TTR protein levels, with 2-3 animals per group. A positive control group (AD-65492, see Table 3 of CN108138182A) and a negative control group (PBS) were set up, along with experimental groups of BPR-30221223, BPR-30221228, BPR-30221617, BPR-30221618, BRP-30221620, BPR-30221678, BPR-30221686, BRP-30222201, and BPR-30222218. The single subcutaneous injection dose was 1 g / kg, and the administration volume was 1 ml / kg. Day 0 was designated as the day of administration. Blood samples were collected at various time points after administration, including Day 7, Day 14, Day 21, Day 28, Day 35, and Day 42, and serum was obtained after centrifugation. Serum TTR protein was quantitatively detected using an ELISA kit (Abcam ab231920). Specific procedures were as described in the kit instructions. The relative inhibition rate of serum TTR protein at each time point after administration to each animal was calculated as follows:

[0370] The relative inhibition rate of serum TTR protein at each time point after administration = 100% × (TTR serum protein level at each time point after administration / TTR serum protein level before administration of the corresponding individual - 1).

[0371] The serum TTR protein content of each group of mice decreased significantly over time. On the 35th day after administration, the maximum inhibition rate of the experimental group compared with that before administration was as high as 90%. Details of some experimental groups are shown in Figure 3.

[0372] The above describes in detail the specific embodiments of the present disclosure. However, the present disclosure is not limited to the specific details of the above embodiments. Within the technical concept of the present disclosure, various simple modifications can be made to the technical solutions of the present disclosure, and these simple modifications all fall within the scope of protection of the present disclosure.

[0373] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any appropriate manner without contradiction. In order to avoid unnecessary repetition, the present disclosure will not further describe various possible combinations.

[0374] In addition, the various embodiments of the present disclosure may be arbitrarily combined, and as long as they do not violate the concept of the present disclosure, they should also be regarded as the contents disclosed by the present disclosure.

Claims

1. A double-stranded RNAi agent, wherein the RNAi comprises a sense strand and an antisense strand that can form a double-stranded region, wherein the antisense strand is complementary to at least a portion of an mRNA encoding a thyroxine transporter (TTR), and wherein the antisense strand comprises at least 15, 16, 17, 18, 19, 20, 21, 22 or 23 consecutive nucleotides that differ from any of the antisense strand nucleotide sequences listed in Table 1 by no more than 5, 4, 3, 2 or 1 nucleotides, Preferably, the antisense strand is complementary to the 118-140 fragment, the 411-433 fragment, the 542-566 fragment, and / or the 581-603 fragment of the TTR mRNA sequence as shown in SEQ ID NO:

884. More preferably, the antisense strand comprises at least 15, 16, 17, 18, 19, 20, 21, 22 or 23 consecutive nucleotides that differ by no more than 3, 2 or 1 nucleotides from any of the following sequences: SEQ ID NO:90, 340, 424, 432, 444, 914 and 915.

2. The double-stranded RNAi agent of claim 1, wherein the RNAi comprises a sense strand and an antisense strand, the sense strand comprising at least 15, 16, 17, 18, 19, 20 or 21 consecutive nucleotides that differ from any one of the nucleotide sequences of the sense strand in Table 1 by no more than 5, 4, 3, 2 or 1 nucleotides, Preferably, the sense strand comprises at least 15, 16, 17, 18, 19, 20 or 21 consecutive nucleotides of the fragment 118-140, the fragment 411-433, the fragment 542-566, and / or the fragment 581-603 of the TTR mRNA sequence as shown in SEQ ID NO: 884, Preferably, the sense strand comprises at least 15, 16, 17, 18, 19, 20 or 21 consecutive nucleotides that differ by no more than 3, 2 or 1 nucleotides from any of the following sequences: SEQ ID NO:89, 339, 423, 431 and 443.

