Glycerol nucleic acid modified oligonucleotide agents and use thereof

Abstract

Provided herein is a double-stranded oligonucleotide agent capable of activating or upregulating expression of a target gene, comprising a sense strand and an antisense strand, each strand having a length of 15 to 35 nucleotides wherein the sense strand and the antisense strand are at least 50% complementary to each other forming a double-stranded structure of at least 8 base pairs, wherein the double-stranded oligonucleotide agent comprises one or more glycerol nucleic acid (GNA) modified nucleotides located in one or both of the sense strand and the antisense strand.

Description

(19) State Intellectual Property Office (12) Invention Patent Application (10) Application Publication Number (43) Application Publication Date (21) Application Number 202480049319.5 (22) Application Date 2024.07.26 (66) Domestic Priority Data PCT / CN2023 / 109510 2023.07.27 CN (85) PCT International Application Entering National Phase Date 2026.01.26 (86) PCT International Application Application Data PCT / CN2024 / 107766 2024.07.26 (87) PCT International Application Publication Data WO2025 / 021186 EN 2025.01.30 (71) Applicant Sino-US Ruikang Nucleic Acid Technology (Nantong) Research Institute Co., Ltd. Address 226400 Life and Health Industry Park, No. 888 Zhujiang Road, Juegang Street, Rudong County, Nantong City, Jiangsu Province (72) Inventors: Li Longcheng, Gan Zubao, Jiang Wulin (74) Patent Agency: Shanghai Patent & Trademark Agency Co., Ltd. 31100 Patent Attorneys: Li Zhengzhang, Qian Wenyu (51) Int.Cl. C12N 15 / 113 (2006.01) A61K 31 / 713 (2006.01) A61K 48 / 00 (2006.01) A61P 43 / 00 (2006.01) (54) Invention Title: Glycerol-modified oligonucleotide agent and its application (57) Abstract: This paper provides a double-stranded oligonucleotide agent capable of activating or upregulating the expression of a target gene, comprising a sense strand and an antisense strand, each strand being 15 to 35 nucleotides in length, wherein the sense strand and the antisense strand are at least 50% complementary to each other, forming a double-stranded structure of at least 8 base pairs, wherein the double-stranded oligonucleotide agent comprises one or more glycerol nucleic acid (GNA) modified nucleotides located in one or both of the sense strand and the antisense strand. Claims (4 pages), Description (50 pages), Sequence Listing (electronic publication), Drawings (10 pages) CN 121569037 A 2026.02.24 CN 1 21 56 90 37 A 1. A double-stranded oligonucleotide agent capable of activating or upregulating the expression of a target gene, comprising a sense strand and an antisense strand, each strand having a length of 15 to 35 nucleotides, wherein the sense strand and the antisense strand form a complementary double-stranded structure of at least 8 base pairs, wherein the double-stranded oligonucleotide agent comprises one or more glycerol nucleic acid (GNA) modified nucleotides located in one or both of the sense strand and the antisense strand. 2. The double-stranded oligonucleotide agent according to claim 1, wherein one or both of the sense strand and the antisense strand is a guide strand mediating RNA activation. 3. The double-stranded oligonucleotide agent according to claim 2, wherein the guide strand has a 5' portion located in the guide strand.4. The double-stranded oligonucleotide agent of claim 3, wherein the seed region begins no more than 3 nucleotides from the 5' end of the leader strand. 5. The double-stranded oligonucleotide agent of claim 3, wherein the seed region comprises nucleotides at positions 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 3 to 6, 2 to 5, 3 to 6, 3 to 5, or 4 to 6, starting from the 5' end of the leader strand. 6. The double-stranded oligonucleotide agent of claim 1, wherein the one or more GNA-modified nucleotides comprise at least one selected from the group consisting of: GNA-modified adenine (GNA-A), GNA-modified thymine (GNA-T), GNA-modified cytosine (GNA-C), GNA-modified guanine (GNA-G), and GNA-modified uracil (GNA-U). 7. The double-stranded oligonucleotide agent of claim 1, wherein the double-stranded oligonucleotide agent comprises 1 to 50 GNA-modified nucleotides. 8. The double-stranded oligonucleotide agent of claim 2, wherein the double-stranded oligonucleotide agent comprises one or more GNA-modified nucleotides located at positions 1 and / or 2 and / or 3 and / or 4 and / or 5 and / or 6 and / or 7 and / or 8 and / or 9 and / or 10 and / or 11 and / or 12 and / or 13 and / or 14 and / or 15 and / or 16 and / or 17 and / or 18 and / or 19 and / or 20 and / or 21 and / or 22 and / or 23 and / or 24 and / or 25, starting from the 5' end of the leader strand. 9. The double-stranded oligonucleotide agent of claim 3, wherein the one or more GNA-modified nucleotides are located within and / or outside the seed region of the leader strand. 10. The double-stranded oligonucleotide agent of claim 3, wherein the one or more GNA-modified nucleotides are located in the guest strand. 11. The double-stranded oligonucleotide agent according to claim 1, wherein the sense strand comprises the nucleotide sequence shown in any one of SEQ ID NO: 1, 3, 12, 14, 15, 17, 18, 20, 21, 23, 24, 27, 29, 52, 54, and the antisense strand comprises the nucleotide sequence shown in any one of SEQ ID NO: 2, 4, 5, 6, 7, 8, 9, 10, 11, 13, 16, 19, 22, 25, 26, 28, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 53, 55. 12. The double-stranded oligonucleotide agent according to claim 1, wherein the leader chain is synthesized using a GNA-modified nucleotide monomer of formula (1) to include the one or more GNA-modified nucleotides: Claims 1 / 4 page 2CN 121569037 A (1) wherein the base is selected from the group consisting of: adenine nucleobases, thymine nucleobases, cytosine nucleobases, guanine nucleobases, uracil nucleobases and analogues thereof. 13. The double-stranded oligonucleotide agent according to claim 12, wherein the base in formula (1) is selected from the following structures: , , , , , , , , Claims 2 / 4 page 3 CN 121569037 A and. 14. The double-stranded oligonucleotide agent of claim 1, wherein the sense strand or the antisense strand has at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 nucleotides complementary to an isometric segment of the coding strand of the target gene. 15. The double-stranded oligonucleotide agent of claim 1, wherein the complementary double-stranded structure of at least 8 base pairs has no more than 5 mismatched nucleotides between the sense strand and the antisense strand, i.e., 5, 4, 3, 2, 1, or 0 mismatched nucleotides. 16. The double-stranded oligonucleotide agent of claim 15, wherein the mismatched nucleotides are located inside or near the 3' or 5' end of the antisense strand. 17. The double-stranded oligonucleotide agent of claim 15, wherein the mismatched nucleotide is located at a GNA position in the sense strand and / or the antisense strand. 18. The double-stranded oligonucleotide agent of claim 1, wherein the double-stranded oligonucleotide agent is a small activating RNA (saRNA) that upregulates the expression of the target gene by at least 10%. 19. The double-stranded oligonucleotide agent of claim 1, wherein at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or about 100% of the non-GNA nucleotides of the double-stranded oligonucleotide agent comprise modifications selected from the group consisting of: 2'-fluoro(2'-F), 2'-O-methyl(2'-OMe), 2'-O-methoxyethyl(2'-MOE), 5'-(E)-vinylphosphonate, thiophosphate backbone modifications, and combinations thereof. 20. A conjugating agent comprising a double-stranded oligonucleotide agent according to any one of claims 1-19 and at least one conjugating moiety, wherein the at least one conjugating moiety is selected from: lipids, fatty acids, fluorophores, ligands, sugars, peptides, and antibodies. 21. The conjugating agent according to claim 20, wherein the conjugating moiety is selected from accessory oligonucleotides (ACOs), lipids / fatty acids, and GalNAc clusters. 22. The conjugating agent according to claim 21, wherein the conjugating moiety is selected from: [The remaining text appears to be incomplete and requires further context.]Claims 3 / 4 pages 4 CN 121569037 A (tC2x6), and (C5x5), wherein represent the carrier material. 23. A cell comprising the double-stranded oligonucleotide agent according to claim 1 or the conjugate agent according to claim 20. 24. A pharmaceutical composition comprising the double-stranded oligonucleotide agent according to claim 1 or the conjugate agent according to claim 20, and at least one pharmaceutically acceptable carrier. 25. A kit for activating or upregulating a target gene in a cell or subject, comprising the double-stranded oligonucleotide agent according to any one of claims 1-19, or the conjugate agent according to any one of claims 20-22, or the pharmaceutical composition according to claim 24. 26. A method for activating or upregulating a target gene in a cell or subject, comprising administering to the cell or subject the double-stranded oligonucleotide agent according to any one of claims 1-19 or the conjugate agent according to any one of claims 20-22. 27. A method for mitigating off-target effects caused by a double-stranded oligonucleotide agent capable of activating or upregulating the expression of a target gene in a cell or subject, comprising administering to said cell or subject the double-stranded oligonucleotide agent according to any one of claims 1-19 or the conjugate agent according to any one of claims 20-22. 28. Use of the double-stranded oligonucleotide agent according to any one of claims 1-19 or the conjugate agent according to any one of claims 20-22 in the preparation of a product for activating or upregulating a target gene in a cell or subject. Claims 4 / 4 Page 5 CN 121569037 A Glycerol-modified oligonucleotide agent and its application technology field

[0001] This disclosure relates to the field of nucleic acid technology, and more particularly to oligonucleotide agents capable of activating gene expression and reducing off-target effects and the pharmaceutical use of their double-stranded RNA (dsRNA). Background Art

[0002] Oligonucleotides are a class of emerging therapeutic agents currently under active development for the treatment of various diseases. The main categories of therapeutic oligonucleotide agents include single-stranded antisense oligonucleotides and dsRNA. dsRNA also includes two main categories: small interfering RNA (siRNA) and small activating RNA (saRNA). Although both require the Argonaute (AGO) protein, siRNA and saRNA differ significantly in their mechanistic framework.

[0003] saRNA-mediated gene activation provides a promising strategy for upregulating target gene expression by promoting endogenous transcription, a phenomenon known as RNA activation (RNAa). saRNAs activate target genes by directly binding to the target gene promoter or by interacting with antisense transcripts transcribed from overlapping promoter sequences. Although this technology has potential for therapeutic applications...While the application of saRNA has great potential, its development process has encountered significant challenges, particularly the emergence of undesirable "off-target" effects and related toxicities. Ensuring that saRNA meets stringent safety requirements during drug discovery and development remains a key issue in this field. Summary of the Invention

[0004] To address the above problems, this disclosure provides oligonucleotide agents, such as saRNA, which maintain or even enhance gene activation efficacy while reducing off-target effects.

[0005] In one aspect, this disclosure provides a double-stranded oligonucleotide agent capable of activating or upregulating the expression of a target gene, comprising a sense strand and an antisense strand, each strand having a length of 15 to 35 nucleotides, wherein the sense strand and the antisense strand form a complementary double-stranded structure of at least 8 base pairs, wherein the double-stranded oligonucleotide agent comprises one or more glycerol nucleic acid (GNA) modified nucleotides.

[0006] In another aspect, this disclosure provides a conjugation agent comprising the double-stranded oligonucleotide agent described herein and at least one conjugation moiety.

[0007] In another aspect, this disclosure provides a cell comprising the double-stranded oligonucleotide agent or conjugation agent described herein.

[0008] In another aspect, this disclosure provides a composition comprising the double-stranded oligonucleotide agent or conjugate described herein.

[0009] In another aspect, this disclosure provides a kit comprising the double-stranded oligonucleotide agent or conjugate described herein.

[0010] In another aspect, this disclosure provides a method for activating or upregulating a target gene in a cell or subject, comprising administering the double-stranded oligonucleotide agent or conjugate described herein to the cell or subject.

[0011] In another aspect, this disclosure provides a method for mitigating off-target effects caused by a double-stranded oligonucleotide agent capable of activating or upregulating the expression of a target gene in a cell or subject, comprising administering the double-stranded oligonucleotide agent or conjugate described herein to the cell or subject.

[0012] In another aspect, this disclosure provides the use of the double-stranded oligonucleotide agent or conjugate described herein in the preparation of a product. Instructions 1 / 50 Page 6 CN 121569037 A

[0013] The double-stranded oligonucleotide agents (such as saRNA) provided herein can effectively and specifically upregulate the expression of target genes at the mRNA or protein level in vitro or in vivo, while reducing off-target effects, and can be used to prepare products for the prevention or treatment of diseases, conditions or symptoms associated with insufficient expression of such genes. Brief Description of the Drawings

[0014] The novel features of the invention are specifically set forth in the appended claims. The features and advantages of the invention can be better understood by referring to the following detailed description, which illustrates illustrative embodiments employing the principles of the invention, and the accompanying drawings (also referred to herein as “Figures”), in which: Figure 1 shows the effect of glycerol-modified saRNA (GNA-saRNA) on full-length SMN2 (SMN2-) in GM03813 cells.The activity of SMN2-FL mRNA expression was assessed. Specific GNA-saRNAs (RD-15977, RD-15978, RD-15979, RD-15980, RD-15981, RD-15982, and RD-15983) were transfected into GM03813 cells for 3 days at specified concentrations (0.78 nM, 1.56 nM, 3.13 nM, 6.25 nM, 12.5 nM, 25 nM, 50 nM, and 100 nM). Cells were transfected with RD-10994 as a non-GNA control. Mock treatment was performed in the absence of oligonucleotides (not shown in the figure). dsCon2 was used as a non-specific duplex control (not shown in the figure). In each PCR reaction, the mRNA level of SMN2-FL was quantified by RT-qPCR using a gene-specific primer set. The geometric mean of SDHA and GAPDH mRNA levels was used as an internal control to normalize the expression data. The data represent the mean expression level of SMN2-FL after normalization with SDHA and GAPDH relative to the Mock treatment (mean of two parallel transfection wells ± SEM).

[0015] Figures 2A to 2H show the activity of GNA-saRNA in the expression of full-length (SMN2-FL) and exon-skipped (SMN2-Δ7) SMN2 mRNA in GM03813 cells. GM03813 cells were transfected with specified GNA-saRNAs (RD-15977, RD-15978, RD-15979, RD-15980, RD-15981, RD-15982, and RD-15983) at specified concentrations (0.78 nM, 1.56 nM, 3.13 nM, 6.25 nM, 12.5 nM, 25 nM, 50 nM, and 100 nM) for 3 days. Cells were transfected with RD-10994 as a non-GNA control. Mock treatment was performed in the absence of oligonucleotides (not shown in the figure). dsCon2 was used as a non-specific duplex control (not shown in the figure). Figures 2A through 2H show the mRNA levels of SMN2-FL and SMN2-Δ7 quantified by RT-qPCR using gene-specific primer sets in each PCR reaction. The geometric mean of SDHA and GAPDH mRNA levels was used as an internal control to normalize the expression data. Data represent the mean expression levels of SMN2-FL or SMN2-Δ7 normalized to SDHA and GAPDH relative to the Mock-treated cells (mean of two parallel transfection wells ± SEM).

[0016] Figures 3A to 3H show the effect of GNA-saRNA on the mRNA expression of SMN2-FL and SMN2-Δ7 in GM22592 cells.Activity. GM22592 cells were transfected with specified GNA-saRNAs (RD-15977, RD-15978, RD-15979, RD-15980, RD-15981, RD-15982, and RD-15983) at specified concentrations (0.78 nM, 1.56 nM, 3.13 nM, 6.25 nM, 12.5 nM, 25 nM, 50 nM, and 100 nM) for 3 days. Cells were transfected with RD-10994 as a non-GNA control. Mock treatment was performed in the absence of oligonucleotides (not shown in the figure). dsCon2 was used as a non-specific duplex control (not shown in the figure). Figures 3A through 3H show the mRNA levels of SMN2-FL and SMN2-Δ7 quantified by RT-qPCR using gene-specific primer sets in each PCR reaction. The geometric mean of SDHA and GAPDH mRNA levels was used as an internal control to normalize the expression data. Data represent the mean expression levels of SMN2-FL or SMN2-Δ7 normalized to SDHA and GAPDH relative to the Mock-treated cells (mean of two parallel transfection wells ± SEM).

[0017] Figures 4A and 4B show the activity of GNA-saRNA against the mRNA expression of SMN2-FL and SMN2-Δ7 in GM03813 cells. Specific GNA-saRNAs (i.e., RD-15977, RD-15978, RD-15979, RD-15980, RD-15981, RD-15982, and RD-15983) were transfected into GM03813 cells at 100 nM for 3 days. Cells were transfected with RD-10994 as a non-GNA control. Mock treatment involved transfection in the absence of oligonucleotides. dsCon2 was used as a nonspecific double-stranded control. Figures 4A and 4B show the mRNA levels of SMN2-FL and SMN2-Δ7 quantified by RT-qPCR using gene-specific primer sets in each PCR reaction. SDHA amplification was used as an internal control to normalize the expression data. Data represent the mean expression level of SMN2-FL or SMN2-Δ7 relative to the Mock treatment (mean of four parallel transfection wells ± SEM) after SDHA normalization.

[0018] Figures 5A and 5B show the off-target effects of GNA-saRNA on the expression of the potential off-target gene P2RY2 in GM03813 cells. Specified GNA-saRNAs (i.e., RD-15977, RD-15978, RD-15981, RD-15982, and RD-15983) were expressed at 6.25 nM andGM03813 cells were transfected with 25 nM for 3 days. Cells were transfected with RD-10994 as a non-GNA control. Mock treatment was performed in the absence of oligonucleotides. dsCon2 was used as a non-specific duplex control (not shown in the figure). Figure 5A shows the "query" (antisense) sequence and "seed" region (italicized, gray highlighted) of RD-10994 and its predicted complementary target site in P2RY2 transcripts containing two mismatched nucleotides (italicized and bolded nucleotides). Figure 5B shows the mRNA level of P2RY2 quantified by RT-qPCR using gene-specific primer sets in each PCR reaction. SDHA amplification was used as an internal control to normalize expression data. Data represent the mean expression level of P2RY2 relative to the Mock treatment (mean of four parallel transfection wells ± SEM) normalized to SDHA.

