WO2025077916A1 - Transthyretin-targeting oligonucleotide and use thereof - Google Patents

Abstract

Provided are a transthyretin-targeting oligonucleotide and the use thereof. The targeting oligonucleotide can significantly inhibit the gene expression level of transthyretin, and has a lasting drug effect.

Description

Oligonucleotides targeting transthyretin and uses thereof Technical Field

The present disclosure relates to an oligonucleotide, in particular to the inhibition of TTR gene expression and the treatment of TTR-related amyloidosis.

Background Art

Transthyretin (TTR), also known as vitamin A binding protein, is an important plasma protein component that is widely distributed in various cells, plasma and tissue fluid. As a carrier protein, TTR is mainly synthesized in the liver and the choroid plexus in the brain, secreted into the blood and cerebrospinal fluid, and plays a role in transporting thyroxine (mainly T4) and retinol (vitamin A) to various tissues and cells throughout the body. Thyroxine is of great significance to human growth and development and to maintaining important functions of the body. Vitamin A deficiency is also the main cause of night blindness. It can be seen that TTR is of great significance in maintaining normal human functions. Under physiological conditions, TTR is a stable protein. After TTR is decomposed into monomers, it can cause amyloidosis. In recent years, a large number of studies have shown that TTR protein is related to infection, inflammation, malnutrition, amyloidosis and tumors. In particular, for the treatment of TTR amyloidosis, a variety of TTR-specific drugs have been developed and approved for marketing. At the same time, more and more TTR-targeted drugs are gradually moving towards clinical use for neurological diseases, endocrine and metabolic diseases, etc.

Amyloidosis is a disease caused by the accumulation of abnormally folded proteins in tissues. The symptoms caused depend on the site of amyloid accumulation, and the lesions mainly occur in organs such as the kidneys, heart, and nervous system. Amyloid deposition of TTR mutants can cause TTR amyloidosis. Specifically, TTR is a tetrameric structural protein with high stability, but TTR tetramers can decompose and degrade into monomers under pathological conditions or abnormal physiological conditions (such as stress and inflammatory response). TTR monomers can generate complex and diverse types of amyloid fibers, leading to abnormal physiological aggregation of amyloid fibers in cells. Abnormal amyloid deposition in cells can cause abnormal metabolism of the cells themselves and even changes and disorders in the function of the entire tissue, thereby causing related diseases, such as the hereditary transthyretin amyloidosis, which is reported more frequently.

As a basic substance for human life activities, TTR's physiological functions are far more than just transporting thyroxine and retinol. TTR in specific locations has specific physiological effects. In the study of TTR-related diseases, the most studied are TTR-related amyloidosis, such as transthyretin cardiac amyloidosis myocardiopathy (ATTR-CM), transthyretin amyloid polyneuropathy (ATTR-PN), and Alzheimer's disease (AD). TTR is also associated with a variety of metabolic diseases, such as type II diabetes and impaired glucose regulation. TTR has also been reported to be related to tumors. There are obvious differences in its expression levels in different tumor cells, and it may be involved in the process of promoting or inhibiting malignant tumors.

As mentioned above, TTR gene mutations can lead to heritable familial amyloidosis (such as ATTR-CM and ATTR-PN). ATTR-CM is a rare cardiomyopathy, mainly due to TTR gene mutations, which cause abnormal depolymerization of TTR protein to form abnormal substances (amyloid substances) deposited in the heart, manifesting as restrictive cardiomyopathy and progressive heart failure. ATTR-PN is a rare hereditary fatal neurodegenerative disease, an autosomal dominant hereditary disease in which TTR gene mutations cause peripheral nerves and multiple organ systems to be affected. Its clinical manifestations are mainly symmetrical sensory disorders in the distal limbs, such as numbness, pain, muscle weakness and muscle atrophy, etc. At present, the treatment methods for ATTR-CM and ATTR-PN are mainly to inhibit the production of mutant TTR gene mRNA or stabilize the structure of TTR protein tetramers.

Familial amyloid polyneuropathy (FAP) is a group of life-threatening multisystem diseases that are inherited in an autosomal dominant pattern. First reported in northern Portugal by Andrade in 1952 and followed in Japan (1968) and Sweden, FAP is a neuropathy caused by the deposition of amyloid fibrils, most commonly caused by mutations in transthyretin (TTR). The precursors of amyloid are mutated apolipoprotein A-1 or gellin. The first identified cause of FAP is the TTR Val30Met mutation, which remains the most common amyloid origin mutations identified worldwide. The penetrance and age of onset in FAP populations carrying the same mutation vary from country to country. The symptomatology and clinical course of FAP can be highly variable. TTR FAP usually causes a nerve length dependent polyneuropathy starting in the feet, with loss of temperature and pain sensitivity, and life-threatening autonomic dysfunction, leading to cachexia and death within a mean of 10 years. TTR is synthesized primarily in the liver, and liver transplantation appears to have a favorable effect on the course of the neuropathies, but not on the heart or eyes. Oral tafamidis meglumine is being evaluated in patients with TTR FAP to prevent misfolding and deposition of mutant TTR.

ATTR-CM is a progressive, fatal, and neglected disease, with 23 articles reporting a weighted average mean and median diagnostic delay of 6.1 and 3.4 years for the wild-type (ATTRwt-CM) and 5.7 and 2.6 years for the genotype (ATTRv-CM). The reported misdiagnosis rate ranged from 34 to 57%. Prior to receiving an ATTR-CM diagnosis, there is evidence that patients receive unnecessary or inappropriate evaluations or treatments when misdiagnosed. Diagnostic “red flags” are reported to be underutilized. Data on the impact of delays on patients and health systems are scarce, but given the progressive nature of ATTR-CM, delays are likely to have adverse consequences.

In addition to hereditary amyloid diseases, another disease that has been studied more with TTR is Alzheimer’s disease (AD). AD is a degenerative disease of the central nervous system, and sticky plaques of amyloid protein are the hallmark of AD. Studies have found that when TTR expression increases, AD symptoms develop more slowly. Other reports show that TTR genetic mutations may serve as risk genes for Alzheimer’s disease. Therefore, TTR may have both protective and harmful effects in AD.

As a routine biochemical test indicator, TTR has long been regarded as a sensitive clinical indicator for evaluating the nutritional status of the body. Many studies have shown that the level of TTR in diabetic patients is closely related to their complications and prognosis. In addition, high plasma TTR levels are significantly positively correlated with an increased risk of type 2 diabetes and impaired glucose regulation. TTR can bind to retinol transporter to form macromolecular compounds to reduce the renal activity of retinol binding protein. The results showed that TTR can regulate the filtration rate of the liver and maintain the plasma retinol binding protein concentration. As a clear adipokine, retinol binding protein is closely related to the occurrence and development of diabetes. These studies provide a theoretical basis for TTR as a potential biomarker for metabolic disease risk.

Several studies have shown that TTR is abnormally expressed in the plasma and tumor cells of patients with various tumors, including pancreatic cancer (Chen, Jiong, et al. 2013), lung cancer (Ding, Hongmei, et al. 2014), liver cancer (Shimura, Tatsuo, et al. 2018), ovarian cancer (Schweigert, Florian J. 2005), and colorectal cancer (Nicklasson, J. 2018). For example, in liver cancer cells, the TTR gene is deleted, and its growth can be inhibited after in vitro transfection of liver cancer cells. Experiments have found that the expression level of TTR in pancreatic ductal carcinoma is upregulated, and the expression of TTR in other tumors such as lung cancer, ovarian cancer and colorectal cancer is also different, suggesting that TTR may play a dual role in different tumors. Most studies now believe that as one of the proteins expressed differently in tumors, TTR is a new tumor marker with good sensitivity and specificity, and has potential value and broad space in clinical applications. It has been reported (Lee, C.C., et al. 2019) that human serum transthyretin (TTR) concentrations are significantly elevated in lung cancer patients. Significant increases in TTR concentrations were observed in serum, bronchoalveolar lavage fluid, alveolar type II epithelial cells, and alveolar myeloid cells in the CCSP-rtTA/(tetO)7-Stat3C lung tumor mouse model. Recombinant TTR mediates the proliferation and growth of lung tumor cells by activating mitogenic and oncogenic molecules. TTR has cytokine function that stimulates myeloid cell differentiation and plays a role in the tumor environment.

At present, there are several TTR targeted drugs developed and marketed, mainly TTR inhibitors and TTR stabilizers, which are used to treat TTR amyloidosis. TTR inhibitors prevent amyloid deposition by inhibiting TTR genes and transthyretin proteins, thereby treating amyloidosis. TTR stabilizers selectively bind to TTR at the thyroxine binding site, stabilize the compound and reduce the dissociation into monomers, limit the rate of TTR amyloid formation, and improve the structural stability of TTR. The marketed TTR stabilizers include: Pfizer's tafamidis. In addition, BridgeBio Pharma's subsidiary Eidos Therapeutics has a newly developed tetramer stabilizer acoramidis (AG-10, BBP-265) with good tolerability and safety, and is undergoing Phase III clinical trials. The marketed TTR inhibitors include: Ionis Pharmaceuticals' Inotersen Sodium and Alnylam Pharmaceuticals' Patisiran Sodium. In addition to TTR inhibitors and TTR stabilizers, more innovative drugs targeting TTR have entered the clinic, such as NI-006 protein folding regulator, a humanized monoclonal antibody under development, which is currently in Phase I clinical trials for myocardial diseases, TTR amyloidosis, and familial amyloid neuropathy. Based on domestic and foreign literature, TTR is also valuable for early prediction of a variety of metabolism and malignant tumors. Therefore, further research on TTR for clinical disease amyloidosis, metabolism, and tumors in the future will be of great significance.

Summary of the invention

In order to solve the problems existing in the prior art, the purpose of the present disclosure is to provide an inhibitor targeting TTR with good efficacy, high safety and long-lasting efficacy.

The present disclosure provides dsRNA and methods of using the dsRNA to inhibit TTR gene expression in cells or mammals, wherein the dsRNA targets the TTR gene. The present disclosure also provides compositions and methods for treating pathological conditions and diseases (e.g., TTR amyloidosis) in mammals caused by TTR gene expression. The dsRNA directs sequence-specific degradation of mRNA through a process called RNA interference (RNAi).

In one aspect, the present disclosure provides an oligonucleotide or a pharmaceutically acceptable salt thereof for reducing the expression of the transthyretin (TTR) gene, wherein the oligonucleotide comprises a sense chain and an antisense chain, wherein the sense chain has a sequence having at least 80% sequence identity with a sequence shown in any one of SEQ ID NOs: 2-4, 6-21, 23-83, or a fragment thereof, or a modified sequence of the aforementioned sequence or a fragment thereof, and preferably has a sequence identity of 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more; the antisense chain has a sequence having at least 80% sequence identity with a sequence shown in any one of SEQ ID NOs: 85-87, 89-104, 106-179, or a fragment thereof, or a modified sequence of the aforementioned sequence or a fragment thereof, and preferably has a sequence identity of 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more.

On the other hand, the present disclosure provides a conjugate for reducing the expression of the transthyretin (TTR) gene or a pharmaceutically acceptable salt thereof, comprising: (i) the aforementioned oligonucleotide or a pharmaceutically acceptable salt thereof, and (ii) a ligand conjugated to the aforementioned oligonucleotide or a pharmaceutically acceptable salt thereof, wherein at least one nucleotide of the oligonucleotide is conjugated to a targeting ligand.

In another aspect, the present disclosure provides a composition comprising the aforementioned oligonucleotide or a pharmaceutically acceptable salt thereof, and optionally a pharmaceutically acceptable carrier.

In another aspect, the present disclosure provides use of the aforementioned oligonucleotide or a pharmaceutically acceptable salt thereof or a composition thereof in the preparation of a medicament for treating and/or preventing a TTR-related disease.

In another aspect, the present disclosure provides a method for treating and/or preventing a TTR-related disorder, disorder, and/or condition in a subject by administering a therapeutic agent (eg, an oligonucleotide or a vector or a transgene encoding the same) to the subject.

In another aspect, the present disclosure provides methods of treating and/or preventing TTR-related disorders, disorders and/or conditions in a subject using the aforementioned oligonucleotides or pharmaceutically acceptable salts thereof, conjugates or compositions in combination with other drugs and/or other therapeutic methods.

Experiments have shown that the candidate compounds disclosed herein can effectively reduce the TTR content in the body and are effective inhibitors of human TTR, thereby effectively treating heritable familial amyloidosis (such as ATTR-CM and ATTR-PN), wherein ATTR-PN is a rare hereditary fatal neurodegenerative disease, an autosomal dominant hereditary disease in which TTR gene mutations cause peripheral nerves and multiple organ systems to be affected. The reduction of TTR can also treat familial amyloid polyneuropathy (FAP), a life-threatening multi-system disease. In addition, there are many reports showing that the inhibition of TTR can effectively slow down the disease progression of type II diabetes and Alzheimer's disease, which will be of great significance for the research of various clinical disease fields, such as amyloidosis, metabolism, and tumors.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 shows the steps of solid phase synthesis of siRNA.

FIG. 2 shows a map of the pFB-AAV-CAG-TTR-MM recombinant plasmid.

Figure 3 shows the efficacy of TTR RNAi in TTR-Gluc mice.

Figure 4 shows the efficacy of hTTR siRNA in V30 hTTR transgenic mice.

Figure 5 shows the efficacy of hTTR siRNA in V30 hTTR transgenic mice.

DETAILED DESCRIPTION

In the present disclosure, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology and laboratory operation procedures used herein are terms and routine procedures widely used in the corresponding fields. At the same time, in order to better understand the present disclosure, the definitions and explanations of the relevant terms are provided below.

As used herein, the term "about" or "approximately" as applied to one or more target values refers to a value similar to the reference value. In certain embodiments, unless otherwise specified or in addition apparent from the context, the term "about" or "approximately" refers to a range of values falling into 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less in the reference value in either direction (greater than or less than) (unless such numerals will exceed 100% of possible values).

