Oligonucleotide targeting sflt-1 gene, and use thereof

Abstract

The present disclosure relates to an RNAi agent, such as a double-stranded small RNA (siRNA) agent, targeting soluble Fms-like tyrosine kinase-1 (sFLT-1). The RNAi agent significantly inhibits the expression level of the sFLT-1 gene, and has a long-lasting efficacy.

Description

Oligonucleotides targeting the sFLT-1 gene and their applications Technical Field

[0001] This disclosure relates to an oligonucleotide, particularly to the inhibition of expression of the soluble FMS-like tyrosine kinase-1 (sFLT-1) gene and the treatment of diseases caused by overexpression of this gene. Background Technology

[0002] Preeclampsia (PE) is an idiopathic pregnancy disorder with an incidence rate of 2%–8%, and is one of the leading causes of adverse maternal and fetal outcomes and death. The pathogenesis of PE is not fully understood, but numerous studies suggest it may be related to placental dysfunction and widespread vascular endothelial damage. The anti-angiogenic factor—soluble FMS-like tyrosine kinase receptor I—is significantly elevated in the blood circulation of pregnant women with PE. It can antagonize vascular endothelial growth factor (VEGF), causing widespread vascular endothelial damage and leading to clinical manifestations such as hypertension and proteinuria. In recent years, sFLT-1 has become one of the predictive indicators of PE, and researchers have begun to focus on its application value as a therapeutic target in the prevention and treatment of PE.

[0003] Structure and subtypes of sFLT-1: sFLT-1, also known as the soluble vascular endothelial growth factor receptor, is a secretory splice variant of vascular endothelial growth factor (VEGFR) 1. It possesses only a ligand-binding domain, lacking both a cytoplasmic and transmembrane domain. sFLT-1 binds to endothelial cell surface receptors VEGFR-1 and VEGFR-2 to form a heterodimer, thereby blocking the biological activity of VEGF and placental growth factor (PIGF), leading to impaired angiogenesis and endothelial damage. In recent years, with ongoing research, different splice variants of sFLT-1 have become increasingly recognized. The FMS-like tyrosine kinase receptor I (Flt-1) gene encodes a 30-exon mRNA. Its precursor mRNA is spliced ​​to form a full-length membrane-bound FLT-1 molecule and four soluble splice variants, namely sFLT-1-i13 (also known as sFLT-1 or sFLT-1_v1, isolated in 1993), sFLT-1-i14, sFLT-1-e15a (also known as sFLT-1_v2 or sFLT-1-14, isolated in 2009, unique to humans and non-human primates, as shown in Figure 1), and sFLT-1-e15b. The expression of sFLT-1-i13 and sFLT-1-e15a exhibits histological and cellular specificity. For example, the expression level of sFLT-1-i13 in the placenta is more than four times that in the heart, kidney, and liver; while sFLT-1-e15a is predominantly placental, with expression levels in the placenta more than 600 times that in other organs. Moreover, among all sFLT-1s expressed in the placenta, the subtype sFLT-1-e15a accounts for more than 80%. Both sFLT-1-i13 and sFLT-1-e15a can bind to VEGF and PIGF, thereby antagonizing their functions (Lin, L.; Yang, H.X., Chinese Journal of Obstetrics and Gynecology, 2019; Jebbink, R. et al, Hypertension, 2011).

[0004] Physical examination (PE) is considered a placental-origin disease. Multiple angiogenic factors derived from the placenta and uterus play a crucial role in initiating and regulating arterial remodeling in rejected arteries, among which sFLT-1's antagonistic effect on VEGF plays a significant role. In pregnant women with PE, circulating sFLT-1 levels are significantly elevated 5 weeks before the onset of typical clinical symptoms, but decrease markedly within 48 hours postpartum, suggesting that excessive sFLT-1 may originate from the placenta. In recent years, with in-depth research on sFLT-1 subtypes, researchers have found that both sFLT-1-i13 and sFLT-1-e15a are upregulated in the placenta of pregnant women with PE, with sFLT-1-e15a showing the most significant upregulation. Its level is correlated with the severity of PE. Furthermore, differential expression of sFLT-1-i13 and sFLT-1-e15a was observed between early-onset and late-onset PE. Researchers pointed out that sFLT-1-e15a was significantly upregulated in early-onset PE, while sFLT-1-i13 was more significantly elevated in pregnant women with late-onset PE. Some researchers have suggested that early-onset PE is mainly related to placental abnormalities leading to impaired vascular remodeling, while late-onset PE is related to pre-existing maternal endothelial dysfunction. Combined with the aforementioned observation that "sFLT-1-e15a is almost entirely produced by the placenta compared to sFLT-1-i13," the differential expression of these two in early-onset and late-onset PE to some extent supports the above theory.

[0005] Currently, there are no effective preventive and therapeutic methods for PE. However, with the deepening research into the pathogenesis of PE, more and more studies are beginning to explore new treatment measures. As a key molecule and early predictive indicator of the occurrence and development of PE, sFLT-1 has attracted attention from scholars as a potential therapeutic target for PE. They are exploring a series of measures to regulate the level of sFLT-1 in the body, thereby achieving the goal of preventing and treating PE.

[0006] In 1998, two American scientists, Andrew Fire and Craig Mello, discovered a biological mechanism: small interfering RNA (siRNA) can mediate the degradation of specific mRNAs (Fire, Andrew, et al. Nature 391.6669(1998):806-811). This mechanism is activated when RNA molecules appear in the cell in double-stranded form, resulting in RNA interference. This discovery heralded the beginning of a new research field, and the two scientists were awarded the 2006 Nobel Prize in Physiology or Medicine for this discovery. When double-stranded RNA binds to the protein complex Dicer, Dicer cleaves the dsRNA into fragments. Then, another protein complex, RISC, binds to these fragments. One strand of the siRNA double strand is removed, but the other strand remains bound to the RISC complex. Guided by single-stranded RNA, RISC recognizes and degrades the mRNA of the target gene, inhibiting the expression of the specific protein and thus specifically leading to gene silencing.

[0007] RNA interference has opened up a new field for the application of gene technology. Double-stranded RNA (dsRNA) molecules have been artificially designed to silence specific genes in humans, animals, or plants. These artificially designed and synthesized small double-stranded interfering RNA molecules (siRNA) for gene silencing are introduced into cells and activate RNA interference mechanisms to degrade the corresponding mRNA. Currently, this method is an important research tool in biology and biomedicine. Furthermore, numerous siRNA drugs have been developed to treat viral infections, cardiovascular diseases, cancer, endocrine disorders, and many other diseases. Most siRNA therapies, either in the research and development stage or already approved for marketing, have shown promising therapeutic effects. Since the first siRNA drug was launched in 2018, at least six siRNAs have been approved for marketing in the EU or the US. Therefore, using RNA interference technology to inhibit the expression of specific target genes has become an effective approach to disease treatment. Summary of the Invention

[0008] The purpose of this disclosure is to provide an inhibitor of sFLT-1 expression that is effective, safe, and has a long-lasting effect.

[0009] This disclosure provides an oligonucleotide or a pharmaceutically acceptable salt thereof and a method for inhibiting sFLT-1 gene expression in cells or mammals using the oligonucleotide or a pharmaceutically acceptable salt thereof, wherein the oligonucleotide targets the sFLT-1 gene. The oligonucleotide is a double-stranded RNA (dsRNA). This document also provides compositions and methods for treating pathological conditions and diseases in mammals caused by sFLT-1 overexpression or mutation. dsRNA directs sequence-specific degradation of mRNA through a process called RNA interference (RNAi).

[0010] In one aspect, this disclosure provides an oligonucleotide or a pharmaceutically acceptable salt thereof for inhibiting sFLT-1 gene expression, the oligonucleotide comprising a sense strand and an antisense strand, the sense strand having at least 80% sequence identity with any sequence or fragment thereof shown in SEQ ID NO. 3-27 and 29-75, or a modified sequence thereof, preferably having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity; the antisense strand having at least 80% sequence identity with any sequence or fragment thereof shown in SEQ ID NO. 78-102 and 104-150, or a modified sequence thereof, preferably having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity.

[0011] In another aspect, this disclosure provides a conjugate or a pharmaceutically acceptable salt thereof for inhibiting sFLT-1 gene expression, comprising: (i) the aforementioned oligonucleotide or a pharmaceutically acceptable salt thereof, 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 ligand.

[0012] In another aspect, this disclosure provides a composition comprising the aforementioned oligonucleotide or a pharmaceutically acceptable salt thereof or the aforementioned conjugate or a pharmaceutically acceptable salt thereof, and optionally a pharmaceutically acceptable carrier.

[0013] In another aspect, this disclosure provides the use of the aforementioned oligonucleotides or pharmaceutically acceptable salts, conjugates or pharmaceutically acceptable salts or compositions thereof in the preparation of medicaments for the treatment and / or prevention of sFLT-1 related conditions.

[0014] In another aspect, this disclosure provides a method for treating and / or preventing sFLT-1-related conditions in a subject by administering a therapeutic agent (e.g., the aforementioned oligonucleotide or a pharmaceutically acceptable salt thereof, or the aforementioned conjugate or a pharmaceutically acceptable salt thereof, or the aforementioned composition, or a vector or transgene encoding an oligonucleotide) to the subject.

[0015] On the other hand, this disclosure provides a method for treating and / or preventing sFLT-1-related conditions in subjects in combination with other drugs and / or other treatments, using the aforementioned oligonucleotides or pharmaceutically acceptable salts or conjugates thereof or combinations thereof.

[0016] Experiments have shown that the oligonucleotides disclosed herein can effectively reduce the level of sFLT-1 in the body and are effective inhibitors of PE-related disease characteristics in animals. Attached Figure Description

[0017] Figure 1 shows a schematic diagram of the shearing variants of sFLT1-i13 and sFLT-e15a.

[0018] Figure 2 shows a flowchart of oligonucleotide solid-phase synthesis.

[0019] Figure 3 shows the efficacy of sFLT-1 RNAi in CD1 pregnant mice. Detailed Implementation

[0020] In this disclosure, unless otherwise stated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the terms and laboratory procedures related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology used herein are all widely used terms and routine procedures in their respective fields. To better understand this disclosure, definitions and explanations of relevant terms are provided below.

[0021] As used herein, the term “about” or “approximately” when applied to one or more target values ​​means a value similar to a reference value. In some embodiments, unless otherwise stated or otherwise apparent from the context, the term “approximately” or “about” means a range of values ​​falling within 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less of the reference value in any direction (unless such a number would exceed 100% of the possible value).

[0022] As used herein, the term "complementary" refers to a structural relationship between nucleotides (e.g., two nucleotides on opposing nucleic acids or on opposing regions of a single nucleic acid strand) that allows the nucleotides to form base pairs with each other. For example, a purine nucleotide complementary to a pyrimidine nucleotide of an opposing nucleic acid can be base-paired together by forming hydrogen bonds with each other. In some embodiments, complementary nucleotides may be base-paired in a Watson-Crick manner or in any other manner that allows the formation of a stable duplex. In some embodiments, the two nucleic acids may have nucleotide sequences that are complementary to each other to form complementary regions, as described herein.

[0023] As used herein, the term "chain" refers to a single, continuous sequence of nucleotides linked together by internucleotide bonds (e.g., phosphodiester bonds, thiophosphate bonds). In some embodiments, the chain has two free ends, such as a 5'-end and a 3'-end.

[0024] As used herein, the term "deoxyribonucleotide" refers to a nucleotide that has a hydrogen atom at the 2' position of its pentose sugar compared to a ribonucleotide. A modified deoxyribonucleotide is a deoxyribonucleotide that has one or more modifications or substitutions (including modifications or substitutions in the sugar, phosphate group, or base) other than at the 2' position.

[0025] As used herein, the term "oligonucleotide" refers to a short nucleic acid, such as a short nucleic acid less than 100 nucleotides in length. Oligonucleotides may comprise ribonucleotides, deoxyribonucleotides, and / or modified nucleotides, including, for example, modified ribonucleotides. Oligonucleotides may be single-stranded or double-stranded. Oligonucleotides may or may not have a double-stranded region. As a set of non-limiting examples, oligonucleotides may be, but are not limited to, small interfering RNA (siRNA), microRNA (miRNA), short hairpin RNA (shRNA), Dicer substrate interfering RNA (dsiRNA), antisense oligonucleotides, short siRNA, or single-stranded siRNA. In some embodiments, the double-stranded oligonucleotide is an RNAi oligonucleotide.

[0026] As used herein, the term "double-stranded oligonucleotide" refers to an oligonucleotide that is substantially in a double-stranded form. In some embodiments, complementary base pairings are formed between antiparallel sequences of nucleotides in covalently separated nucleic acid chains, forming one or more double-stranded regions of the double-stranded oligonucleotide. In some embodiments, complementary base pairings are formed between antiparallel sequences of nucleotides in covalently linked nucleic acid chains. In some embodiments, complementary base pairings of one or more double-stranded regions of the double-stranded oligonucleotide are formed from a single nucleic acid chain folded (e.g., via a hairpin) to provide complementary antiparallel sequences of nucleotides that are base-paired together. In some embodiments, the double-stranded oligonucleotide comprises two covalently separated nucleic acid chains that are fully double-stranded with each other. However, in some embodiments, the double-stranded oligonucleotide comprises two covalently separated nucleic acid chains that are partially double-stranded, for example, having overhangs at one or both ends. In some embodiments, the double-stranded oligonucleotide comprises antiparallel sequences of nucleotides that are partially complementary, and therefore may have one or more mismatches, which may include internal mismatches or terminal mismatches.