3. The double-stranded RNAi agent according to claim 1 or 2, wherein the sense strand / antisense strand comprises SEQ ID NO: 89 / 90, SEQ ID NO: 339 / 340, SEQ ID NO: 417 / 418, SEQ ID NO: 419 / 420, SEQ ID NO: 421 / 422, SEQ ID NO: 423 / 424, SEQ ID NO: 425 / 426, SEQ ID NO: 427 / 428, SEQ ID NO: 429 / 430, SEQ ID NO: 431 / 432, SEQ ID NO: 431 / 914, SEQ ID NO: 431 / 915, SEQ ID NO: 433 / 434, SEQ ID NO: 435 / 436, SEQ ID NO: 437 / 438, SEQ ID NO: 439 / 440, SEQ ID NO: 441 / 442, SEQ ID NO: 443 / 444, SEQ ID NO: NO:445 / 446, SEQ ID NO:447 / 448, SEQ ID NO:449 / 450, SEQ ID NO:451 / 452, SEQ ID NO:453 / 454, SEQ ID NO:455 / 456, SEQ ID NO:457 / 458, SEQ ID NO:459 / 460, SEQ ID NO:461 / 462, SEQ ID NO:463 / 464, SEQ ID NO:465 / 466, SEQ ID NO:467 / 468 or SEQ ID NO:469 / 470, or consist of the said sequences respectively.

4. The double-stranded RNAi agent according to any one of claims 1 to 3, wherein the sense strand / antisense strand comprises the nucleotide sequence shown in SEQ ID NO:89 / 90, SEQ ID NO:339 / 340, SEQ ID NO:423 / 424, SEQ ID NO:431 / 432, SEQ ID NO:431 / 914, SEQ ID NO:431 / 915, or SEQ ID NO:443 / 444, or consists of the said sequence.

5. The double-stranded RNAi agent according to any one of claims 1 to 4, comprising at least one modified nucleotide, Preferably, at least one of the modified nucleotides is selected from one or more of the following: 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-O-methoxyethyl modified nucleotides, L-2'-fluoro modified nucleotides, 2'-deoxyribonucleotides, 2'-amino modified nucleotides, 2'-alkoxy modified nucleotides, 2',3'-broken nucleotide mimics, L-2'-O-methyl modified nucleotides, L-2'-deoxyribonucleotides, locked nucleotides, threose nucleic acids, 4'-modified threose nucleic acids, reverse nucleotides, reverse 2'-O-methyl modified nucleotides, reverse 2'-deoxyribonucleotides, diol nucleotides, 5'-vinyl phosphate nucleotides, More preferably, at least one of the modified nucleotides is selected from one or more of the following: 4'-modified threose nucleic acids, 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-deoxy modified nucleotides.

6. The double-stranded RNAi agent according to any one of claims 1 to 5, comprising at least one 4'-modified threose nucleic acid, Preferably, the 4'-modified threose nucleic acid has the structure of the following formula (A1), in, Base represents a natural or modified nucleoside base, wherein the natural nucleoside base is A, T, C, G or U, and R represents an alkyl group having 1 to 30 carbon atoms. Preferably, the threose nucleic acid is located at the 5' end of the sense strand of the RNAi agent, and R represents an alkyl group having 10 to 30 carbon atoms, More preferably, the threose nucleic acid is a 4'-modified threose nucleic acid having the following structure (A1'): Wherein, Base is a natural nucleoside base A, T, C, G or U.

7. The double-stranded RNAi agent according to any one of claims 1 to 6, wherein at least one nucleotide is linked to one or two adjacent nucleotides via a phosphate group or a phosphorothioate group.

8. A double-stranded RNAi agent for inhibiting the expression of a thyroxine transporter (TTR) gene, comprising a sense strand and an antisense strand, wherein the sense strand and the antisense strand are reversely complementary by at least 18 nucleotides; The sense strand comprises, consists of or consists essentially of the sequence represented by the following general formula (I): 5’-n1-n2-n3-n4-n5-n6-n7-n8-n9-n 10 -n 11 -n 12 -n 13 -n 14 -n 15 -n 16 -n 17 -n 18 -n 19 -n 20 -n 21 -3’ General formula (I); The antisense strand comprises, consists of or consists essentially of the sequence represented by the following general formula (II): 5’-N1-N2-N3-N4-N5-N6-N7-N8-N9-N 10 -N 11 -N 12 -N 13 -N 14 -N 15 -N 16 -N 17 -N 18 -N 19 -N 20 -N 21 -N 22 -N 23 -3’ general formula (II); Among them, in the general formula (I), n1 to n 21 represents 21 consecutive nucleotides contained in the positive strand, and N1 to N 23 represents 23 consecutive nucleotides contained in the antisense strand, and each n and N is independently a modified nucleotide.

