Nucleic acid molecule for inhibiting ALK7 expression and use thereof

Abstract

Provided are a nucleic acid molecule for inhibiting ALK7 gene expression by means of RNAi, and a derivative product of the nucleic acid molecule. Also provided is use of the nucleic acid molecule and the derivative product in inhibiting the expression of ALK7 in a cell or in a subject, and preventing or treating ALK7-mediated diseases or symptoms.

Description

A nucleic acid molecule that inhibits ALK7 expression and its application

[0001] Citation of relevant applications

[0002] This application claims priority to the following applications:

[0003] Chinese Patent Application No. 202510178887.1, filed on February 18, 2025;

[0004] Chinese Patent Application No. 202510837614.3, filed on June 20, 2025; and

[0005] Chinese Patent Application No. 202511855507.X, filed on December 10, 2025;

[0006] The entire contents of the aforementioned patent application are incorporated herein by reference and used for all purposes. Technical Field

[0007] This application relates to the field of molecular biology, particularly RNA interference (RNAi), and specifically to a nucleic acid molecule that can inhibit the expression of the activin receptor-like kinase 7 (ALK7) gene via RNAi and its uses. Background Technology

[0008] RNA interference (RNAi) refers to the highly conserved phenomenon of efficient and specific degradation of homologous mRNA induced by double-stranded RNA (dsRNA) during evolution. dsRNA, typically 19-30 bp in length, is one of the important tools in RNAi technology. In natural organisms, longer dsRNAs, after entering cells, are specifically recognized and cleaved by the Dicer enzyme into small RNA fragments (siRNAs) of approximately 21-23 nucleotides in length. These cleaved dsRNA fragments unwind into single strands and form complexes (RISCs) with certain proteins. RISCs bind to mRNAs complementary to the antisense strand of the dsRNA within the cell and cleave them, causing their degradation and preventing protein synthesis, resulting in gene silencing. In industrial production, the chemical synthesis and modification of dsRNA are preferred to further improve the stability and efficacy of dsRNA drugs.

[0009] Activin receptor-like kinase 7 (ALK7), also known as ACVR1C, is a member of the type I transforming growth factor β (TGF-β) receptor family and plays a crucial role in the regulation of metabolic homeostasis. ALK7 is a single-transmembrane protein, and its expression level varies in different tissues, with the most significant expression in white adipose tissue (WAT) and brown adipose tissue (BAT).

[0010] ALK7-mediated signal transduction primarily relies on the Smad protein family. When ALK7 binds to its specific ligands, it activates Smad2, Smad3, and Smad4, thereby regulating the expression of downstream genes. Smad3 and Smad4 interact with members of the CCAAT / enhancer-binding protein (C / EBP) family and inhibit their transcriptional activation function. C / EBP proteins are key regulators of adipogenesis and adipocyte differentiation, primarily affecting lipid metabolism by regulating the expression of peroxisome proliferator-activated receptor γ (PPARγ) and lipolysis-related enzymes (such as ATGL and HSL). When the transcriptional activity of C / EBP is inhibited, lipolysis decreases, leading to lipid accumulation.

[0011] Previous studies have shown that antibody treatment targeting ALK7 can significantly reduce body weight and improve metabolic disorders such as glucose intolerance and insulin resistance in genetically modified obese mice (TSOD) and high-fat diet-induced obese mouse models.

[0012] Based on the above research results, RNA interference (RNAi) strategies targeting ALK7, such as ALK7 siRNA, are expected to become an effective means of treating obesity and diabetes.

[0013] Invention Overview

[0014] In a first aspect, this application provides a double-stranded RNA (dsRNA) molecule that inhibits the expression of the activin receptor-like kinase 7 (ALK7) gene via RNAi, comprising a sense strand and an antisense strand that are complementary to each other to form a double-stranded region, or composed of a sense strand and an antisense strand that are complementary to each other to form a double-stranded region, wherein the sense strand is 15-30 nt in length, and the antisense strand is 15-30 nt in length, wherein:

[0015] (1) The sense strand sequence of the dsRNA molecule comprises at least 15 consecutive bases of the sequence shown in SEQ ID NO. 31, and the antisense strand sequence of the dsRNA molecule comprises at least 15 consecutive bases of the sequence shown in SEQ ID NO. 32; or

[0016] (2) The sense strand sequence of the dsRNA molecule comprises at least 15 consecutive bases of the sequence shown in SEQ ID NO.1, and the antisense strand sequence of the dsRNA molecule comprises at least 15 consecutive bases of the sequence shown in SEQ ID NO.2; or

[0017] (3) The sense strand sequence of the dsRNA molecule comprises at least 15 consecutive bases of the sequence shown in SEQ ID NO.3, and the antisense strand sequence of the dsRNA molecule comprises at least 15 consecutive bases of the sequence shown in SEQ ID NO.4; or

[0018] (4) The sense strand sequence of the dsRNA molecule comprises at least 15 consecutive bases of the sequence shown in SEQ ID NO. 5, and the antisense strand sequence of the dsRNA molecule comprises at least 15 consecutive bases of the sequence shown in SEQ ID NO. 6; or

[0019] (5) The sense strand sequence of the dsRNA molecule comprises at least 15 consecutive bases of the sequence shown in SEQ ID NO. 7, and the antisense strand sequence of the dsRNA molecule comprises at least 15 consecutive bases of the sequence shown in SEQ ID NO. 8; or

[0020] (6) The sense strand sequence of the dsRNA molecule comprises at least 15 consecutive bases of the sequence shown in SEQ ID NO. 9, and the antisense strand sequence of the dsRNA molecule comprises at least 15 consecutive bases of the sequence shown in SEQ ID NO. 10; or

[0021] (7) The sense strand sequence of the dsRNA molecule comprises at least 15 consecutive bases of the sequence shown in SEQ ID NO. 15, and the antisense strand sequence of the dsRNA molecule comprises at least 15 consecutive bases of the sequence shown in SEQ ID NO. 16; or

[0022] (8) The sense strand sequence of the dsRNA molecule contains at least 15 consecutive bases of the sequence shown in SEQ ID NO.17, and the antisense strand sequence of the dsRNA molecule contains at least 15 consecutive bases of the sequence shown in SEQ ID NO.18.

[0023] Secondly, this application provides engineered nucleic acid molecules comprising the dsRNA molecules described in the first aspect.

[0024] Thirdly, this application provides engineered nucleic acid molecules that can be transcribed or spliced ​​in cells to form the dsRNA molecules described in the first aspect.

[0025] Fourthly, this application provides a delivery system comprising the dsRNA molecule described in the first aspect, the engineered nucleic acid molecule described in the second aspect, or the engineered nucleic acid molecule described in the third aspect.

[0026] Fifthly, this application provides cells that contain the engineered nucleic acid molecules described in the third aspect.

[0027] Sixthly, this application provides tautomers, stereoisomers, solvates, isotope derivatives, or pharmaceutically acceptable salts of the dsRNA molecule described in the first aspect, the engineered nucleic acid molecule described in the second aspect, or the engineered nucleic acid molecule described in the third aspect.

[0028] In a seventh aspect, this application provides a pharmaceutical composition comprising the dsRNA molecule described in the first aspect, the engineered nucleic acid molecule described in the second aspect, the engineered nucleic acid molecule described in the third aspect, the delivery system described in the fourth aspect, the cell described in the fifth aspect, or the tautomer, stereoisomer, solvate, isotope derivative, or pharmaceutically acceptable salt described in the sixth aspect, and a pharmaceutically acceptable carrier or diluent.

[0029] Eighthly, this application provides the use of the dsRNA molecule described in the first aspect, the engineered nucleic acid molecule described in the second aspect, the engineered nucleic acid molecule described in the third aspect, or the tautomer, stereoisomer, solvate, isotope derivative, or pharmaceutically acceptable salt described in the sixth aspect for inhibiting the expression of ALK7 in cells or in a subject.

[0030] Ninthly, this application provides the use of the dsRNA molecule described in the first aspect, the engineered nucleic acid molecule described in the second aspect, the engineered nucleic acid molecule described in the third aspect, or the tautomer, stereoisomer, solvate, isotope derivative, or pharmaceutically acceptable salt described in the sixth aspect for the preparation of a drug that inhibits ALK7 expression in a subject.

[0031] Tenthly, this application provides the use of the dsRNA molecule described in the first aspect, the engineered nucleic acid molecule described in the second aspect, the engineered nucleic acid molecule described in the third aspect, the delivery body described in the fourth aspect, the cell described in the fifth aspect, the tautomer, stereoisomer, solvate, isotope derivative or pharmaceutically acceptable salt described in the sixth aspect, or the pharmaceutical composition described in the seventh aspect for the preparation of a medicament for the prevention or treatment of ALK7-mediated diseases or symptoms.

[0032] In its eleventh aspect, this application provides methods for treating or preventing ALK7-mediated diseases or symptoms in subjects, including:

[0033] The administration to a subject in need of treatment or prevention of an effective amount of the dsRNA molecule of the first aspect, the engineered nucleic acid molecule of the second aspect, the engineered nucleic acid molecule of the third aspect, the delivery body of the fourth aspect, the cell of the fifth aspect, the tautomer, stereoisomer, solvate, isotope derivative or pharmaceutically acceptable salt of the sixth aspect, or the pharmaceutical composition of the seventh aspect.

[0034] In a twelfth aspect, this application provides the use of the dsRNA molecule described in the first aspect, the engineered nucleic acid molecule described in the second aspect, the engineered nucleic acid molecule described in the third aspect, the delivery body described in the fourth aspect, the cell described in the fifth aspect, the tautomer, stereoisomer, solvate, isotope derivative or pharmaceutically acceptable salt described in the sixth aspect, or the pharmaceutical composition described in the seventh aspect, for weight loss in a subject.

[0035] Brief description of the attached diagram

[0036] Figure 1 shows the change in body weight of cynomolgus monkeys over time after administration of Z52-lipophilic fraction 5 and AC006189.

[0037] Figure 2 shows the changes in abdominal fat volume of cynomolgus monkeys at different detection points after administration of Z52-lipophilic fraction 5 and AC006189.

[0038] Figure 3 shows the changes in visceral fat volume of cynomolgus monkeys at different detection points after administration of Z52-lipophilic fraction 5 and AC006189.

[0039] Figure 4 shows the change in serum NEFA content in cynomolgus monkeys over time after administration of Z52-lipophilic fraction 5 and AC006189.

[0040] Figure 5 shows the change in body weight of cynomolgus monkeys over time after administration of D152-lipophilic fraction 5-PEO.

[0041] Figure 6 shows the changes in abdominal and visceral fat volume at different detection points in cynomolgus monkeys after administration of the D152-lipophilic fraction 5-PEO.

[0042] Figure 7 shows the changes in ALK7 gene mRNA expression levels in abdominal fat of cynomolgus monkeys at different detection sites after administration of D152-lipophilic fraction 5-PEO.

[0043] Invention Details

[0044] the term

[0045] For the purposes of this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural forms, and vice versa. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the described technology pertains. All technical and patent disclosures referenced herein are incorporated herein by reference in their entirety.

[0046] Unless otherwise stated, the practice of this application may employ known techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, including techniques described in the prior art literature.

[0047] As used herein, the term "lipophilic moiety" or "lipophilic portion" broadly refers to any compound or chemical part that has an affinity for lipids. One way to characterize the lipophilicity of a lipophilic moiety is through the octanol-water partition coefficient LogK. ow K ow The octanol-water partition coefficient is the ratio of the concentration of a chemical substance in the octanol phase to its concentration in the aqueous phase at equilibrium in a two-phase system. It is a laboratory-measured property of the substance. However, it can also be predicted using coefficients attributed to the structural components of the chemical substance, calculated using first-principles or empirical methods (see, for example, Tetko et al., J. Chem. Inf. Comput. Sci. [Journal of Chemical Information and Computer Science] 41:1407-21 (2001), which is incorporated herein by reference in its entirety). It provides a thermodynamic measure of a substance's tendency to prefer non-aqueous or oily environments over water (i.e., its hydrophilic / lipophilic balance). In principle, when LogK... ow When the value exceeds 0, the chemical substance exhibits lipophilicity. Typically, the logK of the lipophilic portion... ow Exceeding 1, exceeding 1.5, exceeding 2, exceeding 3, exceeding 4, exceeding 5, or exceeding 10. For example, predicting the LogK of, for instance, 6-aminohexanol. ow The value is approximately 0.7. Using the same method, the LogK of cholesterol-based N-(hexyl-6-ol)carbamate was predicted. ow It is 10.7.

[0048] Exemplary lipophilic portions have any of the lipophilic portions listed in CN202510837614.3, the entire text of which is incorporated herein by reference, such as those listed below:

[0049] In this application, the term "5'-cPrp" refers to 5'-cyclopropyl phosphate, whose chemical structure after being linked with a nucleotide is shown below:

[0050] Wherein: Bx1 is a natural or modified base; T2 is a phosphate ester bond or thiophosphate bond that links the above compound to the oligonucleotide chain; and G is a substituent at the 2' position of the corresponding nucleotide, including but not limited to F, hydroxyl, H, methoxy, methoxyethoxy, etc.

[0051] As used herein, "dsRNA" refers to double-stranded RNA. Since siRNA is a double-stranded RNA, the term "dsRNA" encompasses siRNA. dsRNA also includes double-stranded RNAs longer than siRNA, where "longer than siRNA" can mean that its sense strand is longer than siRNA, or its antisense strand is longer than siRNA, or both its sense and antisense strands are longer than siRNA. Typically, double-stranded RNAs longer than the siRNA sequence they contain are cleaved into siRNA by a type III endonuclease called Dicer after entering the cell. In some embodiments, the lengths of the two strands of the dsRNA are each independently 15 to 30 nt (in this application, "nt" refers to nucleotides). When "siRNA" is incorporated into the RNA-induced silencing complex (RISC), one or more helicases in the RISC unwind the siRNA double helix. When it binds to a target mRNA complementary to the antisense strand of the siRNA, one or more endonucleases in the RISC cleave the target, inducing gene silencing. Typically, the majority of nucleotides in each strand of a dsRNA molecule are ribonucleotides, but this does not preclude the inclusion of one or more non-ribonucleotides, such as deoxyribonucleotides and / or non-natural nucleotides, in any one or two strands. In some embodiments, the dsRNA molecule does not contain non-natural nucleotides. In some embodiments, each nucleotide in the dsRNA is a ribonucleotide. As used herein, the dsRNA may contain one or more chemically modified nucleotides or may not contain chemically modified nucleotides.

[0052] The terms "Activin A receptor type 1C", "Activin receptor-like kinase 7", "ALK7", and "ACVR1" are used interchangeably. ALK7 can be mammalian-derived ALK7. In some embodiments, ALK7 is primate-derived ALK7. In some embodiments, ALK7 is human-derived ALK7. In some embodiments, ALK7 is cynomolgus monkey-derived ALK7. As used herein, "ALK7 mRNA" refers to the mRNA encoding the ALK7 protein, which can be transcribed from ALK7 DNA, and can be mature mRNA or pre-mRNA, and therefore may or may not contain introns. Because the ALK7 gene may have a few nucleotide mutations in different individuals, unless otherwise specified, the ALK7 gene mRNA sequence of this application is intended to include all mRNA sequences transcribed from ALK7 gene mutants. The human ALK7 mRNA sequence can be found on NCBI (Gene... The ALK7 mRNA sequence of rhesus monkeys can be found in NCBI (Gene ID: 130399, NM_145259.3, NM_001111031.2, NM_001111032.2 or NM_001111033.2, SEQ ID NO: 333). The ALK7 mRNA sequence of cynomolgus monkeys can be found in NCBI (Gene ID: 697826, NM_001266690.1). The ALK7 mRNA sequence of cynomolgus monkeys can be found in NCBI (Gene ID: 102131774, XM_005573230.4). The ALK7 mRNA sequence of mice can be found in NCBI (Gene ID: 269275, NM_001033369.3). The ALK7 mRNA sequence of rats can be found in NCBI (Gene ID: 269275, NM_001033369.3). The sequence was found in SEQ ID NO: 245921 (NM_139090.2). Other instances of the ALK7 gene mRNA sequence are readily available using publicly available databases such as GenBANK. Unless otherwise specified, the ALK7 mRNA is referenced to SEQ ID NO: 333 (i.e., NM_001111031.2), meaning that the nucleotide positions in the ALK7 mRNA are numbered according to the nucleotide numbers in the 5' to 3' directions of the reference sequence SEQ ID NO: 333.

[0053] As used herein, the term "reference sequence" refers to a standard sequence used for homologous sequence alignment, which can be used to define the position of nucleotides in homologous polynucleotide or polynucleotide sequences. For example, "the position number of the base in the ALK7 mRNA sequence is the corresponding base number in the reference sequence SEQ ID NO: 333" means that after introducing vacancies or deleting nucleotides into the ALK7 mRNA sequence to make the ALK7 mRNA sequence and the reference sequence have as many identical bases as possible, the nucleotides in the reference sequence are numbered sequentially starting from the first nucleotide at the 5' end. The ALK7 mRNA sequence and the reference sequence define the position of the nucleotide by aligning corresponding nucleotides with the same number.

[0054] As used herein, the “complementarity” of nucleic acids refers to the ability of one nucleic acid to form hydrogen bonds with another nucleic acid through conventional Watson-Crick base pairing. Percentage complementarity indicates the percentage of nucleotides in the shorter nucleic acid molecule that can form hydrogen bonds (i.e., Watson-Crick base pairing) with the other nucleic acid molecule (e.g., approximately 50%, 60%, 70%, 80%, 90%, and 100% complementarity out of 10). “Complete complementarity” means that all consecutive residues in the nucleic acid sequence form hydrogen bonds with the same number of consecutive residues in the second nucleic acid sequence. As used herein, "substantially complementary" refers to a degree of complementarity of at least approximately 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% within a region of approximately 40, 50, 60, 70, 80, 100, 150, 200, 250, or more nucleotides, or to two nucleic acids hybridizing under stringent conditions. For a single base or nucleotide, according to the Watson-Crick base pairing rule, A paired with T or U, or C paired with G or I, is called complementary, paired, or matched, and vice versa; all other base pairings are called non-complementary.

[0055] As used herein, nucleic acid “hybridization” refers to a reaction in which one or more polynucleotides react to form a complex that is stabilized by hydrogen bonds between nucleotide residues. Hydrogen bonds can occur through Watson-Crick base pairing, Hoogstein binding, or any other sequence-specific mechanism. The complex may include two strands forming a double-stranded structure, three or more strands forming a multi-stranded complex, a single self-hybridizing strand, or a combination thereof.

[0056] In this application, when referring to two polynucleotide sequences or two nucleic acid strands "hybridizing with maximum complementarity," it means hybridization in which as many nucleotides as possible in the two strands pair with each other through hydrogen bonds. In "hybridization with maximum complementarity," one or more mismatches and one or more bulges in one or more of the two strands are permissible. However, in some embodiments, the two strands "hybridizing with maximum complementarity" may hybridize without mismatches or bulges. As used herein, "complementary region" refers to all hydrogen-bonded base pairs from the 5' end to the last hydrogen-bonded base pair after hybridization. The complementary regions formed by the two hybridized strands can be continuous or spaced apart. In this application, "complementary region 1" specifically refers to the complementary region formed by complementary base pairs in the antisense strand and the ALK7 gene mRNA sequence when the antisense strand hybridizes with the ALK7 gene mRNA at maximum complementarity. "Complementary region 2" refers to the complementary region formed by complementary base pairs in the antisense strand and the sense strand when the antisense strand hybridizes with the sense strand at maximum complementarity. Since both complementary regions 1 and 2 involve antisense strands, the antisense strand portions of complementary regions 1 and 2 typically have partially or completely identical nucleotide compositions. In this application, antisense and sense strands are relative to a third sequence complementary to one of these sequences. For example, in "nucleic acid molecules that inhibit ALK7 gene expression in cells via RNAi," the antisense and sense strands are relative to the third sequence, the ALK7 gene mRNA sequence. That is, the antisense strand refers to a sequence in the nucleic acid molecule that has a complementary region to the ALK7 gene mRNA sequence, and the sense strand refers to a sequence in the nucleic acid molecule that has at least 10 consecutive identical nucleotides to the ALK7 gene mRNA sequence. Furthermore, in this application, the 5' end of any complementary region refers to the position of the nucleotide or base pair closest to the 5' end of the sense strand or the third sequence within its complementary region, while the 5' end of the complementary region refers to one side or end relatively close to the 5' end of the sense strand or the third sequence. Similarly, the 3' end of any complementary region in this application refers to the position of the nucleotide or base pair closest to the 3' end of the sense strand or the third sequence within its complementary region, while the 3' end of the complementary region refers to one side or end relatively close to the 3' end of the sense strand or the third sequence. In this application, when describing nucleotide positions on the same sequence or nucleoside chain, "close," "near," or "far" refers to the number of nucleotides separating the two nucleotide positions.

[0057] As used herein, "protruding nucleotide" refers to a nucleotide located outside complementary region 2, relative to complementary region 2, in the sense and / or antisense strands after hybridization with maximum complementarity. In some embodiments, the protruding nucleotide is located only in the sense strand; in some embodiments, the protruding nucleotide is located only in the antisense strand; and in some embodiments, the protruding nucleotide is located in both the sense and antisense strands. In some embodiments, the protruding nucleotide is present only on the 5' end side of complementary region 2. In some embodiments, the protruding nucleotide is present only on the 5' end side of the sense strand complementary region 2. In some embodiments, the protruding nucleotide is present only on the 5' end side of the antisense strand complementary region 2. In some embodiments, the protruding nucleotide is present only on the 3' end side of complementary region 2. In some embodiments, the protruding nucleotide is present only on the 3' end side of the sense strand complementary region 2. In some embodiments, the protruding nucleotide is present only on the 3' end side of the antisense strand complementary region 2. In some embodiments, the number of the protruding nucleotides on the same side (e.g., the 5' end or the 3' end) of the sense strand or antisense strand does not exceed two (i.e., one or two). In this application, "5' end side" and "3' end side" are used to describe the relative positional relationship between two sequences, two nucleotides, or one nucleotide and a sequence within the same polynucleotide sequence; wherein, "5' end" refers to the end of the polynucleotide sequence containing a 5' free phosphate group or a 5' free hydroxyl group, and "3' end" refers to the end of the polynucleotide sequence containing a free 3'-hydroxyl group or a 3'-phosphate group, and the sequence or nucleotide on the 3' end side of a certain sequence in the nucleic acid chain is closer to the 3' end of the nucleic acid chain than the sequence in question. For example, "on the 5' end side of complementary region 2" also includes a certain sequence or one or more nucleotides, which means that the "certain sequence or one or more nucleotides" is closer to the 5' end of the polynucleotide sequence (e.g., the antisense strand or sense strand) in which they share a common location, relative to the sequence in "complementary region 2".

[0058] The term "nucleotide" hereincludes not only naturally occurring ribonucleotide or deoxyribonucleotide monomers, but also, in this context, its related structural variants, including derivatives and analogs, which are functionally equivalent in the specific context of the use of the nucleotide, unless the context explicitly indicates otherwise. For example, "nucleotide" refers to deoxyribonucleotides or ribonucleotides. Nucleotides can be standard nucleotides (i.e., adenosine, guanosine, cytidine, thymidine, and uridine), nucleotide isomers, or nucleotide analogs. Nucleotide analogs refer to nucleotides having modified purine or pyrimidine bases or modified ribose moieties. Nucleotide analogs can be naturally occurring nucleotides (e.g., inosine, pseudouridine, etc.) or non-naturally occurring nucleotides. Non-limiting examples of modifications to the sugar or base moieties of nucleotides include the addition (or removal) of acetyl, amino, carboxyl, carboxymethyl, hydroxyl, methyl, phosphoryl, and thiol groups, as well as the substitution of carbon and nitrogen atoms of the base by other atoms (e.g., 7-denitropurine). Nucleotide analogs also include dideoxynucleotides, 2'-O-methylnucleotides, locked nucleic acids (LNAs), peptide nucleic acids (PNAs), and morpholino oligonucleotides. In some embodiments, the "nucleotides" of this application do not contain non-natural nucleotides with modified bases. In some embodiments, the "nucleotides" of this application do not contain nucleotides with modified bases. In this application, “G”, “C”, “A”, “T” and “U” generally represent nucleotides, nucleosides or bases with guanine, cytosine, adenine, thymine and uracil as bases, respectively. Unless otherwise specified, “G”, “C”, “A”, “T” and “U” indicate nucleotides, nucleosides or bases that are not limited to those containing modifications. They can be used to represent natural nucleotides or non-natural nucleotides (or nucleosides). The non-natural nucleotides may contain ribose and / or nucleotides with modified bases, as long as the bases therein can still be complementary to their naturally paired (i.e., paired according to the Watson-Crick principle) bases through hydrogen bonds. Similarly, they can also represent natural or non-natural bases or nucleosides. To further clarify, when “G,” “C,” “A,” “T,” and “U” represent natural nucleotides, nucleosides, bases, or their derivatives, the derivatives indicate chemical modifications relative to their corresponding natural nucleotides, nucleosides, or bases, while retaining the ability to complementary pairing with their naturally paired (i.e., pairing according to the Watson-Crick principle) bases via hydrogen bonds, the affinity of this complementary pairing is greater than the affinity with other bases with which they do not naturally pair. Therefore, since T and U are derivatives of each other, unless otherwise specified, T and U are interchangeable in base sequences (e.g., the base sequence portions in the sequence appendix). However, when T or U appears as an unmodified nucleotide or nucleoside in RNA, it only represents uridine; similarly, when T appears as an unmodified nucleotide in DNA, it only represents 2'-deoxythymidine.

[0059] It should be understood that in this application, whenever nucleotide sequences are mentioned, the terms "nucleotide" and "nucleotide residue" are used interchangeably. The number of base pairs is measured in bp, where one bp represents one base pair. The number of nucleotides is measured in nt, where one nt represents one nucleotide.

[0060] Unless otherwise specified, "nucleotide linkage" as used herein refers to a chemical bond (or linking group) between two adjacent nucleotides or between a nucleotide and a target moiety (e.g., a lipophilic moiety). Unless otherwise specified, this chemical bond is a phosphate ester bond. However, when a modification motif specifically indicates "s," signifying the position of a phosphate thioester modification, only the nucleotide linkage at the position of "s" is a phosphate thioester bond; all other unmarked nucleotide linkages are phosphate ester bonds. For example:

[0061] (1) Sense strand: NmNmNmNmNmNmNfNmNfNdNfNmNmNmNmNmNmNmNmNmNm, antisense strand: NmNfNmNmNdNmNdNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmNmNm;

[0062] Then the linkages between the nucleotides in (1) can be either phosphate ester bonds or thiophosphate ester bonds;

[0063] And if it is:

[0064] Sense strand: lipophilic portion s-NmsNmNmNmNmNmNfNmNfNdNfNmNmNmNmNmNmNmNmsNms-lipophilic portion, antisense strand: vp-NmsNfsNmNmNdNmNdNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm. Due to the specially marked "s", except for the following nucleotide linkages which are phosphate ester bonds, all other nucleotide linkages are phosphate ester bonds: the first nucleus starting from the 5' end of the sense strand. The linkages between nucleotides and between the second nucleotide, the linkages between the first and second nucleotides starting from the 3' end of the sense strand, the linkages between the first and second nucleotides starting from the 5' end of the antisense strand, and the linkages between the first and second nucleotides starting from the 3' end of the antisense strand; wherein, for the sense strand, the first nucleotide linkage is a linkage between the lipophilic portion and the nucleotide, that is, as shown in the above modified motifs, the lipophilic portion is also linked to the 3' and 5' nucleotides of the sense strand by a phosphate thioester bond.

[0065] As used herein, unless otherwise specified, the term "base sequence" refers only to the arrangement of bases in a nucleic acid molecule or nucleic acid chain. "Base" does not limit the type of base to natural or non-natural bases, and it does not limit ribose modification in nucleotides, nor does it limit phosphate group modification. Unless otherwise specified, the term "nucleoside sequence" refers only to the arrangement of nucleosides in a nucleic acid molecule or nucleic acid chain. It may specify that the nucleoside has a certain ribose and / or base modification, but it does not limit phosphate group modification. However, it does not exclude the possibility of other modifications to the base or ribose beyond the specified modifications. Unless otherwise specified, the term "nucleotide sequence" refers to the sequence of nucleotides, which may specify that the nucleotide has base, phosphate, and / or ribose modifications. Similarly, it does not exclude the possibility of other modifications to the base, ribose, and phosphate groups beyond the specified modifications.

[0066] As used herein, a "modification motif" refers to a pattern of nucleotide modification along an oligonucleotide (e.g., dsRNA), consisting of sequential modifications to each nucleotide in a nucleic acid sequence that differ from the native nucleotide. The nucleotides in an oligonucleotide modified using a "modification motif" change accordingly with the nucleotide modification at the corresponding position in the "modification motif." In some embodiments, the 2' hydroxyl groups of the 2nd, 14th, and 16th nucleotides of the antisense strand of the dsRNA of this application are substituted with fluorine, and the 2' hydroxyl groups of the other nucleotides of the antisense strand are substituted with methoxy or hydrogen. In some embodiments, the 2' hydroxyl groups of the 9th and 11th nucleotides of the sense strand of the dsRNA molecule of this application are substituted with fluorine, the 2' hydroxyl group of the 10th nucleotide is substituted with fluorine or hydrogen, and the 2' hydroxyl groups of the other nucleotides are substituted with fluorine, methoxy, or hydrogen. In some embodiments, the 2' hydroxyl groups of the 9th and 11th nucleotides of the sense strand in the dsRNA molecule of this application are substituted with fluorine, the 2' hydroxyl group of the 10th nucleotide is substituted with fluorine or hydrogen, the 2' hydroxyl groups of the 7th and 12th nucleotides are substituted with fluorine or methoxy, and the 2' hydroxyl groups of the other nucleotides are substituted with methoxy or hydrogen. In some embodiments, the 2' hydroxyl groups of the 9th and 11th nucleotides of the sense strand in the dsRNA molecule of this application are substituted with fluorine, the 2' hydroxyl group of the 10th nucleotide is substituted with fluorine or hydrogen, and the 2' hydroxyl groups of the other nucleotides are substituted with fluorine, methoxy, or hydrogen. In some embodiments, the 2' hydroxyl groups of the 9th and 11th nucleotides of the sense strand in the dsRNA molecule of this application are substituted with fluorine, the 2' hydroxyl group of the 10th nucleotide is substituted with fluorine or hydrogen, the 2' hydroxyl groups of the 7th and 12th nucleotides are substituted with fluorine or methoxy, and the 2' hydroxyl groups of the other nucleotides are substituted with methoxy or hydrogen. In some embodiments, the 2' hydroxyl groups of the sense strand at positions 9 and 11, starting from the 5' end, of the dsRNA molecule of this application are substituted with fluorine, the 2' hydroxyl group at position 10 is substituted with fluorine or hydrogen, the 2' hydroxyl groups at positions 7 and 12 are substituted with fluorine or methoxy, and the 2' hydroxyl groups of the other nucleotides are substituted with methoxy.

[0067] As used herein, the terms "overhang," "dangling end," and "dangling sequence" are used interchangeably and refer to one or more unpaired nucleotides extending beyond the double-stranded region at the end of a strand. A nucleotide overhang is typically formed when the 3' end of one strand extends beyond the 5' end of another strand, or when the 5' end of one strand extends beyond the 3' end of another strand. The length of a nucleotide overhang is typically between 1 and 6 nucleotides, 1 and 5 nucleotides, 1 and 4 nucleotides, 1 and 3 nucleotides, 2 and 6 nucleotides, 2 and 5 nucleotides, or 2 and 4 nucleotides. In some embodiments, a nucleotide overhang comprises 1, 2, 3, 4, 5, or 6 nucleotides. In one particular embodiment, a nucleotide overhang comprises 1 to 4 nucleotides. In some embodiments, a nucleotide overhang comprises 2 nucleotides. In some other embodiments, a nucleotide overhang comprises a single nucleotide.

[0068] The nucleotide at the overhang can be a ribonucleotide as described herein or a modified nucleotide. In some embodiments, the nucleotide at the overhang is a 2′-modified nucleotide (e.g., a 2′-fluorinated nucleotide, a 2′-O-methylated nucleotide), a deoxyribonucleotide, a reverse nucleotide (e.g., a reverse abase-free nucleotide, a reverse deoxyribonucleotide), or a combination thereof. For example, in one embodiment, the nucleotide at the overhang is a deoxyribonucleotide, such as deoxythymidine. In another embodiment, the nucleotide at the overhang is a 2′-O-methylated nucleotide, a 2′-fluorinated nucleotide, a 2′-methoxyethylated nucleotide, or a combination thereof. In other embodiments, the overhang comprises 5′-uridine-uridine-3′ (5′-UU-3′) dinucleotide. In such embodiments, the UU dinucleotide may comprise a ribonucleotide or a modified nucleotide, such as a 2′-modified nucleotide. In other embodiments, the overhang comprises 5′-deoxythymidine-deoxythymidine-3′ (5′-dTdT-3′) dinucleotide. When a nucleotide overhang is present in the antisense strand, the nucleotide in the overhang can be complementary to the target gene sequence, forming a mismatch with the target gene sequence or containing some other sequences (such as polypyrimidine or polypurine sequences, such as UU, TT, AA, GG, etc.).

[0069] Nucleotide overhangs can be located at the 5' or 3' end of one or both strands. For example, in one embodiment, the RNA molecule includes nucleotide overhangs at both the 5' and 3' ends of the antisense strand. In another embodiment, the RNA molecule includes nucleotide overhangs at both the 5' and 3' ends of the sense strand. In some embodiments, the RNA molecule includes nucleotide overhangs at both the 5' end of the sense strand and the 5' end of the antisense strand. In other embodiments, the RNA molecule includes nucleotide overhangs at both the 3' end of the sense strand and the 3' end of the antisense strand.

[0070] RNA molecules may have nucleotide overhangs at one end of a double-stranded RNA molecule and blunt ends at the other end. A "blunt end" means that the sense and antisense strands are completely base-paired at the ends of the molecule, and there are no unpaired nucleotides extending beyond the double-stranded region. In some embodiments, the RNA molecule has nucleotide overhangs at the 3' end of the sense strand and blunt ends at the 5' end of the sense strand and the 3' end of the antisense strand. In other embodiments, the RNA molecule has nucleotide overhangs at the 3' end of the antisense strand and blunt ends at the 5' end of the antisense strand and the 3' end of the sense strand. In some embodiments, the RNA molecule has blunt ends at both ends of the double-stranded RNA molecule. In these embodiments, the sense and antisense strands have the same length, and the length of the double-stranded region is the same as that of the sense and antisense strands (i.e., the molecule is double-stranded along its entire length).

[0071] It should be understood that the 5' end of the dsRNA in this application refers to the end where the 5' nucleotide of the sense strand and the 3' nucleotide of the antisense strand are located. Unless otherwise specified, this end can be a blunt end or a convex end. When the 5' end of the dsRNA is a convex end, the convex nucleotide can be located at the 5' end of the sense strand or the 3' end of the antisense strand; similarly, when the 3' end of the dsRNA is a convex end, the convex nucleotide can be located at the 3' end of the sense strand or the 5' end of the antisense strand.

[0072] As used herein, “GalNAc” or “N-acetylgalactosamine” refers to 2-(acetylamino)-2-deoxy-D-galactopyranose. Unless otherwise specified, the term “GalNAc” or “N-acetylgalactosamine” includes both the β-form: 2-(acetylamino)-2-deoxy-β-D-galactopyranose and the α-form: 2-(acetylamino)-2-deoxy-α-D-galactopyranose. Preferably, the GalNAc compound of this application is in the β-form, namely 2-(acetylamino)-2-deoxy-β-D-galactopyranose.