[0027] As used herein, the terms “iRNA,” “RNAi,” “RNAi agent,” “iRNA agent,” and “RNA interference agent” are used interchangeably and refer to an RNA containing the terms defined herein and an agent that mediates targeted cleavage of RNA transcripts via the RNA-inducible silencing complex (RISC) pathway. RNA interference (RNAi) is a process that directs the specific degradation of mRNA sequences. RNAi regulates, for example, the inhibition of SFLT-1 expression in cells, such as within an individual, such as a mammalian individual.

[0028] As used herein, “conjugation” refers to the covalent connection between two or more chemical moieties, each with a specific function; correspondingly, “conjugate” refers to a compound formed by the covalent connection of these chemical moieties. Further, “siRNA conjugate” refers to a compound formed by the covalent attachment of one or more chemical moieties with specific functions to siRNA. In the following text, the siRNA conjugates of this disclosure will sometimes be simply referred to as “conjugates.” The term “siRNA conjugate” should be understood, depending on the context, as a general term for siRNA conjugates, including siRNA sense strand conjugates and siRNA antisense strand conjugates.

[0029] As used herein, the term "double-stranded RNA" or "dsRNA" refers to a complex of ribonucleic acid molecules having a double-stranded structure comprising two antiparallel and substantially complementary nucleic acid strands with "sense" and "antisense" orientations relative to the target RNA (i.e., the SFLT-1 gene). In some embodiments of this disclosure, the double-stranded RNA (dsRNA) triggers the degradation of the target RNA (e.g., mRNA) through a post-transcriptional gene silencing mechanism referred to herein as RNA interference or RNAi. Generally, the majority of nucleotides in each strand of the dsRNA molecule are ribonucleotides, but each or both strands may also contain one or more non-ribonucleotides, such as deoxyribonucleotides or modified nucleotides, as described in detail herein. Additionally, as used herein, "RNAi" may contain chemically modified ribonucleotides; RNAi may include substantial modifications at multiple nucleotide sites.

[0030] As used herein, the term "modified nucleotide" refers to a nucleotide that independently has a modified sugar moiety, a modified internucleotide bond, or a modified nucleobase, or any combination thereof. Therefore, the term "modified nucleotide" encompasses substitutions, additions, or removals of, for example, functional groups or atoms, of internucleotide bonds, sugar moieties, or nucleobases. Modifications applicable to pharmaceuticals disclosed herein include all types of modifications disclosed herein or known in the art.

[0031] As used herein, the term "nucleotide overhang" refers to at least one unpaired nucleotide protruding from the double-stranded structure of a double-stranded iRNA. For example, a nucleotide overhang exists when the 3' end of one strand of a dsRNA extends beyond the 5' end of the other strand, or vice versa. A dsRNA may include an overhang of at least one nucleotide; alternatively, an overhang may include at least two, three, four, five, or more nucleotides. A nucleotide overhang may include or consist of nucleotide / nucleoside analogs comprising deoxynucleotides / nucleosides. The overhang may be on the sense strand, antisense strand, or any combination thereof. Furthermore, the overhanging nucleotide may be present at the 5' end, 3' end, or both ends of the antisense strand or sense strand of the dsRNA.

[0032] As used in this article, the term "naked sequence" refers to an unmodified nucleotide sequence.

[0033] As used herein, the term “inhibition” is used interchangeably with “knockdown,” “reduction,” “silence,” “downregulation,” “suppression,” and other similar terms, and includes any degree of inhibition.

[0034] As used herein, the term "lipophilic" or "lipophilic fraction" broadly refers to any compound or chemical fraction that has an affinity for lipids.

[0035] The phrase "inhibit sFLT-1 expression" is intended to refer to the inhibition of the expression of any sFLT-1 gene (e.g., mouse sFLT-1 gene, rat sFLT-1 gene, monkey sFLT-1 gene, or human sFLT-1 gene) and variants or mutants of the sFLT-1 gene. Therefore, in the context of gene manipulation cells, cell populations, or organisms, the sFLT-1 gene can be a wild-type sFLT-1 gene, a mutant sFLT-1 gene, or a transgenic sFLT-1 gene.

[0036] "Suppression of sFLT-1 gene expression" includes suppression of the sFLT-1 gene at any level, such as at least partial suppression of sFLT-1 gene expression. sFLT-1 gene expression can be assessed based on the level or changes in the level of any variable associated with sFLT-1 gene expression, such as sFLT-1 mRNA levels or sFLT-1 protein levels, or indirectly by suppressing the mRNA levels of the Gluc and sFLT-1 fusion protein gene, thereby suppressing the Gluc protein level.

[0037] This level can be assessed in individual cells or cell populations, including, for example, samples from subjects. It is understood that sFLT-1 is a soluble protein primarily expressed in the placenta.

[0038] Inhibition can be assessed by a reduction in the absolute or relative level of one or more variables associated with sFLT-1 expression compared to a control level. The control level can be any type of control level used in the art, such as baseline levels before administration, or levels determined from similar subjects who were untreated or treated with a control (e.g., a buffer-only control or an inactive agent control).

[0039] The term "pharmaceutically acceptable salt" refers to salts that retain the bioavailability and properties of a free base or acid, and are not biologically or otherwise undesirable. These salts are formed using inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid (especially 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, methanesulfonic acid, ethylsulfonic acid, p-toluenesulfonic acid, salicylic acid, and N-acetylcysteine. Furthermore, these salts can be prepared by adding an inorganic or organic base to a free acid. Salts derived from inorganic bases include, but are not limited to, alkali metal salts (such as sodium, potassium, and lithium salts), ammonium salts, and alkaline earth metal salts (such as calcium and magnesium salts). Salts derived from organic bases include, but are not limited to, salts formed with organic bases such as organic amines: 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, and polyamine resins. The oligonucleotides of this disclosure may also exist in zwitterionic form. Particularly preferred pharmaceutical salts of this disclosure are sodium salts, lithium salts, potassium salts, and trialkylammonium salts.

[0040] As used herein, the term "subject" refers to an animal that expresses the target gene endogenously or heterologously, such as a mammal, including primates (e.g., humans, non-human primates such as monkeys and chimpanzees), non-primates (e.g., cattle, pigs, horses, goats, rabbits, sheep, hamsters, guinea pigs, cats, dogs, rats, or mice), or birds. In one embodiment, the subject is a human.

[0041] As used herein, the term "treating" or "treatment" refers to a beneficial or desired outcome, such as a reduction in at least one sign or symptom of sFLT-1-related disorder in a subject. Treatment also includes reducing one or more signs or symptoms associated with undesired sFLT-1 expression; reducing the degree of undesired sFLT-1 activation or stabilization; and improving or mitigating undesired sFLT-1 activation or stabilization. Treatment also includes reducing one or more signs or symptoms associated with undesired sFLT-1 expression. "Treatment" can also mean prolonged survival compared to expected survival without treatment.

[0042] As used in this article, the terms “prevention” or “preventing” when referring to a disease or condition will benefit from a reduction in sFLT-1 gene expression or sFLT-1 protein production.

[0043] As used herein, the term "therapeutic effective amount" is intended to encompass the amount of RNAi agent that, when administered to a subject with sFLT-1-related conditions, is sufficient to affect the treatment of the disease (e.g., by reducing, improving, or maintaining existing disease or symptoms of one or more diseases). "Therapeutic effective amount" may vary depending on the RNAi agent, how it is administered, the disease and its severity, as well as medical history, age, weight, family history, genetic makeup, type of prior or concomitant treatment (if any), and other individual characteristics of the subject being treated.

[0044] As used herein, the term "preventive effective dose" refers to the amount of RNAi agent sufficient to prevent or improve the condition or one or more symptoms of the condition when administered to a subject with sFLT-1-related disease. Improving the disease includes slowing its progression or reducing the severity of later-stage disease. The "preventive effective dose" may vary depending on the RNAi agent, how it is administered, the degree of disease risk, and factors such as medical history, age, weight, family history, genetic makeup, type of prior or concomitant treatment (if any), and other individual characteristics of the patient to be treated.

[0045] As used herein, the term "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition, or medium, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, magnesium talc, calcium stearate, zinc stearate, or stearic acid), or solvent encapsulation material (involving the carrying or delivery of a subject compound from one organ or part of the body to another organ or part of the body). Each carrier must be "acceptable" in the sense that it is compatible with the other components of the formulation and harmless to the treated subject. Such carriers are known in the art. Pharmaceutically acceptable carriers include carriers intended for administration by injection.

[0046] In one aspect, this disclosure provides an oligonucleotide or a pharmaceutically acceptable salt thereof for inhibiting sFLT-1 expression, the oligonucleotide comprising a sense strand and an antisense strand, the sense strand having at least 80% sequence identity with a sequence or fragment thereof shown in any of SEQ ID NO. 3-27 and 29-75, or a modified sequence thereof, preferably having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity; the antisense strand having at least 80% sequence identity with a sequence or fragment thereof shown in any of SEQ ID NO. 78-102 and 104-150, or a modified sequence thereof, preferably having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity.

[0047] In some implementations, the sense strand and / or antisense strand are 15-30, 17-25, or 19 to 25-27 nucleotides in length.

[0048] In some implementations, each chain is independently 15-30, 17-25, or 19 to 25-27 nucleotides in length.

[0049] In some implementations, each chain is independently 19 to 25 nucleotides in length.

[0050] In some implementations, the antisense strand is 19 to 23 nucleotides in length.

[0051] In some implementations, the length of the positive chain is 19 to 23 nucleotides.

[0052] In some embodiments, the oligonucleotide comprises a 5' and / or 3'-overhang sequence of one or more nucleotides in length, wherein the 5' and / or 3'-overhang sequence is present on the antisense strand and / or the sense strand. In one embodiment, the antisense strand of the oligonucleotide has one to ten nucleotides at the 3' or 5' overhang, for example, one, two, three, four, five, six, seven, eight, nine, or ten nucleotides. In one embodiment, the sense strand of the dsRNA has one to ten nucleotides at the 3' or 5' overhang, for example, one, two, three, four, five, six, seven, eight, nine, or ten nucleotides. In another embodiment, one or more nucleotides in the overhang are replaced by a nucleoside thiophosphate.

[0053] In some implementations, the antisense chain has one or two protruding ends.

[0054] In some implementations, the justice chain has one or two protruding ends.

[0055] In some implementations, the oligonucleotide comprises a 3'-overhang sequence of one or two nucleotides in length.

[0056] In some implementations, the oligonucleotide comprises a 5'-protruding sequence of one or two nucleotides in length.

[0057] In some implementations, the 3'-protruding sequence is present on the antisense strand. In some implementations, the protruding sequence is selected from: AA, AG, AU, C, CA, CC, CG, CU, G, GA, GC, GG, GU, U, UA, UC, UG, UU.

[0058] In some implementations, the 5'-protrusion sequence is present on the antisense strand. In some implementations, the protrusion sequence is selected from A and G.

[0059] In some implementations, the oligonucleotide comprises an antisense strand and a sense strand, each ranging in length from 19 to 25 nucleotides.

[0060] In some implementations, the oligonucleotide comprises an antisense strand and a sense strand, each ranging in length from 19 to 23 nucleotides.

[0061] In some implementations, the justice chain and the antisense chain form a dual-chain region.

[0062] In some implementations, the justice chain and the antisense chain are bichain structures with 19 / 21 pairing, 21 / 21 pairing, 21 / 23 pairing, or 23 / 23 pairing, respectively.

[0063] In some embodiments, the oligonucleotide comprises a 5' overhang and a 3' overhang sequence of length 1 nucleotide, wherein the 5' overhang and the 3' overhang sequence are present on the antisense strand, and wherein the sense strand is 19 nucleotides long and the antisense strand is 21 nucleotides long, such that the sense strand and the antisense strand form a double helix of length 19 nucleotides.

[0064] In some embodiments, the oligonucleotide includes a 3'-overhang sequence of 2 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 double helix of 19 nucleotides in length.

[0065] In some embodiments, the oligonucleotide includes a 3'-overhang sequence of 2 nucleotides in length, wherein the 3'-overhang sequence is present on both the antisense and sense strands, and wherein the sense strand is 21 nucleotides in length and the antisense strand is 21 nucleotides in length, such that the sense and antisense strands form a doublet of 19 nucleotides in length.

[0066] In some embodiments, the oligonucleotide includes a 3'-overhang sequence of 2 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 double helix of 21 nucleotides in length.

[0067] In some embodiments, the oligonucleotide includes a 3'-overhang sequence of 2 nucleotides in length, wherein the 3'-overhang sequence is present on both the antisense and sense strands, and wherein the sense strand is 23 nucleotides in length and the antisense strand is 23 nucleotides in length, such that the sense and antisense strands form a doublet of 21 nucleotides in length.

[0068] In some embodiments, pharmaceutically acceptable salts of oligonucleotides can be prepared by adding an inorganic or organic base to a free acid. Salts derived from inorganic bases include, but are not limited to, alkali metal salts (such as sodium, potassium, and lithium salts), ammonium salts, and alkaline earth metal salts (such as calcium and magnesium salts). Salts derived from organic bases (e.g., 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, and polyamine resins.

[0069] In some embodiments, pharmaceutically acceptable salts of oligonucleotides include, but are not limited to, ammonium salts, such as salts of tertiary alkylamine compounds (e.g., triethylamine salts), metal salts such as sodium, potassium, and magnesium salts, etc.

[0070] In some implementations, the oligonucleotide or its salt may be in the form of a hydrate or a solvate.

[0071] In some implementations, the oligonucleotide contains at least one modified nucleotide.

[0072] In some implementations, the oligonucleotide contains at least one 2'-modified nucleotide.