As used herein, the term "transthyretin" ("TTR") is also referred to as ATTR, HsT2651, PALB, CTS1, prealbumin, TBPA, and transthyretin (prealbumin, amyloidosis type I). The sequence of the human TTR mRNA transcript can be found at NM_000371. The sequence of the cynomolgus monkey TTR mRNA can be found at XM_045377903.1. The sequence of the mouse TTR mRNA can be found at NM_013697.2, and the sequence of the rat TTR mRNA can be found at NM_012681.1.

As used herein, the term "complementary" refers to a structural relationship between nucleotides (e.g., on two nucleotides on a relative nucleic acid or on a relative region of a single nucleic acid chain) that allows nucleotides to form base pairs with each other. For example, a purine nucleotide complementary to a pyrimidine nucleotide of a nucleic acid relative to a nucleic acid can be base-paired together by forming hydrogen bonds with each other. In some embodiments, complementary nucleotides can be base-paired in a Watson-Crick manner or in any other manner that allows the formation of a stable duplex. In some embodiments, two nucleic acids can have a nucleotide sequence that is complementary to each other so as to form a complementary region, as described herein.

As used herein, the term "strand" refers to a single continuous sequence of nucleotides linked together by internucleotide bonds (eg, phosphodiester bonds, phosphorothioate bonds). In some embodiments, the strand has two free ends, eg, a 5'-end and a 3'-end.

As used herein, the term "deoxyribonucleotide" refers to a nucleotide having a hydrogen at the 2' position of its pentose compared to a ribonucleotide. A modified deoxyribonucleotide is a deoxyribonucleotide having a modification or substitution of one or more atoms other than at the 2' position (including a modification or substitution in or of a sugar, a phosphate group, or a base).

As used herein, the term "oligonucleotide" refers to a short nucleic acid, such as a short nucleic acid less than 100 nucleotides in length. The oligonucleotide may comprise ribonucleotides, deoxyribonucleotides and/or modified nucleotides, including, for example, modified ribonucleotides. The oligonucleotide may be single-stranded or double-stranded. The oligonucleotide may or may not have a duplex region. As a group of non-limiting examples, the oligonucleotide may be, but is not limited to, small interfering RNA (siRNA), microRNA (miRNA), short hairpin RNA (shRNA), Dicer substrate interfering RNA (dsiRNA), antisense oligonucleotide, short siRNA or single-stranded siRNA. In some embodiments, the double-stranded oligonucleotide is an RNAi oligonucleotide.

As used herein, the terms "iRNA", "RNAi agent", "iRNA agent", "RNA interfering agent" are used interchangeably herein and refer to an agent that comprises RNA as such terms are defined herein and mediates targeted cleavage of RNA transcripts through the RNA-induced silencing complex (RISC) pathway. RNA interference (RNAi) is a process that directs sequence-specific degradation of mRNA. RNAi modulates, for example, inhibits expression of TTR in a cell, for example, a cell within an individual, for example, a mammalian individual.

As used herein, the term "double-stranded oligonucleotide" refers to an oligonucleotide that is substantially in the form of a duplex. In some embodiments, complementary base pairing of one or more duplex regions of a double-stranded oligonucleotide is formed between the antiparallel sequences of the nucleotides of the covalently separated nucleic acid chains. In some embodiments, complementary base pairing of one or more duplex regions of a double-stranded oligonucleotide is formed between the antiparallel sequences of the nucleotides of the covalently attached nucleic acid chains. In some embodiments, complementary base pairing of one or more duplex regions of a double-stranded oligonucleotide is formed from a single nucleic acid chain, and the single nucleic acid chain is folded (e.g., via a hairpin) to provide a complementary antiparallel sequence of the nucleotides of the base pairing together. In some embodiments, a double-stranded oligonucleotide comprises two covalently separated nucleic acid chains that are completely duplexed from each other. However, in some embodiments, a double-stranded oligonucleotide comprises partially duplexed, for example, two covalently separated nucleic acid chains with overhangs at one or both ends. In some embodiments, a double-stranded oligonucleotide comprises an antiparallel sequence of nucleotides, which is partially complementary, and therefore, one or more mispairings may be present, and the mispairings may include internal mispairings or terminal mispairings.

As used herein, "conjugation" refers to the covalent connection between two or more chemical moieties each having a specific function; accordingly, "conjugate" refers to a compound formed by covalent connection between the chemical moieties. Further, "siRNA conjugate" means a compound formed by covalently connecting one or more chemical moieties having a specific function to siRNA. In the following, the siRNA conjugate of the present invention is sometimes referred to as a "conjugate". The siRNA conjugate should be understood as a general term for siRNA conjugates, the first siRNA conjugate or the second siRNA conjugate, or the siRNA sense chain conjugate or the siRNA antisense chain conjugate, depending on the context.

As used herein, the term "double-stranded RNA" or "dsRNA" refers to a complex of ribonucleic acid molecules having a duplex structure comprising two antiparallel and substantially complementary nucleic acid strands having "sense" and "antisense" orientations relative to the target RNA (i.e., TTR gene). In some embodiments of the present disclosure, double-stranded RNA (dsRNA) triggers the transcription of a target RNA (e.g., TTR gene) through a post-transcriptional gene silencing mechanism referred to herein as RNA interference or RNAi. mRNA). Generally speaking, most of the nucleotides of each strand of a dsRNA molecule are ribonucleotides, but as described in detail herein, each strand or both strands may also contain one or more non-ribonucleotides, such as deoxyribonucleotides or modified nucleotides. In addition, as used herein, "iRNA" may contain ribonucleotides with chemical modifications; iRNA may contain substantial modifications at multiple nucleotides.

As used herein, the term "modified nucleotide" refers to a nucleotide independently having a modified sugar moiety, a modified internucleotide bond, or a modified nucleobase, or any combination thereof. Thus, the term "modified nucleotide" encompasses substitutions, additions, or removals of internucleoside bonds, sugar moieties, or nucleobases, such as functional groups or atoms. Modifications suitable for use with the disclosed agents include all types of modifications disclosed herein or known in the art.

As used herein, the term "nucleotide overhang" refers to at least one unpaired nucleotide protruding from the duplex structure of a double-stranded iRNA. For example, when the 3' end of one strand of a dsRNA extends beyond the 5' end of the other strand, or vice versa, there is a nucleotide overhang. The dsRNA may include an overhang of at least one nucleotide; alternatively, the overhang may include at least two nucleotides, at least three nucleotides, at least four nucleotides, at least five nucleotides or more. The nucleotide overhang may include or be composed of: nucleotide/nucleoside analogs, including deoxynucleotides/nucleosides. The overhang may be on the sense strand, the antisense strand, or any combination thereof. In addition, the nucleotides of the overhang may be present on the 5' end, the 3' end, or both ends of the antisense strand or the sense strand of the dsRNA.

As used herein, the term "naked sequence" refers to an unmodified nucleotide sequence.

As used herein, the term "inhibit" is used interchangeably with "knockdown," "reduction," "silencing," "downregulate," "suppression," and other similar terms, and includes any degree of inhibition.

The phrase "inhibiting the expression of TTR" is intended to refer to inhibiting the expression of any TTR gene (such as, for example, 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. Thus, in the context of a genetically manipulated cell, cell population, or organism, the TTR gene can be a wild-type TTR gene, a mutant TTR gene, or a transgenic TTR gene.

"Inhibiting the expression of the TTR gene" includes any level of inhibition of the TTR gene, for example, at least partial inhibition of the expression of the TTR gene. The expression of the TTR gene can be assessed based on the level or change in the level of any variable associated with the expression of the TTR gene, for example, the mRNA level or the TTR protein level, or by inhibiting the mRNA level of the Gluc and TTR fusion protein genes, thereby inhibiting the level of the Gluc protein, which indirectly reflects the inhibition of the TTR protein level.

The term "pharmaceutically acceptable salt" refers to salts that retain the biological effectiveness and characteristics of free alkali or free acid, which are not biologically or otherwise undesirable. These salts are formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid (particularly hydrochloric acid) and organic acids such as acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methylsulfonic acid, ethylsulfonic acid, p-toluenesulfonic acid, salicylic acid, N-acetylcysteine. In addition, these salts can be prepared by adding inorganic bases or organic bases to free acids. Salts derived from inorganic bases include but are not limited to alkali metal salts (such as sodium salts, potassium salts and lithium salts), ammonium salts, alkaline earth metal salts (such as calcium salts and magnesium salts). Salts derived from organic bases include, but are not limited to, salts formed with the following organic bases (e.g., organic amines): primary amines, secondary amines, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, and basic ion exchange resins, such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, lysine, arginine, N-ethylpiperidine, piperidine, polyamine resins. The oligonucleotides of the present invention may also exist in the form of zwitterions. Particularly preferred pharmaceutical salts of the present invention are sodium salts, lithium salts, potassium salts, and trialkylammonium salts.

As used herein, the term "subject" is an animal that expresses the target gene endogenously or heterologously, such as a mammal, including primates (such as humans, non-human primates, such as monkeys and chimpanzees), non-primates (such as cows, pigs, horses, goats, rabbits, sheep, hamsters, guinea pigs, cats, dogs, rats or mice) or birds. In one embodiment, the subject is a human.

As used herein, the term "treating" or "treatment" refers to a beneficial or desired result, such as reducing at least one sign or symptom of a TTR-related disorder in a subject. Treatment also encompasses reducing one or more signs or symptoms associated with undesirable TTR expression; reducing the extent of undesirable TTR activation or stabilization; ameliorating or alleviating undesirable TTR activation or stabilization. Treatment also encompasses reducing one or more signs or symptoms associated with undesirable TTR expression. "Treatment" may also mean prolonging survival compared to expected survival without treatment.

As used herein, the terms "prevention" or "preventing" when used in reference to a disease or condition that would benefit from a decrease in TTR gene expression or TTR protein production.

As used herein, the term "therapeutically effective amount" is intended to encompass an amount of a RNAi agent that, when administered to a subject with a TTR-related disorder, is sufficient to affect treatment of the disease (e.g., by reducing, ameliorating, or maintaining an existing disease or one or more disease symptoms). The "therapeutically effective amount" may vary depending on the RNAi agent, how the agent is administered, the disease and its severity, as well as medical history, age, weight, family history, genetic makeup, type of previous or concomitant treatment (if any), and other individual characteristics of the subject to be treated.

As used herein, the term "prophylactically effective amount" is intended to include an amount of a RNAi agent that, when administered to a subject with a TTR-related disorder, is sufficient to prevent or ameliorate the disorder or one or more symptoms of the disorder. Amelioration of the disease includes slowing the progression of the disease or reducing the severity of the disease that later develops. A "prophylactically effective amount" may vary depending on the RNAi agent, how the agent is administered, the degree of disease risk, and medical history, age, weight, family history, genetic makeup, type of previous or concomitant therapy (if any), and other individual characteristics of the patient to be treated.

In one aspect, the present disclosure provides an oligonucleotide or a pharmaceutically acceptable salt thereof for reducing the expression of the transthyretin (TTR) gene, wherein the oligonucleotide comprises a sense chain and an antisense chain, wherein the sense chain has a sequence having at least 80% sequence identity with a sequence shown in any one of SEQ ID NOs: 2-4, 6-21, 23-83, or a fragment thereof, or a modified sequence of the aforementioned sequence or a fragment thereof, and preferably has a sequence identity of 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more; the antisense chain has a sequence having at least 80% sequence identity with a sequence shown in any one of SEQ ID NOs: 85-87, 89-104, 106-179, or a fragment thereof, or a modified sequence of the aforementioned sequence or a fragment thereof, and preferably has a sequence identity of 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more.

In some embodiments, wherein each strand is independently 19 to 25 nucleotides in length.

In some embodiments, the antisense strand is 19 to 23 nucleotides in length.

In some embodiments, the sense strand is 19 to 23 nucleotides in length.

In some embodiments, the aforementioned oligonucleotides include 5' and/or 3'-overhang sequences of one or more nucleotides in length, wherein the aforementioned 5' and/or 3'-overhang sequences are present on antisense strands and/or sense strands. In one embodiment, the antisense strand of the aforementioned oligonucleotides has 1 to 10 nucleotides, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides at the overhang at the 3' end or 5' end. In one embodiment, the sense strand of dsRNA has 1 to 10 nucleotides, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides at the overhang at the 3' end or 5' end. In another embodiment, one or more nucleotides in the overhang are replaced by nucleoside thiophosphates.

In some embodiments, the antisense strand has an overhang.

In some embodiments, the sense strand has an overhang.

In some embodiments, the aforementioned oligonucleotide comprises a 3'-overhang sequence of two nucleotides in length.

In some embodiments, the aforementioned 3'-overhang sequence is present on the aforementioned sense strand; preferably, the aforementioned overhang sequence is selected from: GG, GA, GC, UC, UG, UU, UA, CA, CC, CG, CU, AA, AG, AU, AC.

In some embodiments, the aforementioned 3'-overhang sequence is present on the aforementioned antisense strand; preferably, the aforementioned overhang sequence is selected from: AA, AG, AU, CA, CC, CG, CU, GA, GC, GG, GU, UA, UG, UU; more preferably, the aforementioned overhang sequence is UU, AA or AU.

In some embodiments, the oligonucleotide comprises an antisense strand and a sense strand each ranging from 19 to 23 nucleotides in length.

In some embodiments, the sense strand and the antisense strand form a duplex region.

In some embodiments, the sense strand and the antisense strand are respectively in a 19/21 paired, 21/21 paired, 21/23 paired, or 23/23 paired duplex structure.

In some embodiments, the oligonucleotide comprises a 3'-overhang sequence that is two nucleotides in length, wherein the 3'-overhang sequence is present on the antisense strand, and wherein the sense strand is 19 nucleotides in length and the antisense strand is 21 nucleotides in length, such that the sense strand and the antisense strand form a duplex that is 19 nucleotides in length.