[0019] Figures 6A and 6B show the activity of GNA-saRNA against the mRNA expression of SMN2-FL and SMN2-Δ7 in GM03813 cells. GM03813 cells were transfected with designated GNA-saRNAs (RD-19658, RD-19659, RD-19660, RD-19663, RD-19664, RD-19665, RD-19666, RD-19667, RD-19668, RD-19669, RD-19670, and RD-19672) at 2.5 nM for 3 days. Cells were transfected with RD-19040 as a non-GNA control. Cells were transfected with RD-10004 (ASO-1027) at 25 nM as a positive control. Mock treatment was performed in the absence of oligonucleotides. dsCon2M13v was used as a non-specific double-stranded control. Figures 6A and 6B show the mRNA levels of SMN2-FL and SMN2-Δ7 quantified by RT-qPCR using gene-specific primer sets in each PCR reaction. TBP amplification was used as an internal control to normalize the expression data. The data represent the mean expression level of SMN2-FL or SMN2-Δ7 (mean of four parallel transfection wells ± SEM) normalized by TBP relative to the Mock treatment.

[0020] Figures 7A and 7B show the off-target effects of GNA-saRNA on the expression of the potential off-target gene ARPIN in GM03813 cells. The specified GNA-saRNAs (RD-19650, RD-19651, RD-19652, RD-19653, RD-19654, RD-19655, RD-19657, RD-19661, RD-19662, RD-19674 and RD-19675) were transfected into GM03813 cells at 2.5 nM for 3 days.Cells were transfected with RD-19040 as a non-GNA control. Cells were transfected with RD-10004 (ASO-1027) at 25 nM as a positive control. Mock treatment was performed in the absence of oligonucleotides. dsCon2M13v was used as a nonspecific duplex control. Figure 7A shows the “retrieval” (antisense) sequence and “seed” region (italicized, gray highlighted) of RD-19040 and its predicted complementary target site in ARPIN transcripts containing two mismatched nucleotides (italicized and bolded nucleotides). Figure 7B shows the ARPIN mRNA levels quantified by RT-qPCR using gene-specific primer sets in each PCR reaction. TBP amplification was used as an internal control to normalize expression data. Data represent mean ARPIN expression levels (mean of four parallel transfection wells ± SEM) normalized to TBP relative to the Mock treatment.

[0021] Figures 8A to 8E compare the activity of GNA-saRNA and non-GNA-saRNA on SERPING1 mRNA expression in Hep3B cells. Specified non-GNA-saRNAs (RD-17229, RD-17235, RD-17238, RD-17241, and RD-17244) and their corresponding GNA-saRNAs (RD-17074, RD-17082, RD-17086, RD-17096, and RD-17099) were transfected into Hep3B cells at specified concentrations (0.02 nM, 0.07 nM, 0.21 nM, 0.62 nM, 1.85 nM, 5.56 nM, 16.67 nM, and 50 nM) for 3 days. Mock treatment was performed in the absence of oligonucleotides (not shown in the figures). dsCon2 was used as a non-specific duplex control (not shown in the figures). Figures 8A to 8E show the mRNA levels of SERPING1 quantified by RT-qPCR using gene-specific primer sets in each PCR reaction, according to the manual (page 3 / 50, CN 121569037 A). The geometric mean of HPRT1 and TBP mRNA levels was used as an internal control to normalize the expression data. The data represent the mean expression level of SERPING1 relative to the Mock treatment (mean of four parallel transfection wells ± SEM) after normalization by HPRT1 and TBP.

[0022] Figures 9A to 9C compare the activity of GNA-saRNA and non-GNA-saRNA on SERPING1 mRNA expression in HepG2 cells. Specified non-GNA-saRNAs (i.e., RD-17229, RD-17235, and RD-17241) and their corresponding GNA-saRNAs (i.e.,RD-17074, RD-17082, and RD-17096 were transfected into HepG2 cells for 3 days at specified concentrations (i.e., 0.02 nM, 0.07 nM, 0.21 nM, 0.62 nM, 1.85 nM, 5.56 nM, 16.67 nM, and 50 nM). Mock treatment was performed in the absence of oligonucleotides (not shown in the figures). dsCon2 was used as a nonspecific duplex control (not shown in the figures). Figures 9A through 9C show the mRNA levels of SERPING1 quantified by RT-qPCR using gene-specific primer sets in each PCR reaction. The geometric mean of HPRT1 and TBP mRNA levels was used as an internal control to normalize the expression data. Data represent the mean expression level of SERPING1 relative to the Mock treatment, normalized for HPRT1 and TBP (mean of four parallel transfection wells ± SEM). Detailed Description

[0023] 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 this invention pertains. Furthermore, all publications, patent applications, patents, and other references mentioned herein are incorporated herein by reference.

[0024] Definitions

[0025] As used herein, “and / or” means “and, or as an alternative”. If any numerical range is provided, the range includes any numerical value within that range (including upper and lower limits), and any subranges within that range. For example, a range from 1 to 3 may include any one of the numerical values ​​1, 2, and 3, and subranges from 1 to 2 and from 2 to 3.

[0026] As used herein, the terms “oligonucleotide” or “polynucleotide” are interchangeable and refer to polymers of nucleotides, particularly single-stranded nucleic acid molecules of DNA, RNA, or DNA / RNA hybrids, oligonucleotide chains containing regular and irregular alternations of deoxyribosyl and ribosyl moieties, and modified and naturally or non-naturally occurring frameworks of such oligonucleotides, such as phosphoryldiamine morpholino oligomers (PMOs). The oligonucleotides described herein for activating target gene transcription may be or may include small activating nucleic acid molecules (saRNA).

[0027] As used herein, the term “complementary” refers to the ability to form base pairs between two oligonucleotide chains. These base pairs are typically formed by hydrogen bonds between nucleotides in antiparallel oligonucleotide chains. The bases of complementary oligonucleotide chains may be paired in a Watson-Crick manner (such as A to T, A to U, and C to G) or in any other manner that allows for the formation of a duplex (such as Hoogsteen or reverse Hoogsteen base pairing).

[0028] Complementarity includes complete complementarity and incomplete complementarity. “Complete complementarity” or “100% complementarity” means from the first oligonucleotide.Each nucleotide of the acid chain can form a hydrogen bond with a corresponding nucleotide in the second oligonucleotide chain of the double-stranded region of the double-stranded oligonucleotide molecule, and no base pairs are “mismatched”. “Incomplete complementarity” means that not all nucleotide units of the two chains are linked to each other by hydrogen bonds.

[0029] As used herein, the terms “oligonucleotide chain,” “chain,” and “oligonucleotide sequence” are used interchangeably and are a general term for short nucleotide sequences (including nucleotides in deoxyribonucleic acid (DNA) or ribonucleic acid (RNA)) having fewer than 35 bases. In a non-limiting example, the length of the chain can be any length from 15 to 35 nucleotides.

[0030] As used herein, the term “target gene” can refer to a nucleic acid sequence, transgenic, viral or bacterial sequence, chromosome, or exogenous gene that is naturally present in an organism and / or can be transiently or stably transfected or incorporated into cells and / or their chromatin. Target genes can be protein-coding genes or non-protein-coding genes (such as microRNA genes and long non-coding RNA genes). Target genes typically contain a promoter sequence, and positive regulation of the target gene can be achieved by designing a saRNA that has sequence identity (also known as homology) with the promoter sequence, characterized by upregulating the expression of the target gene. The terms “target sequence” and “target site” are used interchangeably and refer to a sequence fragment in the sequence of the target gene, such as the target gene promoter, that is homologous to or complementary to the sense or antisense strand of the saRNA. Target genes may also include one or more regulatory elements, wherein one or more saRNAs are designed to have sequence identity with the regulatory element. Non-limiting examples of one or more regulatory elements include: promoters, enhancers, silencers, insulators, TATA boxes, GC boxes, CAAT boxes, transcription start sites, DNA-binding motifs of transcription factors or other proteins that regulate transcription, and 5' untranslated regions.

[0031] As used herein, the term “guide strand” or “G strand” refers to the strand in a small RNA duplex that assembles with the Argonaute protein. The other strand, which is partially or completely complementary to the guide strand, is called the "guest strand" or "P strand." Without being bound by any particular theory, the strand carrying a sequence complementary to the target is the antisense strand and, if properly designed, will be preferentially chosen as the guide strand. In this case, the guest strand is the sense strand. However, a strand cannot be called a guide strand unless its 5' end is captured by the MID domain of AGO2. Therefore, the sense strand can be chosen as the guide strand to generate an antisense guest strand. In some embodiments, either the antisense strand or the sense strand can be chosen as the guide strand. In some embodiments, both the antisense strand and the sense strand can assemble with the Argonaute (AGO) protein, such that each of the sense and antisense strands in the RNA duplex functions independently or simultaneously as the guide strand.

[0032] As used herein, in saRNA duplexes, the term "sense strand" of saRNA refers to a strand that has sequence homology or sequence identity with a segment of the coding strand of the target gene sequence.

[0033] As used herein, in saRNA duplexes, the term "antisense strand" of saRNA refers to a strand that is complementary to the sequence of the sense strand. The antisense strand may interact with a target region of the target gene to activate or upregulate gene expression, the target region being a segment of the coding strand of the target gene sequence.

[0034] As used herein, the term "coding strand" refers to a DNA strand in the target gene that cannot be used for transcription, and whose nucleotide sequence is identical to the nucleotide sequence of the transcribed RNA (in RNA, T in DNA is replaced by U). The coding strand of the double-stranded DNA sequence of the target gene promoter described herein refers to the promoter sequence located on the same DNA strand as the coding strand of the target gene.

[0035] As used herein, the term “template strand” refers to the strand complementary to the coding strand of the double-stranded DNA of the target gene, i.e., the strand that serves as a template for transcription into RNA, and which is complementary to the transcribed RNA (A pairs with U and G pairs with C). During transcription, RNA polymerase binds to the template strand, moves along the 3'→5' direction of the template strand, and catalyzes RNA synthesis along the 5'→3' direction. The template strand of the double-stranded DNA sequence of the target gene promoter described herein refers to the promoter sequence located on the same DNA strand as the DNA template strand of the target gene.

[0036] As used herein, the term “promoter” refers to a sequence that is spatially associated with a protein-coding or RNA-coding nucleic acid sequence and regulates the transcription of that sequence. Typically, eukaryotic gene promoters contain 100 to 5000 base pairs, although this length range is not intended to limit the term “promoter” as used herein. Although promoter sequences are typically located at the 5' end of a protein-coding or RNA-coding sequence, promoter sequences are also present in exons and introns.

[0037] As used herein, the term “GNA” also refers to glycerol nucleic acid, which is a nucleic acid similar to DNA or RNA but with a different sugar-phosphodiester backbone composition, wherein propylene glycol replaces ribose or deoxyribose. As used herein, the term “LNA” refers to locked nucleic acid, wherein the 2'-oxygen and 4'-carbon atoms are linked by an additional bridge. As used herein, the term “BNA” refers to a nucleic acid that may contain a 2'-O and 4'-aminoethylene-bridged structure with N-O bonds. As used herein, the term “PNA” refers to a nucleic acid mimic with a pseudopeptide backbone, as described on page 5 / 50 of the specification, CN 121569037 A, which is composed of N-(2-aminoethyl)glycine units, wherein the nucleobases are linked to glycine nitrogen via carbonylmethylene linkers.

[0038] As used herein, the term "identity" or "homology" means that one oligonucleotide chain (sense or antisense chain) of the saRNA has sequence similarity to the coding or template chain in a region of the target gene. As used herein, "identity" or "homology" can be at least about 75%, about 79%, about 80%, about 85%, about 90%, about 95%, or 99%.

[0039] As used herein, the term "protrusion" refers to a non-base-paired nucleotide at the end (5' or 3') of an oligonucleotide chain, formed by one strand of a double-stranded oligonucleotide extending beyond the other strand. A single-stranded region extending beyond the 3' end and / or 5' end of the double-stranded oligonucleotide is called a protrusion.

[0040] As used herein, the term "natural protrusion" refers to a protrusion consisting of one or more nucleotides that are identical or complementary to the corresponding position on the target sequence. A natural protrusion on the sense strand consists of one or more nucleotides that are identical to the corresponding position on the DNA target. The natural overhang on the antisense strand consists of one or more nucleotides complementary to the corresponding position on the DNA target.

[0041] As used herein, the term "isolated" refers to material removed from its original or natural environment (e.g., the natural environment if it is naturally occurring). For example, naturally occurring polynucleotides or polypeptides present in living animals are not isolated, but the same polynucleotides or polypeptides isolated from some or all of the coexisting substances in the natural system through human intervention are isolated. Such polynucleotides may be part of a carrier, and / or such polynucleotides or polypeptides may be part of a composition and are still isolated because such carriers or compositions are not part of the environment in which they are found in nature. Isolated molecules can be obtained, for example, by extraction from a natural source, by expression of recombinant nucleic acids, or by chemical synthesis of the molecule. For example, the term "isolated RNA" refers to an RNA molecule that is substantially free of other cellular material or culture medium when produced by recombinant technology, or substantially free of chemical precursors or other chemicals when chemically synthesized. In some embodiments, the materials of this application, such as the polynucleotides, oligonucleotides, and / or saRNAs of this application, are isolated.

[0042] As used herein, the terms “gene activation” or “activated gene expression” and “gene upregulation” or “upregulated gene expression” are used interchangeably and refer to an increase in the transcription, translation, expression, or activity of a nucleic acid, determined by measuring transcriptional levels, mRNA levels, protein levels, enzyme activity, methylation state, chromatin state or conformation, translational level, or the activity or state of a gene in a cell or biological system. These activities or states can be determined directly or indirectly. Furthermore, “gene activation,” “activated gene expression,” “gene upregulation,” or “upregulated gene expression” refers to an increase in activity associated with a nucleic acid sequence, and not...Regardless of the mechanism of such activation. For example, gene activation occurs at the transcriptional level to increase transcription to RNA, and the RNA is translated into protein, thereby increasing protein expression.

[0043] As used herein, the terms “small activating RNA,” “saRNA,” and “small activating nucleic acid molecule” are used interchangeably and refer to a nucleic acid molecule that can upregulate the expression of a target gene and can be composed of a first nucleic acid fragment (sense strand) and a second nucleic acid fragment (antisense strand), the first nucleic acid fragment containing a nucleotide sequence that has sequence identity with a non-coding nucleic acid sequence (e.g., promoter or enhancer) of the target gene, and the second nucleic acid fragment containing a nucleotide sequence complementary to the first nucleic acid fragment, wherein the first nucleic acid fragment and the second nucleic acid fragment form a double strand. saRNA can also be composed of a synthetic or vector-expressed single-stranded RNA molecule that can form a hairpin structure through two complementary regions within the molecule, wherein the first region contains a nucleotide sequence that has sequence identity with a target region of the gene’s promoter, and the second region contains a nucleotide sequence complementary to the first region. The length of the double-stranded region of a saRNA molecule is typically about 15 to about 35 base pairs, about 16 to about 32 base pairs, about 17 to about 30 base pairs, about 18 to about 28 base pairs, about 19 to about 26 base pairs, about 20 to about 24 base pairs, and about 21 to about 22 base pairs, and is typically about 15 base pairs, about 16 base pairs, about 17 base pairs, about 18 base pairs, about 19 base pairs, about 20 base pairs, about 21 base pairs, about 22 base pairs, or about 23 base pairs. Furthermore, the terms "saRNA," "small activating RNA," and "small activating nucleic acid molecule" also include nucleic acids other than ribonucleotides, including but not limited to modified nucleotides or the like.

[0044] As used herein, the term "seed region" refers to the region at or near the 5' end of the guide strand (G strand) of a double-stranded oligonucleotide that plays a crucial role in target recognition of the oligonucleotide. As mentioned above, the guide strand can be the sense strand, antisense strand, or both sense and antisense strands of the double-stranded oligonucleotide. Typically, the seed region is 2 to 10 nucleotides in length.

[0045] As used herein, the term "accessory oligonucleotide (ACO)" means a non-targeting single-stranded oligonucleotide having at least 6 nucleotides, with or without one or more linker portions conjugated to another oligonucleotide. The ACO component is not designed to specifically target any complementary nucleic acid sequence of the target. The ACO component may be present in its backbone, nucleotide, or other locations (e.g., thiophosphate, methanesulfonylaminophosphate, or boron phosphate).The oligonucleotide can be chemically modified on its ester backbone, 2'-fluoro-2'-deoxynucleotide (2'-F), 2'-O-methyl (2'-O-Me), 2'-O-(2-methoxyethyl) (2'-O-MOE), locked nucleic acid (LNA), bridging nucleic acid (BNA), peptide nucleic acid (PNA), 5'-(E)-vinylphosphonate moiety, 5'-methylcytosine moiety, etc., to impart physiological and chemical properties that are beneficial to improving the bioavailability and delivery of the oligonucleotide. The covalent linker can be a natural or non-natural nucleotide, ethylene glycol, carbohydrate, alkyl chain, or any other linker used to covalently link any two oligonucleotides located at the 3'- or 5'-terminus of one or both chains within the oligonucleotide agent.

[0046] As used herein, the term "oligonucleotide agent" refers to a substance containing at least one or more saRNAs of the present invention or oligonucleotides thereof, and having activity in regulating target gene expression or enhancing the effect of saRNA, and may also include other oligonucleotide portions / components (such as ASO) or non-oligonucleotide portions / components conjugated, combined with, or mixed with saRNA. In some embodiments, the oligonucleotide agent includes RNA (such as saRNA of the present invention), DNA, BNA, LNA, GNA, or peptide nucleic acid (PNA).

[0047] As used herein, the term "prevention" means slowing the progression of a disease, symptom, or condition from its present state to a more harmful state.

[0048] As used herein, the term "treatment" means preventing, improving, reversing, curing, and / or delaying a disease, symptom, or condition.

[0049] As used herein, the capital letters "SMN2-FL" or "SMN2-FL gene" and "SMN2-Δ7" or "SMN2-Δ7 gene" refer to human genes. As used herein, the terms “SMN2 mRNA,” “SMN2-FL mRNA,” or “SMN2-Δ7 mRNA” refer to messenger RNA (mRNA) generated from the expression of or transcription of the SMN2 gene.

[0050] As used herein, the term “SERPING1 mRNA” refers to messenger RNA (mRNA) generated from the expression of or transcription of the SERPING1 gene.