[0073] As used herein, a "5'-phosphate mimic" generally refers to a phosphate mimic linked to the 5' end nucleotide of a nucleic acid sequence. This mimic can be a phosphate ester, phosphonate ester, or other similar substance, linked to the 4' or 5' position of the 5' end nucleotide of the nucleic acid sequence. For example, it can be linked to the 4' or 5' position of the 5' end nucleotide of the nucleic acid sequence via a carbon-containing or carbon-free linker. It can maintain or increase the phosphorylation of the 5' end nucleotide of the nucleic acid sequence, reducing or preventing its degradation from the 5'-3' position by phosphatases and exonucleases. Non-limiting examples of the "5'-phosphate mimic" include, for example, vinylphosphonates and PEO, which are also referred to herein as "VP," "E-VP," or "5'-vp."

[0074] As used in this article, "VP modification" or "E-VP" is a modification that places a vinylphosphonate at the 5' position of a nucleotide, and the structure formed after its linkage with the nucleotide is shown below:

[0075] in:

[0076] Bx1 is a natural or modified base; T2 is a phosphate ester or thiophosphate nucleotide linker that connects the above compound to the oligonucleotide chain; and G is a substituent at the 2' position of the corresponding nucleotide, including but not limited to F, hydroxyl, H, methoxy, methoxyethoxy, etc.

[0077] In this application, "PEO" is also a 5'-phosphate mimic, and when a nucleotide is modified with PEO (or the nucleotide is "PEO modified"), it is attached to the 4' position of the nucleotide; PEO has the structure shown below:

[0078] The ribose in the modified nucleotide can be of D or L configuration. In some embodiments, the ribose in the nucleotide modified by PEO is of D configuration. Furthermore, this application also provides a structure in which the hydrogen in one of the hydroxyl groups of PEO is replaced by a methyl group, thus also providing an equivalent scheme for dsRNA modified by this structure; that is, in any PEO-modified dsRNA, the PEO can be replaced by this structure to form an equivalent scheme. In this application, "3' end / terminus," "3' side," "5' end / terminus," and "5' side" have their conventional meanings in the art. Only in reference are "3' end / terminus" and "5' end / terminus" more often used to indicate specific nucleotide positions; for example, "3' end / terminus" is often used to refer to the position of the first nucleotide or base pair at the 3' end of a single nucleotide sequence or double-stranded polynucleotide; "5' end" is often used to refer to the position of the first nucleotide or base pair at the 5' end of a single nucleotide sequence or double-stranded polynucleotide. "3' side" and "5' side" are used to describe the relative positions of nucleotides.

[0079] As used in this application, the term "nucleic acid molecule" may be used to refer to any molecule having a nucleotide sequence consisting of two or more nucleotides linked by phosphate ester bonds, or modified phosphate ester bonds (e.g., thiophosphate bonds).

[0080] As used herein, the term "nucleotide sequence" refers to a polynucleotide chain composed of nucleotides arranged in a specific sequence. This polynucleotide chain can constitute a nucleic acid molecule or a segment of a chain within a nucleic acid molecule. Therefore, "nucleotide sequence" can be represented as a precise polynucleotide sequence composed of various nucleotides (e.g., ATCG) (e.g., any one of SEQ ID NO: 1 to 100), or as nucleotides at specific positions within a sequence, such as "nucleotides at positions 1430 to 1450 in the ALK7 gene mRNA sequence." In this application, unless otherwise specified, in sequences or nucleic acid molecules containing "nucleotides at specific positions within a sequence," the sequence of these nucleotides is consistent with the sequence of the aforementioned nucleotides within the sequence. Without limitation, a "nucleotide sequence" can be RNA or DNA, or a hybrid of RNA and DNA, and may also incorporate non-natural or artificially modified nucleotides.

[0081] In RNA contexts (e.g., mRNA, siRNA, dsRNA, or shRNA), N represents ribonucleotide, dN represents deoxyribonucleotide (DNA), Nm represents a 2'-O-Me modified nucleotide or a 2'-O-methyl modified nucleotide; Nf represents a 2'-F modified ribonucleotide or a 2'-fluoro modified ribonucleotide; and s represents a phosphothioester modification, i.e., a 5'-thio modified phosphoester. When this modification occurs between two nucleotides or between a nucleotide and a target moiety (e.g., a lipophilic moiety), it replaces the phosphate ester bond between the natural nucleotides and is called a phosphothioester bond. As used herein, unless otherwise specified, "phosphothioester bond" and "phosphothiodiester bond" are used interchangeably; similarly, "phosphate ester bond" and "phosphodiester bond" are used interchangeably.

[0082] Furthermore, as used herein, when describing a nucleotide whose 2' hydroxyl group is substituted with a methoxy, fluorine, or hydrogen, it is equivalent to describing the nucleotide as being modified with 2'-O-Me, 2'-F, or as a deoxyribonucleotide (2'-OH deoxy).

[0083] It should be understood that, in this application, whenever a chain, nucleic acid molecule, or sequence is mentioned as containing X (X is an integer) consecutive bases of another chain, another nucleic acid molecule, or another sequence, the X consecutive bases are also X consecutive bases arranged in the same order in the chain, nucleic acid molecule, or sequence.

[0084] As used herein, the term "about" refers to the general range of error for various values ​​that is readily known to those skilled in the art. References to a value or parameter "about" herein include (and describe) an embodiment for that value or parameter itself. As used herein, when the term "about" precedes a numerical value, it indicates a range of 10% above or below that value. For example, "about 100" encompasses both 90 and 110.

[0085] As used herein, unless otherwise indicated, the singular forms “a,” “a,” and “the” include the plural forms.

[0086] Furthermore, unless otherwise specified, all abbreviations or symbols appearing in this application as shown in the table below shall be interpreted as follows:

[0087] It should be understood that a nucleoside or nucleotide with "2'-OMe modification" indicates that the 2' position of the nucleoside or nucleotide is directly linked to a methoxy group; that is, when the nucleoside or nucleotide is a ribonucleoside or ribonucleotide, its 2' hydroxyl group is replaced by a methoxy group; when the nucleoside or nucleotide is a deoxyribonucleoside or deoxyribonucleotide, its 2' hydrogen is replaced by a methoxy group. Similarly, a nucleoside or nucleotide with "2'-F modification" indicates that the 2' position of the nucleoside or nucleotide is directly linked to a fluorine group. Furthermore, a nucleoside or nucleotide with "lipophilic moiety modification" indicates that the nucleoside or nucleotide is linked to a lipophilic moiety. In some embodiments, the lipophilic moiety is located in the middle of the nucleic acid chain and is linked to the 2' position of the nucleotide. In some embodiments, the lipophilic moiety is located at the end of the nucleic acid chain, for example, linked to the 3' position of the 3' terminal nucleotide of the nucleic acid chain.

[0088] Unless otherwise specified, the terms "pharmaceutically acceptable salt" or "medicinal salt" refer to a salt that, within the bounds of reasonable medical judgment, is suitable for contact with the tissues of mammals, particularly humans, without excessive toxicity, irritation, allergic reactions, etc., and is commensurate with a reasonable benefit / risk ratio. Medically acceptable salts of amines, carboxylic acids, and other types of compounds are well known in the art. The salt can be prepared in situ during the final separation and purification of the compounds of this application, or solely by reacting a free base or free acid with a suitable reagent.

[0089] Unless otherwise specified, the term "isotope derivative" refers to a compound of this application that can exist in an isotopically traced or enriched form, containing one or more atoms whose atomic weights or mass numbers differ from the atomic weights or mass numbers of the most abundant atoms found in nature. Isotopes can be radioactive or non-radioactive. Commonly used isotopes for isotopic labeling include: hydrogen isotopes. 2 H and 3 H; Carbon isotopes: 13 C and 14C; Chlorine isotopes: 35 Cl and 37 Cl; Fluorine isotopes: 18 F; Iodine isotopes: 123 I and 125 I; Nitrogen isotopes: 13 N and 15 N; oxygen isotopes: 15 O、 17 O and 18 O and sulfur isotopes 35 S. These isotope-labeled compounds can be used to study the distribution of pharmaceutical molecules in tissues. Especially 2 H and 13 C, because they are easy to label and convenient to detect, are more widely used. Some heavy isotopes, such as deuterium (… 2 Substitution with H can enhance metabolic stability and prolong the half-life, thereby achieving the goal of reducing dosage and providing therapeutic advantages. Isotope-labeled compounds are generally synthesized from labeled starting materials using known synthetic techniques, just like non-isotope-labeled compounds.

[0090] Unless otherwise specified, the terms "solvent" or "solvent compound" refer to the physical association of the compound of this application with one or more solvent molecules (organic or inorganic). This physical association includes hydrogen bonding. In some cases, such as when one or more solvent molecules are incorporated into the crystal lattice of a crystalline solid, the solvate can be separated. The solvent molecules in the solvate may be present in a regular and / or disordered arrangement. The solvate may contain stoichiometric or non-stoichiometric solvent molecules. "Solvent compound" encompasses both solution phases and separable solvates. Exemplary solvates include, but are not limited to, hydrates, ethanol compounds, methanol compounds, and isopropanol compounds. Solvation methods are well known in the art.

[0091] Unless otherwise specified, the term "stereoisomer" refers to compounds having the same chemical structure but with different spatial arrangements of atoms or groups. Stereoisomers include enantiomers, diastereomers, conformational isomers (rotational isomers), geometric isomers (cis / trans) isomers, and inhibited isomers. Any mixture of stereoisomers obtained can be separated into pure or substantially pure geometric isomers, enantiomers, and diastereomers based on differences in the physicochemical properties of the components, for example, by chromatography and / or fractional crystallization.

[0092] Unless otherwise specified, the term "tautomer" refers to structural isomers with different energies that can interconvert through a low energy barrier. If tautomerism is possible (e.g., in solution), chemical equilibrium can be achieved in the tautomer. For example, proton tautomers (also called proton transfer tautomers) involve interconversions via proton migration, such as keto-enol isomerization and imine-enamine isomerization. Valence tautomers involve interconversions via the rearrangement of some bonding electrons.

[0093] Unless otherwise indicated, the structural formulas described in this application include all isomers (such as enantiomers, diastereomers, and geometric isomers (or conformational isomers)): for example, R and S configurations containing an asymmetric center, (Z) and (E) isomers of double bonds, and (Z) and (E) conformational isomers. Therefore, any single stereochemical isomer of the compound of this application, or its enantiomers, diastereomers, or mixtures of geometric isomers (or conformational isomers), are within the scope of this application.

[0094] As used herein, the term "pharmaceutical composition" means a combination of at least one drug, optionally a pharmaceutically acceptable carrier or excipient, used together to achieve a particular purpose. Pharmaceutically acceptable carriers may include, for example, water, saline, glucose, buffer solutions (such as PBS), excipients, diluents, disintegrants, binders, lubricants, sweeteners, flavorings, preservatives, or combinations thereof.

[0095] As used in this article, the term "treatment" refers to a clinical intervention aimed at altering the natural course of disease in the individual or cells receiving the treatment during the clinicopathological process. Ideal outcomes of treatment include slowing or reducing the rate of disease progression, improving or alleviating the disease state, and mitigating or improving prognosis.

[0096] As used in this article, the term "prevention" refers to preventive treatment of a disease, disorder, or ailment; or delaying the onset or progression of a disease, disorder, or ailment.

[0097] In this application, the terms "effective amount" or "therapeutic effective amount" are used interchangeably and refer to an amount that has a therapeutic effect on a subject, such as: in subjects who have been given the amount, the symptoms or state of the disease are alleviated, reduced, or eliminated, or the development of the symptoms or state of the disease is delayed or suppressed compared to subjects who have not been given the amount.

[0098] As used herein, a subject, patient, or individual is considered "needed" for treatment if the subject would benefit biologically, medically, or in terms of quality of life from the treatment. In this text, the terms "subject," "patient," and "individual" are used interchangeably in certain contexts and have the same meaning.

[0099] The terms “subject,” “object,” or “individual” as used in this article refer to mammals, such as humans, but may also refer to other animals, such as wild animals, livestock, or laboratory animals (e.g., chimpanzees, monkeys, rats, mice, rabbits, guinea pigs, marmots, ground squirrels, etc.).

[0100] This application provides a dsRNA molecule capable of inducing RNA-induced silencing complex (RISC)-mediated cleavage of the activin receptor-like kinase 7 (ALK7) gene RNA transcript, and derivatives thereof. The derivatives include, but are not limited to, engineered nucleic acid molecules (also referred to herein as "first engineered nucleic acid molecule") or compounds containing the dsRNA molecule, engineered nucleic acid molecules capable of being transcribed or cleaved to form the dsRNA molecule (also referred to herein as "second engineered nucleic acid molecule"), delivery systems containing the dsRNA molecule, the first engineered nucleic acid molecule, or the second engineered nucleic acid molecule, viruses or cells capable of transcribing or expressing the engineered nucleic acid molecule or the dsRNA molecule, and uses of the dsRNA molecule, the first engineered nucleic acid molecule, and the second engineered nucleic acid molecule. Examples of this application demonstrate the high inhibitory efficiency of the dsRNA molecule on ALK7 mRNA expression.

[0101] In its initial aspect, this application provides a double-stranded RNA (dsRNA) molecule that inhibits the expression of the activin receptor-like kinase 7 (ALK7) gene via RNAi. The dsRNA molecule comprises a sense strand and an antisense strand that are complementary to each other to form a double-stranded region, or is composed of a sense strand and an antisense strand that are complementary to each other to form a double-stranded region. When the antisense strand hybridizes with ALK7 mRNA (e.g., shown in SEQ ID NO. 333) at maximum complementarity, complementary region 1 is formed by complementary base pairs between the antisense strand and ALK7 mRNA. When the antisense strand hybridizes with the sense strand at maximum complementarity, complementary region 2 is formed by complementary base pairs between the antisense strand and the sense strand. The antisense strand base sequence comprises at least 15 consecutive bases selected from the antisense strand of any dsRNA molecule selected from Table A, excluding the *Gynostemma pentaphyllum* molecule.

[0102] The antisense strand base sequence comprises at least 15 consecutive bases of a homologous sequence selected from the antisense strand of any dsRNA molecule other than the ginseng molecule in "Table A". In some embodiments, the sense strand base sequence comprises at least 15 consecutive bases of a homologous sequence selected from the sense strand of any dsRNA molecule other than the ginseng molecule in "Table A".

[0103] "Homologous sequence" refers to a nucleic acid or amino acid sequence that originates from a common ancestor and has a similar nucleotide or amino acid sequence. Those skilled in the art can easily determine homology based on methods such as sequence alignment analysis, and can identify homologous sequences through specific sequence similarity thresholds (such as a certain proportion of sequence identity) or functional conservation. Unless otherwise stated, "homologous sequence" may include sequences with a certain degree of sequence variation but still retaining similar structure or function. For example, in this application, the "homologous sequence" of the antisense strand refers to the sequence in the complementary sequence of different ALK mRNA variants that is homologous to the antisense strand of any of the dsRNA molecules; for example, the base sequence of ALK7-Z15 is as shown in SEQ ID NO: 1, which is complementary to positions 637 to 659 of the ALK7 mRNA transcript NM_001111033.2 (SEQ ID NO. 333). The homologous segment corresponding to this segment includes at least the complementary sequence of nucleotides 868 to 890 of the variant transcript NM_001111033.2 and the complementary sequence of nucleotides 958 to 980 of the variant transcript NM_001111033.2. Two sequences that are homologous to each other can have exactly the same bases or can have one or more base differences. For example, two homologous sequences can have 80% or more homology. Similarly, the "homogeneous sequence" of the sense strand is the sequence in different ALK mRNA variants that is homologous to the sense strand of any of the dsRNA molecules.

[0104] More specifically, in a first aspect, this application provides a double-stranded RNA (dsRNA) molecule that inhibits the expression of the activin receptor-like kinase 7 (ALK7) gene via RNAi, comprising a sense strand and an antisense strand capable of complementing each other to form a double-stranded region, or composed of a sense strand and an antisense strand capable of complementing each other to form a double-stranded region, wherein the sense strand is 15-30 nt in length, and the antisense strand is 15-30 nt in length, wherein:

[0105] (1) The sense strand sequence of the dsRNA molecule comprises at least 15 consecutive bases of the sequence shown in SEQ ID NO. 31, and the antisense strand sequence of the dsRNA molecule comprises at least 15 consecutive bases of the sequence shown in SEQ ID NO. 32; or

[0106] (2) The sense strand sequence of the dsRNA molecule comprises at least 15 consecutive bases of the sequence shown in SEQ ID NO.1, and the antisense strand sequence of the dsRNA molecule comprises at least 15 consecutive bases of the sequence shown in SEQ ID NO.2; or

[0107] (3) The sense strand sequence of the dsRNA molecule comprises at least 15 consecutive bases of the sequence shown in SEQ ID NO.3, and the antisense strand sequence of the dsRNA molecule comprises at least 15 consecutive bases of the sequence shown in SEQ ID NO.4; or

[0108] (4) The sense strand sequence of the dsRNA molecule comprises at least 15 consecutive bases of the sequence shown in SEQ ID NO. 5, and the antisense strand sequence of the dsRNA molecule comprises at least 15 consecutive bases of the sequence shown in SEQ ID NO. 6; or

[0109] (5) The sense strand sequence of the dsRNA molecule comprises at least 15 consecutive bases of the sequence shown in SEQ ID NO. 7, and the antisense strand sequence of the dsRNA molecule comprises at least 15 consecutive bases of the sequence shown in SEQ ID NO. 8; or

[0110] (6) The sense strand sequence of the dsRNA molecule comprises at least 15 consecutive bases of the sequence shown in SEQ ID NO. 9, and the antisense strand sequence of the dsRNA molecule comprises at least 15 consecutive bases of the sequence shown in SEQ ID NO. 10; or

[0111] (7) The sense strand sequence of the dsRNA molecule comprises at least 15 consecutive bases of the sequence shown in SEQ ID NO. 15, and the antisense strand sequence of the dsRNA molecule comprises at least 15 consecutive bases of the sequence shown in SEQ ID NO. 16; or

[0112] (8) The sense strand sequence of the dsRNA molecule contains at least 15 consecutive bases of the sequence shown in SEQ ID NO.17, and the antisense strand sequence of the dsRNA molecule contains at least 15 consecutive bases of the sequence shown in SEQ ID NO.18.

[0113] As a supplement to the first aspect, this application also provides a double-stranded RNA (dsRNA) molecule that inhibits the expression of the activin receptor-like kinase 7 (ALK7) gene via RNAi, comprising a sense strand and an antisense strand that are complementary to each other to form a double-stranded region, or consisting of a sense strand and an antisense strand that are complementary to each other to form a double-stranded region, wherein the sense strand is 15-30 nt in length and the antisense strand is 15-30 nt in length, and the base sequences of the sense and antisense strands of the dsRNA molecule comprise at least 15 consecutive bases of the sense and antisense strand sequences of any dsRNA molecule in “Table A” except for the Yangshen molecule. Examples of any dsRNA molecule other than the *Codonopsis pilosula* molecule in "Table A" include, but are not limited to: ALK7-Z01, ALK7-Z02, ALK7-Z15, ALK7-Z16, ALK7-Z17, ALK7-Z25, ALK7-Z35, ALK7-Z37, ALK7-Z38, ALK7-Z47, ALK7-Z49, ALK7-Z50, ALK7-Z52, ALK7-Z56, ALK7-Z59, ALK7-Z65, ALK7-Z66, ALK7-Z73, ALK7-Z106, ALK7-Z130, ALK7-Z190, ALK7-Z01, ALK7-Z02, ALK7-Z15, ALK7-Z16, ALK7-Z17, ALK7-Z18, ALK7-Z190, ALK7-Z106, ALK7-Z130 ... K7-Z195, ALK7-Z200, ALK7-D03, ALK7-D04, ALK7-D09, ALK7-D25, ALK7-D37, ALK7-D38, ALK7-D45, ALK7-D46, ALK7-D71, ALK7-D85, ALK7 -D88, ALK7-D91, ALK7-D95, ALK7-D127, ALK7-D138, ALK7-D152, ALK7-D168, ALK7-D169, ALK7-D173, ALK7-D183, ALK7-D192, ALK7-D196.

[0114] The detailed descriptions of the implementation schemes and technical features of the dsRNA molecule in the first aspect, which follow below, also apply to the aforementioned supplementary aspects of the first aspect, provided there are no contradictions. Similarly, the detailed descriptions of the implementation schemes and technical features of the second to twelfth aspects derived from the first aspect, which follow below, also apply to the respective supplementary aspects of the second to twelfth aspects derived from the aforementioned supplementary aspects of the first aspect, provided there are no contradictions.

[0115] In some embodiments, the complementary regions of the sense and antisense strands of the dsRNA molecule contain at least 15 consecutive base pairs selected from the complementary regions of any dsRNA molecule in "Table A".

[0116] In some embodiments, the antisense strand of the dsRNA molecule is 100% complementary to the following sequence of the ALK7 mRNA:

[0117] The 15th, 16th, 17th, 18th, 19th, 21st, 22nd, or 23rd consecutive nucleotides from position 90 to 112.

[0118] The 15th, 16th, 17th, 18th, 19th, 21st, 22nd, or 23rd nucleotides from position 233 to 255.

[0119] The 15, 16, 17, 18, 19, 21, 22, or 23 consecutive nucleotides from position 326 to 348

[0120] The 15, 16, 17, 18, 19, 21, 22, or 23 consecutive nucleotides from position 480 to 503

[0121] The 15, 16, 17, 18, 19, 21, 22, or 23 consecutive nucleotides from position 585 to 607

[0122] The 15, 16, 17, 18, 19, 21, 22, or 23 consecutive nucleotides from position 637 to 659

[0123] The 15, 16, 17, 18, 19, 21, 22, or 23 consecutive nucleotides from position 662 to 685

[0124] The 15th, 16th, 17th, 18th, 19th, 21st, 22nd, or 23rd consecutive nucleotides from positions 701 to 723

[0125] The 15, 16, 17, 18, 19, 21, 22, or 23 consecutive nucleotides from position 834 to 856

[0126] The 15th, 16th, 17th, 18th, 19th, 21st, 22nd, or 23rd nucleotides in positions 939 to 961

[0127] The 15, 16, 17, 18, 19, 21, 22 or 23 consecutive nucleotides from position 941 to 963

[0128] The 15, 16, 17, 18, 19, 21, 22, or 23 consecutive nucleotides from position 944 to 966

[0129] The 15, 16, 17, 18, 19, 21, 22, or 23 consecutive nucleotides from position 954 to 976

[0130] The 15, 16, 17, 18, 19, 21, 22 or 23 consecutive nucleotides from position 1053 to 1075, or

[0131] The 15, 16, 17, 18, 19, 21, 22 or 23 consecutive nucleotides from position 939 to 1075, wherein the nucleotide site number in the ALK7 mRNA sequence is the corresponding nucleotide number in the reference sequence SEQ ID NO: 333.

[0132] In some embodiments, the sense and antisense base sequences each consist of 15-30 consecutive bases independently.

[0133] In some embodiments, (1) the sense strand sequence of the dsRNA molecule comprises 19-21 consecutive bases (e.g., 21 consecutive bases) of the sequence shown in SEQ ID NO. 31 and / or the antisense strand sequence of the dsRNA molecule comprises 21-23 consecutive bases (e.g., 23 consecutive bases) of the sequence shown in SEQ ID NO. 32; or

[0134] (2) The sense strand sequence of the dsRNA molecule comprises 19-21 consecutive bases (e.g., 21 consecutive bases) of the sequence shown in SEQ ID NO.1 and / or the antisense strand sequence of the dsRNA molecule comprises 21-23 consecutive bases (e.g., 23 consecutive bases) of the sequence shown in SEQ ID NO.2; or

[0135] (3) The sense strand sequence of the dsRNA molecule comprises 19-21 consecutive bases (e.g., 21 consecutive bases) of the sequence shown in SEQ ID NO.3 and / or the antisense strand sequence of the dsRNA molecule comprises 21-23 consecutive bases (e.g., 23 consecutive bases) of the sequence shown in SEQ ID NO.4; or

[0136] (4) The sense strand sequence of the dsRNA molecule comprises 19-21 consecutive bases (e.g., 21 consecutive bases) of the sequence shown in SEQ ID NO. 5 and / or the antisense strand sequence of the dsRNA molecule comprises 21-23 consecutive bases (e.g., 23 consecutive bases) of the sequence shown in SEQ ID NO. 6; or

[0137] (5) The sense strand sequence of the dsRNA molecule comprises 19-21 consecutive bases (e.g., 21 consecutive bases) of the sequence shown in SEQ ID NO. 7 and / or the antisense strand sequence of the dsRNA molecule comprises 21-23 consecutive bases (e.g., 23 consecutive bases) of the sequence shown in SEQ ID NO. 8; or

[0138] (6) The sense strand sequence of the dsRNA molecule comprises 19-21 consecutive bases (e.g., 21 consecutive bases) of the sequence shown in SEQ ID NO. 9 and / or the antisense strand sequence of the dsRNA molecule comprises 21-23 consecutive bases (e.g., 23 consecutive bases) of the sequence shown in SEQ ID NO. 10; or

[0139] (7) The sense strand sequence of the dsRNA molecule comprises 19-21 consecutive bases (e.g., 21 consecutive bases) of the sequence shown in SEQ ID NO. 15 and / or the antisense strand sequence of the dsRNA molecule comprises 21-23 consecutive bases (e.g., 23 consecutive bases) of the sequence shown in SEQ ID NO. 16; or

[0140] (8) The sense strand sequence of the dsRNA molecule comprises 19-21 consecutive bases (e.g., 21 consecutive bases) of the sequence shown in SEQ ID NO.17 and / or the antisense strand sequence of the dsRNA molecule comprises 21-23 consecutive bases (e.g., 23 consecutive bases) of the sequence shown in SEQ ID NO.18.

[0141] In some embodiments, the sense strand of the dsRNA molecule of the first aspect comprises 19, 20, or 21 consecutive bases of the sense strand of any dsRNA molecule selected from "Table A" excluding the ginseng molecule. In some embodiments, the antisense strand of the dsRNA molecule of the first aspect comprises 21, 22, or 23 consecutive bases of the antisense strand of the said dsRNA molecule. In some embodiments, the sense strand of the dsRNA molecule of the first aspect comprises 19, 20, or 21 consecutive bases of the sense strand of any dsRNA molecule selected from "Table A" excluding the ginseng molecule, and the antisense strand of the dsRNA molecule of the first aspect comprises 21, 22, or 23 consecutive bases of the antisense strand of the said dsRNA molecule.

[0142] In some embodiments, the sense strand of the dsRNA molecule of the first aspect comprises 21 consecutive bases of the sense strand of any dsRNA molecule selected from "Table A" excluding the ginseng molecule. In some embodiments, the antisense strand of the dsRNA molecule of the first aspect comprises 23 consecutive bases of the antisense strand of the said dsRNA molecule. In some embodiments, the sense strand of the dsRNA molecule of the first aspect comprises 21 consecutive bases of the sense strand of any dsRNA molecule selected from "Table A" excluding the ginseng molecule, and the antisense strand of the dsRNA molecule of the first aspect comprises 23 consecutive bases of the antisense strand of the said dsRNA molecule.

[0143] In some embodiments, more than 80%, 85%, 90%, or 95% of the antisense strand of the dsRNA molecule of the first aspect is complementary to ALK7 mRNA (e.g., the sequence shown in SEQ ID NO. 333), or the antisense strand of the dsRNA molecule is completely complementary to ALK7 mRNA. In some embodiments, more than 80%, 85%, 90%, or 95% of the base sequence of the sense strand of the dsRNA molecule of the first aspect is contained in the base sequence of ALK7 mRNA (e.g., the sequence shown in SEQ ID NO. 333), or the full-length base sequence of the sense strand of the dsRNA molecule is contained in the base sequence of ALK7 mRNA. In some embodiments, more than 80%, 85%, 90%, or 95% of the antisense strand of the dsRNA molecule of the first aspect is complementary to ALK7 mRNA, or the antisense strand of the dsRNA molecule is completely complementary to ALK7 mRNA, and the base sequence of more than 80%, 85%, 90%, or 95% of the sense strand of the dsRNA molecule of the first aspect is contained in the ALK7 mRNA base sequence, or the full-length base sequence of the sense strand of the dsRNA molecule is contained in the ALK7 mRNA base sequence. In some embodiments, more than 95% of the antisense strand of the dsRNA molecule of the first aspect is complementary to ALK7 mRNA, and the base sequence of more than 95% of the sense strand of the dsRNA molecule of the first aspect is contained in the ALK7 mRNA base sequence.

[0144] In some embodiments, the sense strand of the dsRNA molecule of the first aspect includes a salient terminus composed of 1, 2, or 3 nucleotides. In some embodiments, the antisense strand of the dsRNA molecule of the first aspect includes a salient terminus composed of 1, 2, or 3 nucleotides. In some embodiments, the sense strand of the dsRNA molecule of the first aspect includes a salient terminus, and the antisense strand of the dsRNA molecule of the first aspect does not include a salient terminus. In some embodiments, the sense strand of the dsRNA molecule of the first aspect does not include a salient terminus, and the antisense strand of the dsRNA molecule of the first aspect includes a salient terminus. In some embodiments, both the sense strand and the antisense strand of the dsRNA molecule of the first aspect include salient terms. In some embodiments, the salient terminus of the dsRNA molecule of the first aspect is located on the antisense strand. In some embodiments, the salient terminus of the dsRNA molecule of the first aspect is located only on the antisense strand. In some embodiments, the salient terminus of the dsRNA molecule of the first aspect is located on the sense strand. In some embodiments, the salient terminus of the dsRNA molecule of the first aspect is located only on the sense strand. In some embodiments, the dsRNA molecule of the first aspect has a convex 5' end and a blunt 3' end. In some embodiments, the dsRNA molecule of the first aspect has a convex 3' end and a blunt 5' end. In some embodiments, both the 3' and 5' ends of the dsRNA molecule of the first aspect are blunt ends. In some embodiments, both the 3' and 5' ends of the dsRNA molecule of the first aspect are convex ends. In some embodiments, the dsRNA molecule of the first aspect includes a convex end, and the convex end is located only at the 3' end of the antisense strand. In some embodiments, the dsRNA molecule of the first aspect includes a convex end, and the convex end is located only at the 5' end of the antisense strand. In some embodiments, the dsRNA molecule of the first aspect includes a convex end, and the convex end is located only at the 3' end of the sense strand. In some embodiments, the dsRNA molecule of the first aspect includes a convex end, and the convex end is located only at the 5' end of the sense strand. In some embodiments, the convex end of the dsRNA molecule is located at both the 5' end of the sense strand and the 5' end of the antisense strand. In some embodiments, the dsRNA molecule has a convex terminus located at the 3' end of both the sense strand and the antisense strand. In some embodiments, the convex terminus consists of two nucleotides. In some embodiments, the dsRNA has only one convex terminus located at the 3' end of the antisense strand and consisting of two nucleotides.

[0145] In some embodiments, the complementary region 1 of the nucleic acid molecule has 16, 17, 18, 19, 20, 21, 22, or 23 bp base pairs. In some embodiments, the complementary region 2 of the nucleic acid molecule has 15, 16, 17, 18, 19, 20, or 21 bp base pairs. In some embodiments, the complementary region 1 has 22 bp base pairs, and the complementary region 2 has 21 bp base pairs. In some embodiments, the complementary region 1 has 22 bp base pairs, and the complementary region 2 has 21 bp base pairs. In some embodiments, the complementary region 1 has 23 bp base pairs, and the complementary region 2 has 21 bp base pairs. In some embodiments, the complementary region 1 and the complementary region 2 have 15, 16, 17, 18, 19, 20, 21, 22, or 23 identical base pairs. In some embodiments, complementary regions 1 and 2 have 19 to 25, 20 to 24, 21 to 23, or 22 to 23 identical base pairs.

[0146] In some embodiments, complementary region 1 and complementary region 2 have 21 identical base pairs.

[0147] In some implementations, the length of the sense sequence of the first aspect nucleic acid molecule is 21 nt, and the length of the antisense sequence is 23 nt.

[0148] In some embodiments, the sense and / or antisense sequences of the nucleic acid molecule of the first aspect further include one or two overhanging nucleotides outside the complementary region 2. In some embodiments, there are two overhanging nucleotides located in the antisense sequence, adjacent to the 5' end of the complementary region 2, and the sense sequence does not contain any overhanging nucleotides. In some embodiments, the antisense sequence contains only 0, 1, or 2 nucleotides in the region outside the complementary region 1, and the sense sequence contains only 0, 1, or 2 nucleotides outside the complementary region 2. In some embodiments, the 3' end of the complementary region 2 is an AU base pair. "The 3' end of the complementary region 2 is an AU base pair" includes the following two cases: (1) the sense nucleotide in the 3' end of the complementary region 2 is A, and the antisense nucleotide is U; (2) the sense nucleotide in the 3' end of the complementary region 2 is U, and the antisense nucleotide is A. In some embodiments, the antisense nucleotide in the 3' end of the complementary region 2 is complementary to the target mRNA in the case of "the 3' end of the complementary region 2 is an AU base pair". In some embodiments, the antisense nucleotide at the 3' end of complementary region 2, where "the 3' end of complementary region 2 is an AU base pair," is not complementary to the target mRNA. In some embodiments, the 5' end of the antisense strand in the nucleic acid molecule forms a blunt end with the 3' end of the sense strand, and this blunt end is an AU base pair; that is, the 3' end of complementary region 2 is this blunt end, and this blunt end is an AU base pair. In some embodiments, the 3' end of the dsRNA molecule of the first aspect is a blunt end, and this 3' end base pair is an AU base pair. In some embodiments, the AU base pair consists of an A at the 3' end of the sense strand of the dsRNA and a U at the 5' end of the antisense strand of the dsRNA. In some embodiments, the AU base pair consists of a U at the 3' end of the sense strand of the dsRNA and an A at the 5' end of the antisense strand of the dsRNA. In some embodiments, the sense strand sequence of the dsRNA molecule of the first aspect consists of 21 bases, and the antisense strand sequence consists of 23 bases.

[0149] In some embodiments, (1) the sense sequence of the dsRNA molecule comprises or is the sequence shown in SEQ ID NO.1, and the antisense sequence of the dsRNA molecule comprises or is the sequence shown in SEQ ID NO.2; or

[0150] (2) The sense strand sequence of the dsRNA molecule includes or is the sequence shown in SEQ ID NO.3, and the antisense strand sequence of the dsRNA molecule includes or is the sequence shown in SEQ ID NO.4; or

[0151] (3) The sense strand sequence of the dsRNA molecule includes or is the sequence shown in SEQ ID NO. 5, and the antisense strand sequence of the dsRNA molecule includes or is the sequence shown in SEQ ID NO. 6; or

[0152] (4) The sense strand sequence of the dsRNA molecule includes or is the sequence shown in SEQ ID NO. 7, and the antisense strand sequence of the dsRNA molecule includes or is the sequence shown in SEQ ID NO. 8; or

[0153] (5) The sense sequence of the dsRNA molecule comprises or is the sequence shown in SEQ ID NO. 9, and the antisense sequence of the dsRNA molecule comprises or is the sequence shown in SEQ ID NO. 10; or

[0154] (6) The sense strand sequence of the dsRNA molecule includes or is the sequence shown in SEQ ID NO. 15, and the antisense strand sequence of the dsRNA molecule includes or is the sequence shown in SEQ ID NO. 16; or

[0155] (7) The sense sequence of the dsRNA molecule comprises or is the sequence shown in SEQ ID NO. 17, and the antisense sequence of the dsRNA molecule comprises or is the sequence shown in SEQ ID NO. 18; or

[0156] (8) The sense sequence of the dsRNA molecule includes or is the sequence shown in SEQ ID NO. 31, and the antisense sequence of the dsRNA molecule includes or is the sequence shown in SEQ ID NO. 32; or

[0157] (9) The sense strand sequence of the dsRNA molecule includes or is the sequence shown in SEQ ID NO. 33, and the antisense strand sequence of the dsRNA molecule includes or is the sequence shown in SEQ ID NO. 34; or

[0158] (10) The sense strand sequence of the dsRNA molecule comprises or is the sequence shown in SEQ ID NO. 35, and the antisense strand sequence of the dsRNA molecule comprises or is the sequence shown in SEQ ID NO. 36; or

[0159] (11) The sense strand sequence of the dsRNA molecule comprises or is the sequence shown in SEQ ID NO. 37, and the antisense strand sequence of the dsRNA molecule comprises or is the sequence shown in SEQ ID NO. 38; or

[0160] (12) The sense strand sequence of the dsRNA molecule includes or is the sequence shown in SEQ ID NO. 39, and the antisense strand sequence of the dsRNA molecule includes or is the sequence shown in SEQ ID NO. 40; or

[0161] (13) The sense strand sequence of the dsRNA molecule includes or is the sequence shown in SEQ ID NO.41, and the antisense strand sequence of the dsRNA molecule includes or is the sequence shown in SEQ ID NO.42; or

[0162] (14) The sense strand sequence of the dsRNA molecule includes or is the sequence shown in SEQ ID NO.47, and the antisense strand sequence of the dsRNA molecule includes or is the sequence shown in SEQ ID NO.48; or

[0163] (15) The sense strand sequence of the dsRNA molecule comprises or is the sequence shown in SEQ ID NO.49, and the antisense strand sequence of the dsRNA molecule comprises or is the sequence shown in SEQ ID NO.50; or

[0164] (16) The sense strand sequence of the dsRNA molecule includes or is the sequence shown in SEQ ID NO. 63, and the antisense strand sequence of the dsRNA molecule includes or is the sequence shown in SEQ ID NO. 64; or

[0165] (17) The sense strand sequence of the dsRNA molecule comprises or is the sequence shown in SEQ ID NO. 65, and the antisense strand sequence of the dsRNA molecule comprises or is the sequence shown in SEQ ID NO. 66; or

[0166] (18) The sense strand sequence of the dsRNA molecule includes or is the sequence shown in SEQ ID NO.69, and the antisense strand sequence of the dsRNA molecule includes or is the sequence shown in SEQ ID NO.70.

