Glycerol nucleic acid modified oligonucleotide agent and application 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

Technical Field

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

[0002] Oligonucleotides are an emerging class of therapeutic agents currently under active development for treating a variety of diseases. The main categories of therapeutic oligonucleotides include single-stranded antisense oligonucleotides and dsRNAs. dsRNAs further include two main classes: small interfering RNAs (siRNAs) and small activating RNAs (saRNAs). Although both require the Argonaute (AGO) protein, siRNAs and saRNAs differ significantly in their mechanistic framework.

[0003] saRNA-mediated gene activation offers 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. Despite the potential for therapeutic applications, the development of this technology has faced significant challenges, particularly the emergence of undesirable off-target effects and associated toxicities. Ensuring that saRNAs meet stringent safety requirements during drug discovery and development remains a critical issue in this field. Summary of the Invention

[0004] To address the aforementioned issues, 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 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.

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

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

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

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

[0010] In another respect, 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 respect, 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 a cell or subject.

[0012] On the other hand, this disclosure provides the use of the double-stranded oligonucleotide agents or conjugates described herein in the preparation of products.

[0013] The double-stranded oligonucleotide agents (such as saRNA) described 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. Attached Figure Description

[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 of illustrative embodiments employing the principles of the invention, along with the accompanying drawings (also referred to herein as “Figures”), in which: Figure 1 The image shows the effect of glycerol-modified saRNA (GNA-saRNA) on the full-length RNA in GM03813 cells. SMN2 ( SMN2- FL The activity of 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 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). In each PCR reaction, gene-specific primer sets were used for RT-qPCR to assess the expression of the target GNA-saRNA. SMN2-FL The mRNA level was quantified. SDHA and GAPDH The geometric mean of mRNA levels was used as an internal control to normalize the expression data. Data representation... SDHA and GAPDH Normalization, relative to Mock processing SMN2-FL The average expression level (mean of two parallel transfection wells ± SEM).

[0015] Figure 2 A to Figure 2 H shows the full-length GNA-saRNA in GM03813 cells. SMN2-FL ) and skip exon 7 ( SMN2-Δ7 )of SMN2 mRNA expression activity was assessed. GM03813 cells were transfected with designated GNA-saRNAs (RD-15977, RD-15978, RD-15979, RD-15980, RD-15981, RD-15982, and RD-15983) at designated 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). Figure 2 A to Figure 2 H shows the quantification by RT-qPCR using gene-specific primer sets in each PCR reaction. SMN2-FL and SMN2-Δ7 mRNA levels. SDHA and GAPDH The geometric mean of mRNA levels was used as an internal control to normalize the expression data. Data representation... SDHA and GAPDH Normalization, relative to Mock processing SMN2-FL or SMN2-Δ7 The average expression level (mean of two parallel transfection wells ± SEM).

[0016] Figure 3 A to Figure 3 H shows the effect of GNA-saRNA on GM22592 cells. SMN2-FL and SMN2-Δ7The activity of 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 GM22592 cells 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). Figure 3 A to Figure 3 H shows the quantification by RT-qPCR using gene-specific primer sets in each PCR reaction. SMN2-FL and SMN2-Δ7 mRNA levels. SDHA and GAPDH The geometric mean of mRNA levels was used as an internal control to normalize the expression data. Data representation... SDHA and GAPDH Normalization, relative to Mock processing SMN2-FL or SMN2-Δ7 The average expression level (mean of two parallel transfection wells ± SEM).

[0017] Figure 4A and Figure 4B The image shows the effect of GNA-saRNA on GM03813 cells. SMN2-FL and SMN2-Δ7 The activity of 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 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 non-specific duplex control. Figure 4A and Figure 4B This demonstrates the quantification achieved by RT-qPCR using gene-specific primer sets in each PCR reaction. SMN2-FL and SMN2-Δ7 mRNA levels. SDHA The amplified data served as an internal control to normalize the expression data. Data representation... SDHA Normalization, relative to Mock processing SMN2-FL or SMN2-Δ7 The average expression level (mean of four parallel transfection wells ± SEM).