[0051] Double-stranded oligonucleotide agents

[0052] Oligonucleotides offer great potential for the prevention or treatment of a variety of diseases, symptoms, or conditions by regulating, for example, the upregulation or downregulation of protein expression of disease-related genes and their variants. Among therapeutic oligonucleotides, saRNAs as upregulators have emerged as an emerging class of therapeutic agents and are under active development. However, their undesirable side effects, such as “off-target” effects, currently hinder the realization of their full therapeutic potential. There remains an unmet need in the art for oligonucleotide agents that simultaneously possess good gene-activating efficacy and low off-target effects.

[0053] Although sequences containing only natural structural units have been found to have important uses, oligonucleotide synthesis technology still offers the possibility of various nucleotide modifications. It has been surprisingly found that certain chemical modifications of oligonucleotide agents (such as saRNA) provide a way to effectively mitigate undesirable off-target effects while retaining the excellent gene-activating function of the agent. These modified oligonucleotide agents can overcome their current limitations and can be used more broadly in the medical and pharmaceutical fields.

[0054] Therefore, in one aspect, this document provides a double-stranded oligonucleotide agent capable of activating or upregulating the expression of a target gene, comprising a sense strand and an antisense strand, each strand being 15 to 35 nucleotides in length, wherein the sense strand and the antisense strand form a complementary double-stranded structure of at least 8 base pairs, wherein the double-stranded oligonucleotide agent comprises one or more glycerol nucleic acid (GNA) modified nucleotides located in one or both of the sense strand and the antisense strand.

[0055] GNA is a DNA / RNA analog wherein the (deoxy)ribose in the sugar-phosphodiester backbone of the DNA / RNA is replaced by propylene glycol. Similarly, GNA-modified nucleotides include GNA forms of non-GNA DNA / RNA nucleotides, such as naturally occurring DNA / RNA nucleotides adenine (A), thymine (T), cytosine (C), guanine (G), and uracil (U), wherein their (deoxy)ribose moieties are replaced by propylene glycol.

[0056] In some embodiments, one or more GNA-modified nucleotides include at least one selected from the group consisting of: GNA-modified adenine (GNA-A), GNA-modified thymine (GNA-T), GNA-modified cytosine (GNA-C), GNA-modified guanine (GNA-G), and GNA-modified uracil (GNA-U).

[0057] The sense and / or antisense strands of the double-stranded oligonucleotide agent include one or more GNA-modified nucleotides replacing their corresponding non-GNA nucleotides. In other words, one or more non-GNA nucleotides on the sense and / or antisense strands of the double-stranded oligonucleotide agent are modified or replaced by their GNA-modified forms.

[0058] In some embodiments, the double-stranded oligonucleotide agent comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, or more.At least 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 GNA-modified nucleotides. In some embodiments, the double-stranded oligonucleotide agent comprises 1 to 50 (e.g., 1 to 50, 1 to 45, 1 to 40, 1 to 35, 1 to 30, 1 to 25, 1 to 20, 1 to 15, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2) GNA-modified nucleotides.

[0059] In some embodiments, at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39 or at least 40 GNA-modified nucleotides are located in the sense strand and / or antisense strand of the double-stranded oligonucleotide agent. In some embodiments, 1 to 50 (e.g., 1 to 50, 1 to 45, 1 to 40, 1 to 35, 1 to 30, 1 to 25, 1 to 20, 1 to 15, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2) GNA-modified nucleotides are located in the sense strand and / or antisense strand of the double-stranded oligonucleotide agent.

[0060] In some embodiments, either or both of the sense strand and antisense strand of the double-stranded oligonucleotide agent may be a guide strand mediating RNA activation.

[0061] In some embodiments, the leader strand of the double-stranded oligonucleotide agent comprises 1 to 50 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or any range thereof) GNA-modified nucleotides.

[0062] In some embodiments, one or more GNA-modified nucleotides are located at a position starting from the 5' end of the leader strand. (Specification page 8 / 50, 13 CN 121569037 A)Positions 1 and / or 2 and / or 3 and / or 4 and / or 5 and / or 6 and / or 7 and / or 8 and / or 9 and / or 10 and / or 11 and / or 12 and / or 13 and / or 14 and / or 15 and / or 16 and / or 17 and / or 18 and / or 19 and / or 20 and / or 21 and / or 22 and / or 23 and / or 24 and / or 25 of the guide strand may be one or both of the sense and antisense strands. In some embodiments, one or more GNA-modified nucleotides are located at positions 1 to 25 (e.g., 2 to 25, 2 to 24, 2 to 23, or 2 to 22) of the guide strand, which may be one or both of the sense and antisense strands. Typically, the guide strand of a double-stranded oligonucleotide agent includes a seed region that functions in the recognition of the target gene. In some embodiments, the seed region is 2 to 10 nucleotides long (e.g., 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, or 2 to 4).

[0063] The seed region is located at or near the 5' end of the leader strand. In some embodiments, the seed region begins no more than 3 nucleotides (e.g., 3, 2, 1, or 0) from the 5' end of the leader strand. In some embodiments, the 5' end nucleotide of the seed region is located no more than 3 nucleotides (e.g., 3, 2, 1, or 0) from the 5' end of the leader strand. In some embodiments, the 3' end nucleotide of the seed region is at least 6 nucleotides (e.g., 6, 7, 8, 9, or 10) from the 5' end of the leader strand. In some embodiments, the seed region comprises consecutive nucleotides at positions 2 to 10, or 2 to 9, or 2 to 8, or 2 to 7, or 2 to 6, or 3 to 6, or 2 to 5, or 3 to 6, or 3 to 5, or 4 to 6, starting from the 5' end of the leader strand.

[0064] In some embodiments, the seed region has at least 80%, at least 85%, at least 90%, at least 95%, or 100% complementarity to an isolength segment of the positive strand of the double-stranded oligonucleotide agent. In one specific embodiment, the seed region is 100% complementary to an isolength segment of the positive strand of the double-stranded oligonucleotide agent.

[0065] One or more GNA-modified nucleotides may be located within and / or outside the seed region. In some embodiments, the seed region comprises 1 to 8 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or any range thereof) GNA-modified nucleotides. In some embodiments, the seed region comprises 1 to 6 GNA-modified nucleotides. In some embodiments, the seed region comprises 1 to 5 GNA-modified nucleotides. In some embodiments, the seed region comprises 1 to 4 GNA-modified nucleotides. In some embodiments, the seed region comprises 1 to 3 GNA-modified nucleotides. In some embodiments, the seed region comprises 1 or 2 GNA-modified nucleotides. In some embodiments, the seed region comprises at least 3%, at least 5%, at least 10%, at least 20%, at least 30%, or at least...40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% of the nucleotides are GNA-modified nucleotides.

[0066] In some embodiments, all or part of the GNA-modified nucleotides in the double-stranded oligonucleotide agent are located in the seed region. In some embodiments, all or part of the GNA-modified nucleotides in the leader strand of the double-stranded oligonucleotide agent are located in the seed region.

[0067] The double-stranded oligonucleotide agent may include one or more GNA-modified nucleotides located at or near either or both of the 5' and 3' ends of the seed region. In an exemplary embodiment, the double-stranded oligonucleotide agent includes one or more GNA-modified nucleotides located in the first three (e.g., the first two or one) nucleotides counting from either or both of the 5' and 3' ends of the seed region.

[0068] In some embodiments, the double-stranded oligonucleotide agent also includes one or more GNA-modified nucleotides located outside the seed region. In some embodiments, the double-stranded oligonucleotide agent also includes one or more GNA-modified nucleotides located in the guest strand (P strand). In some embodiments, the double-stranded oligonucleotide agent comprises one or more GNA-modified nucleotides on one or both of the sense and antisense strands.

[0069] The sense and antisense strands of the double-stranded oligonucleotide agent can be obtained by chemical synthesis, for example by phosphoramide process (e.g., solid-phase phosphoramide process). In some embodiments, either or both of the sense and antisense strands are synthesized to comprise one or more GNA-modified nucleotides. In some embodiments, the antisense strand is synthesized to comprise one or more GNA-modified nucleotides. The synthesized sense and antisense strands are then annealed to form a double-stranded structure. Specification 9 / 50 pages 14 CN 121569037 A

[0070] In some embodiments, the GNA-modified nucleotide monomer of formula (1) can be used to synthesize: (1) wherein the bases may be selected from the group consisting of: adenine nucleobases, thymine nucleobases, cytosine nucleobases, guanine nucleobases, uracil nucleobases and analogues thereof.

[0071] In some embodiments, the bases in formula (1) may be selected from the following structures: , , , , , , and.

[0072] The positive strand of the double-stranded oligonucleotide agent has sequence homology or identity with a segment of the coding strand of the target gene. In some embodiments, the positive strand has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence homology or identity with an iso-length segment of the coding strand of the target gene. In some embodiments, the positive strand has 5 or fewer nucleotide differences relative to an iso-length segment of the coding strand of the target gene, i.e., 5, 4, 3, 2, 1, or 0 nucleotide differences.

[0073] Typically, the antisense strand of a double-stranded oligonucleotide agent is complementary to a segment of the coding strand of the target gene, particularly a segment in the promoter region of the target gene, and thus can interact with the gene (segment) to activate or increase gene transcription and further activate or increase gene expression at the protein level. In some embodiments, the sense strand or the antisense strand has at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 nucleotides complementary to an iso-length segment of the coding strand of the target gene. In some embodiments, the antisense strand has 5 or fewer nucleotide differences or mismatches relative to an iso-length segment of the coding strand of the target gene, i.e., 5, 4, 3, 2, 1, or 0 nucleotide differences or mismatches. In some embodiments, the difference or mismatch is located inside or near the 3' end of the antisense strand. Specification 11 / 50 pages 16 CN 121569037 A

[0074] The sense and antisense strands of the double-stranded oligonucleotide agent include complementary regions forming a double-stranded structure of at least 8 base pairs, for example, complementary regions of a double-stranded structure of at least 10, at least 12, at least 15, at least 18, or at least 20 base pairs. In some embodiments, the sense and antisense strands are at least 50% complementary to each other, for example, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% complementary. In some embodiments, there are no more than 5 mismatched nucleotides in the complementary region between the sense and antisense strands, i.e., 5, 4, 3, 2, 1, or 0 mismatched nucleotides. In some embodiments, the complementary double-stranded structure of at least 8 base pairs has no more than 5 mismatched nucleotides between the sense and antisense strands, i.e., 5, 4, 3, 2, 1, or 0 mismatched nucleotides. In some embodiments, the mismatched nucleotides are located inside or near the 3' or 5' end of the antisense strand. In some embodiments, the mismatched nucleotides are located at the GNA position in the sense and / or antisense strands.

[0075] The sense and antisense strands of the double-stranded oligonucleotide agent described herein may be present on two different nucleic acid strands or on one nucleic acid strand (e.g., a continuous nucleic acid sequence). When the sense and antisense strands are located on two different strands, at least one strand of the double-stranded oligonucleotide agent has a 3' overhang of 0 to 6 nucleotides in length, such that the overhang is 0, 1, 2, 3, 4, 5, or 6 nucleotides in length, and in some cases, both strands have a 3' overhang of 2 or 3 nucleotides in length. In some embodiments, the nucleotides of the overhang may be selected from corresponding positions on a DNA target.(i.e., the natural overhang) or a complementary nucleotide thereto. In some cases, when the sense and antisense strands are located on a single nucleic acid strand, the double-stranded oligonucleotide agent can be a hairpin single-stranded nucleic acid molecule, wherein the complementary regions of the sense and antisense strands form a double-stranded structure with each other.

[0076] The sense and antisense strands of the double-stranded oligonucleotide agents described herein each have a length of 15 to 35 nucleotides. For example, in some embodiments, the sense and antisense strands each have a length of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 nucleotides or any range thereof.

[0077] In one exemplary embodiment, the sense strand of the double-stranded oligonucleotide agent may include the nucleotide sequence shown in any one of SEQ ID NO: 1, 3, 12, 14, 15, 17, 18, 20, 21, 23, 24, 27, 29, 52, 54, and the antisense strand of the double-stranded oligonucleotide agent may include the nucleotide sequence shown in any one of SEQ ID NO: 2, 4, 5, 6, 7, 8, 9, 10, 11, 13, 16, 19, 22, 25, 26, 28, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 53, 55.

[0078] In some embodiments, the double-stranded oligonucleotide agent may include RNA, DNA, BNA, LNA, GNA, or peptide nucleic acid (PNA).

[0079] In some embodiments, the double-stranded oligonucleotide agent is a saRNA that upregulates the expression of the target gene.

[0080] In a non-limiting example, the saRNA is designed at least in part based on the following criteria: (1) having a GC content of 35% to 70%; (2) having fewer than 5 consecutive identical nucleotides; (3) having 3 or fewer dinucleotide repeats; and (4) having 3 or fewer trinucleotide repeats. In some embodiments, the saRNA is designed or selected at least in part based on criteria that enable the production of functional saRNA. For example, in some cases, the sequence upstream of the TSS may include a sequence that, although located in a hotspot region, is unfavorable for saRNA synthesis.

[0081] In some embodiments, the saRNA is designed or selected at least in part based on the following criteria: including sequences having a specific GC content (e.g., 25% to 75% GC content) and lacking consecutive identical nucleotides, consecutive dinucleotides, or consecutive trinucleotides. In some embodiments, the saRNA sequence includes the following sequences: (1) having a GC content of 35% to 70%; (2) having fewer than 5 consecutive identical nucleotides; (3) having 3 or fewer dinucleotide repeats; and (4) having 3 or fewer trinucleotide repeats.

[0082] In some embodiments, the saRNA sequence comprises a sequence having a GC content of 25% to 75%, 30% to 70%, 35% to 70%, 40% to 60%, or 45% to 55%. In some embodiments, the saRNA comprises a sequence having a GC content of 35% to 70%.

[0083] In some embodiments, the saRNA sequence comprises a sequence having fewer than 7 consecutive identical nucleotides, fewer than 6 consecutive identical nucleotides, fewer than 5 consecutive identical nucleotides, fewer than 4 consecutive identical nucleotides, or fewer than 3 consecutive identical nucleotides. In some embodiments, the saRNA comprises a sequence having fewer than 5 consecutive identical nucleotides.

[0084] In some embodiments, the saRNA sequence comprises a sequence having 5 or fewer dinucleotide repeats, 4 or fewer dinucleotide repeats, 3 or fewer dinucleotide repeats, or 2 or fewer dinucleotide repeats. In some embodiments, the saRNA comprises a sequence having 3 or fewer dinucleotide repeats.

[0085] In some embodiments, the saRNA sequence comprises a sequence having 5 or fewer trinucleotide repeats, 4 or fewer trinucleotide repeats, 3 or fewer trinucleotide repeats, or 2 or fewer trinucleotide repeats. In some embodiments, the saRNA comprises a sequence having 3 or fewer trinucleotide repeats.

[0086] The methods and principles of saRNA molecule design are well known to those skilled in the art and are described in detail in, for example, Place et al., Molecular Therapy–Nucleic Acids (2012) 1, e15; and Li et al., PNAS, 2006, vol. 103, no. 46, 17337–17342, which are incorporated herein by reference in their entirety.

[0087] In the double-stranded oligonucleotide agents described herein, all nucleotides may be natural or unmodified nucleotides, or at least one nucleotide may be a chemically modified nucleotide. Non-limiting examples of chemical modifications include one or more combinations of the following: (1) modification of the phosphodiester bond of the nucleotide in the nucleotide sequence of the double-stranded oligonucleotide agent; (2) modification of the 2'-OH of the ribose in the nucleotide sequence of the double-stranded oligonucleotide agent; (3) modification of the bases in the nucleotide of the double-stranded oligonucleotide agent; and (4) at least one nucleotide in the nucleotide sequence of the double-stranded oligonucleotide agent is a locked nucleic acid, a bridging nucleic acid, DNA, GNA, or a peptide nucleic acid (PNA).

[0088] The chemical modifications described herein are well known to those skilled in the art. The modifications described herein can stabilize the structure of the reagent and maintain high specificity and high affinity for base pairing.

[0089] In some embodiments, the double-stranded oligonucleotide agent described herein comprises at least one chemically modified nucleotide that is modified at the 2'-OH position of the pentose sugar of the nucleotide, i.e., by introducing certain substituents at the hydroxyl position of the ribose, such as 2'-fluorine modification, 2'-oxymethyl modification, 2'-oxyethylidene methoxy modification, 2,4'-dinitrophenol modification, locked nucleic acid (LNA), 2'-amino modification, or 2'-deoxy modification, for example, nucleotides modified with 2'-deoxy-2'-fluorine, nucleotides modified with 2'-deoxy.

[0090] In some embodiments, the double-stranded oligonucleotide agent described herein comprises at least one chemically modified nucleotide that is modified at the base of the nucleotide, for example, 5'-bromouracil modification, 5'-iodouracil modification, N-methyluracil modification, or 2,6-diaminopurine modification.

[0091] In some embodiments, the chemical modification of the double-stranded oligonucleotide agent is the addition of an (E)-vinylphosphonate moiety to the 5' end of the sense or antisense sequence. In some embodiments, the chemical modification of at least one chemically modified nucleotide is the addition of a 5-methylcytosine moiety or 5-methyluracil to the 5' end of the sense or antisense sequence.

[0092] In some embodiments, the double-stranded oligonucleotide agents described herein include at least one chemically modified nucleic acid nucleotide in the nucleotide sequence of the reagent, such as locked nucleotides, abase nucleotides, 2'-amino-modified nucleotides, 2'-alkyl-modified nucleotides, morpholinonucleotides, aminophosphates, and nucleotides containing non-natural bases. In some embodiments, the double-stranded oligonucleotide agents described herein include "endo-light" modifications with a 2'-O-methyl-modified nucleotide and a nucleotide containing a 5'-thiophosphate group. Specification 13 / 50 pages 18 CN 121569037 A

[0093] In some embodiments, the double-stranded oligonucleotide agents described herein are chemically modified to enhance stability or other beneficial characteristics. The specific nucleic acids described in this disclosure can be synthesized and / or modified using conventional methods, such as those described in Current Protocols in Nucleic Acid Chemistry, Beaucage, SL et al. (Edrs.), John Wiley & Sons, Inc., New York, USA, which are incorporated herein by reference. Modifications include, for example, (a) terminal modifications, such as 5' modifications (phosphorylation, conjugation, reverse linkage, etc.), 3' modifications (conjugation, DNA nucleotides, reverse linkage, etc.), (b) base modifications, such as substitution with a stabilizing base, a destabilizing base, or a base paired with a base from an expanded ligand library, removal of a base (de-base nucleotide) or conjugated base, and (c) sugar modifications (e.g., at the 2' or 4' position) or sugar substitutions.(d) Backbone modifications, including modifications or substitutions of phosphodiester bonds. Specific examples of double-stranded oligonucleotides that can be used in this disclosure include, but are not limited to, RNA containing a modified backbone or RNA without native nucleoside interchains. In some embodiments, RNA with a modified backbone includes RNA without phosphorus atoms in its backbone. In some embodiments, modified RNA without phosphorus atoms in its internucleotide backbone may also be considered an oligonucleotide. In some embodiments, the modified oligonucleotide will have phosphorus atoms in its internucleotide backbone.