[0167] In some embodiments, the sense strand sequence of the dsRNA molecule of the first aspect comprises the sense strand selected from any dsRNA molecule other than the *Gynostemma pentaphyllum* molecule in "Table A", and the antisense strand of the dsRNA molecule of the first aspect is identical to the antisense strand of the aforementioned dsRNA molecule. In some embodiments, the antisense strand sequence of the dsRNA molecule of the first aspect comprises the antisense strand selected from any dsRNA molecule other than the *Gynostemma pentaphyllum* molecule in "Table A", and the sense strand of the dsRNA molecule of the first aspect is identical to the sense strand of the aforementioned dsRNA molecule.

[0168] In some embodiments, the 3' terminal base pair of the dsRNA molecule of the first aspect is an AU base pair (optionally, the 3' terminal base of the sense strand is A and the 5' terminal base of the antisense strand is U), and:

[0169] (1) The sense strand sequence of the dsRNA molecule comprises 20 consecutive bases starting from the 5' end of the sequence shown in SEQ ID NO.1, and the antisense strand sequence of the dsRNA molecule comprises 22 consecutive bases starting from the 3' end of the sequence shown in SEQ ID NO.2; or

[0170] (2) The sense strand sequence of the dsRNA molecule comprises 20 consecutive bases starting from the 5' end of the sequence shown in SEQ ID NO.3, and the antisense strand sequence of the dsRNA molecule comprises 22 consecutive bases starting from the 3' end of the sequence shown in SEQ ID NO.4; or

[0171] (3) The sense strand sequence of the dsRNA molecule comprises 20 consecutive bases starting from the 5' end of the sequence shown in SEQ ID NO.5, and the antisense strand sequence of the dsRNA molecule comprises 22 consecutive bases starting from the 3' end of the sequence shown in SEQ ID NO.6; or

[0172] (4) The sense strand sequence of the dsRNA molecule comprises 20 consecutive bases starting from the 5' end of the sequence shown in SEQ ID NO.7, and the antisense strand sequence of the dsRNA molecule comprises 22 consecutive bases starting from the 3' end of the sequence shown in SEQ ID NO.8; or

[0173] (5) The sense strand sequence of the dsRNA molecule comprises 20 consecutive bases starting from the 5' end of the sequence shown in SEQ ID NO.9, and the antisense strand sequence of the dsRNA molecule comprises 22 consecutive bases starting from the 3' end of the sequence shown in SEQ ID NO.10; or

[0174] (6) The sense strand sequence of the dsRNA molecule comprises 20 consecutive bases starting from the 5' end of the sequence shown in SEQ ID NO. 15, and the antisense strand sequence of the dsRNA molecule comprises 22 consecutive bases starting from the 3' end of the sequence shown in SEQ ID NO. 16; or

[0175] (7) The sense strand sequence of the dsRNA molecule comprises 20 consecutive bases starting from the 5' end of the sequence shown in SEQ ID NO. 17, and the antisense strand sequence of the dsRNA molecule comprises 22 consecutive bases starting from the 3' end of the sequence shown in SEQ ID NO. 18; or

[0176] (8) The sense strand sequence of the dsRNA molecule comprises 20 consecutive bases starting from the 5' end of the sequence shown in SEQ ID NO.31, and the antisense strand sequence of the dsRNA molecule comprises 22 consecutive bases starting from the 3' end of the sequence shown in SEQ ID NO.32.

[0177] In some embodiments, the 3' end base pair of the dsRNA molecule of the first aspect is an AU base pair, its sense strand base sequence comprises 20 consecutive bases from the 5' end of the sense strand of any dsRNA molecule selected from "Table A" excluding the ginseng molecule, and its antisense strand base sequence comprises 22 consecutive bases from the 3' end of the antisense strand of the said dsRNA molecule, and the sense strand base sequence of the dsRNA molecule of the first aspect consists of 21 bases, and the antisense strand base sequence of the dsRNA molecule of the first aspect consists of 23 bases.

[0178] In some implementations, each nucleotide in the dsRNA molecule of the first aspect independently contains any of the following modifications:

[0179] Modifications include locked nucleic acid modifications, ring-opening or non-locked nucleic acid modifications, 2′-methoxyethyl modifications, 2′-O-methyl modifications (2′-OMe), 2′-O-allyl modifications, 2′-C-alkyl modifications, 2′-C-allyl modifications, 2′-fluoro modifications (2′-F), 2′-deoxy modifications, thiophosphate modifications, 2′-amino-modification, morpholino modifications, aminophosphate modifications, methylphosphonate modifications, tetrahydropyran modifications, 1,5-dehydrated hexitol modifications, 5′-phosphate, 5′-phosphate mimic modifications, cyclohexenyl modifications, and invAb (reverse debased deoxyribonucleotide) modifications. The “phosphate mimic” modifications include, but are not limited to, aminophosphate modifications, methylphosphonate modifications, thiophosphate modifications, and vinylphosphonate modifications. In some embodiments, the 5′-phosphate mimic modification is a 5′-vinylphosphonate modification (5′-VP) or a 5′-cyclopropyl phosphate modification (5′-cPrp).

[0180] In some implementations, the 2' hydroxyl groups of the nucleotides at positions 2, 14, and 16 of the antisense strand of the dsRNA molecule in the first aspect are replaced with fluorine, and the 2' hydroxyl groups of the other nucleotides in the antisense strand are replaced with methoxy or hydrogen.

[0181] In some embodiments, the 2' hydroxyl groups of the 2nd, 14th, and 16th nucleotides of the antisense strand in the dsRNA molecule of the first aspect are replaced with fluorine, and the 2' hydroxyl groups of the other nucleotides of the antisense strand are replaced with methoxy groups.

[0182] In some embodiments, in the dsRNA molecule of the first aspect, the 2' hydroxyl groups of the nucleotides at positions 2, 14, and 16 of the antisense strand starting from the 5' end are replaced with fluorine, the 2' hydroxyl groups of the nucleotides at positions 5 and 7 are replaced with hydrogen, and the 2' hydroxyl groups of the other nucleotides of the antisense strand are all replaced with methoxy groups.

[0183] In some embodiments, the 2' hydroxyl groups of the sense strand at positions 9 and 11, starting from the 5' end, in the dsRNA molecule of the first aspect are replaced by fluorine, the 2' hydroxyl group at position 10 is replaced by fluorine or hydrogen, and the 2' hydroxyl groups of the other nucleotides in the sense strand are replaced by fluorine, methoxy, or hydrogen.

[0184] In some embodiments, in the dsRNA molecule of the first aspect, the 2' hydroxyl groups of the sense strand at positions 9 and 11, starting from the 5' end, are replaced by fluorine; the 2' hydroxyl group of the sense strand at position 10 is replaced by fluorine or hydrogen; the 2' hydroxyl groups of the sense strand at positions 7 and 12 are replaced by fluorine or methoxy; and the 2' hydroxyl groups of the other nucleotides in the sense strand are replaced by methoxy or hydrogen.

[0185] In some embodiments, in the dsRNA molecule of the first aspect, the 2' hydroxyl groups of the sense strand at positions 9 and 11, starting from the 5' end, are replaced by fluorine; the 2' hydroxyl group of the sense strand at position 10 is replaced by fluorine or hydrogen; the 2' hydroxyl groups of the sense strand at positions 7 and 12 are replaced by fluorine or methoxy; and the 2' hydroxyl groups of the other nucleotides in the sense strand are all replaced by methoxy.

[0186] In some implementations, the first aspect of the dsRNA molecule:

[0187] The 2' hydroxyl groups of the 9th and 11th nucleotides of the sense chain starting from the 5' end are replaced by fluorine, the 2' hydroxyl group of the 10th nucleotide is replaced by hydrogen, and the 2' hydroxyl groups of the other nucleotides of the sense chain are all replaced by methoxy groups.

[0188] The 2' hydroxyl groups of the 7th, 9th and 11th nucleotides of the sense chain starting from the 5' end are replaced by fluorine, the 2' hydroxyl group of the 10th nucleotide is replaced by hydrogen, and the 2' hydroxyl groups of the other nucleotides of the sense chain are replaced by methoxy groups.

[0189] In the sense chain, the 2' hydroxyl groups at positions 7, 9, 10, 11, and 12 (starting from the 5' end) are substituted with fluorine, while the 2' hydroxyl groups at the other nucleotides in the sense chain are substituted with methoxy groups; or

[0190] The 2' hydroxyl groups of the 7th, 9th, 10th, and 11th nucleotides of the sense chain, starting from the 5' end, are replaced by fluorine, while the 2' hydroxyl groups of the other nucleotides in the sense chain are replaced by methoxy groups.

[0191] In some embodiments, the 2' hydroxyl groups of the nucleotides at positions 2, 14, and 16 of the antisense strand of the dsRNA molecule from the 5' end are replaced with fluorine, and the 2' hydroxyl groups of the other nucleotides in the antisense strand are replaced with methoxy or hydrogen. In the sense strand, the 2' hydroxyl groups of the nucleotides at positions 9 and 11 from the 5' end are replaced with fluorine, the 2' hydroxyl group of the nucleotide at position 10 is replaced with fluorine or hydrogen, and the 2' hydroxyl groups of the other nucleotides in the sense strand are replaced with fluorine, methoxy, or hydrogen.

[0192] In some embodiments, in the dsRNA molecule of the first aspect, the 2' hydroxyl groups of the nucleotides at positions 2, 14, and 16 of the antisense strand starting from the 5' end are replaced with fluorine, the 2' hydroxyl groups of the other nucleotides of the antisense strand are all replaced with methoxy groups, and the 2' hydroxyl groups of the nucleotides at positions 9 and 11 of the sense strand starting from the 5' end are replaced with fluorine, the 2' hydroxyl group of the nucleotide at position 10 is replaced with fluorine or hydrogen, and the 2' hydroxyl groups of the other nucleotides of the sense strand are all replaced with fluorine, methoxy, or hydrogen.

[0193] In some embodiments, in the dsRNA molecule of the first aspect, the 2' hydroxyl groups of the nucleotides at positions 2, 14, and 16 of the antisense strand starting from the 5' end are replaced with fluorine, the 2' hydroxyl groups of the nucleotides at positions 5 and 7 are replaced with hydrogen, the 2' hydroxyl groups of the other nucleotides of the antisense strand are all replaced with methoxy groups, and the 2' hydroxyl groups of the nucleotides at positions 9 and 11 of the sense strand starting from the 5' end are replaced with fluorine, the 2' hydroxyl group of the nucleotide at position 10 is replaced with fluorine or hydrogen, and the 2' hydroxyl groups of the other nucleotides of the sense strand are all replaced with fluorine, methoxy, or hydrogen.

[0194] In some implementations, the 2' hydroxyl groups of the 7th, 9th, 10th, 11th, and 12th nucleotides of the sense strand of the first aspect dsRNA molecule are substituted with fluorine, and the 2' hydroxyl groups of the other nucleotides of the sense strand are substituted with methoxy groups. In addition, the 2' hydroxyl groups of the 2nd, 14th, and 16th nucleotides of the antisense strand are substituted with fluorine, and the 2' hydroxyl groups of the other nucleotides of the antisense strand are substituted with methoxy groups.

[0195] In some implementations, the 2' hydroxyl groups of the 7th, 9th, 10th, and 11th nucleotides of the sense strand starting from the 5' end of the dsRNA molecule of the first aspect are replaced with fluorine, and the 2' hydroxyl groups of the other nucleotides of the sense strand are all replaced with methoxy groups. In addition, the 2' hydroxyl groups of the 2nd, 14th, and 16th nucleotides of the antisense strand starting from the 5' end are replaced with fluorine, and the 2' hydroxyl groups of the other nucleotides of the antisense strand are all replaced with methoxy groups.

[0196] In some embodiments, the 2' hydroxyl groups of the sense strand of the first aspect dsRNA molecule at positions 9 and 11, starting from the 5' end, are substituted with fluorine, and the 2' hydroxyl group of the sense strand at position 10 is substituted with hydrogen. The 2' hydroxyl groups of the other nucleotides in the sense strand are substituted with methoxy groups. In the antisense strand, the 2' hydroxyl groups of the antisense strand at positions 2, 14, and 16, starting from the 5' end, are substituted with fluorine, and the 2' hydroxyl groups of the 5th and 7th nucleotides are substituted with hydrogen. The 2' hydroxyl groups of the other nucleotides in the antisense strand are substituted with methoxy groups.

[0197] In some embodiments, the 2' hydroxyl groups of the 7th, 9th, and 11th nucleotides of the sense strand starting from the 5' end of the dsRNA molecule of the first aspect are replaced with fluorine, the 2' hydroxyl group of the 10th nucleotide is replaced with hydrogen, and the 2' hydroxyl groups of the other nucleotides of the sense strand are all replaced with methoxy groups. In the antisense strand, the 2' hydroxyl groups of the 2nd, 14th, and 16th nucleotides starting from the 5' end of the antisense strand are replaced with fluorine, the 2' hydroxyl groups of the 5th and 7th nucleotides are replaced with hydrogen, and the 2' hydroxyl groups of the other nucleotides of the antisense strand are all replaced with methoxy groups.

[0198] In some embodiments, the antisense strand of the dsRNA molecule of the first aspect contains a phosphate ester or phosphate ester mimic modification at its 5' end. In some embodiments, the antisense strand of the dsRNA molecule of the first aspect contains a 5'-VP modification or a 5'-cPrp modification at its 5' end.

[0199] In some implementations, the dsRNA molecule of the first aspect comprises sense strand 5' and / or 3' nucleotides linked to invAb monomers.

[0200] In some embodiments, the dsRNA molecule of the first aspect contains a phosphate thioester modification at at least one of the following positions, or at all of the following positions:

[0201] Between the first and second nucleotides starting at the 5' end of the sense strand,

[0202] Between the second and third nucleotides starting from the 5' end of the sense strand,

[0203] Between the first and second nucleotides starting at the 3' end of the sense strand,

[0204] Between the second and third nucleotides starting from the 3' end of the sense strand,

[0205] Between the first and second nucleotides starting at the 5' end of the antisense strand,

[0206] Between the second and third nucleotides starting from the 5' end of the antisense strand,

[0207] Between the first and second nucleotides starting from the 3' end of the antisense strand, and

[0208] Between the second and third nucleotides starting from the 3' end of the antisense strand.

[0209] In some implementations, the dsRNA molecule of the first aspect contains one or more lipophilic moieties.

[0210] In some implementations, the octanol-water partition coefficient logK is used. ow The measured lipophilicity of the lipophilic portion exceeds 0. The logK of the lipophilic portion... ow It can exceed 1, 1.5, 2, 3, 4, 5 or 10.

[0211] In some embodiments, after the lipophilic portion of the dsRNA of the first aspect is attached, the hydrophobicity of the dsRNA molecule, as measured by the unbound fraction in a plasma protein binding assay of the dsRNA molecule, is greater than 0.2. In some embodiments, the plasma protein binding assay uses an electrophoretic mobility shift assay (EMSA) of human serum albumin, and the hydrophobicity of the dsRNA molecule of the first aspect, as measured by the fraction of unbound dsRNA in the plasma protein binding assay, is greater than 0.15, greater than 0.2, greater than 0.25, greater than 0.3, greater than 0.35, greater than 0.4, greater than 0.45, or greater than 0.5 to enhance in vivo delivery of the dsRNA.

[0212] In some embodiments, each of the lipophilic portions independently comprises one or more structures selected from: saturated or unsaturated C4-C. 30 Hydrocarbon chain, saturated or unsaturated C6-C 30 Acid, saturated or unsaturated C6-C 30 Alcohols, saturated or unsaturated C6-C 30 Amines, cholesterol, bile acids, vitamins, peptides, and steroids;

[0213] Optionally, one or more carbon atoms in the above structure and the hydrogen atoms directly connected to the one or more carbon atoms may be independently replaced with one or more of the following: -O-, -S-, -NH-, -C(O)-, -C(S)-, -S(O)-, -S(O)2-, -SS-, -C(O)O-, -OC(O)-, -C(O)NH-, -NHC(O)-, -OP(O)(OH)O-, -OP(O)(SH)O-, -S(O)2NH-, -NHS(O)2-, -N≡N-, -C≡C-.

[0214] In some embodiments, the lipophilic moiety is aliphatic, such as cyclic (e.g., monocyclic or polycyclic) aliphatic (e.g., polycyclic compounds), such as steroids (e.g., sterols), or straight-chain or branched aliphatic hydrocarbons. Examples of lipophilic moieties include cholesterol, retinoic acid, cholic acid, adamantaneacetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis-O-(hexadecyl)glycerol, geraniol, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecanyl, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, ibuprofen, naproxen, dimethoxytriphenylmethyl, or phenoxazine.

[0215] In some embodiments, the lipophilic moiety is saturated or unsaturated C4-C. 30 Hydrocarbon chains (e.g., C4-C) 30 Alkyl or alkenyl groups, optionally wherein one or more carbon atoms and the hydrogen atoms directly attached thereto may be independently replaced by one or more -O-, -S-, -NH-, -C(O)-, -C(S)-, -S(O)-, -S(O)2-, -SS-, -C(O)O-, -OC(O)-, -C(O)NH-, -NHC(O)-, -OP(O)(OH)O-, -OP(O)(SH)O-, -S(O)2NH-, -NHS(O)2-, -N≡N- (i.e., azide group), -C≡C- (i.e., alkynyl group). In some embodiments, the lipophilic moiety contains saturated or unsaturated C6-C6 groups. 26 Hydrocarbon chains (e.g., straight-chain or branched C6-C) 26 Alkyl or alkenyl groups). In some embodiments, the lipophilic moiety contains saturated or unsaturated C8-C6 groups. 24 Hydrocarbon chains (e.g., straight-chain or branched C8-C) 24 Alkyl or alkenyl groups). In some embodiments, the lipophilic moiety contains saturated or unsaturated C64 groups. 10 -C 22 Hydrocarbon chains (e.g., straight or branched) C6 -C 26 Alkyl or alkenyl groups). In some embodiments, the lipophilic moiety contains saturated or unsaturated C64 groups. 12 -C 22 Hydrocarbon chains (e.g., straight-chain or branched C) 12- C 22 Alkyl or alkenyl groups). In some embodiments, the lipophilic moiety contains saturated or unsaturated C64 groups. 16 Hydrocarbon chains (e.g., straight-chain or branched C) 16 alkyl or alkenyl), C 17 Hydrocarbon chains (e.g., straight-chain or branched C) 17 alkyl or alkenyl), C 18 Hydrocarbon chains (e.g., straight-chain or branched C) 18 alkyl or alkenyl), C 19Hydrocarbon chains (e.g., straight-chain or branched C) 19 alkyl or alkenyl), C 20 Hydrocarbon chains (e.g., straight-chain or branched C) 20 alkyl or alkenyl), C 21 Hydrocarbon chains (e.g., straight-chain or branched C) 21 alkyl or alkenyl), C 22 Hydrocarbon chains (e.g., straight-chain or branched C) 22 Alkyl or alkenyl). In some embodiments, one or more carbon atoms and the hydrogen atoms directly attached thereto in the lipophilic moiety described above may be independently replaced with one or more -O-, -S-, -NH-, -C(O)-, -SS-, -C(O)O-, -OC(O)-, -C(O)NH-, -NHC(O)-.

[0216] In some embodiments, the lipophilic portion is saturated or unsaturated C6-C. 30 Acids (e.g., hexanoic acid, heptanoic acid, octanoic acid, nonanoic acid, decanoic acid, undecanoic acid, dodecanoic acid, tridecanoic acid, tetradecanoic acid, pentadecanoic acid, hexadecanoic acid, heptadecanoic acid, octadecanoic acid, oleic acid, linoleic acid, arachidonic acid, cis-4,7,10,13,16,19-docosahexaenoic acid, vitamin A, vitamin E, cholesterol, etc.) or saturated or unsaturated C6-C 30 Alcohols (e.g., hexanol, heptanol, octanol, nonanol, decanol, undecanol, dodecanol, tridecanol, tetradecanol, pentadecanol, hexadecanol, heptadecanol, octadecanol, oleyl alcohol, linolenic acid, arachidonic acid, cis-4,7,10,13,16,19-docosahexaenoic acid, retinol, vitamin E, cholesterol, etc.), optionally, the above C6-C 30 Acid or C6-C 30 One or more carbon atoms and their directly attached hydrogen atoms in an alcohol can be independently replaced by one or more of the following groups: -O-, -S-, -NH-, -C(O)-, -C(S)-, -S(O)-, -S(O)2-, -SS-, -C(O)O-, -OC(O)-, -C(O)NH-, -NHC(O)-, -OP(O)(OH)O-, -OP(O)(SH)O-, -S(O)2NH-, -NHS(O)2-, -N≡N- (i.e., azide group), and -C≡C- (i.e., alkynyl group).

[0217] In some embodiments, the lipophilic moiety is independently selected from one or more of the following structures: cholesterol, bile acid, vitamin, peptide, and steroid. Optionally, one or more carbon atoms and the hydrogen atoms directly attached thereto in the above structures may be independently replaced by one or more of the following groups: -O-, -S-, -NH-, -C(O)-, -C(S)-, -S(O)-, -S(O)2-, -SS-, -C(O)O-, -OC(O)-, -C(O)NH-, -NHC(O)-, -OP(O)(OH)O-, -OP(O)(SH)O-, -S(O)2NH-, -NHS(O)2-, -N≡N- (i.e., azide group), and -C≡C- (i.e., alkynyl group).

[0218] In some embodiments, the lipophilic portion comprises one or more molecules with the following structures, or the lipophilic portion is one or more molecules with the following structures: One of the above structures This indicates a nucleotide conjugation.

[0219] In some embodiments, the lipophilic portion comprises one or more molecules with the following structures, or the lipophilic portion is one or more molecules with the following structures: Each of the above structures contains two The structure, two Both indicate conjugation to a nucleotide in the sense or antisense strand, or one This indicates a nucleotide conjugation to either the sense or antisense strand, another It indicates that it is connected to hydrogen.

[0220] In some embodiments, the lipophilic portion may be directly attached to the ribose of the sense and / or antisense strands of the dsRNA, thereby conjugating the sense and / or antisense strands. Alternatively, the lipophilic portion may be conjugated to the sense and / or antisense strands of the dsRNA via a linker and / or a vector.

[0221] In some embodiments, the lipophilic portion is conjugated to the sense and / or antisense strands of the dsRNA via one or more linkers.

[0222] In some embodiments, the lipophilic portion is conjugated to the sense and / or antisense strand of the dsRNA molecule via a linker containing an ether, thioether, urea, carbonate, amine, amide, maleimide-thioether, disulfide, phosphodiester, sulfonamide bond, click reaction product (e.g., triazole from azide-alkyne cycloaddition), or carbamate. In some embodiments, at least one linker is a redox-cleavable linker (such as a reductant linker, e.g., a disulfide group), an acid-cleavable linker (e.g., an hydrazone, ester, acetal, or ketal group), an esterase-cleavable linker (e.g., an ester group), a phosphatase-cleavable linker (e.g., a phosphate ester), or a peptidase-cleavable linker (e.g., a peptide bond).

[0223] In some embodiments, at least one adapter is a biolytic adapter selected from one or more of the following: DNA, RNA, disulfides, amides, functionalized monosaccharides or oligosaccharides of galactosamine, glucosamine, glucose, galactose, mannose, and combinations thereof.

[0224] In some embodiments, the lipophilic portion is conjugated to the sense and / or antisense strands of the dsRNA via phosphate or thiophosphate links.

[0225] In some embodiments, the lipophilic portion is conjugated to the sense and / or antisense strand of the dsRNA molecule of the first aspect via a carrier, said carrier being a cyclic or acyclic group. In some embodiments, the cyclic group is selected from any one or more of the following: pyrrolidinyl, pyrazolinyl, pyrazolinyl, imidazolinyl, imidazolinyl, piperidinyl, piperazinyl, [1,3]dioxolane, oxazolinyl, isoxazolinyl, morpholinyl, thiazolinyl, isothiazolinyl, quinoxolinyl, pyridazinone, tetrahydrofuranyl, and decahydronaphthalene, and their substitutes substituted with one or more conventional groups (such as hydroxyl, mercapto, amino, methoxy, ethoxy, methoxyethoxy, ethoxymethoxy, F, Cl, methyl, ethyl, trifluoromethyl, oxymethyl, oxyethyl, oxyvinyl, etc.). In some embodiments, the cyclic group is selected from: pyrrolyl, pyrazolinyl, pyrazolinyl, imidazolinyl, imidazolinyl, piperidinyl, and tetrahydrofuranyl, and its derivatives substituted with one or more of the groups selected from hydroxyl, methoxy, ethoxy, methoxyethoxy, F, methyl, ethyl, and oxymethyl. In some embodiments, the acyclic group is a portion based on the serine or diethanolamine backbone, and its derivatives substituted with one or more conventional groups (such as hydroxyl, mercapto, amino, methoxy, ethoxy, methoxyethoxy, ethoxymethoxy, F, Cl, methyl, ethyl, trifluoromethyl, oxymethyl, oxyethyl, oxyvinyl, etc.).

[0226] In some embodiments, the lipophilic portion is conjugated to the dsRNA via one or more adapters and one or more vectors.

[0227] In some embodiments, the lipophilic moiety is conjugated to the dsRNA via a phosphate ester or thiophosphate linker and a carrier selected from: pyrrolyl, pyrazolinyl, pyrazolinyl, imidazolinyl, imidazolinyl, piperidinyl, piperazinyl, and tetrahydrofuranyl, and thereof substituted with one or more of the following: hydroxyl, methoxy, ethoxy, methoxyethoxy, F, methyl, ethyl, or oxymethyl. In some embodiments, the lipophilic moiety is conjugated to the dsRNA via a thiophosphate linker and a carrier selected from: pyrrolyl, pyrazolinyl, pyrazolinyl, imidazolinyl, imidazolinyl, piperidinyl, piperazinyl, and tetrahydrofuranyl, and thereof substituted with one or more of the following: hydroxyl, methoxy, ethoxy, methoxyethoxy, F, methyl, ethyl, or oxymethyl.

[0228] In some embodiments, the lipophilic portion is conjugated to one or more nucleotides at an internal location in the dsRNA molecule of the first aspect.

[0229] In some embodiments, the lipophilic moiety is conjugated to one or more nucleotides at the end of the sense or antisense strand. In some embodiments, the lipophilic moiety is conjugated to a terminal nucleotide at the 3' end of the sense strand, thereby acting as an end cap protecting the 3' end of the sense strand. In some embodiments, the lipophilic moiety is conjugated to one or more nucleotides at the end of the sense or antisense strand via a phosphate ester or thiophosphate linker and a carrier, specifically, the carrier may be pyrrolidinyl, pyrazolinyl, pyrazolinyl, imidazolinyl, imidazolinyl, piperidinyl, piperazinyl, and tetrahydrofuranyl, and its substitutes thereof substituted with one or more conventional groups (such as hydroxyl, mercapto, amino, methoxy, ethoxy, methoxyethoxy, ethoxymethoxy, F, Cl, methyl, ethyl, trifluoromethyl, oxymethyl, oxyethyl, oxyvinyl, etc.).

[0230] In some embodiments, the lipophilic portion is conjugated to one or more internal positions on at least one strand of the dsRNA, the internal positions covering all positions except the two terminal positions at each end of the strand.

[0231] In some embodiments, at least one (e.g., 1, 2, 3, 4, 5, or 6) of the lipophilic moieties is conjugated to one or more positions at at least one end of the sense strand and / or antisense strand of the dsRNA. In some embodiments, at least one of the lipophilic moieties is conjugated to the last nucleotide at at least one end of the sense strand and / or antisense strand of the dsRNA. In some embodiments, at least one of the lipophilic moieties is conjugated to the last nucleotide at at least one end of the sense strand of the dsRNA. In some embodiments, at least one of the lipophilic moieties is conjugated to the last nucleotide at the 3' end of the sense strand of the dsRNA. In some embodiments, at least one of the lipophilic moieties is conjugated to the last nucleotide at the 5' end of the sense strand of the dsRNA. In some embodiments, at least one of the lipophilic moieties is conjugated to the last nucleotide at both the 3' and 5' ends of the sense strand of the dsRNA.

[0232] In some embodiments, at least one of the lipophilic moieties is conjugated to the first five nucleotides of the sense strand at the 3' and / or 5' ends of the dsRNA.

[0233] In some embodiments, at least one lipophilic moiety is conjugated to the first four nucleotides at the 3' and / or 5' ends of the sense strand of the dsRNA.

[0234] In some embodiments, at least one lipophilic moiety is conjugated to the first three nucleotides at the 3' and / or 5' ends of the sense strand of the dsRNA.

[0235] In some embodiments, at least one lipophilic moiety is conjugated to the first two nucleotides at the 3' and / or 5' ends of the sense strand of the dsRNA.

[0236] In some embodiments, at least one lipophilic moiety is conjugated to the first nucleotide at the 3' and / or 5' end of the sense strand of the dsRNA.

[0237] In some embodiments, at least one lipophilic moiety is conjugated to the first nucleotide at the 3' and 5' ends of the sense strand of the dsRNA, respectively.

[0238] In some embodiments, the lipophilic portion is conjugated to one or more internal positions on at least one strand of the dsRNA that do not include cleavage sites on the sense strand. For example, the internal positions do not include positions 9-12 counted from the 5' end of the sense strand. For example, the internal positions do not include positions 9-11 counted from the 5' end of the sense strand. Optionally, the internal positions do not include positions 11-13 counted from the 3' end of the sense strand.

[0239] In some embodiments, the lipophilic portion is conjugated to one or more internal positions on at least one strand that do not include cleavage sites of the antisense strand. For example, the internal positions do not include positions 12-14 counted from the 5' end of the sense strand.

[0240] In some embodiments, the lipophilic portion is attached to one or more internal positions on at least one chain, excluding positions 11-13 on the sense chain counting from the 3' end and positions 12-14 on the antisense chain counting from the 5' end.

[0241] In some embodiments, one or more of the lipophilic portions are conjugated to one or more of the following internal positions: counting from the 5' end of each chain, positions 4-8 and 13-18 on the sense chain, and positions 6-10 and 15-18 on the antisense chain.

[0242] In some embodiments, one or more of the lipophilic portions are conjugated to one or more of the following internal locations: counting from the 5' end of each chain, positions 5, 6, 7, 15 and 17 on the sense chain, and positions 15 and 17 on the antisense chain.

[0243] In some embodiments, the lipophilic portion is attached to the 5' end of the sense strand of the dsRNA of the first aspect. In some embodiments, the lipophilic portion is attached to the 3' end of the sense strand of the dsRNA of the first aspect. In some embodiments, the lipophilic portion is attached to both the 5' and 3' ends of the sense strand of the dsRNA of the first aspect. In some embodiments, the lipophilic portion is attached to the 5' end of the antisense strand of the dsRNA of the first aspect. In some embodiments, the lipophilic portion is attached to the 3' end of the antisense strand of the dsRNA of the first aspect. In some embodiments, the lipophilic portion is attached to both the 5' and 3' ends of the antisense strand of the dsRNA of the first aspect.

[0244] In some embodiments, the lipophilic portion is attached only to the 5' end of the sense strand of the dsRNA of the first aspect. In some embodiments, the lipophilic portion is attached only to the 3' end of the sense strand of the dsRNA of the first aspect. In some embodiments, the lipophilic portion is attached only to both the 5' and 3' ends of the sense strand of the dsRNA of the first aspect. In some embodiments, the lipophilic portion is attached only to the 5' end of the antisense strand of the dsRNA of the first aspect. In some embodiments, the lipophilic portion is attached only to the 3' end of the antisense strand of the dsRNA of the first aspect. In some embodiments, the lipophilic portion is attached only to both the 5' and 3' ends of the antisense strand of the dsRNA of the first aspect.

[0245] In some embodiments, the lipophilic portion is attached to the 9th, 10th, 11th, and / or 12th nucleotides counting from the 5' end of the sense strand of the dsRNA in the first aspect. In some embodiments, the lipophilic portion is attached to the 19th, 20th, and / or 21st nucleotides counting from the 5' end of the sense strand of the dsRNA in the first aspect. In some embodiments, the lipophilic portion is attached to the 9th, 10th, 11th, and / or 12th nucleotides counting from the 5' end of the sense strand of the dsRNA in the first aspect; and the lipophilic portion is also attached to the 19th, 20th, and / or 21st nucleotides counting from the 5' end of the sense strand of the dsRNA in the first aspect.

[0246] In some embodiments, the dsRNA molecule of the first aspect is further linked to one or more of the aforementioned lipophilic moieties, and the dsRNA molecule of the first aspect further contains a phosphate thioester modification at at least one of the following positions, or contains a phosphate thioester modification at all of the following positions:

[0247] Between the first and second nucleotides starting at the 5' end of the sense strand,

[0248] Between the second and third nucleotides starting from the 5' end of the sense strand,

[0249] Between the first and second nucleotides starting at the 3' end of the sense strand,

[0250] Between the second and third nucleotides starting from the 3' end of the sense strand,

[0251] Between the first and second nucleotides starting at the 5' end of the antisense strand,

[0252] Between the second and third nucleotides starting from the 5' end of the antisense strand,

[0253] Between the first and second nucleotides starting from the 3' end of the antisense strand, and

[0254] Between the second and third nucleotides starting from the 3' end of the antisense strand.

[0255] In some embodiments, the dsRNA molecule of the first aspect is further linked to one or more of the aforementioned lipophilic moieties, and the dsRNA molecule of the first aspect further contains a phosphate thioester modification at at least one of the following positions, or contains a phosphate thioester modification at all of the following positions:

[0256] Between the first and second nucleotides starting at the 5' end of the sense strand,

[0257] Between the second and third nucleotides starting from the 5' end of the sense strand,

[0258] Between the first and second nucleotides starting at the 3' end of the sense strand,

[0259] Between the second and third nucleotides starting from the 3' end of the sense strand,

[0260] Between the first and second nucleotides starting at the 5' end of the antisense strand,

[0261] Between the second and third nucleotides starting from the 5' end of the antisense strand,

[0262] Between the first and second nucleotides starting from the 3' end of the antisense strand, and

[0263] Between the second and third nucleotides starting from the 3' end of the antisense strand;

[0264] In this embodiment, even if the lipophilic portion contains nucleotides or nucleotide analogs, when determining the position of the thiophosphate ester, the nucleotides or nucleotide analogs in the lipophilic portion are not counted as nucleotides in the sense or antisense strand.