9. The RNAi agent of claim 8, wherein the sequence of the antisense strand comprises a sequence motif that is complementary to a portion of a TTR gene mRNA sequence.

10. The RNAi agent of claim 8 or 9, wherein the complementation is partial, substantial or complete reverse complementation.

11. The RNAi agent according to claim 9 or 10, wherein the sequence motif is complementary to the 118-140 fragment, the 411-433 fragment, the 542-566 fragment, and / or the 581-603 fragment of the TTR mRNA sequence as shown in SEQ ID NO: 884, Preferably, the motif comprises at least 15, 16, 17, 18, 19, 20, 21, 22 or 23 consecutive nucleotides that differ by no more than 3, 2 or 1 nucleotides from any of the following sequences: SEQ ID NO:90, 340, 424, 432, 444, 914 and 915.

12. The RNAi agent of any one of claims 8 to 11, wherein the modified nucleotides are selected from: 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-O-methoxyethyl modified nucleotides, L-2'-fluoro modified nucleotides, 2'-deoxyribonucleotides, 2'-amino modified nucleotides, 2'-alkoxy modified nucleotides, 2',3'-bromonucleotide mimics, L-2'-O-methyl modified nucleotides, L-2'-deoxyribonucleotides, locked nucleotides, threose nucleic acids, 4'-modified threose nucleic acids, reverse nucleotides, reverse 2'-O-methyl modified nucleotides, reverse 2'-deoxyribonucleotides, diol nucleotides, 5'-vinyl phosphate nucleotides.

13. The RNAi agent according to any one of claims 8 to 12, wherein n1 and n 21 The nucleotide at the position is a threose nucleotide, a 4'-modified threose nucleotide, an inverted 2'-deoxyribonucleotide, an inverted nucleotide, an L-2'-O-methyl modified nucleotide, an L-2'-deoxyribonucleotide or a 2'-O-methyl modified nucleotide.

14. The RNAi agent according to any one of claims 8 to 13, wherein n7, n9, n 10 and n 11 The nucleotide position is a 2'-deoxyribonucleotide or a 2'-fluoro-modified nucleotide.

15. The RNAi agent according to any one of claims 8 to 14, wherein n2, n3, n4, n5, n6, n8, n9, n10, n11, n12, n13, n14, n15, n16, n17, n18, n19, n20, n21, n3, n4, n5 12 、n 13 、n 14 、n 15 、n 16 、n 17 、n 18 、n 19 and n 20 The nucleotides are 2'-O-methyl modified.

16. The RNAi agent according to any one of claims 8 to 15, wherein the 5' end of the sequence represented by general formula (I) contains at least one phosphorothioate group, and preferably one or more of n1 and n2, and n2 and n3 are connected by a phosphorothioate group.

17. The RNAi agent according to any one of claims 8 to 16, wherein the N1 position is a 5'-vinyl phosphate-2'-O-methyl modified nucleotide, a 2'-O-methyl modified nucleotide, a threose nucleic acid, a 4'-modified threose nucleic acid or an inverted nucleotide.

18. The RNAi agent according to any one of claims 8 to 17, wherein the N3 position is a 2'-O-methoxyethyl modified nucleotide or a 2'-O-methyl modified nucleotide.

19. The RNAi agent according to any one of claims 8 to 18, wherein the N5 position is a 2'-deoxyribonucleotide or a 2'-O-methyl modified nucleotide.

20. The RNAi agent according to any one of claims 8 to 19, wherein N6, N 11 The nucleotide position is a 2'-O-methyl modified nucleotide, a 2'-O-methoxyethyl modified nucleotide or a 2'-fluoro modified nucleotide.

21. The RNAi agent according to any one of claims 8 to 20, wherein the N7 position is a 2'-deoxyribonucleotide, a 2'-O-methyl modified nucleotide, a 2',3'-tetranucleotide mimic or a diol nucleotide.

22. The RNAi agent according to any one of claims 8 to 21, wherein the RNAi agent comprises a 4'-modified threose nucleic acid, for example, a 4'-modified threose nucleic acid having the following formula (A1), in, Base represents a natural or modified nucleoside base, wherein the natural nucleoside base is A, T, C, G or U, and R represents an alkyl group having 1 to 30 carbon atoms. Preferably, the threose nucleic acid is located at the 5' end of the sense strand of the RNAi agent, and R represents an alkyl group having 10 to 30 carbon atoms, More preferably, the threose nucleic acid is a 4'-modified threose nucleic acid having the following structure (A1'): Wherein, Base is a natural nucleoside base A, T, C, G or U.