In some embodiments, the oligonucleotide comprises a 3'-overhang sequence that is two nucleotides in length, wherein the 3'-overhang sequence is present on the antisense strand and the sense strand, and wherein the sense strand is 21 nucleotides in length and the antisense strand is 21 nucleotides in length, such that the sense strand and the antisense strand form a duplex that is 19 nucleotides in length.

In some embodiments, the oligonucleotide comprises a 3'-overhang sequence that is two nucleotides in length, wherein the 3'-overhang sequence is present on the antisense strand, and wherein the sense strand is 21 nucleotides in length and the antisense strand is 23 nucleotides in length, such that the sense strand and the antisense strand form a duplex that is 21 nucleotides in length.

In some embodiments, the oligonucleotide comprises a 3'-overhang sequence that is two nucleotides in length, wherein the 3'-overhang sequence is present on the antisense strand and the sense strand, and wherein the sense strand is 23 nucleotides in length and the antisense strand is 23 nucleotides in length, such that the sense strand and the antisense strand form a duplex that is 21 nucleotides in length.

In some embodiments, the pharmaceutically acceptable salt of the oligonucleotide can be prepared by adding an inorganic base or an organic base to the free acid. Salts derived from inorganic bases include, but are not limited to, alkali metal salts (such as sodium salts, potassium salts and lithium salts), ammonium salts, alkaline earth metal salts (such as calcium salts and magnesium salts). Salts derived from organic bases (such as organic amines) include, but are not limited to, salts formed with the following organic bases: primary amines, secondary amines and tertiary amines, substituted amines include naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, lysine, arginine, N-ethylpiperidine, piperidine, polyamine resins.

In some embodiments, examples of pharmaceutically acceptable salts of oligonucleotides include, but are not limited to, ammonium salts, such as salts of tertiary alkylamine compounds (eg, triethylamine salts), metal salts such as sodium salts, potassium salts, and magnesium salts, and the like.

In some embodiments, the oligonucleotide or its salt may be in the form of a hydrate or a solvate.

In some embodiments, the aforementioned oligonucleotides comprise at least one modified nucleotide.

In some embodiments, the aforementioned oligonucleotides comprise at least one 2'-modified nucleotide.

In some embodiments, the aforementioned 2'-modified nucleotides are selected from one or more of 2'-alkoxy-modified nucleotides, 2'-substituted alkoxy-modified nucleotides, 2'-alkyl-modified nucleotides, 2'-substituted alkyl-modified nucleotides, 2'-amino-modified nucleotides, 2'-substituted amino-modified nucleotides, 2'-fluoro-modified nucleotides, and 2'-deoxynucleotides.

In some embodiments, the aforementioned 2'-modification is a modification selected from the group consisting of 2'-methoxy, 2'-acetylamino, 2'-aminoethyl, 2'-fluoro, 2'-O-methoxyethyl.

In some embodiments, the aforementioned oligonucleotides have a 5'-phosphate analog modified nucleotide at the 5' terminal nucleotide.

In some embodiments, the aforementioned 5'-phosphate analog modified nucleotide has a vinyl phosphonate modified nucleotide shown in formula (I), wherein R is selected from H, OH, fluorine, 2'-methoxy, 2'-acetylamino, 2'-aminoethyl and 2'-O-methoxyethyl, and Base represents a nucleic acid base selected from A, G, C, T and U.

In some embodiments, the aforementioned '-phosphate analog modified nucleotide has a vinyl phosphate modified nucleotide shown in formula (II), wherein R is selected from H, OH, fluorine, 2'-methoxy, 2'-acetylamino, 2'-aminoethyl and 2'-O-methoxyethyl.

In some embodiments, the aforementioned 5'-phosphonate analog modified nucleotide is APU shown in formula (II-1) or VPUm shown in formula (II-2).

In some embodiments, the oligonucleotide comprises at least one modified internucleotide linkage.

In some embodiments, at least one modified internucleotide bond is a phosphorothioate bond. The phosphorothioate internucleotide bond modification can occur on any nucleotide of the sense strand, antisense strand, or two strands in any position of the strand. For example, the internucleotide bond modification can occur on each nucleotide on the sense strand or antisense strand; each internucleotide bond modification can occur in an alternating pattern on the sense strand or antisense strand; or the sense strand or antisense strand can contain two internucleotide bond modifications in an alternating pattern. The alternating pattern of the internucleotide bond modification on the sense strand can be the same or different from the antisense strand, and the alternating pattern of the internucleotide bond modification on the sense strand can have an offset relative to the alternating pattern of the internucleotide bond on the antisense strand. In one embodiment, the double-stranded RNAi agent includes 6 to 8 phosphorothioate internucleotide bonds. In some embodiments, the antisense strand includes two phosphorothioate internucleotide bonds at the 5' end, and includes two phosphorothioate internucleotide bonds at the 3' end, and the sense strand includes at least two phosphorothioate internucleotide bonds at the 5' end or the 3' end.

In some embodiments, the aforementioned positive chain is selected from any of the aforementioned unmodified oligonucleotides in SEQ ID NO: 3, 6, 26-35, 37-40, 43-47, 49-53, 76-83, or any of the aforementioned modified oligonucleotides in SEQ ID NO: 181-183, 185-200, 202-216; the aforementioned antisense chain is selected from any of the aforementioned unmodified oligonucleotides in SEQ ID NO: 86, 89, 109-118, 120-123, 126-130, 132, 135-136, 159-175, 177-179, or any of the aforementioned modified oligonucleotides in SEQ ID NO: 218-220, 222-237, 239-257.

In some embodiments, the aforementioned oligonucleotide is selected from any one of the following sense strand and antisense strand combinations:

(1) the sense strand shown in SEQ ID NO:3, and the antisense strand shown in SEQ ID NO:86;

(2) the sense strand shown in SEQ ID NO:6, and the antisense strand shown in SEQ ID NO:89;

(3) the sense strand shown in SEQ ID NO:26, and the antisense strand shown in SEQ ID NO:109;

(4) the sense strand represented by SEQ ID NO:27, and the antisense strand represented by SEQ ID NO:110;

(5) the sense strand represented by SEQ ID NO:28, and the antisense strand represented by SEQ ID NO:111;

(6) the sense strand represented by SEQ ID NO:29, and the antisense strand represented by SEQ ID NO:112;

(7) the sense strand represented by SEQ ID NO:30, and the antisense strand represented by SEQ ID NO:113;

(8) the sense strand represented by SEQ ID NO:31, and the antisense strand represented by SEQ ID NO:114;

(9) the sense strand represented by SEQ ID NO:32, and the antisense strand represented by SEQ ID NO:115;

(10) the sense strand represented by SEQ ID NO:33, and the antisense strand represented by SEQ ID NO:116;

(11) the sense strand represented by SEQ ID NO:34, and the antisense strand represented by SEQ ID NO:117;

(12) the sense strand represented by SEQ ID NO:35, and the antisense strand represented by SEQ ID NO:118;

(13) the sense strand represented by SEQ ID NO:37, and the antisense strand represented by SEQ ID NO:120;

(14) the sense strand represented by SEQ ID NO:38, and the antisense strand represented by SEQ ID NO:121;

(15) the sense strand represented by SEQ ID NO:39, and the antisense strand represented by SEQ ID NO:122;

(16) the sense strand represented by SEQ ID NO:40, and the antisense strand represented by SEQ ID NO:123;

(17) the sense strand represented by SEQ ID NO:43, and the antisense strand represented by SEQ ID NO:126;

(18) the sense strand represented by SEQ ID NO:44, and the antisense strand represented by SEQ ID NO:127;

(19) the sense strand represented by SEQ ID NO:45, and the antisense strand represented by SEQ ID NO:128;

(20) the sense strand represented by SEQ ID NO:46, and the antisense strand represented by SEQ ID NO:129;

(21) the sense strand represented by SEQ ID NO:47, and the antisense strand represented by SEQ ID NO:130;

(22) the sense strand shown in SEQ ID NO:49, and the antisense strand shown in SEQ ID NO:132;

(23) the sense strand represented by SEQ ID NO:52, and the antisense strand represented by SEQ ID NO:135;

(24) the sense strand represented by SEQ ID NO:53, and the antisense strand represented by SEQ ID NO:136;

(25) the sense strand represented by SEQ ID NO:22, and the antisense strand represented by SEQ ID NO:105;

(26) the sense strand represented by SEQ ID NO:76, and the antisense strand represented by SEQ ID NO:159;

(27) the sense strand represented by SEQ ID NO:77, and the antisense strand represented by SEQ ID NO:160;

(28) the sense strand represented by SEQ ID NO:77, and the antisense strand represented by SEQ ID NO:161;

(29) the sense strand represented by SEQ ID NO:77, and the antisense strand represented by SEQ ID NO:162;

(30) the sense strand represented by SEQ ID NO:78, and the antisense strand represented by SEQ ID NO:163;

(31) the sense strand represented by SEQ ID NO:79, and the antisense strand represented by SEQ ID NO:164;

(32) the sense strand represented by SEQ ID NO:80, and the antisense strand represented by SEQ ID NO:165;

(33) the sense strand represented by SEQ ID NO:81, and the antisense strand represented by SEQ ID NO:166;

(34) the sense strand represented by SEQ ID NO:82, and the antisense strand represented by SEQ ID NO:167;

(35) the sense strand represented by SEQ ID NO:83, and the antisense strand represented by SEQ ID NO:168;

(36) the sense strand represented by SEQ ID NO:83, and the antisense strand represented by SEQ ID NO:169;

(37) the sense strand represented by SEQ ID NO:83, and the antisense strand represented by SEQ ID NO:170;

(38) the sense strand represented by SEQ ID NO:26, and the antisense strand represented by SEQ ID NO:109;

(39) the sense strand represented by SEQ ID NO:43, and the antisense strand represented by SEQ ID NO:126;

(40) the sense strand represented by SEQ ID NO:27, and the antisense strand represented by SEQ ID NO:110;

(41) the sense strand represented by SEQ ID NO:44, and the antisense strand represented by SEQ ID NO:171;

(42) the sense strand represented by SEQ ID NO:44, and the antisense strand represented by SEQ ID NO:172;

(43) the sense strand represented by SEQ ID NO:28, and the antisense strand represented by SEQ ID NO:111;

(44) the sense strand represented by SEQ ID NO:45, and the antisense strand represented by SEQ ID NO:173;

(45) the sense strand represented by SEQ ID NO:45, and the antisense strand represented by SEQ ID NO:174;

(46) the sense strand represented by SEQ ID NO:29, and the antisense strand represented by SEQ ID NO:112;

(47) the sense strand represented by SEQ ID NO:46, and the antisense strand represented by SEQ ID NO:175;

(48) the sense strand represented by SEQ ID NO:31, and the antisense strand represented by SEQ ID NO:114;

(49) the sense strand represented by SEQ ID NO:32, and the antisense strand represented by SEQ ID NO:115;

(50) the sense strand represented by SEQ ID NO:49, and the antisense strand represented by SEQ ID NO:177;

(51) the sense strand represented by SEQ ID NO:33, and the antisense strand represented by SEQ ID NO:116;

(52) the sense strand represented by SEQ ID NO:50, and the antisense strand represented by SEQ ID NO:178;

(53) the sense strand shown in SEQ ID NO:34, and the antisense strand shown in SEQ ID NO:117; and

(54) the sense strand represented by SEQ ID NO:51, and the antisense strand represented by SEQ ID NO:179;

wherein each strand is independently 19 to 25 nucleotides in length.

In some embodiments, the aforementioned oligonucleotide comprises any one selected from the following sense strand and antisense strand combinations:

(1) the sense strand represented by SEQ ID NO:181, and the antisense strand represented by SEQ ID NO:218;

(2) the sense strand represented by SEQ ID NO:182, and the antisense strand represented by SEQ ID NO:219;

(3) the sense strand represented by SEQ ID NO:183, and the antisense strand represented by SEQ ID NO:220;

(4) the sense strand represented by SEQ ID NO:185, and the antisense strand represented by SEQ ID NO:222;

(5) the sense strand represented by SEQ ID NO:186, and the antisense strand represented by SEQ ID NO:223;

(6) the sense strand represented by SEQ ID NO:187, and the antisense strand represented by SEQ ID NO:224;

(7) the sense strand represented by SEQ ID NO:188, and the antisense strand represented by SEQ ID NO:225;

(8) the sense strand represented by SEQ ID NO:189, and the antisense strand represented by SEQ ID NO:226;

(9) the sense strand represented by SEQ ID NO: 190, and the antisense strand represented by SEQ ID NO: 227;

(10) the sense strand represented by SEQ ID NO:191, and the antisense strand represented by SEQ ID NO:228;

(11) the sense strand represented by SEQ ID NO:192, and the antisense strand represented by SEQ ID NO:229;

(12) the sense strand represented by SEQ ID NO:193, and the antisense strand represented by SEQ ID NO:230;

(13) the sense strand represented by SEQ ID NO:194, and the antisense strand represented by SEQ ID NO:231;

(14) the sense strand represented by SEQ ID NO:195, and the antisense strand represented by SEQ ID NO:232;

(15) the sense strand represented by SEQ ID NO:196, and the antisense strand represented by SEQ ID NO:233;

(16) the sense strand represented by SEQ ID NO:197, and the antisense strand represented by SEQ ID NO:234;

(17) the sense strand represented by SEQ ID NO:198, and the antisense strand represented by SEQ ID NO:235;

(18) the sense strand represented by SEQ ID NO:199, and the antisense strand represented by SEQ ID NO:236;

(19) the sense strand represented by SEQ ID NO: 200, and the antisense strand represented by SEQ ID NO: 237;

(20) the sense strand represented by SEQ ID NO: 202, and the antisense strand represented by SEQ ID NO: 239;

(21) the sense strand shown in SEQ ID NO:203, and the antisense strand shown in SEQ ID NO:240;

(22) the sense strand represented by SEQ ID NO: 204, and the antisense strand represented by SEQ ID NO: 241;

(23) the sense strand represented by SEQ ID NO:205, and the antisense strand represented by SEQ ID NO:242;

(24) the sense strand represented by SEQ ID NO: 206, and the antisense strand represented by SEQ ID NO: 243;

(25) the sense strand represented by SEQ ID NO: 207, and the antisense strand represented by SEQ ID NO: 244;

(26) the sense strand represented by SEQ ID NO:208, and the antisense strand represented by SEQ ID NO:245;

(27) the sense strand represented by SEQ ID NO: 209, and the antisense strand represented by SEQ ID NO: 246;

(28) the sense strand represented by SEQ ID NO:210, and the antisense strand represented by SEQ ID NO:247;

(29) the sense strand represented by SEQ ID NO:210, and the antisense strand represented by SEQ ID NO:248;

(30) the sense strand represented by SEQ ID NO:211, and the antisense strand represented by SEQ ID NO:249;

(31) the sense strand represented by SEQ ID NO:212, and the antisense strand represented by SEQ ID NO:250;

(32) the sense strand represented by SEQ ID NO:213, and the antisense strand represented by SEQ ID NO:251;

(33) the sense strand represented by SEQ ID NO:213, and the antisense strand represented by SEQ ID NO:252;

(34) the sense strand represented by SEQ ID NO:206, and the antisense strand represented by SEQ ID NO:253;

(35) the sense strand represented by SEQ ID NO:206, and the antisense strand represented by SEQ ID NO:254;

(36) the sense strand represented by SEQ ID NO:209, and the antisense strand represented by SEQ ID NO:255;

(37) the sense strand represented by SEQ ID NO:214, and the antisense strand represented by SEQ ID NO:247;

(38) the sense strand represented by SEQ ID NO:215, and the antisense strand represented by SEQ ID NO:256; and

(39) the sense strand represented by SEQ ID NO:216, and the antisense strand represented by SEQ ID NO:257;

wherein each strand is independently 19 to 25 nucleotides in length.