[0094] The modified oligonucleotide backbone includes, for example, thiophosphates, chiral thiophosphates, dithiophosphates, phosphate triesters, aminoalkyl phosphate triesters, methyl and other alkylphosphonates (including 3'-alkylphosphonates and chiral phosphonates), phosphonates, aminophosphates (including 3'-aminophosphates and aminoalkylaminophosphates), thioaminophosphates, thioalkylphosphonates, thioalkylaminophosphate triesters, and borophosphates having a normal 3'-5' bond, analogs of these borophosphates with 2'-5' linkages, and those compounds having opposite polarities, wherein adjacent nucleoside unit pairs are 3'-5' linked to 5'-3' or 2'-5' linked to 5'-2'. Various salts, mixed salts, and free acid forms are also included.

[0095] In some embodiments, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or about 100% of the non-GNA modified nucleotides (non-GNA nucleotides) of the double-stranded oligonucleotide agent include modifications selected from the group consisting of: 2'-fluoro(2'-F), 2'-O-methyl(2'-OMe), 2'-O-methoxyethyl(2'-MOE), 5'-(E)-vinylphosphonate, thiophosphate backbone modifications, and combinations thereof.

[0096] Conjugation Agents

[0097] Furthermore, in order to promote the entry of the double-stranded oligonucleotide agent into the cell, based on the above modifications, a chemical conjugation portion may be introduced into the end of the sense or antisense strand of the double-stranded oligonucleotide agent to promote action through the cell membrane composed of a lipid bilayer, as well as the nuclear membrane and gene promoter regions within the cell nucleus. Therefore, this disclosure also provides conjugators comprising a double-stranded oligonucleotide agent and at least one conjugation moiety.

[0098] In some embodiments, the conjugator comprises the double-stranded oligonucleotide agent described herein and one or more conjugation moieties covalently linked to the oligonucleotide agent. In some embodiments, the conjugation moiety alters one or more properties of the linked oligonucleotide, including but not limited to pharmacodynamics, pharmacokinetics, stability, binding, absorption, tissue distribution, cellular uptake, charge, and clearance. In some embodiments, the conjugation moiety imparts new properties to the linked oligonucleotide, such as...It can detect the fluorophore or reporter group of oligonucleotides. Some of the conjugated moieties have been previously described, such as: accessory oligonucleotides (ACO, WO2023280190A1 and PCT / CN2024 / 084814), lipids / fatty acids (WO2024002046A1), cholesterol moieties (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556); bile acids (Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4, 1053-1060); thioethers, such as hexyl-S-triphenylmethylthiol (Manoharan et al., Ann. NY Acad. Sci., 1992, 660, 306-309; Manoharan et al., Bioorg. Med. Chem. Lett., 1993, 3, 2765-2770); Thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20, 533-538); Fatty chains, such as dodecyldiol or undecyl residues (Saison-Behmoaras et al., EMBO 1, 1991, 10, 1111-1118; Kabanov et al., FEBS Lett., 1990, 259, 327-330; Svinarchuk et al., Biochimie, 1993, 75, 49-54); Phospholipids, such as hexadecyl-racemic-glycerol or triethyl-ammonium 1,2-di-O-hexadecyl-racemic-glycerol-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, ... 36, 3651-3654; Shea et al., Nucl. Acids Res., 1990, 18, 3777-3783; polyamine or polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973) or adamantaneacetic acid; palmitoyl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229-237), stearylamine or hexano-carbonyl-hydroxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277, 923-937), tocopherol group (Nishina et al., Molecular Therapy Nucleic Acid).Acids, 2015, 4, e220; and Nishina et al., Molecular Therapy, 2008, 16, 734-740), or GalNAc clusters (e.g., WO2024002046A1).

[0099] In some embodiments, the sense or antisense strand of the double-stranded oligonucleotide agent is conjugated to one or more conjugation moieties selected from the following: intercalating agents, reporter molecules, polyamines, polyamides, peptides, carbohydrates, vitamin moieties, polyethylene glycols, thioethers, polyethers, cholesterol, thiocholesterol, bile acid moieties, folic acid, lipids, phospholipids, biotin, phenazine, phenanthridine, anthraquinones, adamantane, acridine, fluorescein, rhodamine, coumarin, fluorophores, and dyes.

[0100] In some embodiments, the conjugated portion includes an active pharmaceutical substance, such as aspirin, warfarin, phenylbutazone, ibuprofen, sulprofen, fenbufen, ketoprofen, (S)-(+)-pranoprofen, carbofen, tansylsarcosinate, 2,3,5-triiodobenzoic acid, fingolimod, flufenamic acid, leucovorin, benzothiazide, chlorothiazide, diazoxide, indomethacin, barbiturates, cephalosporins, sulfonamides, antidiabetic drugs, antimicrobial agents, or antibiotics.

[0101] In some embodiments, the double-stranded oligonucleotide agent described herein is conjugated to one or more conjugated portions selected from the group consisting of lipids, fatty acids, fluorophores, ligands, sugars, peptides, and antibodies.

[0102] In some embodiments, the sense or antisense strand of the double-stranded oligonucleotide agent is conjugated to one or more conjugation moieties selected from the group consisting of: cell-penetrating peptides, polyethylene glycol, alkaloids, tryptophan, benzimidazole, quinolones, amino acids, cholesterol, glucose, and N-acetylgalactosamine.

[0103] In some embodiments of the double-stranded oligonucleotide agent, the sense or antisense strand of the agent disclosed in this application is conjugated to one or more conjugation moieties selected from the group consisting of: cell-penetrating peptides, polyethylene glycol, alkaloids, tryptophan, benzimidazole, quinolones, amino acids, cholesterol, glucose, and N-acetylgalactosamine. In some embodiments, the double-stranded oligonucleotide agent is conjugated to a lipid selected from C4-30 fatty acids. In some embodiments, the conjugation moieties are lipids / fatty acids having a saturated or unsaturated straight-chain or branched C16 carbon chain.

[0104] According to another embodiment, the double-stranded oligonucleotide agent further includes at least one auxiliary oligonucleotide (ACO) conjugated to the oligonucleotide agent. The term "accessory oligonucleotide (ACO)" in this document refers to a non-targeting single-stranded oligonucleotide having at least six nucleotides, with or without one or more linker portions conjugated to another oligonucleotide. ACO components are not designed to specifically target any complementary nucleic acid sequence in the target organism.The oligonucleotide can be chemically modified on its backbone, nucleotides, or other sites (e.g., thiophosphate, methanesulfonylaminophosphate, or borophosphate backbones, 2'-fluoro-2'-deoxynucleotide (2'-F), 2'-O-methyl (2'-O-Me), 2'-O-(2-methoxyethyl) (2'-O-MOE), locked nucleic acid (LNA), bridging nucleic acid (BNA), peptide nucleic acid (PNA), 5'-(E)-vinylphosphonate moiety, 5'-methylcytosine moiety, etc.) to impart physiological and chemical properties that improve the bioavailability and delivery of the oligonucleotide. The covalent linker can be a natural or non-natural nucleotide, ethylene glycol, carbohydrate, alkyl chain, or any other linker for covalently linking any two oligonucleotides located at the 3'- or 5'-terminus of one or both chains within the oligonucleotide agent. The ACO can be prepared according to the description disclosed in WO2023280190A1, the entire text of which is incorporated herein by reference. Specification 15 / 50 pages 20 CN 121569037 A

[0105] In some embodiments, the double-stranded oligonucleotide agent of this application relates to a sense or antisense strand of a double-stranded oligonucleotide agent conjugated to one or more conjugation moieties selected from: cell-penetrating peptides, polyethylene glycol, alkaloids, tryptophan, benzimidazole, quinolones, amino acids, cholesterol, glucose, and N-acetylgalactosamine. In some embodiments, the double-stranded oligonucleotide agent is conjugated to two conjugation moieties. In some embodiments, the two conjugation moieties are lipids and N-acetylgalactosamine. In some embodiments, one or more conjugation moieties are derived from tC2x6, C5x5, or combinations thereof shown in this application: (tC2x6) and (C5x5), where (tC2x6) represents a carrier material.

[0106] In some embodiments, the conjugation moieties conjugated to the double-stranded oligonucleotide agent are tC2x6 and C5x5 shown in this application. In some embodiments, tC2x6 is conjugated to the 3' end of the positive strand; C5x5 is conjugated to the 5' end of the positive strand. The conjugated portions can be synthesized by procedures known in the art, such as WO2024002046A1, which is incorporated herein by reference, for the synthesis of tC2x6 and C5x5.

[0107] In some embodiments, the conjugated portions are lipids selected from fatty acids with a carbon chain length of 4 to 30 carbon atoms. In some embodiments, the conjugated portions are fatty acids with a carbon chain length of 16 carbon atoms. In some embodiments, the conjugated portions are selected from lipophilic portions as described in WO2021092371A2. In some embodiments, the double-stranded oligonucleotide agent may comprise one, two, three, four, five, six, or even more oligonucleotides conjugated to one, two, three, four, five, six, or even more conjugated portions respectively via one, two, three, four, five, six, or even more linker portions.

[0108] According to one embodiment, when a connecting portion is present, the connecting portion may be selected from the group consisting of: -O-, -S-, -C(O)-, -NH-, -N((C1-C12)alkyl)-, -N((C1-C12)alkyl)-C(O)-O-, -O-C(O)-, -C(O)-, -O-C(O)-, -O-C(O)-O-, -C(O)-NH-, -OP(O)2O-, -P(O)(O-)O-, -OP(O)O-, -OP(O)(S)O-, -O-S(O)2O- O-、-S(O)2-O-、-S(O)-O-、-(C1-C22)alkylene-、-(C1-C22)alkylene-NH-、-NH-(C1-C22)alkylene-、-(C1-C22)alkylene-NH-C(O)-、-(C1-C22)alkylene-C(O)-、-C(O)-(C1- C22)alkylene-, -NH-C(O)-(C1-C22)alkylene-, -C(O)-NH-(C1-C22)alkylene-, -C(O)-(C1-C22)alkylene-NH-, -NH-(C1-C22)alkylene-C(O)-, -C(O)-(C1-C22)alkylene-C(O)-, -NH-(C1-C22)alkylene-NH-, -C(O)-(C1-C22)alkylene-C(O)O-, -O-C(O)-(C1-C22)alkylene-C(O)-O-, -C(O)-O-(C1-C22) Alkylene -O-C(O)-, -C(O)-(C1-C22)alkylene-NH-C(O)-, -NH-C(O)-(C1-C22)alkylene-C(O)-, -NH-C(O)-(C1-C22)alkylene-C(O)-NH-, -C(O)-NH-(C1-C22)alkylene-NH-C(O)-, -(C1-C22)alkylene-OP(O)2O-, -(C1-C22)alkylene-OP(O)(O-)O-, -(C1-C22)alkylene-OP(O)(O-)O-(C1-C22)alkylene-, - (C1-C22)alkylene-OP(O)O-, -(C1-C22)alkylene-OP(O)(S)O-, -(C1-C22)alkylene-O-S(O)2-O-, -(C1-C22)alkylene-S(O)2-O-, -(C1-C22)alkylene-S(O)-O-, -O-P(O)2-O-(C1-C22)alkylene-OP(O)2O-, -O-P(O)-O-(C1-C22)alkylene-OP(O)O-, -OP(O)(S)O-(C1-C22)alkylene-OP(O)(S)O-, -O-S (O)2-O-(C1-C22)alkylene-O-S(O)2-O-, -S(O)2-O-(C1-C22)alkylene-S(O)2-O- and -O-S(O)-(C1-C22)alkylene-S(O)-O-; wherein the -(C1-C22)alkylene- contained in the linking portion can be an alkylene containing 1 to 22 carbon atoms, for example 2 to 20 carbon atoms, or 3 to 18 carbon atoms, or 4 to 16 carbon atoms, or 5 to 12 carbon atoms, or 6 to 10 carbon atoms. In one embodiment, when the linking portion is a direct bond, the conjugation portion is directly linked to the oligonucleotide.

[0109] In some embodiments, the double-stranded oligonucleotide agent conjugated to one or more conjugated portions disclosed in the embodiments is directly contacted, transferred to, delivered to, or administered to cells or a subject. The terms “patient,” “individual,” or “subject,” used interchangeably herein, can refer to a non-human (e.g., mammalian) subject or a human subject.

[0110] The double-stranded oligonucleotide agent activates or upregulates the expression of a target gene in a cell via an RNAa mechanism. As used herein, the RNAa mechanism (also known as RNA activation) refers to the mechanism by which a double-stranded nucleic acid structure upregulates a target gene at the transcriptional level in a sequence-specific manner.

[0111] In some embodiments, the double-stranded oligonucleotide agent described herein upregulates the expression of a target gene by at least 10%, for example, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 300%, at least 500%, at least 800%, at least 1000%, at least 2000%, or at least 5000%, compared to the expression of an unregulated gene.

[0112] In some embodiments, the expression of target genes upregulated by the double-stranded oligonucleotide agent described herein is at least the same as, or at least 10% higher than, the expression regulated by the same but non-GNA double-stranded oligonucleotide agent, for example, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 300%, or at least 500%.

[0113] In some embodiments, the double-stranded oligonucleotide agent described herein reduces at least the same or at least 10% of the off-target effects compared to the same but non-GNA double-stranded oligonucleotide agent, for example, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100%.

[0114] In some embodiments, the double-stranded oligonucleotide agent described herein interacts with and activates a target gene associated with a disease, symptom, or condition. In some embodiments, the disease, symptom, or condition is caused by insufficient expression of the target gene. In some embodiments, upregulation of the target gene is beneficial for the prevention of the disease, symptom, or condition. In some embodiments, upregulation of the target gene promotes the relief of symptoms of the disease, symptom, or condition, or the treatment of the disease, symptom, or condition. Specification 17 / 50 pages 22 CN 121569037 A

[0115] An exemplary target gene used herein is the Survival Motor Neuron 2 (SMN2) gene. The Survival Motor Neuron (SMN) protein produced by this gene maintains the health and normal function of motor neurons, while insufficient SMN protein due to mutations in the SMN1 gene leads to spinal muscular atrophy (SMA). Higher or activated expression of the SNM2 gene is known to be associated with paralogous salvage therapy for less severe SMA symptoms.

[0116] Another exemplary target gene used herein is the SERPING1 gene. SERPING1 is primarily expressed in the liver and encodes a C1 repressor protein, which is secreted into the bloodstream and participates in the normal function of the contact, coagulation, and fibrinolytic systems. It encodes a C1 esterase inhibitor (C1EI or C1INH), the largest member of the serine protease inhibitor (SERPIN) superfamily. Expression of the SERPING1 gene is upregulated via RNA activation, and related diseases (particularly HAE) are treated by increasing the expression level of the C1IHN protein (i.e., the C1 repressor protein). Since the SERPING1 gene encodes the C1IHN protein, increased SERPING1 mRNA expression increases the level of the C1IHN protein.

[0117] However, it should be understood that the selection and use of the SMN2 gene, the SERPING1 gene, and double-stranded oligonucleotide agents (e.g., saRNA) for such genes are for illustrative purposes only and are not intended to limit the scope of the invention in any way.

[0118] Cells comprising oligonucleotide agents

[0119] In another aspect, this document provides a cell comprising the double-stranded oligonucleotide agents or conjugates described herein.

[0120] Upon contact with the cell, the double-stranded oligonucleotide agents or conjugates described herein can effectively activate or upregulate the expression of a target gene in the cell, for example, activating or upregulating the expression of the target gene by at least 10% compared to the expression of an unregulated gene, or activating or upregulating the expression of the target gene to a level equal to or at least 10% higher than that obtained by the same but non-GNA double-stranded oligonucleotide agent or conjugate, while producing a reduced off-target effect compared to the same but non-GNA double-stranded oligonucleotide agent.

[0121] The cell may comprise one or more of the double-stranded oligonucleotide agents or conjugates described herein.

[0122] In some embodiments, this disclosure relates to cells comprising the double-stranded oligonucleotide agents or conjugates described herein. In some embodiments, the cells are mammalian cells. In some embodiments, the cells are human cells. The cells described herein may be in vitro or ex vivo, such as cell lines or cell strains, or may be present in mammals, such as humans.

[0123] Compositions comprising oligonucleotide agents or conjugates

[0124] In another aspect, this document provides a composition comprising the double-stranded oligonucleotide agents or conjugates described herein.

[0125] The composition may comprise one or more of the double-stranded oligonucleotide agents or conjugates described herein.

[0126] In some embodiments, the composition is a pharmaceutical composition. In some embodiments, the composition comprises at least one pharmaceutically acceptable carrier. In some embodiments, the composition includes at least one pharmaceutically acceptable carrier selected from aqueous carriers, liposomes or LNPs, polymers, micelles, colloids, metal nanoparticles, non-metal nanoparticles, bioconjugates (e.g., GalNAc, lipids, antibodies, peptides, or single- or double-stranded oligonucleotides), polypeptides, antibodies, and any combination thereof. In one embodiment, the aqueous carrier may be, for example, RNase-free water or RNase-free buffer.