23. The RNAi agent according to any one of claims 8 to 22, wherein the N 22 ~N 23 One or two of the positions are L-2'-O-methyl modified nucleotides, L-2'-deoxyribonucleotides, threose nucleic acids, 4'-modified threose nucleic acids, reverse 2'-deoxyribonucleotides, reverse nucleotides, reverse 2'-O-methyl modified nucleotides or 2'-O-methyl modified nucleotides, Preferably, the 4'-modified threose nucleic acid is as defined in claim 6 or 22.

24. The RNAi agent according to any one of claims 8 to 23, wherein N2, N 14 、N 16 The nucleotide position is a 2'-deoxyribonucleotide or a 2'-fluoro-modified nucleotide.

25. The RNAi agent according to any one of claims 8 to 24, wherein N4, N8, N9, N 10 、N 12 、N 13 、N 15 、N 17 、N 18 、N 19 、N 20 、N 21 It is a 2'-O-methyl modified nucleotide.

26. The RNAi agent according to any one of claims 8 to 25, wherein the 5' end and the 3' end of the sequence represented by the general formula (II) each contain at least one phosphorothioate group, preferably N1 and N2, N2 and N3, N 21 With N 22 、N 22 With N 23 One or more of the residues are linked by a phosphorothioate group.

27. The RNAi agent according to any one of claims 1 to 26, wherein the antisense strand comprises at least 15 consecutive nucleotides (preferably at least 16, 17, 18, 19 consecutive nucleotides, more preferably at least 20, 21, 22 or 23 consecutive nucleotides) of any modified antisense strand listed in Table 6; or differs from the modified antisense strand by no more than 3, 2 or 1 nucleotides in at least 15 consecutive nucleotides (preferably at least 16, 17, 18, 19 consecutive nucleotides, more preferably at least 20, 21, 22 or 23 consecutive nucleotides); or comprises at least 15 consecutive nucleotides that differ from the modified antisense strand by no more than 3, 2 or 1 nucleotides, Preferably, the antisense strand comprises, consists of, or consists essentially of any one of the modified antisense strands listed in Table 6.

28. The RNAi agent according to any one of claims 1 to 26, wherein the sense strand comprises at least 15 consecutive nucleotides (preferably at least 16, 17, 18, 19 consecutive nucleotides, more preferably at least 20 or 21 consecutive nucleotides) of any modified sense strand listed in Table 7; or differs from the modified sense strand by no more than 3, 2 or 1 nucleotides in at least 15 consecutive nucleotides (preferably at least 16, 17, 18, 19 consecutive nucleotides, more preferably at least 20 or 21 consecutive nucleotides); or comprises at least 15 consecutive nucleotides that differ from the modified sense strand by no more than 3, 2 or 1 nucleotides, Preferably, the sense strand comprises, consists of, or consists essentially of any one of the modified sense strands listed in Table 7.

29. The RNAi agent according to any one of claims 1 to 28, wherein the sense strand consists of 21 nucleotides, and the antisense strand consists of 23 nucleotides.

30. The RNAi agent according to any one of claims 1 to 29, which is obtained by independently modifying the nucleotides at each site of the sense strand and the antisense strand shown in Table 2.

31. The RNAi agent according to any one of claims 1-30, wherein the sense strand and the antisense strand are as shown in Table 7 and Table 6, respectively.

32. The RNAi agent of any one of claims 1 to 31, wherein the antisense strand comprises, consists of, or consists essentially of a sequence obtained by independently modifying the nucleotide at each position of a nucleotide sequence that differs from one of the following nucleotide sequences by 0, 1, 2, or 3 nucleotides: 5'-AUCUAGAACUUUGACCAUCAGAG-3'(SEQ ID NO:90) 5'-UAGGAGUAGGGGCUCAGCAGGGC-3'(SEQ ID NO:340) 5'-UAGGUGAAAACACUGCUUUAGGG-3'(SEQ ID NO:424) 5'-AUAUGAGGUGAAAACACUGCUGG-3'(SEQ ID NO:432) 5'-AAUGUUUUAUUGUCUCUGCCUGG-3'(SEQ ID NO:444) 5'-AUAUGAGGUGAAAACACUGCUUU-3'(SEQ ID NO:914) 5'-AUAUGAGGUGAAAACACUGCUAU-3' (SEQ ID NO:915).