[0073] In some embodiments, at least one of the modified nucleotides is selected from the group consisting of: deoxynucleotides, 3'-terminal deoxythymidine (dT) nucleotides, 2'-O-methyl modified nucleotides, 2'-fluorine modified nucleotides, 2'-deoxy modified nucleotides, locked nucleotides, 2'-5'-linked ribonucleotides (3'-RNA), unlocked nucleotides, conformation-restricted nucleotides, restricted ethyl nucleotides, base-free nucleotides, 2'-amino modified nucleotides, 2'-O-allyl modified nucleotides, 2'-O-alkyl modified nucleotides, 2'-hydroxyl modified nucleotides, 2'- Nucleotides modified with methoxyethyl, 2'-O-alkyl, morpholinonucleotides, aminophosphates, nucleotides including non-natural bases, tetrahydropyran-modified nucleotides, 1,5-dehydrohexyl-modified nucleotides, cyclohexenyl-modified nucleotides, nucleotides including thiophosphate groups, nucleotides including methylphosphonate groups, nucleotides including 5'-phosphates, nucleotides including 5'-phosphate mimics, vinyl-phosphonate nucleotides, heat-labile nucleotides, ethylene glycol-modified nucleotides, nucleotides including 2'-phosphates, and nucleotides modified with 2-O-(N-methylacetamide); and combinations thereof.

[0074] In some embodiments, the 2'-modified nucleotide is selected from one or more of the following: 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.

[0075] In some embodiments, the 2'-modification is selected from the following: 2'-methoxy, 2'-acetamido, 2'-aminoethyl, 2'-fluorine, 2'-O-methoxyethyl.

[0076] In some embodiments, the oligonucleotide includes a modification at the 5' end, the modification comprising 5'-(E)-vinylphosphonate (5'-VP) or 6-(3-(2-carboxyethyl)phenyl)purine (6-mCEPh-purine);

[0077] In some embodiments, the nucleotide with a 5' end containing a 5'-(E)-vinylphosphonate modified nucleotide has the structure shown in formula (I); wherein, Base represents a nucleic acid base selected from A, G, C, and U; and R is selected from H, fluorine, 2'-methoxy, 2'-acetamido, 2'-aminoethyl, and 2'-O-methoxyethyl.

[0078] In some embodiments, the oligonucleotide comprises a 5'-(E)-vinylphosphonate-modified nucleotide at its 5' end, having the structure shown in formula (II-1) or (II-2); wherein R is selected from H, fluorine, 2'-methoxy, 2'-acetamido, 2'-aminoethyl, and 2'-O-methoxyethyl; preferably, the oligonucleotide has an APU at its 5' end, which is uridine monophosphate (2'-acetamido-5'-vinylphosphonate-uridine monophosphate) modified with a 5'-phosphate analog as shown in formula (III); preferably, the oligonucleotide has a VPUm at its 5' end, which is uridine monophosphate (2'-methoxy-5'-vinylphosphonate-uridine monophosphate) modified with a 5'-phosphate analog as shown in formula (IV-1); preferably, the oligonucleotide has a VPAm at its 5' end, which is uridine monophosphate (2'-methoxy-5'-vinylphosphonate-adenosine monophosphate) modified with a 5'-phosphate analog as shown in formula (IV-2);

[0079] In some embodiments, the oligonucleotide comprises a 6-(3-(2-carboxyethyl)phenyl)purine-modified nucleotide at its 5' end; preferably, the oligonucleotide comprises formula M, which is 2'-O-methyl-6-(3-(2-carboxyethyl)phenyl)purine nucleotide as shown in formula (V);

[0080] In some embodiments, the oligonucleotide comprises uridine-2'-phosphate (U-2'5') as shown in formula (VI), guanosine-2'-phosphate (G-2'5') as shown in formula (VII); cytidine-2'-phosphate (C-2'5') as shown in formula (VIII); adenosine-2'-phosphate (A-2'5') as shown in formula (IX); and thymidine-2'-phosphate (T-2'5') as shown in formula (X).

[0081] In some embodiments of this disclosure, the oligonucleotide contains at least one modified nucleotide inter-bond.

[0082] In some embodiments of this disclosure, at least one modified nucleotide inter-bond is a phosphate-thioester bond. Phospho-thioester nucleotide inter-bond modification can occur at any position on any nucleotide of the sense strand, antisense strand, or both strands. For example, nucleotide inter-bond modification can occur on each nucleotide of the sense strand or antisense strand; each nucleotide inter-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 nucleotide inter-bond modifications in an alternating pattern. The alternating pattern of nucleotide inter-bond modifications on the sense strand can be the same as or different from that on the antisense strand, and the alternating pattern of nucleotide inter-bond modifications on the sense strand can be offset relative to the alternating pattern of nucleotide inter-bonds on the antisense strand. In one embodiment, the double-stranded RNAi agent comprises 6 to 8 phosphate-thioester nucleotide inter-bonds. In some embodiments, the antisense strand comprises two phosphate-thioester nucleotide inter-bonds at the 5' end and two phosphate-thioester nucleotide inter-bonds at the 3' end, and the sense strand comprises at least two phosphate-thioester nucleotide inter-bonds at the 5' end and / or the 3' end.

[0083] In some embodiments of this disclosure, the sense strand is selected from any unmodified oligonucleotide of SEQ ID NO. 3, 6, 7, 10, 11, 12, 13, 19, 20, 21, 34, 35, 38, 39, 41, 50, 51, 53, 55, 57, 58, 59, 60, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, and 75, or any modified oligonucleotide of SEQ ID NO. 153-177 and 179-225; the antisense strand is selected from SEQ ID NO. Unmodified oligonucleotides of any of NO. 78, 81, 82, 85, 86, 87, 88, 94, 95, 96, 109, 110, 113, 114, 116, 125, 126, 128, 130, 132, 133, 134, 135, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, and 150, or modified oligonucleotides of any of SEQ ID NO. 228-252 and 254-300.

[0084] In some embodiments of this disclosure, the oligonucleotide comprises any of the following combinations of sense and antisense strands:

[0085] (1) The positive chain contains the sequence shown in SEQ ID NO.3, and the negative chain contains the sequence shown in SEQ ID NO.78;

[0086] (2) The sense chain contains the sequence shown in SEQ ID NO.6, and the antisense chain contains the sequence shown in SEQ ID NO.81;

[0087] (3) The sense chain contains the sequence shown in SEQ ID NO.7, and the antisense chain contains the sequence shown in SEQ ID NO.82;

[0088] (4) The sense chain contains the sequence shown in SEQ ID NO.10, and the antisense chain contains the sequence shown in SEQ ID NO.85;

[0089] (5) The sense chain contains the sequence shown in SEQ ID NO.11, and the antisense chain contains the sequence shown in SEQ ID NO.86;

[0090] (6) The sense chain contains the sequence shown in SEQ ID NO.12, and the antisense chain contains the sequence shown in SEQ ID NO.87;

[0091] (7) The positive chain contains the sequence shown in SEQ ID NO.13, and the negative chain contains the sequence shown in SEQ ID NO.88;

[0092] (8) The positive chain contains the sequence shown in SEQ ID NO.19, and the negative chain contains the sequence shown in SEQ ID NO.94;

[0093] (9) The sense chain contains the sequence shown in SEQ ID NO.20, and the antisense chain contains the sequence shown in SEQ ID NO.95;

[0094] (10) The sense chain contains the sequence shown in SEQ ID NO.21, and the antisense chain contains the sequence shown in SEQ ID NO.96;

[0095] (11) The sense chain contains the sequence shown in SEQ ID NO.34, and the antisense chain contains the sequence shown in SEQ ID NO.109;

[0096] (12) The sense chain contains the sequence shown in SEQ ID NO.35, and the antisense chain contains the sequence shown in SEQ ID NO.110;

[0097] (13) The sense chain contains the sequence shown in SEQ ID NO.38, and the antisense chain contains the sequence shown in SEQ ID NO.113;

[0098] (14) The sense chain contains the sequence shown in SEQ ID NO.39, and the antisense chain contains the sequence shown in SEQ ID NO.114;

[0099] (15) The sense chain contains the sequence shown in SEQ ID NO.41, and the antisense chain contains the sequence shown in SEQ ID NO.116;

[0100] (16) The sense chain contains the sequence shown in SEQ ID NO.50, and the antisense chain contains the sequence shown in SEQ ID NO.125;

[0101] (17) The sense chain contains the sequence shown in SEQ ID NO.51, and the antisense chain contains the sequence shown in SEQ ID NO.126;

[0102] (18) The sense chain contains the sequence shown in SEQ ID NO.53, and the antisense chain contains the sequence shown in SEQ ID NO.128;

[0103] (19) The sense chain contains the sequence shown in SEQ ID NO.55, and the antisense chain contains the sequence shown in SEQ ID NO.130;

[0104] (20) The sense chain contains the sequence shown in SEQ ID NO.57, and the antisense chain contains the sequence shown in SEQ ID NO.132;

[0105] (21) The sense chain contains the sequence shown in SEQ ID NO.58, and the antisense chain contains the sequence shown in SEQ ID NO.133;

[0106] (22) The sense chain contains the sequence shown in SEQ ID NO.59, and the antisense chain contains the sequence shown in SEQ ID NO.134;

[0107] (23) The sense chain contains the sequence shown in SEQ ID NO.60, and the antisense chain contains the sequence shown in SEQ ID NO.135;

[0108] (24) The sense chain contains the sequence shown in SEQ ID NO.62, and the antisense chain contains the sequence shown in SEQ ID NO.137;

[0109] (25) The sense chain contains the sequence shown in SEQ ID NO.63, and the antisense chain contains the sequence shown in SEQ ID NO.138;

[0110] (26) The sense chain contains the sequence shown in SEQ ID NO.64, and the antisense chain contains the sequence shown in SEQ ID NO.139;

[0111] (27) The sense chain contains the sequence shown in SEQ ID NO. 65, and the antisense chain contains the sequence shown in SEQ ID NO. 140;

[0112] (28) The sense chain contains the sequence shown in SEQ ID NO.66, and the antisense chain contains the sequence shown in SEQ ID NO.141;

[0113] (29) The sense chain contains the sequence shown in SEQ ID NO.67, and the antisense chain contains the sequence shown in SEQ ID NO.142;

[0114] (30) The sense chain contains the sequence shown in SEQ ID NO.68, and the antisense chain contains the sequence shown in SEQ ID NO.143;

[0115] (31) The sense chain contains the sequence shown in SEQ ID NO.69, and the antisense chain contains the sequence shown in SEQ ID NO.144;

[0116] (32) The sense chain contains the sequence shown in SEQ ID NO.70, and the antisense chain contains the sequence shown in SEQ ID NO.145;

[0117] (33) The positive chain contains the sequence shown in SEQ ID NO.71, and the negative chain contains the sequence shown in SEQ ID NO.146;

[0118] (34) The sense chain contains the sequence shown in SEQ ID NO.72, and the antisense chain contains the sequence shown in SEQ ID NO.147;

[0119] (35) The sense chain contains the sequence shown in SEQ ID NO.73, and the antisense chain contains the sequence shown in SEQ ID NO.148;

[0120] (36) The sense chain contains the sequence shown in SEQ ID NO. 74, and the antisense chain contains the sequence shown in SEQ ID NO. 149; and

[0121] (37) The sense chain contains the sequence shown in SEQ ID NO.75, and the antisense chain contains the sequence shown in SEQ ID NO.150;

[0122] Preferably, the oligonucleotide comprises any combination of the following sense and antisense strands:

[0123] (1) The positive chain contains the sequence shown in SEQ ID NO.3, and the negative chain contains the sequence shown in SEQ ID NO.78;

[0124] (2) The sense chain contains the sequence shown in SEQ ID NO.6, and the antisense chain contains the sequence shown in SEQ ID NO.81;

[0125] (3) The sense chain contains the sequence shown in SEQ ID NO.10, and the antisense chain contains the sequence shown in SEQ ID NO.85;

[0126] (4) The sense chain contains the sequence shown in SEQ ID NO.12, and the antisense chain contains the sequence shown in SEQ ID NO.87;

[0127] (5) The sense chain contains the sequence shown in SEQ ID NO.19, and the antisense chain contains the sequence shown in SEQ ID NO.94;

[0128] (6) The sense chain contains the sequence shown in SEQ ID NO.21, and the antisense chain contains the sequence shown in SEQ ID NO.96;

[0129] (7) The sense chain contains the sequence shown in SEQ ID NO.34, and the antisense chain contains the sequence shown in SEQ ID NO.109;

[0130] (8) The sense chain contains the sequence shown in SEQ ID NO.35, and the antisense chain contains the sequence shown in SEQ ID NO.110;

[0131] (9) The sense chain contains the sequence shown in SEQ ID NO.51, and the antisense chain contains the sequence shown in SEQ ID NO.126;

[0132] (10) The sense chain contains the sequence shown in SEQ ID NO.58, and the antisense chain contains the sequence shown in SEQ ID NO.133;

[0133] (11) The sense chain contains the sequence shown in SEQ ID NO.59, and the antisense chain contains the sequence shown in SEQ ID NO.134;

[0134] (12) The sense chain contains the sequence shown in SEQ ID NO.62, and the antisense chain contains the sequence shown in SEQ ID NO.137;

[0135] (13) The sense chain contains the sequence shown in SEQ ID NO.63, and the antisense chain contains the sequence shown in SEQ ID NO.138;

[0136] (14) The sense chain contains the sequence shown in SEQ ID NO.67, and the antisense chain contains the sequence shown in SEQ ID NO.142;

[0137] (15) The sense chain contains the sequence shown in SEQ ID NO.69, and the antisense chain contains the sequence shown in SEQ ID NO.144;

[0138] (16) The sense chain contains the sequence shown in SEQ ID NO.71, and the antisense chain contains the sequence shown in SEQ ID NO.146;

[0139] (17) The sense chain contains the sequence shown in SEQ ID NO. 72, and the antisense chain contains the sequence shown in SEQ ID NO. 147; and

[0140] (18) The sense strand contains the sequence shown in SEQ ID NO.74, and the antisense strand contains the sequence shown in SEQ ID NO.149; wherein each strand is independently 19 to 25 nucleotides in length.