[0127] In some embodiments, the composition may include 0.001 nM to 200 nM (e.g., 0.01 nM to 100 nM, 0.1 nM to 50 nM, 1 nM to 150 nM, 1 nM to 200 nM, 1 nM to 20 nM, 0.001 nM to 1 nM, 1 nM to 10 nM, 10 nM to 100 nM, 10 nM to 50 nM, 20 nM to 50 nM, 20 nM to 100 nM, 25 nM to 100 nM, or 30 nM to 100 nM) of a double-stranded oligonucleotide agent or conjugate as described herein. In some embodiments, the composition includes 20 nM of a double-stranded oligonucleotide agent or conjugate as described herein. In some embodiments, the composition includes 25 nM of a double-stranded oligonucleotide agent or conjugate as described herein. In some embodiments, as described on pages 18 / 50 of CN 121569037 A, the composition comprises 30 nM of a double-stranded oligonucleotide agent or conjugate as described herein. In some embodiments, the composition comprises 50 nM of a double-stranded oligonucleotide agent or conjugate as described herein. In some embodiments, the composition comprises 100 nM of a double-stranded oligonucleotide agent or conjugate as described herein.

[0128] Kits comprising oligonucleotide agents

[0129] In another aspect, a kit comprising a double-stranded oligonucleotide agent or conjugate as described herein is provided herein.

[0130] As used herein, “kit” is generally defined as comprising one or more of the components or embodiments of this disclosure.Packaging, components, or containers (e.g., insulated containers) of other components related to this disclosure, such as those described above. Any agent or component of the kit may be provided in liquid form (e.g., solution) or in solid form (e.g., dry powder, frozen, etc.).

[0131] The kit may include one or more of the double-stranded oligonucleotide agents or conjugates described herein.

[0132] In some embodiments, the kit is used to activate or upregulate the expression of a target gene in cells or a subject. In some embodiments, the kit is used to increase the level of mRNA or protein encoded by a target gene in cells or a subject. In some embodiments, the kit is used to prevent or treat a disease, symptom, or condition associated with insufficient expression of the target gene.

[0133] In some embodiments, the kit includes a composition (e.g., a pharmaceutical composition) comprising the double-stranded oligonucleotide agent or conjugate described herein.

[0134] In some embodiments, the kit further includes means for administering the double-stranded oligonucleotide agent or conjugate to a subject. In some embodiments, the kit is in a labeled package, and the label on the package indicates that the double-stranded oligonucleotide agent or conjugate or composition is available for the prevention or treatment of a disease, symptom, or condition induced by insufficient expression of a target gene in a subject.

[0135] In other embodiments, the kit may include instructions for use of the kit in any form or from a website or other source. For example, instructions may include instructions for the use, modification, mixing, dilution, preservation, assembly, storage, packaging, and / or preparation of the components and / or other components associated with the kit. In some cases, instructions may also include instructions for the delivery of the components (e.g., transport or storage at room temperature, sub-zero temperatures, cryogenic conditions, etc.). Instructions may be provided in any form available to the user of the kit (such as written or oral (e.g., telephone), digital, optical, visual (e.g., videotape, DVD, etc.), and / or electronic communication (including the Internet or network-based communication)).

[0136] Use of Oligonucleotide Agents or Conjugates

[0137] In another aspect, this document provides a method for activating or upregulating a target gene in a cell or subject, comprising administering to the cell or subject the double-stranded oligonucleotide agent or conjugate described herein.

[0138] In another aspect, this document also provides a method for mitigating off-target effects caused by a double-stranded oligonucleotide agent or conjugate capable of activating the expression of a target gene in a cell or subject, comprising administering to the cell or subject the double-stranded oligonucleotide agent or conjugate described herein.

[0139] In some embodiments, when, for example, the double-stranded oligonucleotide agent or conjugate described herein is administered to a cell or subject, the expression of the target gene is activated / upregulated by at least 10% compared to the expression of an unregulated gene, for example, at least20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 300%, at least 500%, at least 800%, at least 1000%, at least 2000%, or at least 5000%.

[0140] In some embodiments, when, for example, the double-stranded oligonucleotide agent or combination thereof described herein is administered to cells or a subject, the expression of the target gene is at least the same as or at least 10% higher than the expression regulated by the same but non-GNA double-stranded oligonucleotide agent, for example, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 300%, or at least 500%.

[0141] In some embodiments, when the double-stranded oligonucleotide agent or conjugate described herein is administered, for example to cells or subjects, at least the same or at least 10% of the off-target effects are mitigated compared to the same but non-GNA double-stranded oligonucleotide agent, for example at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100%.

[0142] In some embodiments, the expression of a target gene is activated / upregulated by administering to cells at a concentration of at least 0.01 nM, for example, at least 0.02 nM, at least 0.05 nM, at least 0.08 nM, at least 0.1 nM, at least 0.2 nM, at least 0.3 nM, at least 0.4 nM, at least 0.5 nM, at least 0.6 nM, at least 0.8 nM, at least 1 nM, at least 2 nM, at least 3 nM, at least 4 nM, at least 5 nM, at least 6 nM, at least 7 nM, at least 8 nM, at least 9 nM, at least 10 nM, at least 25 nM, at least 50 nM, at least 75 nM, at least 100 nM, at least 150 nM, or at least 200 nM of a double-stranded oligonucleotide agent or conjugate described herein.

[0143] In some embodiments, activation of the target gene is beneficial for the prevention and / or treatment of diseases, symptoms, or conditions associated with or induced by insufficient expression of the target gene.

[0144] In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a human cell. The cells described herein may be in vitro or ex vivo, such as cell lines or cell strains, or may be present in mammals, such as humans.

[0145] In some embodiments, the subject is a mammal. In some embodiments, the subject is a human. In some embodiments, the subject is a person who has or is at risk of having a disease, symptom, or condition associated with insufficient expression of the gene (i.e., the target gene).

[0146] In some embodiments, the double-stranded oligonucleotide agent or conjugate is administered to a subject or transfected into cells at a concentration of at least 0.01 nM, for example, at least 0.02 nM, at least 0.05 nM, at least 0.08 nM, at least 0.1 nM, at least 0.2 nM, at least 0.3 nM, at least 0.4 nM, at least 0.5 nM, at least 0.6 nM, at least 0.8 nM, at least 1 nM, at least 2 nM, at least 3 nM, at least 4 nM, at least 5 nM, at least 6 nM, at least 7 nM, at least 8 nM, at least 9 nM, at least 10 nM, at least 25 nM, at least 50 nM, at least 75 nM, at least 100 nM, at least 150 nM, or at least 200 nM.

[0147] In some embodiments, the double-stranded oligonucleotide agent or conjugate is administered to the subject or transfected into cells at concentrations ranging from 0.01 nM to 500 nM, for example, within the range obtained by combining any two of the following endpoints: 0.01 nM, 0.02 nM, 0.05 nM, 0.08 nM, 0.1 nM, 0.2 nM, 0.3 nM, 0.4 nM, 0.5 nM, 0.6 nM, 0.8 nM, 1 nM, 2 nM, 3 nM, 4 nM, 5 nM, 6 nM, 7 nM, 8 nM, 9 nM, 10 nM, 15 nM, 20 nM, 25 nM, 30 nM, 35 nM, 40 nM, 45 nM, 50 nM, 55 nM. 60nM, 65nM, 70nM, 75nM, 80nM, 85nM, 90nM, 95nM, 100nM, 105nM, 110nM, 115nM, 120nM, 125nM, 130nM, 135nM, 140nM, 145nM, 150nM, 155nM, 160nM, 165nM, 170nM, 175nM, 180nM, 185nM, 190nM, 195nM, 200nM, 220nM, 240nM, 260nM, 280nM, 300nM, 320nM, 340nM, 360nM, 380nM, 400 nM, 420 nM, 440 nM, 460 nM, 480 nM, and 500 nM.

[0148] In some embodiments, administration of the double-stranded oligonucleotide agent or conjugate described herein to a subject activates / upregulates the expression of a target gene and is beneficial for the prevention and / or treatment of the subject's disease, condition, or illness.

[0149] In some embodiments, administration of the double-stranded oligonucleotide agent or conjugate described herein to a subject comprises administration of a composition (e.g., a pharmaceutical composition) including the double-stranded oligonucleotide agent described herein in an amount effective in preventing or treating a disease, condition, or illness.

[0150] In some embodiments, the route of administration is selected from one or more of the following: parenteral infusion, oral administration, intranasal administration, inhalation administration, vaginal administration, and rectal administration. In some embodiments, the route of administration is selected from the following.The specification, pages 20 / 50, 25 CN 121569037 A, comprises the following groups: intrathecal, intramuscular, intravenous, intraarterial, intraperitoneal, intravesical, intraventricular, intravitreal, subcutaneous, and combinations thereof.

[0151] The dosage of the double-stranded oligonucleotide agents or compositions disclosed herein can vary widely and will meet the individual needs of each case.

[0152] A single dose of the double-stranded oligonucleotide agent may range from 0.01 mg / kg to 1000 mg / kg of the subject's body weight, for example, about 0.01 mg / kg, 0.02 mg / kg, 0.05 mg / kg, 0.1 mg / kg, 0.2 mg / kg, 0.5 mg / kg, 1 mg / kg, 2 mg / kg, 2.5 mg / kg, 5 mg / kg, 7.5 mg / kg, 10 mg / kg, 12.5 mg / kg, 15 mg / kg, 17.5 mg / kg, 20 mg / kg, 25 mg / kg, 30 mg / kg, 40 mg / kg, 50 mg / kg, 75 mg / kg, 100 mg / kg, 120 mg / kg, 150 mg / kg, 200 mg / kg, 250 mg / kg, A single dose of 300 mg / kg, 400 mg / kg, 500 mg / kg, 750 mg / kg, or 1000 mg / kg of subject weight. Doses described herein may include one or more of any of the double-stranded oligonucleotides or conjugates described herein.

[0153] In some embodiments, the dose will be adjusted based on the subject's age, subject weight, and / or other factors that may require adjustment of injection parameters.

[0154] Examples of other compositions or components associated with the double-stranded oligonucleotides, conjugates, compositions, kits, and methods described herein include, but are not limited to: diluents, salts, buffers, chelating agents, preservatives, desiccants, antimicrobial agents, needles, syringes, packaging materials, tubes, bottles, flasks, beakers, etc., for example, components used for use, modification, assembly, storage, packaging, preparation, mixing, dilution, and / or preservation for a specific purpose. In embodiments using any component in liquid form, the liquid form may be concentrated or ready for direct use.

[0155] In some embodiments, the pharmaceutical composition includes a delivery system. Examples of delivery systems include, but are not limited to, liposomes and emulsions. Certain delivery systems can be used to prepare certain pharmaceutical compositions, including those comprising hydrophobic compounds. In some embodiments, certain organic solvents, such as dimethyl sulfoxide, are used.

[0156] In some embodiments, the pharmaceutical composition comprises one or more tissue-specific delivery molecules designed to deliver one or more agents of this disclosure to a specific tissue or cell type. For example, in some embodiments, the pharmaceutical composition comprises liposomes coated with tissue-specific antibodies.

[0157] The formulations, pharmaceutical compositions, or drugs disclosed herein are formulated, administered, and applied in accordance with "good medical practice." Factors considered in this context include the specific condition being treated, the specific mammal being treated, the individual subject's clinical condition, the cause of the condition, the site of delivery of the drug, the method of administration, the timing of administration, and other factors known to the physician.

[0158] Typical formulations of the double-stranded oligonucleotide agents or conjugates of this disclosure are prepared by mixing the reagents described herein with a carrier or excipient. Suitable carriers and excipients are well known to those skilled in the art and are described in detail, for example, Ansel HC et al., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems (2004), Lippincott, Williams & Wilkins, Philadelphia; Gennaro A. R. et al., Remington: The Science and Practice of Pharmacy (2000), Lippincott, Williams & Wilkins, Philadelphia; and Rowe R. C., Handbook of Pharmaceutical Excipients (2005), Pharmaceutical Press, Chicago. The formulation may also include one or more buffers, stabilizers, surfactants, wetting agents, lubricants, emulsifiers, suspending agents, preservatives, antioxidants, opacifiers, flow aids, processing aids, colorants, sweeteners, flavorings, diluents, and other known additives to provide an elegant presentation of the medicament (i.e., the double-stranded oligonucleotide agent or pharmaceutical composition thereof described herein) or to facilitate the preparation of the pharmaceutical product (i.e., the drug).

[0159] In another aspect, the use of double-stranded oligonucleotide agents or conjugates in the preparation of products is provided herein.

[0160] The product may be a composition, a drug, or a kit. Specification 21 / 50 pages 26 CN 121569037 A

[0161] In some embodiments, the product is used to activate or upregulate the expression of a target gene in cells or a subject. In some embodiments, the product is used to increase the level of mRNA or protein encoded by the target gene in cells or a subject. In some embodiments, the product is used to prevent or treat a disease, symptom, or condition in a subject that is associated with or induced by insufficient expression of the target gene.

[0162] In some embodiments, the product includes a double-stranded oligonucleotide agent or conjugate that effectively activates or upregulates the expression of a target gene in cells or a subject. In some embodiments, the product includes an amount that effectively increases the expression of a target gene in cells or a subject.A double-stranded oligonucleotide agent or conjugate at the level of gene-encoded mRNA or protein. In some embodiments, the product comprises a double-stranded oligonucleotide agent or conjugate that effectively prevents or treats a disease, symptom, or condition in a subject that is associated with or induced by insufficient expression of the target gene.

[0163] Specific Embodiments

[0164] Embodiment 1. A double-stranded oligonucleotide agent capable of activating or upregulating the expression of a target gene, comprising a sense strand and an antisense strand, each strand being 15 to 35 nucleotides in length, wherein the sense strand and the antisense strand form a complementary double-stranded structure of at least 8 base pairs, wherein the double-stranded oligonucleotide agent comprises one or more glycerol nucleic acid (GNA) modified nucleotides located in one or both of the sense strand and the antisense strand.

[0165] Embodiment 2. The double-stranded oligonucleotide agent according to Embodiment 1, wherein one or both of the sense strand and the antisense strand are guide strands mediating RNA activation.

[0166] Embodiment 3. The double-stranded oligonucleotide agent according to Embodiment 2, wherein the guide strand has a seed region of 2 to 10 nucleotides in length located at or near the 5' end of the guide strand.

[0167] Embodiment 4. The double-stranded oligonucleotide agent according to Embodiment 3, wherein the seed region begins no more than 3 nucleotides from the 5' end of the guide strand.

[0168] Embodiment 5. The double-stranded oligonucleotide agent according to Embodiment 3, wherein the seed region comprises nucleotides at positions 2 to 10, or 2 to 9, or 2 to 8, or 2 to 7, or 2 to 6, or 3 to 6, or 2 to 5, or 3 to 6, or 3 to 5, or 4 to 6, starting from the 5' end of the guide strand.

[0169] Embodiment 6. The double-stranded oligonucleotide agent according to Embodiment 1, wherein the one or more GNA-modified nucleotides comprise at least one selected from the group consisting of: GNA-modified adenine (GNA-A), GNA-modified thymine (GNA-T), GNA-modified cytosine (GNA-C), GNA-modified guanine (GNA-G), and GNA-modified uracil (GNA-U).

[0170] Embodiment 7. The double-stranded oligonucleotide agent according to Embodiment 1, wherein the double-stranded oligonucleotide agent comprises 1 to 50 GNA-modified nucleotides.

[0171] Embodiment 8. The double-stranded oligonucleotide agent according to Embodiment 2, the double-stranded oligonucleotide agent comprising one or more GNA-modified nucleotides located at positions 1 and / or 2 and / or 3 and / or 4 and / or 5 and / or 6 and / or 7 and / or 8 and / or 9 and / or 10 and / or 11 and / or 12 and / or 13 and / or 14 and / or 15 and / or 16 and / or 17 and / or 18 and / or 19 and / or 20 and / or 21 and / or 22 and / or 23 and / or 24 and / or 25, starting from the 5' end of the leader strand.

[0172] Embodiment 9. The double-stranded oligonucleotide agent according to Embodiment 3, wherein the one or more GNA-modified nucleotides are located within and / or outside the seed region of the leader strand.

[0173] Embodiment 10. The double-stranded oligonucleotide agent according to Embodiment 3, wherein the one or more GNA-modified nucleotides are located in the transit strand.

[0174] Embodiment 11. The double-stranded oligonucleotide agent according to Embodiment 1, wherein the sense strand comprises the nucleotide sequence shown in any one of SEQ ID NO: 1, 3, 12, 14, 15, 17, 18, 20, 21, 23, 24, 27, 29, 52, 54, and the antisense strand comprises the nucleotide sequence shown in any one of SEQ ID NO: 2, 4, 5, 6, 7, 8, 9, 10, 11, 13, 16, 19, 22, 25, 26, 28, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 53, 55.

[0175] Embodiment 12. The double-stranded oligonucleotide agent according to Embodiment 1, wherein the leader chain is synthesized by using a GNA-modified nucleotide monomer of formula (1) to include the one or more GNA-modified nucleotides: (1) wherein the base is selected from the group consisting of: adenine nucleobases, thymine nucleobases, cytosine nucleobases, guanine nucleobases, uracil nucleobases and analogs thereof.

[0176] Embodiment 13. The double-stranded oligonucleotide agent according to Embodiment 12, wherein the base in formula (1) is selected from the following structures: , , , and .

[0177] Embodiment 14. The double-stranded oligonucleotide agent according to Embodiment 1, wherein the sense strand or the antisense strand has at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 nucleotides complementary to an isometric segment of the coding strand of the target gene.

[0178] Embodiment 15. The double-stranded oligonucleotide agent according to Embodiment 1, wherein the complementary double-stranded structure of at least 8 base pairs has no more than 5 mismatched nucleotides between the sense strand and the antisense strand, i.e., 5, 4, 3, 2, 1, or 0 mismatched nucleotides.

[0179] Embodiment 16. The double-stranded oligonucleotide agent according to Embodiment 15, wherein the mismatched nucleotides are located at...The antisense strand is located inside or near the 3' or 5' end.

[0180] Embodiment 17. The double-stranded oligonucleotide agent according to Embodiment 15, wherein the mismatched nucleotide is located at the GNA position in the sense strand and / or the antisense strand.

[0181] Embodiment 18. The double-stranded oligonucleotide agent according to Embodiment 1, wherein the double-stranded oligonucleotide agent is a small activating RNA (saRNA) that upregulates the expression of the target gene by at least 10%.