In some embodiments, the aforementioned oligonucleotide comprises any one selected from the following sense strand and antisense strand combinations:

(1) the sense strand shown in SEQ ID NO: 202, and the antisense strand shown in SEQ ID NO: 239;

(2) the sense strand shown in SEQ ID NO: 203, and the antisense strand shown in SEQ ID NO: 240;

(3) the sense strand represented by SEQ ID NO: 210, and the antisense strand represented by SEQ ID NO: 247;

(4) the sense strand represented by SEQ ID NO: 211, and the antisense strand represented by SEQ ID NO: 249;

(5) the sense strand represented by SEQ ID NO:212, and the antisense strand represented by SEQ ID NO:250;

(6) the sense strand represented by SEQ ID NO:213, and the antisense strand represented by SEQ ID NO:251;

(7) the sense strand represented by SEQ ID NO:214, and the antisense strand represented by SEQ ID NO:247;

(8) the sense strand represented by SEQ ID NO:215, and the antisense strand represented by SEQ ID NO:256;

(9) the sense strand represented by SEQ ID NO: 216, and the antisense strand represented by SEQ ID NO: 257;

wherein each strand is independently 19 to 25 nucleotides in length.

On the other hand, the present disclosure provides a conjugate or a pharmaceutically acceptable salt thereof for reducing transthyretin (TTR) gene expression, comprising: (i) the aforementioned oligonucleotide or a pharmaceutically acceptable salt thereof, and (ii) a ligand conjugated to the aforementioned oligonucleotide or a pharmaceutically acceptable salt thereof, wherein at least one nucleotide of the oligonucleotide is conjugated to a targeting ligand. In some embodiments, at least one nucleotide of the aforementioned oligonucleotide or a salt thereof is conjugated to a targeting ligand to form an siRNA conjugate. The aforementioned siRNA conjugate contains the aforementioned siRNA and a conjugated group conjugated to the siRNA. The term "oligonucleotide salt" refers to an oligonucleotide compound in the form of a salt. Oligonucleotide salts include salts of oligonucleotide conjugated compounds and salts of unconjugated oligonucleotide compounds. Oligonucleotide salts are advantageously present in the form of solid powders.

In general, the aforementioned conjugated group includes at least one pharmaceutically acceptable targeting ligand and an optional linker, and the aforementioned siRNA, the aforementioned linker and the aforementioned targeting ligand are connected in sequence. The targeting group can be a ligand conventionally used in the field of siRNA administration, such as the various ligands described in WO2009082607A2, and all of its disclosures are incorporated herein by reference. In some embodiments, the aforementioned targeting ligand is 2-4. The aforementioned siRNA molecule can be non-covalently or covalently conjugated to the aforementioned conjugated group, for example, it can be covalently conjugated to the aforementioned conjugated group. The conjugation site of siRNA and the conjugated group can be at the 3' end or 5' end of the siRNA sense strand or antisense strand, and can also be in the internal sequence of siRNA. In some embodiments, the conjugation site of the aforementioned siRNA and the conjugated group is at the 3' end or 5' end of the siRNA sense strand. In some embodiments, the conjugation site of the aforementioned siRNA and the conjugated group is at the 3' end or 5' end of the siRNA antisense strand. In some preferred embodiments, the conjugation site between the aforementioned siRNA and the conjugation group is at the 3' end of the siRNA sense strand.

In some embodiments, the targeting ligand comprises an asialoglycoprotein receptor ligand. In some embodiments, the asialoglycoprotein receptor ligand comprises or consists of one or more galactose derivatives. As used herein, the term "galactose derivative" includes galactose and lactose derivatives whose affinity for the asialoglycoprotein receptor is equal to or greater than that of galactose. Galactose derivatives include, but are not limited to, galactose, galactosamine, N-formylgalactosamine, N-acetylgalactosamine, N-propionyl-galactosamine, N-n-butyryl-galactosamine, and N-isobutyrylgalactosamine. Galactose derivatives and galactose derivative clusters that can be used for oligonucleotides and other molecules to target the liver in vivo are known in the art. Galactose derivatives have been used to target molecules to hepatocytes in vivo by binding to asialoglycoprotein receptors (ASGPRs) expressed on the surface of hepatocytes. The binding of ASGPR ligands to ASGPR (s) facilitates cell-specific targeting to hepatocytes and endocytosis of molecules into hepatocytes. ASGPR ligands can be monomeric (eg, with a single galactose derivative) or polymeric (eg, with multiple galactose derivatives). Galactose derivatives or clusters of galactose derivatives can be linked to the 3' or 5' end of the siRNA using methods known in the art.

In some embodiments, the pharmaceutically acceptable targeting ligand in the aforementioned siRNA conjugate may be galactose or N-acetylgalactosamine (GalNAc), wherein the galactose or N-acetylgalactosamine molecule may be monovalent, divalent, trivalent, or tetravalent. Valency, divalency, trivalency, and tetravalency refer to the molar ratio of siRNA molecules to galactose or N-acetylgalactosamine molecules in the siRNA conjugate after the siRNA molecule forms a siRNA conjugate with a conjugated group containing galactose or N-acetylgalactosamine molecules as a targeting ligand. 1: 1, 1: 2, 1: 3 or 1: 4. In some embodiments, the pharmaceutically acceptable targeting ligand is N-acetylgalactosamine. In some embodiments, when the siRNA of the present invention is conjugated to a conjugated group containing N-acetylgalactosamine, the N-acetylgalactosamine molecule is trivalent or tetravalent. In some embodiments, when the siRNA of the present invention is conjugated to a conjugated group containing N-acetylgalactosamine, the N-acetylgalactosamine molecule is trivalent.

In some embodiments, the aforementioned targeting ligand comprises a carbohydrate, an amino sugar, cholesterol, a polypeptide, or a lipid.

In some embodiments, the aforementioned targeting ligand comprises an N-acetylgalactosamine (GalNAc) moiety.

In some embodiments, the aforementioned GalNac moiety is a monovalent GalNAc moiety, a divalent GalNAc moiety, a trivalent GalNAc moiety, or a tetravalent GalNAc moiety.

In some embodiments, the aforementioned targeting ligand is A1 represented by formula (III):

A1

In some embodiments, the aforementioned targeting ligand is L96 represented by formula (IV):

L96

The present disclosure also provides a conjugate for inhibiting TTR expression or a pharmaceutically acceptable salt thereof, comprising: (i) the aforementioned oligonucleotide or a pharmaceutically acceptable salt thereof, and (ii) one or more lipophilic moieties conjugated to one or more internal positions on at least one chain and/or one or more lipophilic moieties conjugated to the 3' and/or 5' termini on at least one chain, optionally via a linker or a carrier.

In certain embodiments, the lipophilic part is aliphatic, cyclic, for example, alicyclic or polycyclic, for example, alicyclic compounds, such as steroids (e.g., sterols) or straight or branched aliphatic hydrocarbons. The lipophilic part can generally include a hydrocarbon chain, which can be cyclic or acyclic. The hydrocarbon chain can include various substituents and/or one or more heteroatoms, such as oxygen or nitrogen atoms. Such lipophilic aliphatic parts include, but are not limited to, saturated or unsaturated C ₄ -C ₃₀ hydrocarbons (e.g., C ₆ -C ₂₂ hydrocarbons), saturated or unsaturated fatty acids, waxes (e.g., monohydric alcohol esters of fatty acids and fatty diamides), terpenes (e.g., C ₁₀ terpenes, C ₁₅ sesquiterpenes, C ₂₀ diterpenes, C ₃₀ triterpenes and C ₄₀ tetraterpenes) and other polyalicyclic hydrocarbons. For example, the lipophilic part can include C ₄ to C ₃₀ hydrocarbon chains (e.g., C ₄ to C ₃₀ alkyl or alkenyl). In some embodiments, the lipophilic moiety comprises a saturated or unsaturated C ₆ to C ₁₈ hydrocarbon chain (e.g., a straight chain C ₆ to C ₂₂ alkyl or alkenyl). In one embodiment, the lipophilic moiety comprises a saturated or unsaturated C ₁₆ hydrocarbon chain (e.g., a straight chain C ₁₆ alkyl or alkenyl). The lipid portion is a 2'-O-alkyl, including single chain and or branched 10-30 hydrocarbon chains.

The lipophilic moiety is conjugated to the double-stranded RNAi agent via a linker comprising an ether, thioether, urea, carbonate, amine, amide, maleimide-thioether, disulfide, phosphodiester, sulfonamide bond, click reaction (e.g., triazole from azide-alkyne cycloaddition), or carbamate.

In another aspect, the present disclosure provides a composition comprising the aforementioned oligonucleotide or a pharmaceutically acceptable salt thereof or a conjugate thereof or A salt of a conjugate thereof, and optionally a pharmaceutically acceptable carrier.

In some embodiments, the composition is in the form of an oral agent, an intravenous injection, a subcutaneous injection or an intramuscular injection, preferably a subcutaneous injection.

In some embodiments, the aforementioned combination further comprises other drugs for treating and/or preventing TTR-related diseases.

In some embodiments, the aforementioned TTR-related disease is selected from amyloidosis, metabolic disease, Alzheimer's disease and tumors.

In some embodiments, the aforementioned amyloidosis is familial amyloidosis.

In some embodiments, the aforementioned amyloidosis is selected from transthyretin amyloid cardiomyopathy (ATTR-CM) and transthyretin amyloid polyneuropathy (ATTR-PN).

In some embodiments, the aforementioned amyloidosis is familial amyloid polyneuropathy (FAP).

In some embodiments, the aforementioned amyloidosis is Alzheimer's disease.

In some embodiments, the aforementioned metabolic diseases are diabetes and impaired glucose regulation.

In some embodiments, the aforementioned diabetes is type II diabetes.

In another aspect, the present disclosure provides use of the aforementioned oligonucleotide or a pharmaceutically acceptable salt thereof or a composition thereof in the preparation of a medicament for treating and/or preventing a TTR-related disease.

In some embodiments, the aforementioned TTR-related disease is selected from amyloidosis, metabolic disease, Alzheimer's disease and tumors.

In some embodiments, the aforementioned amyloidosis is familial amyloidosis.

In some embodiments, the aforementioned amyloidosis is selected from transthyretin amyloid cardiomyopathy (ATTR-CM) and transthyretin amyloid polyneuropathy (ATTR-PN).

In some embodiments, the aforementioned amyloidosis is familial amyloid polyneuropathy (FAP).

In some embodiments, the aforementioned amyloidosis is Alzheimer's disease.

In some embodiments, the aforementioned metabolic disease is selected from diabetes and impaired glucose regulation.

In some embodiments, the aforementioned diabetes is type II diabetes.

In some embodiments, the aforementioned tumor is selected from lung cancer and pancreatic ductal carcinoma.

In some embodiments of the disclosed methods, expression of the TTR gene is inhibited by at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95%, or to below the level of detection in an assay. In a preferred embodiment, expression of TTR is inhibited by at least 70%. It should also be understood that it may be desirable to inhibit the expression of TTR in certain tissues without significantly inhibiting expression in other tissues.

In some embodiments, inhibition of in vivo expression is determined by knocking down a human gene in a rodent expressing a human gene, e.g., an AAV-infected mouse expressing a human target gene (i.e., TTR), e.g., when administered as a single dose, e.g., at a nadir of TTR expression following subcutaneous injection at 3 mg/kg to confirm the inhibitory effect on the human gene. Such a system is useful when the nucleic acid sequences of the human gene and the model animal gene are sufficiently close so that the human iRNA provides effective knockdown of the model animal gene.

Inhibition of TTR gene expression can be represented by a decrease in the amount of mRNA expressed by a cell line (such cells can be present, for example, in a sample derived from a subject) in which the TTR gene is transcribed and which is treated (e.g., by contacting one or more cells with an iRNA of the disclosure, or by administering an iRNA of the disclosure to a subject in which cells are or were present) such that expression of the TTR gene is inhibited compared to a cell line that is substantially identical to the cell line but has not been treated (control cells that have not been treated with the iRNA or have not been treated with an iRNA targeting the target gene). In a preferred embodiment, inhibition is assessed using a 10 nM siRNA concentration in a species-matched cell line using the method provided in Example 2, represented by 2^-ΔΔCT of the amount of mRNA expression in the treated cells relative to the mRNA level in the control cells, using the following formula, wherein a housekeeping gene (e.g., GAPDH) is used as an internal reference for normalization:

△CT＝CT _TTR -CT _GAPDH

△△CT＝△CT _{treated cells-} △CT _{control cells}

mRNA level = 2^-△△CT

In other embodiments, inhibition of TTR gene expression can be assessed based on a decrease in a parameter associated with TTR gene expression function, e.g., TTR protein levels in blood or serum from a subject. TTR gene silencing can be determined in any cell expressing TTR, whether endogenous or heterologous from an expression construct, and by any assay known in the art.