[0141] In some embodiments of this disclosure, the oligonucleotide comprises any of the following combinations of sense and antisense strands:

[0142] (1) The positive chain contains the sequence shown in SEQ ID NO.153, and the negative chain contains the sequence shown in SEQ ID NO.228;

[0143] (2) The sense chain contains the sequence shown in SEQ ID NO.156, and the antisense chain contains the sequence shown in SEQ ID NO.231;

[0144] (3) The positive chain contains the sequence shown in SEQ ID NO.157, and the negative chain contains the sequence shown in SEQ ID NO.232;

[0145] (4) The sense chain contains the sequence shown in SEQ ID NO.160, and the antisense chain contains the sequence shown in SEQ ID NO.235;

[0146] (5) The positive chain contains the sequence shown in SEQ ID NO.161, and the negative chain contains the sequence shown in SEQ ID NO.236;

[0147] (6) The positive chain contains the sequence shown in SEQ ID NO.162, and the negative chain contains the sequence shown in SEQ ID NO.237;

[0148] (7) The positive chain contains the sequence shown in SEQ ID NO.163, and the negative chain contains the sequence shown in SEQ ID NO.238;

[0149] (8) The positive chain contains the sequence shown in SEQ ID NO.169, and the negative chain contains the sequence shown in SEQ ID NO.244;

[0150] (9) The sense chain contains the sequence shown in SEQ ID NO.170, and the antisense chain contains the sequence shown in SEQ ID NO.245;

[0151] (10) The sense chain contains the sequence shown in SEQ ID NO.171, and the antisense chain contains the sequence shown in SEQ ID NO.246;

[0152] (11) The sense chain contains the sequence shown in SEQ ID NO.184, and the antisense chain contains the sequence shown in SEQ ID NO.259;

[0153] (12) The sense chain contains the sequence shown in SEQ ID NO.185, and the antisense chain contains the sequence shown in SEQ ID NO.260;

[0154] (13) The sense chain contains the sequence shown in SEQ ID NO.188, and the antisense chain contains the sequence shown in SEQ ID NO.263;

[0155] (14) The sense chain contains the sequence shown in SEQ ID NO.189, and the antisense chain contains the sequence shown in SEQ ID NO.264;

[0156] (15) The sense chain contains the sequence shown in SEQ ID NO.191, and the antisense chain contains the sequence shown in SEQ ID NO.266;

[0157] (16) The sense chain contains the sequence shown in SEQ ID NO.200, and the antisense chain contains the sequence shown in SEQ ID NO.275;

[0158] (17) The sense chain contains the sequence shown in SEQ ID NO.201, and the antisense chain contains the sequence shown in SEQ ID NO.276;

[0159] (18) The sense chain contains the sequence shown in SEQ ID NO.203, and the antisense chain contains the sequence shown in SEQ ID NO.278;

[0160] (19) The sense chain contains the sequence shown in SEQ ID NO.205, and the antisense chain contains the sequence shown in SEQ ID NO.280;

[0161] (20) The sense chain contains the sequence shown in SEQ ID NO.207, and the antisense chain contains the sequence shown in SEQ ID NO.282;

[0162] (21) The sense chain contains the sequence shown in SEQ ID NO.208, and the antisense chain contains the sequence shown in SEQ ID NO.283;

[0163] (22) The sense chain contains the sequence shown in SEQ ID NO.209, and the antisense chain contains the sequence shown in SEQ ID NO.284;

[0164] (23) The sense chain contains the sequence shown in SEQ ID NO.210, and the antisense chain contains the sequence shown in SEQ ID NO.285;

[0165] (24) The sense chain contains the sequence shown in SEQ ID NO.212, and the antisense chain contains the sequence shown in SEQ ID NO.287;

[0166] (25) The sense chain contains the sequence shown in SEQ ID NO.213, and the antisense chain contains the sequence shown in SEQ ID NO.288;

[0167] (26) The sense chain contains the sequence shown in SEQ ID NO.214, and the antisense chain contains the sequence shown in SEQ ID NO.289;

[0168] (27) The sense chain contains the sequence shown in SEQ ID NO.215, and the antisense chain contains the sequence shown in SEQ ID NO.290;

[0169] (28) The sense chain contains the sequence shown in SEQ ID NO.216, and the antisense chain contains the sequence shown in SEQ ID NO.291;

[0170] (29) The sense chain contains the sequence shown in SEQ ID NO.217, and the antisense chain contains the sequence shown in SEQ ID NO.292;

[0171] (30) The sense chain contains the sequence shown in SEQ ID NO.218, and the antisense chain contains the sequence shown in SEQ ID NO.293;

[0172] (31) The positive chain contains the sequence shown in SEQ ID NO.219, and the negative chain contains the sequence shown in SEQ ID NO.294;

[0173] (32) The sense chain contains the sequence shown in SEQ ID NO.220, and the antisense chain contains the sequence shown in SEQ ID NO.295;

[0174] (33) The sense chain contains the sequence shown in SEQ ID NO.221, and the antisense chain contains the sequence shown in SEQ ID NO.296;

[0175] (34) The sense chain contains the sequence shown in SEQ ID NO.222, and the antisense chain contains the sequence shown in SEQ ID NO.297;

[0176] (35) The sense chain contains the sequence shown in SEQ ID NO.223, and the antisense chain contains the sequence shown in SEQ ID NO.298;

[0177] (36) The sense chain contains the sequence shown in SEQ ID NO. 224, and the antisense chain contains the sequence shown in SEQ ID NO. 299; and

[0178] (37) The positive strand comprises the sequence shown in SEQ ID NO. 225, and the antisense strand comprises the sequence shown in SEQ ID NO. 300; preferably, the oligonucleotide comprises any of the following combinations of positive and antisense strands:

[0179] (1) The positive chain contains the sequence shown in SEQ ID NO.153, and the negative chain contains the sequence shown in SEQ ID NO.228;

[0180] (2) The sense chain contains the sequence shown in SEQ ID NO.156, and the antisense chain contains the sequence shown in SEQ ID NO.231;

[0181] (3) The sense chain contains the sequence shown in SEQ ID NO.160, and the antisense chain contains the sequence shown in SEQ ID NO.235;

[0182] (4) The positive chain contains the sequence shown in SEQ ID NO.162, and the negative chain contains the sequence shown in SEQ ID NO.237;

[0183] (5) The sense chain contains the sequence shown in SEQ ID NO.169, and the antisense chain contains the sequence shown in SEQ ID NO.244;

[0184] (6) The positive chain contains the sequence shown in SEQ ID NO.171, and the negative chain contains the sequence shown in SEQ ID NO.246;

[0185] (7) The sense chain contains the sequence shown in SEQ ID NO.184, and the antisense chain contains the sequence shown in SEQ ID NO.259;

[0186] (8) The sense chain contains the sequence shown in SEQ ID NO.185, and the antisense chain contains the sequence shown in SEQ ID NO.260;

[0187] (9) The sense chain contains the sequence shown in SEQ ID NO.201, and the antisense chain contains the sequence shown in SEQ ID NO.276;

[0188] (10) The sense chain contains the sequence shown in SEQ ID NO.208, and the antisense chain contains the sequence shown in SEQ ID NO.283;

[0189] (11) The sense chain contains the sequence shown in SEQ ID NO.209, and the antisense chain contains the sequence shown in SEQ ID NO.284;

[0190] (12) The sense chain contains the sequence shown in SEQ ID NO.212, and the antisense chain contains the sequence shown in SEQ ID NO.287;

[0191] (13) The sense chain contains the sequence shown in SEQ ID NO.213, and the antisense chain contains the sequence shown in SEQ ID NO.288;

[0192] (14) The positive chain contains the sequence shown in SEQ ID NO.217, and the negative chain contains the sequence shown in SEQ ID NO.292;

[0193] (15) The sense chain contains the sequence shown in SEQ ID NO.219, and the antisense chain contains the sequence shown in SEQ ID NO.294;

[0194] (16) The sense chain contains the sequence shown in SEQ ID NO.221, and the antisense chain contains the sequence shown in SEQ ID NO.296;

[0195] (17) The sense chain contains the sequence shown in SEQ ID NO. 222, and the antisense chain contains the sequence shown in SEQ ID NO. 297; and

[0196] (18) The sense chain contains the sequence shown in SEQ ID NO.224, and the antisense chain contains the sequence shown in SEQ ID NO.299;

[0197] Each of these chains is independently 19 to 25 nucleotides in length.

[0198] In some embodiments, at least one nucleotide of an oligonucleotide or a salt thereof is conjugated to one or more ligands to form an siRNA conjugate. The siRNA conjugate contains the aforementioned siRNA and a conjugating group attached to the siRNA. The term "oligonucleotide salt" refers to an oligonucleotide compound in salt form. Oligonucleotide salts include salts of oligonucleotide conjugated compounds and salts of unconjugated oligonucleotide compounds. Oligonucleotide salts are advantageously present in solid powder form.

[0199] Generally, the conjugation group comprises at least one pharmaceutically acceptable ligand and an optional linker, with the siRNA, linker, and ligand sequentially linked. The targeting group can be a ligand conventionally used in the field of siRNA drug delivery, such as the various ligands described in WO2009082607A2, the entire disclosure of which is incorporated herein by reference. In some embodiments, there are 2-4 ligands. The siRNA molecule can be non-covalently or covalently conjugated to the conjugation group, for example, it can be covalently conjugated to the conjugation group. The conjugation site of the siRNA to the conjugation group can be at the 3' or 5' end of the siRNA's sense or antisense strand, or it can be within the siRNA's internal sequence. In some embodiments, the conjugation site of the siRNA to the conjugation group is at the 3' or 5' end of the siRNA's sense strand. In some embodiments, the conjugation site of the siRNA to the conjugation group is at the 3' or 5' end of the siRNA's antisense strand. In some preferred embodiments, the conjugation site of the siRNA to the conjugation group is at the 3' end of the siRNA's sense strand.

[0200] In some embodiments of this disclosure, another modification of the RNAi agent chemically links one or more ligands, portions or conjugates that enhance RNAi activity, cellular distribution or cellular uptake to the RNA. Such moieties include, but are not limited to, lipid moieties, such as cholesterol moieties (Letsinger et al., (1989) Proc. Natl. Acid. Sci. USA, 86: 6553-6556), bile acids (Manohara et al., (1994) Biorg. Med. Chem. Let., 4: 1053-1060), thioethers, such as beryl-S-triphenylmethylthiol (Manoharan et al., (1992) Ann. NY Acad. Sci., 660: 306-309; Manoharan et al., (1993) Biorg. Med. Chem. Let., 3: 2765-2770), sulfur cholesterol (Oberhauser et al., (1992) Nucl. Acids Res., 20: 533-538), and fatty acid chains, such as dodecanediol or undecyl residues (Saison-Behmoaras et al., (1991) EMBO J, 10:1111-1118; Kabanov et al., (1990) FEBS Lett, 259:327-330; Svinarchuk et al., (1993) Biochimie, 75:49-54), phospholipids, such as di-hexadecyl-racemic-glycerol or triethyl-ammonium 1,2-di-O-hexadecyl-racemic-glycerol-3-phosphate (Manoharan et al., (1995) Tetrahedron Lett., 36:3651-3654; Shea et al., (1990) Nucl. Acids Res., 18:3777-3783), polyamines or polyethylene glycol chains (Manoharan et al., (1995) Nucleosides & Nucleotides, 14:969-973), or adamantane acetic acid (Manoharan et al., (1995) Tetrahedron Lett., 36:3651-3654), palmitic moiety (Mishra et al., (1995) Biochim. Biophys. Acta, 1264:229-237), or octadecylamine or hexylamino-carbonyl-oxocholesterol moiety (Crooke et al., (1996) J. Pharmacol. Exp. Ther., 277:923-937).

[0201] In some embodiments, the lipophilic moiety is an aliphatic, cyclic (e.g., alicyclic) or polycyclic (e.g., polyalicyclic) compound, such as a steroid (e.g., a sterol) or a straight-chain or branched aliphatic hydrocarbon. The lipophilic moiety may typically comprise a hydrocarbon chain, which may be cyclic or acyclic. The hydrocarbon chain may contain various substituents and / or one or more heteroatoms, such as oxygen or nitrogen atoms. Such lipophilic aliphatic moietyes include, but are not limited to, saturated or unsaturated C4-C... 30 Hydrocarbons (e.g., C6-C) 22 Hydrocarbons), saturated or unsaturated fatty acids, waxes (e.g., monohydric alcohol esters of fatty acids and fatty diamides), terpenes (e.g., C464 ... 10 Terpenes, C 15 Sesquiterpenes, C 20 Diterpenes, C 30 Triterpenes and C 40 Tetraterpenes and other polycyclic hydrocarbons. For example, the lipophilic moiety can contain C4 to C5. 30 Hydrocarbon chains (e.g., C4 to C5) 30 Alkyl or alkenyl). In some embodiments, the lipophilic moiety comprises saturated or unsaturated C6 to C6 groups. 18 Hydrocarbon chains (e.g., straight-chain C6 to C15) 22 (alkyl or alkenyl). In one embodiment, the lipophilic moiety comprises saturated or unsaturated C. 16 Hydrocarbon chains (e.g., straight-chain C) 16 Alkyl or alkenyl). The lipid moiety is 2'-O-alkyl, consisting of 10-30 hydrocarbon chains, including single and / or branched chains.