[0265] In some embodiments, the dsRNA molecule of the first aspect has a lipophilic moiety attached to the 5' end and 3' end of the sense strand, respectively, and the dsRNA molecule of the first aspect contains phosphate thioester modifications at all of the following positions:

[0266] Between the first and second nucleotides starting at the 5' end of the sense strand,

[0267] Between the first nucleotide at the 5' end of the sense strand and the lipophilic moiety,

[0268] Between the first and second nucleotides starting at the 3' end of the sense strand,

[0269] Between the first nucleotide at the 3' end of the sense strand and the lipophilic moiety,

[0270] Between the first and second nucleotides starting at the 5' end of the antisense strand,

[0271] Between the second and third nucleotides starting from the 5' end of the antisense strand,

[0272] Between the first and second nucleotides starting from the 3' end of the antisense strand, and

[0273] Between the second and third nucleotides starting from the 3' end of the antisense strand, in this embodiment, even if the lipophilic portion contains nucleotides or nucleotide analogs, they are not counted as nucleotides in the sense strand when determining the position of the thiophosphate.

[0274] In some implementations, the nucleotide modifications in the dsRNA molecule of the first aspect are each independently selected from only one or more of the following: 2'-OMe modification, 2'-F modification, 2'-deoxy modification, thiophosphate modification, 5'-VP modification, 5'-cPrp modification, invAb modification, and lipophilic moiety modification.

[0275] In some implementations, the sense and antisense strands of the dsRNA molecule of the first aspect contain modifications defined by a modification motif selected from any of the following (1)-(6):

[0276] (1) Sense chain: Lipophilic part s-NmsNmNmNmNmNmNfNmNfNdNfNmNmNmNmNmNmNmNmNmsNms-Lipophilic part, Antisense chain: vp / PEO-NmsNfsNmNmNdNmNdNmNmNmNmNmNmNmNfNmNfNmNfNmNmNmNmNmNmsNmsNm;

[0277] (2) Sensitive strand: lipophilic part s-NmsNmNmNmNmNmNmNmNmNfNdNfNmNmNmNmNmNmNmNmNmsNms-lipophilic part, antisense strand: vp / PEO-NmsNfsNmNmNdNmNdNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmNmNm;

[0278] (3) Sense chain: Lipophilic part s-NmsNmNmNmNmNmNmNfNmNfNfNfNfNmNmNmNmNmNmNmNmsNms-Lipophilic part, Antisense chain: vp / PEO-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNmNmNfNmNmNmNmNmNfNmNfNmNmNmNmNmNmNmNmNmNmNmNm;

[0279] (4) Sense chain: lipophilic moiety-carrier-s-NmsNmNmNmNmNmNfNmNfNdNfNmNmNmNmNmNmNmNmNmsNms-carrier-lipophilic moiety, antisense chain: vp / PEO-NmsNfsNmNmNdNmNdNmNmNmNmNmNmNmNf ...

[0280] (5) Sense strand: Lipophilic moiety-carrier-s-NmsNmNmNmNmNmNmNmNmNmNfNdNfNmNmNmNmNmNmNmNmNmsNms-carrier-lipophilic moiety, Antisense strand: vp / PEO-NmsNfsNmNmNdNmNdNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmsNmsNm; and

[0281] (6) Sense chain: lipophilic moiety-carrier-s-NmsNmNmNmNmNmNfNmNfNfNfNfNmNmNmNmNmNmNmNmsNms-carrier-lipophilic moiety, antisense chain: vp / PEO-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNmNf ...

[0282] In each of the modified motifs (1)-(6), each N represents a nucleotide from 5' to 3' of the sense or antisense strand, Nm represents a 2'-OMe modified ribonucleotide, Nf represents a 2'-F modified ribonucleotide, s indicates that the two nucleotides on either side of "s" are linked by a phosphate thioester bond, and Nd represents a deoxy-modified ribonucleotide (i.e., the deoxyribonucleotide corresponding to the ribonucleotide). When the base portion of N in Nd is A, G, or C, Nd is associated with... The base moiety should be a deoxyribonucleotide with a base moiety of A, G, or C (i.e., Ad, Gd, or Cd). When the base moiety of N in Nd is U, Nd is Ud or Td. vp / PEO- indicates 5'-VP modification or 4'-PEO modification. The lipophilic moiety s- or s-lipophilic moiety indicates that the lipophilic moiety is linked to the adjacent nucleoside through a thiophosphate bond. The lipophilic moiety-carrier-s- or s-carrier-lipophilic moiety indicates that the lipophilic moiety is linked to the carrier, and the carrier is linked to the adjacent nucleoside through a thiophosphate bond.

[0283] In some implementations, the dsRNA molecule of the first aspect has a feature selected from any of the following:

[0284] (1) Sensitive chain modification motif: lipophilic part -(carrier)-s-NmsNmNmNmNmNmNfNmNfNdNfNmNmNmNmNmNmNmNmNmsNms-(carrier)-lipophilic part, antisense chain modification motif: vp / PEO-NmsNfsNmNmNdNmNdNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmsNmsNm, base sequence is the same as ALK7-Z15 (see Table A, the same below);

[0285] (2) Sensitive chain modification motif: lipophilic part -(carrier) -s-NmsNmNmNmNmNmNmNmNmNfNdNfNmNmNmNmNmNmNmNmsNms-(carrier)-lipophilic part, antisense chain modification motif: vp / PEO-NmsNfsNmNmNdNmNdNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm, base sequence is the same as ALK7-Z15;

[0286] (3) Sensitive chain modification motif: lipophilic part -(carrier) -s-NmsNmNmNmNmNmNfNmNfNfNfNfNmNmNmNmNmNmNmsNms-(carrier)-lipophilic part, antisense chain modification motif: vp / PEO-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmsNmsNm, base sequence is the same as ALK7-Z15;

[0287] (4) Sensitive chain modification motif: lipophilic part -(carrier) -s-NmsNmNmNmNmNmNfNmNfNdNfNmNmNmNmNmNmNmNmsNms-(carrier)-lipophilic part, antisense chain modification motif: vp / PEO-NmsNfsNmNmNdNmNdNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm, base sequence is the same as ALK7-Z52;

[0288] (5) Sensitive chain modification motif: lipophilic part -(carrier) -s-NmsNmNmNmNmNmNmNmNmNfNdNfNmNmNmNmNmNmNmNmsNms-(carrier)-lipophilic part, antisense chain modification motif: vp / PEO-NmsNfsNmNmNdNmNdNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm, base sequence is the same as ALK7-Z52;

[0289] (6)Sense chain modification motif: lipophilic part -(carrier) -s-NmsNmNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmsNms-(carrier)-lipophilic part, antisense chain modification motif: vp / PEO-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmsNmsNm, base sequence is the same as ALK7-Z52;

[0290] (7)Sense chain modification motif: lipophilic part -(carrier)-s-NmsNmNmNmNmNmNfNmNfNdNfNmNmNmNmNmNmNmNmsNms-(carrier)-lipophilic part, antisense chain modification motif: vp / PEO-NmsNfsNmNmNdNmNdNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmsNmsNm, base sequence is the same as ALK7-Z38;

[0291] (8) Sensitive chain modification motif: lipophilic part -(carrier) -s-NmsNmNmNmNmNmNmNmNfNdNfNmNmNmNmNmNmNmNmsNms-(carrier)-lipophilic part, antisense chain modification motif: vp / PEO-NmsNfsNmNmNdNmNdNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm, base sequence is the same as ALK7-Z38;

[0292] (9) Sensitive chain modification motif: lipophilic part -(carrier) -s-NmsNmNmNmNmNmNfNmNfNfNfNfNmNmNmNmNmNmNmsNms-(carrier)-lipophilic part, antisense chain modification motif: vp / PEO-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmsNmsNm, base sequence is the same as ALK7-Z38;

[0293] (10)Sense chain modification motif: lipophilic part -(carrier) -s-NmsNmNmNmNmNmNfNmNfNdNfNmNmNmNmNmNmNmNmsNms-(carrier)-lipophilic part, antisense chain modification motif: vp / PEO-NmsNfsNmNmNdNmNdNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmsNmsNm, base sequence is the same as ALK7-D127;

[0294] (11)Sense chain modification motif: lipophilic part -(carrier)-s-NmsNmNmNmNmNmNmNmNfNdNfNmNmNmNmNmNmNmNmNmsNms-(carrier)-lipophilic part, antisense chain modification motif: vp / PEO-NmsNfsNmNmNdNmNdNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm, base sequence is the same as ALK7-D127;

[0295] (12)Sense chain modification motif: lipophilic part -(carrier) -s-NmsNmNmNmNmNmNfNmNfNfNfNfNmNmNmNmNmNmNmsNms-(carrier)-lipophilic part, antisense chain modification motif: vp / PEO-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmsNmsNm, base sequence is the same as ALK7-D127;

[0296] (13)Sense chain modification motif: lipophilic part -(carrier) -s-NmsNmNmNmNmNmNfNmNfNdNfNmNmNmNmNmNmNmNmsNms-(carrier)-lipophilic part, antisense chain modification motif: vp / PEO-NmsNfsNmNmNdNmNdNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmsNmsNm, base sequence is the same as ALK7-D71;

[0297] (14)Sense chain modification motif: lipophilic part -(carrier) -s-NmsNmNmNmNmNmNmNmNmNfNdNfNmNmNmNmNmNmNmNmsNms-(carrier)-lipophilic part, antisense chain modification motif: vp / PEO-NmsNfsNmNmNdNmNdNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm, base sequence is the same as ALK7-D71;

[0298] (15)Sense chain modification motif: lipophilic part -(carrier) -s-NmsNmNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmsNms-(carrier)-lipophilic part, antisense chain modification motif: vp / PEO-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmsNmsNm, base sequence is the same as ALK7-D71;

[0299] (16)Sense chain modification motif: lipophilic part -(carrier) -s-NmsNmNmNmNmNmNfNmNfNdNfNmNmNmNmNmNmNmNmNmsNms-(carrier)-lipophilic part, antisense chain modification motif: vp / PEO-NmsNfsNmNmNdNmNdNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmsNmsNm, base sequence is the same as ALK7-Z16;

[0300] (17)Sense chain modification motif: lipophilic part -(carrier) -s-NmsNmNmNmNmNmNmNmNmNfNdNfNmNmNmNmNmNmNmNmsNms-(carrier)-lipophilic part, antisense chain modification motif: vp / PEO-NmsNfsNmNmNdNmNdNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm, base sequence is the same as ALK7-Z16;

[0301] (18)Sense chain modification motif: lipophilic motif -(carrier) -s-NmsNmNmNmNmNmNmNmNfNdNfNmNmNmNmNmNmNmNmsNms-(carrier)-lipophilic motif, antisense chain modification motif: vp / PEO-NmsNfsNmNmNdNmNdNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm, base sequence is the same as ALK7-Z16;

[0302] (19)Sense chain modification motif: lipophilic part -(carrier) -s-NmsNmNmNmNmNmNfNmNfNdNfNmNmNmNmNmNmNmNmNmsNms-(carrier)-lipophilic part, antisense chain modification motif: vp / PEO-NmsNfsNmNmNdNmNdNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmsNmsNm, base sequence is the same as ALK7-Z37;

[0303] (20)Sense chain modification motif: lipophilic motif -(carrier) -s-NmsNmNmNmNmNmNmNmNfNdNfNmNmNmNmNmNmNmNmNmsNms-(carrier)-lipophilic motif, antisense chain modification motif: vp / PEO-NmsNfsNmNmNdNmNdNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm, base sequence is the same as ALK7-Z37;

[0304] (21)Sense chain modification motif: lipophilic part -(carrier) -s-NmsNmNmNmNmNmNmNmNmNfNdNfNmNmNmNmNmNmNmNmsNms-(carrier)-lipophilic part, antisense chain modification motif: vp / PEO-NmsNfsNmNmNdNmNdNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm, base sequence is the same as ALK7-Z37;

[0305] (22)Sense chain modification motif: lipophilic motif -(carrier) -s-NmsNmNmNmNmNmNmNmNfNdNfNmNmNmNmNmNmNmNmNmsNms-(carrier)-lipophilic motif, antisense chain modification motif: vp / PEO-NmsNfsNmNmNdNmNdNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm, base sequence is the same as ALK7-Z47;

[0306] (23)Sense chain modification motif: lipophilic part -(carrier)-s-NmsNmNmNmNmNmNmNmNfNdNfNmNmNmNmNmNmNmNmsNms-(carrier)-lipophilic part, antisense chain modification motif: vp / PEO-NmsNfsNmNmNdNmNdNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm, base sequence is the same as ALK7-Z47;

[0307] (24)Sense chain modification motif: lipophilic motif -(carrier) -s-NmsNmNmNmNmNmNmNmNfNdNfNmNmNmNmNmNmNmNmNmsNms-(carrier)-lipophilic motif, antisense chain modification motif: vp / PEO-NmsNfsNmNmNdNmNdNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm, base sequence is the same as ALK7-Z47;

[0308] (25)Sense chain modification motif: lipophilic motif -(carrier) -s-NmsNmNmNmNmNmNmNmNmNfNdNfNmNmNmNmNmNmNmNmsNms-(carrier)-lipophilic motif, antisense chain modification motif: vp / PEO-NmsNfsNmNmNdNmNdNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm, base sequence is the same as ALK7-Z50;

[0309] (26)Sense chain modification motif: lipophilic motif -(carrier) -s-NmsNmNmNmNmNmNmNmNfNdNfNmNmNmNmNmNmNmNmsNms-(carrier)-lipophilic motif, antisense chain modification motif: vp / PEO-NmsNfsNmNmNdNmNdNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm, base sequence is the same as ALK7-Z50;

[0310] (27)Sense chain modification motif: lipophilic motif -(carrier) -s-NmsNmNmNmNmNmNmNmNmNfNdNfNmNmNmNmNmNmNmNmNmsNms-(carrier)-lipophilic motif, antisense chain modification motif: vp / PEO-NmsNfsNmNmNdNmNdNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm, base sequence is the same as ALK7-Z50;

[0311] (28)Sense chain modification motif: lipophilic part -(carrier) -s-NmsNmNmNmNmNmNmNmNfNdNfNmNmNmNmNmNmNmNmNmsNms-(carrier)-lipophilic part, antisense chain modification motif: vp / PEO-NmsNfsNmNmNdNmNdNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm, base sequence is the same as ALK7-Z59;

[0312] (29)Sense chain modification motif: lipophilic part -(carrier) -s-NmsNmNmNmNmNmNmNmNmNfNdNfNmNmNmNmNmNmNmNmsNms-(carrier)-lipophilic part, antisense chain modification motif: vp / PEO-NmsNfsNmNmNdNmNdNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm, base sequence is the same as ALK7-Z59;

[0313] (30)Sense chain modification motif: lipophilic part -(carrier) -s-NmsNmNmNmNmNmNmNmNfNdNfNmNmNmNmNmNmNmNmsNms-(carrier)-lipophilic part, antisense chain modification motif: vp / PEO-NmsNfsNmNmNdNmNdNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm, base sequence is the same as ALK7-Z59;

[0314] (31)Sense chain modification motif: lipophilic part -(carrier) -s-NmsNmNmNmNmNmNmNmNmNfNdNfNmNmNmNmNmNmNmNmsNms-(carrier)-lipophilic part, antisense chain modification motif: vp / PEO-NmsNfsNmNmNdNmNdNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm, base sequence is the same as ALK7-Z66;

[0315] (32)Sense chain modification motif: lipophilic part -(carrier) -s-NmsNmNmNmNmNmNmNmNfNdNfNmNmNmNmNmNmNmNmsNms-(carrier)-lipophilic part, antisense chain modification motif: vp / PEO-NmsNfsNmNmNdNmNdNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm, base sequence is the same as ALK7-Z66;

[0316] (33)Sense chain modification motif: lipophilic part -(carrier) -s-NmsNmNmNmNmNmNmNmNfNdNfNmNmNmNmNmNmNmNmsNms-(carrier)-lipophilic part, antisense chain modification motif: vp / PEO-NmsNfsNmNmNdNmNdNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm, base sequence is the same as ALK7-Z66;

[0317] (34)Sense chain modification motif: lipophilic part -(carrier) -s-NmsNmNmNmNmNmNmNmNmNfNdNfNmNmNmNmNmNmNmNmsNms-(carrier)-lipophilic part, antisense chain modification motif: vp / PEO-NmsNfsNmNmNdNmNdNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm, base sequence is the same as ALK7-Z73;

[0318] (35)Sense chain modification motif: lipophilic part -(carrier) -s-NmsNmNmNmNmNmNmNmNfNdNfNmNmNmNmNmNmNmNmNmsNms-(carrier)-lipophilic part, antisense chain modification motif: vp / PEO-NmsNfsNmNmNdNmNdNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm, base sequence is the same as ALK7-Z73;

[0319] (36)Sense chain modification motif: lipophilic part -(carrier) -s-NmsNmNmNmNmNmNmNmNfNdNfNmNmNmNmNmNmNmNmNmsNms-(carrier)-lipophilic part, antisense chain modification motif: vp / PEO-NmsNfsNmNmNdNmNdNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm, base sequence is the same as ALK7-Z73;

[0320] (37)Sense chain modification motif: lipophilic part -(carrier) -s-NmsNmNmNmNmNmNmNmNfNdNfNmNmNmNmNmNmNmNmsNms-(carrier)-lipophilic part, antisense chain modification motif: vp / PEO-NmsNfsNmNmNdNmNdNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm, base sequence is the same as ALK7-Z130;

[0321] (38)Sense chain modification motif: lipophilic motif -(carrier) -s-NmsNmNmNmNmNmNmNmNfNdNfNmNmNmNmNmNmNmNmNmsNms-(carrier)-lipophilic motif, antisense chain modification motif: vp / PEO-NmsNfsNmNmNdNmNdNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm, base sequence is the same as ALK7-Z130;

[0322] (39)Sense chain modification motif: lipophilic part -(carrier)-s-NmsNmNmNmNmNmNmNmNfNdNfNmNmNmNmNmNmNmNmsNms-(carrier)-lipophilic part, antisense chain modification motif: vp / PEO-NmsNfsNmNmNdNmNdNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm, base sequence is the same as ALK7-Z130;

[0323] (40)Sense chain modification motif: lipophilic part -(carrier) -s-NmsNmNmNmNmNmNmNmNfNdNfNmNmNmNmNmNmNmNmsNms-(carrier)-lipophilic part, antisense chain modification motif: vp / PEO-NmsNfsNmNmNdNmNdNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm, base sequence is the same as ALK7-D45;

[0324] (41)Sense chain modification motif: lipophilic part -(carrier) -s-NmsNmNmNmNmNmNmNmNfNdNfNmNmNmNmNmNmNmNmsNms-(carrier)-lipophilic part, antisense chain modification motif: vp / PEO-NmsNfsNmNmNdNmNdNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm, base sequence is the same as ALK7-D45;

[0325] (42)Sense chain modification motif: lipophilic motif -(carrier) -s-NmsNmNmNmNmNmNmNmNfNdNfNmNmNmNmNmNmNmNmNmsNms-(carrier)-lipophilic motif, antisense chain modification motif: vp / PEO-NmsNfsNmNmNdNmNdNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm, base sequence is the same as ALK7-D45;

[0326] (43)Sense chain modification motif: lipophilic part -(carrier) -s-NmsNmNmNmNmNmNmNmNfNdNfNmNmNmNmNmNmNmNmNmsNms-(carrier)-lipophilic part, antisense chain modification motif: vp / PEO-NmsNfsNmNmNdNmNdNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm, base sequence is the same as ALK7-D85;

[0327] (44)Sense chain modification motif: lipophilic part -(carrier) -s-NmsNmNmNmNmNmNmNmNmNfNdNfNmNmNmNmNmNmNmNmNmsNms-(carrier)-lipophilic part, antisense chain modification motif: vp / PEO-NmsNfsNmNmNdNmNdNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm, base sequence is the same as ALK7-D85;

[0328] (45)Sense chain modification motif: lipophilic part -(carrier) -s-NmsNmNmNmNmNmNmNmNfNdNfNmNmNmNmNmNmNmNmsNms-(carrier)-lipophilic part, antisense chain modification motif: vp / PEO-NmsNfsNmNmNdNmNdNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm, base sequence is the same as ALK7-D85;

[0329] (46)Sense chain modification motif: lipophilic part -(carrier) -s-NmsNmNmNmNmNmNmNmNfNdNfNmNmNmNmNmNmNmNmsNms-(carrier)-lipophilic part, antisense chain modification motif: vp / PEO-NmsNfsNmNmNdNmNdNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm, base sequence is the same as ALK7-D152;

[0330] (47)Sense chain modification motif: lipophilic motif -(carrier) -s-NmsNmNmNmNmNmNmNmNfNdNfNmNmNmNmNmNmNmNmNmsNms-(carrier)-lipophilic motif, antisense chain modification motif: vp / PEO-NmsNfsNmNmNdNmNdNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm, base sequence is the same as ALK7-D152;

[0331] (48)Sense chain modification motif: lipophilic part -(carrier) -s-NmsNmNmNmNmNmNmNmNfNdNfNmNmNmNmNmNmNmNmNmsNms-(carrier)-lipophilic part, antisense chain modification motif: vp / PEO-NmsNfsNmNmNdNmNdNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm, base sequence is the same as ALK7-D152;

[0332] (49)Sense chain modification motif: lipophilic part -(carrier) -s-NmsNmNmNmNmNmNmNmNmNfNdNfNmNmNmNmNmNmNmNmsNms-(carrier)-lipophilic part, antisense chain modification motif: vp / PEO-NmsNfsNmNmNdNmNdNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm, base sequence is the same as ALK7-Z15-1;

[0333] (50)Sense chain modification motif: lipophilic part -(carrier) -s-NmsNmNmNmNmNmNmNmNfNdNfNmNmNmNmNmNmNmNmNmsNms-(carrier)-lipophilic part, antisense chain modification motif: vp / PEO-NmsNfsNmNmNdNmNdNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm, base sequence is the same as ALK7-Z15-1;

[0334] (51)Sense chain modification motif: lipophilic part -(carrier)-s-NmsNmNmNmNmNmNmNmNfNdNfNmNmNmNmNmNmNmNmNmsNms-(carrier)-lipophilic part, antisense chain modification motif: vp / PEO-NmsNfsNmNmNdNmNdNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm, base sequence is the same as ALK7-Z15-1;

[0335] (52)Sense chain modification motif: lipophilic part -(carrier) -s-NmsNmNmNmNmNmNmNmNmNfNdNfNmNmNmNmNmNmNmNmsNms-(carrier)-lipophilic part, antisense chain modification motif: vp / PEO-NmsNfsNmNmNdNmNdNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm, base sequence is the same as ALK7-Z52-1;

[0336] (53)Sense chain modification motif: lipophilic part -(carrier)-s-NmsNmNmNmNmNmNmNmNfNdNfNmNmNmNmNmNmNmNmNmsNms-(carrier)-lipophilic part, antisense chain modification motif: vp / PEO-NmsNfsNmNmNdNmNdNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm, base sequence is the same as ALK7-Z52-1;

[0337] (54)Sense chain modification motif: lipophilic part -(carrier) -s-NmsNmNmNmNmNmNmNmNfNdNfNmNmNmNmNmNmNmNmsNms-(carrier)-lipophilic part, antisense chain modification motif: vp / PEO-NmsNfsNmNmNdNmNdNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm, base sequence is the same as ALK7-Z52-1;

[0338] (55)Sense chain modification motif: lipophilic part -(carrier) -s-NmsNmNmNmNmNmNmNmNfNdNfNmNmNmNmNmNmNmNmsNms-(carrier)-lipophilic part, antisense chain modification motif: vp / PEO-NmsNfsNmNmNdNmNdNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm, base sequence is the same as ALK7-Z38-1;

[0339] (56)Sense chain modification motif: lipophilic part -(carrier) -s-NmsNmNmNmNmNmNmNmNfNdNfNmNmNmNmNmNmNmNmsNms-(carrier)-lipophilic part, antisense chain modification motif: vp / PEO-NmsNfsNmNmNdNmNdNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm, base sequence is the same as ALK7-Z38-1;

[0340] (57)Sense chain modification motif: lipophilic part -(carrier) -s-NmsNmNmNmNmNmNmNmNmNfNdNfNmNmNmNmNmNmNmNmsNms-(carrier)-lipophilic part, antisense chain modification motif: vp / PEO-NmsNfsNmNmNdNmNdNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm, base sequence is the same as ALK7-Z38-1;

[0341] (58)Sense chain modification motif: lipophilic part -(carrier) -s-NmsNmNmNmNmNmNmNmNfNdNfNmNmNmNmNmNmNmNmsNms-(carrier)-lipophilic part, antisense chain modification motif: vp / PEO-NmsNfsNmNmNdNmNdNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm, base sequence is the same as ALK7-D127-1;

[0342] (59)Sense chain modification motif: lipophilic part -(carrier)-s-NmsNmNmNmNmNmNmNmNfNdNfNmNmNmNmNmNmNmNmsNms-(carrier)-lipophilic part, antisense chain modification motif: vp / PEO-NmsNfsNmNmNdNmNdNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm, base sequence is the same as ALK7-D127-1;

[0343] (60)Sense chain modification motif: lipophilic motif -(carrier) -s-NmsNmNmNmNmNmNmNmNfNdNfNmNmNmNmNmNmNmNmsNms-(carrier)-lipophilic motif, antisense chain modification motif: vp / PEO-NmsNfsNmNmNdNmNdNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm, base sequence is the same as ALK7-D127-1;

[0344] (61)Sense chain modification motif: lipophilic part -(carrier)-s-NmsNmNmNmNmNmNfNmNfNdNfNmNmNmNmNmNmNmNmNmNmsNms-(carrier)-lipophilic part, antisense chain modification motif: vp / PEO-NmsNfsNmNmNdNmNdNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmsNmsNm, base sequence is the same as ALK7-D71-1;

[0345] (62)Sense chain modification motif: lipophilic part -(carrier)-s-NmsNmNmNmNmNmNmNmNfNdNfNmNmNmNmNmNmNmNmNmsNms-(carrier)-lipophilic part, antisense chain modification motif: vp / PEO-NmsNfsNmNmNdNmNdNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm, base sequence is the same as ALK7-D71-1;

[0346] (63)Sense chain modification motif: lipophilic part -(carrier) -s-NmsNmNmNmNmNmNfNmNfNfNfNfNmNmNmNmNmNmNmsNms-(carrier)-lipophilic part, antisense chain modification motif: vp / PEO-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmsNmsNm, base sequence is the same as ALK7-D71-1;

[0347] (64)Sense chain modification motif: lipophilic part -(carrier)-s-NmsNmNmNmNmNmNfNmNfNdNfNmNmNmNmNmNmNmNmNmNmsNms-(carrier)-lipophilic part, antisense chain modification motif: vp / PEO-NmsNfsNmNmNdNmNdNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmsNmsNm, base sequence is the same as ALK7-Z16-1;

[0348] (65)Sense chain modification motif: lipophilic motif -(carrier) -s-NmsNmNmNmNmNmNmNmNfNdNfNmNmNmNmNmNmNmNmNmsNms-(carrier)-lipophilic motif, antisense chain modification motif: vp / PEO-NmsNfsNmNmNdNmNdNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm, base sequence is the same as ALK7-Z16-1;

[0349] (66)Sense chain modification motif: lipophilic motif -(carrier) -s-NmsNmNmNmNmNmNfNmNfNfNfNfNmNmNmNmNmNmNmsNms-(carrier)-lipophilic motif, antisense chain modification motif: vp / PEO-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmNmNmNmNmNmNmNmNmsNmsNm, base sequence is the same as ALK7-Z16-1;

[0350] (67)Sense chain modification motif: lipophilic part -(carrier) -s-NmsNmNmNmNmNmNfNmNfNdNfNmNmNmNmNmNmNmNmNmNmsNms-(carrier)-lipophilic part, antisense chain modification motif: vp / PEO-NmsNfsNmNmNdNmNdNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm, base sequence is the same as ALK7-Z37-1;

[0351] (68)Sense chain modification motif: lipophilic part -(carrier) -s-NmsNmNmNmNmNmNmNmNfNdNfNmNmNmNmNmNmNmNmNmsNms-(carrier)-lipophilic part, antisense chain modification motif: vp / PEO-NmsNfsNmNmNdNmNdNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm, base sequence is the same as ALK7-Z37-1;

[0352] (69)Sense chain modification motif: lipophilic part -(carrier)-s-NmsNmNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmsNms-(carrier)-lipophilic part, antisense chain modification motif: vp / PEO-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmsNmsNm, base sequence is the same as ALK7-Z37-1;

[0353] (70)Sense chain modification motif: lipophilic part -(carrier) -s-NmsNmNmNmNmNmNfNmNfNdNfNmNmNmNmNmNmNmNmNmNmsNms-(carrier)-lipophilic part, antisense chain modification motif: vp / PEO-NmsNfsNmNmNdNmNdNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmsNmsNm, base sequence is the same as ALK7-Z47-1;

[0354] (71)Sense chain modification motif: lipophilic part -(carrier) -s-NmsNmNmNmNmNmNmNmNfNdNfNmNmNmNmNmNmNmNmNmsNms-(carrier)-lipophilic part, antisense chain modification motif: vp / PEO-NmsNfsNmNmNdNmNdNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm, base sequence is the same as ALK7-Z47-1;

[0355] (72)Sense chain modification motif: lipophilic motif -(carrier) -s-NmsNmNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmsNms-(carrier)-lipophilic motif, antisense chain modification motif: vp / PEO-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmsNmsNm, base sequence is the same as ALK7-Z47-1;

[0356] (73)Sense chain modification motif: lipophilic motif -(carrier) -s-NmsNmNmNmNmNmNfNmNfNdNfNmNmNmNmNmNmNmNmNmsNms-(carrier)-lipophilic motif, antisense chain modification motif: vp / PEO-NmsNfsNmNmNdNmNdNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmsNmsNm, base sequence is the same as ALK7-Z50-1;

[0357] (74)Sense chain modification motif: lipophilic motif -(carrier) -s-NmsNmNmNmNmNmNmNmNfNdNfNmNmNmNmNmNmNmNmsNms-(carrier)-lipophilic motif, antisense chain modification motif: vp / PEO-NmsNfsNmNmNdNmNdNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm, base sequence is the same as ALK7-Z50-1;

[0358] (75)Sense chain modification motif: lipophilic motif -(carrier) -s-NmsNmNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmsNms-(carrier)-lipophilic motif, antisense chain modification motif: vp / PEO-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmNmsNmsNm, base sequence is the same as ALK7-Z50-1;

[0359] (76)Sense chain modification motif: lipophilic part -(carrier) -s-NmsNmNmNmNmNmNfNmNfNdNfNmNmNmNmNmNmNmNmNmsNms-(carrier)-lipophilic part, antisense chain modification motif: vp / PEO-NmsNfsNmNmNdNmNdNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmsNmsNm, base sequence is the same as ALK7-Z59-1;

[0360] (77)Sense chain modification motif: lipophilic motif -(carrier) -s-NmsNmNmNmNmNmNmNmNfNdNfNmNmNmNmNmNmNmNmsNms-(carrier)-lipophilic motif, antisense chain modification motif: vp / PEO-NmsNfsNmNmNdNmNdNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm, base sequence is the same as ALK7-Z59-1;

[0361] (78)Sense chain modification motif: lipophilic motif -(carrier) -s-NmsNmNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmsNms-(carrier)-lipophilic motif, antisense chain modification motif: vp / PEO-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmNmNmNmsNmsNm, base sequence is the same as ALK7-Z59-1;

[0362] (79)Sense chain modification motif: lipophilic part -(carrier)-s-NmsNmNmNmNmNmNfNmNfNdNfNmNmNmNmNmNmNmNmNmNmsNms-(carrier)-lipophilic part, antisense chain modification motif: vp / PEO-NmsNfsNmNmNdNmNdNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm, base sequence is the same as ALK7-Z66-1;

[0363] (80)Sense chain modification motif: lipophilic motif -(carrier) -s-NmsNmNmNmNmNmNmNmNfNdNfNmNmNmNmNmNmNmNmsNms-(carrier)-lipophilic motif, antisense chain modification motif: vp / PEO-NmsNfsNmNmNdNmNdNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm, base sequence is the same as ALK7-Z66-1;

[0364] (81)Sense chain modification motif: lipophilic motif -(carrier) -s-NmsNmNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmsNms-(carrier)-lipophilic motif, antisense chain modification motif: vp / PEO-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmNmNmNmsNmsNm, base sequence is the same as ALK7-Z66-1;

[0365] (82)Sense chain modification motif: lipophilic part -(carrier)-s-NmsNmNmNmNmNmNfNmNfNdNfNmNmNmNmNmNmNmNmNmsNms-(carrier)-lipophilic part, antisense chain modification motif: vp / PEO-NmsNfsNmNmNdNmNdNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm, base sequence is the same as ALK7-Z73-1;

[0366] (83)Sense chain modification motif: lipophilic part -(carrier)-s-NmsNmNmNmNmNmNmNmNfNdNfNmNmNmNmNmNmNmNmNmsNms-(carrier)-lipophilic part, antisense chain modification motif: vp / PEO-NmsNfsNmNmNdNmNdNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm, base sequence is the same as ALK7-Z73-1;

[0367] (84)Sense chain modification motif: lipophilic motif -(carrier) -s-NmsNmNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmsNms-(carrier)-lipophilic motif, antisense chain modification motif: vp / PEO-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmNmNmsNmsNm, base sequence is the same as ALK7-Z73-1;

[0368] (85)Sense chain modification motif: lipophilic part -(carrier)-s-NmsNmNmNmNmNmNfNmNfNdNfNmNmNmNmNmNmNmNmNmsNms-(carrier)-lipophilic part, antisense chain modification motif: vp / PEO-NmsNfsNmNmNdNmNdNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmsNmsNm, base sequence is the same as ALK7-Z130-1;

[0369] (86)Sense chain modification motif: lipophilic part -(carrier) -s-NmsNmNmNmNmNmNmNmNfNdNfNmNmNmNmNmNmNmNmsNms-(carrier)-lipophilic part, antisense chain modification motif: vp / PEO-NmsNfsNmNmNdNmNdNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm, base sequence is the same as ALK7-Z130-1;

[0370] (87)Sense chain modification motif: lipophilic part -(carrier) -s-NmsNmNmNmNmNmNfNmNfNfNfNfNmNmNmNmNmNmNmsNms-(carrier)-lipophilic part, antisense chain modification motif: vp / PEO-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmsNmsNm, base sequence is the same as ALK7-Z130-1;

[0371] (88)Sense chain modification motif: lipophilic part -(carrier) -s-NmsNmNmNmNmNmNfNmNfNdNfNmNmNmNmNmNmNmNmNmsNms-(carrier)-lipophilic part, antisense chain modification motif: vp / PEO-NmsNfsNmNmNdNmNdNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmsNmsNm, base sequence is the same as ALK7-D45-1;

[0372] (89) Sense chain modification motif: lipophilic motif -(carrier) -s-NmsNmNmNmNmNmNmNmNfNdNfNmNmNmNmNmNmNmNmNmsNms-(carrier)-lipophilic motif, antisense chain modification motif: vp / PEO-NmsNfsNmNmNdNmNdNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm, base sequence is the same as ALK7-D45-1;

[0373] (90)Sense chain modification motif: lipophilic motif -(carrier) -s-NmsNmNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmsNms-(carrier)-lipophilic motif, antisense chain modification motif: vp / PEO-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmsNmsNm, base sequence is the same as ALK7-D45-1;