Inhibition of TTR protein expression can be demonstrated by a decrease in the level of TTR protein expressed by a cell or cell population or in a sample from a subject (e.g., the level of protein in a blood sample from a subject). As described above, to assess mRNA inhibition, inhibition of protein expression levels in treated cells or cell populations can be similarly expressed as a percentage of the protein level in a control cell or cell population, or as a change in protein level in a sample from a subject (e.g., blood or serum therefrom).

mRNA inhibition percentage = (protein expression level _{treated cells} - protein expression level _{control cells} ) / protein expression level _{control cells} * 100%

Control cells, cell populations, or subject samples that can be used to assess inhibition of TTR gene expression include cells, cell populations, or subject samples that have not been contacted with the RNAi agents disclosed herein. For example, control cells, cell lines, or subject samples can be from an individual subject (e.g., a human or animal subject) before treating the subject or an appropriately matched group control with an RNAi agent.

In some embodiments of the disclosed methods, the iRNA is administered to a subject so that the iRNA is delivered to a specific site within the subject. Inhibition of TTR expression can be assessed by measuring the level or change of TTR mRNA or TTR protein or fusion secreted luciferase in a sample of fluid or tissue from a specific site (e.g., liver or blood) of the subject.

The present disclosure also provides methods of using the iRNA of the present disclosure or compositions comprising the iRNA of the present disclosure to inhibit TTR expression, thereby preventing or treating The invention relates to the treatment of the following diseases, disorders and/or conditions, including but not limited to: amyloidosis (e.g., familial amyloid polyneuropathy (FAP), Alzheimer's disease), metabolic diseases (e.g., diabetes, impaired glucose regulation) and tumors (e.g., lung cancer, pancreatic ductal carcinoma), as well as complications of each of the above conditions.

Various formulations have been developed to facilitate the use of oligonucleotides. For example, an oligonucleotide, a salt thereof, a conjugate thereof, or a salt thereof of a conjugate thereof can be delivered to a subject or a cell environment using a formulation that minimizes degradation, facilitates delivery and/or uptake, or provides another beneficial property for an oligonucleotide, a salt thereof, a conjugate thereof, or a salt thereof in a formulation. In some embodiments, provided herein are compositions comprising an oligonucleotide (e.g., a single-stranded or double-stranded oligonucleotide), a salt thereof, a conjugate thereof, or a salt thereof of a conjugate thereof to reduce the expression of TTR. Such compositions can be suitably formulated so that when applied to a subject (to the direct environment of a target cell or systemically), a sufficient portion of the oligonucleotide, a salt thereof, a conjugate thereof, or a salt thereof of a conjugate thereof enters the cell to reduce TTR expression. Any of a variety of suitable oligonucleotide formulations can be used to deliver an oligonucleotide, a salt thereof, a conjugate thereof, or a salt thereof of a conjugate thereof for reducing TTR as disclosed herein. In some embodiments, an oligonucleotide, a salt thereof, a conjugate thereof, or a salt thereof of a conjugate thereof is formulated in a buffer solution, such as a phosphate-buffered saline solution, a liposome, a micellar structure, and a shell. The buffer solution can be selected from a solution of acetate, citrate, prolamin, carbonate or phosphate or any combination thereof. In some embodiments, the naked (i.e., without a delivery agent) oligonucleotide or its conjugate is formulated in water or an aqueous solution (e.g., pH-adjusted water). In some embodiments, the naked oligonucleotide or its conjugate is formulated in an alkaline buffered aqueous solution (e.g., PBS).

The preparation of oligonucleotides with cationic lipids can be used to facilitate transfection of oligonucleotides into cells. For example, cationic lipids such as lipofectin, cationic glycerol derivatives and polycationic molecules (e.g., polylysine) can be used.

Thus, in some embodiments, the formulation comprises lipid nanoparticles. In some embodiments, the excipient comprises a liposome, lipid, lipid complex, microsphere, microparticle, nanosphere or nanoparticle, or may be otherwise formulated for administration to a cell, tissue, organ or body of a subject in need thereof.

In some embodiments, the preparation as disclosed herein includes excipients. In some embodiments, excipients give the stability of the improvement of active ingredient, the absorption of improvement, the solubility of improvement and/or therapeutic enhancement to the composition. In some embodiments, excipients are buffers (e.g., sodium citrate, sodium phosphate, tris alkali or sodium hydroxide) or vehicles (e.g., buffered solutions, petrolatum, dimethyl sulfoxide or mineral oil). In some embodiments, oligonucleotides are freeze-dried for extending their shelf life, and then solutions are made before use (e.g., being applied to a subject). Therefore, the excipient in the composition comprising any one of the oligonucleotides described herein, their salts or their conjugates or the salts of their conjugates can be lyoprotectants (e.g., mannitol, lactose, polyethylene glycol or polyvinyl pyrrolidone) or collapse temperature modifiers (e.g., dextran, ficoll or gelatin).

In some embodiments, the pharmaceutical composition is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, such as intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration. Typically, the route of administration is intravenous or subcutaneous.

Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous or subcutaneous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL.TM. (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). The aforementioned carrier may be a solvent or dispersion medium containing, for example, water, ethanol, a polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.) and a suitable mixture thereof. In many cases, it is preferred to include an isotonic agent, such as a sugar, a polyol such as mannitol, sorbitol, and sodium chloride in the composition. A sterile injectable solution can be prepared by incorporating the desired amount of an oligonucleotide, a salt thereof, or a conjugate thereof, or a salt of its conjugate into a selected solvent with one or a combination of the desired ingredients listed above, followed by filtration sterilization.

In some embodiments, the composition may contain at least about 0.1% or more of a therapeutic agent (e.g., an oligonucleotide for reducing TTR expression, a salt thereof, or a conjugate thereof, or a salt of a conjugate thereof), although the percentage of one or more active ingredients may be between about 1% and about 80% or more by weight or volume of the total composition. Those skilled in the art of preparing such pharmaceutical formulations will consider factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, and other pharmacological considerations, and therefore various doses and treatment regimens may be desired.

Cells suitable for treatment using the methods of the present disclosure can be any cell expressing the TTR gene, for example, a liver cell, a brain cell, or a kidney cell, but preferably a liver cell. Cells suitable for use in the methods of the present disclosure can be mammalian cells, for example, primate cells (such as human cells, including human cells in chimeric non-human animals, or non-human primate cells, for example, monkey cells or chimpanzee cells) or non-primate cells. In some embodiments, the cell is a human cell, for example, a human liver cell. In the methods of the present disclosure, the expression of TTR in the cell is inhibited by at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95%, or below the detection level of the assay.

The in vivo method of the present disclosure may include administering to a subject a composition comprising an iRNA, wherein the iRNA comprises a nucleotide sequence complementary to at least a portion of an RNA transcript of a TTR gene of a mammal to which the RNAi agent is administered. The composition may be administered by any means known in the art, including but not limited to oral, intraperitoneal or parenteral routes, including intracranial (e.g., intraventricular, intraparenchymal and intrathecal), intravenous, intramuscular, subcutaneous, transdermal, airway (aerosol), nasal, rectal and topical (including oral and sublingual) administration. In some embodiments, the composition is administered by intravenous infusion or injection. In some embodiments, the composition is administered subcutaneously. In some embodiments, the composition is administered by intramuscular injection.

In one aspect, the present disclosure also provides a method for inhibiting mammalian TTR gene expression. The aforementioned method includes administering an oligonucleotide or a pharmaceutically acceptable salt thereof, a conjugate thereof, or a salt of its conjugate, or a composition thereof to a mammal. The aforementioned oligonucleotide is a double-stranded RNA (dsRNA), and the aforementioned dsRNA targets the TTR gene in mammalian cells and maintains the mammal for a sufficient time to obtain degradation of the mRNA transcript of the TTR gene, thereby inhibiting the expression of the TTR protein in the cell. The reduction in gene expression can be assessed by any method known in the art and by methods, such as qRT-PCR described herein, for example, in Example 2. The reduction in protein products can be assessed by any method known in the art (e.g., ELISA). In other embodiments, a blood sample is used as a subject sample to monitor the reduction in TTR protein expression.

The present disclosure also provides methods of treating in a subject in need thereof, e.g., a subject diagnosed with a TTR-related disorder, including but not limited to: amyloidosis (e.g., familial amyloid polyneuropathy (FAP), Alzheimer's disease), metabolic diseases (e.g., diabetes, glucose-regulated receptors, damage) and tumors (e.g. lung cancer, pancreatic ductal carcinoma).

The iRNA of the present disclosure can be administered as "free iRNA". Free iRNA is administered without a pharmaceutical composition. Naked iRNA can be in a suitable buffer solution. The buffer solution can contain acetate, citrate, lactate, tartrate, carbonate or phosphate, or any combination thereof. In one embodiment, the buffer solution is phosphate buffered saline (PBS). The pH and osmotic pressure of the buffer solution containing the iRNA can be adjusted to make it suitable for administration to a subject.

The iRNA of therapeutic amount can be applied to the subject, such as the dosage in the range of about 0.01mg/kg to about 200mg/kg (for example, about 0.1mg/kg to about 100mg/kg). In some embodiments, the salt of oligonucleotide, its salt or its conjugate or its conjugate is in the range of about 0.1mg/kg to about 50mg/kg, preferably about 0.1mg/kg to about 20mg/kg, 0.3mg/kg to about 18mg/kg, 0.5mg/kg to about 15mg/kg or 0.8mg/kg to about 12mg/kg, more preferably about 1mg/kg to about 10mg/kg. The iRNA is preferably administered subcutaneously, that is, by subcutaneous injection. One or more injections can be used to deliver the iRNA of the desired dose to the subject. The injection can be repeated over a period of time.

In some embodiments, the iRNA is administered at a fixed dose of about 50 mg to about 800 mg. In some embodiments, the iRNA is administered to a subject at a fixed dose of about 50 mg to about 200 mg, about 200 mg to about 400 mg, or about 400 mg to about 800 mg. In some embodiments, the iRNA is administered to a subject at a fixed dose of about 100 mg, about 150 mg, about 200 mg, about 300 mg, about 400 mg, 500 mg, about 600 mg, about 700 mg, or about 800 mg.

Can be regularly repeated administration. In some embodiments, after the initial treatment regimen, treatment can be carried out at a lower frequency. Repeated dosing regimens may include regular administration of the iRNA of the therapeutic amount, such as once a month to once a year. In some embodiments, the iRNA is administered about once a month to about once every three months, or about once every three months to about once every six months, or even once a year.

In some embodiments, the fixed dose is administered to the subject at intervals of once a month. In some embodiments, the fixed dose is administered to the subject at intervals of once every six months.

In some embodiments, a subject is administered a fixed dose of about 150 mg approximately once every six months. In some embodiments, a subject is administered a fixed dose of about 200 mg approximately once every six months. In some embodiments, a subject is administered a fixed dose of about 300 mg approximately once every six months. In some embodiments, a subject is administered a fixed dose of about 600 mg approximately once every six months. In some embodiments, a subject is administered a fixed dose of about 800 mg approximately once every six months. In some embodiments, a subject is administered a fixed dose of about 800 mg approximately once every six months.

In some embodiments, the subject to be treated is a human (e.g., a human patient) or a non-human primate or other mammalian subject. Other exemplary subjects include domestic animals such as dogs and cats; livestock such as horses, cattle, pigs, sheep, goats, and chickens; and animals such as mice, rats, guinea pigs, and hamsters.

The present disclosure further provides that the combination therapy of iRNA agents or their pharmaceutical compositions with other drugs and/or other treatment methods (e.g., known drugs and/or known treatment methods, e.g., those currently used to treat these conditions) will benefit patients with amyloidosis, metabolic diseases, Alzheimer's disease, and tumors, etc. The combination therapy can be an oral drug, etc.

For purposes of clarity and concise description, features are described herein as part of the same or separate embodiments, however, it will be understood that the scope of the present disclosure may include embodiments having a combination of all or some of the described features.

Hereinafter, the present disclosure will be described in more detail with reference to specific examples; however, the examples are only for illustrative purposes and have no limiting effect on the present disclosure.

Example

In the absence of specific information on the source of reagents, such reagents can be obtained from any molecular biology reagent supplier at quality/purity standards appropriate for molecular biology.

Abbreviations for nucleotide monomers used in nucleic acid sequence representations. It will be understood that these monomers, when present in an oligonucleotide, are interconnected by 5'-3' phosphodiester bonds unless otherwise specified.

Table A. Nucleotide monomer abbreviations used in nucleic acid sequence representation

Preparation of targeting ligands

(i) L96 was prepared according to the method described in patent CN104717982B.