[0202] The lipophilic moiety is bound to the double-stranded RNAi agent via a linker, which may contain ether, thioether, urea, carbonate, amine, amide, maleimide-thioether, disulfide, phosphate diester, sulfonamide bond, click reaction (e.g., triazole from azide-alkynyl cycloaddition), or carbamate.

[0203] In some embodiments of this disclosure, the ligand that enhances RNAi activity, cell distribution, or cellular uptake comprises C 12 Alkyl, C 16 Alkyl, C 18 Alkyl, C 22 Alkyl groups, or branched lipids such as DDA (cationic dimethyl dioctadecyl ammonium), TDB (trehalose 6,6,9-disorbate), and lipid-cholesterol linkages, or cholesterol, steroids, etc.

[0204] The straight-chain lipophilic portion (C16) attached to one position on the chain has the following structure:

[0205] Base is a nucleotide base or a nucleotide base analogue, optionally, wherein Base is selected from the group consisting of adenine, guanine, cytosine, thymine, and uracil.

[0206] For example, 2'-O-hexadecyluridine has the following structure:

[0207] This disclosure also provides a conjugate or a pharmaceutically acceptable salt thereof for inhibiting sFLT-1 expression, comprising a lipid conjugate having the following structure:

[0208] A is a modified double-stranded oligonucleotide or a modified single-stranded oligonucleotide, wherein the modified double-stranded oligonucleotide or the modified single-stranded oligonucleotide is conjugated to a lipid-containing moiety at the 3' end of one strand of the modified double-stranded oligonucleotide or the 3' end of the modified single-stranded nucleic acid.

[0209] X1 is:

[0210] L1 is -(CH2)n-, -(CH2) n L2(CH2) n - or key;

[0211] L2 is -C(=O)NH-, -C(=O)O-, -OC(=O)O-, -NHC(=O)O-, -NHC(=O)NH-, -C(=S)NH-, -C(=O)S-, -NH-, O (oxygen) or S (sulfur).

[0212] Each m is an integer from 10 to 18, and each n is an integer from 1 to 6.

[0213] For example, DTX-1

[0214] This disclosure also provides a conjugate or a pharmaceutically acceptable salt thereof for inhibiting sFLT-1 expression, wherein the lipid conjugate compound PCDCA has the following structure:

[0215] In some embodiments of the methods disclosed herein, the expression of the sFLT-1 gene is suppressed by at least 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, or below the detection level. In a preferred embodiment, the expression of sFLT-1 is suppressed by at least 70%. It should also be understood that it may be desirable to suppress the expression of sFLT-1 in certain tissues (e.g., placenta) without significantly suppressing its expression in other tissues. In a preferred embodiment, the expression level is determined in suitable species-matched cell lines using the assay method provided in Example 2 with siRNA concentrations of 0.5 nM, 0.1 nM, and 0.05 nM.

[0216] In some embodiments, inhibition of in vivo expression is determined by knocking down human gene expression in rodents, for example, AAV-infected mice expressing the human target gene (i.e., sFLT-1), for example, by confirming the inhibitory effect on the human gene when administered as a single dose, for example, by identifying the lowest point of sFLT-1 expression after a subcutaneous injection of 10 mg / kg. Such systems are useful when the nucleic acid sequences of the human gene and the model animal gene are sufficiently similar so that human RNAi provides effective knockdown of the model animal gene. RNA expression in cells is determined using the PCR method provided in Example 2.

[0217] Inhibition of sFLT-1 gene expression can be represented by a reduction in the amount of mRNA expressed in a cell line (such cells may be present in, for example, a sample derived from a subject), or by a decrease in the expression of luciferase fused to sFLT-1. The sFLT-1 gene or luciferase-sFLT-1 fusion gene is transcribed in cells or cell populations and is treated (e.g., by contacting one or more cells with the RNAi of this disclosure, or by administering the RNAi of this disclosure to a subject in which cells are present or were previously present), such that the expression of the sFLT-1 gene or luciferase-sFLT-1 fusion gene is inhibited compared to a substantially identical cell line that is untreated (control cells not treated with RNAi or not treated with RNAi targeting the target gene). In a preferred embodiment, inhibition was assessed in species-matched cell lines using 0.5 nM, 0.1 nM, and 0.05 nM siRNA concentrations as described in Example 2, expressed as 2^-ΔΔCT of the mRNA expression level in the treated cells relative to the mRNA level in the control cells, using the following formula: ΔCT = CT sFLT-1 -CT GAPDH △△CT=△CT 处理细胞 -△CT 对照细胞 mRNA level = 2^-△△CT

[0218] Or relative luciferase ratio: Relative luciferase ratio = Renalis luciferase activity (RL) / Firefly luciferase activity (FL)

[0219] Inhibition efficiency (%) = 1 - (Ratio) 给药组 / Ratio 对照组 )*100%.

[0220] In other embodiments, inhibition of sFLT-1 gene expression can be assessed based on a decrease in parameters associated with sFLT-1 gene expression function, such as sFLT-1 protein levels or sFLT-1 mRNA levels in the blood or serum of a subject. sFLT-1 gene silencing can be determined in any cell expressing sFLT-1, whether endogenous or heterologous from the expression construct, and by any assay known in the art.

[0221] Inhibition of sFLT-1 protein expression can be demonstrated by a decrease in the level of sFLT-1 mRNA or secreted luciferase expressed in cells or cell populations, or in a subject sample (e.g., protein levels in a blood sample from the subject). As described above, to assess mRNA inhibition, inhibition of protein expression levels in treated cells or cell populations can similarly be expressed as a percentage of protein levels in control cells or cell populations, or as a change in protein levels in a subject sample (e.g., blood or serum from it). Inhibition is assessed using the method provided in Example 3, using the following formula, expressed as a percentage of the amount of sFLT-1 mRNA expressed in the treated sample (e.g., from its placenta) relative to the amount of sFLT-1 mRNA or Gluc expressed in the control group. Percentage of mRNA inhibition = (mRNA amount...) 给药组 -mRNA amount 对照组 ) / mRNA amount 对照组 *100%

[0222] Control cells, cell populations, or subject samples that can be used to assess inhibition of sFLT-1 gene expression include cells, cell populations, or subject samples that have not yet been exposed to the RNAi agent of this disclosure. For example, control cells, cell lines, or subject samples may be derived from individual subjects (e.g., human or animal subjects) before treatment of subjects or appropriately matched cohorts with the RNAi agent.

[0223] In some embodiments of the methods disclosed herein, iRNA is administered to a subject to deliver iRNA to a specific site within the subject. Inhibition of sFLT-1 expression can be assessed by measuring or changing levels of sFLT-1 mRNA or fusion secreted luciferase from a specific site or tissue of the subject (e.g., sciatic nerve, gastrocnemius muscle, or blood).

[0224] This disclosure also provides methods for using the RNAi of this disclosure or compositions containing the RNAi of this disclosure to inhibit sFLT-1 expression, thereby preventing or treating sFLT-1-related conditions, such as diseases involving physiological and pathological processes of angiogenesis, angiogenesis, and / or angiogenesis. Therefore, these pharmaceutical compositions can be used to treat diseases, disorders, and conditions requiring inhibition of angiogenesis, angiogenesis, or angiogenesis, including but not limited to cancer tumor growth and metastasis, tumors, ocular neovascularization (including macular degeneration, diabetic retinopathy, ischemic retinopathy, retinopathy of prematurity, choroidal neovascularization), rheumatoid arthritis, osteoarthritis, chronic asthma, septic shock, inflammatory diseases, synovitis, bone and cartilage destruction, pannus growth, osteophyte formation, osteomyelitis, psoriasis, obesity, hemangioma, and keratosis pilaris. Bosie's sarcoma, atherosclerosis (including ruptured atherosclerotic plaques), endometriosis, warts, hypertrichosis, keloids, allergic edema, dysfunctional uterine bleeding, follicular cysts, ovarian hyperstimulation syndrome, endometriosis, osteomyelitis, inflammatory and infectious processes (hepatitis, pneumonia, glomerulonephritis), asthma, nasal polyps, transplantation, liver regeneration, leukomalacia, thyroiditis, goiter, lymphoproliferative disorders, hematologic malignancies, vascular malformations, preeclampsia, eclampsia, and / or HELLP syndrome.

[0225] Cells suitable for treatment using the methods of this disclosure can be any cell expressing the sFLT-1 gene, such as trophoblast cells, epidermal cells, and melanoma cells, but preferably trophoblast cells and melanoma cells. Cells suitable for use in the methods of this disclosure can be mammalian cells, such as primate cells (e.g., human cells, including human cells in chimeric nonhuman animals, or nonhuman primate cells, such as monkey cells or chimpanzee cells) or non-primate cells. In some embodiments, the cells are human cells, such as human melanoma cells. In the methods of this disclosure, the expression of sFLT-1 in the cells is inhibited by at least 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, or below a measured detection level.

[0226] The in vivo methods of this disclosure may include administering a composition comprising RNAi to a subject, wherein the RNAi comprises a nucleotide sequence complementary to at least a portion of the RNA transcript of the sFLT-1 gene of the mammal to which the RNAi agent is administered. The composition may be administered in any manner 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 local (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 intrathecal injection.

[0227] In one aspect, this disclosure also provides a method for inhibiting the expression of the mammalian sFLT-1 gene. The method comprises administering an oligonucleotide or a pharmaceutically acceptable salt thereof, or a combination thereof, to a mammal. The oligonucleotide is a double-stranded RNA (dsRNA) that targets the sFLT-1 gene in mammalian cells and sustains the mammal for sufficient time to allow for degradation of the mRNA transcript of the sFLT-1 gene, thereby inhibiting the expression of the sFLT-1 protein in the cells. The reduction in gene expression can be assessed by any method known in the art and by methods such as qRT-PCR as described herein, for example, as in Example 2. The reduction in protein product 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 sFLT-1 protein expression.

[0228] This disclosure also provides certain embodiments of the invention relating to the treatment of one or more angiogenesis disorders. "Treatment of angiogenesis disorders" means the use of the oligonucleotides (e.g., siRNA) of the invention in the pharmaceutical compositions for the treatment of diseases involving the physiological and pathological processes of angiogenesis, vasculogenesis, and / or angiogenesis. Therefore, these pharmaceutical compositions can be used to treat diseases, ailments, and conditions requiring inhibition of angiogenesis, vasculogenesis, or angiogenesis, including but not limited to cancer tumor growth and metastasis, tumors, ocular neovascularization (including macular degeneration, diabetic retinopathy, ischemic retinopathy, retinopathy of prematurity, choroidal neovascularization), rheumatoid arthritis, osteoarthritis, chronic asthma, septic shock, inflammatory diseases, synovitis, bone and cartilage destruction, pannus growth, osteophyte formation, osteomyelitis, psoriasis, obesity, hemangioma, and vascular malformation. Bosie's sarcoma, atherosclerosis (including ruptured atherosclerotic plaques), endometriosis, warts, hypertrichosis, keloids, allergic edema, dysfunctional uterine bleeding, follicular cysts, ovarian hyperstimulation syndrome, endometriosis, osteomyelitis, inflammatory and infectious processes (hepatitis, pneumonia, glomerulonephritis), asthma, nasal polyps, transplantation, liver regeneration, leukomalacia, thyroiditis, goiter, lymphoproliferative disorders, hematologic malignancies, vascular malformations, preeclampsia, eclampsia, and / or HELLP syndrome. In some implementations, the disease or condition is preeclampsia. In some implementations, the disease or condition is postpartum preeclampsia. In some implementations, the disease or condition is eclampsia. In some implementations, the disease or condition is HELLP syndrome.

[0229] Preeclampsia ("PE") refers to a multisystem disorder characterized by hypertension with proteinuria or edema or both, and one or more of the following: glomerular dysfunction, cerebral edema, hepatic edema or coagulation abnormalities due to pregnancy or recent pregnancy. PE usually occurs after the 20th week of pregnancy. PE is usually defined as a combination of the following symptoms: (1) systolic blood pressure (BP) >140 mmHg and diastolic blood pressure >90 mmHg after the 20th week of pregnancy (usually measured twice, 4–168 hours apart), (2) new-onset proteinuria (urinalysis showed 1+ by dipstick, protein >300 mg in a 24-hour urine collection, or a protein / creatinine ratio >v0.3 in a single random urine sample), and (3) resolution of hypertension and proteinuria 12 weeks postpartum.

[0230] The RNAi disclosed herein can be administered as “free RNAi.” Free RNAi is administered in the absence of a pharmaceutical composition. Naked RNAi can be administered in a suitable buffer solution. The buffer solution may 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 RNAi can be adjusted to suit its administration to the subject.

[0231] The administration of RNAi according to the method of this disclosure can lead to the prevention or treatment of sFLT-1 related conditions, such as PE. A therapeutic dose of RNAi can be administered to the subject, such as from about 0.1 mg / kg to about 200 mg / kg. Preferably, it is from 1 mg / kg to about 30 mg / kg. RNAi is preferably administered subcutaneously, i.e., by intramuscular injection. One or more injections can be used to deliver the desired dose of iRNA to the subject. Injections can be repeated over a period of time.