[0374] (91)Sense chain modification motif: lipophilic part -(carrier)-s-NmsNmNmNmNmNmNfNmNfNdNfNmNmNmNmNmNmNmNmNmNmsNms-(carrier)-lipophilic part, antisense chain modification motif: vp / PEO-NmsNfsNmNmNdNmNdNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm, base sequence is the same as ALK7-D85-1;

[0375] (92)Sense chain modification motif: lipophilic part -(carrier) -s-NmsNmNmNmNmNmNmNmNmNfNdNfNmNmNmNmNmNmNmNmNmsNms-(carrier)-lipophilic part, antisense chain modification motif: vp / PEO-NmsNfsNmNmNdNmNdNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm, base sequence is the same as ALK7-D85-1;

[0376] (93)Sense chain modification motif: lipophilic part -(carrier) -s-NmsNmNmNmNmNmNfNmNfNfNfNfNmNmNmNmNmNmNmsNms-(carrier)-lipophilic part, antisense chain modification motif: vp / PEO-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmNmNmNmNmNmNmsNmsNm, base sequence is the same as ALK7-D85-1;

[0377] (94)Sense chain modification motif: lipophilic part -(carrier) -s-NmsNmNmNmNmNmNfNmNfNdNfNmNmNmNmNmNmNmNmNmsNms-(carrier)-lipophilic part, antisense chain modification motif: vp / PEO-NmsNfsNmNmNdNmNdNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmsNmsNm, base sequence is the same as ALK7-D152-1;

[0378] (95)Sense chain modification motif: lipophilic part -(carrier) -s-NmsNmNmNmNmNmNmNmNfNdNfNmNmNmNmNmNmNmNmsNms-(carrier)-lipophilic part, antisense chain modification motif: vp / PEO-NmsNfsNmNmNdNmNdNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm, base sequence is the same as ALK7-D152-1;

[0379] (96)Sense chain modification motif: lipophilic part -(carrier) -s-NmsNmNmNmNmNmNfNmNfNfNfNfNmNmNmNmNmNmNmsNms-(carrier)-lipophilic part, antisense chain modification motif: vp / PEO-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmNmNmNmNmNmNmsNmsNm, base sequence is the same as ALK7-D152-1;

[0380] (97)Sense chain modification motif: lipophilic part -(carrier) -s-NmsNmNmNmNmNmNfNmNfNdNfNmNmNmNmNmNmNmNmNmsNms-(carrier)-lipophilic part, antisense chain modification motif: vp / PEO-NmsNfsNmNmNdNmNdNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm, base sequence is the same as ALK7-Z15-2;

[0381] (98)Sense chain modification motif: lipophilic motif -(carrier) -s-NmsNmNmNmNmNmNmNmNfNdNfNmNmNmNmNmNmNmNmNmsNms-(carrier)-lipophilic motif, antisense chain modification motif: vp / PEO-NmsNfsNmNmNdNmNdNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm, base sequence is the same as ALK7-Z15-2;

[0382] (99)Sense chain modification motif: lipophilic part -(carrier)-s-NmsNmNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmsNms-(carrier)-lipophilic part, antisense chain modification motif: vp / PEO-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmNmNmNmNmNmNmsNmsNm, base sequence is the same as ALK7-Z15-2;

[0383] (100)Sense chain modification motif: lipophilic motif -(carrier) -s-NmsNmNmNmNmNmNfNmNfNdNfNmNmNmNmNmNmNmNmNmsNms-(carrier)-lipophilic motif, antisense chain modification motif: vp / PEO-NmsNfsNmNmNdNmNdNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmsNmsNm, base sequence is the same as ALK7-Z37-2;

[0384] (101)Sense chain modification motif: lipophilic motif -(carrier) -s-NmsNmNmNmNmNmNmNmNfNdNfNmNmNmNmNmNmNmNmNmsNms-(carrier)-lipophilic motif, antisense chain modification motif: vp / PEO-NmsNfsNmNmNdNmNdNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm, base sequence is the same as ALK7-Z37-2;

[0385] (102)Sense chain modification motif: lipophilic motif -(carrier) -s-NmsNmNmNmNmNmNfNmNfNfNfNfNmNmNmNmNmNmNmsNms-(carrier)-lipophilic motif, antisense chain modification motif: vp / PEO-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmNmNmsNmsNm, base sequence is the same as ALK7-Z37-2;

[0386] (103)Sense chain modification motif: lipophilic part -(carrier) -s-NmsNmNmNmNmNmNfNmNfNdNfNmNmNmNmNmNmNmNmNmNmsNms-(carrier)-lipophilic part, antisense chain modification motif: vp / PEO-NmsNfsNmNmNdNmNdNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmsNmsNm, base sequence is the same as ALK7-Z47-2;

[0387] (104)Sense chain modification motif: lipophilic part -(carrier) -s-NmsNmNmNmNmNmNmNmNfNdNfNmNmNmNmNmNmNmNmNmsNms-(carrier)-lipophilic part, antisense chain modification motif: vp / PEO-NmsNfsNmNmNdNmNdNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm, base sequence is the same as ALK7-Z47-2;

[0388] (105)Sense chain modification motif: lipophilic motif -(carrier) -s-NmsNmNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmsNms-(carrier)-lipophilic motif, antisense chain modification motif: vp / PEO-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmNmNmsNmsNm, base sequence is the same as ALK7-Z47-2;

[0389] (106)Sense chain modification motif: lipophilic motif -(carrier) -s-NmsNmNmNmNmNmNfNmNfNdNfNmNmNmNmNmNmNmNmNmsNms-(carrier)-lipophilic motif, antisense chain modification motif: vp / PEO-NmsNfsNmNmNdNmNdNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmsNmsNm, base sequence is the same as ALK7-Z73-2;

[0390] (107) Sense chain modification motif: lipophilic motif -(carrier) -s-NmsNmNmNmNmNmNmNmNmNfNdNfNmNmNmNmNmNmNmNmsNms-(carrier)-lipophilic motif, antisense chain modification motif: vp / PEO-NmsNfsNmNmNdNmNdNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm, base sequence same as ALK7-Z73-2; and

[0391] (108)Sense chain modification motif: lipophilic motif -(carrier) -s-NmsNmNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmsNms-(carrier)-lipophilic motif, antisense chain modification motif: vp / PEO-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmNmNmNmNmNmNmNmNmsNmsNm, base sequence is the same as ALK7-Z73-2;

[0392] In each of the modified motifs (1)-(6), each N represents a nucleotide from 5' to 3' of the sense or antisense strand, Nm represents a 2'-OMe modified ribonucleotide, Nf represents a 2'-F modified ribonucleotide, s indicates that the two nucleotides on both sides of "s" are linked by a phosphate thioester bond, and Nd represents a deoxy-modified ribonucleotide (i.e., the deoxyribonucleotide corresponding to the ribonucleotide). When the base part of N in Nd is A, G or C, Nd is correspondingly an A base part. Deoxyribonucleotides of type G or C (i.e., Ad, Gd or Cd). When the base part of N in Nd is U, Nd is Ud or Td. vp / PEO- indicates 5'-VP modification or 4'-PEO modification. The lipophilic part s- or s-lipophilic part indicates that the lipophilic part is linked to the adjacent nucleoside through a thiophosphate bond. The lipophilic part-carrier-s- or s-carrier-lipophilic part indicates that the lipophilic part is linked to the carrier. The carrier is linked to the adjacent nucleoside through a thiophosphate bond. -(carrier)- indicates that the carrier is present or absent.

[0393] Unless otherwise specified, if the dsRNA molecule of the first aspect of this application contains a PEO-modified nucleotide, then the nucleotide is a D-configuration nucleotide.

[0394] In some embodiments, the sense and antisense strands of the dsRNA molecule of the first aspect each comprise a sequence that differs from the sense and antisense nucleoside sequences of any dsRNA selected from Table B, or from a truncated form of the sense or antisense strand, by no more than two nucleoside modifications. In some embodiments, the nucleosides in Table B do not include modifications not listed therein.

[0395] Table B. Exemplary dsRNA molecule modified nucleoside sequences (without limitation on whether they contain 5'-phosphate mimics and targeting ligands, and without limitation on whether they have modified internucleotide linkages)

[0396] In some embodiments, the sense strand and antisense strand of the dsRNA molecule of the first aspect are each independently 19-25 nt long, comprising a continuous 19 nt nucleotide selected from any dsRNA sense strand nucleotide sequence in Table B and a continuous 21 nt nucleotide from a paired antisense strand nucleotide sequence. In some embodiments, the sense strand of the dsRNA molecule of the first aspect is 19-25 nt long, comprising a continuous 19 nt nucleotide selected from any dsRNA sense strand nucleotide sequence in Table B and a continuous 21 nt nucleotide from the 5' end of a paired antisense strand nucleotide sequence. In some embodiments, the sense strand of the dsRNA molecule of the first aspect is 19-25 nt long, comprising a continuous 19 nt nucleotide from the 3' end of any dsRNA sense strand nucleotide sequence in Table B and a continuous 21 nt nucleotide from the 5' end of a paired antisense strand nucleotide sequence.

[0397] In some embodiments, the sense strand and antisense strand of the dsRNA molecule of the first aspect are each independently 19-25 nt long, comprising a continuous 21 nt nucleotide selected from any dsRNA sense strand nucleotide sequence in Table B and a continuous 21 nt nucleotide from the paired antisense strand nucleotide sequence. In some embodiments, the sense strand of the dsRNA molecule of the first aspect is 19-25 nt long, comprising a continuous 21 nt nucleotide selected from any dsRNA sense strand nucleotide sequence in Table B and a continuous 21 nt nucleotide from the 5' end of the paired antisense strand nucleotide sequence.

[0398] In some embodiments, the sense strand and antisense strand of the first aspect dsRNA molecule are each independently 19-25 nt long, comprising a continuous 21 nt nucleotide selected from any dsRNA sense strand nucleotide sequence in Table B and a continuous 23 nt nucleotide paired with an antisense strand nucleotide sequence.

[0399] In some embodiments, the sense strand and antisense strand of the dsRNA molecule of the first aspect are each independently 19-23 nt long, comprising a continuous 19 nt nucleotide selected from any dsRNA sense strand nucleotide sequence in Table B and a continuous 21 nt nucleotide from a paired antisense strand nucleotide sequence. In some embodiments, the sense strand and antisense strand of the dsRNA molecule of the first aspect are each independently 19-23 nt long, comprising a continuous 19 nt nucleotide selected from any dsRNA sense strand nucleotide sequence in Table B and a continuous 21 nt nucleotide from the 5' end of a paired antisense strand nucleotide sequence. In some embodiments, the sense strand and antisense strand of the dsRNA molecule of the first aspect are each independently 19-23 nt long, comprising a continuous 19 nt nucleotide from the 3' end of any dsRNA sense strand nucleotide sequence in Table B and a continuous 21 nt nucleotide from the 5' end of a paired antisense strand nucleotide sequence.

[0400] In some embodiments, the sense strand and antisense strand of the dsRNA molecule of the first aspect are each independently 19-23 nt long, comprising a continuous 21 nt nucleotide selected from any dsRNA sense strand nucleotide sequence in Table B and a continuous 21 nt nucleotide from the paired antisense strand nucleotide sequence. In some embodiments, the sense strand and antisense strand of the dsRNA molecule of the first aspect are each independently 21-23 nt long, comprising a continuous 21 nt nucleotide selected from any dsRNA sense strand nucleotide sequence in Table B and a continuous 21 nt nucleotide from the 5' end of the paired antisense strand nucleotide sequence.

[0401] In some embodiments, the sense strand and antisense strand of the first aspect dsRNA molecule are each independently 19-23 nt long, comprising a continuous 21 nt nucleotide selected from any dsRNA sense strand nucleotide sequence in Table B and a continuous 23 nt nucleotide paired with an antisense strand nucleotide sequence.

[0402] In some embodiments, the sense strand and antisense strand of the dsRNA molecule of the first aspect are each independently 21-25 nt long, comprising a continuous 19 nt nucleotide selected from any dsRNA sense strand nucleotide sequence in Table B and a continuous 21 nt nucleotide from a paired antisense strand nucleotide sequence. In some embodiments, the sense strand and antisense strand of the dsRNA molecule of the first aspect are each independently 21-25 nt long, comprising a continuous 19 nt nucleotide selected from any dsRNA sense strand nucleotide sequence in Table B and a continuous 21 nt nucleotide from the 5' end of a paired antisense strand nucleotide sequence. In some embodiments, the sense strand and antisense strand of the dsRNA molecule of the first aspect are each independently 21-25 nt long, comprising a continuous 19 nt nucleotide from the 3' end of any dsRNA sense strand nucleotide sequence in Table B and a continuous 21 nt nucleotide from the 5' end of a paired antisense strand nucleotide sequence.

[0403] In some embodiments, the sense strand and antisense strand of the dsRNA molecule of the first aspect are each independently 21-25 nt long, comprising a continuous 21 nt nucleotide selected from any dsRNA sense strand nucleotide sequence in Table B and a continuous 21 nt nucleotide from the paired antisense strand nucleotide sequence. In some embodiments, the sense strand and antisense strand of the dsRNA molecule of the first aspect are each independently 21-25 nt long, comprising a continuous 21 nt nucleotide selected from any dsRNA sense strand nucleotide sequence in Table B and a continuous 21 nt nucleotide from the 5' end of the paired antisense strand nucleotide sequence.

[0404] In some embodiments, the sense strand and antisense strand of the first aspect dsRNA molecule are each independently 21-25 nt long, comprising a continuous 21 nt nucleotide selected from any of the dsRNA sense strand nucleotide sequences in Table B and a continuous 23 nt nucleotide paired with an antisense strand nucleotide sequence.

[0405] In some embodiments, the sense strand and antisense strand of the dsRNA molecule of the first aspect are each independently 21-23 nt long, comprising a continuous 19 nt nucleotide selected from any dsRNA sense strand nucleotide sequence in Table B and a continuous 21 nt nucleotide from a paired antisense strand nucleotide sequence. In some embodiments, the sense strand and antisense strand of the dsRNA molecule of the first aspect are each independently 21-23 nt long, comprising a continuous 19 nt nucleotide selected from any dsRNA sense strand nucleotide sequence in Table B and a continuous 21 nt nucleotide from the 5' end of a paired antisense strand nucleotide sequence. In some embodiments, the sense strand and antisense strand of the dsRNA molecule of the first aspect are each independently 21-23 nt long, comprising a continuous 19 nt nucleotide from the 3' end of any dsRNA sense strand nucleotide sequence in Table B and a continuous 21 nt nucleotide from the 5' end of a paired antisense strand nucleotide sequence.

[0406] In some embodiments, the sense strand and antisense strand of the dsRNA molecule of the first aspect are each independently 21-23 nt long, comprising a continuous 21 nt nucleotide selected from any dsRNA sense strand nucleotide sequence in Table B and a continuous 21 nt nucleotide from the paired antisense strand nucleotide sequence. In some embodiments, the sense strand and antisense strand of the dsRNA molecule of the first aspect are each independently 21-23 nt long, comprising a continuous 21 nt nucleotide selected from any dsRNA sense strand nucleotide sequence in Table B and a continuous 21 nt nucleotide from the 5' end of the paired antisense strand nucleotide sequence.

[0407] In some embodiments, the sense strand and antisense strand of the first aspect dsRNA molecule are each independently 21-23 nt long, comprising a continuous 21 nt nucleotide selected from any of the dsRNA sense strand nucleotide sequences in Table B and a continuous 23 nt nucleotide paired with an antisense strand nucleotide sequence.

[0408] In some embodiments, the dsRNA molecule of the first aspect has a sense strand of 19 nt and an antisense strand of 21 nt, each correspondingly comprising a continuous 19 nt nucleotide selected from any dsRNA sense strand nucleotide sequence in Table B and a continuous 21 nt nucleotide from a paired antisense strand nucleotide sequence. In some embodiments, the dsRNA molecule of the first aspect has a sense strand of 19 nt and an antisense strand of 21 nt, each correspondingly comprising a continuous 19 nt nucleotide selected from any dsRNA sense strand nucleotide sequence in Table B and a continuous 21 nt nucleotide from the 5' end of a paired antisense strand nucleotide sequence. In some embodiments, the dsRNA molecule of the first aspect has a sense strand of 19 nt and an antisense strand of 21 nt, each correspondingly comprising a continuous 19 nt nucleotide from the 3' end of any dsRNA sense strand nucleotide sequence in Table B and a continuous 21 nt nucleotide from the 5' end of a paired antisense strand nucleotide sequence.

[0409] In some embodiments, the dsRNA molecule of the first aspect has a sense strand of 21 nt and an antisense strand of 21 nt, each correspondingly comprising a continuous 19 nt nucleotide selected from any dsRNA sense strand nucleoside sequence in Table B and a continuous 21 nt nucleotide from a paired antisense strand nucleoside sequence. In some embodiments, the dsRNA molecule of the first aspect has a sense strand of 21 nt and an antisense strand of 21 nt, each correspondingly comprising a continuous 19 nt nucleotide selected from any dsRNA sense strand nucleoside sequence in Table B and a continuous 21 nt nucleotide from the 5' end of a paired antisense strand nucleoside sequence. In some embodiments, the dsRNA molecule of the first aspect has a sense strand of 21 nt and an antisense strand of 21 nt, each correspondingly comprising a continuous 19 nt nucleotide from the 3' end of any dsRNA sense strand nucleoside sequence in Table B and a continuous 21 nt nucleotide from the 5' end of a paired antisense strand nucleoside sequence.

[0410] In some embodiments, the dsRNA molecule of the first aspect has a sense strand of 21 nt and an antisense strand of 23 nt, each correspondingly comprising a continuous 19 nt nucleotide selected from any dsRNA sense strand nucleoside sequence in Table B and a continuous 21 nt nucleotide from a paired antisense strand nucleoside sequence. In some embodiments, the dsRNA molecule of the first aspect has a sense strand of 21 nt and an antisense strand of 23 nt, each correspondingly comprising a continuous 19 nt nucleotide selected from any dsRNA sense strand nucleoside sequence in Table B and a continuous 21 nt nucleotide from the 5' end of a paired antisense strand nucleoside sequence. In some embodiments, the dsRNA molecule of the first aspect has a sense strand of 21 nt and an antisense strand of 23 nt, each correspondingly comprising a continuous 19 nt nucleotide from the 3' end of any dsRNA sense strand nucleoside sequence in Table B and a continuous 21 nt nucleotide from the 5' end of a paired antisense strand nucleoside sequence.

[0411] In some embodiments, the dsRNA molecule of the first aspect has a sense strand of 23 nt and an antisense strand of 23 nt, each correspondingly comprising a continuous 19 nt nucleotide selected from any dsRNA sense strand nucleotide sequence in Table B and a continuous 21 nt nucleotide from the paired antisense strand nucleotide sequence. In some embodiments, the dsRNA molecule of the first aspect has a sense strand of 23 nt and an antisense strand of 23 nt, each correspondingly comprising a continuous 19 nt nucleotide selected from any dsRNA sense strand nucleotide sequence in Table B and a continuous 21 nt nucleotide from the 5' end of the paired antisense strand nucleotide sequence. In some embodiments, the dsRNA molecule of the first aspect has a sense strand of 23 nt and an antisense strand of 23 nt, each correspondingly comprising a continuous 19 nt nucleotide from the 3' end of any dsRNA sense strand nucleotide sequence in Table B and a continuous 21 nt nucleotide from the 5' end of the paired antisense strand nucleotide sequence.

[0412] In some embodiments, the dsRNA molecule of the first aspect has a sense strand of 19 nt and an antisense strand of 21 nt, each correspondingly comprising a consecutive 21 nt nucleotide selected from any dsRNA sense strand nucleotide sequence in Table B and a consecutive 21 nt nucleotide from the paired antisense strand nucleotide sequence. In some embodiments, the dsRNA molecule of the first aspect has a sense strand of 19 nt and an antisense strand of 21 nt, each correspondingly comprising a consecutive 21 nt nucleotide selected from any dsRNA sense strand nucleotide sequence in Table B and a consecutive 21 nt nucleotide from the 5' end of the paired antisense strand nucleotide sequence.

[0413] In some embodiments, the dsRNA molecule of the first aspect has a sense strand of 21 nt and an antisense strand of 21 nt, each correspondingly comprising a continuous 21 nt nucleotide selected from any dsRNA sense strand nucleotide sequence in Table B and a continuous 21 nt nucleotide from the paired antisense strand nucleotide sequence. In some embodiments, the dsRNA molecule of the first aspect has a sense strand of 21 nt and an antisense strand of 21 nt, each correspondingly comprising a continuous 21 nt nucleotide selected from any dsRNA sense strand nucleotide sequence in Table B and a continuous 21 nt nucleotide from the 5' end of the paired antisense strand nucleotide sequence.

[0414] In some embodiments, the dsRNA molecule of the first aspect has a sense strand of 21 nt and an antisense strand of 23 nt, each correspondingly comprising a continuous 21 nt nucleotide selected from any dsRNA sense strand nucleotide sequence in Table B and a continuous 21 nt nucleotide from the paired antisense strand nucleotide sequence. In some embodiments, the dsRNA molecule of the first aspect has a sense strand of 21 nt and an antisense strand of 23 nt, each correspondingly comprising a continuous 21 nt nucleotide selected from any dsRNA sense strand nucleotide sequence in Table B and a continuous 21 nt nucleotide from the 5' end of the paired antisense strand nucleotide sequence.

[0415] In some embodiments, the dsRNA molecule of the first aspect has a sense strand of 23 nt and an antisense strand of 23 nt, each correspondingly comprising a continuous 21 nt nucleotide selected from any dsRNA sense strand nucleotide sequence in Table B and a continuous 21 nt nucleotide from the paired antisense strand nucleotide sequence. In some embodiments, the dsRNA molecule of the first aspect has a sense strand of 23 nt and an antisense strand of 23 nt, each correspondingly comprising a continuous 21 nt nucleotide selected from any dsRNA sense strand nucleotide sequence in Table B and a continuous 21 nt nucleotide from the 5' end of the paired antisense strand nucleotide sequence.

[0416] In some embodiments, the dsRNA molecule of the first aspect has a sense strand of 21 nt and an antisense strand of 23 nt, each correspondingly comprising a consecutive 21 nt nucleotide selected from any dsRNA sense strand nucleotide sequence in Table B and a consecutive 23 nt nucleotide selected from a paired antisense strand nucleotide sequence.

[0417] In some embodiments, the sense and antisense strands of the dsRNA molecule of the first aspect each comprise a nucleotide sequence selected from any dsRNA in Table C, or the nucleotide sequence is identical to any dsRNA selected from Table C. In some embodiments, the nucleotide sequences in Table C do not contain any nucleotide modifications not explicitly stated therein.

[0418] The second aspect of this application provides an engineered nucleic acid molecule (also referred to herein as a "first engineered nucleic acid molecule") comprising one or more dsRNA molecules of the first aspect. In some embodiments, the first engineered nucleic acid molecule comprises two or more nucleic acid molecules from the dsRNA molecules of the first aspect whose sense strands and / or antisense strands are linked to each other. In some embodiments, the first engineered nucleic acid molecule is a dual-targeting or multi-targeting siRNA molecule, i.e., the first engineered nucleic acid molecule comprises at least one dsRNA molecule from the dsRNA molecules of the first aspect, and the sense and / or antisense strands of the at least one dsRNA molecule are linked to siRNA targeting other target sites of ALK7 mRNA or siRNA targeting other gene mRNAs. In some embodiments, the first engineered nucleic acid molecule is or comprises a hairpin structure, the double-stranded portion of which comprises any one of the dsRNA molecules of the first aspect. In some embodiments, the first engineered nucleic acid molecule is or comprises a hairpin structure, the double-stranded portion of which is composed of any one of the dsRNA molecules of the first aspect.

[0419] This application also provides compounds comprising one or more of the dsRNA molecules described in the first aspect. It should be understood that the scope of this application does not exclude other compounds or compositions comprising the dsRNA molecules of the first aspect, provided that such compounds or compositions contain the dsRNA molecules of the first aspect and that the dsRNA molecules still function as ALK7 RNAi, then such compounds or compositions fall within the scope of this application.

[0420] A third aspect of this application provides an engineered nucleic acid molecule (also referred to herein as a "second engineered nucleic acid molecule") that can be transcribed or spliced ​​in cells to form any of the aforementioned first aspect's unmodified dsRNA molecules. In some embodiments, the second engineered nucleic acid molecule can be transcribed into a shRNA precursor or spliced ​​to form mature shRNA or dsRNA (i.e., any of the first aspect's unmodified dsRNA molecules). In some embodiments, the second engineered nucleic acid molecule is a circular or linear nucleic acid molecule. In some embodiments, the second engineered nucleic acid molecule is a circular or linear plasmid. In some embodiments, the second engineered nucleic acid molecule is single-stranded. In some embodiments, the second engineered nucleic acid molecule is double-stranded or single-stranded. In some embodiments, the nucleic acid molecule belongs to an artificially constructed viral genome, selected from, but not limited to, lentiviral vectors or other retroviral vectors, adenovirus vectors, AAV vectors, poxvirus vectors, baculovirus vectors, and herpes simplex virus vectors. In some embodiments, the nucleic acid molecule belongs to a cellular genome, such as a nuclear genome, mitochondrial nucleic acid, or cytoplasmic free nucleic acid. In some implementations, the second engineered nucleic acid molecule is an RNA molecule, a DNA molecule, or a chimeric molecule of RNA and DNA.

[0421] A fourth aspect of this application also provides a nucleic acid delivery body comprising any of the dsRNA molecules of the first aspect, any of the first engineered nucleic acid molecules of the second aspect, and any of the second engineered nucleic acid molecules of the third aspect. In some embodiments, the nucleic acid delivery body is a liposome, lipid nanoparticles or other polymers, endosomes, exosomes, or vesicles.

[0422] This application also provides viral particles (which may also fall under the category of "delivery vehicles") comprising any of the second engineered nucleic acid molecules described in the third aspect above. In some embodiments, the viral particles are enveloped or capped viral particles. In some embodiments, the viral particles are pseudovirus particles. In some embodiments, the viral particles are AAV, baculovirus, poxvirus, herpesvirus, alphavirus, lentivirus, or other retroviruses.

[0423] The fifth aspect of this application provides a cell comprising the second engineered nucleic acid molecule of the third aspect described above. In some embodiments, the cell is a prokaryotic cell. In some embodiments, the cell is a eukaryotic cell, such as a stem cell, such as a mesenchymal stem cell, mesenchymal cell, etc.

[0424] Sixthly, this application provides tautomers, stereoisomers, solvates, isotope derivatives, or pharmaceutically acceptable salts of the dsRNA molecule described in the first aspect, the engineered nucleic acid molecule described in the second aspect, or the engineered nucleic acid molecule described in the third aspect.

[0425] A seventh aspect of this application provides a pharmaceutical composition comprising any of the dsRNA molecules of the first aspect, any of the first engineered nucleic acid molecules of the second aspect, any of the second engineered nucleic acid molecules of the third aspect, or the tautomer, stereoisomer, solvate, isotope derivative, or pharmaceutically acceptable salt described in the sixth aspect, and a pharmaceutically acceptable carrier or diluent. In some embodiments, the pharmaceutical composition is used to treat or prevent ALK7-mediated diseases or symptoms in a subject; in some embodiments, the diseases or symptoms are related to fat or glucose metabolism. In some embodiments, the fat metabolism-related diseases or symptoms are type 2 diabetes or obesity. In some embodiments, the fat or glucose metabolism-related diseases or symptoms are obesity accompanied by type 2 diabetes.

[0426] The eighth to twelfth aspects of this application provide the use of the dsRNA molecule described in the first aspect, the engineered nucleic acid molecule described in the second aspect, the engineered nucleic acid molecule described in the third aspect, the delivery body described in the fourth aspect, the cell described in the fifth aspect, the tautomer, stereoisomer, solvate, isotope derivative or pharmaceutically acceptable salt described in the sixth aspect, or the pharmaceutical composition described in the seventh aspect.

[0427] Eighthly, this application provides the use of the dsRNA molecule described in the first aspect, the engineered nucleic acid molecule described in the second aspect, the engineered nucleic acid molecule described in the third aspect, or the tautomer, stereoisomer, solvate, isotope derivative, or pharmaceutically acceptable salt described in the sixth aspect for inhibiting the expression of ALK7 in cells or in a subject. Inhibition of ALK7 expression in cells or in a subject includes inhibiting or reducing the transcription of ALK7 mRNA or the expression of ALK7 protein in target cells.

[0428] Ninthly, this application provides the use of the dsRNA molecule described in the first aspect, the engineered nucleic acid molecule described in the second aspect, the engineered nucleic acid molecule described in the third aspect, or the tautomer, stereoisomer, solvate, isotope derivative, or pharmaceutically acceptable salt described in the sixth aspect for the preparation of a medicament for inhibiting ALK7 expression in a subject. Inhibition of intracellular or in vivo ALK7 expression includes inhibiting or reducing the transcription of ALK7 mRNA or the expression of ALK7 protein in target cells.

[0429] Tenthly, this application provides the use of the dsRNA molecule described in the first aspect, the engineered nucleic acid molecule described in the second aspect, the engineered nucleic acid molecule described in the third aspect, the delivery body described in the fourth aspect, the cell described in the fifth aspect, the tautomer, stereoisomer, solvate, isotope derivative, or pharmaceutically acceptable salt described in the sixth aspect, or the pharmaceutical composition described in the seventh aspect, for the preparation of a medicament for the prevention or treatment of ALK7-mediated diseases or symptoms. In some embodiments, the ALK7-mediated diseases or symptoms are diseases or symptoms related to fat or glucose metabolism. In some embodiments, the fat metabolism-related diseases or symptoms are type 2 diabetes or obesity. In some embodiments, the fat or glucose metabolism-related diseases or symptoms are obesity accompanied by type 2 diabetes.

[0430] In its eleventh aspect, this application provides methods for treating or preventing ALK7-mediated diseases or symptoms in subjects, including:

[0431] The treatment or preventative effective amount of the dsRNA molecule described in the first aspect, the engineered nucleic acid molecule described in the second aspect, the engineered nucleic acid molecule described in the third aspect, the delivery body described in the fourth aspect, the cell described in the fifth aspect, the tautomer, stereoisomer, solvate, isotope derivative, or pharmaceutically acceptable salt described in the sixth aspect, or the pharmaceutical composition described in the seventh aspect, is administered to a subject in need. In some embodiments, the ALK7-mediated disease or symptom is a disease or symptom related to lipid or glucose metabolism. In some embodiments, the lipid metabolism-related disease or symptom is type 2 diabetes or obesity. In some embodiments, the lipid or glucose metabolism-related disease or symptom is obesity accompanied by type 2 diabetes.

[0432] In a twelfth aspect, this application provides the use of the dsRNA molecule described in the first aspect, the engineered nucleic acid molecule described in the second aspect, the engineered nucleic acid molecule described in the third aspect, the delivery system described in the fourth aspect, the cell described in the fifth aspect, the tautomer, stereoisomer, solvate, isotope derivative, or pharmaceutically acceptable salt described in the sixth aspect, or the pharmaceutical composition described in the seventh aspect, for weight loss in a subject. In some embodiments, the subject is a patient with type 2 diabetes.

[0433] This application also provides the use of the dsRNA molecule described in the first aspect, the engineered nucleic acid molecule described in the second aspect, the engineered nucleic acid molecule described in the third aspect, the delivery system described in the fourth aspect, the cell described in the fifth aspect, the tautomer, stereoisomer, solvate, isotope derivative, or pharmaceutically acceptable salt described in the sixth aspect, or the pharmaceutical composition described in the seventh aspect for regulating lipid metabolism in patients with diseases requiring control of blood lipids or body fat content. In some embodiments, the patients with diseases requiring control of blood lipids or body fat content are patients with type 2 diabetes and / or obesity.

[0434] This application also provides a method for regulating lipid metabolism in diseases requiring control of blood lipids or body fat content, comprising administering to a subject or cell in need an effective amount of the dsRNA molecule described in the first aspect, the engineered nucleic acid molecule described in the second aspect, the engineered nucleic acid molecule described in the third aspect, the delivery body described in the fourth aspect, the cell described in the fifth aspect, the tautomer, stereoisomer, solvate, isotope derivative, or pharmaceutically acceptable salt described in the sixth aspect, or the pharmaceutical composition described in the seventh aspect. In some embodiments, the patient with the disease requiring control of blood lipids or body fat content is a patient with type 2 diabetes and / or obesity.

[0435] The preferred embodiments of this application have been described in detail above; however, this application is not limited thereto. Within the scope of the technical concept of this application, various simple modifications can be made to the technical solutions of this application, including combining various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in this application and are all within the protection scope of this application. The aspects and embodiments of this application described herein include aspects and embodiments described as "comprising," "forming," and "substantially consisting of." Example

[0436] The inventions of this application are further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of this application. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer. Furthermore, any methods and materials similar to or equivalent to those described herein can be applied to the methods of this application. The preferred embodiments and materials shown herein are for illustrative purposes only.

[0437] In the following specific embodiments of this application, each dsRNA molecule does not contain any modifications not specified in these embodiments. That is, each dsRNA molecule used in the following specific embodiments of this application is a naked molecule (i.e., a polynucleotide composed of natural nucleotides without any modifications) or contains only the modifications specified in the modification motif used (e.g., specified by Nm, Nf, Nd, s, etc.).

[0438] Example 1: Sequence Design and Synthesis of dsRNA

[0439] 1.1 dsRNA Design

[0440] Based on the human ALK7 gene (Gene ID, 130399) mRNA sequence (NM_145259.3, SEQ ID NO. 333), multiple ALK7 dsRNAs were designed at different sites. The designed dsRNA sequences were compared with all other non-target gene sequences using sequence similarity software and showed the lowest homology. The naked sequences of the aforementioned dsRNA sequences and positive control sequences are shown in Table A at the end of this document. In this application, the dsRNA molecules disclosed in International Application Publication No. WO 2024 / 148329 A1 (named AC006189 and AC006188 in WO 2024 / 148329 A1) were selected as positive controls.

[0441] 1.2 Synthesis and purification of dsRNA and its conjugates

[0442] All phosphoramidite monomers used in the preparation of dsRNA in the examples (including unmodified and phosphoramidite nucleoside monomers modified with 2'-methoxy, 2'-fluoro, 5'-cyclopropyl phosphate (5'-cPrp), 4'-PEO (PEO) and similar phosphate mimics and / or 5'-vinylphosphonate (5'-VP or VP), and nucleoside monomers modified with targeting ligands (e.g., lipophilic moieties)) were commercially available or synthesized in-house; the 4'-PEO-modified phosphoramidite nucleoside monomer was also referred to as compound 1H). Synthesis was performed on a CPG using an oligo 48 synthesizer. An acetonitrile solution of 5-ethimercaptotetrazole (0.6 M) was used as the activating agent. The coupling time was 300 seconds (2′OMe and 2′F). Thiophosphate or phosphate modifications were introduced into the sequence via an oxidation step in a cyclic reaction. After solid-phase synthesis, the dried solid support was treated with ammonia solution at 45-55°C for 16 hours, then purified, desalted, and lyophilized to obtain chemically modified nucleotide conjugates other than PEO-L and PEO in Table 15. If ethyl or methyl-protected 4'-PEO and similar phosphate ester analogs were used to modify the nucleoside monomers, after solid-phase synthesis, the solid support was treated with 7% trimethylbromosilane acetonitrile solution for 2 hours. Afterward, the silane groups deethylated or methylated were quenched with 1M mercaptoethanol solution for 1 hour. The deethylation or methylation step was then completed, and the dried solid support was treated with ammonia solution at 45-55°C for 16 hours, then purified, desalted, and lyophilized to obtain nucleotide conjugates without all protecting groups (e.g., PEO-L and PEO in Table 15). The crude product was then purified by reversed-phase HPLC. Buffer A was 100 mM TEAA, pH 7.5, containing 5% acetonitrile, and buffer B was 100% acetonitrile. Record the UV trace at 260 nm and collect appropriate fractions. Use an ELISA reader to determine the concentration using UV light. Mix equimolar amounts of sense and antisense strands into a new EP tube, heat at 95°C for 5 min, and slowly anneal to room temperature. Finally, use a vacuum concentrator to evaporate to dryness at room temperature to obtain the final product.