(ii) The preparation steps of A1 are as follows:

(1) Synthesis of intermediate E1

F1 (199.46 mg, 0.41 mmol) was dissolved in DMF and activated by adding HBTU (155.39 mg, 0.41 mmol), followed by adding DIEA (67.81 L, 0.41 mmol), stirring for 30 min, and then adding intermediate G1 (100.0 mg, 0.14 mmol) to the reaction system. After stirring overnight, TLC detected that the reaction was complete, and after reduced pressure concentration, column chromatography separation (PE: EA = 10: 1) was performed to obtain intermediate I-F1 (174.98 mg, yield 58%). Intermediate I-F1 was debenzylated with palladium carbon under hydrogen catalysis to obtain intermediate II-F1 (156.34, 95%). The HBTU activated intermediate II-F1 was coupled with (3R,5S)-5-[[bis(4-methoxyphenyl)phenylmethoxy]methyl]-3-pyrrolidinol in DMF to give intermediate E1 ¹ HNMR (400 MHz, DMSO-D ₆ ): δ7.91-7.86(m,8H),7.51-7.45(m,6H),7.40-7.32(m,10H),7.02-6.95(m,3H),6.03-5.89(m,4H),5.31(d,3 H),5.07-4.99(m,4H),4.43-4.37(m,3H),4.23-4.19(m,4H),4.17-4.13(m,6H),4.05-4.00(m,6H),3.88-3.81( m,6H),3.77(s,6H),3.69-3.66(m,9H),3.50-3.47(m,12H),3.40-3.37(m,6H),3.20-3.16(m,16H),2.60-2.57( m,2H),2.46(s,9H),2.30(s,9H),2.10(m,6H),2.07(s,9H),1.98(s,9H),1.56-1.45(m,8H),1.32-1.27(m,4H). HRMS[M+H] ⁺ m/z 2467 (calcd for C ₁₁₅ H ₁₆₈ N ₁₄ O ₄₅ , 2466).

(2) Synthesis of solid phase support A1 with protective groups

Intermediate E1 was treated with succinic anhydride in the presence of DMAP and Et3N to obtain the hemisuccinate intermediate E1-1 in quantitative yield. The hemisuccinate intermediate E1-1 was coupled with the amino group of a 60% divinylbenzene (DVB) cross-linked (aminomethyl) polystyrene resin having an amine content of 250 mol/g to obtain a solid support A1 having a loading of 100 mol/g and having a protective group.

Preparation of oligonucleotides

(1) Preparation of siRNA

The siRNA sequence is synthesized separately on a solid support via a sense strand (SS) and an antisense strand (AS), and is obtained after deprotection, cleavage, purification, annealing, purification and freeze-drying.

Solid phase synthesis (Figure 1): The sense strand and antisense strand are synthesized separately on a solid support using an oligonucleotide automatic synthesizer using phosphoramidite technology. The synthesizers include AKTA Oligopilot (Cytiva) and Dr.Oligo 192XLc (Kunshan Berleke Precision Instrument Co., Ltd.). Solid phase synthesis starts from the 3' end of the sequence and couples the monomers into the sequence in sequence. Each coupling of a phosphoramidite monomer includes four chemical steps: 1) unblocking or deprotection (removal of hydroxyl protecting group); 2) coupling; 3) oxidation; 4) end-capping. The phosphoramidite monomers, reagents, and purification consumables used are all commercial circulating reagents and consumables, such as various phosphoramidite monomers (such as 5'-O-(4,4'-Dimethoxytrityl)-2'-O-methyl-Uridine-3'-CE-Phosphoramidite) purchased from Shanghai Zhaowei Technology Development Co., Ltd., and reaction reagents (such as 40wt% methylamine aqueous solution, 28wt% ammonium hydroxide aqueous solution, etc.) purchased from Sigma-Aldrich LLC. The siRNA synthesis and purification methods used in this article are described in US20130178612A1, US2015100197A1, etc.; the synthesis method containing VPUm and APU structure sequences is described in J.Med.Chem.2018,61,734-744.

(2) Preparation of double-stranded RNA agents

(a) Synthesis of the positive strand

The solid phase phosphoramidite method is a mature oligonucleotide synthesis method. The reaction is carried out in a stainless steel synthesis column using a computer-controlled synthesizer. In the synthesis of the positive chain, a solid phase support loaded with a targeting ligand (such as L96 and A1) is used as the starting point, or the solid phase support is used directly as the starting point. Different raw materials, reagents and solvents are injected into different pipelines in the order of sequence 3' to 5' through the solid phase synthesizer to connect the phosphoramidite nucleoside monomers one by one. The reaction process includes four cycles of DMT protection group removal reaction, condensation reaction, oxidation or thiolation reaction, and end-capping reaction. Each cycle connects a nucleotide unit to obtain an oligonucleotide sequence of 19 or 21 nucleotide units. After the synthesis is completed, the protecting group (2-cyanoethyl) is removed on the solid phase synthesis column, and the synthesized sequence is cut from the solid phase support by aminolysis reaction, filtered, the filter cake is washed with ethanol, the filtrate and washing liquid are collected, and concentrated to obtain the crude positive chain. The crude product is purified by chromatography (SOURCE 15Q) and freeze-dried to obtain the target product positive chain. Among them, in the synthesizer, the siRNA positive chain conjugate is synthesized starting from the solid support loaded with the targeting ligand (such as L96); and the siRNA is synthesized directly starting from the solid support.

(b) Synthesis of antisense strand

The synthesis of the antisense strand is similar to that of the sense strand. Different raw materials, reagents and solvents are injected into different pipelines in the order of 3' to 5' of the sequence through a solid phase synthesizer to connect phosphoramidite nucleoside monomers one by one. The reaction process includes four cycles of DMT protection group removal reaction, condensation reaction, oxidation or thiolation reaction, and end-capping reaction. Each cycle connects a nucleotide unit to obtain an oligonucleotide sequence of 21 or 23 nucleotide units. After the synthesis is completed, the protecting group (2-cyanoethyl) is removed on the solid phase synthesis column, and then the synthesized sequence is cut from the solid phase support by aminolysis reaction, filtered, and the filter cake is washed with ethanol. The filtrate and washing liquid are collected and concentrated to obtain the crude antisense strand. The crude product is purified by chromatography (SOURCE 15Q), ultrafiltered, and freeze-dried to obtain the target product antisense strand siRNA.

(c) Preparation of double-stranded siRNA

The AS chain and the SS chain were dissolved in injection water respectively, mixed in a determined ratio (1.01:1.0-1.2:1.0), incubated at 30-50°C for 30-90 minutes, cooled to room temperature, and freeze-dried to obtain a double-stranded siRNA product.

According to the same method, the double-stranded siRNA agents shown in Tables 2, 3 and 4 below were prepared.

Example 1. Activity screening of naked TTR-siRNA sequences in Hep3B cells

First, a computer-based algorithm was used to generate candidate oligonucleotide sequences complementary to human TTR mRNA (NM_000371.4, Table 1), some of which were also complementary to cynomolgus monkey TTR mRNA (XM_045377903.1, Table 1) or had no more than 2 mismatches. Some of them were designed as double-stranded siRNAs with 19/21 pairings of the sense and antisense strands, respectively, and the antisense strand had two overhangs complementary to the mRNA sequence, and in some cases the overhangs of the antisense strand were non-complementary UU; some of them were designed as double-stranded siRNAs with 21/23 pairings of the sense and antisense strands, respectively, and the antisense strand had two overhangs complementary to the mRNA sequence; some of them were designed as double-stranded siRNAs with 21/21 and 23/23 pairings. In some of the complementary paired sequences, the first base at the 5' end of the antisense strand (the last base at the 3' end of the sense strand) was replaced with a base that did not match the TTR mRNA.

Table 1 Human and cynomolgus monkey TTR mRNA sequences

In Tables 2, 3 and 4, "G", "C", "A", "U", "T" and "I" generally represent nucleotides with guanine, cytosine, adenine, uracil, thymine and hypoxanthine as bases, respectively. dT represents 2'-deoxythymidine-3'-phosphate.

For the modification of nucleotides: m represents 2'-methoxy; f represents 2'-deoxy-2'-fluoro; s represents thiophosphate; APU is uridine acid (2'-acetylamino-5'-vinylphosphonate-uridine acid) modified by a 5'-phosphate analogue as shown in formula (II-1); A1 is a GalNAc targeting ligand as shown in formula (III); L96 is N-[tri(GalNAc-alkyl)amidodecanoyl]-4-hydroxyprolinol (Hyp-(GalNAc-alkyl)3) as shown in formula (IV).

Table 2 Naked oligonucleotide sequences

Table 3 Modified oligonucleotides

Table 4 siRNA sequences with targeting ligands

(1) Hep3B cell culture and transfection:

Human liver cancer cell line Hep3B (Shanghai Fushen Biotechnology Co., Ltd.) was used and placed in an incubator at 37°C and 5% _CO2 . DMEM medium (Hyclone, SH30022.01, 2g/L Glucose) was used and supplemented with 10% FBS (aqlabteech, AQ-MV-06600) and 1% penicillin-streptomycin (Keygen Biotechnology, KGY0023) for culture. When the cell confluence reached 90%, the cells were digested with trypsin (Amresco, 0458-250G) and counted with a counter (Nexcelom, cellometer Mini). 150μl of cell suspension/well was inoculated in a 96-well plate, and the cell number was 2* ¹⁰⁴ cells/well. The cells were allowed to adhere to the plate the next day for transfection.

Lipofectamine ^TM RNAiMAX (thermofisher, 13778150) was used for transfection. 10 μl (50 nM or 5 nM) of the diluted compound was dispersed in 20 μL Opti-MEM (thermofisher, 1105821), and 0.2 μL RNAiMAX was dispersed in 25 μL Opti-MEM. The final concentration of siRNA was 10 nM or 1 nM. After incubation for 5 min, the cells were mixed with the compound dispersion and incubated for 10 min. The cells were added to the transfection complex (n=2) and cultured in a 37°C, 5% CO2 incubator for 24 hours.

(2) RNA extraction and detection

(i) Total RNA was extracted using RNA-Quick Purification Kit (RN001, Yishan Biotechnology):

Take out the 12-well plate from the incubator, drain the culture medium, wash once with an appropriate amount of PBS, add 500μl of lysis buffer to each well, and transfer the supernatant to a new 1.5ml centrifuge tube. Add 500μl of anhydrous ethanol to the lysed cells and mix thoroughly. Invert the centrifuge tube several times, or use a pipette to blow and aspirate vigorously 10 times to disperse the precipitate, then add the liquid to the centrifuge column, place the centrifuge tube symmetrically in a centrifuge (eppendorf, 5430), and centrifuge at 4000×g for 1min. Take out the centrifuge tube and add 500μl of wash buffer to the column, centrifuge at 12000×g for 1min, take out the column after the centrifugation, pour out the waste liquid, put the RNA column back into the collection tube, centrifuge the empty tube once to remove the residual wash buffer. Place the column in a clean 1.5ml centrifuge tube without RNase, open the lid and dry for 2min. Add 30μl of elution buffer to the center of the membrane of the RNA column, let it stand at room temperature for 2min, centrifuge at 2000×g for 1min, and the RNA is eluted. After eluting, place on ice. Measure the concentration of eluted RNA for subsequent experiments. The extracted RNA can be used immediately for subsequent experiments or stored at -80℃ for later use.

(ii) Use cDNA was synthesized using IIQ RT SuperMix for qPCR (+gDNA wiper) reverse transcription kit (Novozyme, R223-01):

Prepare a mixture of 4μl 4×gDNA wiper Mix, 1μg template RNA, and 16μl RNase-free ddH ₂ O in an RNase-free centrifuge tube to remove genomic DNA. Use a pipette to gently mix and incubate at 42℃ for 2min. Then add 4μl 5×HiScript II qRT SuperMix II directly to the reaction tube and mix gently with a pipette. Place in a PCR instrument (Applied Biosystems, 9700) at 50℃ for 15min; 85℃ for 5sec, and maintain at 4℃. The product can be used for qPCR reaction immediately, or stored at -20℃ and used within half a year. For long-term storage, it needs to be stored at -80℃ after aliquoting. Repeated freezing and thawing of cDNA should be avoided.

(iii) qPCR quantification using ChamQ SYBR qPCR Master Mix (Novozymes, Q311-02):

A mixed solution of 10 μl 2×ChamQ SYBR qPCR Master Mix, 0.5 μl Forword primer (Ruiboxin), 0.5 μl Reverse primer (Ruiboxin), 1 μl Template cDNA, and 8 μl ddH ₂ O was prepared to a 20 μl system. Each sample was replicated three times. The 96-well plate was placed in a qPCR instrument (ROCGENE, Archimed) and the following program was executed: pre-denaturation, 95°C, 30 sec; amplification, 95°C, 10 sec, 60°C, 30 sec, 40 cycles; melting curve, 95°C, 15 sec, 60°C, 60 sec, 95°C, 15 sec.

(3) Data statistical analysis:

The data were exported to EXCEL format and normalized to the control group using CT _TTR -CT _hTBP . In order to calculate the fold change of relative silencing efficiency, the data were analyzed using the △△CT method. The results are shown in Table 5.

At a dosage of 10 nM, the inhibition rate of naked sequences AL0051001-AL0051041 and AL0051044-AL0051080 on the TTR gene expression level of Hep3B cell lines was between about 10-90%, among which the inhibition rate of naked sequences AL0051003, AL0051006, AL0051022, AL0051026-AL0051035, AL0051037-AL0051040, AL0051045-AL0051049, AL0051051, AL0051054 and AL0051055 on the TTR gene expression level of Hep3B cell lines was above 70%. At a low concentration of 1 nM, the inhibition rate of TTR mRNA decreased.