33. The RNAi agent of any one of claims 1 to 32, wherein the sense strand comprises, consists of, or consists essentially of a sequence obtained by independently modifying the nucleotide at each position of a nucleotide sequence that differs from one of the following nucleotide sequences by 0, 1, 2, or 3 nucleotides: 5'-CUGAUGGUCAAAGUUCUAGAU-3'(SEQ ID NO:89) 5'-CCUGCUGAGCCCCUACUCCUA-3'(SEQ ID NO:339) 5'-CUAAAGCAGUGUUUUCACCUA-3'(SEQ ID NO:423) 5'-AGCAGUGUUUUCACCUCAUAU-3'(SEQ ID NO:431) 5'-AGGCAGAGACAAUAAAACAUU-3' (SEQ ID NO: 443).

34. The RNAi agent according to any one of claims 1 to 33, comprising a duplex obtained by independently modifying the nucleotides at each site of each strand of any of the following pairs of double strands: Sense strand: 5'-CUGAUGGUCAAAGUUCUAGAU-3' (SEQ ID NO: 89) and Antisense strand: 5′-AUCUAGAACUUUGACCAUCAGAG-3′ (SEQ ID NO: 90); Sense strand: 5'-CCUGCUGAGCCCCUACUCCUA-3' (SEQ ID NO: 339) and Antisense strand: 5′-UAGGAGUAGGGGCUCAGCAGGGC-3′ (SEQ ID NO: 340); Sense strand: 5'-CUAAAGCAGUGUUUUCACCUA-3' (SEQ ID NO: 423) and Antisense strand: 5'-UAGGUGAAAACACUGCUUUAGGG-3' (SEQ ID NO: 424) Sense strand: 5'-AGCAGUGUUUUCACCUCAUAU-3' (SEQ ID NO: 431) and Antisense strand: 5'-AUAUGAGGUGAAAACACUGCUGG-3' (SEQ ID NO: 432); Sense strand: 5'-AGCAGUGUUUUCACCUCAUAU-3' (SEQ ID NO: 431) and Antisense strand: 5'-AUAUGAGGUGAAAACACUGCUUU-3' (SEQ ID NO: 914); Sense strand: 5'-AGCAGUGUUUUCACCUCAUAU-3' (SEQ ID NO: 431) and Antisense strand: 5'-AUAUGAGGUGAAAACACUGCUAU-3' (SEQ ID NO: 915); Sense strand: 5'-AGGCAGAGACAAUAAAACAUU-3' (SEQ ID NO: 443) and Antisense strand: 5'-AAUGUUUUAUUGUCUCUGCCUGG-3' (SEQ ID NO: 444).