[0018] Figure 5A and Figure 5B The image shows potential off-target genes of GNA-saRNA in GM03813 cells. P2RY2 Off-target effects of expression. GM03813 cells were transfected with designated GNA-saRNAs (RD-15977, RD-15978, RD-15981, RD-15982, and RD-15983) at 6.25 nM and 25 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 non-specific duplex control (not shown in the figure). Figure 5A The image shows the "Query" (antisense) sequence and "Seed" region (italicized, gray highlighted) of RD-10994, as well as its position within a region containing two mismatched nucleotides (italicized and bolded). P2RY2 Predicted complementary target sites in transcripts. Figure 5B This demonstrates the quantification achieved by RT-qPCR using gene-specific primer sets in each PCR reaction. P2RY2 mRNA levels. SDHA The amplified data served as an internal control to normalize the expression data. Data representation... SDHA Normalization, relative to Mock processing P2RY2 The average expression level (mean of four parallel transfection wells ± SEM).

[0019] Figure 6A and Figure 6B The image shows the effect of GNA-saRNA on GM03813 cells. SMN2-FL and SMN2-Δ7 The activity of mRNA expression was assessed. Specific 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) 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 non-specific duplex control. Figure 6A and Figure 6B This demonstrates the quantification achieved by RT-qPCR using gene-specific primer sets in each PCR reaction. SMN2-FL and SMN2-Δ7mRNA levels. TBP The amplified data served as an internal control to normalize the expression data. Data representation... TBP Normalization, relative to Mock processing SMN2-FL or SMN2-Δ7 The average expression level (mean of four parallel transfection wells ± SEM).

[0020] Figure 7A and Figure 7B The image shows potential off-target genes of GNA-saRNA in GM03813 cells. ARPIN Off-target effects of expression. GM03813 cells were transfected with designated 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) 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 duplex control. Figure 7A The image shows the "retrieval" (antisense) sequence and "seed" region (italicized, gray highlighted) of RD-19040, as well as its position within a region containing two mismatched nucleotides (italicized and bolded nucleotides). ARPIN Predicted complementary target sites in transcripts. Figure 7B This demonstrates the quantification achieved by RT-qPCR using gene-specific primer sets in each PCR reaction. ARPIN mRNA levels. TBP The amplified data served as an internal control to normalize the expression data. Data representation... TBP Normalization, relative to Mock processing ARPIN The average expression level (mean of four parallel transfection wells ± SEM).

[0021] Figure 8 A to Figure 8 E compared the effects of GNA-saRNA and non-GNA-saRNA on Hep3B cells. SERPING1mRNA expression activity was assessed. 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 figure). dsCon2 was used as a nonspecific duplex control (not shown in the figure). Figure 8 A to Figure 8 E demonstrates the quantification achieved by RT-qPCR using gene-specific primer sets in each PCR reaction. SERPING1 mRNA levels. HPRT1 and TBP The geometric mean of mRNA levels was used as an internal control to normalize the expression data. Data representation... HPRT1 and TBP Normalization, relative to Mock processing SERPING1 The average expression level (mean of four parallel transfection wells ± SEM).

[0022] Figures 9A to 9C The effects of GNA-saRNA and non-GNA-saRNA on HepG2 cells were compared. SERPING1 mRNA expression activity was assessed. Specified non-GNA-saRNAs (RD-17229, RD-17235, and RD-17241) and their corresponding GNA-saRNAs (RD-17074, RD-17082, and RD-17096) were transfected into HepG2 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 figure). dsCon2 was used as a nonspecific duplex control (not shown in the figure). Figures 9A to 9C This demonstrates the quantification achieved by RT-qPCR using gene-specific primer sets in each PCR reaction. SERPING1 mRNA levels. HPRT1 and TBP The geometric mean of mRNA levels was used as an internal control to normalize the expression data. Data representation... HPRT1 and TBP Normalization, relative to Mock processing SERPING1The average expression level (mean of four parallel transfection wells ± SEM). Detailed Implementation

[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] definition

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

[0026] As used herein, the terms “oligonucleotide” and “polynucleotide” are used interchangeably and refer to polymers of nucleotides, particularly single-stranded nucleic acid molecules of DNA, RNA, or DNA / RNA hybrids, containing oligonucleotide chains with regular and irregular alternations of deoxyribosyl and ribosyl moieties, as well as 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 can be or may include small activating nucleic acid molecules (saRNAs).

[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 can pair 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 anti-Hoogsteen base pairing).