[0182] Embodiment 19. The double-stranded oligonucleotide agent according to Embodiment 1, wherein the double-stranded oligonucleotide agent, as described in the specification 24 / 50 pages 29 CN 121569037 A, comprises at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or about 100% of non-GNA nucleotides, including modifications selected from the group consisting of: 2'-fluoro(2'-F), 2'-O-methyl(2'-OMe), 2'-O-methoxyethyl(2'-MOE), 5'-(E)-vinylphosphonate, thiophosphate backbone modifications, and combinations thereof.

[0183] Embodiment 20. A conjugating agent comprising the double-stranded oligonucleotide agent according to any one of Embodiments 1-19 and at least one conjugating moiety, wherein the at least one conjugating moiety is selected from: lipids, fatty acids, fluorophores, ligands, sugars, peptides, and antibodies.

[0184] Embodiment 21. The conjugating agent according to Embodiment 20, wherein the conjugating portion is selected from accessory oligonucleotides (ACO), lipids / fatty acids, and GalNAc clusters.

[0185] Embodiment 22. The conjugating agent according to Embodiment 21, wherein the conjugating portion is selected from:

[0186] (tC2x6), and

[0187] (C5x5), wherein representing a carrier material.

[0188] Embodiment 23. A cell comprising a double-stranded oligonucleotide agent according to Embodiment 1 or a conjugating agent according to Embodiment 20.

[0189] Embodiment 24. A pharmaceutical composition comprising a double-stranded oligonucleotide agent according to Embodiment 1 or a conjugating agent according to Embodiment 20, and at least one pharmaceutically acceptable carrier.

[0190] Embodiment 25. A kit for activating or upregulating a target gene in a cell or subject, comprising a double-stranded oligonucleotide agent according to any one of Embodiments 1-19, or a conjugating agent according to any one of Embodiments 20-22, or a pharmaceutical composition according to Embodiment 24.

[0191] Embodiment 26. A method for activating or upregulating a target gene in a cell or subject, comprising: [Detailed specification 25 / 50 pages 30 CN 121569037 A]The administration of a double-stranded oligonucleotide agent according to any one of embodiments 1-19 or a conjugate agent according to any one of embodiments 20-22 to a cell or subject.

[0192] Embodiment 27. A method for mitigating off-target effects caused by a double-stranded oligonucleotide agent capable of activating or upregulating the expression of a target gene in a cell or subject, comprising administering to the cell or subject a double-stranded oligonucleotide agent according to any one of embodiments 1-19 or a conjugate agent according to any one of embodiments 20-22.

[0193] Embodiment 28. Use of a double-stranded oligonucleotide agent according to any one of embodiments 1-19 or a conjugate agent according to any one of embodiments 20-22 in the preparation of a product for activating or upregulating a target gene in a cell or subject.

[0194] Examples

[0195] The present application will now be further described with reference to specific embodiments and accompanying drawings. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the present application. In the following embodiments, research methods not specifically specified are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer.

[0196] The following embodiments are presented to provide a complete disclosure and description of how to prepare and use the invention to those skilled in the art, and are not intended to limit the scope of what the inventors consider to be their invention, nor to represent that the following experiments are all or only the experiments performed. Efforts have been made to ensure the accuracy of the figures used (e.g., quantities, temperatures, etc.), but some experimental errors and biases should be taken into account. Unless otherwise stated, parts are parts by weight, molecular weight is weight-average molecular weight, temperature is in degrees Celsius, and pressure is atm or near atm. Standard abbreviations may be used, such as bp for base pair, kb for kilobase, nM for nanomolar, s or sec for second, min for minute, h or hr for hour, aa for amino acid, nt for nucleotide, im. for intramuscular, ip for intraperitoneal, s.c. for subcutaneous, ivt or IVT for intravenous, iv or IV for tail vein, icv or ICV for intraventricular, etc.

[0197] Unless otherwise stated, all raw materials, reagents and solvents used below are commercially available and used as received. Purification of the reaction products was performed by column chromatography comprising silica gel (200-300 mesh) and hexane / ethyl acetate, DCM / MeOH eluent. Thin-layer chromatography (TLC) was performed using pre-coated silica gel GF plates and KMnO4 staining agent.Color development. ¹H NMR spectra were recorded using CDCl₃ with TMS at 400 or 500 MHz (Varian). High-resolution mass spectrometry (HRMS) was recorded by ESI or matrix-assisted laser desorption / ionization (MALDI) on LC / MS (Agilent Technologies 1260 Infinity II / 6120 Quadrupole) and time-of-flight mass spectrometer.

[0198] Example 1. Preparation of the compound GNA-U of this disclosure

[0199] In this example, compound GNA-U was prepared by using the following method. Instructions for Use, Page 26 / 50, 31 CN 121569037 A

[0200]

[0201] (1) Preparation of Compound 2

[0202] DMTrCl (64.5 g, 190 mmol) was added to a CH2Cl2 (340 mL) solution of Compound 1 (10 mL, 151 mmol, 1.0 eq) and Et3N (42 mL, 302 mmol, 2.0 eq). After 12 hours, the reaction mixture was poured into a saturated NaHCO3 aqueous solution (500 mL). The organic layer was extracted with ethyl acetate, washed with brine, dried over anhydrous Na2SO4 and concentrated under reduced pressure. The residue was purified by rapid chromatography (silica gel, gradient eluent: 1%–10% ethyl acetate / hexane, 1% Et3N) to give Compound 2 (52.2 g, 90% yield) as a yellow oil. The product was characterized by 1H NMR. 1H NMR (400 MHz, CDCl3) δ 7.48 – 7.42 (m, 2H), 7.39 – 7.23 (m, 7H), 6.85 – 6.80 (m, 4H), 3.78 (s, 6H), 3.36 – 3.27 (m, 1H), 3.17 – 3.08 (m, 2H), 2.81 – 2.59 (m, 2H).

[0203] (2) Preparation of compound 3

[0204] NaH (148 mg, 3.7 mmol, 60% mineral oil solution, 0.2 eq) was added to 45 mL of anhydrous DMF solution of compound 11 (2.5 g, 22.3 mmol, 1.2 eq), and the mixture was stirred for 1 hour under nitrogen protection. 5.0 mL of anhydrous DMF solution of compound 2 (7.0 g, 18.6 mmol, 1.0 eq) was added to the above solution, and the reaction mixture was heated to 110 °C overnight. The solution was cooled in an ice bath and quenched with saturated ammonium chloride. The reaction mixture was extracted twice with ethyl acetate, and the organic phase was washed three times with saturated LiCl solution and once with brine. The mixture was then dried over anhydrous Na₂SO₄ and concentrated under reduced pressure. The resulting residue was purified by rapid chromatography.The product was purified by elution with silica gel (gradient eluent: 50%–100% ethyl acetate / hexane, 1% Et3N) to give compound 3 (6.14 g, 67% yield). The product was characterized by mass spectrometry and 1H NMR. Calculated MW: 488.19; Measured MW: 487.63 [M-H]+. 1H NMR (400 MHz, CDCl3) δ 7.42 – 7.38 (m, 2H), 7.33 – 7.24 (m, 7H), 7.16 (d, J = 7.9 Hz, 1H), 6.87 – 6.79 (m, 4H), 5.55 (d, J = 7.9 Hz, 1H), 4.08 – 4.02 (m, 2H), 3.78 (s, 6H), 3.68 – 3.59 (m, 1H), 3.23 – 3.14 (m, 2H).

[0205] (3) Preparation of compound GNA-U

[0206] Under a nitrogen atmosphere, at room temperature, compound 3 (2.0 g, 4.1 mmol, 1 2-Cyanoethyl N,N-diisopropylphosphonamide (2.7 mL, 12.3 mmol, 3.0 eq) was added to an anhydrous DCM (40 mL) solution of 2.2 mL (12.3 mmol, 3.0 eq) and DIPEA (2.2 mL, 12.3 mmol, 3.0 eq). The reaction mixture was stirred for 1.5 h. The mixture was extracted twice with DCM, washed with saturated NaHCO3 and brine, and dried over anhydrous Na2SO4. The organic layer was concentrated under reduced pressure, and the residue was purified by rapid chromatography (silica gel, gradient eluent: 20%–50% ethyl acetate / hexane, 1% Et3N) to give compound GNA-U (3.4 g, 72% yield). The product was characterized by 1H NMR. 1H NMR (400 MHz, CDCl3) δ 7.48 – 7.41 (m, 2H), 7.35 – 7.21 (m, 7H), 7.17 (dd, J = 17.2 Hz, 1H), 6.87 – 6.78 (m, 4H), 5.54 (dd, J = 13.1 Hz, 1H), 4.27 – 4.18 (m, 2H), 4.17 – 4.08 (m, 2H), 3.79 (d, J = 3.4 Hz, 6H), 3.74 – 3.68 (m, 1H), 3.62 – 3.45 (m, 4H) , 3.34 – 3.12 (m, 2H), 1.18 – 1.06 (m, 12H).

[0207] Example 2. Preparation of compound GNA-T of this disclosure

[0208] In this example, compound GNA-T was prepared by the following method.

[0209] Specification 28 / 50 pages 33 CN 121569037 A

[0210] (1) Preparation of compound 4

[0211] NaH (256 mg, 6.4 mmol, 60% mineral oil solution, 0.2 eq) was added to 122.0 mL of anhydrous DMF solution of compound 12 (4.8 g, 38.3 mmol, 1.2 eq), and the mixture was stirred for 1 hour under nitrogen protection. Anhydrous DMF solution of compound 2 (12.0 g, 31.9 mmol, 1.0 eq) was added to the above solution, and the reactants were heated to 110 °C overnight. The solution was cooled in an ice bath and quenched with saturated ammonium chloride. The reaction was extracted twice with ethyl acetate, the organic phase was washed three times with saturated LiCl solution, and washed once with brine. The product was then dried over anhydrous Na₂SO₄ and concentrated under reduced pressure. The residue was purified by rapid chromatography (silica gel, gradient eluent: 50%–100% ethyl acetate / hexane, 1% Et₃N) to give compound 4 (8.5 g, 53% yield). The product was characterized by mass spectrometry and 1H NMR. Calculated MW: 502.21; Measured MW: 500.74 [M-H]⁺. 1H NMR (400 MHz, CDCl3) δ 7.43 – 7.38 (m, 2H), 7.33 – 7.26 (m, 7H), 7.04 (s, 1H), 6.87 – 6.80 (m, 4H), 4.10 – 3.98 (m, 2H), 3.79 (s, 6H), 3.69 – 3.62 (m, 1H), 3.18 (d, J = 5.4 Hz, 2H), 1.84 (s, 3H).

[0212] (2) Preparation of compound GNA-T

[0213] Under a nitrogen atmosphere, at room temperature, compound 4 (8.5 g, 16.9 mmol, 1 2-Cyanoethyl N,N-diisopropylphosphonamide (11.3 mL, 50.7 mmol, 3.0 eq) was added to anhydrous DCM (169.0 mL) solution of DIPEA (8.4 mL, 50.7 mmol, 3.0 eq) and N,N-diisopropylphosphonamide (11.3 mL, 50.7 mmol, 3.0 eq). The reaction mixture was stirred for 1.5 hours. The mixture was extracted twice with DCM, washed with saturated NaHCO3 and brine, and dried over anhydrous Na2SO4. The organic layer was concentrated under reduced pressure, and the residue was subjected to rapid chromatography (silica gel, gradient eluent: 20%–50% ethyl acetate / hexane, 1%).The product was purified by Et3N to give compound GNA-T (10.0 g, 84% yield). The product was characterized by mass spectrometry and 1H NMR. Calculated MW: 702.32; Measured MW: 617.66 [M-diisopropyl-H]+. 1H NMR (400 MHz, CDCl3) δ 7.48 – 7.41 (m, 2H), 7.36 – 7.26 (m, 7H), 29 / 50 pages, 34 CN 121569037 A 7.05 (s, 1H), 6.87 – 6.79 (m, 4H), 4.26 – 4.17 (m, 2H), 4.16 – 4.01 (m, 2H), 3.80 (d, 6H), 3.74 – 3.68 (m, 1H), 3.67 – 3.49 (m, 4H), 3.29 – 3.15 (m, 2H), 1.83 (s, 3H), 1.19 – 1.04 (m, 12H).

[0214] Example 3. Preparation of compound GNA-C of the present disclosure

[0215] In this example, compound GNA-C was prepared by the following method.

[0216]

[0217] (1) Preparation of compound 5

[0218] NaH (136 mg, 3.7 mmol, 60% mineral oil solution, 0.2 eq) was added to 55.0 mL of anhydrous DMF solution of compound 13 (2.68 g, 17.5 mmol, 1.1 eq), and the mixture was stirred for 1 hour under nitrogen protection. Anhydrous DMF solution of compound 2 (6.0 g, 15.9 mmol, 1.0 eq) was added to the above solution, and the reactants were heated to 110 °C overnight. The solution was cooled in an ice bath and quenched with saturated ammonium chloride. The reaction was extracted twice with ethyl acetate, and the organic phase was washed three times with saturated LiCl solution and once with brine. The mixture was then dried over anhydrous Na₂SO₄ and concentrated under reduced pressure. The residue was purified by rapid chromatography (silica gel, gradient eluent: 50%–100% ethyl acetate / hexane, 1% Et₃N) to give compound 5 (2.5 g, 30% yield). The product was characterized by ¹H NMR. 1H NMR (400 MHz, CDCl3) δ 9.04 (d, 1H) , 7.57 – 7.50 (m, 1H) , 7.45 – 7.37 (m, 2H) , 7.32 – 7.26 (m, 7H) , 6.86 – 6.79 (m, 4H) , 4.35 – 4.27 (m, 1H) , 3.86 – 3.79 (m, 2H) ,3.78 (s, 6H), 3.28 – 3.21 (m, 1H), 3.12 (page 35 of the specification 30 / 50 CN 121569037 A – 3.06 (m, 1H), 2.21 (s, 3H).

[0219] (2) Preparation of compound GNA-C

[0220] Under a nitrogen atmosphere, 2-cyanoethyl N,N-diisopropylphosphoramide (1.8 mL, 8.03 mmol, 2.5 eq) was added to an anhydrous DCM (38 mL) solution of compound 5 (1.7 g, 3.21 mmol, 1.0 eq) and DIPEA (1.4 mL, 8.03 mmol, 2.5 eq) at room temperature. The reaction mixture was stirred for 1.0 h. The mixture was extracted twice with DCM, washed with saturated NaHCO3 and brine, and dried with anhydrous Na2SO4. The organic layer was concentrated under reduced pressure, and the resulting residue was purified by rapid chromatography (silica gel, gradient eluent: 20%–50% ethyl acetate / hexane, 1% Et3N) to give compound GNA-C (2.2 g, 94% yield). The product was characterized by 1H NMR. 1H NMR (400 MHz, CDCl3) δ 9.64 (d, J = 65.8 Hz, 1H) , 7.55 (dd, J = 9.2, 7.2 Hz, 1H) , 7.48 – 7.43 (m, 2H) , 7.37 – 7.26 (m, 7H) , 6.86 – 6.78 (m, 4H) , 4.42 – 4.26 (m, 2H) , 4.23 – 4.09 (m, 1H) , 3.78 (s, 6H) , 3.68 – 3.56 (m, 2H) , 3.56 – 3.74 (m, 2H) , 3.28 – 3.08 (m, 2H) , 2.73 – 2.50 (m, 2H), 2.24 (s, 3H), 1.18 – 1.07 (m, 12H).

[0221] Example 4. Preparation of compound GNA-A of the present disclosure

[0222] In this example, compound GNA-A was prepared by the following method.

[0223]

[0224] (1) Preparation of compound 6, specification 31 / 50 pages 36 CN 121569037 A

[0225] NaH (140 mg, 3.51 mmol, 60% mineral oil solution, 0.22 eq) was added to an anhydrous DMF solution of compound 14 (2.28 g, 16.9 mmol, 1.1 eq) in 65.0 mL, and the mixture was stirred for 2 hours under nitrogen protection. 5.0 mL of compound 2 (6.0An anhydrous DMF solution (15.9 g, 1.0 eq) was added to the above solution, and the reactants were heated to 105 °C overnight. The solution was cooled in an ice bath and quenched with saturated ammonium chloride. The reaction mixture was extracted twice with ethyl acetate, and the organic phase was washed three times with saturated LiCl solution and once with brine. The mixture was then dried over anhydrous Na₂SO₄ and concentrated under reduced pressure. The residue was purified by rapid chromatography (silica gel, gradient eluent: 1%–5% MeOH / DCM, 1% Et₃N) to give compound 6 (5.4 g, 66% yield). The product was characterized by mass spectrometry and 1H NMR. Calculated MW: 511.58; Measured MW: 512.37 [M+H]⁺. 1H NMR (400 MHz, CDCl3) δ 8.26 (s, 1H), 7.73 (s, 1H), 7.45 – 7.39 (m, 2H), 7.34 – 7.20 (m, 7H), 6.87 – 6.80 (m, 4H), 5.99 (s, 2H), 4.46 – 4.38 (m, 1H), 4.33 – 4.17 (m, 2H), 3.80 (s, 6H), 3.32 – 3.24 (m, 1H), 3.12 – 3.04 (m, 1H).

[0226] (2) Preparation of compound 7

[0227] To 44.0 mL of anhydrous DMF solution of compound 6 (4.4 g, 8.6 mmol, 1.0 eq), dimethylformamide dimethyl acetal (4.1 mL, 30.1 mmol, 3.5 eq) was added, and the mixture was heated to 60 °C for 1 h. The solution was cooled in an ice bath and extracted twice with ethyl acetate. The organic phase was washed three times with saturated LiCl solution and once with brine. The solution was then dried over anhydrous Na₂SO₄ and concentrated under reduced pressure. The residue was purified by rapid chromatography (silica gel, gradient eluent: 1%–5% MeOH / DCM, 1% Et₃N) to give compound 7 (4.0 g, 82% yield). The product was characterized by mass spectrometry and 1H NMR. Calculated MW: 566.66; Measured MW: 567.62 [M+H]⁺. 1H NMR (400 MHz, CDCl3) δ 8.91 (s, 1H) , 8.44 (s, 1H) , 7.83 (s, 1H) , 7.44 – 7.37 (m, 2H) , 7.31 – 7.22 (m, 7H) , 6.84 – 6.77 (m, 4H) , 4.48 – 4.40 (m, 1H) , 4.32 – 4.18 (m, 2H) , 3.78 (s, 6H) ,3.29 – 3.24 (m, 1H), 3.22 (d, J = 14.0 Hz, 6H), 3.11 – 3.05 (m, 1H).