[0232] A therapeutic amount of RNAi or its salt, or its conjugate, or a salt of a conjugate, or a combination thereof, may be administered to the subject, such as at a dose ranging from about 0.01 mg / kg to about 200 mg / kg (e.g., about 0.1 mg / kg to about 100 mg / kg). In some embodiments, the oligonucleotide or its salt, or its conjugate, or a salt of a conjugate, or a combination thereof, is administered to the subject at a dose ranging from about 0.1 mg / kg to about 50 mg / kg, preferably from about 0.1 mg / kg to about 20 mg / kg, 0.3 mg / kg to about 18 mg / kg, 0.5 mg / kg to about 15 mg / kg, or 0.5 mg / kg to about 12 mg / kg, more preferably from about 1 mg / kg to about 10 mg / kg. Preferably, it is from 1 mg / kg to about 50 mg / kg. RNAi or its salt, or its conjugate, or a salt of a conjugate, or a combination thereof is preferably administered subcutaneously, i.e., by subcutaneous injection. One or more injections may be used to deliver the desired dose of RNAi to the subject. Injections may be repeated over a period of time.

[0233] In some embodiments, an RNAi or its salt, or a conjugate thereof, or a salt of a conjugate thereof, or a combination thereof, is administered to a subject at a fixed dose of about 50 mg to about 800 mg. In some embodiments, an RNAi or its salt, or a conjugate thereof, or a salt of a conjugate thereof, or a combination thereof, 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, an RNAi or its salt, or a conjugate thereof, or a salt of a conjugate thereof, or a combination thereof, is administered to a subject at a fixed dose of about 100 mg, about 200 mg, about 300 mg, about 400 mg, 500 mg, about 600 mg, about 700 mg, or about 800 mg.

[0234] It can be administered repeatedly at regular intervals. In some embodiments, treatment can be administered at a lower frequency after the initial treatment regimen. Repeated dosing regimens may include periodic administration of therapeutic amounts of RNAi or its salts, or conjugates thereof, or salts of conjugates thereof, or combinations thereof, such as once monthly to once a year. In some embodiments, RNAi or its salts, or conjugates thereof, or salts of conjugates thereof, or combinations thereof are administered approximately once monthly to approximately every three months, or approximately every three months to approximately every six months, or even once a year.

[0235] In some embodiments, a fixed dose is administered to the subject at monthly intervals. In some embodiments, a fixed dose is administered to the subject at six-month intervals.

[0236] In some embodiments, the subject is administered a fixed dose of about 50 mg approximately every six months. In some embodiments, the subject is administered a fixed dose of about 100 mg approximately every six months. In some embodiments, the subject is administered a fixed dose of about 150 mg approximately every six months. In some embodiments, the subject is administered a fixed dose of about 300 mg approximately every six months. In some embodiments, the subject is administered a fixed dose of about 300 mg approximately every six months. In some embodiments, the subject is administered a fixed dose of about 600 mg approximately every six months. In some embodiments, the subject is administered a fixed dose of about 800 mg approximately every six months. In some embodiments, the subject is administered a fixed dose of about 800 mg approximately every six months.

[0237] 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. This disclosure further provides RNAi agents or pharmaceutical compositions thereof in combination with other drugs and / or other treatments (e.g., known drugs and / or known treatments, such as those currently used to treat these conditions) to treat subjects who would benefit from reduced and / or suppressed sFLT-1 gene expression, such as subjects with sFLT-1-related diseases. Other therapeutic agents and treatments suitable for treating subjects who would benefit from reduced sFLT-1 expression (e.g., subjects with sFLT-1-related diseases) include rehabilitation therapy, symptomatic drug treatment, drugs for the prevention of hypertension, low-dose aspirin, corticosteroids, and magnesium sulfate, etc.

[0238] For the purpose of clarity and concise description, the features are described herein as part of some identical or separate embodiments; however, it will be understood that the scope of this disclosure may include some embodiments having a combination of all or some of the features described.

[0239] The present disclosure will now be described in more detail with reference to specific embodiments. However, the embodiments are for illustrative purposes only and are not intended to limit the scope of the present disclosure.

[0240] Example

[0241] Example 1. Preparation of ligands and siRNA

[0242] Abbreviations for nucleotide monomers used in nucleic acid sequence representation.

[0243] Table A. Abbreviations for nucleotide monomers used in nucleic acid sequence representation

[0244] Preparation of ligands

[0245] C16 was purchased from Chengdu Pioneer Pharmaceuticals Co., Ltd.

[0246] DTX was purchased from Suzhou Ouli Biomedical Technology Co., Ltd.

[0247] The synthesis of PCDCA is based on patent US10633653B2.

[0248] Preparation of oligonucleotides

[0249] (1) Preparation of siRNA

[0250] First, a computer-based algorithm was used to generate candidate oligonucleotide sequences complementary to human sFLT1-i13 and sFLT1-e15a mRNAs (Table 1). Some were designed as double-stranded siRNAs with 19 / 21 pairings on the sense and antisense strands, with the antisense strand having two dangling ends complementary to the mRNA sequence. In some cases, the dangling ends of the antisense strand were non-complementary UU. Other sequences were designed as double-stranded siRNAs with 19 / 21 pairings on the sense and antisense strands, with the antisense strand having two dangling ends complementary to the mRNA sequence. In some complementary pairing sequences, the 5' end of the antisense strand (the last 3' end of the sense strand) was replaced with a base that did not match the sFLT-1 mRNA.

[0251] Table 1. Human and cynomolgus monkey sFLT-1 mRNA sequences

[0252] The siRNA sequence was synthesized separately on a solid support via the sense strand (SS) and antisense strand (AS), and was obtained after deprotection, cleavage, purification, annealing, purification and lyophilization.

[0253] Solid-phase synthesis (Figure 2): The sense and antisense strands were synthesized separately on a solid support using an automated oligonucleotide synthesizer, employing phosphoramide technology. The synthesizer used was, for example, the AKTA Oligopilot (Cytiva) or Dr. Oligo 192XLc (Kunshan Berlik Precision Instruments Co., Ltd.). Solid-phase synthesis began at the 3' end of the sequence, with monomers sequentially coupled into the sequence. Each coupling of a phosphoramide monomer involved four chemical steps: 1) unblocking or deprotection (deprotection of hydroxyl protecting groups); 2) coupling; 3) oxidation; and 4) end-capping. All phosphoramidite monomers, reagents, and purification consumables used were commercially available. For example, various phosphoramidite monomers (such as 5'-O-(4,4'-Dimethoxytrityl)-2'-O-methyl-Uridine-3'-CE-Phosphoramidite) were purchased from Shanghai Zhaowei Technology Development Co., Ltd., and reaction reagents (such as 40wt% methylamine aqueous solution and 28wt% ammonium hydroxide aqueous solution) were purchased from Sigma-Aldrich LLC. The siRNA synthesis and purification methods used in this paper are as described in US20130178612A1 and US2015100197A1; the synthesis methods for VPUm and APU structural sequences are as described in J.Med.Chem.2018,61,734-744.

[0254] (2) Preparation of double-stranded RNA reagent

[0255] (a) The synthesis of the chain of justice

[0256] Solid-phase phosphoramide synthesis is a mature method for synthesizing oligonucleotides. A computer-controlled synthesizer is used, and the reaction takes place in a stainless steel column. The positive chain synthesis begins with a solid support loaded with ligands (e.g., L96 or PCDCA), or directly with the solid support. Different starting materials, reagents, and solvents are injected sequentially from the 3' to 5' positions through different tubing lines controlled by the solid-phase synthesizer, linking phosphoramide nucleoside monomers one by one. The reaction process involves four cyclic steps: DMT protection removal, condensation, oxidation or thiolation, and end-capping. One nucleotide unit is linked in each cycle, yielding an oligonucleotide sequence of 19 or 21 nucleotides. After synthesis, the protecting group (2-cyanoethyl) is removed on the solid-phase column, and the synthesized sequence is cleaved from the solid support via ammonolysis. The sequence is filtered, the filter cake is washed with ethanol, and the filtrate and washings are collected and concentrated to obtain the crude positive chain. The crude product is purified by chromatography (SOURCE 15Q) and lyophilized to obtain the target product, the positive chain. In the synthesizer, siRNA positive strand conjugates were synthesized starting with a solid support loaded with ligands (e.g., L96); siRNA was synthesized directly starting with a solid support.

[0257] (b) Synthesis of antisense chains

[0258] Similar to the sense strand synthesis, the antisense strand is synthesized using a solid-phase synthesizer. Different starting materials, reagents, and solvents are injected sequentially from the 3' to 5' ends of the sequence through different tubing, linking phosphoramidine nucleoside monomers one by one. The reaction process involves four cyclic steps: DMT protection removal, condensation, oxidation or thiolation, and end-capping. One nucleotide unit is linked in each cycle, yielding an oligonucleotide sequence of 21 or 23 nucleotides. After synthesis, the protecting group (2-cyanoethyl) is removed on a solid-phase column, and the synthesized sequence is cleaved from the solid support via ammonolysis. The sequence is filtered, the filter cake is washed with ethanol, and the filtrate and washings are collected and concentrated to obtain the crude antisense strand. The crude product is purified by chromatography (SOURCE 15Q), ultrafiltered, and lyophilized to obtain the target product, antisense siRNA.

[0259] (c) Preparation of double-stranded siRNA

[0260] The AS and SS strands were dissolved separately in injection water and mixed in a defined ratio (1.01:1.0-1.2:1.0). The mixture was incubated at 30-50°C for 30-90 minutes and then cooled to room temperature. The double-stranded siRNA product was obtained by freeze-drying.

[0261] The double-stranded siRNA reagents listed in Tables 2, 3, and 4 below were prepared using the same method.

[0262] In Tables 2, 3, and 4, “G”, “C”, “A”, “U”, and “T” typically represent nucleotides with guanine, cytosine, adenine, uracil, and thymine as bases, respectively. The naked sequences in Tables 2, 3, and 4 refer to unmodified oligonucleotide sequences.

[0263] Modifications: m represents 2'-methoxy; f represents 2'-deoxy-2'-fluorine; s represents thiophosphate; VPUm represents 2'-methoxy-modified uridine; M represents 2'-O-methyl-6-(3-(2-carboxyethyl)phenyl)-adenosine; dA represents 2'-deoxyadenosine-3'-phosphate; dG represents 2'-deoxyguanosine-3'-phosphate; dC represents 2'-deoxycytidine-3'-phosphate; dU represents 2'-deoxyuridine-3'-phosphate.

[0264] VPUm:

[0265] M:

[0266] DTX-1

[0267] PCDCA:

[0268] Table 2. Naked Oligonucleotide Sequences

[0269] Table 3 Oligonucleotide Modification Sequences

[0270] Table 4. Oligonucleotide vector sequences

[0271] Example 2. In vitro activity screening of sFLT-1-siRNA

[0272] (1) Cell culture and transfection:

[0273] WM115 cells (SNL-544, Shangen Biotechnology) were incubated at 37°C in a 5% CO2 incubator using DMEM medium (Shanghai Biotechnology Co., Ltd., catalog number iCell-0001) supplemented with 10% FBS (GIBCO, 12483020) and 1% penicillin-streptomycin (GIBCO, 15140-122). Once cell confluence reached 90%, cells were digested with trypsin-EDTA (Thermo, 25200-072), counted using a Countstar (IC1000), and seeded at 190 μl cell suspension per well in 96-well plates. The seeding density of WM115 cells was 2*10n. 4 Cells / wells will adhere to the culture vessel the following day for transfection.

[0274] Use Lipofectamine TM Transfection was performed using RNAiMAX (thermofisher, 13778150). 2.2 μl (2 μM) of the diluted compound, 19.1 μl of Opti-MEM (thermofisher, 1105821), and 0.7 μl of RNAiMAX were mixed to prepare the transfection complex. After incubation for 5 minutes, the transfection complex was added to the cells (two replicates per complex), 10 μl per well, with a final siRNA concentration of 10 nM. The cells were incubated at 37°C in a 5% CO2 incubator for 24 hours.

[0275] (2) RNA extraction and detection

[0276] (i) Total RNA was extracted using the RNA-Quick Purification Kit (RN001, Yishan Biotechnology):

[0277] Remove the 12-well plate from the incubator, aspirate the culture medium, wash once with an appropriate amount of PBS, add 500 μl of lysis buffer to each well, and transfer the supernatant to a new 1.5 ml centrifuge tube. Add 500 μl of anhydrous ethanol to the lysed cells and mix thoroughly. Invert the centrifuge tube several times, or use a pipette to vigorously pipette 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 1 min. Remove the centrifuge tube, add 500 μl of wash buffer to the column, and centrifuge at 12000 × g for 1 min. After centrifugation, remove the column, discard the waste liquid, and put the RNA column back into the collection tube. Centrifuge the empty tube once to remove any possible residual wash buffer. Place the column on a clean, RNase-free 1.5 ml centrifuge tube and allow it to air dry for 2 minutes. Add 30 μl of elution buffer to the center of the RNA column membrane, incubate at room temperature for 2 minutes, centrifuge at 2000×g for 1 min to elute the RNA, and then place on ice. Measure the concentration of the eluted RNA for subsequent experiments. The extracted RNA can be used immediately for subsequent experiments or stored at -80℃ for later use.

[0278] (ii) Use II Q RT SuperMix for qPCR (+gDNA wiper) reverse transcription kit (Novizan, R223-01) for cDNA synthesis:

[0279] Prepare a mixture in an RNase-free centrifuge tube: 4 μl 4×g DNA wiper Mix, 1 μg template RNA, and RNase-free ddH2O to a final volume of 16 μl to remove genomic DNA. Gently pipette to mix and incubate at 42°C for 2 min. Then, directly add 4 μl 5×HiScript II qRT SuperMix II to the reaction tube and gently pipette to mix. Incubate in a PCR instrument (Applied Biosystems, 9700) at 50°C for 15 min, then at 85°C for 5 sec, and finally at 4°C. The product can be used immediately for qPCR or stored at -20°C and used within six months. For long-term storage, aliquot and store at -80°C. Avoid repeated freeze-thaw cycles for cDNA.