[0443] Synthesis of compound 1H (when 1H appears alone, and not, for example, 1H in 1H-tetrazole)

[0444] In the nucleic acid molecule of this application embodiment, the 4'-PEO modified nucleotide is linked to PEO at the 4' position, and the PEO has the following structural formula:

[0445] In the nucleic acid molecules of this application embodiment, the 4'-PEO modified nucleotide U (for example) has the following structural formula:

[0446] The phosphoramidine nucleoside monomer used to generate 4'-PEO-modified nucleotides is called compound 1H, and for example, when the base is U, it has the structure shown below:

[0447] The following example, using a 1H compound with a U base, illustrates the synthesis process of a 4'-PEO-modified nucleoside monomer:

[0448] ① Synthesis of compound 1B

[0449] Compound 1A (150 g), (5'-oxy-(4,4'-dimethoxytriphenyl)-2'-methoxy-uridine, 5'-O-DMTr-2'-OMe-U, commercially available, CAS No.: 103285-22-9), DMAP (48.9 g), and pyridine (1500 ml) were added to a 5 L single-necked flask. The mixture was stirred at 0-5 °C for 5 minutes under nitrogen protection. Benzoyl chloride (150.6 g) was slowly added dropwise at 0-5 °C. After the addition was complete, the mixture was heated to room temperature and reacted overnight. The reaction solution was quenched in 2 L of ice water, and a solid precipitated. The solid was filtered, and the filter cake was washed with n-heptane. The wet product was dried in a 50 °C oven to obtain 195.0 g of compound 1B sample, with a yield of 95%. The theoretical calculated value of LCMS(ESI)C45H40O10N2[M+H]+m / z is 769.3, and the measured value is 770.7.

[0450] ② Synthesis of compound 1C

[0451] Compound 1B (180.0 g) and dichloromethane (1800 ml) were added to a 5 L single-necked flask. The temperature was controlled at 0-5 °C. Dichloroacetic acid (301.8 g) was added, and the mixture was heated to room temperature and reacted overnight. The reaction solution was cooled to 0-5 °C and extracted successively with 10% sodium bicarbonate solution and saturated brine. The organic phase was dried over anhydrous sodium sulfate and then evaporated to dryness to obtain the crude product. The crude product was purified by silica gel column chromatography to obtain 45.0 g of pure compound 1C, with a yield of 41.2%. The theoretical calculated value of LCMS(ESI)C24H23O8N2[M+H]+m / z is 467.1, and the measured value is 468.5.

[0452] ③ Synthesis of compound 1D

[0453] Compound 1C (36.0 g), acetonitrile (210 ml), and H2O (90 ml) were added to a 1000 ml single-necked flask and stirred at room temperature for 5 minutes. Then, TEMPO (3.63 g) and BAIB (16.6 g) were added sequentially, and the mixture was reacted overnight at room temperature. The reaction solution was washed and extracted with ethyl acetate and water. The organic phase was dried over anhydrous sodium sulfate and then evaporated to dryness to obtain the crude product. The crude product was purified by silica gel column chromatography to obtain 33.0 g of pure compound 1D, with a yield of 88.9%. The theoretical calculated value of LCMS(ESI)C24H21N2O9[M+H]+ m / z is 481.1, and the measured value is 482.4.

[0454] ④ Synthesis of Compound 1E

[0455] Compound 1D (30.0 g), 1,2-dichloroethane (300 ml), and H2O (90 ml) were added to a 1000 ml single-necked flask. AcOH (3.6 g), Mn(OAc)2 (2.4 g), and DIB (40.2 g) were then added sequentially. The mixture was purged three times with nitrogen at room temperature, then heated to 80 °C and stirred for 25 h. The organic phase was dried over anhydrous sodium sulfate and then evaporated to dryness to obtain the crude product. The crude product was purified by silica gel column chromatography to obtain 15.0 g of pure compound 1E, with a yield of 48.6%. The theoretical calculated value of LCMS(ESI)C25H23O9N2[M+H]+ m / z is 495.1, and the measured value is 496.4.

[0456] ⑤ Synthesis of compound 1F

[0457] Compound 1E (3.0 g) and dichloromethane (50 ml) were added to a 250 ml single-necked flask. The mixture was cooled to 0 °C, and then 1E-1 (4.42 g) and BF3·Et2O (4.06 g) were added sequentially. The mixture was stirred for 16 h. The reaction solution was washed with water for extraction. The organic phase was dried over anhydrous sodium sulfate and then evaporated to dryness to obtain the crude product. The crude product was purified by silica gel column chromatography to obtain 3.2 g of pure compound 1F, with a yield of 85.5%. The theoretical calculated value of LCMS(ESI)C29H34O11N2P[M+H]+m / z is 617.18, and the measured value is 618.56.

[0458] ⑥ Synthesis of Compound 1G

[0459] Compound 1F (3.0 g) and MeOH (40 ml) were added to a 100 ml single-necked flask, followed by K2CO3 (2.01 g). The mixture was stirred at room temperature for 1 h. The reaction solution was washed with water for extraction. The organic phase was dried over anhydrous sodium sulfate and then evaporated to dryness to obtain the crude product. The crude product was purified by silica gel column chromatography to obtain 1.5 g of pure compound 1G, with a yield of 75.3%. The theoretical calculated value of LCMS(ESI)C15H25O9N2P[M+H]+m / z is 408.13, and the measured value is 409.34.

[0460] ⑦ Synthesis of compound 1H

[0461] Compound 1G (1.5 g) and dichloromethane (20 ml) were added to a 50 ml single-necked flask, followed by the addition of 2-cyanoethyl N,N,N',N'-tetraisopropylphosphonamide (1.33 g) and 4,5-dicyanimidazole (0.35 g). The mixture was stirred at room temperature for 2 h. The reaction solution was washed and extracted with saturated NaHCO3, water, and brine. The organic phase was dried over anhydrous sodium sulfate and then evaporated to dryness to obtain the crude product. The crude product was purified by silica gel column chromatography to obtain 1.5 g of pure compound 1H, with a yield of 67.0%. The theoretical calculated value of LCMS(ESI)C24H42O10N4P2[M+H]+m / z is 608.24, and the measured value is 609.57.

[0462] Example 2: High-throughput screening and detection of in vitro inhibitory activity of ALK7 gene-dsRNA (unmodified)

[0463] The dsRNA molecules tested in this embodiment are unmodified; that is, their sense and antisense strands are composed of natural nucleotides. Such molecules are also referred to as "naked molecules" in this application, and their nucleotide sequences are called "naked sequences."

[0464] 2.1 ALK7 dsRNA transfection of hTERT A41hBAT-SVF cells

[0465] 2.1.1 Cell Culture

[0466] hTERT A41hBAT-SVF cells (human brown adipocyte-hTERT immortalized cell line, KORBIE, catalog number CBP61655) were cultured in DMEM medium containing 10% fetal bovine serum under 5% CO2 and 37°C conditions. When the cells were in the logarithmic growth phase and in good condition (70% confluence), they were digested and plated for transfection assays.

[0467] 2.1.2 Preparation of the transfection complex

[0468] Mix 100 μL of Opti-MEM with 1 μL of RNAiMax transfection reagent, then add 0.6 μL of dsRNA stock solution (dsRNA concentration 0.1 μM), and use enzyme-free water as a blank control. Gently pipette to mix, then transfer to a 24-well plate and incubate for 15 minutes to ensure complete complex formation.

[0469] 2.2 Cell transfection procedure

[0470] hTERT A41hBAT-SVF cells were removed from the incubator, rinsed with PBS buffer, and trypsin digested to adjust the cell density to 1.6 × 10⁻⁶. 5Cells / mL. Based on experimental requirements, 500 μL of cell suspension was dispensed into each well of a 24-well plate, ensuring a final dsRNA concentration of 0.1 nM. The treated 24-well plates were then incubated in a 5% CO2, 37°C incubator for 48 hours.

[0471] 2.3 Real-time quantitative PCR analysis

[0472] Forty-eight hours after transfection, cells were collected and total RNA was extracted using the FastPure Cell / Tissue Total RNA Isolation Kit V2 (Vazyme, catalog number RC112-01). The extracted RNA was then reverse transcribed into cDNA using the Takara PrimeScript RT Master Mix (Perfect Real Time) reverse transcription kit. Subsequently, TB... Premix Ex Taq TM Quantitative fluorescence PCR was performed on cDNA using (Tli RNaseH Plus). The human GAPDH gene was used as an internal reference gene. The qPCR primer sequences (GAPDH-H (SEQ ID NO: 319 and 320) and ALK7-H (SEQ ID NO: 321 and 322)) are shown in Table A at the end of the document.

[0473] After the PCR reaction, the 2–ΔΔCt (Livak) method was used to normalize the expression of ALK7 mRNA in the blank control group (without enzyme water) to 1, with GAPDH gene as the internal reference gene and blank control (without enzyme water) group as the control. Relative quantitative analysis was then performed.

[0474] 2.4 Data Analysis and Results

[0475] After the PCR reaction was completed, relative quantitative analysis was performed using the reference gene as a standard and CFX96 software, and statistical analysis was performed using GarphPad software.

[0476] Using the methods described in 2.1 to 2.4 above, a total of three rounds of screening were conducted.

[0477] First round of screening: ALK7-Z01 to ALK7-Z200 and ALK7-D01 to ALK7-D201 were synthesized (the sequences of dsRNA molecules that entered the second round of screening and some dsRNA molecules that did not enter the second round of screening are shown in Table A at the end of the article), a total of 400 dsRNAs were screened, and the results are not shown;

[0478] Second round of screening: 131 dsRNA molecules with the highest ALK7 mRNA expression inhibition efficiency in the first round of screening were selected for a second round of confirmation and screening. The results are shown in Table 1.

[0479] Third round of screening: 45 dsRNA molecules with the highest ALK7 mRNA expression inhibition efficiency in the second round of screening were selected for three rounds of confirmation and screening. The results are shown in Table 2.

[0480] Table 1: Results of high-throughput screening of Top 131 dsRNA molecules (unmodified) at 0.1 nM

[0481] Table 2: Results of High-Throughput Screening of Top 45 Unmodified dsRNAs at 0.1 nM

[0482] Example 3: Screening and detection of in vitro inhibitory activity of ALK7 gene-dsRNA (E20 modified)

[0483] Based on the third round of activity screening results of naked dsRNA molecules in Example 2, the Top 16 naked dsRNA molecules with the highest inhibitory activity (see Table 3) and the Yangshen molecule AC006188 naked molecule were selected and modified using the E20 modification motif shown below. The dsRNA synthesis steps are described in Example 1.

[0484] E20 modification:

[0485] Sense strand: NmsNmsNmNmNmNmNfNmNfNfNfNfNfNmNmNmNmNmNmNmsNmsNm;

[0486] Antisense strand: NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm;

[0487] In each dsRNA molecule, the nucleotides in the sense and antisense strands correspond one-to-one with the N in the above sense and antisense strand modification motifs from the 5' end to the 3' end. Nm indicates that the nucleotide has a 2'-OMe modification, Nf indicates that the nucleotide has a 2'-F modification, and s indicates that the two nucleotides before and after it are linked by a phosphate thioester bond.

[0488] It should be understood that in this embodiment and in subsequent embodiments, each dsRNA molecule does not contain any modifications not specified in the modification motif.

[0489] The inhibition activity detection procedure was the same as in Example 2, with the working concentration of dsRNA molecules being 0.1 nM.

[0490] The screening results are shown in Table 3. In the transfection experiment with a final concentration of 0.1 nM, the inhibitory activity of ALK7 mRNA expression of 16 candidate dsRNA molecules containing E20 modification was evaluated. Among them, the five dsRNA molecules with the strongest inhibitory activity were ALK7-Z15-E20, ALK7-Z52-E20, ALK7-D127-E20, ALK7-D71-E20, and ALK7-Z38-E20. The inhibitory effects of these molecules were all better than the positive control AC006188-E20, and they could reduce the ALK7 mRNA expression level to about 40-50%. In addition, according to sequence analysis, all 400 dsRNA molecules tested in the first round of screening in Example 2 of this application simultaneously target ALK7 mRNA in humans and cynomolgus monkeys. Among them, ALK7-Z38 can simultaneously target ALK7 mRNA in humans, cynomolgus monkeys, and mice.

[0491] Table 3. Screening results of Top 16 dsRNAs (E20 modified) at 0.1 nM

[0492] Example 4: Screening of the lipophilic fraction

[0493] This embodiment aims to test the effect of introducing lipophilic modifications into dsRNA molecules on enhancing dsRNA molecule activity.

[0494] 4.1 Synthesis of Modified Nucleoside Monomer Compounds

[0495] The exemplary lipophilic portions of this application and the lipophilic portions used in this embodiment are shown in Table 4. The manner in which the lipophilic portions are linked to nucleotides to form conjugates is described in Example 1.

[0496] Table 4: Note: The monomers in this application need to carry relevant protecting groups before synthesizing dsRNA. During the synthesis of dsRNA, the protecting groups are removed to form the specific structure in the oligonucleotide.

[0497] The synthetic methods for each lipophilic moiety in Table 4 are shown below. 4.1.1: Synthesis of Compound 15

[0498] ① Synthesis of compound 11:

[0499] 11 g of compound 4 (CAS #: 51468-42-9, β-D-Ribofuranose, 2-O-methyl-,1-acetate3,5-dibenzoate, 1-acetoxy-2-methoxy-3,5-dibenzoyloxy-1-beta-D-ribofuranosyl), 5.27 g of TMSCN, and 110 mL of DCM were added to a three-necked flask. After stirring to dissolve, the mixture was cooled to -17 °C, and 3.11 mL of anhydrous SnCl4 was added under nitrogen protection. The mixture was stirred at -17 °C for 2 min. Then, 110 mL of 5% sodium bicarbonate aqueous solution was carefully added dropwise. The mixture was heated to 0 °C, stirred for 30 min, and then allowed to stand for separation. The organic phase was washed successively with 110 mL of 5% sodium bicarbonate aqueous solution and 110 mL of saturated saline solution. The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was subjected to column chromatography to obtain 6 g of compound 11, a colorless oil, with a yield of 59.3%. ESI+MS: m / z 399.3 [M+NH4] + ] + .

[0500] ② Synthesis of compound 12:

[0501] HCl-EtOH (7.87 mL, 4 M) was added to 6 g of compound 11, and the mixture was stirred at 60 °C for 0.5 h. The solution was then concentrated under vacuum to obtain crude compound 12, which was used directly in the next step. ESI+MS: m / z 446.3 [M+NH4] + ] + .

[0502] ③ Synthesis of compound 13:

[0503] 3 g of crude compound 12 was dissolved in 30 mL of 1,4-dioxane, followed by the addition of 6.26 g of hexadecylamine and 9.73 mL of Et3N. The mixture was then heated to 110 °C and stirred at 110 °C for 12 hours. The reaction solution was cooled to room temperature and concentrated under vacuum. The crude product was chromatographically analyzed with EA:hept. = 1:10-4:1 eluent. After concentration and drying, 1.5 g of compound 13 was obtained as a white solid powder. The two-step yield was 34.3%. ESI+MS: m / z 624.6 [M+H] + .

[0504] ④ Synthesis of compound 14:

[0505] 1.5 g of compound 13 was dissolved in a mixed solution of DCM / MeOH (3 mL / 12 mL). 433 mg of MeONa / MeOH solution (approximately 30% by mass) was carefully added dropwise at room temperature, and stirring was continued for 1 hour at room temperature. The reaction mixture was cooled to 0 °C, and 3 mL of 10% NH4Cl aqueous solution was slowly added dropwise. Stirring was continued at 0 °C for 15 min, followed by concentration under vacuum. The crude product was redissolved in EA (30 mL), washed successively with 15 mL of 5% sodium bicarbonate aqueous solution and 15 mL of saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was used directly in the next step. ESI+MS: m / z 416.6 [M+H] + .

[0506] The crude product was dissolved in pyridine (15 mL), and 488.8 mg DMTrCl was added. The mixture was stirred at room temperature for 1 hour, and then quenched with MeOH (1.5 mL). After stirring for 15 min, 600 mg NaHCO3 solid was added, and stirring was continued for another 15 min. The mixture was then concentrated under vacuum, and the resulting crude product was redissolved in DCM (15 mL). It was washed successively with 5% sodium bicarbonate aqueous solution (7.5 mL) and saturated brine (7.5 mL). The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was chromatographically analyzed with EA:hept. = 10:1-1:1 eluent. After concentration and drying, 0.71 g of compound 14 was obtained as a colorless oil. The two-step yield was 41.0%. ESI-MS: m / z 776.6 [M+OAc] - ] - .

[0507] ⑤ Synthesis of Compound 15:

[0508] 0.71 g of compound 14 was dissolved in DCM (7 mL) and cooled at 0 ± 2 °C. 357.7 mg of 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphonic diamine was added, followed by 55.4 mg of 1H-tetrazole. The reaction mixture was stirred at 0 ± 2 °C for 15 min, then brought to room temperature and stirred for another 12 hours. The reaction mixture was cooled at 0 ± 2 °C, quenched with 5% NaHCO3 (7 mL), and the organic phase was separated. The organic phase was washed with saturated brine (7 mL) at 0 ± 2 °C, dried over anhydrous sodium sulfate, and filtered. The collected organic phase was concentrated under vacuum, and the crude product was chromatographically analyzed with EA:hept. = 10:1-2:1 eluent. After concentration and drying, 0.65 g of compound 15 was obtained as a colorless oil, with a yield of 71.6% and a phosphorus spectrum purity of 93.1%. ESI-MS: m / z 976.8 [M+OAc] - ] - .

[0509] 4.1.2 Synthesis of Compound 23

[0510] ① Synthesis of compound 17:

[0511] 5 g of compound 16 (CAS #: 506-13-8, 16-Hydroxyhexadecanoic acid, 16-hydroxypalmitic acid) was dissolved in DMSO (75 mL), followed by the addition of 2.54 g of potassium carbonate and 1.37 mL of iodomethane. The mixture was then heated to 50 °C and stirred at 50 °C for 12 hours. The reaction mixture was cooled to room temperature. 150 mL of DCM was added to the reaction mixture, followed by washing with water (75 mL × 2) and saturated brine (75 mL). The organic phase was dried over anhydrous sodium sulfate and filtered. The collected organic phase was concentrated under vacuum to obtain crude compound 17, which was used directly in the next step.

[0512] ② Synthesis of compound 18:

[0513] Add 4.4 g of compound 4 and 44 mL of DCM to a three-necked flask. After stirring to dissolve, cool to -17 °C. Under nitrogen protection, carefully add 3.54 g of TMSOTf dropwise and stir at -17 °C for 15 min. Then carefully add 3.65 g of a DCM solution of compound 17 (8.8 mL). After the addition is complete, continue stirring at -17 °C for 2 hours. Heat the reaction solution to 0-5 °C and carefully add 5% sodium bicarbonate aqueous solution (44 mL). After stirring for 15 min, allow to stand and separate the liquid. Separate the organic phase and wash successively with 5% sodium bicarbonate aqueous solution (44 mL) and saturated brine (44 mL). Dry the organic phase with anhydrous sodium sulfate, filter, and concentrate. 6 g of compound 18, a colorless oily crude product, is obtained and used directly in the next step. ESI+MS: m / z 658.6 [M+NH4+]+.

[0514] ③ Synthesis of compound 19:

[0515] 6 g of crude compound 18 was dissolved in a mixed solution of DCM / MeOH (12 mL / 48 mL). 1.69 g of MeONa / MeOH solution (approximately 30% by mass) was carefully added dropwise at room temperature, and stirring continued for 1 hour at room temperature. The reaction mixture was cooled to 0°C, and 12 mL of 10% NH4Cl aqueous solution was slowly added dropwise. Stirring continued at 0°C for 15 min, followed by concentration under vacuum. The resulting crude product was redissolved in EA (60 mL), washed successively with 12 mL of 5% sodium bicarbonate aqueous solution and 12 mL of saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The resulting crude compound 19 was used directly in the next step. ESI-MS: m / z 491.4 [M+OAc-]-.

[0516] ④ Synthesis of compound 20:

[0517] The crude compound 19 was dissolved in pyridine (40 mL), and 3.37 g of DMTrCl was added. The mixture was stirred at room temperature for 1 hour, and then quenched with MeOH (1.5 mL). After stirring for 15 min, 1.25 g of NaHCO3 solid was added, and stirring was continued for another 15 min. The mixture was then concentrated under vacuum, and the resulting crude product was redissolved in DCM (40 mL). It was washed successively with 5% sodium bicarbonate aqueous solution (20 mL) and saturated brine (20 mL). The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated. The crude compound 20 was used directly in the next step. ESI-MS: m / z 793.7 [M+OAc-]-.

[0518] ⑤ Synthesis of compound 21:

[0519] The crude compound 20 was dissolved in a mixed solution of THF / MeOH / water (14 mL / 56 mL / 7 mL), and 625.2 mg of LiOH·H₂O was added. The mixture was stirred at room temperature for 16 hours, followed by concentration under vacuum. The resulting crude product was redissolved in DCM (70 mL), washed successively with water (20 mL) and saturated brine (20 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. MeOH (1.5 mL) was added to quench the reaction. After stirring for 15 min, 1.25 g of NaHCO₃ solid was added, and stirring was continued for another 15 min. The mixture was then concentrated under vacuum, and the resulting crude product was redissolved in DCM (40 mL), washed successively with 5% sodium bicarbonate aqueous solution (20 mL) and saturated brine (20 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The resulting crude compound 21 was used directly in the next step. ESI-MS: m / z 719.6 [MH]-.

[0520] ⑥ Synthesis of compound 22:

[0521] To a DCM (65 mL) solution of the crude compound 21, 2.07 g of EDCI·HCl, 1.83 g of HOBt, and 8.0 mL of Et3N were added sequentially. The mixture was stirred at room temperature for 0.5 hours under nitrogen protection. Then, 659.4 mg of n-butylamine was added, and the mixture was stirred at room temperature for another 16 hours under nitrogen protection. The reaction was quenched with 30 mL of saturated brine, and the organic phase was separated. The organic phase was dried over anhydrous sodium sulfate and filtered. The collected organic phase was concentrated under vacuum. The crude product was chromatographically analyzed using EA:hept. = 1:10-4:1 eluent. After concentration and drying, 4 g of compound 22, a colorless oil, was obtained, with a five-step yield of 57.1%. ESI-MS: m / z 834.7 [M+OAc-]-.

[0522] ⑦ Synthesis of compound 23:

[0523] 4 g of compound 22 was dissolved in DCM (40 mL) and cooled at 0 ± 2 °C. 1.86 g of 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphonic diamine was added, followed by 288.9 mg of 1H-tetrazole. The reaction mixture was stirred at 0 ± 2 °C for 15 min, then brought to room temperature and stirred for another 12 hours. The reaction mixture was cooled at 0 ± 2 °C, quenched with 40 mL of 5% NaHCO3, and the organic phase was separated. The organic phase was washed with saturated brine (40 mL) at 0 ± 2 °C, dried over anhydrous sodium sulfate, and filtered. The collected organic phase was concentrated under vacuum, and the crude product was chromatographically analyzed with EA:hept. = 1:10-1:2 eluent. After concentration and drying, 3.5 g of compound 23, a colorless oil, was obtained, with a yield of 70% and a phosphorus spectrum purity of 88.2%. ESI-MS: m / z 1034.8[M+OAc-]-.

[0524] 4.1.3 Synthesis of Compound 30

[0525] ① Synthesis of compound 24:

[0526] Add 75 mL of Et3N to 20 g of 1,3-propanediamine in MeOH (400 mL), and carefully add 38.33 g of ethyl trifluoroacetate solution dropwise while cooling in an ice bath. After the addition is complete, bring the mixture to room temperature and stir for 1 hour. Then concentrate the reaction solution, and use the crude compound 24 directly in the next step.

[0527] ② Synthesis of compound 25:

[0528] 4 g of crude compound 12 was dissolved in 40 mL of 1,4-dioxane, followed by 4.47 g of crude compound 24 and 6.49 mL of Et3N. The mixture was then heated to 110 °C and stirred at 110 °C for 20 hours. The reaction solution was cooled to room temperature and concentrated under vacuum. The resulting crude compound 25 was used directly in the next step. ESI+MS: m / z 553.0 [M+H] + .

[0529] ③ Synthesis of compound 26:

[0530] The crude compound 25 was dissolved in a mixed solution of DCM / MeOH (8 mL / 32 mL). After thorough stirring, 1.68 g of MeONa was carefully added dropwise under nitrogen protection, and the mixture was stirred at room temperature for 1 hour. The solution was quenched with 10% NH4Cl aqueous solution (8 mL) and then concentrated. The resulting crude product was redissolved in a mixed solution of DCM / i-PrOH (40 mL / 20 mL), washed successively with 5% sodium bicarbonate aqueous solution (10 mL) and saturated brine (10 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The crude compound 26 was used directly in the next step. ESI-MS: m / z 345.2 [M+H] + .

[0531] ④ Synthesis of compound 27:

[0532] The crude compound 26 was dissolved in 30 mL of pyridine. After stirring and dissolving, 2.52 g of DMTrCl was added under nitrogen protection. After the addition was complete, the mixture was stirred at room temperature for 1 hour. Methanol (3 mL) was slowly added to the reaction mixture to quench the reaction. After stirring at room temperature for 15 min, 937 mg of NaHCO3 was added. The mixture was stirred for another 15 min, concentrated, and the crude product was obtained. DCM / H2O (30 mL / 15 mL) was added, stirred and dissolved, and washed with saturated brine (15 mL). The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated. The crude compound 27 was then subjected to column chromatography to give 1 g of a colorless oil. The four-step yield was 16.7%. ESI-MS: m / z 645.4 [MH] - .

[0533] ⑤ Synthesis of compound 28:

[0534] 1 g of compound 27 was dissolved in 3 mL of ACN, and NaOH solution (123.7 mg NaOH dissolved in 1.5 mL of water) was added. The mixture was stirred at room temperature for 12 hours. EA (10 mL) was added to the reaction mixture, and after stirring for 10 min, the organic phase was separated. The organic phase was washed with saturated brine (2 x 3 mL), dried over anhydrous sodium sulfate, and filtered. The collected organic phase was concentrated under vacuum, and the crude compound 28 was used directly in the next step. ESI-MS: m / z 609.4 [M+OAc] - ] - .

[0535] ⑥ Synthesis of compound 29:

[0536] To a DCM (8 mL) solution of 360 mg palmitic acid, 403.7 mg EDCI·HCl, 341.5 mg HOBt, and 1.25 mL Et3N were added sequentially. The mixture was stirred at room temperature for 0.5 hours under nitrogen protection. Then, the crude compound 28 was added, and the mixture was stirred at room temperature for another 16 hours under nitrogen protection. The reaction was quenched with 3 mL saturated brine, and the organic phase was separated. The organic phase was dried over anhydrous sodium sulfate and filtered. The collected organic phase was concentrated under vacuum. The crude product was chromatographically analyzed using EA:hept. = 1:10-4:1 eluent. After concentration and drying, 0.5 g of compound 29, a colorless oil, was obtained. The two-step yield was 45.0%. ESI-MS: m / z 847.7 [M+OAc] - ] - .

[0537] ⑦ Synthesis of compound 30:

[0538] 0.5 g of compound 29 was dissolved in 5 mL of DCM and cooled at 0 ± 2 °C. 229.2 mg of 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphonic diamine was added, followed by 35.5 mg of 1H-tetrazole. The reaction mixture was stirred at 0 ± 2 °C for 15 min, then brought to room temperature and stirred for another 12 hours. The reaction mixture was cooled at 0 ± 2 °C, quenched with 5 mL of 5% NaHCO3, and the organic phase was separated. The organic phase was washed with saturated brine (5 mL) at 0 ± 2 °C, dried over anhydrous sodium sulfate, and filtered. The collected organic phase was concentrated under vacuum, and the crude product was chromatographically analyzed with EA:hept. = 1:10-1:2 eluent. After concentration and drying, 0.38 g of compound 30 was obtained as a colorless oil, with a yield of 60.6% and a phosphorus spectrum purity of 92.69%. ESI-MS: m / z 1047.9 [M+OAc] - ] - .

[0539] Following the synthetic route and procedures described above, and using conventional raw materials and synthetic methods in this field, nucleoside monomers with other 1'-position modifications and other 3' / 5' protecting groups on the sugar ring can be prepared. Table 4 shows representative modified nucleoside monomers prepared and used in this application.

[0540] This embodiment uses several dsRNA molecular modification motifs as shown in Table 5:

[0541] Table 5 Note: In Table 5, each N in each motif independently corresponds from left to right to the nucleotides from 5' to 3' of one strand of the modified dsRNA molecule. Nm represents 2'-OMe modified ribonucleoside, Nf represents 2'-F modified ribonucleoside, and Nd represents deoxyribonucleoside. When N in Nd corresponds to A, G, or C, Nd represents a deoxyribonucleoside with bases A, G, or C. When N in Nd corresponds to U or T, Nd represents deoxythymidine. s indicates that the two nucleosides before and after it are linked by a phosphate thioester bond or that the nucleoside is linked to the target ligand. Nucleosides not marked with s are linked by a phosphodiester bond. vp- represents... The 5'-VP (i.e., 5'-(E)-vinylphosphonate) modification is shown, that is, the vinylphosphonate modification at the 5' position of the antisense strand of the dsRNA molecule. The lipophilic moiety 1-s, lipophilic moiety 2-s, and lipophilic moiety 5-s represent the 3' positions of lipophilic moiety 1, lipophilic moiety 2, and lipophilic moiety 5 as shown in Table 4, which are connected to the 5' position of the nucleotide on the 3' side of "s" via a thiophosphate bond. The s-lipophilic moiety 1, s-lipophilic moiety 2, and s-lipophilic moiety 5 represent the 5' positions of lipophilic moiety 1, lipophilic moiety 2, and lipophilic moiety 5 as shown in Table 4, which are connected to the 3' position of the nucleotide on the 5' side of "s" via a thiophosphate bond. Other structures are shown below:

[0542] invAb:

[0543] LP379-a:

[0544] L6:

[0545] (NH-C6)s:

[0546] C6-S:

[0547] LP-371-a:

[0548] cPrpNms:

[0549] It can be obtained commercially or synthesized by referring to Examples 1-3 in WO 2024 / 148329 A1 patent.

[0550] 4.2 Assay of the inhibitory activity of dsRNA on ALK7 mRNA expression at the cellular level

[0551] Based on the molecular structure information shown in Table 6-1 (modified motifs are shown in Table 5), two dsRNA molecules, Z38-lipophilic moiety 5 and Z52-lipophilic moiety 5, were prepared.

[0552] Using the same method as in Example 2, the expression level of ALK7 mRNA in hTERT A41hBAT-SVF cells transfected with dsRNA at final concentrations of 1 nM and 0.1 nM, relative to the blank control group, was tested 48 hours after transfection. The results are shown in Table 6-1.

[0553] Table 6-1 Inhibitory activity of dsRNA on ALK7 mRNA expression

[0554] As can be seen from the data in Table 2, the Z38 and Z52 molecules modified with the lipophilic part 5 retained a strong inhibitory effect on ALK7 mRNA expression.

[0555] 4.3 In vivo inhibition test of dsRNA on ALK7 mRNA expression in WT mice

[0556] Based on the molecular structure information shown in Table 6-2 (modification motifs are shown in Table 5), five dsRNA molecules were prepared: Z38-lipophilic moiety 5, Z38-lipophilic moiety 2, Z38-lipophilic moiety 1, AC005181-lipophilic moiety 5, and AC005181.

[0557] This experiment used 6-8 week old male C57BL / 6 mice (Shanghai Southern Model Biotechnology Co., Ltd.). Five mice were randomly assigned to each group based on body weight and received a single subcutaneous injection of dsRNA at a dose of 1.5 mg / kg. On day 14 post-administration, the mice were sacrificed, and approximately 100 mg of inguinal white adipose tissue (iWAT) and perigonial white adipose tissue (pgWAT) were collected. RNA was extracted and analyzed by qPCR (methods are described in sections 2.3 to 2.4 of Example 1; mouse primers used are shown in Table A at the end of this document as GAPDH-M (SEQ ID NO: 325 and 326) and ALK7-M (SEQ ID NO: 327 and 328)). The expression level of ALK7 mRNA in the adipose tissue samples was analyzed. GAPDH was used as an internal reference gene, the saline group (same volume as the drug treatment group) was used as a blank control, and AC005181 molecule was used as a positive control. AC005181 is disclosed in WO 20241 / 48329A1, and its naked sequence is shown in Table A at the end of the article.

[0558] Table 6-2. Relative expression levels of ALK7 mRNA in mouse adipose tissue

[0559] As shown in Table 6-2, all modified dsRNA molecules can reduce the expression level of ALK7 mRNA, and some modified dsRNA molecules are more effective than the yangshen molecule AC005181. For example, the nucleotide sequence of AC005181-lipophilic motif 5 is exactly the same as that of AC005181. The main difference is the lipophilic motif used in both. It can be seen that the lipophilic motif 5 introduced in AC005181-lipophilic motif 5 has a better mRNA expression inhibition effect than the lipophilic motif in AC005181. In addition, Z38-lipophilic motif 5, Z38-lipophilic motif 2, and Z38-lipophilic motif 1 have the same base sequence. The mRNA expression inhibition effect of Z38-lipophilic motif 5 (motif 5-lipophilic modification 4-vp) is better than that of Z38-lipophilic motif 2 and Z38-lipophilic motif 1 (motif 4-lipophilic modification 5-vp and motif 4-lipophilic modification 8-vp).

[0560] Example 5: Detection of the effectiveness of DIO in cynomolgus monkeys

[0561] The Z52 molecule (with sense and antisense strand base sequences as shown in SEQ ID NO. 3 and SEQ ID NO. 4, respectively) was modified with motif 5-lipophilic modification 4-vp as shown in Table 5 to obtain the Z52-lipophilic motif 5 as shown in Table 7. The structure of (GN75) is the same as lipophilic motif 5, which is linked to the 5' or 3' position of the 5' or 3' end nucleotide via a thiophosphate bond. The AC006189 molecule from WO 2024 / 148329 A1 (i.e., base sequence see Table A, modified with motif 6-lipophilic modification 21-cPrp as shown in Table 5) was used as a positive control; its specific structure is shown in Table 7.

[0562] Table 7. Structure of dsRNA molecules tested in DIO cynomolgus monkeys

[0563] The experiment used four 8-year-old male cynomolgus macaques (Kunming Keling Biotechnology Co., Ltd. (KBI)) weighing approximately 11-13 kg (diet-induced obesity). After screening, adaptation, and training, two macaques were enrolled in each group. On day 0, a single subcutaneous administration of dsRNA at a dose of 3 MPk was administered. Serum free fatty acids (NEFA) were measured 7 days before administration and at days 0, 7, 14, 21, 28, 42, and 56 after administration. Simultaneously with weight monitoring, MRI scans were performed before administration (-2 weeks) and at weeks 4, 8, and 14 after administration to quantitatively measure liver fat, abdominal fat, and visceral fat.

[0564] The results showed that, compared with pre-drug administration, the body weight of both groups of cynomolgus monkeys decreased significantly over time after a single dose (Figure 1) for more than 3 months, with the Z52-lipophilic fraction 5 group showing a slightly greater reduction than the AC006189 group. MRI scans (Figures 2 and 3) showed that the abdominal fat volume and visceral fat volume of both groups of cynomolgus monkeys decreased to some extent over time, consistent with the trend of body weight changes. In addition, the serum NEFA levels of both groups of cynomolgus monkeys increased over time (Figure 4), indicating increased lipolysis and the production of more NEFA after administration. These results confirm that the Z52-lipophilic fraction 5 molecule can exert a weight-loss effect by reducing the content of adipose tissue.