[0228] (3) Preparation instructions for compound GNA-A, pages 32 / 50, CN 121569037 A

[0229] Under a nitrogen atmosphere, at room temperature, 2-cyanoethyl N,N-diisopropylphosphoramide (1.97 mL, 8.82 mmol, 2.5 eq) was added to an anhydrous DCM (40 mL) solution of compound 7 (2.0 g, 3.53 mmol, 1.0 eq) and DIPEA (1.54 mL, 8.82 mmol, 2.5 eq). The reaction mixture was stirred for 0.5 hours. The mixture was extracted twice with DCM, washed with saturated NaHCO3 and brine, and dried over anhydrous Na2SO4. The organic layer was concentrated under reduced pressure, and the residue was purified by rapid chromatography (silica gel, gradient eluent: 1%–5% MeOH / DCM, 1% Et3N) to give compound GNA-A (2.4 g, 89% yield). The product was characterized by 1H NMR. 1H NMR (400 MHz, CDCl3) δ 8.95 (s, 1H), 8.52 (s, 1H), 7.90 (s, 1H), 7.48 – 7.42 (m, 2H), 7.35 – 7.25 (m, 7H), 6.87 – 6.76 (m, 4H) , 4.57 – 4.47 (m, 1H) , 4.46 – 4.29 (m, 2H) , 4.21 – 4.12 (m, 2H) , 3.79 (s, 6H) , 3.54 – 3.47 (m, 4H) , 3.34 – 3.26 (m, 1H) , 3.23 (d , J = 18.8 Hz, 6H), 3.19 – 3.12 (m, 1H), 1.13 – 0.99 (m, 12H).

[0230] Example 5. Preparation of compound GNA-G of the present disclosure

[0231] In this example, compound GNA-G was prepared by the following method.

[0232]

[0233] (1) Preparation of compound 8, specification 33 / 50 pages 38 CN 121569037 A

[0234] NaH (140 mg, 3.50 mmol, 60% mineral oil solution, 0.22 eq) was added to an anhydrous DMF solution of 6-(benzyloxy)-9H-purine-2-amine compound 15 (4.20 g, 17.5 mmol, 1.05 eq) in 55.0 mL, and the mixture was stirred under nitrogen protection.Stir for 1 hour. Add 5.0 mL of anhydrous DMF solution of compound 2 (6.0 g, 15.9 mmol, 1.0 eq) to the above solution, and heat the reaction mixture to 90 °C overnight. Cool the solution in an ice bath and quench with saturated ammonium chloride. Extract the reaction mixture twice with ethyl acetate, wash the organic phase three times with saturated LiCl solution, and wash once with brine. Then dry with anhydrous Na₂SO₄ and concentrate under reduced pressure. Purify the residue by rapid chromatography (silica gel, gradient eluent: 1%–5% MeOH / DCM, 1% Et₃N) to give compound 8 (5.0 g, 51% yield). Characterize the product by mass spectrometry and 1H NMR. Calculated MW: 617.26; Measured MW: 618.75 [M+H]⁺. 1H NMR (400 MHz, CDCl3) δ 7.52 (s, 1H), 7.52 – 7.47 (m, 2H), 7.42 – 7.37 (m, 2H), 7.34 – 7.17 (m, 10H), 6.84 – 6.78 (m, 4H), 5.52 (s, 2H), 4.32 – 4.23 (m, 1H), 4.20 – 4.09 (m, 2H), 3.77 (s, 6H), 3.25 – 3.17 (m, 1H), 3.06 – 2.97 (m, 1H).

[0235] (2) Preparation of compound 9

[0236] Compound 8 (4.80 g, 7.80 mmol) and Pd / C (2.40 g, 10%) were suspended in EtOAc (192.0 mL). The solution was purged with nitrogen and then hydrogen, and stirred under a hydrogen atmosphere. After 3 h, TLC showed that the reaction was complete. The mixture was filtered through diatomaceous earth and washed with 5% MeOH / DCM to give crude compound 9. The product was characterized by mass spectrometry. Calculated MW: 527.22; Measured MW: 528.60 [M+H]+.

[0237] (3) Preparation of Compound 10

[0238] Dimethylformamide (3.4 mL, 25.1 mmol, 3.5 eq) was added to anhydrous DMF solution of compound 9 (3.8 g, 7.2 mmol, 1.0 eq) in 30.0 mL of dimethylformamide (3.4 mL, 25.1 mmol, 3.5 eq) and the mixture was heated to 60 °C for 1 hour. The solution was cooled in an ice bath and extracted twice with ethyl acetate. The organic phase was washed three times with saturated LiCl solution and once with brine. Then it was dried with anhydrous Na2SO4 and concentrated under reduced pressure. The residue was purified by rapid chromatography (silica gel, gradient eluent: 1%–5% MeOH / DCM, 1% Et3N) to give compound 7 (2.8 g, 7.2 mmol, 1.0 eq).g, 67% yield). The product was characterized by mass spectrometry. Calculated MW: 582.26; Measured MW: 583.45 [M+H]+.

[0239] (4) Preparation of compound GNA-G

[0240] Under a nitrogen atmosphere, 2-cyanoethyl N,N-diisopropylphosphoramide (1.83 mL, 6.44 mmol, 2.5 eq) was added to an anhydrous DCM (15.0 mL) solution of compound 10 (1.5 g, 2.58 mmol, 1.0 eq) and DIPEA (0.97 mL, 6.44 mmol, 2.5 eq) at room temperature. The reaction mixture was stirred for 0.5 hours. The mixture was extracted twice with DCM, washed with saturated NaHCO3 and brine, and dried with anhydrous Na2SO4. The organic layer was concentrated under reduced pressure, and the resulting residue was purified by rapid chromatography (silica gel, gradient eluent: 1%–5% MeOH / DCM, 1% Et3N) to give compound GNA-G (1.2 g, 59% yield). The product was characterized by 1H NMR. 1H NMR (400 MHz, CDCl3) δ 8.51 (s, 1H), 7.62 (s, 1H), 7.46 (d, J = 7.1 Hz, 2H), 7.33 – 7.19 (m, 7H), 6.86 – 6.76 (m, 4H) , 4.56 – 4.43 (m, 1H) , 4.42 – 4.31 (m, 1H) , 4.25 – 4.12 (m, 1H) , 3.78 (s, 6H) , 3.68 – 3.54 (m, 2H) , 3.53 – 3.46 (m, 2H) , 3.37 – 3.19 (m, 2H), 3.17 – 3.08 (m, 1H), 3.07 (s, 3H), 3.05 – 3.01 (m, 1H), 2.81 (s, 3H), 2.56 – 2.40 (m, 2H), 1.13 – 0.97 (m, 12H).

[0241] Example 6. Synthesis and design of GNA-modified oligonucleotides

[0242] The oligonucleotides tested in subsequent examples are shown in Table 1.

[0243] RD-12318 is a sequence designed to target the promoter of the SMN2 gene to activate the transcription of the allele via the RNA activation (RNAa) mechanism. By transfecting cell lines with chemically modified saRNA derived from RD-12318, the expression of SMN2-FL (i.e., full-length SMN2 including exon 7) and SMN2-Δ7 (i.e., SMN2 excluding exon 7) mRNAs can be regulated.The level of SMN protein encoded by SMN2-FL mRNA is increased. The “seed” region (GUUGCUU) is part of the antisense sequence (SEQ ID NO: 2) of RD-12318.

[0244] RD-10994 is synthesized based on RD-12318 with chemical modifications, namely 2'-fluorine, 2'-O-methyl (2'-OMe), 5'-(E)-vinylphosphonate and thiophosphate (PS) backbone modifications. The synthesis of RD-15977, RD-15978, RD-15979, RD-15980, RD-15981, RD-15982 and RD-15983 is the same as that of RD-10994, except that they have glycerol nucleic acid (GNA) modifications at positions 2, 3, 4, 5, 6, 7 and 8, counting from the 5' end of their antisense strand. “nG”, “nU” and “nC” represent GNA-modified nucleotides.

[0245] RD-19588 is a sequence designed to target the promoter of the SMN2 gene to activate the transcription of the allele via the RNAa mechanism. The “seed” region (GCAGGCC) is part of the antisense sequence (SEQ ID NO: 28) of RD-19588. RD-19040 is synthesized based on RD-19588 and has chemical modifications, namely 2'-fluoro, 2'-O-methyl (2'-OMe), 5'-(E)-vinylphosphine ester and thiophosphate (PS) backbone modifications. The synthesis of RD-19650, RD-19651, RD-19652, RD-19653, RD-19654, RD-19655, RD-19657, RD-19658, RD-19659, RD-19660, RD-19661, RD-19662, RD-19663, RD-19664, RD-19665, RD-19666, RD-19667, RD-19668, RD-19669, and RD-19670 is the same as that of RD-10994, except that the positions 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, and 14 are counted starting from the 5' end of their antisense chains. Positions 15, 16, 17, 18, 19, 20, 21, and 22 contain GNA modifications. The synthesis of RD-19672 is the same as RD-10994, except that it has two GNA modifications at positions 8 and 17, counting from the 5' end of its antisense chain. The synthesis of RD-19674 is the same as RD-10994, except that it has a GNA modification at position 9, counting from the 5' end of its antisense chain, and a GNA modification at position 13, counting from the 5' end of its positive chain. The synthesis of RD-19675 is the same as RD-10994, except that...The GNA modification is present at position 10, counting from the 5' end of the antisense strand, and at position 12, counting from the 5' end of the sense strand. “nG”, “nU”, “nA”, and “nC” represent GNA-modified nucleotides.

[0246] Non-GNA-saRNAs (i.e., RD-17229, RD-17235, RD-17238, RD-17241, and RD-17244) were engineered to target the promoter of the SERPING1 gene to activate allele transcription via the RNAa mechanism. Their corresponding GNA-saRNAs (i.e., RD-17074, RD-17082, RD-17086, RD-17096, and RD-17099) were synthesized in the same manner as the non-GNA-saRNAs, except that they have a GNA modification at position 7, counting from the 5' end of either the sense or antisense strand. The "seed" regions of RD-17074, RD-17082, RD-17086, and RD-17096 are located at positions 2 to 8, counting from the 5' end of their sense strand. The "seed" region of RD-17099 is located at positions 2 to 8, counting from the 5' end of its antisense strand. "nG", "nA", and "nC" indicate GNA-modified nucleotides.

[0247] Table 1. Oligonucleotide Sequences and Compositions Specification 36 / 50 Page 41 CN 121569037 A Specification 37 / 50 Page 42 CN 121569037 A Specification 38 / 50 Page 43 CN 121569037 A Specification 39 / 50 Page 44 CN 121569037 A Specification 40 / 50 Page 45 CN 121569037 A

[0248] Note: Uppercase letters indicate RNA; indicate phosphate thioester (PS) backbone modification; f indicates 2'-fluorine; m indicates 2'-O-methyl (2'-OMe); Vp indicates 5'-(E)-vinylphosphonate; me indicates 2'-O-methoxyethyl (2'MOE); meC indicates Specification 41 / 50 Page 46 CN 121569037 A 2'-O-methoxyethyl-5-methylcytosine; meU represents 2'-O-methoxyethyl-5-methyluracil; n represents ethylene glycol nucleic acid; nG, nU, nC and nA represent GNA-modified nucleotides, italics, bold, gray highlighted GUUGCUU and GCAGGCC represent "seed" regions; n / a: not applicable.

[0249] Example 7. In vitro activity of GNA-saRNA in regulating the conversion of SMN2-Δ7 to SMN2-FL mRNA in GM03813 cells

[0250] In order to evaluate the in vitro activity of GNA-saRNA at various nucleotide positions, a specified GNA-saRNA (i.e. RD-RD-15977, RD-15978, RD-15979, RD-15980, RD-15981, RD-15982, and RD-15983 were transfected into GM03813 cells at specified concentrations (i.e., 0.78 nM, 1.56 nM, 3.13 nM, 6.25 nM, 12.5 nM, 25 nM, 50 nM, and 100 nM) for 3 days. Cells were transfected using RD-10994 as a non-GNA control. In each PCR reaction, the mRNA level of SMN2-FL was quantified by RT-qPCR using a gene-specific primer set, and the results are plotted in Figure 1. Table 2 summarizes the maximum power (Emax) and area under the curve (AUC) of SMN2-FL expression. As described in the Materials and Methods section, the area under the curve (AUC) of SERPING1 mRNA expression was calculated using GraphPad Prism software. The results showed that GNA-saRNA enhanced the activity against SMN2-FL mRNA expression compared with RD-10994 without GNA-saRNA.

[0251] Table 2. Emax and AUC of SMN2-FL expression after GNA-saRNA treatment in GM03813 cells

[0252] Note: "SEM" represents the standard error of the mean, n / a: not applicable.

[0253] As shown in Figures 2A to 2H, GNA-saRNA showed higher SMN2-FL induction activity in GM03813 cells, while reducing SMN2-Δ7 mRNA. As shown in Figures 3A to 3H, GNA-saRNA showed higher SMN2-FL induction activity in GM22592 cells, while reducing SMN2-Δ7 mRNA. All GNA-saRNAs increased SMN2-FL mRNA levels, indicating that GNA-saRNAs can enhance the activity of SMN2-FL mRNA expression by regulating the conversion of SMN2-Δ7 to SMN2-FL mRNA.

[0254] Example 8. In vitro activity of GNA-saRNAs in regulating the conversion of SMN2-Δ7 to SMN2-FL mRNA in GM03813 cells

[0255] To confirm the in vitro activity of GNA modifications at various nucleotide positions, designated GNA-saRNAs (i.e., RD-15977, RD-15978, RD-15979, RD-15980, RD-15981, RD-15982, and RD-15983) were transfected into GM03813 cells at 100 nM for 3 days. Cells were transfected with RD-10994 and used as a non-GNA control. In each PCR reaction, the mRNA levels of SMN2-FL and SMN2-Δ7 were quantified by RT-qPCR using gene-specific primer sets. (See Figures 4A and 4B)As shown, compared to RD-10994 without GNA saRNA, all GNA-saRNAs exhibited higher SMN2-FL induction activity at 100 nM, while reducing SMN2-Δ7 mRNA. The expression of SMN2-FL and SMN2-Δ7 mRNA is summarized in Table 3. The results indicate that GNA-saRNA can enhance the activity of SMN2-FL mRNA expression by regulating the conversion of SMN2-Δ7 to SMN2-FL mRNA.

[0256] Table 3. Expression of SMN2-FL and SMN2-Δ7 mRNA in GM03813 cells

[0257] Note: "SEM" represents the standard error of the mean.

[0258] Example 9. GNA-saRNA alleviates the off-target effect of potential off-target gene P2RY2 expression in GM03813 cells.

[0259] To evaluate the off-target mitigation effect of GNA-saRNA, the antisense strand of RD-10994 was used as a "retrieval" sequence to search for potential off-target genes by computer analysis and to predict P2RY2 as a potential off-target gene. Figure 5A shows the "retrieval" sequence (antisense strand) and "seed" region (highlighted in gray) of RD-10994 and its predicted complementary sequence at the target site in a P2RY2 transcript containing two mismatched nucleotides (italicized and bolded nucleotides). The specified GNA-saRNAs (i.e., RD-15977, RD-15978, RD-15981, RD-15982, and RD-15983) were transfected into GM03813 cells at 6.25 nM and 25 nM for 3 days. Cells were transfected with RD-10994 and used as a non-GNA control. In each PCR reaction, the mRNA level of P2RY2 was quantified by RT-qPCR using a gene-specific primer set. As shown in Figure 5B, all GNA-saRNAs reduced off-target effects compared to the non-GNA control RD-10994, except for RD-15981 at 25 nM treatment. The expression of P2RY2 mRNA is summarized in Table 4. The results show that GNA modification at different positions can reduce off-target effects to varying degrees.

[0260] Table 4. Expression of P2RY2 mRNA in GM03813 cells

[0261] Note: " / " indicates not detected. "SEM" indicates the standard error of the mean.

[0262] Example 10. In vitro activity of GNA-saRNA in inducing SMN2-FL and SMN2-Δ7 mRNA expression in GM03813 cells

[0263] In order to evaluate the in vitro activity of GNA modification at various nucleotide positions, the specified GNA-saRNA (i.e., RD-RD-19658, RD-19659, RD-19660, RD-19663, RD-19664, RD-19665, RD-19666, RD-19667, RD-19668, RD-19669, RD-19670, and RD-19672) were transfected into GM03813 cells at 2.5 nM for 3 days. Cells were transfected with RD-19040 as a non-GNA control. Cells were transfected with RD-10004 (ASO-1027) at 25 nM as a positive control. As shown in Figures 6A and 6B, compared to the non-GNA saRNA RD-19040, all GNA-saRNAs showed higher activity in inducing SMN2-FL and SMN2-Δ7 mRNA at 2.5 nM. The expression of SMN2-FL and SMN2-Δ7 mRNA is summarized in Table 5. The results show that GNA modification of SMN2 saRNA can enhance their targeting activity in inducing the expression of SMN2-FL and SMN2-Δ7 mRNA.

[0264] Table 5. Expression of SMN2-FL and SMN2-Δ7 mRNA in GM03813 cells

[0265] Note: “SEM” represents the standard error of the mean.

[0266] Example 11. GNA-saRNA alleviates the off-target effect on the expression of ARPIN, a potential off-target gene, in GM03813 cells.

[0267] To evaluate the off-target mitigation effect of GNA-saRNA, the antisense strand of RD-19040 was used as the “search” sequence to search for potential off-target genes by computer analysis and ARPIN was predicted to be a potential off-target gene. Figure 7A shows the “retrieval” sequence (antisense strand) and “seed” region (highlighted in gray) of RD-19040 and its predicted complementary sequence at the target site in an ARPIN transcript containing two mismatched nucleotides (italicized and bolded nucleotides). Designated GNA-saRNAs (i.e., RD-19650, RD-19651, RD-19652, RD-19653, RD-19654, RD-19655, RD-19657, RD-19661, RD-19662, RD-19674, and RD-19675) were transfected into GM03813 cells at 2.5 nM for 3 days. Cells were transfected with RD-19040 and used as a non-GNA control. In each PCR reaction, ARPIN mRNA levels were quantified by RT-qPCR using a set of gene-specific primers. As shown in Figure 7B, all GNA-saRNAs mitigated off-target effects compared to RD-19040, which is a non-GNA saRNA. ARPINThe expression of mRNA is summarized in Table 6. The results show that GNA modification at different positions can alleviate off-target effects to varying degrees.