35. The RNAi agent of any one of claims 1-34, wherein the antisense strand comprises, consists of, or consists essentially of a modified nucleotide sequence that differs by 0, 1, 2, or 3 nucleotides from one of the following modified nucleotide sequences: 5'-UmsAfsGmGmUmGfAmAmAmAmCmAmCmUfGmCfUmUmUmAmGmsiGsiG-3' (SEQ ID NO: 597) 5'-UmsAfsGmGmdTGmdAAmAmAmCmAmCmUfGmCfUmUmUmAmGmsGmsGm-3' (SEQ ID NO: 604) 5'-AmsUfsAmUmGmAfGmGmUmGmAmAmAmAfCmAfCmUmGmCmUmsGmsGm-3' (SEQ ID NO: 606) 5'-AmsUfsAmUmdGAmdGGmUmGmAmAmAmAfCmAfCmUmGmCmUmsGmsGm-3' (SEQ ID NO: 620) 5'-VpUmsUfsAmUmdGAmdGGmUmGmAmAmAmAfCmAfCmUmGmCmUmsGmsGm-3'(SEQ ID NO:621) 5'-AmsAfsUmGmUmUfUmUmAmUmU(moe)GmUmCfUmCfUmGmCmCmUmsGmsGm-3'(SEQ ID NO:644) 5'-AmsAfsUmGmUmUfdTUmAmUmUmGmUmCfUmCfUmGmCmCmUmsGmsGm-3'(SEQ ID NO:655) 5'-AmsUfsAmUmdGAmdGGmUmGmAmAmAmAfCmAfCmUmGmCmUmsUmsUm-3'(SEQID NO:889) 5'-AmsUfsAmUmdGAmdGGmUmGmAmAmAmAfCmAfCmUmGmCmUmsAmsUm-3' (SEQ ID NO:891) in, The capital letters "G", "C", "A" and "U" represent ribonucleotides containing guanine, cytosine, adenine and uracil as bases, respectively; "T" represents a deoxyribonucleotide containing thymine as a base; the lowercase letter "m" represents that the nucleotide adjacent to the left of the letter m is a 2'-O-methyl modified nucleotide; the lowercase letter "f" represents that the nucleotide adjacent to the left of the letter f is a 2'-fluoro modified nucleotide; "moe" represents that the nucleotide adjacent to the left of the letter moe is a 2'-O-methoxyethyl modified nucleotide; "Vp" represents that the nucleotide adjacent to the right of the letter Vp is a 5'-vinyl phosphate nucleotide; the lowercase letter "i" represents that the nucleotide adjacent to the right of the letter i is an inverted nucleotide; the lowercase letter "d" represents that the nucleotide adjacent to the right of the letter d is a 2'-deoxyribonucleotide; the lowercase letter "s" represents that the connection between the two adjacent nucleotides on the left and right of the letter s is a phosphorothioate connection.

36. The RNAi agent of any one of claims 1-35, wherein the sense strand comprises, consists of, or consists essentially of a modified nucleotide sequence that differs by 0, 1, 2, or 3 nucleotides from one of the following modified nucleotide sequences: composition: 5'-ChsUmsAmAmAmGmCfAmGfUfGfUmUmUmUmCmAmCmCmUmAm-3' (SEQ ID NO: 601) 5'-iCsUmsAmAmAmGmCfAmGfUfGfUmUmUmUmCmAmCmCmUmAm-3' (SEQ ID NO: 603) 5'-AmsGmsCmAmGmUmGfUmUfUfUfCmAmCmCmUmCmAmUmAmUm-3' (SEQ ID NO: 605) 5'-AhsGmsCmAmGmUmGfUmUfUfUfCmAmCmCmUmCmAmUmAmUm-3' (SEQ ID NO: 617) 5'-AmsGmsGmCmAmGmAfGmAfCfAfAmUmAmAmAmAmCmAmUmUm-3' (SEQ ID NO: 641) 5'-AhsGmsGmCmAmGmAfGmAfCfAfAmUmAmAmAmAmCmAmUmUm-3' (SEQ ID NO: 653) 5'-AhsGmsCmAmGmUmGfUmUfUfUfCmAmCmCmUmCmAmUmAmAm-3' (SEQ ID NO:744) Among them, the capital letters "G", "C", "A" and "U" represent ribonucleotides containing guanine, cytosine, adenine and uracil as bases, respectively; the lowercase letter "m" indicates that the nucleotide adjacent to the left of the letter m is a 2'-O-methyl modified nucleotide; the lowercase letter "f" indicates that the nucleotide adjacent to the left of the letter f is a 2'-fluoro modified nucleotide; the lowercase letter "h" indicates that the nucleotide adjacent to the left of the letter h is a 4'-modified threose nucleic acid; "i" indicates that the nucleotide adjacent to the right of the letter i is an inverted nucleotide; the lowercase letter "s" indicates that the connection between the two adjacent nucleotides on the left and right of the letter s is a phosphorothioate connection.

37. The RNAi agent of any one of claims 1-36, comprising any one of the following groups: SEQ ID NOs: 601 and 597, SEQ ID NOs: 601 and 604, SEQ ID NOs: 603 and 597, SEQ ID NOs: 603 and 604, SEQ ID NOs: 605 and 606, SEQ ID NOs: 605 and 620, SEQ ID NOs: 605 and 889, SEQ ID NOs: 617 and 606, SEQ ID NOs: 617 and 620, SEQ ID NOs: 744 and 621, SEQ ID NOs: 641 and 644, SEQ ID NOs: 641 and 655, SEQ ID NOs: 653 and 644, SEQ ID NOs: 653 and 655, SEQ ID NOs: 751 and 620, SEQ ID NOs: 751 and 891.