[0028] Complementarity includes perfect complementarity and imperfect complementarity. "Perfect complementarity" or "100% complementarity" means that each nucleotide from the first oligonucleotide chain can form a hydrogen bond with the corresponding nucleotide in the second oligonucleotide chain within the double-stranded region of the double-stranded oligonucleotide molecule, and no base pairs are "mismatched". "Imperfect 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 chain length 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, chromosomal and / or extrachromosomal 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 a target gene can be achieved by designing a saRNA that has sequence identity (also known as homology) with the promoter sequence, characterized by upregulation of target gene expression. The terms "target sequence" and "target site" are used interchangeably and refer to a sequence fragment of the target gene sequence, such as the target gene promoter, that is homologous 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, partially or fully 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, a sense strand can be chosen as the guide strand, resulting in 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 the saRNA duplex, the “sense strand” of the term 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 the saRNA duplex, the term "antisense strand" of saRNA refers to the strand complementary to the sense strand sequence. The antisense strand can interact with a target region of a target gene to activate or upregulate gene expression; the target region can be 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 which the T in the 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; that is, 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 target gene's template DNA strand.

[0036] As used herein, the term "promoter" refers to a sequence that is spatially associated with a protein- or RNA-coding nucleic acid sequence and that 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 use of the term "promoter" herein. While promoter sequences are typically located at the 5' end of a protein- or RNA-coding sequence, they can also be found in exons and introns.

[0037] As used herein, the term “GNA,” also known as glycerol nucleic acid, is a nucleic acid similar to DNA or RNA but with a different sugar-phosphodiester backbone composition, in which propylene glycol replaces ribose or deoxyribose. As used herein, the term “LNA” refers to locked nucleic acid, in which 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 pentagonal or hexavalent bridging structure with NO bonds, with 2'-O and 4'-aminoethylene bridging. As used herein, the term “PNA” refers to a nucleic acid mimic with a pseudopeptide backbone composed of N-(2-aminoethyl)glycine units, in which 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. The single-stranded region extending beyond the 3' and / or 5' ends of the double-stranded oligonucleotide is called a protrusion.

[0040] As used herein, the term "natural overhang" refers to an overhang consisting of one or more nucleotides that are identical to or complementary to the corresponding position on the target sequence. Natural overhangs on the sense strand consist of one or more nucleotides that are identical to the corresponding position on the DNA target. Natural overhangs on the antisense strand consist of one or more nucleotides that are 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 a 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 to 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 levels, 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, regardless of the mechanism of such activation. For example, gene activation occurs at the transcriptional level to increase transcription to RNA, and 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 upregulates the expression of a target gene and may consist of a first nucleic acid fragment (sense strand) and a second nucleic acid fragment (antisense strand). The first nucleic acid fragment contains a nucleotide sequence that is sequence-identical to a non-coding nucleic acid sequence (e.g., promoter or enhancer) of the target gene, and the second nucleic acid fragment contains a nucleotide sequence complementary to the first nucleic acid fragment, wherein the first and second nucleic acid fragments form a double strand. saRNA may also consist of a synthetic or vector-expressed single-stranded RNA molecule that forms a hairpin structure through two complementary regions within the molecule, wherein the first region contains a nucleotide sequence that is sequence-identical to 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 the target recognition of the oligonucleotide. As mentioned above, the guide strand can be the sense strand, antisense strand, or both of the 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)” refers to a non-targeting single-stranded oligonucleotide having at least six nucleotides, with or without one or more linker motifs conjugated to another oligonucleotide. ACO components are not designed to specifically target any complementary nucleic acid sequence in the target organism. ACO components may be chemically modified on their 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 used to covalently link any two oligonucleotides located at the 3'- or 5'-terminus of one or both chains within an oligonucleotide agent.

[0046] As used herein, the term "oligonucleotide agent" refers to a substance containing oligonucleotides that includes at least one or more saRNAs of the present invention or composed thereof, and has the activity of 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 the saRNA of the present invention), DNA, BNA, LNA, GNA, or peptide nucleic acid (PNA).

[0047] As used in this article, the term "prevention" refers to slowing the progression of a disease, symptom, or condition from its current state to a more harmful state.

[0048] As used in this article, the term “treatment” means the prevention, improvement, reversal, cure, and / or delay of a disease, symptom, or condition.

[0049] As used in this article, the capital letter " SMN2-FL "or" SMN2-FL "Genes" and " SMN2-Δ7 "or" SMN2-Δ7 "Gene" refers to human genes. As used in this article, the term "gene"... SMN2 mRNA SMN2-FL mRNA or SMN2-Δ7 "mRNA" refers to the RNA produced by... SMN2 Gene expression or SMN2 Messenger RNA (mRNA) produced by gene transcription.

[0050] As used in this article, the term " SERPING1 "mRNA" refers to the RNA produced by... SERPING1 Gene expression or SERPING1 Messenger RNA (mRNA) produced by gene transcription.