[0268] Table 6. Expression of ARPIN mRNA in GM03813 cells 44 / 50 pages 49 CN 121569037 A

[0269] Note: “SEM” represents the standard error of the mean.

[0270] Example 12. In vitro activity of GNA-saRNA in inducing SERPING1 mRNA expression in Hep3B and HepG2 cells

[0271] To verify the in vitro activity of GNA-saRNA, specified non-GNA-saRNAs (i.e., RD-17229, RD-17235, RD-17238, RD-17241 and RD-17244) and their corresponding GNA-saRNAs (i.e., RD-17074, RD-17082, RD-17086, RD-17096 and RD-17099) were transfected into Hep3B cells at specified concentrations (i.e. 0.02 nM, 0.07 nM, 0.21 nM, 0.62 nM, 1.85 nM, 5.56 nM, 16.67 nM and 50 nM) for 3 days. The SERPING1 mRNA levels of non-GNA-saRNA and GNA-saRNA are shown in Figures 8A to 8E.

[0272] Similarly, specified non-GNA-saRNAs (i.e., RD-17229, RD-17235, and RD-17241) and their corresponding GNA-saRNAs (i.e., RD-17074, RD-17082, and RD-17096) were transfected into HepG2 cells at specified concentrations (i.e., 0.02 nM, 0.07 nM, 0.21 nM, 0.62 nM, 1.85 nM, 5.56 nM, 16.67 nM, and 50 nM) for 3 days. The SERPING1 mRNA levels of non-GNA-saRNA and GNA-saRNA are shown in Figures 9A to 9C.

[0273] Table 7 summarizes the Emax and AUC of SERPING1 expression. As described in the Materials and Methods section, the AUC of SERPING1 mRNA expression was calculated using GraphPad Prism software. The results showed that GNA-saRNA could enhance the targeting activity of non-GNA-saRNA in inducing SERPING1 mRNA expression.

[0274] Table 7. Emax and AUC of SERPING1 expression after GNA-saRNA treatment in Hep3B and HepG2 cells Specification 45 / 50 pages 50 CN 121569037 A

[0275] Note: “n / a” indicates not applicable, “ / ” indicates not detected.

[0276] Materials and Methods

[0277] saRNA Synthesis

[0278] (1) Single-stranded synthesis

[0279] Single-stranded oligonucleotides were synthesized using solid-phase synthesis techniques on a K&A DNA synthesizer (K&A Laborgeraete GbR, Schafheim, Germany).

[0280] The starting material was a general-purpose solid-phase support or a special solid-phase support, which was commercially available or synthesized as disclosed above. Typically, in a DNA synthesizer, phosphoramide monomers (0.1 M, acetonitrile or dichloromethane solution) comprising various linkers and conjugates were sequentially added to the solid-phase support to generate the desired full-length oligonucleotide.

[0281] Phosphoramide addition: Each phosphoramide addition cycle consisted of four chemical reactions, including detriphenylmethylation, coupling, oxidation / thiolization, and capping. In the first step, detriphenylmethylation was performed for 45 seconds using DCM in 3% dichloroacetic acid (DCA). In the second step, all phosphoramides were coupled at 12 eq for 6 minutes. In the third step, oxidation was performed for 1 minute using a 0.02 M iodine solution of THF:pyridine:water (70:20:10, v / v / v); if thiophosphate modification was required, thiolation was performed for 3 minutes using a 0.1 M hydroflavin solution of pyridine:ACN (50:50, v / v) instead of oxidation. In the fourth step, end-capping was performed for 20 seconds using a mixture of THF:acetic anhydride:pyridine (80:10:10, v / v / v) (CAP A) and N-methylimidazole:THF (10:90, v / v) (CAP B). The cycle of the four chemical reactions depended on the length of the individual oligonucleotide.

[0282] Deprotection I (nucleobase deprotection): After synthesis, the solid support was transferred to a screw-capped microcentrifuge tube. For a 1 μmol synthesis scale, 1 mL of a mixture of methylamine and ammonium hydroxide was added. The tube containing the solid support was then heated in an oven at 60°C to 65°C for 15 minutes, and then cooled to room temperature. The lysis solution was collected and evaporated to dryness in a SpeedVac to obtain crude single-stranded oligonucleotides.

[0283] Deprotection II (removal of 2'-TBDMS group): If the crude RNA oligonucleotides still carried the 2'-TBDMS group, they were dissolved in 0.1 mL DMSO. After adding 1 mL triethylamine trihydrofluoride, the tube was capped and the mixture was vigorously shaken to ensure complete dissolution, and then heated in an oven at 65°C for 15 minutes. The tube was removed from the oven and cooled to room temperature. The solution containing the fully desilylated oligonucleotides was cooled on dry ice. 2 mL of ice-cold n-butanol (-20°C) was carefully added in 0.5 mL increments to precipitate the oligonucleotides. The precipitate was filtered, washed with 1 mL of ice-cold n-butanol, and then dissolved in 0.01 M tris(hydroxymethyl)aminomethanol hydrochloride buffer.

[0284] (2) Single-chain purification instructions, pages 46 / 50, CN 121569037 A

[0285] The oligonucleotides were purified on an AKTA explorer 10 equipped with a Source 15Q 4.6 / 100 PE column under the following conditions: Buffer A: 10 mM Tris-HCl, 1 mM EDTA, pH 7.5; Buffer B: 10 mM Tris-HCl, 1 mM EDTA, 2 M NaCl, pH 7.5; gradient: 10% B to 60% B, over 25 minutes; flow rate: 1 mL / min. The purified oligonucleotides were collected and desalted using a HiPrep 26 / 10 desalting column.

[0286] (3) Annealing to form duplexes

[0287] For the duplexes, after generating a desalted purified single-stranded solution, the sense and antisense strands were mixed in equal volumes at equimolar concentrations in a tube. The tubes were placed in a heating block at 95°C for 5 minutes and then cooled to room temperature. The resulting duplexes were then lyophilized into powder.

[0288] Cell Culture and Processing

[0289] Fibroblasts from SMA patients were obtained from the Corriere Institute (Camden, NJ, USA) and included GM03813 (SMA type II, with 3 copies of the SMN2 gene) and GM22592 (SMA type II, with 3 copies of the SMN2 gene). They were cultured at 37°C and 5% CO2 in modified MEM medium (Gibco, Thermo Fisher Scientific, Carl Pasteur, California) supplemented with 15% fetal bovine serum (Sigma-Aldrich), 1% NEAA (Gibco), and 1% penicillin / streptomycin (Gibco). HepG2 cells (SCSP-510, National Center for Cell Culture Collection, China) were cultured at 37°C and 5% CO2 in modified DMEM medium (Gibco) supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin. Hep3B cells (CBP60197, Kebai Biotechnology, China) were cultured in modified MEM medium supplemented with 10% fetal bovine serum, 1% NEAA and 1% penicillin / streptomycin at 37°C and 5% CO2. Transfection was performed in the growth medium using Lipofectamine RNAiMax (Thermo Fisher Scientific, Waltham, Massachusetts, USA) according to the manufacturer's protocol.

[0290] RNA Isolation and Reverse Transcription-Quantitative Polymerase Chain Reaction (RT-qPCR)

[0291] (1) RNA Isolation and Two-Step RT-qPCR

[0292] To quantify mRNA expression in cells, total cellular RNA was isolated from treated cells using the RNeasy Plus Mini kit (Qiagen, Hilden, Germany) according to its instructions. The RNA was then isolated using PrimeScript™ containing a gDNA Eraser.The obtained RNA (1 μg) was reverse transcribed into cDNA using an RT kit (Takara, RR047A, Shiga, Japan). The obtained cDNA was amplified using TB Green® Premix Ex Taq™ II reagents (Takara, RR820A, Shiga, Japan) and primers specifically designed for amplifying the target gene in a Roche LightCycler 480 Multiwell Plate 384 (Roche, reference: 4729749001, USA).

[0293] The reaction conditions were as follows: reverse transcription (stage 1): 42°C for 5 min, 95°C for 10 s; PCR (stage 2): 95°C for 5 s, 60°C for 30 s, 72°C for 10 s; 40 amplification cycles; and melting curve (stage 3). The PCR reaction conditions are shown in Tables 8 and 9. The primer sequences are shown in Table 10.

[0294] Table 8. RT reaction instructions 47 / 50 pages 52 CN 121569037 A

[0295] Table 9. RT-qPCR reaction

[0296] Table 10. Primer sequences for RT-qPCR assay instructions 48 / 50 pages 53 CN 121569037 A

[0297] A reference gene

[0298] In order to calculate the expression level (Erel) of target mRNA in the saRNA-treated sample relative to the control treatment, the Ct values ​​of the target gene and the internal reference gene are substituted into Formula I,

[0299] (Formula I)

[0300] Where CtTm is the Ct value of the target gene from the control-treated sample; CtTs is the Ct value of the target gene from the saRNA-treated sample; CtRm is the Ct value of the internal reference gene from the control-treated sample; CtRs is the Ct value of the internal reference gene from the saRNA-treated sample.

[0301] Two reference genes

[0302] To calculate the expression level (Erel) of the target gene mRNA relative to the control treatment in the saRNA-treated sample, the Ct values ​​of the target gene and two internal reference genes were substituted into the following Formula II,

[0303] (Formula II)

[0304] Where CtTm is the Ct value of the target gene from the control-treated sample; CtTs is the Ct value of the target gene from the saRNA-treated sample; CtR1m is the Ct value of internal reference gene 1 from the control-treated sample; CtR1s is the Ct value of internal reference gene 1 from the saRNA-treated sample; CtR2m is the Ct value of internal reference gene 2 from the control-treated sample; and CtR2s is the Ct value of internal reference gene 2 from the saRNA-treated sample.

[0305] Calculation of area under the curve (AUC)

[0306] The area under the curve (AUC) of a specific test product is derived from a curve plotted from dose-response data at eight doses.The curve reflects the cumulative effect of the test product at all test doses. Essentially, AUC quantifies the overall response to treatment, integrating the responses at individual doses into a single metric, such as average efficacy.

[0307] To calculate AUC, we first plot the agonist versus response curve based on the dose-response data. The curve is then evaluated using the Area Under the Curve (AUC) plugin in the GraphPad Prism software. The AUC value is dimensionless but can be used as a comparative metric to determine the relative response compared to other test products. Instruction manual 50 / 50 pages 55 CN 121569037 A Figure 1 Instruction manual Figure 1 / 10 pages 56 CN 121569037 A Figure 2 Instruction manual Figure 2 / 10 pages 57 CN 121569037 A Figure 3 Instruction manual Figure 3 / 10 pages 58 CN 121569037 A Figure 4A Figure 4B Instruction manual Figure 4 / 10 pages 59 CN 121569037 A Figure 5A Figure 5B Instruction manual Figure 5 / 10 pages 60 CN 121569037 A Figure 6A Figure 6B Figure 7A Instruction manual Figure 6 / 10 pages 61 CN 121569037 A Figure 7B Instruction manual Figure 7 / 10 pages 62 CN 121569037 A Figure 8 Instruction manual Figure 8 / 10 pages 63 CN 121569037 A Figure 9A Instruction manual Figure 9 / 10 pages 64 CN 121569037 A Figure 9B Figure 9C Instruction Manual Drawings 10 / 10 Page 65 CN 121569037 A

Claims

1. A double-stranded oligonucleotide agent capable of activating or upregulating the expression of a target gene, comprising a sense strand and an antisense strand, each strand having a length of 15 to 35 nucleotides, wherein the sense strand and the antisense strand form a complementary double-stranded structure of at least 8 base pairs, wherein the double-stranded oligonucleotide agent comprises one or more glycerol nucleic acid (GNA) modified nucleotides located in one or both of the sense strand and the antisense strand.

2. The double-stranded oligonucleotide agent according to claim 1, wherein one or both of the sense strand and the antisense strand are guide strands mediating RNA activation.

3. The double-stranded oligonucleotide agent according to claim 2, wherein the guide strand has a seed region of 2 to 10 nucleotides in length located at or near the 5' end of the guide strand.

4. The double-stranded oligonucleotide agent according to claim 3, wherein the seed region begins no more than 3 nucleotides from the 5' end of the guide strand.

5. The double-stranded oligonucleotide agent according to claim 3, wherein the seed region comprises nucleotides at positions 2 to 10, or 2 to 9, or 2 to 8, or 2 to 7, or 2 to 6, or 3 to 6, or 2 to 5, or 3 to 6, or 3 to 5, or 4 to 6, starting from the 5' end of the guide strand.

6. The double-stranded oligonucleotide agent according to claim 1, wherein the one or more GNA-modified nucleotides comprise at least one selected from the group consisting of: GNA-modified adenine (GNA-A), GNA-modified thymine (GNA-T), GNA-modified cytosine (GNA-C), GNA-modified guanine (GNA-G), and GNA-modified uracil (GNA-U).

7. The double-stranded oligonucleotide agent according to claim 1, wherein the double-stranded oligonucleotide agent comprises 1 to 50 GNA-modified nucleotides.

8. The double-stranded oligonucleotide agent according to claim 2, wherein the double-stranded oligonucleotide agent comprises one or more GNA-modified nucleotides located at positions 1 and / or 2 and / or 3 and / or 4 and / or 5 and / or 6 and / or 7 and / or 8 and / or 9 and / or 10 and / or 11 and / or 12 and / or 13 and / or 14 and / or 15 and / or 16 and / or 17 and / or 18 and / or 19 and / or 20 and / or 21 and / or 22 and / or 23 and / or 24 and / or 25, starting from the 5' end of the leader strand.

9. The double-stranded oligonucleotide agent according to claim 3, wherein one or more GNA-modified nucleotides are located within and / or outside the seed region of the leader strand.

10. The double-stranded oligonucleotide agent according to claim 3, wherein one or more GNA-modified nucleotides are located in the transit chain.

11. The double-stranded oligonucleotide agent according to claim 1, wherein the sense strand comprises the nucleotide sequence shown in any one of SEQ ID NO: 1, 3, 12, 14, 15, 17, 18, 20, 21, 23, 24, 27, 29, 52, 54, and the antisense strand comprises the nucleotide sequence shown in any one of SEQ ID NO: 2, 4, 5, 6, 7, 8, 9, 10, 11, 13, 16, 19, 22, 25, 26, 28, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 53, 55.

12. The double-stranded oligonucleotide agent according to claim 1, wherein the leader chain is synthesized using a GNA-modified nucleotide monomer of formula (1) to include the one or more GNA-modified nucleotides: （1） The bases mentioned herein are selected from the group consisting of: adenine nucleobases, thymine nucleobases, cytosine nucleobases, guanine nucleobases, uracil nucleobases, and analogues thereof.

13. The double-stranded oligonucleotide agent according to claim 12, wherein the base in formula (1) is selected from the following structures: , , , , , , and .

14. The double-stranded oligonucleotide agent according to claim 1, wherein the sense strand or the antisense strand has at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 nucleotides complementary to an isometric segment of the coding strand of the target gene.

15. The double-stranded oligonucleotide agent according to claim 1, wherein the complementary double-stranded structure of at least 8 base pairs has no more than 5 mismatched nucleotides between the sense strand and the antisense strand, i.e., 5, 4, 3, 2, 1 or 0 mismatched nucleotides.

16. The double-stranded oligonucleotide agent of claim 15, wherein the mismatched nucleotide is located inside or near the 3' or 5' end of the antisense strand.

17. The double-stranded oligonucleotide agent of claim 15, wherein the mismatched nucleotide is located at the GNA position in the sense strand and / or the antisense strand.

18. The double-stranded oligonucleotide agent according to claim 1, wherein the double-stranded oligonucleotide agent is a small activating RNA (saRNA) that upregulates the expression of the target gene by at least 10%.

19. The double-stranded oligonucleotide agent of claim 1, wherein at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or about 100% of the non-GNA nucleotides of the double-stranded oligonucleotide agent comprise modifications selected from the group consisting of: 2'-fluoro(2'-F), 2'-O-methyl(2'-OMe), 2'-O-methoxyethyl(2'-MOE), 5'-( E )-Vinylphosphonates, thiophosphate backbone modifications, and combinations thereof.

20. A conjugating agent comprising a double-stranded oligonucleotide agent according to any one of claims 1-19 and at least one conjugating moiety, wherein the at least one conjugating moiety is selected from: lipids, fatty acids, fluorophores, ligands, sugars, peptides and antibodies.

21. The conjugating agent of claim 20, wherein the conjugating moiety is selected from auxiliary oligonucleotides (ACO), lipids / fatty acids, and GalNAc clusters.

22. The binding agent of claim 21, wherein the binding portion is selected from: (tC2x6), and (C5x5) in Represents the carrier material.

23. A cell comprising the double-stranded oligonucleotide agent according to claim 1 or the conjugating agent according to claim 20.

24. A pharmaceutical composition comprising a double-stranded oligonucleotide agent according to claim 1 or a conjugating agent according to claim 20, and at least one pharmaceutically acceptable carrier.

25. A kit for activating or upregulating a target gene in cells or a subject, comprising a double-stranded oligonucleotide agent according to any one of claims 1-19, or a conjugate agent according to any one of claims 20-22, or a pharmaceutical composition according to claim 24.

26. A method for activating or upregulating a target gene in a cell or subject, comprising administering to the cell or subject a double-stranded oligonucleotide agent according to any one of claims 1-19 or a conjugate agent according to any one of claims 20-22.

27. A method for mitigating off-target effects caused by a double-stranded oligonucleotide agent capable of activating or upregulating the expression of a target gene in a cell or subject, comprising administering to the cell or subject the double-stranded oligonucleotide agent according to any one of claims 1-19 or the conjugate agent according to any one of claims 20-22.

28. Use of the double-stranded oligonucleotide agent according to any one of claims 1-19 or the conjugate agent according to any one of claims 20-22 in the preparation of a product for activating or upregulating a target gene in a cell or subject.