[0565] Example 6: In vivo efficacy test in humanized mice

[0566] Based on the results of Example 3, five dsRNA molecules—Z15, Z47, Z50, D71, and D152—were selected and modified with the motif 5-lipophilic modification 4-vp shown in Table 5 to obtain the five modified dsRNA molecules shown in Table 8. Their effectiveness in mice was then tested. The AC006189 molecule (same as in Example 5) was used as a positive control, and the saline group served as a blank control.

[0567] Six- to eight-week-old humanized male ALK7 mice (Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd.) were randomly divided into groups of 6-8 mice per group according to body weight (6 mice per group for Z47-lipophilic fraction 5, Z50-lipophilic fraction 5, D71-lipophilic fraction 5, and D152-lipophilic fraction 5, and 8 mice per group for the remaining groups). A single subcutaneous injection of dsRNA at 3 mg / kg was administered on day 0. On day 14, inguinal white adipose tissue (iWAT) and epididymal white adipose tissue (eWAT) were collected. RNA was extracted from the tissues using the RNA Easy Fast Animal Tissue / Cell Total RNA Extraction Kit (Tiangen Biotech, catalog number DP451). The extracted RNA was then reverse transcribed into cDNA using the Takara PrimeScript RT Master Mix (Perfect Real Time) reverse transcription kit. Subsequently, TB... Pre mix Ex Taq TM (Tli RNaseH Plus) was used to perform quantitative fluorescent PCR detection of cDNA. Elf1 and Rpl30 were used as internal reference genes, and the qPCR primer sequence information is shown in Table A as Elf1-M (SEQ ID: 329 and 330), Rpl30-M (SEQ ID: 331 and 332), and ALK7-H2 (SEQ ID: 323 and 324).

[0568] Table 8. Structure of dsRNA molecules tested in humanized mice

[0569] Table 9-1. Results of qPCR detection of iWAT tissue in humanized mice.

[0570] Table 9-2. Results of qPCR detection of eWAT tissue in humanized mice.

[0571] The experimental results (Tables 9-1 and 9-2) showed that, compared with the blank control group, the expression levels of human ALK7 gene mRNA in iWAT and eWAT tissues of each treatment group were reduced on day 14. Among them, the expression level of D152-lipophilic fraction 5 was reduced the most, decreasing by about 65% compared with the blank control group, which was better than that of Yangshen molecule AC006189.

[0572] Example 7: Detection of DIO in Cynomolgus Monkeys

[0573] Based on the results of Example 6, the 5'-phosphate mimicry in the D152-lipophilic moiety 5 molecule was changed (i.e., VP (vinylphosphonate) was replaced with PEO (oxyethylphosphonate)), and named "D152-lipophilic moiety 5-PEO". Its effectiveness was further evaluated in DIO cynomolgus monkeys. PEO is attached to the 4' position of the 5' terminal nucleotide of the antisense strand. The specific structure of "D152-lipophilic moiety 5-PEO" is shown in Table 10. The structure and synthesis method of PEO are as described in Example 1.

[0574] Table 10. Structure of dsRNA molecules tested in DIO cynomolgus monkeys

[0575] Two DIO cynomolgus monkeys (Kunming Keling Biotechnology Co., Ltd. (KBI)) were used in the experiment. After screening, adaptation, and training, they were administered dsRNA subcutaneously on day 0 at a dose of 3 mpk. Weight was monitored weekly after administration. Abdominal fat aspiration was performed before administration (day -13) and at days 15, 28, and 58 post-administration, and the expression level of the target gene ALK7 mRNA in abdominal adipose tissue was detected by qPCR. Simultaneously with weight monitoring, MRI scans were performed before administration (day -13) and at days 28 and 58 post-administration to quantify liver fat, abdominal fat, and visceral fat.

[0576] The results showed that the body weight of cynomolgus monkeys decreased significantly over time after a single injection compared to pre-administration levels (Figure 5). MRI scans (Figure 6) showed a reduction in both abdominal and visceral fat volume over time, consistent with the trend in body weight changes. Furthermore, the D152-lipophilic 5-PEO molecule significantly reduced the level of ALK gene mRNA in the abdominal fat of cynomolgus monkeys (Figure 7) (commercial internal control gene and ALK7 amplification primers: Primer probe for Human GAPDH (VIC-MGB), Thermo, catalog number: Hs 02786624g1; Primer probe for Human ALK7 (FAM-MGB), HelyxQuest, catalog number: Hs00899854m1). These results confirm that the D152-lipophilic 5-PEO molecule can indeed exert a significant weight-loss effect in primates by inhibiting the ALK7 gene and reducing adipose tissue content.

[0577] Example 8: Study of ALK7-D152 target

[0578] Following the method described in Example 2, hTERT A41hBAT-SVF cells were transfected with ALK7-D152-related dsRNAs (dsRNAs generated by shifting the ALK7-D152 target region onto the target mRNA to the 5' or 3' region, or dsRNAs generated by truncating ALK7-D152) listed in Table 11. All dsRNAs were unmodified, and their base sequences are shown in Table A for the corresponding dsRNA molecules. The transfection complex was prepared as follows: dsRNA was diluted with Opti-MEM to obtain 50 μL of dsRNA-Opti-MEM mixtures of different concentrations. Simultaneously, 49 μL of Opti-MEM and 1 μL of RNAiMax transfection reagent were mixed and incubated for 5 min each. Then, the two mixtures were combined and incubated for 20 min to form the transfection complex. The working concentration of dsRNA was 1 nM. Real-time quantitative PCR analysis was performed 48 h after transfection. The results are shown in Table 11.

[0579] Table 11: Results of in vitro activity assay of unmodified dsRNA (1 nM)

[0580] As shown in Table 11, without any modification to the dsRNA, ALK7-D152 and the dsRNA corresponding to the target region obtained by shifting its target region (the sequence complementary to the antisense sequence on the mRNA) 1 to 3 nucleotides towards the 5' and 3' ends of the mRNA respectively (dsRNA containing 18 nt of sense strand from the 5' end and 20 nt of antisense strand from the 3' end; and dsRNA containing 19 nt of sense strand from the 3' end and 21 nt of antisense strand from the 5' end) can achieve better inhibitory effects on the target mRNA. Moreover, ALK7-D152, ALK7-D152+[1] and ALK7-D152-short are significantly better than Yangshen molecules.

[0581] Furthermore, the nucleotide sequences of ALK7-D152 after being modified with various modification motifs are shown in Table 12.

[0582] Table 12.

[0583] Using the method described in Example 2, the inhibitory effect of the dsRNA molecules listed in Table 12 on the target genes was tested, with AC0006189 designated as a positive control (structure and modifications are shown in Table 7). hTERT A41hBAT-SVF cells were transfected at working concentrations of 1 nM and 0.02 nM. After 48 h, cells were collected and ALK7 mRNA expression was detected by quantitative real-time PCR. The results are shown in Tables 13-1 and 13-2.

[0584] Table 13-1: Results of in vitro activity assay of dsRNA (1 nM)

[0585] Table 13-2: Results of in vitro activity assay of dsRNA (0.02 nM)

[0586] As shown in Tables 13-1 and 13-2, the knockdown effect of the naked ALK7-D152 molecule with different modifications at various concentrations was still superior to that of the positive control molecule AC0006189, exhibiting higher repressive activity against the target gene. Furthermore, among the three modifications, the E20 modification resulted in a better repressive effect. Additionally, the truncated form of ALK7-D152-E20 also showed superior repressive activity.

[0587] Example 9: Testing the effects of different phosphonic acid group modifications

[0588] Using the method shown in Example 8, dsRNAs modified with different 5'-phosphate mimics were tested. hTERT A41hBAT-SVF cells were transfected with different concentration gradients of each dsRNA, with the highest working concentration being 100 nM. Different concentration gradients were obtained by serially diluting the dsRNAs six times in a 10-fold series. Real-time quantitative PCR analysis was performed 48 h after transfection. The structures and sequences of the tested dsRNAs are shown in Tables 14-15, and the results are shown in Tables 16-17.

[0589] Table 14. Nucleotide sequence structure

[0590] Table 15. Structures of 5'-phosphate ester mimics in Table 14

[0591] Table 16. IC50 of ALK7 dsRNA after different phosphate mimics 50 Result-1

[0592] Table 17. IC50 of ALK7 dsRNA after different phosphate mimics 50 Result-2

[0593] In vitro results showed that the IC50 of the PEO-modified dsRNA of this application inhibiting the target gene ALK7 was lower than that of VP-modified and other 5'-phosphate mimics modified with similar structures (e.g., L isomers or other minor group substitutions). The PEO-modified dsRNA had higher inhibitory activity against the target gene and was a better modification method.

[0594] The synthesis processes of phosphoramide monomers for each nucleotide monomer in Table 15 are as follows: (1) Phosphoramide monomers of PEO-Lc-Am and PEO-L-Am: Synthesis process of compound 4P

[0595] ① Synthesis of compound 4B:

[0596] 195.96 g of 4,4'-dimethoxytriphenylmethyl chloride was added in portions to a pyridine (1 L) solution of compound 4A (100 g) (CAS #: 37077-81-9,1,2-O-isopropylidene-α-D-xylose-furanose), and the mixture was stirred at room temperature for 2 hours under nitrogen protection. Excess 4,4'-dimethoxytriphenylmethyl chloride was quenched by slowly adding methanol (50 mL) to the reaction mixture, and the mixture was stirred at room temperature for 15 min. Then, sodium bicarbonate (53 g) was added, and the mixture was concentrated to obtain a crude product. Dichloromethane / water was added, the mixture was stirred to dissolve, and the product was washed with water. The organic phase was dried over anhydrous sodium sulfate and filtered. The collected organic phase was concentrated under vacuum, and the resulting crude compound 4B was used directly in the next step. LCMS(ESI)C 29 H 32 O7[M+H] + Theoretical calculated value of m / z: 493.6, measured value [M+OAc] - ] - =551.2.

[0597] ② Synthesis of compound 4C:

[0598] The crude compound 4B was dissolved in 4.25 L of N,N-dimethylformamide and cooled at 0±2 °C. 20.71 g of sodium hydride (stored in 60% mineral oil) was carefully added, and the mixture was stirred at 0±2 °C for half an hour, followed by the addition of 58.79 g of iodomethane. The reaction mixture was stirred at 0±2 °C for 15 min, then brought to room temperature and stirred for another 16 hours. The reaction mixture was cooled at 0±2 °C, carefully quenched with water, extracted with dichloromethane, and the organic phase was separated. The organic phase was washed with saturated brine at 0±2 °C, dried over anhydrous sodium sulfate, and filtered. The collected organic phase was concentrated under vacuum, and the crude compound 4C was used directly in the next step. LCMS(ESI)C 30 H 34 O7[M+H] + Theoretical calculated value of m / z: 507.6; measured value: [M+Na] + =529.1.

[0599] ③ Synthesis of compound 4D:

[0600] Compound 4C (21 g) and dichloromethane (210 mL) were added to a flask. After stirring to dissolve, the mixture was cooled to 0 ± 2 °C, and dichloroacetic acid (5.34 g) was carefully added dropwise. The reaction mixture was stirred at 0 ± 2 °C for 15 min, then brought to room temperature and stirred for another hour. The reaction mixture was cooled to 0 ± 2 °C, and 5% sodium bicarbonate aqueous solution was carefully added dropwise. After stirring for 15 min, the mixture was allowed to stand and separated. The organic phase was dried with anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. The crude compound 4D was used directly in the next step. LCMS(ESI)C9H 16O5[M+H] + Theoretical calculated m / z value: 205.2, measured value: [M+NH4] + ] + =222.1.

[0601] ④ Synthesis of compound 4E:

[0602] Compound 4D (50 g) was dissolved in tetrahydrofuran (500 mL) and cooled at 0 ± 2 °C. 19.58 g of sodium hydride (stored in 60% mineral oil) was carefully added, and the mixture was stirred at 0 ± 2 °C for half an hour, followed by the addition of 62.81 g of benzyl bromide. The reaction mixture was stirred at 0 ± 2 °C for 15 min, then brought to room temperature and stirred for another 16 hours. The reaction mixture was cooled at 0 ± 2 °C, carefully quenched with 10% ammonium chloride, extracted with ethyl acetate, and the organic phase was separated. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and filtered. The collected organic phase was concentrated under vacuum, and the crude compound 4E was used directly in the next step. LCMS(ESI)C 16 H 22 O5[M+H] + Theoretical calculated m / z value: 295.4, measured value: [M+NH4] + ] + =312.2.

[0603] ⑤ Synthesis of compound 4F:

[0604] Water (80 mL) and trifluoroacetic acid (120 mL) were added sequentially to a flask and stirred until homogeneous. Then, compound 4E (20 g) was added. After dissolution, the mixture was stirred at room temperature for 4 hours. Water (60 mL) was added to dilute the reaction mixture, and the temperature was lowered to 0 ± 2 °C. The pH was then adjusted to 3-4 with solid sodium hydroxide and finally neutralized to 7-8 with 5% sodium bicarbonate solution. The mixture was then extracted with dichloromethane / isopropanol. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and filtered. The collected organic phase was concentrated under vacuum, and the crude compound 4F was used directly in the next step. LCMS(ESI)C 13 H 18 O5[M+H] + Theoretical calculated value of m / z is 255.3, measured value [M+NH4] + ] + =272.2.

[0605] ⑥ Synthesis of compound 4G:

[0606] Compound 4F (15 g) was dissolved in pyridine (75 mL), and acetic anhydride (30.11 g) was added. The mixture was stirred at room temperature for 1.5 hours. The reaction solution was concentrated under vacuum at 40-45 °C. The resulting crude compound 4G was used directly in the next step. LCMS(ESI)C 17 H 22 O7[M+H] + Theoretical calculated value of m / z: 339.4; Measured value: [M+NH4] + ] + =356.3.

[0607] ⑦ Synthesis of compound 4G-1:

[0608] 10 g of 4G-2 was slowly added to a 1 mol / L solution of boranetetrahydrofuran in a 500 mL three-necked flask. The mixture was stirred at 25 °C for 16 hours. After the reaction was complete, ethanol was slowly added dropwise to quench the reaction. The mixture was then concentrated under reduced pressure. The residue was purified by silica gel column chromatography to obtain 7 g of 4G-1. 1 H NMR (CDCl3, 400MHz) δ3.93-3.84(m,2H),3.78(s,3H),3.75(s,3H),2.14-2.05(m,2H).

[0609] ⑧ Synthesis of compound 4H:

[0610] Compound 4G (10.0 g) and dichloromethane (100 mL) were added to a three-necked flask. After stirring to dissolve, the mixture was cooled to -17°C. Under nitrogen protection, trimethylsilyl trifluoromethanesulfonate (13.14 g) was carefully added dropwise while stirring at -17°C for 15 min. Subsequently, compound 4G-1 (6.83 g) was carefully added dropwise. After the addition was complete, stirring was continued at -17°C for 1 hour. The reaction mixture was then heated to 0-5°C, and triethylamine was carefully added dropwise to quench the reaction. After stirring for 15 min, the mixture was allowed to stand and separated. The organic phase was washed successively with 5% sodium bicarbonate aqueous solution and saturated brine. The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated. Methanol (100 mL) was added to the concentrated residue, and after stirring to dissolve, potassium carbonate (6.13 g) was added. The mixture was stirred at room temperature for 3 hours. The residue was filtered, and the pH of the filtrate was adjusted to 7-8 with glacial acetic acid, followed by concentration to dryness under reduced pressure.

[0611] The concentrated residue was diluted once with pyridine, then redissolved in pyridine (100 mL). The solution was cooled to 0±2 °C, and benzoyl chloride (6.23 g) was carefully added dropwise. The reaction mixture was stirred at 0±2 °C for 10 min, then brought to room temperature and stirred for another 16 hours. The reaction was quenched with solid sodium bicarbonate, and then concentrated under reduced pressure. The solution was redissolved with dichloromethane / water, stirred for 15 min, and allowed to stand before separation. The organic phase was separated, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was subjected to column chromatography to give 8 g of compound 4H oil, with a yield of 46.9%. LCMS(ESI)C 24 H 31 O9P[M+H] + Theoretical calculated value of m / z: 495.5, measured value: [M+NH4] + ] + =512.4.

[0612] Synthesis of compound 4J:

[0613] Add compound 4H (8 g), acetic acid (1 mL), ethanol (72 mL), and water (8 mL) to a three-necked flask. After stirring to dissolve, purge with nitrogen for 3-5 min each time. Under nitrogen protection, carefully add palladium on carbon (1.6 g), then purge with hydrogen for 3-5 min each time. Stir at room temperature for 16 hours under a hydrogen atmosphere. Filter, and wash the residue with ethanol. Concentrate the filtrate under reduced pressure. The crude compound 4J obtained is used directly in the next step. LCMS(ESI)C 17 H 25 O9P[M+H] + Theoretical calculated value of m / z: 405.4, measured value: [M+OAc] - ] - =463.1.

[0614] ⑩ Synthesis of compound 4K:

[0615] Compound 4J (2.5 g) was dissolved in acetonitrile / water (15 mL / 10 mL). The solution was first cooled to 0 ± 2 °C, and then 4.38 g of diacetoxyiodobenzene (4.38 g) and 2,2,6,6-tetramethylpiperidine oxide (298.8 mg) were added sequentially. The reaction mixture was stirred at 0 ± 2 °C for 10 min, then brought to room temperature and stirred for another 16 hours. The solution was diluted with water and extracted with dichloromethane. The organic phases were combined and washed with saturated brine. The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was then subjected to column chromatography to give 2 g of compound 4K as a colorless oil, with a yield of 77.3%. LCMS(ESI)C 17 H 23 O 10 P[M+H] + Theoretical calculated m / z value: 419.3, measured value: [M+NH4] +] + =436.3.

[0616] Synthesis of compound 4L:

[0617] Compound 4K (2 g) was dissolved in tetrahydrofuran (8 mL), followed by the addition of pyridine (1.93 mL) and lead tetraacetate (4.24 g), and stirred at room temperature for 1 hour. The mixture was quenched with water (20 mL), then extracted with dichloromethane (20 mL), stirred for 15 min, allowed to stand, and separated into organic and inorganic phases. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was then subjected to column chromatography to give 1.6 g of compound 4L as an oil, with a yield of 77.4%. LCMS(ESI)C 18 H 25 O 10 P[M+H] + Theoretical calculated m / z value: 433.4, measured value: [M+NH4] + ] + =450.3.

[0618] Synthesis of compound 4M:

[0619] 4 L (1.6 g) of compound was dissolved in acetonitrile (16 mL), cooled to -17 °C, and tin tetrachloride (2.11 g) was carefully added dropwise under nitrogen protection while stirring at -17 °C for 15 minutes. Then, N6-benzoyladenine (646 mg) (CAS #: 4005-49-6) was added, and stirring continued at -17 °C for 1 hour. The reaction solution was heated to 0-5 °C, and sodium bicarbonate was added in portions until pH = 7-8. The solution was diluted with dichloromethane, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was subjected to column chromatography to give 280 mg of compound 4M as a colorless oil, with a yield of 12.4%. LCMS(ESI)C 28 H 30 O9N5P[M+H] + Theoretical calculated value of m / z: 612.6; Measured value [M+H] + =612.4.

[0620] Synthesis of compound 4N:

[0621] Compound 4M (230 mg) was dissolved in methanol (10 mL), and then potassium carbonate (104 mg) was added. The mixture was stirred at room temperature for 1 hour. The solution was filtered, and the pH of the filtrate was adjusted to 7-8 with glacial acetic acid. The solution was then concentrated to dryness under reduced pressure. The crude product was subjected to column chromatography to give 100 mg of compound 4N as a colorless oil, with a yield of 52.4%. LCMS(ESI)C 21 H26 O8N5P[M+H] + Theoretical calculated value of m / z: 508.4; measured value [M+H] + =508.3.

[0622] Synthesis of compound 4P:

[0623] Compound 4N (100 mg) was dissolved in dichloromethane (10 mL) and cooled at -5°C. 2-Cyanoethyl-N,N,N',N'-tetraisopropylphosphonic diamine (118 mg) was added, followed by 4,5-dicyanimidazole (35 mg). The reaction mixture was stirred at -5°C for 10 minutes, then brought to room temperature and stirred for another 0.5 hours. The reaction mixture was then cooled at -5°C, quenched with 5% NaHCO3 aqueous solution, stirred for 10 minutes, allowed to stand, and separated. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and filtered. The collected organic phase was concentrated under vacuum to give 130 mg of compound 4P as a colorless oil, with a yield of 93.2%. LCMS(ESI)C 30 H 43 O9N7P2[M+H] + Theoretical calculated value of m / z: 708.7; measured value [M+H] + =708.4. 1 H NMR (500MHz, DMSO) δ11.27(s,1H),8.76(dd,J=29.0,1.5Hz,2H),8.05(d,J=7.3Hz,2H),7.61(dt ,J=15.3,7.4Hz,3H),6.27(dd,J=10.4,7.1Hz,1H),5.20(d,J=34.9Hz,1H),4.90(ddd,J=20.9,7 .0,4.1Hz,1H),4.51(ddd,J=15.7,10.4,4.1Hz,1H),4.01–3.57(m,10H),3.33(d,J=27.2Hz,3H),2.86(t,J=5.9Hz,2H),2.20–2.03(m,2H),1.49–0.90(m,20H).(2) The synthesis process of the phosphorous amide monomer of PEO-Am: compound 5D

[0624] ① Synthesis of compound 3B

[0625] Compound 3A (100 g), 2,2,6,6-tetramethylpiperidine oxide (30.26 g), and sodium bicarbonate (130.14 g) were added to a 2 L three-necked flask. Acetonitrile / water ratio was 1:1 (1 L). The mixture was cooled to 0 °C, and m-chloroperoxybenzoic acid (267.35 g) was slowly added. The reaction mixture was stirred at 25 °C for 4 hours. After the reaction was complete, 20% sodium bisulfite was added to quench the reaction, and the mixture was diluted with ethyl acetate and adjusted to pH 1-2 with concentrated hydrochloric acid. The mixture was extracted twice with ethyl acetate, and the combined organic phases were concentrated under reduced pressure. The residue was slurried with methyl tert-butyl ether and filtered to obtain the first batch of product. The aqueous phase was concentrated under reduced pressure to approximately 300 mL, filtered, and the filter cake was washed twice with water to obtain the second batch of product. The two batches were combined and dried under reduced pressure to obtain 100 g of compound 3B as a white solid, with a yield of 94.8%. LCMS(ESI)C 10 H 12 O7N2[M+H] + Theoretical m / z value: 273.2; Measured value: [M+H] + =273.1.

[0626] ② Synthesis of compound 3C

[0627] Compound 3B (100 g), 4-dimethylaminopyridine (5.84 g), pyridine (95.9 g), and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (112.69 g), along with acetonitrile (1 L), were added to a 2 L three-necked flask. The mixture was cooled to 0 °C, and dimethylhydroxylamine hydrochloride (53.75 g) was added. The reaction mixture was stirred at 25 °C for 2 hours. Imidazole (75.03 g) and tert-butyldimethylchlorosilane (138.42 g) were added, and the reaction mixture was stirred at 25 °C for another 14 hours. After the reaction was complete, the mixture was diluted with water and concentrated under reduced pressure to 300 mL. The mixture was extracted twice with ethyl acetate, and the combined organic phases were washed successively with 1 N hydrochloric acid and saturated sodium bicarbonate, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to 300 mL. Adding petroleum ether (1 L) precipitated a solid, which was filtered and the filter cake dried to give 50 g of compound 3C, a white solid, with a yield of 31.68%. LCMS(ESI)C 18 H 31 O7N3Si[M+H] + Theoretical calculated value of m / z: 430.5; Measured value: [M+H] + =430.2.

[0628] 1H-NMR(DMSO-d6,400MHz)δ11.39(s,1H),8.49(d,J=8.0Hz,1H),6.06(d,J=6.8Hz,1H),5.76(d,J=8.0Hz,1H),4.76(s,1H),4.48 (dd,J=4.0,2.0Hz,1H),3.84(dd,J=6.8,4.0Hz,1H),3.71(s,3H),3.30(s,3H),3.18(s,3H),0.89(s,9H),0.10(d,J=1.2Hz,6H).

[0629] ③ Synthesis of Compound 3D

[0630] Compound 3C (50 g) and 2-methyltetrahydrofuran (750 mL) were added to a 2 L three-necked flask. The mixture was cooled to 0 °C, and 3 M methylmagnesium chloride (89.2 mL) was added dropwise under a nitrogen atmosphere. The reaction mixture was stirred at 25 °C for 16 hours. After the reaction was complete, saturated ammonium chloride was added to quench the reaction, and the mixture was extracted with ethyl acetate. The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography to give 30 g of compound 3D as a yellow solid, with a yield of 67.01%.

[0631] 1 H-NMR(DMSO-d6,400MHz)δ11.49(d,J=1.2Hz,1H),8.09(d,J=8.0Hz,1H),5.97(d,J=6.0Hz,1H),5.81(dd,J=8.0,2.0Hz,1H),4.6 2-4.61(m,1H),4.55(d,J=3.2Hz,1H),3.88(dd,J=5.6,4.8Hz,1H),3.36(s,3H),2.26(s,3H),0.96(s,9H),0.19(d,J=3.2Hz,6H).

[0632] ④ Synthesis of compound 3E

[0633] Compound 3D (30 g), m-chloroperoxybenzoic acid (26.92 g), sodium bicarbonate (19.66 g), and acetonitrile (300 mL) were added to a 1 L three-necked flask. The mixture was stirred at 25 °C for 6 hours. After the reaction was complete, saturated sodium bicarbonate was added, and the mixture was extracted three times with dichloromethane. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography to give 24 g of compound 3E as a yellow solid, with a yield of 76.7%.

[0634] 1H-NMR(DMSO-d6,400MHz)δ11.50(d,J=1.6Hz,1H),7.55(d,J=8.0Hz,1H),5.10(d,J=7.2Hz,1H),5.82-5.78(m,2H) ,4.41(d,J=3.6Hz,1H),4.17(dd,J=7.2,4.0Hz,1H),3.31(s,3H),2.13(s,3H),0.89(s,9H),0.12(d,J=2.8Hz,6H).

[0635] ⑤ Synthesis of compound 3F

[0636] Compound 3E (24 g), triethylamine hydrofluoric acid (28.97 g), triethylamine (36.37 g), and acetonitrile (240 mL) were added to a 1 L three-necked flask. The mixture was stirred at 25 °C for 16 hours. Then, 4-dimethylaminopyridine (0.73 g) and benzoic anhydride (20.33 g) were added, and the reaction mixture was stirred at 25 °C for another 2 hours. The reaction was quenched with saturated sodium bicarbonate solution, extracted twice with dichloromethane, and the organic phases were combined. The mixture was washed twice with saturated sodium bicarbonate, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography to give 20 g of compound 3F as a yellow solid, with a yield of 85.4%. LCMS(ESI)C 18 H 18 O8N2[MH] - Theoretical m / z value: 389.4; Measured value: [MH] - =389.1.

[0637] 1 H-NMR (DMSO-d) 6, 400MHz) δ11.56(d,J=1.2Hz,1H),8.04(d,J=7.4Hz,2H),7.73(t,J=7.4Hz,1H),7.64-7.57(m,3H),6.24(s,1H),6.2 2(s,1H),5.86(dd,J=8.0,2.0Hz,1H),5.67(d,J=4.4Hz,1H),4.64(dd,J=7.2,4.4Hz,1H),3.34(s,3H),2.18(s,3H).

[0638] ⑥ Synthesis of compound 3G

[0639] Compound 3F (100 g) was added to a reactor. Under stirring at -10 to -20 °C, trimethylsilyl trifluoromethanesulfonate (137 g) and 1E-1 (117 g) were added. The mixture was heated to room temperature and stirred continuously. Extraction was then performed. The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated. Ethyl acetate was added to the concentrate, and the mixture was heated and stirred until dissolved. Heptane was slowly added dropwise to precipitate the product. The precipitate was filtered and dried to obtain 70 g of the target product, compound 3G, with a yield of 53.3%. LCMS(ESI)C 22 H 29 O 10 N2P[M+H] + Theoretical m / z value: 513.5; Measured value: [M+H] + =513.50.

[0640] ⑦ Synthesis of Compound 5A

[0641] Compound 3G (100 g) and acetic anhydride (500 mL) were added to a flask, followed by trifluoroacetic acid (1 mL). After stirring to dissolve, the mixture was heated to 95 °C and stirred at 100 °C until the reaction was complete. The reaction solution was cooled to room temperature and concentrated to obtain a crude product. Reversed-phase chromatography was used to prepare 10.5 g of pure compound 5A, with a yield of 11.7%. 20 H 29 O 10 P[M+H] + Theoretical m / z value: 461.4; Measured value: [M+H] + =461.4.

[0642] ⑧ Synthesis of compound 5B

[0643] Compound 5A (13 g) and acetonitrile (325 mL) were added to a three-necked flask, and the mixture was cooled to 0 °C. N6-benzoyladenine (10.1 g) and tin tetrachloride (22.1 g) were then added, and the mixture was heated to room temperature and stirred for 1 hour. The mixture was then extracted with dichloromethane. The organic phase was dried over anhydrous sodium sulfate and concentrated to obtain a crude product. The crude product was then subjected to reversed-phase chromatography to obtain 2.3 g of pure compound 5B, with a yield of 12.7%. LCMS(ESI)C 30 H 34 O9N5P[M+H] + Theoretical m / z value: 640.6; Measured value: [M+H] + =640.37.

[0644] ⑨ Synthesis of compound 5C

[0645] Compound 5B (3.2 g) was added to a three-necked flask and dissolved in anhydrous methanol (32 mL). Under nitrogen protection, potassium carbonate (0.66 g) was added. After the addition was complete, the temperature was raised to 0-5 °C, and the reaction was quenched with hydrochloric acid and extracted. The organic phase was dried over anhydrous sodium sulfate and concentrated to obtain a crude product. The crude product was then subjected to reversed-phase chromatography to obtain 1.3 g of pure compound 5C, with a yield of 48.5%. LCMS(ESI)C 23 H 30 O8N5P[M+H] + Theoretical m / z value: 536.5; Measured value: [M+H] + =536.33.

[0646] 1 H-NMR(DMSO-d6,400MHz)δ11.49(d,J=1.2Hz,1H),8.09(d,J=8.0Hz,1H),5.97(d,J=6.0Hz,1H),5.81(dd,J=8.0,2.0Hz,1H),4.6 2-4.61(m,1H),4.55(d,J=3.2Hz,1H),3.88(dd,J=5.6,4.8Hz,1H),3.36(s,3H),2.26(s,3H),0.96(s,9H),0.19(d,J=3.2Hz,6H).

[0647] ⑩ Synthesis of compound 5D

[0648] Compound 5C (1.6 g) was dissolved in dichloromethane (20 mL) in a single-necked flask. Then, 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphonic diamine (1.3 g) and 1H-tetrazole (0.167 g) were added. The mixture was stirred at room temperature until complete. After the reaction was finished, the mixture was washed with saturated brine. The organic phase was dried over anhydrous sodium sulfate and concentrated to obtain a crude product. The crude product was then dissolved in dichloromethane, and n-heptane was slowly added dropwise to precipitate the product. Finally, the mixture was concentrated and dried to obtain the target product, compound 5D, 1.6 g, with a yield of 72.7%. LCMS(ESI)C 32 H 47 O9N7P2[M+H] + Theoretical m / z value: 736.7; Measured value: [M+H] + =736.49.

[0649] 1H NMR(500MHz,DMSO)δ11.27(s,1H),8.76(dd,J=29.0,1.5Hz,2H),8.05(d,J=7.3Hz,2H), 7.61(dt,J=15.3,7.4Hz,3H),6.27(dd,J=10.4,7.1Hz,1H),5.20(d,J=34.9Hz,1H),4.9 0(ddd,J=20.9,7.0,4.1Hz,1H),4.51(ddd,J=15.7,10.4,4.1Hz,1H),4.01–3.57(m,10H ),3.33(d,J=27.2Hz,3H),2.86(t,J=5.9Hz,2H),2.20–2.03(m,2H),1.49–0.90(m,20H).

[0650] (3) The phosphoramide monomer of VP-Am, namely 5'(E)-VP-2'-OMe-A(Bz) phosphoramide monomer (CAS:2419895-65-9), is a commercially available compound.

[0651] The base sequences of the dsRNA involved in the above embodiments of this application are shown in Table A below. It should be understood that the following sequences are merely exemplary sequences for the embodiments of this application and are not intended to limit the invention of this application in any way.

[0652] Table A: Sequence List

Claims

1. A double-stranded RNA (dsRNA) molecule that inhibits the expression of the activin receptor-like kinase 7 (ALK7) gene via RNAi, comprising a sense strand and an antisense strand that are complementary to each other to form a double-stranded region, or composed of a sense strand and an antisense strand that are complementary to each other to form a double-stranded region, wherein the sense strand is 15-30 nt in length, and the antisense strand is 15-30 nt in length, wherein: (1) The sense strand sequence of the dsRNA molecule comprises at least 15 consecutive bases of the sequence shown in SEQ ID NO. 31, and the antisense strand sequence of the dsRNA molecule comprises at least 15 consecutive bases of the sequence shown in SEQ ID NO. 32; or (2) The sense strand sequence of the dsRNA molecule comprises at least 15 consecutive bases of the sequence shown in SEQ ID NO.1, and the antisense strand sequence of the dsRNA molecule comprises at least 15 consecutive bases of the sequence shown in SEQ ID NO.2; or (3) The sense strand sequence of the dsRNA molecule comprises at least 15 consecutive bases of the sequence shown in SEQ ID NO.3, and the antisense strand sequence of the dsRNA molecule comprises at least 15 consecutive bases of the sequence shown in SEQ ID NO.4; or (4) The sense strand sequence of the dsRNA molecule comprises at least 15 consecutive bases of the sequence shown in SEQ ID NO. 5, and the antisense strand sequence of the dsRNA molecule comprises at least 15 consecutive bases of the sequence shown in SEQ ID NO. 6; or (5) The sense strand sequence of the dsRNA molecule comprises at least 15 consecutive bases of the sequence shown in SEQ ID NO. 7, and the antisense strand sequence of the dsRNA molecule comprises at least 15 consecutive bases of the sequence shown in SEQ ID NO. 8; or (6) The sense strand sequence of the dsRNA molecule comprises at least 15 consecutive bases of the sequence shown in SEQ ID NO. 9, and the antisense strand sequence of the dsRNA molecule comprises at least 15 consecutive bases of the sequence shown in SEQ ID NO. 10; or (7) The sense strand sequence of the dsRNA molecule comprises at least 15 consecutive bases of the sequence shown in SEQ ID NO. 15, and the antisense strand sequence of the dsRNA molecule comprises at least 15 consecutive bases of the sequence shown in SEQ ID NO. 16; or (8) The sense strand sequence of the dsRNA molecule contains at least 15 consecutive bases of the sequence shown in SEQ ID NO.17, and the antisense strand sequence of the dsRNA molecule contains at least 15 consecutive bases of the sequence shown in SEQ ID NO.

18.