38. The RNAi agent of any one of claims 1-37, wherein the sense strand and / or antisense strand is linked to a targeting ligand to form a conjugate.

39. The RNAi agent of claim 38, wherein the targeting ligand comprises one or more (e.g., 1, 2, 3 or 4) N-acetylgalactosamine and / or its derivatives, wherein the N-acetylgalactosamine structural formula is as shown in formula (BI):

40. The RNAi agent according to claim 38 or 39, wherein the structural formula of the targeting ligand is as shown in formula (B-II):

41. The RNAi agent according to any one of claims 38-40, wherein the targeting ligand is conjugated to the sense strand, preferably, the targeting ligand is conjugated to the 3' end or the 5' end of the sense strand.

42. The RNAi agent according to any one of claims 38 to 41, wherein the structural formula of the conjugate is as shown in formula (B-III): in represents the double-stranded oligonucleotide of the RNAi, 3' represents the 3' end of the sense strand, and X is O or S.

43. The RNAi agent according to any one of claims 38 to 42, wherein the conjugate is as shown in Table 5, preferably BPR-30221218, BPR-30221221, BPR-30221223, BPR-30221228, BPR-30221686, BPR-30221617, BPR-30221618, BPR-30221620, BPR-30221672, BPR-30221676, BPR-30221678, BPR-30222203, BPR-30222212, BPR-30222218.

44. A cell comprising the RNAi agent of any one of claims 1-43.

45. A composition comprising the RNAi agent of any one of claims 1-43 and a pharmaceutically acceptable diluent, carrier and / or excipient; preferably, the diluent is PBS buffer, saline or water; more preferably, the composition further comprises one or more additional therapeutic agents.

46. ​​A pharmaceutical combination comprising the RNAi agent of any one of claims 1 to 43 and one or more other therapeutic agents, such as any therapeutic agent effective in preventing or treating diseases and / or disorders mediated by TTR gene expression.

47. A method for inhibiting TTR gene expression in a cell in vivo or in vitro, the method comprising introducing into the cell an effective amount of the RNAi agent of any one of claims 1 to 43, the composition of claim 45, or the pharmaceutical combination of claim 46.

48. The method of claim 47, wherein the cell is in a subject, preferably, the subject is a human.

49. The method of claim 47 or 48, wherein the TTR gene expression is inhibited by at least about 50%.

50. Use of the RNAi agent of any one of claims 1-43 or the composition of claim 45 for the preparation of a medicament for treating and / or preventing a disease, disorder or symptom mediated at least in part by TTR gene expression.

51. The use according to claim 50, wherein the disease comprises ATTR-PN (transthyretin amyloidosis polyneuropathy), ATTR-CM (transthyretin amyloidosis cardiomyopathy), senile systemic amyloidosis, and leptomeningeal / central nervous system amyloidosis.

52. A method for treating and / or preventing a disease, disorder or symptom mediated at least in part by TTR gene expression, comprising administering a therapeutically effective amount of the RNAi agent of any one of claims 1-38 or the composition of claim 40 to a patient in need thereof, preferably the patient is a human.

53. The method of claim 52, wherein the disease comprises ATTR-PN (transthyretin amyloidosis polyneuropathy), ATTR-CM (transthyretin amyloidosis cardiomyopathy), senile systemic amyloidosis, leptomeningeal / central nervous system amyloidosis.

54. The method of claim 52 or 53, wherein the RNAi agent or the composition is administered to the patient by subcutaneous, intravenous, intramuscular, intrabronchial, intrapleural, intraperitoneal, intraarterial, lymphatic and / or cerebrospinal administration.

55. The method of any one of claims 52-54, wherein the RNAi agent or the composition is delivered to the liver, choroid plexus, retina and / or pancreas of the patient.

56. The method of any one of claims 52-55, wherein the RNAi agent is administered at a dose of about 1-300 mg / kg body weight.

57. The method of any one of claims 52-56, wherein the RNAi agent is administered once or more every day, every week, every two weeks, every three weeks, every 1 month, every 2 months, every 3 months, every 4 months, every 5 months, every 6 months, every 7 months, every 8 months, every 9 months, every 10 months, every 11 months, or every 12 months.

58. A kit comprising the RNAi agent of any one of claims 1-43 or the composition of claim 45.

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