[0280] (iii) Quantitative analysis using ChamQ SYBR qPCR Master Mix (Novazia, Q311-02):

[0281] Prepare a 20 μl mixture by adding 10 μl of 2×ChamQ SYBR qPCR Master Mix, 0.5 μl of Forward primer (Ruiboxingke), 0.5 μl of Reverse primer (Ruiboxingke), 1 μl of Template cDNA, and 8 μl of ddH2O. Each sample was tested in triplicate. The 96-well plate was placed in a qPCR instrument (ROCGENE, Archimed). The following program was executed: pre-denaturation, 95℃, 30 sec; amplification, 95℃, 10 sec, 60℃, 30 sec, 40 cycles; melting curve, 95℃, 15 sec, 60℃, 60 sec, 95℃, 15 sec.

[0282] (3) Data statistical analysis:

[0283] Export the data to Excel format using CT. sFLT1 -CT GAPDH The control group was normalized. To calculate the fold change in relative silencing efficiency, the data were analyzed using the ΔΔCT method. The mean and standard deviation of the three parallel replicates were calculated.

[0284] The cell screening results are shown in Tables 5 and 6.

[0285] As shown in Table 5, at a dosage of 0.5 nM, 10 siRNAs exhibited an inhibition rate of over 80% on sFLT1-i13 mRNA, such as AL0315002, AL0315005, AL0315006, AL0315009, AL0315010, AL0315011, AL0315012, AL0315018, AL0315019, and AL0315020. When the dosage was reduced to 0.1 nM, 6 siRNAs showed an inhibition rate exceeding 50% on the target gene mRNA, such as AL0315002, AL0315005, AL0315009, AL0315011, AL0315018, and AL0315020.

[0286] As shown in Table 6, at a dosage of 0.05 nM, the inhibition rate of 26 siRNA sequences against sFLT1-e15a mRNA can reach over 50%, such as AL0315032, AL0315033, AL0315036, AL0315037, AL0315039, AL0315048, AL0315049, AL0315051, AL0315053, AL0315055, AL0315056, AL0315057, AL0315058, AL0315060, AL0315061, AL0315062, AL0315063, AL0315064, AL0315065, AL0315066, AL0315067, AL0315068, AL0315069 ... AL0315067, AL0315068, AL0315069, AL0315070, AL0315071, AL0315072, and AL0315073, and even some siRNA sequences can achieve an inhibition rate of over 70% against target gene mRNA, such as AL0315032, AL0315033, AL0315049, AL0315056, AL0315057, AL0315060, AL0315061, AL0315065, AL0315067, AL0315069, AL0315070, and AL0315072.

[0287] Table 5. Knockdown levels of sFLT1-i13 by sFLT-1 siRNA sequence in WM115 cells

[0288] Table 6 shows the knockdown level of sFLT1-e15a by sFLT1 siRNA sequence in WM115 cells.

[0289] Example 3. In vivo testing of sFLT-1 RNAi agent in CD1 pregnant mice

[0290] SPF-grade male CD1 pregnant mice, aged 8–9 weeks (purchased from SPAF (Beijing) Biotechnology Co., Ltd.), were used in the experiment. Mice were randomly assigned to groups according to body weight on day 0 of drug administration. Mice were administered a single subcutaneous dose of 20 mg / kg of the RNAi agents AL0317000, AL0317000-1, AL0317001, and AL0317002. A control group (NC) was set up as the solvent group, receiving the same volume of 0.9% physiological saline. One week after drug administration, placentas from the pregnant mice were collected, with the physiological saline group serving as the control, to detect the sFLT1 mRNA knockdown level. During the experiment, no animals showed signs of death or near-death. No significant abnormalities were observed in any animal during clinical observation. The changes in sFLT1 levels are shown in Figure 3.

[0291] As shown in Figure 3, compared with the control group (NC), all siRNA drug groups can effectively knock down sFLT1 protein in mice. At a dose of 20 mg / kg, all groups can reduce the mRNA of the target protein by at least 50% or more.

Claims

1. An oligonucleotide or a pharmaceutically acceptable salt thereof for inhibiting sFLT-1 expression, said oligonucleotide comprising a sense strand and an antisense strand, said sense strand having at least 80% sequence identity with a sequence or fragment thereof shown in any of SEQ ID NO. 3-27 and 29-75, or a modified sequence thereof, preferably having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity; said antisense strand having at least 80% sequence identity with a sequence or fragment thereof shown in any of SEQ ID NO. 78-102 and 104-150, or a modified sequence thereof, preferably having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity.

2. The oligonucleotide or a pharmaceutically acceptable salt thereof according to claim 1, wherein, The oligonucleotide or its pharmaceutically acceptable salt 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 its sodium or potassium salt; 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.

3. The oligonucleotide or a pharmaceutically acceptable salt thereof according to claim 1 or 2, wherein, The oligonucleotide contains at least one modified nucleotide; Preferably, at least one of the modified nucleotides is selected from the group consisting of: deoxynucleotides, 3'-terminal deoxythymidine (dT) nucleotides, 2'-O-methyl modified nucleotides, 2'-fluorine modified nucleotides, 2'-deoxy modified nucleotides, locked nucleotides, 2'-5'-linked ribonucleotides (3'-RNA), unlocked nucleotides, conformation-restricted nucleotides, restricted ethyl nucleotides, base-free nucleotides, 2'-amino modified nucleotides, 2'-O-allyl modified nucleotides, 2'-O-alkyl modified nucleotides, 2'-hydroxy modified nucleotides, 2'-methoxy... Nucleotides modified with ethyl groups, nucleotides modified with 2'-O-alkyl groups, morpholinonucleotides, aminophosphates, nucleotides including non-natural bases, tetrahydropyran-modified nucleotides, nucleotides modified with 1,5-dehydrohexyl alcohol, nucleotides modified with cyclohexenyl groups, nucleotides including thiophosphate groups, nucleotides including methylphosphonate groups, nucleotides including 5'-phosphate groups, nucleotides including 5'-phosphate mimics, vinyl-phosphonate nucleotides, heat-labile nucleotides, ethylene glycol-modified nucleotides, nucleotides including 2'-phosphate groups, and nucleotides modified with 2-O-(N-methylacetamide); and combinations thereof; Preferably, the oligonucleotide comprises at least one 2'-modified nucleotide; Preferably, the 2'-modified nucleotide is selected from one or more of the following: 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. Preferably, the 2'-modification is selected from the following modifications: 2'-methoxy, 2'-acetamido, 2'-aminoethyl, 2'-fluorine, 2'-O-methoxyethyl; Preferably, the oligonucleotide includes a modification at the 5' end, the modification comprising 5'-(E)-vinylphosphonate (5'-VP) or 6-(3-(2-carboxyethyl)phenyl)purine (6-mCEPh-purine); Preferably, the nucleotide with 5'-(E)-vinylphosphonate modification at the 5' end has the structure shown in formula (I); wherein, Base represents a nucleic acid base selected from A, G, C and U; R is selected from H, fluorine, 2'-methoxy, 2'-acetamido, 2'-aminoethyl and 2'-O-methoxyethyl; Preferably, the oligonucleotide comprises a 5'-(E)-vinylphosphonate-modified nucleotide at its 5' end, having the structure shown in formula (II-1) or (II-2); wherein R is selected from H, fluorine, 2'-methoxy, 2'-acetamido, 2'-aminoethyl, and 2'-O-methoxyethyl; preferably, the oligonucleotide has an APU at its 5' end, which is uridine monophosphate (2'-acetamido-5'-vinylphosphonate-uridine monophosphate) modified with a 5'-phosphate analog as shown in formula (III); preferably, the oligonucleotide has a VPUm at its 5' end, which is uridine monophosphate (2'-methoxy-5'-vinylphosphonate-uridine monophosphate) modified with a 5'-phosphate analog as shown in formula (IV-1); preferably, the oligonucleotide has a VPAm at its 5' end, which is uridine monophosphate (2'-methoxy-5'-vinylphosphonate-adenosine monophosphate) modified with a 5'-phosphate analog as shown in formula (IV-2); Preferably, the oligonucleotide comprises a 6-(3-(2-carboxyethyl)phenyl)purine-modified nucleotide at its 5' end; preferably, the oligonucleotide comprises formula M, which is a 2'-O-methyl-6-(3-(2-carboxyethyl)phenyl)purine nucleotide as shown in formula (V); 4. The oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of claims 1-3, wherein, The oligonucleotide contains at least one modified internucleotide bond; Preferably, the at least one modified nucleotide inter-bond is a phosphate thioester bond.

5. The oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of claims 1-4, wherein, The sense strand is selected from any of the unmodified oligonucleotides described in SEQ ID NO. 3, 6, 7, 10, 11, 12, 13, 19, 20, 21, 34, 35, 38, 39, 41, 50, 51, 53, 55, 57, 58, 59, 60, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, and 75, or any of the modified oligonucleotides described in SEQ ID NO. 153-177 and 179-225; the antisense strand is selected from SEQ ID NO. The unmodified oligonucleotides described in any of NO. 78, 81, 82, 85, 86, 87, 88, 94, 95, 96, 109, 110, 113, 114, 116, 125, 126, 128, 130, 132, 133, 134, 135, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, and 150, or the modified oligonucleotides described in any of SEQ ID NO. 228-252 and 254-300.

6. The oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of claims 1-5, wherein, The oligonucleotide comprises any of the following combinations of sense and antisense strands: (1) The positive chain contains the sequence shown in SEQ ID NO.3, and the negative chain contains the sequence shown in SEQ ID NO.78; (2) The sense chain comprises the sequence shown in SEQ ID NO.6, and the antisense chain comprises the sequence shown in SEQ ID NO.81; (3) The sense chain comprises the sequence shown in SEQ ID NO.7, and the antisense chain comprises the sequence shown in SEQ ID NO.82; (4) The sense chain comprises the sequence shown in SEQ ID NO.10, and the antisense chain comprises the sequence shown in SEQ ID NO.85; (5) The positive chain contains the sequence shown in SEQ ID NO.11, and the negative chain contains the sequence shown in SEQ ID NO.86; (6) The sense chain comprises the sequence shown in SEQ ID NO.12, and the antisense chain comprises the sequence shown in SEQ ID NO.87; (7) The positive chain comprises the sequence shown in SEQ ID NO.13, and the negative chain comprises the sequence shown in SEQ ID NO.88; (8) The positive chain contains the sequence shown in SEQ ID NO.19, and the negative chain contains the sequence shown in SEQ ID NO.94; (9) The sense chain comprises the sequence shown in SEQ ID NO.20, and the antisense chain comprises the sequence shown in SEQ ID NO.95; (10) The sense chain comprises the sequence shown in SEQ ID NO.21, and the antisense chain comprises the sequence shown in SEQ ID NO.96; (11) The sense chain comprises the sequence shown in SEQ ID NO.34, and the antisense chain comprises the sequence shown in SEQ ID NO.109; (12) The sense chain comprises the sequence shown in SEQ ID NO.35, and the antisense chain comprises the sequence shown in SEQ ID NO.110; (13) The positive chain comprises the sequence shown in SEQ ID NO.38, and the negative chain comprises the sequence shown in SEQ ID NO.113; (14) The positive chain contains the sequence shown in SEQ ID NO.39, and the negative chain contains the sequence shown in SEQ ID NO.114; (15) The positive chain comprises the sequence shown in SEQ ID NO.41, and the negative chain comprises the sequence shown in SEQ ID NO.116; (16) The sense chain comprises the sequence shown in SEQ ID NO.50, and the antisense chain comprises the sequence shown in SEQ ID NO.125; (17) The sense chain comprises the sequence shown in SEQ ID NO.51, and the antisense chain comprises the sequence shown in SEQ ID NO.126; (18) The sense chain comprises the sequence shown in SEQ ID NO.53, and the antisense chain comprises the sequence shown in SEQ ID NO.128; (19) The sense chain comprises the sequence shown in SEQ ID NO.55, and the antisense chain comprises the sequence shown in SEQ ID NO.130; (20) The sense chain comprises the sequence shown in SEQ ID NO.57, and the antisense chain comprises the sequence shown in SEQ ID NO.132; (21) The sense chain comprises the sequence shown in SEQ ID NO.58, and the antisense chain comprises the sequence shown in SEQ ID NO.133; (22) The sense chain comprises the sequence shown in SEQ ID NO.59, and the antisense chain comprises the sequence shown in SEQ ID NO.134; (23) The sense chain comprises the sequence shown in SEQ ID NO.60, and the antisense chain comprises the sequence shown in SEQ ID NO.135; (24) The sense chain comprises the sequence shown in SEQ ID NO.62, and the antisense chain comprises the sequence shown in SEQ ID NO.137; (25) The sense chain comprises the sequence shown in SEQ ID NO.63, and the antisense chain comprises the sequence shown in SEQ ID NO.138; (26) The sense chain comprises the sequence shown in SEQ ID NO.64, and the antisense chain comprises the sequence shown in SEQ ID NO.139; (27) The sense chain comprises the sequence shown in SEQ ID NO. 65, and the antisense chain comprises the sequence shown in SEQ ID NO. 140; (28) The sense chain comprises the sequence shown in SEQ ID NO.66, and the antisense chain comprises the sequence shown in SEQ ID NO.141; (29) The sense chain comprises the sequence shown in SEQ ID NO.67, and the antisense chain comprises the sequence shown in SEQ ID NO.142; (30) The sense chain comprises the sequence shown in SEQ ID NO.68, and the antisense chain comprises the sequence shown in SEQ ID NO.143; (31) The sense chain comprises the sequence shown in SEQ ID NO.69, and the antisense chain comprises the sequence shown in SEQ ID NO.144; (32) The sense chain comprises the sequence shown in SEQ ID NO.70, and the antisense chain comprises the sequence shown in SEQ ID NO.145; (33) The positive chain comprises the sequence shown in SEQ ID NO.71, and the negative chain comprises the sequence shown in SEQ ID NO.146; (34) The sense chain comprises the sequence shown in SEQ ID NO.72, and the antisense chain comprises the sequence shown in SEQ ID NO.147; (35) The sense chain comprises the sequence shown in SEQ ID NO.73, and the antisense chain comprises the sequence shown in SEQ ID NO.148; (36) The sense chain comprises the sequence shown in SEQ ID NO. 74, and the antisense chain comprises the sequence shown in SEQ ID NO. 149; and (37) The sense chain comprises the sequence shown in SEQ ID NO.75, and the antisense chain comprises the sequence shown in SEQ ID NO.150; Preferably, the oligonucleotide comprises any combination of the following sense and antisense strands: (1) The positive chain contains the sequence shown in SEQ ID NO.3, and the negative chain contains the sequence shown in SEQ ID NO.78; (2) The sense chain comprises the sequence shown in SEQ ID NO.6, and the antisense chain comprises the sequence shown in SEQ ID NO.81; (3) The sense chain comprises the sequence shown in SEQ ID NO.10, and the antisense chain comprises the sequence shown in SEQ ID NO.85; (4) The sense chain comprises the sequence shown in SEQ ID NO.12, and the antisense chain comprises the sequence shown in SEQ ID NO.87; (5) The sense chain comprises the sequence shown in SEQ ID NO.19, and the antisense chain comprises the sequence shown in SEQ ID NO.94; (6) The sense chain comprises the sequence shown in SEQ ID NO.21, and the antisense chain comprises the sequence shown in SEQ ID NO.96; (7) The sense chain comprises the sequence shown in SEQ ID NO.34, and the antisense chain comprises the sequence shown in SEQ ID NO.109; (8) The sense chain comprises the sequence shown in SEQ ID NO.35, and the antisense chain comprises the sequence shown in SEQ ID NO.110; (9) The sense chain comprises the sequence shown in SEQ ID NO.51, and the antisense chain comprises the sequence shown in SEQ ID NO.126; (10) The sense chain comprises the sequence shown in SEQ ID NO.58, and the antisense chain comprises the sequence shown in SEQ ID NO.133; (11) The positive chain comprises the sequence shown in SEQ ID NO.59, and the negative chain comprises the sequence shown in SEQ ID NO.134; (12) The sense chain comprises the sequence shown in SEQ ID NO.62, and the antisense chain comprises the sequence shown in SEQ ID NO.137; (13) The positive chain comprises the sequence shown in SEQ ID NO.63, and the negative chain comprises the sequence shown in SEQ ID NO.138; (14) The sense chain comprises the sequence shown in SEQ ID NO.67, and the antisense chain comprises the sequence shown in SEQ ID NO.142; (15) The positive chain comprises the sequence shown in SEQ ID NO.69, and the negative chain comprises the sequence shown in SEQ ID NO.144; (16) The sense chain comprises the sequence shown in SEQ ID NO.71, and the antisense chain comprises the sequence shown in SEQ ID NO.146; (17) The sense chain comprises the sequence shown in SEQ ID NO. 72, and the antisense chain comprises the sequence shown in SEQ ID NO. 147; and (18) The sense chain comprises the sequence shown in SEQ ID NO.74, and the antisense chain comprises the sequence shown in SEQ ID NO.149; Each of these chains is independently 19 to 25 nucleotides in length.