2. The dsRNA molecule according to claim 1, wherein: (1) The sense strand sequence of the dsRNA molecule comprises 19-21 consecutive bases (e.g., 21 consecutive bases) of the sequence shown in SEQ ID NO. 31 and / or the antisense strand sequence of the dsRNA molecule comprises 21-23 consecutive bases (e.g., 23 consecutive bases) of the sequence shown in SEQ ID NO. 32; or (2) The sense strand sequence of the dsRNA molecule comprises 19-21 consecutive bases (e.g., 21 consecutive bases) of the sequence shown in SEQ ID NO.1 and / or the antisense strand sequence of the dsRNA molecule comprises 21-23 consecutive bases (e.g., 23 consecutive bases) of the sequence shown in SEQ ID NO.2; or (3) The sense strand sequence of the dsRNA molecule comprises 19-21 consecutive bases (e.g., 21 consecutive bases) of the sequence shown in SEQ ID NO.3 and / or the antisense strand sequence of the dsRNA molecule comprises 21-23 consecutive bases (e.g., 23 consecutive bases) of the sequence shown in SEQ ID NO.4; or (4) The sense strand sequence of the dsRNA molecule comprises 19-21 consecutive bases (e.g., 21 consecutive bases) of the sequence shown in SEQ ID NO. 5 and / or the antisense strand sequence of the dsRNA molecule comprises 21-23 consecutive bases (e.g., 23 consecutive bases) of the sequence shown in SEQ ID NO. 6; or (5) The sense strand sequence of the dsRNA molecule comprises 19-21 consecutive bases (e.g., 21 consecutive bases) of the sequence shown in SEQ ID NO. 7 and / or the antisense strand sequence of the dsRNA molecule comprises 21-23 consecutive bases (e.g., 23 consecutive bases) of the sequence shown in SEQ ID NO. 8; or (6) The sense strand sequence of the dsRNA molecule comprises 19-21 consecutive bases (e.g., 21 consecutive bases) of the sequence shown in SEQ ID NO. 9 and / or the antisense strand sequence of the dsRNA molecule comprises 21-23 consecutive bases (e.g., 23 consecutive bases) of the sequence shown in SEQ ID NO. 10; or (7) The sense strand sequence of the dsRNA molecule comprises 19-21 consecutive bases (e.g., 21 consecutive bases) of the sequence shown in SEQ ID NO. 15 and / or the antisense strand sequence of the dsRNA molecule comprises 21-23 consecutive bases (e.g., 23 consecutive bases) of the sequence shown in SEQ ID NO. 16; or (8) The sense strand sequence of the dsRNA molecule comprises 19-21 consecutive bases (e.g., 21 consecutive bases) of the sequence shown in SEQ ID NO.17 and / or the antisense strand sequence of the dsRNA molecule comprises 21-23 consecutive bases (e.g., 23 consecutive bases) of the sequence shown in SEQ ID NO.

18.

3. The dsRNA molecule according to any one of claims 1-2, wherein more than 80%, more than 85%, more than 90%, or more than 95% of the antisense strand of the dsRNA molecule is complementary to human ALK7 mRNA, or the antisense strand of the dsRNA molecule is completely complementary to ALK7 mRNA.

4. The dsRNA molecule according to any one of claims 1-3, wherein the base sequence of more than 80%, more than 85%, more than 90%, or more than 95% of the sense strand of the dsRNA molecule is contained in the ALK7 mRNA base sequence, or the full-length base sequence of the sense strand of the dsRNA molecule is contained in the ALK7 mRNA base sequence.

5. The dsRNA molecule according to any one of claims 1-4, wherein the sense strand and / or antisense strand of the dsRNA molecule comprises a protruding end, the protruding end being composed of 1, 2 or 3 nucleotides.

6. The dsRNA molecule according to claim 5, wherein the overhang is located on the antisense strand.

7. The dsRNA molecule of claim 6, wherein the overhang is located at the 3' end of the antisense strand.

8. The dsRNA molecule according to any one of claims 5-7, wherein the overhang is composed of two nucleotides.

9. The dsRNA molecule according to any one of claims 1-8, wherein the 3' end of the dsRNA molecule is a blunt end.

10. The dsRNA molecule according to claim 9, wherein the 3' end base pair is an AU base pair.

11. The dsRNA molecule according to any one of claims 1-10, wherein the sense strand of the dsRNA molecule consists of 21 bases and the antisense strand consists of 23 bases.

12. The dsRNA molecule according to any one of claims 1-11, wherein the 3' terminal base pair of the dsRNA molecule is an AU base pair, optionally, the 3' terminal base of the sense strand is A, the 5' terminal base of the antisense strand is U, and wherein: (1) The sense strand sequence of the dsRNA molecule comprises 20 consecutive bases starting from the 5' end of the sequence shown in SEQ ID NO.1, and the antisense strand sequence of the dsRNA molecule comprises 22 consecutive bases starting from the 3' end of the sequence shown in SEQ ID NO.2; or (2) The sense strand sequence of the dsRNA molecule comprises 20 consecutive bases starting from the 5' end of the sequence shown in SEQ ID NO.3, and the antisense strand sequence of the dsRNA molecule comprises 22 consecutive bases starting from the 3' end of the sequence shown in SEQ ID NO.4; or (3) The sense strand sequence of the dsRNA molecule comprises 20 consecutive bases starting from the 5' end of the sequence shown in SEQ ID NO.5, and the antisense strand sequence of the dsRNA molecule comprises 22 consecutive bases starting from the 3' end of the sequence shown in SEQ ID NO.6; or (4) The sense strand sequence of the dsRNA molecule comprises 20 consecutive bases starting from the 5' end of the sequence shown in SEQ ID NO.7, and the antisense strand sequence of the dsRNA molecule comprises 22 consecutive bases starting from the 3' end of the sequence shown in SEQ ID NO.8; or (5) The sense strand sequence of the dsRNA molecule comprises 20 consecutive bases starting from the 5' end of the sequence shown in SEQ ID NO.9, and the antisense strand sequence of the dsRNA molecule comprises 22 consecutive bases starting from the 3' end of the sequence shown in SEQ ID NO.10; or (6) The sense strand sequence of the dsRNA molecule comprises 20 consecutive bases starting from the 5' end of the sequence shown in SEQ ID NO. 15, and the antisense strand sequence of the dsRNA molecule comprises 22 consecutive bases starting from the 3' end of the sequence shown in SEQ ID NO. 16; or (7) The sense strand sequence of the dsRNA molecule comprises 20 consecutive bases starting from the 5' end of the sequence shown in SEQ ID NO. 17, and the antisense strand sequence of the dsRNA molecule comprises 22 consecutive bases starting from the 3' end of the sequence shown in SEQ ID NO. 18; or (8) The sense strand sequence of the dsRNA molecule comprises 20 consecutive bases starting from the 5' end of the sequence shown in SEQ ID NO.31, and the antisense strand sequence of the dsRNA molecule comprises 22 consecutive bases starting from the 3' end of the sequence shown in SEQ ID NO.

32.

13. The dsRNA molecule according to any one of claims 1-12, wherein: (1) The sense sequence of the dsRNA molecule comprises or is the sequence shown in SEQ ID NO.1, and the antisense sequence of the dsRNA molecule comprises or is the sequence shown in SEQ ID NO.2; or (2) The sense strand sequence of the dsRNA molecule includes or is the sequence shown in SEQ ID NO.3, and the antisense strand sequence of the dsRNA molecule includes or is the sequence shown in SEQ ID NO.4; or (3) The sense sequence of the dsRNA molecule includes or is the sequence shown in SEQ ID NO. 5, and the antisense sequence of the dsRNA molecule includes or is the sequence shown in SEQ ID NO. 6; or (4) The sense strand sequence of the dsRNA molecule includes or is the sequence shown in SEQ ID NO. 7, and the antisense strand sequence of the dsRNA molecule includes or is the sequence shown in SEQ ID NO. 8; or (5) The sense sequence of the dsRNA molecule comprises or is the sequence shown in SEQ ID NO. 9, and the antisense sequence of the dsRNA molecule comprises or is the sequence shown in SEQ ID NO. 10; or (6) The sense sequence of the dsRNA molecule includes or is the sequence shown in SEQ ID NO. 15, and the antisense sequence of the dsRNA molecule includes or is the sequence shown in SEQ ID NO. 16; or (7) The sense strand sequence of the dsRNA molecule comprises or is the sequence shown in SEQ ID NO. 17, and the antisense strand sequence of the dsRNA molecule comprises or is the sequence shown in SEQ ID NO. 18; or (8) The sense strand sequence of the dsRNA molecule includes or is the sequence shown in SEQ ID NO. 31, and the antisense strand sequence of the dsRNA molecule includes or is the sequence shown in SEQ ID NO. 32; or (9) The sense strand sequence of the dsRNA molecule includes or is the sequence shown in SEQ ID NO. 33, and the antisense strand sequence of the dsRNA molecule includes or is the sequence shown in SEQ ID NO. 34; or (10) The sense strand sequence of the dsRNA molecule comprises or is the sequence shown in SEQ ID NO. 35, and the antisense strand sequence of the dsRNA molecule comprises or is the sequence shown in SEQ ID NO. 36; or (11) The sense strand sequence of the dsRNA molecule comprises or is the sequence shown in SEQ ID NO. 37, and the antisense strand sequence of the dsRNA molecule comprises or is the sequence shown in SEQ ID NO. 38; or (12) The sense strand sequence of the dsRNA molecule includes or is the sequence shown in SEQ ID NO. 39, and the antisense strand sequence of the dsRNA molecule includes or is the sequence shown in SEQ ID NO. 40; or (13) The sense sequence of the dsRNA molecule includes or is the sequence shown in SEQ ID NO.41, and the antisense sequence of the dsRNA molecule includes or is the sequence shown in SEQ ID NO.42; or (14) The sense strand sequence of the dsRNA molecule includes or is the sequence shown in SEQ ID NO.47, and the antisense strand sequence of the dsRNA molecule includes or is the sequence shown in SEQ ID NO.48; or (15) The sense strand sequence of the dsRNA molecule comprises or is the sequence shown in SEQ ID NO.49, and the antisense strand sequence of the dsRNA molecule comprises or is the sequence shown in SEQ ID NO.50; or (16) The sense strand sequence of the dsRNA molecule includes or is the sequence shown in SEQ ID NO. 63, and the antisense strand sequence of the dsRNA molecule includes or is the sequence shown in SEQ ID NO. 64; or (17) The sense strand sequence of the dsRNA molecule comprises or is the sequence shown in SEQ ID NO. 65, and the antisense strand sequence of the dsRNA molecule comprises or is the sequence shown in SEQ ID NO. 66; or (18) The sense strand sequence of the dsRNA molecule includes or is the sequence shown in SEQ ID NO.69, and the antisense strand sequence of the dsRNA molecule includes or is the sequence shown in SEQ ID NO.

70.

14. The dsRNA molecule according to any one of claims 1-13, wherein each nucleotide in the sense strand and / or antisense strand of the dsRNA molecule independently comprises a modification selected from any one of the following: Locked nucleic acid modification, ring-opening or non-locked nucleic acid modification, 2′-methoxyethyl modification, 2′-O-methyl modification (2′-OMe), 2′-O-allyl modification, 2′-C-alkyl modification, 2′-C-allyl modification, 2′-fluorinated modification (2′-F), 2′-deoxy modification, thiophosphate modification, 2′-amino-modification, morpholino modification, aminophosphate modification, methylphosphonate modification, tetrahydropyran modification, 1,5-dehydrated hexitol modification, 5′-vinylphosphonate modification (5′-VP), 4′-oxyethylphosphonate modification (4′-PEO), cyclohexenyl modification, invAb (reverse debased deoxyribonucleotide) modification, and 5′-cyclopropyl phosphate modification (5′-cPrp), among which: The 5'-VP is attached to the 4' position of the nucleotide and has the structure shown below: 4'-PEO is attached to the 4' position of the nucleotide and has the structure shown below:

15. The dsRNA molecule according to claim 14, wherein the 2' hydroxyl group of the nucleotides at positions 2, 14, and 16 of the antisense strand starting from the 5' end is replaced by fluorine, and the 2' hydroxyl group of the other nucleotides in the antisense strand is replaced by methoxy or hydrogen.

16. The dsRNA molecule according to claim 15, wherein: The 2' hydroxyl groups of the nucleotides at positions 2, 14, and 16 of the antisense strand, starting from the 5' end, are replaced with fluorine, while the 2' hydroxyl groups of the other nucleotides in the antisense strand are replaced with methoxy groups; or The 2' hydroxyl groups of the nucleotides at positions 2, 14, and 16 of the antisense strand, starting from the 5' end, are replaced with fluorine, and the 2' hydroxyl groups of the nucleotides at positions 5 and 7 are replaced with hydrogen. The 2' hydroxyl groups of the other nucleotides in the antisense strand are all replaced with methoxy groups.

17. The dsRNA molecule according to claim 14, wherein the 2' hydroxyl groups of the 9th and 11th nucleotides of the sense strand starting from the 5' end are replaced by fluorine, the 2' hydroxyl group of the 10th nucleotide is replaced by fluorine or hydrogen, and the 2' positions of the other nucleotides of the sense strand are replaced by fluorine, methoxy, or hydrogen.

18. The dsRNA molecule according to claim 17, wherein: The 2' hydroxyl groups of the 9th and 11th nucleotides of the sense chain, starting from the 5' end, are replaced by fluorine, the 2' hydroxyl group of the 10th nucleotide is replaced by hydrogen, and the 2' positions of the other nucleotides of the sense chain are all replaced by methoxy groups. The 2' hydroxyl groups of the 7th, 9th and 11th nucleotides of the sense chain starting from the 5' end are replaced by fluorine, the 2' hydroxyl group of the 10th nucleotide is replaced by hydrogen, and the 2' positions of the other nucleotides of the sense chain are all replaced by methoxy groups. In the sense chain, the 2' hydroxyl group at positions 7, 9, 10, 11, and 12 (starting from the 5' end) is replaced by fluorine, and the 2' position of all other nucleotides in the sense chain is replaced by a methoxy group; or The 2' hydroxyl group of the 7th, 9th, 10th and 11th nucleotides of the sense chain starting from the 5' end is replaced by fluorine, and the 2' position of the other nucleotides of the sense chain is replaced by methoxy group.

19. The dsRNA molecule according to any one of claims 1-18, wherein the antisense strand of the dsRNA molecule comprises a phosphate ester or phosphate ester mimicry modification at the 5' end; optionally, the 5' phosphate ester mimicry is a 5'-VP modification or a 5'-cPrp modification.

20. The dsRNA molecule according to any one of claims 1-19, wherein the sense strand of the dsRNA molecule is linked to an invAb monomer at its 5' and / or 3' ends.

21. The dsRNA molecule according to any one of claims 1-20, wherein the dsRNA molecule comprises a phosphate thioester modification at at least one of the following positions, or comprises a phosphate thioester modification at all of the following positions: Between the first and second nucleotides starting at the 5' end of the sense strand, Between the second and third nucleotides starting from the 5' end of the sense strand, Between the first and second nucleotides starting at the 3' end of the sense strand, Between the second and third nucleotides starting from the 3' end of the sense strand, Between the first and second nucleotides starting at the 5' end of the antisense strand, Between the second and third nucleotides starting from the 5' end of the antisense strand, Between the first and second nucleotides starting from the 3' end of the antisense strand, and Between the second and third nucleotides starting from the 3' end of the antisense strand.

22. The dsRNA molecule according to any one of claims 1-21, wherein the dsRNA molecule comprises one or more lipophilic moieties.

23. The dsRNA molecule of claim 22, wherein the lipophilic portion is attached to the 5' end and / or 3' end of the sense strand.

24. The dsRNA molecule of claim 22, wherein the lipophilic portion is attached to any one or more of the nucleotides at positions 9-12 and 19-21, counting from the 5' end of the sense strand.

25. The dsRNA molecule according to any one of claims 22-24, wherein each of the lipophilic moieties independently comprises one or more structures selected from the group consisting of saturated or unsaturated C4-C4. 30 Hydrocarbon chain, saturated or unsaturated C6-C 30 Acid, saturated or unsaturated C6-C 30 Alcohols, saturated or unsaturated C6-C 30 Amines, cholesterol, bile acids, vitamins, peptides, and steroids; Optionally, one or more carbon atoms in the above structure and the hydrogen atoms directly connected to the one or more carbon atoms may be independently replaced with one or more of the following: -O-, -S-, -NH-, -C(O)-, -C(S)-, -S(O)-, -S(O)2-, -SS-, -C(O)O-, -OC(O)-, -C(O)NH-, -NHC(O)-, -OP(O)(OH)O-, -OP(O)(SH)O-, -S(O)2NH-, -NHS(O)2-, -N≡N-, -C≡C-.

26. The dsRNA molecule of claim 25, wherein the lipophilic portion comprises one or more molecules with the following structures, or the lipophilic portion is one or more molecules with the following structures: Optionally, the above structures include At least one of them represents a nucleotide conjugation to the sense or antisense strand; further optionally, each of the above structures contains two In the structure, two Both indicate conjugation to a nucleotide in the sense or antisense strand, or one This indicates a nucleotide conjugation to either the sense or antisense strand, another It indicates that it is connected to hydrogen.

27. The dsRNA molecule according to any one of claims 22-26, wherein the lipophilic moiety is conjugated to the sense strand and / or antisense strand via one or more linkers; optionally, the lipophilic moiety is conjugated to the sense strand and / or antisense strand via a phosphate ester or thiophosphate linker, or the lipophilic moiety is conjugated to the sense strand and / or antisense strand via one or more linkers and one or more carriers; further optionally, the lipophilic moiety is conjugated to the sense strand and / or antisense strand via a phosphate ester or thiophosphate linker and a carrier, wherein the carrier is selected from: pyrrolidinyl, pyrazolinyl, pyrazolinyl, imidazolinyl, imidazolinyl, piperidinyl, piperazinyl, and tetrahydrofuranyl, and its derivatives substituted with one or more of the following: hydroxyl, methoxy, ethoxy, methoxyethoxy, fluorine, methyl, ethyl, and oxymethyl.

28. The nucleic acid molecule according to any one of claims 1-27, wherein at least one nucleotide in the sense strand and / or antisense strand of the dsRNA molecule contains a modification, and all modifications contained in the nucleotides in the sense strand and / or antisense strand are of one or more of the following types: 2'-OMe modification, 2'-F modification, 2'-deoxy modification, phosphate thioester modification, 5'-VP modification, 5'-cPrp modification, invAb modification, and lipophilic moiety modification.

29. The dsRNA molecule according to any one of claims 1-28, wherein the sense strand and antisense strand comprise a modification defined by a modification motif selected from any one of the following (1)-(6): (1) Sense chain: Lipophilic part s-NmsNmNmNmNmNmNfNmNfNdNfNmNmNmNmNmNmNmNmNmsNms-Lipophilic part, Antisense chain: vp / PEO-NmsNfsNmNmNdNmNdNmNmNmNmNmNmNmNfNmNfNmNfNmNmNmNmNmNmsNmsNm; (2) Sensitive strand: lipophilic part s-NmsNmNmNmNmNmNmNmNmNfNdNfNmNmNmNmNmNmNmNmNmsNms-lipophilic part, antisense strand: vp / PEO-NmsNfsNmNmNdNmNdNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmNmNm; (3) Sense chain: Lipophilic part s-NmsNmNmNmNmNmNmNfNmNfNfNfNfNmNmNmNmNmNmNmNmsNms-Lipophilic part, Antisense chain: vp / PEO-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNmNmNfNmNmNmNmNmNfNmNfNmNmNmNmNmNmNmNmNmNmNmNm; (4) Sense chain: lipophilic moiety-carrier-s-NmsNmNmNmNmNmNfNmNfNdNfNmNmNmNmNmNmNmNmNmsNms-carrier-lipophilic moiety, antisense chain: vp / PEO-NmsNfsNmNmNdNmNdNmNmNmNmNmNmNmNf ... (5) Sense strand: Lipophilic moiety-carrier-s-NmsNmNmNmNmNmNmNmNmNmNfNdNfNmNmNmNmNmNmNmNmNmsNms-carrier-lipophilic moiety, Antisense strand: vp / PEO-NmsNfsNmNmNdNmNdNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmsNmsNm; and (6) Sense chain: lipophilic moiety-carrier-s-NmsNmNmNmNmNmNfNmNfNfNfNfNmNmNmNmNmNmNmNmsNms-carrier-lipophilic moiety, antisense chain: vp / PEO-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNmNf ... in, In each of the modified motifs (1)-(6), each N represents a nucleotide from 5' to 3' of the sense or antisense strand, Nm represents a 2'-OMe modified ribonucleotide, Nf represents a 2'-F modified ribonucleotide, s represents the two nucleotides on either side of "s" linked by a phosphate thioester bond, and Nd represents a deoxy-modified ribonucleotide (i.e., the deoxyribonucleotide corresponding to the ribonucleotide). When the base portion of N in Nd is A, G, or C, Nd corresponds to... It is a deoxyribonucleotide with a base moiety of A, G or C (i.e. Ad, Gd or Cd). When the base moiety of N in Nd is U, Nd is Ud or Td. vp / PEO- indicates 5'-VP modification or 4'-PEO modification. The lipophilic part s- or s-lipophilic part indicates that the lipophilic part is linked to the adjacent nucleoside through a thiophosphate bond. The lipophilic part-carrier-s- or s-carrier-lipophilic part indicates that the lipophilic part is linked to the carrier, and the carrier is linked to the adjacent nucleoside through a thiophosphate bond.

30. The dsRNA molecule according to any one of claims 1-29, wherein the sense strand and antisense strand of the dsRNA molecule each comprise a sequence having no more than two nucleoside modification differences from any pair of sense and antisense nucleoside sequences selected from (1)-(8) or a truncated form of the sense or antisense nucleoside sequence, optionally, the truncated form of the sense strand comprises 19 consecutive nucleosides from the 3' end of any sense strand from (1)-(8), and the truncated form of the antisense strand comprises 21 consecutive nucleosides from the 5' end of any antisense strand from (1)-(8): (1) Sense chain: UmCmCmUmGmAmAfAmUfGfCfUfUmGmAmUmGmAmUmAmAm(SEQ ID NO: 349), Antisense chain: UmUfAmUmCmAmUmCmAmAmGmCmAmUfUmUfCmAmGmGmAmGmCm(SEQ ID NO: 350); (2) Sense chain: UmUmUmUmGmUmGfUmGfAfUfUfCmUmUmCmAmAmAmCmUm(SEQ ID NO: 351), Antisense chain: AmGfUmUmUmGmAmAmGmAmAmUmCmAfCmAfCmAfCmAmAmAmGmAm(SEQ ID NO: 352); (3) Sense chain: UmAmUmUmCmUmGfUmUfGfGfUfCmUmGmGmUmUmUmAmAm(SEQ ID NO: 353), Antisense chain: UmUfAmAmAmCmCmAmGmAmCmCmAmAfCmAfGmAmAmUmAmGmAm(SEQ ID NO: 354); (4) Sense chain: AmAmCmAmAmUmGfUmUfAfCfCfAmAmAmAmCmCmGmAmAm(SEQ ID NO: 355), Antisense chain: UmUfCmGmGmUmUmUmUmGmGmUmAmAfCmAfUmUmGmUmUmGmGm(SEQ ID NO: 356); (5) Sense chain: UmGmGmCmUmGmGfUmAfUfCfUfGmAmAmUmAmUmCmAmUm(SEQ ID NO: 357), Antisense chain: AmUfGmAmUmAmUmUmCmAmGmAmUmAfCmCfAmGmCmCmAmAmAm(SEQ ID NO: 358); (6) Sense chain: CmGmCmCmUmAmAfCmUfGfCfUfCmUmUmCmGmUmAmUmUm(SEQ ID NO: 359), Antisense chain: AmAfUmAmCmGmAmAmGmAmGmCmAmGfUmUfAmGmGmCmGmGmGm(SEQ ID NO: 360); (7) Sense chain: UmCmCmAmUmUmUfUmCfCfCfCfUmUmUmAmUmGmUmGmAm (SEQ ID NO: 361), Antisense chain: UmCfAmCmAmUmAmAmAmGmGmGmGmGmAfAmAfAmUmGmGmAmAmAm (SEQ ID NO: 362); and (8) Sensitive chain: UmUmCmGmUmAmUfUmAfAfGfAfAmGmAmCmUmAmUmAmUm (SEQ ID NO: 363), antisense chain: AmUfAmUmAmGmUmCmUmUmCmUmUmAfAmUfAmCmGmAmAmGmAm (SEQ ID NO: 364).

31. The dsRNA molecule according to claim 30, wherein the dsRNA molecule comprises a structure selected from any pair of sense strands and antisense strands shown in (1)-(16), or the structure of the dsRNA molecule is as shown in any pair of sense strands and antisense strands selected from (1)-(16): (1) Sense chain: (GN75)sUmsCmCmUmGmAmAfAmUfGfCfUfUmGmAmUmGmAmUmAmsAms(GN75)(SEQ ID NO: 338), Antisense chain: vp-UmsUfsAmUmCmAmUmCmAmAmGmCmAmUfUmUfCmAmGmGmAmUfUfCmAmGmGmAmsGmsCm(SEQ ID NO: 339); (2) Sense chain: (GN75)sUmsUmUmUmGmUmGfUmGfAfUfUfCmUmUmCmAmAmAmCmsUms(GN75)(SEQ ID NO: 365), Antisense chain: vp-AmsGfsUmUmUmGmAmAmGmAmAmUmCmAfCmAfCmAfCmAmAmAmAmsGmsAm(SEQ ID NO: 366); (3) Sense chain: (GN75)sUmsAmUmUmCmUmGfUmUfGfGfGfUfCmUmGmGmUmUmUmAmsAms(GN75)(SEQ ID NO: 340), Antisense chain: vp-UmsUfsAmAmAmCmCmAmGmAmCmCmAmAfCmAfGmAmAmUmAmsGmsAm(SEQ ID NO: 341); (4) Sense chain: (GN75)sAmsAmCmAmAmUmGfUmUfAfCfCfAmAmAmAmCmCmGmAmsAms(GN75) (SEQ ID NO: 342), Antisense chain: vp-UmsUfsCmGmGmUmUmUmUmGmGmUmAmAfCmAfUmUmGmUmUmsGmsGm(SEQ ID NO: 343); (5) Sense chain: (GN75)sUmsGmGmCmUmGmGfUmAfUfCfUfGmAmAmUmAmCmAmsUms(GN75)(SEQ ID NO: 344), Antisense chain: vp-AmsUfsGmAmUmAmUmUmCmAmGmAmUmAfCmCfAmGmCmCmAmsAmsAm(SEQ ID NO: 345); (6) Sense chain: (GN75)sCmsGmCmCmUmAmAfCmUfGfCfUfCmUmUmCmGmUmAmUmsUms(GN75)(SEQ ID NO: 367), Antisense chain: vp-AmsAfsUmAmCmGmAmAmGmAmGmCmAmGfUmUfAmGmGmCmGmsGmsGm(SEQ ID NO: 368); (7) Sense chain: (GN75)sUmsCmCmAmUmUmUfUmCfCfCfCfUmUmUmAmUmGmUmGmsAms(GN75)(SEQ ID NO: 346), Antisense chain: vp-UmsCfsAmCmAmUmAmAmAmGmGmGmGmGmAfAmAfAmUmGmGmAmsAmsAm(SEQ ID NO: 347); (8) Sense chain: (GN75)sUmsUmCmGmUmAmUfUmAfAfGfAfAmGmAmCmUmAmUmAmsUms(GN75)(SEQ ID NO: 334), Antisense chain: vp-AmsUfsAmUmAmGmUmCmUmUmCmUmUmUmAfAmUfAmCmGmAmAmsGmsAm(SEQ ID NO: 335); (9) Sense chain: (GN75)sUmsCmCmUmGmAmAfAmUfGfCfUfUmGmAmUmGmAmUmAmsAms(GN75)(SEQ ID NO: 338), Antisense chain: PEO-UmsUfsAmUmCmAmUmCmAmAmGmCmAmUfUmUfCmAmGmGmAmsGmsCm(SEQ ID NO: 369); (10) Sense chain: (GN75)sUmsUmUmUmGmUmGfUmGfAfUfUfCmUmUmCmAmAmAmCmsUms(GN75)(SEQ ID NO: 365), Antisense chain: PEO-AmsGfsUmUmUmGmAmAmGmAmAmUmCmAfCmAfCmAfCmAmAmAmAmsGmsAm(SEQ ID NO: 370); (11) Sense chain: (GN75)sUmsAmUmUmCmUmGfUmUfGfGfGfUfCmUmGmGmUmUmUmAmsAms(GN75)(SEQ ID NO: 340), Antisense chain: PEO-UmsUfsAmAmAmCmCmAmGmAmCmCmAmAfCmAfGmAmAmUmAmsGmsAm(SEQ ID NO: 371); (12) Sense chain: (GN75)sAmsAmCmAmAmUmGfUmUfAfCfCfAmAmAmAmCmCmGmAmsAms(GN75) (SEQ ID NO: 342), Antisense chain: PEO-UmsUfsCmGmGmUmUmUmUmGmGmUmAmAfCmAfUmUmGmUmUmsGmsGm(SEQ ID NO: 372); (13) Sense chain: (GN75)sUmsGmGmCmUmGmGfUmAfUfCfUfGmAmAmUmAmUmCmAmsUms(GN75)(SEQ ID NO: 344), Antisense chain: PEO-AmsUfsGmAmUmAmUmUmCmAmGmAmUmAfCmCfAmGmCmCmAmsAmsAm(SEQ ID NO: 373); (14) Sense chain: (GN75)sCmsGmCmCmUmAmAfCmUfGfCfUfCmUmUmCmGmUmAmUmsUms(GN75)(SEQ ID NO: 367), Antisense chain: PEO-AmsAfsUmAmCmGmAmAmGmAmGmCmAmGfUmUfAmGmGmCmGmsGmsGm(SEQ ID NO: 374); (15) Sense chain: (GN75)sUmsCmCmAmUmUmUfUmCfCfCfCfUmUmUmAmUmGmUmGmsAms(GN75) (SEQ ID NO: 346), Antisense chain: PEO-UmsCfsAmCmAmUmAmAmAmGmGmGmGmGmAfAmAfAmUmGmGmAmsAmsAm(SEQ ID NO: 348); and (16) Sense chain: (GN75)sUmsUmCmGmUmAmUfUmAfAfGfAfAmGmAmCmUmAmUmAmsUms(GN75)(SEQ ID NO: 334), Antisense chain: PEO-AmsUfsAmUmAmGmUmCmUmUmCmUmUmAfAmUfAmCmGmAmAmsGmsAm(SEQ ID NO: 375); Where vp- represents the 4' position of adjacent nucleotides linked by vp, the structure is as follows: PEO- indicates that a PEO is linked at the 4' position of an adjacent nucleotide, as shown in the following structure: (GN75) represents a targeting ligand linked to an adjacent nucleotide, which has the following structure: One of them Indicates conjugation with the adjacent nucleotide, another Indicates connection to hydrogen; (GN75)s indicates that the 3' position of (GN75) is connected to the 5' position of the adjacent nucleotide via a thiophosphate bond, and s(GN75) indicates that the 5' position of (GN75) is connected to the 3' position of the adjacent nucleotide via a thiophosphate bond.

32. An engineered nucleic acid molecule comprising a dsRNA molecule according to any one of claims 1-31.

33. The engineered nucleic acid molecule according to claim 32, wherein the engineered nucleic acid molecule has a hairpin structure, and the double-stranded portion of the hairpin structure is composed of the dsRNA molecule according to any one of claims 1-31.

34. The engineered nucleic acid molecule of claim 32, wherein the engineered nucleic acid molecule comprises two or more dsRNA molecules, wherein at least one of the two or more dsRNAs is a dsRNA molecule according to any one of claims 1-31.

35. The engineered nucleic acid molecule according to claim 34, wherein the engineered nucleic acid molecule is a dual-target or multi-target nucleic acid molecule, and further comprises a siRNA molecule targeting the mRNA of another gene other than the ALK7 gene, or a siRNA molecule targeting ALK7 mRNA that is different from the dsRNA molecule of any one of claims 1-31.

36. An engineered nucleic acid molecule that can be transcribed or spliced ​​in a cell to form a dsRNA molecule according to any one of claims 1-31.

37. A delivery body comprising a dsRNA molecule according to any one of claims 1-31, an engineered nucleic acid molecule according to any one of claims 32-35, or an engineered nucleic acid molecule according to claim 36, optionally wherein the delivery body is a virus, plasmid, liposome, lipid nanoparticle, endosome, exosome, or vesicle.

38. A cell comprising the engineered nucleic acid molecule according to claim 36.

39. The dsRNA molecule according to any one of claims 1-31, the engineered nucleic acid molecule according to any one of claims 32-35, or a tautomer, stereoisomer, solvate, isotope derivative, or pharmaceutically acceptable salt of the engineered nucleic acid molecule according to claim 36.

40. Use of the dsRNA molecule according to any one of claims 1-31, the engineered nucleic acid molecule according to any one of claims 32-35, the engineered nucleic acid molecule according to claim 36, or the tautomer, stereoisomer, solvate, isotope derivative, or pharmaceutically acceptable salt according to claim 39 for the inhibition of ALK7 expression in cells or in a subject.

41. The use of the dsRNA molecule according to any one of claims 1-31, the engineered nucleic acid molecule according to any one of claims 32-35, the engineered nucleic acid molecule according to claim 36, or the tautomer, stereoisomer, solvate, isotope derivative, or pharmaceutically acceptable salt according to claim 39, for the preparation of a drug that inhibits ALK7 expression in a subject.

42. A pharmaceutical composition comprising a dsRNA molecule according to any one of claims 1-31, an engineered nucleic acid molecule according to any one of claims 32-35, an engineered nucleic acid molecule according to claim 36, a delivery medium according to claim 37, a cell according to claim 38, or a tautomer, stereoisomer, solvate, isotope derivative, or pharmaceutically acceptable salt according to claim 39, and a pharmaceutically acceptable carrier or diluent.

43. The pharmaceutical composition according to claim 42, for the prevention or treatment of ALK7-mediated diseases or symptoms.

44. The pharmaceutical composition of claim 43, wherein the ALK7-mediated disease or symptom is a disease or symptom related to fat or sugar metabolism.

45. The pharmaceutical composition of claim 44, wherein the disease or symptom related to fat or sugar metabolism is type 2 diabetes or obesity.

46. ​​The pharmaceutical composition of claim 44, wherein the disease or symptom related to fat or sugar metabolism is obesity with type 2 diabetes.

47. Use of the dsRNA molecule according to any one of claims 1-31, the engineered nucleic acid molecule according to any one of claims 32-35, the engineered nucleic acid molecule according to claim 36, the delivery body according to claim 37, the cell according to claim 38, the tautomer, stereoisomer, solvate, isotope derivative or pharmaceutically acceptable salt according to claim 39, or the pharmaceutical composition according to claim 42 for the preparation of a medicament for the prevention or treatment of ALK7-mediated diseases or symptoms.

48. The use according to claim 47, wherein the ALK7-mediated disease or symptom is a disease or symptom related to fat or sugar metabolism.

49. The use according to claim 48, wherein the disease or symptom related to fat or sugar metabolism is type 2 diabetes or obesity.

50. The use according to claim 48, wherein the disease or symptom related to fat or sugar metabolism is obesity with type 2 diabetes.

51. Methods for treating or preventing ALK7-mediated diseases or symptoms in subjects, including: Administer to a subject in need a therapeutic or preventative effective amount of the dsRNA molecule according to any one of claims 1-31, the engineered nucleic acid molecule according to any one of claims 32-35, the engineered nucleic acid molecule according to claim 36, the delivery body according to claim 37, the cell according to claim 38, the tautomer, stereoisomer, solvate, isotope derivative or pharmaceutically acceptable salt according to claim 39, or the pharmaceutical composition according to claim 42.

52. The method of claim 51, wherein the ALK7-mediated disease or symptom is a disease or symptom related to fat or glucose metabolism.

53. The method of claim 52, wherein the disease or symptom related to fat or sugar metabolism is type 2 diabetes or obesity.

54. The method of claim 52, wherein the disease or symptom related to fat or sugar metabolism is obesity with type 2 diabetes.

55. The use of the dsRNA molecule according to any one of claims 1-31, the engineered nucleic acid molecule according to any one of claims 32-35, the engineered nucleic acid molecule according to claim 36, the delivery body according to claim 37, the cell according to claim 38, the tautomer, stereoisomer, solvate, isotope derivative or pharmaceutically acceptable salt according to claim 39, or the pharmaceutical composition according to claim 42 for weight loss in a subject.

56. The use according to claim 55, wherein the subject is a patient with type 2 diabetes.

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