Table 5 TTR siRNA naked sequence knockdown levels in Hep3B cells

Example 2. Screening of TTR-siRNA activity in primary cynomolgus monkey hepatocytes

(1) Cynomolgus monkey hepatocyte cell culture and transfection:

Before adding cells, take out the culture medium (Beijing Red Biotech Co., Ltd., HEPO24) and preheat it in a biosafety cabinet. Add 4 ml of FBS to 36 ml of culture medium (TPCS, HEPO24) to prepare complete culture medium, and heat it in a 37°C water bath for 10 min. Treat with coating medium (Beijing Red Biotech Co., Ltd., HEPO44) in a _CO2 incubator at 37°C for 0.5 h. Take out the primary hepatocytes of cynomolgus monkeys (Beijing Red Biotech Co., Ltd., cmTCSC) from liquid nitrogen, revive the cells in a 37°C water bath, take them out after about 2 min, transfer the cell suspension to 40 ml of preheated complete culture medium, wash the cell cryopreservation tube with 2 ml of complete culture medium, centrifuge the cell suspension at 180 × g for 1 min, discard the supernatant, add 2 ml of preheated CM seeding medium (Beijing Red Biotech Co., Ltd., CMHEP054), gently blow to mix the cell suspension, and take 20 μl of cell suspension for counting. According to the counting results, the cells were diluted to 3*10 ⁵ /ml, inoculated into 12-well plates at 3*10 ⁵ /well, and cultured in an incubator at 37°C, 5% CO _2. After 4-5 hours of adherence, the CM seeding medium was aspirated and replaced with preheated medium (Beijing Red Biotech Co., Ltd., CMHEP064), 0.9ml/well, and transfected after 6 hours of adherence. Lipofectamine ^TM 3000 Transfection Reagent (thermofisher, L3000150) was used for transfection. System ① was diluted with 50 μl Opti-MEM (thermofisher, 1105821) to a concentration of 1 μM, 0.2 μM or 20 nM siRNA (Suzhou Ouli Biopharmaceutical Technology Co., Ltd.), and system ② was diluted with 50 μl Opti-MEM to 3 μl Lipo3000. After standing for 5 min, system ① and ② were mixed. The final concentration of siRNA was 0.5 μM or 0.1 μM. After standing for another 15 min, the cells were added dropwise to a 12-well plate. The final concentration of siRNA was 50 nM, 10 nM or 1 nM. DMEM/F12 complete medium was replaced 4 h after transfection, and the 12-well plate was placed in an incubator and incubated for 24 h.

(2) RNA extraction and detection

The steps are the same as (2) RNA extraction and detection in Example 1.

(3) Data statistical analysis:

The data were exported in EXCEL format and normalized to the control group using CT _TTR -CT _GAPDH . In order to calculate the fold change of relative silencing efficiency, the data were analyzed using the △△CT method. The mean and standard deviation of the three parallel repeated data were calculated. The screening results of monkey primary cells are shown in Table 6.

As can be seen from Table 6, after sequence translation design and base mutation of the sequences with better efficacy in Example 1, when the dosage was 10nM, except for AL0051116, the inhibitory effect of all siRNAs in AL0051082-AL0051122 on TTR mRNA was ≥90%.

Table 6 TTR-siRNA naked sequence knockdown level in cynomolgus monkey primary hepatocytes

Example 3. Activity screening of chemically modified TTR-siRNA in primary hepatocytes of cynomolgus monkeys

(1) Cynomolgus monkey hepatocyte cell culture and transfection:

Before adding cells, take out the culture medium (Beijing Red Biotech Co., Ltd., HEPO24) and preheat it in a biosafety cabinet. Add 4 mL of FBS to 36 ml of culture medium (TPCS, HEPO24) to prepare complete culture medium, and heat it in a 37°C water bath for 10 min. Treat with coating medium (Beijing Red Biotech Co., Ltd., HEPO44) in a _CO2 incubator at 37°C for 0.5 h. Take out the primary hepatocytes of cynomolgus monkeys (Beijing Red Biotech Co., Ltd., cmTCSC) from liquid nitrogen, revive the cells in a 37°C water bath, take out after about 2 min, transfer the cell suspension to 40 ml of preheated complete culture medium, wash the cell cryopreservation tube with 2 ml of complete culture medium, centrifuge the cell suspension at 180 × g for 1 min, discard the supernatant, add 2 ml of preheated CM seeding medium (Beijing Red Biotech Co., Ltd., CMHEP054), gently blow to mix the cell suspension, and take 20 μl of cell suspension for counting. According to the counting results, the cells were diluted to 3*10 ⁵ /ml, inoculated into 12-well plates at 3*10 ⁵ /well, and cultured in an incubator at 37°C, 5% CO _2. After 4-5 hours of adherence, the CM seeding medium was aspirated and replaced with preheated medium (Beijing Red Biotech Co., Ltd., CMHEP064), 0.9ml/well, and transfected after 6 hours of adherence. Lipofectamine ^TM 3000 Transfection Reagent (thermofisher, L3000150) was used for transfection. Modified siRNA (Suzhou Ouli Biopharmaceutical Technology Co., Ltd.) was diluted to 200 nM and 20 nM in system ① with 50 μl Opti-MEM (thermofisher, 1105821). 3 μl Lipo3000 was diluted with 50 μl Opti-MEM in system ②. After standing for 5 min, systems ① and ② were mixed and stood for 15 min, and then added dropwise to a 12-well plate. The final concentration of siRNA was 10 nM or 1 nM. DMEM/F12 complete medium was replaced 4 h after transfection, and the 12-well plate was placed in an incubator and incubated for 24 h.

(2) RNA extraction and detection

The steps are the same as (2) RNA extraction and detection in Example 1.

(3) Data statistical analysis:

The data were exported to EXCEL format and normalized to the control group using CT _TTR -CT _GAPDH . In order to calculate the fold change of relative silencing efficiency, the data were analyzed using the △△CT method. The mean and standard deviation of the three parallel repeated data were calculated. The screening results of monkey primary cells are shown in Tables 7 and 8. Tables 7 and 8 are the results of experiments using different batches of monkey primary cells at different times.

When the drug dosage was a low concentration of 1 nM, AL0055001, AL0055005, AL0055013, AL0055018, AL0055019, AL0055022, AL0055023, AL0055024, AL0055025, AL0055026, AL0055027, AL0055028, AL0055029, AL0055030, AL0055031, AL0055032, AL0055033, AL0055034, AL0055035, AL0055036, AL0055041, AL0055042, and AL0055043 siRNA had an effect on The inhibitory effect of TTR mRNA is basically above 75%; in some sequences, when the dosage is a high concentration of 10nM, the inhibition rate of siRNA is above 85%, such as AL0055025, AL0055026, AL0055027, AL0055028, AL0055029, AL0055030, AL0055031, AL0055032, AL0055033, AL0055034, AL0055035, AL0055036, AL0055037, AL0055038, AL0055039, AL0055040, AL0055041, AL0055042 and AL0055043.

Table 7 Knockdown level of chemically modified TTR-siRNA in primary hepatocytes of cynomolgus monkeys

Table 8 Knockdown level of chemically modified TTR-siRNA in primary hepatocytes of cynomolgus monkeys

Example 4. In vivo testing of TTR-RNAi agents in HDI mice

The mRNA sequence of the human TTR gene was obtained from the NCBI database (GENBANK NO.NM_000371.4). The TTR gene 27-470 base sequence (SEQ ID NO.258) was selected to obtain a recombinant plasmid by conventional molecular biology techniques such as restriction digestion and ligation. First, the sequence containing the CAG promoter (SEQ ID NO.259) was inserted between EcoRI and XhoI of the pFB vector (purchased from Agilent, catalog number 013001) to obtain pFB-AAV-CAG, and then the target gene (27-470 of TTR mRNA) was inserted between EcoRI and BamHI by restriction digestion and ligation to obtain pFB-AAV-CAG-TTR-MM recombinant plasmid (Figure 2). The plasmid was injected into the tail vein by hydrodynamics for transient transfection in vivo, which was administered at least 29 days before the administration of the TTR RNAi reagent or control.

The experiment used SPF male C57BL/6 mice (Beijing Biotechnology Co., Ltd.), 6 to 8 weeks old, weighing 20-24g. Each mouse was injected with 15μg/mL pFB-AAV-CAG-TTR-MM recombinant plasmid via the tail vein, with a volume of 2mL/mouse. Blood was collected from the eye socket 14 days after injection. After serum separation, the TTR concentration was detected using an ELISA kit (Assaypro, EP3010-1). According to the TTR detection value, the mice were randomly divided into a vehicle control group (N group), a test group of AL0057001, AL0057002, and AL0057003, a total of 3 groups, with 5 mice in each group. After grouping, the mice were subcutaneously administered a single dose of the corresponding TTR RNAi agent and vehicle control. Blood was collected from the mice on the 8th and 15th days after administration, and the serum was separated to detect the TTR concentration. The inhibitory effect of siRNA on exogenous gene mRNA was evaluated. The results are shown in Figure 3. As shown in Figure 3, although the concentration of TTR decreased over time, the knockdown effect of TTR in the AL0057002 and AL0057003 groups was better than that in the N group on the 15th day after administration.

Example 5. In vivo testing of TTR-RNAi agents in V30 hTTR transgenic mice

The experiment used SPF male, 8-9 weeks old TTR K1V30 transgenic mice (source: Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.). Pre-dose serum samples were obtained on day 0 of administration, and the mice were randomly divided into groups according to TTR levels. TTR K1 V30 transgenic mice were subcutaneously administered a single 3 mg/kg dose of TTR RNAi agents AL0057004, AL0057005, AL0057006, AL0057007, AL0057008, AL0057009, AL0057010, and AL0057011. Blood was collected from mice at 1 week, 2 weeks, 3 weeks, 4 weeks and 5 weeks or at 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks and 10 weeks after administration (blood was collected from the eyeball and sent for inspection within 1 hour after blood collection). The hTTR expression level was measured, and the level of hTTR knockdown was detected (the log value of TTR content in serum) with the control before administration. During the experiment, no animals showed death or dying symptoms. No obvious abnormalities were observed in all animals during clinical observation. The level of TTR changes is shown in Figures 4 and 5.

It can be concluded from Figures 4 and 5 that compared with before administration, the knockdown effect of all siRNAs reached the highest on the 22nd or 29th day after drug intervention, and then gradually recovered slowly. Drug intervention can significantly reduce the hTTR level in the blood of mice. The two groups of experiments were controlled by AL0057008. The AL0057004, AL0057005, AL0057006, AL0057007, AL0057009, AL0057010, and AL0057011 groups had better TTR knockdown effects and a longer sustained reduction time. Among them, as shown in Figure 4, within 1-36 days after administration, compared with the AL0057008 group, the knockdown effect of the AL0057004 and AL0057006 groups was better, with statistical difference (p < 0.03, *); the knockdown effect of the AL0057005 and AL0057007 groups was equivalent to that of the AL0057008 group, with no statistical difference (p < 0.1, ns). As shown in Figure 5, within 1-70 days after administration, compared with the AL0057008 group, the knockdown effect of the AL0057004, AL0057009 and AL0057010 groups was better, with significant statistical difference (p < 0.0001, ****); the knockdown effect of the AL00570011 group was equivalent to that of the AL0057008 group, with no statistical difference (p < 0.1, ns).

Claims (10)