7. The oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of claims 1-6, wherein, The oligonucleotide comprises any of the following combinations of sense and antisense strands: (1) The positive chain comprises the sequence shown in SEQ ID NO.153, and the negative chain comprises the sequence shown in SEQ ID NO.228; (2) The sense chain comprises the sequence shown in SEQ ID NO.156, and the antisense chain comprises the sequence shown in SEQ ID NO.231; (3) The sense chain comprises the sequence shown in SEQ ID NO.157, and the antisense chain comprises the sequence shown in SEQ ID NO.232; (4) The sense chain comprises the sequence shown in SEQ ID NO.160, and the antisense chain comprises the sequence shown in SEQ ID NO.235; (5) The sense chain comprises the sequence shown in SEQ ID NO.161, and the antisense chain comprises the sequence shown in SEQ ID NO.236; (6) The sense chain comprises the sequence shown in SEQ ID NO.162, and the antisense chain comprises the sequence shown in SEQ ID NO.237; (7) The sense chain comprises the sequence shown in SEQ ID NO.163, and the antisense chain comprises the sequence shown in SEQ ID NO.238; (8) The sense chain comprises the sequence shown in SEQ ID NO.169, and the antisense chain comprises the sequence shown in SEQ ID NO.244; (9) The sense chain comprises the sequence shown in SEQ ID NO.170, and the antisense chain comprises the sequence shown in SEQ ID NO.245; (10) The sense chain comprises the sequence shown in SEQ ID NO.171, and the antisense chain comprises the sequence shown in SEQ ID NO.246; (11) The sense chain comprises the sequence shown in SEQ ID NO.184, and the antisense chain comprises the sequence shown in SEQ ID NO.259; (12) The sense chain comprises the sequence shown in SEQ ID NO.185, and the antisense chain comprises the sequence shown in SEQ ID NO.260; (13) The sense chain comprises the sequence shown in SEQ ID NO.188, and the antisense chain comprises the sequence shown in SEQ ID NO.263; (14) The sense chain comprises the sequence shown in SEQ ID NO.189, and the antisense chain comprises the sequence shown in SEQ ID NO.264; (15) The sense chain comprises the sequence shown in SEQ ID NO.191, and the antisense chain comprises the sequence shown in SEQ ID NO.266; (16) The sense chain comprises the sequence shown in SEQ ID NO.200, and the antisense chain comprises the sequence shown in SEQ ID NO.275; (17) The sense chain comprises the sequence shown in SEQ ID NO.201, and the antisense chain comprises the sequence shown in SEQ ID NO.276; (18) The sense chain comprises the sequence shown in SEQ ID NO.203, and the antisense chain comprises the sequence shown in SEQ ID NO.278; (19) The sense chain comprises the sequence shown in SEQ ID NO.205, and the antisense chain comprises the sequence shown in SEQ ID NO.280; (20) The sense chain comprises the sequence shown in SEQ ID NO.207, and the antisense chain comprises the sequence shown in SEQ ID NO.282; (21) The sense chain comprises the sequence shown in SEQ ID NO.208, and the antisense chain comprises the sequence shown in SEQ ID NO.283; (22) The sense chain comprises the sequence shown in SEQ ID NO.209, and the antisense chain comprises the sequence shown in SEQ ID NO.284; (23) The sense chain comprises the sequence shown in SEQ ID NO.210, and the antisense chain comprises the sequence shown in SEQ ID NO.285; (24) The sense chain comprises the sequence shown in SEQ ID NO.212, and the antisense chain comprises the sequence shown in SEQ ID NO.287; (25) The sense chain comprises the sequence shown in SEQ ID NO.213, and the antisense chain comprises the sequence shown in SEQ ID NO.288; (26) The sense chain comprises the sequence shown in SEQ ID NO.214, and the antisense chain comprises the sequence shown in SEQ ID NO.289; (27) The sense chain comprises the sequence shown in SEQ ID NO.215, and the antisense chain comprises the sequence shown in SEQ ID NO.290; (28) The sense chain comprises the sequence shown in SEQ ID NO.216, and the antisense chain comprises the sequence shown in SEQ ID NO.291; (29) The sense chain comprises the sequence shown in SEQ ID NO.217, and the antisense chain comprises the sequence shown in SEQ ID NO.292; (30) The sense chain comprises the sequence shown in SEQ ID NO.218, and the antisense chain comprises the sequence shown in SEQ ID NO.293; (31) The sense chain comprises the sequence shown in SEQ ID NO.219, and the antisense chain comprises the sequence shown in SEQ ID NO.294; (32) The sense chain comprises the sequence shown in SEQ ID NO.220, and the antisense chain comprises the sequence shown in SEQ ID NO.295; (33) The sense chain comprises the sequence shown in SEQ ID NO.221, and the antisense chain comprises the sequence shown in SEQ ID NO.296; (34) The sense chain comprises the sequence shown in SEQ ID NO.222, and the antisense chain comprises the sequence shown in SEQ ID NO.297; (35) The sense chain comprises the sequence shown in SEQ ID NO.223, and the antisense chain comprises the sequence shown in SEQ ID NO.298; (36) The sense chain comprises the sequence shown in SEQ ID NO. 224, and the antisense chain comprises the sequence shown in SEQ ID NO. 299; and (37) The sense chain comprises the sequence shown in SEQ ID NO.225, and the antisense chain comprises the sequence shown in SEQ ID NO.300; Preferably, the oligonucleotide comprises any combination of the following sense and antisense strands: (1) The positive chain comprises the sequence shown in SEQ ID NO.153, and the negative chain comprises the sequence shown in SEQ ID NO.228; (2) The sense chain comprises the sequence shown in SEQ ID NO.156, and the antisense chain comprises the sequence shown in SEQ ID NO.231; (3) The sense chain comprises the sequence shown in SEQ ID NO.160, and the antisense chain comprises the sequence shown in SEQ ID NO.235; (4) The sense chain comprises the sequence shown in SEQ ID NO.162, and the antisense chain comprises the sequence shown in SEQ ID NO.237; (5) The sense chain comprises the sequence shown in SEQ ID NO.169, and the antisense chain comprises the sequence shown in SEQ ID NO.244; (6) The sense chain comprises the sequence shown in SEQ ID NO.171, and the antisense chain comprises the sequence shown in SEQ ID NO.246; (7) The sense chain comprises the sequence shown in SEQ ID NO.184, and the antisense chain comprises the sequence shown in SEQ ID NO.259; (8) The sense chain comprises the sequence shown in SEQ ID NO.185, and the antisense chain comprises the sequence shown in SEQ ID NO.260; (9) The sense chain comprises the sequence shown in SEQ ID NO.201, and the antisense chain comprises the sequence shown in SEQ ID NO.276; (10) The sense chain comprises the sequence shown in SEQ ID NO.208, and the antisense chain comprises the sequence shown in SEQ ID NO.283; (11) The sense chain comprises the sequence shown in SEQ ID NO.209, and the antisense chain comprises the sequence shown in SEQ ID NO.284; (12) The sense chain comprises the sequence shown in SEQ ID NO.212, and the antisense chain comprises the sequence shown in SEQ ID NO.287; (13) The sense chain comprises the sequence shown in SEQ ID NO.213, and the antisense chain comprises the sequence shown in SEQ ID NO.288; (14) The sense chain comprises the sequence shown in SEQ ID NO.217, and the antisense chain comprises the sequence shown in SEQ ID NO.292; (15) The sense chain comprises the sequence shown in SEQ ID NO.219, and the antisense chain comprises the sequence shown in SEQ ID NO.294; (16) The sense chain comprises the sequence shown in SEQ ID NO.221, and the antisense chain comprises the sequence shown in SEQ ID NO.296; (17) The sense chain comprises the sequence shown in SEQ ID NO. 222, and the antisense chain comprises the sequence shown in SEQ ID NO. 297; and (18) The sense chain comprises the sequence shown in SEQ ID NO.224, and the antisense chain comprises the sequence shown in SEQ ID NO.299; Each of these chains is independently 19 to 25 nucleotides in length.

8. Conjugates or pharmaceutically acceptable salts thereof for inhibiting sFLT-1 expression, comprising: (i) the oligonucleotide or a pharmaceutically acceptable salt thereof as described in any one of claims 1-7, 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 ligand; Preferably, the ligand comprises carbohydrates, amino sugars, cholesterol, polypeptides, or lipids; Preferably, the ligand is a lipid; Preferably, the lipids are C16, C18, C22, and PC-DCA; Preferably, the ligand is 3'-C7-phosphocholine-docosahexaenoic acid (PC-DCA); Preferably, the ligand PC-DCA is located at the 3' end of the positive chain; The structure of 3'-C7-phosphocholine-docosahexaenoic acid (PC-DCA) is as follows:

9. A composition comprising the oligonucleotide of any one of claims 1-7 or a pharmaceutically acceptable salt thereof, or the conjugate of claim 8 or a pharmaceutically acceptable salt thereof, and optionally a pharmaceutically acceptable carrier; Preferably, the dosage form of the composition is an oral preparation, an intravenous injection, a subcutaneous injection, an intramuscular injection, or an intrathecal injection, preferably a subcutaneous injection.

10. The oligonucleotide of any one of claims 1-7 or a pharmaceutically acceptable salt thereof, or the conjugate of claim 8 or a pharmaceutically acceptable salt thereof, or the composition of claim 9, in the preparation of a treatment for and / or prevention of sFLT-1 related conditions; Preferably, the sFLT-1 related conditions are selected from preeclampsia and clinical symptoms such as hypertension and proteinuria caused by preeclampsia; Preferably, the medicine for treating and / or preventing sFLT-1-related conditions is also prepared in combination with other medicines for treating and / or preventing sFLT-1-related conditions.

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