An oligonucleotide or a pharmaceutically acceptable salt thereof for reducing the expression of the transthyretin (TTR) gene, wherein the oligonucleotide comprises a sense chain and an antisense chain, wherein the sense chain has a sequence having at least 80% sequence identity with a sequence shown in any one of SEQ ID NOs: 2-4, 6-21, 23-83, or a fragment thereof, or a modified sequence of the sequence or its fragment, and preferably has a sequence identity of 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more; and the antisense chain has a sequence having at least 80% sequence identity with a sequence shown in any one of SEQ ID NOs: 85-87, 89-104, 106-179, or a fragment thereof, or a modified sequence of the sequence or its fragment, and preferably has a sequence identity of 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more. The oligonucleotide or a pharmaceutically acceptable salt thereof according to claim 1, wherein the oligonucleotide or a pharmaceutically acceptable salt thereof is selected from carboxylates, alkali metal salts, ammonium salts, alkaline earth metal salts, salts formed with organic bases and other pharmaceutically acceptable salts; Preferably, the salt is an alkali metal salt, more preferably a sodium salt or a potassium salt thereof; Preferably, the salt is an alkaline earth metal salt, more preferably a magnesium salt or a calcium salt; Preferably, the salt is an ammonium salt, more preferably a triethylamine salt. The oligonucleotide or a pharmaceutically acceptable salt thereof according to claim 1 or 2, wherein the oligonucleotide comprises at least one modified nucleotide; Preferably, the oligonucleotide comprises at least one 2'-modified nucleotide; Preferably, the 2'-modified nucleotide is selected from one or more of 2'-alkoxy modified nucleotides, 2'-substituted alkoxy modified nucleotides, 2'-alkyl modified nucleotides, 2'-substituted alkyl modified nucleotides, 2'-amino modified nucleotides, 2'-substituted amino modified nucleotides, 2'-fluoro modified nucleotides, and 2'-deoxy nucleotides; Preferably, the 2'-modification is a modification selected from the group consisting of: 2'-methoxy, 2'-acetylamino, 2'-aminoethyl, 2'-fluoro, 2'-O-methoxyethyl; Preferably, the oligonucleotide has a 5'-phosphate analog modified nucleotide at the 5'end; preferably, the 5'-phosphate analog modified nucleotide has a vinyl phosphonate modified nucleotide shown in formula (I), wherein R is selected from H, OH, fluorine, 2'-methoxy, 2'-acetylamino, 2'-aminoethyl and 2'-O-methoxyethyl, and Base represents a nucleic acid base selected from A, G, C, T and U; preferably, the 5'-phosphate analog modified nucleotide has a vinyl phosphate modified nucleotide shown in formula (II), wherein R is selected from H, OH, fluorine, 2'-methoxy, 2'-acetylamino, 2'-aminoethyl and 2'-O-methoxyethyl; more preferably, the 5'-phosphonate analog modified nucleotide is APU shown in formula (II-1) or VPUm shown in formula (II-2);
The oligonucleotide or a pharmaceutically acceptable salt thereof of any one of claims 1 to 3, wherein the oligonucleotide comprises at least one modified internucleotide bond; Preferably, said at least one modified internucleotide bond is a phosphorothioate bond. The oligonucleotide or pharmaceutically acceptable salt thereof according to any one of claims 1 to 4, wherein the sense strand is selected from the unmodified oligonucleotide described in any one of SEQ ID NOs: 3, 6, 26-35, 37-40, 43-47, 49-53, 76-83, or the modified oligonucleotide described in any one of SEQ ID NOs: 181-183, 185-200, 202-216; the antisense strand is selected from the unmodified oligonucleotide described in any one of SEQ ID NOs: 86, 89, 109-118, 120-123, 126-130, 132, 135-136, 159-175, 177-179, or the modified oligonucleotide described in any one of SEQ ID NOs: 218-220, 222-237, 239-257. The oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 5, wherein the oligonucleotide is selected from any one of the following sense strand and antisense strand combinations: (1) the sense strand shown in SEQ ID NO:3, and the antisense strand shown in SEQ ID NO:86; (2) the sense strand shown in SEQ ID NO:6, and the antisense strand shown in SEQ ID NO:89; (3) the sense strand shown in SEQ ID NO:26, and the antisense strand shown in SEQ ID NO:109; (4) the sense strand represented by SEQ ID NO:27, and the antisense strand represented by SEQ ID NO:110; (5) the sense strand represented by SEQ ID NO:28, and the antisense strand represented by SEQ ID NO:111; (6) the sense strand represented by SEQ ID NO:29, and the antisense strand represented by SEQ ID NO:112; (7) the sense strand represented by SEQ ID NO:30, and the antisense strand represented by SEQ ID NO:113; (8) the sense strand represented by SEQ ID NO:31, and the antisense strand represented by SEQ ID NO:114; (9) the sense strand represented by SEQ ID NO:32, and the antisense strand represented by SEQ ID NO:115; (10) the sense strand represented by SEQ ID NO:33, and the antisense strand represented by SEQ ID NO:116; (11) the sense strand represented by SEQ ID NO:34, and the antisense strand represented by SEQ ID NO:117; (12) the sense strand represented by SEQ ID NO:35, and the antisense strand represented by SEQ ID NO:118; (13) the sense strand represented by SEQ ID NO:37, and the antisense strand represented by SEQ ID NO:120; (14) the sense strand represented by SEQ ID NO:38, and the antisense strand represented by SEQ ID NO:121; (15) the sense strand represented by SEQ ID NO:39, and the antisense strand represented by SEQ ID NO:122; (16) the sense strand represented by SEQ ID NO:40, and the antisense strand represented by SEQ ID NO:123; (17) the sense strand represented by SEQ ID NO:43, and the antisense strand represented by SEQ ID NO:126; (18) the sense strand represented by SEQ ID NO:44, and the antisense strand represented by SEQ ID NO:127; (19) the sense strand represented by SEQ ID NO:45, and the antisense strand represented by SEQ ID NO:128; (20) the sense strand represented by SEQ ID NO:46, and the antisense strand represented by SEQ ID NO:129; (21) the sense strand represented by SEQ ID NO:47, and the antisense strand represented by SEQ ID NO:130; (22) the sense strand represented by SEQ ID NO:49, and the antisense strand represented by SEQ ID NO:132; (23) the sense strand represented by SEQ ID NO:52, and the antisense strand represented by SEQ ID NO:135; (24) the sense strand represented by SEQ ID NO:53, and the antisense strand represented by SEQ ID NO:136; (25) the sense strand represented by SEQ ID NO:22, and the antisense strand represented by SEQ ID NO:105; (26) the sense strand represented by SEQ ID NO:76, and the antisense strand represented by SEQ ID NO:159; (27) the sense strand represented by SEQ ID NO:77, and the antisense strand represented by SEQ ID NO:160; (28) the sense strand represented by SEQ ID NO:77, and the antisense strand represented by SEQ ID NO:161; (29) the sense strand represented by SEQ ID NO:77, and the antisense strand represented by SEQ ID NO:162; (30) the sense strand represented by SEQ ID NO:78, and the antisense strand represented by SEQ ID NO:163; (31) the sense strand represented by SEQ ID NO:79, and the antisense strand represented by SEQ ID NO:164; (32) the sense strand represented by SEQ ID NO:80, and the antisense strand represented by SEQ ID NO:165; (33) the sense strand represented by SEQ ID NO:81, and the antisense strand represented by SEQ ID NO:166; (34) the sense strand represented by SEQ ID NO:82, and the antisense strand represented by SEQ ID NO:167; (35) the sense strand represented by SEQ ID NO:83, and the antisense strand represented by SEQ ID NO:168; (36) the sense strand represented by SEQ ID NO:83, and the antisense strand represented by SEQ ID NO:169; (37) the sense strand represented by SEQ ID NO:83, and the antisense strand represented by SEQ ID NO:170; (38) the sense strand represented by SEQ ID NO:26, and the antisense strand represented by SEQ ID NO:109; (39) the sense strand represented by SEQ ID NO:43, and the antisense strand represented by SEQ ID NO:126; (40) the sense strand represented by SEQ ID NO:27, and the antisense strand represented by SEQ ID NO:110; (41) the sense strand represented by SEQ ID NO:44, and the antisense strand represented by SEQ ID NO:171; (42) the sense strand represented by SEQ ID NO:44, and the antisense strand represented by SEQ ID NO:172; (43) the sense strand represented by SEQ ID NO:28, and the antisense strand represented by SEQ ID NO:111; (44) the sense strand represented by SEQ ID NO:45, and the antisense strand represented by SEQ ID NO:173; (45) the sense strand represented by SEQ ID NO:45, and the antisense strand represented by SEQ ID NO:174; (46) the sense strand represented by SEQ ID NO:29, and the antisense strand represented by SEQ ID NO:112; (47) the sense strand represented by SEQ ID NO:46, and the antisense strand represented by SEQ ID NO:175; (48) the sense strand represented by SEQ ID NO:31, and the antisense strand represented by SEQ ID NO:114; (49) the sense strand represented by SEQ ID NO:32, and the antisense strand represented by SEQ ID NO:115; (50) the sense strand represented by SEQ ID NO:49, and the antisense strand represented by SEQ ID NO:177; (51) the sense strand represented by SEQ ID NO:33, and the antisense strand represented by SEQ ID NO:116; (52) the sense strand represented by SEQ ID NO:50, and the antisense strand represented by SEQ ID NO:178; (53) the sense strand shown in SEQ ID NO:34, and the antisense strand shown in SEQ ID NO:117; and (54) the sense strand represented by SEQ ID NO:51, and the antisense strand represented by SEQ ID NO:179; wherein each strand is independently 19 to 25 nucleotides in length. The oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 6, wherein the oligonucleotide comprises any one selected from the following sense strand and antisense strand combinations: (1) the sense strand represented by SEQ ID NO:181, and the antisense strand represented by SEQ ID NO:218; (2) the sense strand shown in SEQ ID NO:182, and the antisense strand shown in SEQ ID NO:219; (3) the sense strand represented by SEQ ID NO:183, and the antisense strand represented by SEQ ID NO:220; (4) the sense strand represented by SEQ ID NO:185, and the antisense strand represented by SEQ ID NO:222; (5) the sense strand represented by SEQ ID NO:186, and the antisense strand represented by SEQ ID NO:223; (6) the sense strand represented by SEQ ID NO:187, and the antisense strand represented by SEQ ID NO:224; (7) the sense strand represented by SEQ ID NO:188, and the antisense strand represented by SEQ ID NO:225; (8) the sense strand represented by SEQ ID NO:189, and the antisense strand represented by SEQ ID NO:226; (9) the sense strand represented by SEQ ID NO: 190, and the antisense strand represented by SEQ ID NO: 227; (10) the sense strand represented by SEQ ID NO:191, and the antisense strand represented by SEQ ID NO:228; (11) the sense strand represented by SEQ ID NO:192, and the antisense strand represented by SEQ ID NO:229; (12) the sense strand represented by SEQ ID NO:193, and the antisense strand represented by SEQ ID NO:230; (13) the sense strand represented by SEQ ID NO:194, and the antisense strand represented by SEQ ID NO:231; (14) the sense strand represented by SEQ ID NO:195, and the antisense strand represented by SEQ ID NO:232; (15) the sense strand represented by SEQ ID NO:196, and the antisense strand represented by SEQ ID NO:233; (16) the sense strand represented by SEQ ID NO:197, and the antisense strand represented by SEQ ID NO:234; (17) the sense strand represented by SEQ ID NO:198, and the antisense strand represented by SEQ ID NO:235; (18) the sense strand represented by SEQ ID NO:199, and the antisense strand represented by SEQ ID NO:236; (19) the sense strand represented by SEQ ID NO: 200, and the antisense strand represented by SEQ ID NO: 237; (20) the sense strand represented by SEQ ID NO: 202, and the antisense strand represented by SEQ ID NO: 239; (21) the sense strand represented by SEQ ID NO: 203, and the antisense strand represented by SEQ ID NO: 240; (22) the sense strand represented by SEQ ID NO: 204, and the antisense strand represented by SEQ ID NO: 241; (23) the sense strand represented by SEQ ID NO:205, and the antisense strand represented by SEQ ID NO:242; (24) the sense strand represented by SEQ ID NO: 206, and the antisense strand represented by SEQ ID NO: 243; (25) the sense strand represented by SEQ ID NO: 207, and the antisense strand represented by SEQ ID NO: 244; (26) the sense strand represented by SEQ ID NO:208, and the antisense strand represented by SEQ ID NO:245; (27) the sense strand represented by SEQ ID NO: 209, and the antisense strand represented by SEQ ID NO: 246; (28) the sense strand represented by SEQ ID NO:210, and the antisense strand represented by SEQ ID NO:247; (29) the sense strand represented by SEQ ID NO:210, and the antisense strand represented by SEQ ID NO:248; (30) the sense strand represented by SEQ ID NO:211, and the antisense strand represented by SEQ ID NO:249; (31) the sense strand represented by SEQ ID NO:212, and the antisense strand represented by SEQ ID NO:250; (32) the sense strand represented by SEQ ID NO:213, and the antisense strand represented by SEQ ID NO:251; (33) the sense strand represented by SEQ ID NO:213, and the antisense strand represented by SEQ ID NO:252; (34) the sense strand represented by SEQ ID NO:206, and the antisense strand represented by SEQ ID NO:253; (35) the sense strand represented by SEQ ID NO:206, and the antisense strand represented by SEQ ID NO:254; (36) the sense strand represented by SEQ ID NO:209, and the antisense strand represented by SEQ ID NO:255; (37) the sense strand represented by SEQ ID NO:214, and the antisense strand represented by SEQ ID NO:247; (38) the sense strand represented by SEQ ID NO:215, and the antisense strand represented by SEQ ID NO:256; and (39) the sense strand represented by SEQ ID NO:216, and the antisense strand represented by SEQ ID NO:257; Preferably, the oligonucleotide comprises any one selected from the following sense strand and antisense strand combinations: (1) the sense strand shown in SEQ ID NO: 202, and the antisense strand shown in SEQ ID NO: 239; (2) the sense strand shown in SEQ ID NO: 203, and the antisense strand shown in SEQ ID NO: 240; (3) the sense strand represented by SEQ ID NO: 210, and the antisense strand represented by SEQ ID NO: 247; (4) the sense strand represented by SEQ ID NO: 211, and the antisense strand represented by SEQ ID NO: 249; (5) the sense strand represented by SEQ ID NO: 212, and the antisense strand represented by SEQ ID NO: 250; (6) the sense strand represented by SEQ ID NO:213, and the antisense strand represented by SEQ ID NO:251; (7) the sense strand represented by SEQ ID NO: 214, and the antisense strand represented by SEQ ID NO: 247; (8) the sense strand represented by SEQ ID NO:215, and the antisense strand represented by SEQ ID NO:256; (9) the sense strand represented by SEQ ID NO: 216, and the antisense strand represented by SEQ ID NO: 257; wherein each strand is independently 19 to 25 nucleotides in length. A conjugate for reducing transthyretin (TTR) gene expression or a pharmaceutically acceptable salt thereof, comprising: (i) the compound of claim 1-7 The oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of the preceding claims, and (ii) a ligand conjugated to the oligonucleotide or a pharmaceutically acceptable salt thereof, wherein at least one nucleotide of the oligonucleotide is conjugated to a targeting ligand; Preferably, the targeting ligand comprises a carbohydrate, an amino sugar, cholesterol, a polypeptide or a lipid; Preferably, the targeting ligand comprises an N-acetylgalactosamine (GalNAc) moiety; Preferably, the GalNac moiety is a monovalent GalNAc moiety, a divalent GalNAc moiety, a trivalent GalNAc moiety or a tetravalent GalNAc moiety; Preferably, the targeting ligand is A1 represented by formula (III); A1
Preferably, the targeting ligand is L96 represented by formula (IV): L96
A composition comprising the oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 7 or the conjugate or a pharmaceutically acceptable salt thereof according to claim 8, and optionally a pharmaceutically acceptable carrier; Preferably, the composition is in the form of an oral agent, an intravenous injection, a subcutaneous injection or an intramuscular injection, preferably a subcutaneous injection; Preferably, the combination further comprises other drugs for treating and/or preventing TTR-related diseases; Preferably, the TTR-related disease is selected from amyloidosis, metabolic disease, Alzheimer's disease and tumor; Preferably, the amyloidosis is familial amyloidosis; Preferably, the amyloidosis is selected from transthyretin amyloid cardiomyopathy (ATTR-CM) and transthyretin amyloid polyneuropathy (ATTR-PN); Preferably, the amyloidosis is familial amyloid polyneuropathy (FAP); Preferably, the amyloidosis is Alzheimer's disease; Preferably, the metabolic disease is diabetes and impaired glucose regulation; preferably, the diabetes is type II diabetes. Use of the oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 7, the conjugate or a pharmaceutically acceptable salt thereof according to claim 8, or the composition according to claim 9 in the preparation of a medicament for treating and/or preventing a TTR-related disease; Preferably, the TTR-related disease is selected from amyloidosis, metabolic disease, Alzheimer's disease and tumor; Preferably, the amyloidosis is familial amyloidosis; Preferably, the amyloidosis is selected from transthyretin amyloid cardiomyopathy (ATTR-CM) and transthyretin amyloid polyneuropathy (ATTR-PN); Preferably, the amyloidosis is familial amyloid polyneuropathy (FAP); Preferably, the amyloidosis is Alzheimer's disease; Preferably, the metabolic disease is selected from diabetes and impaired glucose regulation; preferably, the diabetes is type II diabetes; Preferably, the tumor is selected from lung cancer and pancreatic ductal carcinoma.