[0051] Double-stranded oligonucleotide agents

[0052] Oligonucleotides offer significant potential for the prevention or treatment of a wide range of diseases, conditions, or illnesses by regulating, for example, the protein expression of disease-related genes and their variants. Among therapeutic oligonucleotides, saRNAs, as upregulators, have emerged as a new 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] While sequences containing only natural structural units have been found to have important uses, oligonucleotide synthesis technology offers the possibility of various nucleotide modifications. Surprisingly, certain chemical modifications to oligonucleotide agents (such as saRNA) have been found to provide effective ways to mitigate undesirable off-target effects while retaining the agent's excellent gene-activating function. These modified oligonucleotide agents overcome their current limitations and can be used more broadly in the medical and pharmaceutical fields.

[0054] Therefore, in one aspect, this article 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 in which the (deoxy)ribose in the sugar-phosphodiester backbone of DNA / RNA is replaced by propylene glycol. Similarly, GNA-modified nucleotides include the GNA form of non-GNA DNA / RNA nucleotides, such as naturally occurring DNA / RNA nucleotides adenine (A), thymine (T), cytosine (C), guanine (G), and uracil (U), in which their (deoxy)ribose portions 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 a double-stranded oligonucleotide agent comprise one or more GNA-modified nucleotides that replace their corresponding non-GNA nucleotides. In other words, one or more non-GNA nucleotides on the sense and / or antisense strands of a 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, 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. 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 implementations, 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 and / or antisense strands of the double-stranded oligonucleotide agent. In some implementations, 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 and / or antisense strands of the double-stranded oligonucleotide agent.

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

[0061] In some embodiments, the leader strand of the double-stranded oligonucleotide agent comprises 1 to 50 GNA-modified nucleotides (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).

[0062] In some embodiments, one or more GNA-modified nucleotides are 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 guide strand, which can 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 can 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 implementations, the seed region is 2 to 10 nucleotides in length (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 guide 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 guide strand. In some embodiments, the 5' 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 guide strand. In some embodiments, the 3' nucleotide of the seed region is at least 6 nucleotides (e.g., 6, 7, 8, 9, or 10) from the 5' end of the guide strand. In some embodiments, the seed region comprises consecutive 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 guide strand.

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

[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 GNA-modified nucleotides (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or any range thereof). 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, at least 3%, at least 5%, at least 10%, 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 at least 100% of the nucleotides in the seed region 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 one 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 further includes one or more GNA-modified nucleotides located outside the seed region. In some embodiments, the double-stranded oligonucleotide agent further includes one or more GNA-modified nucleotides located in the guest strand (P strand). In some embodiments, the double-stranded oligonucleotide agent includes 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 a phosphoramide process (e.g., a solid-phase phosphoramide process). In some embodiments, either or both of the sense and antisense strands are synthesized to include one or more GNA-modified nucleotides. In some embodiments, the antisense strand is synthesized to include one or more GNA-modified nucleotides. The synthesized sense and antisense strands can then be annealed to form a double-stranded structure.

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

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

[0072] The sense 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 sense 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 isochronous segment of the coding strand of the target gene. In some embodiments, the sense strand has 5 or fewer nucleotide differences relative to an isochronous 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 (or its 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 implementations, the difference or mismatch is located inside or near the 3' end of the antisense chain.

[0074] The positive and negative strands of the double-stranded oligonucleotide agent include complementary regions forming a double-stranded structure of at least 8 base pairs, such as complementary regions of at least 10, 12, 15, 18, or 20 base pairs. In some embodiments, the positive and negative strands are at least 50% complementary to each other, such as at least 60%, 70%, 80%, 90%, 95%, or 100%. In some embodiments, the complementary region between the positive and negative strands contains no more than 5 mismatched nucleotides, 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 positive and negative 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 negative strand. In some implementations, the mismatched nucleotide is located at the GNA position in the sense strand and / or antisense strand.

[0075] The sense and antisense strands of the double-stranded oligonucleotide agent described herein may reside on two different nucleic acid strands or on a single nucleic acid strand (e.g., a continuous nucleic acid sequence). When the sense and antisense strands are 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 3' overhangs of 2 or 3 nucleotides in length. In some embodiments, the nucleotides of the overhang may be selected from the corresponding position on the DNA target (i.e., the natural overhang) or complementary nucleotides thereto. In some cases, when the sense and antisense strands are on a single nucleic acid strand, the double-stranded oligonucleotide agent may 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 double-stranded oligonucleotide agents described herein each have a length of 15 to 35 nucleotides in both the sense and antisense strands. 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 implementations, the double-stranded oligonucleotide agent may include RNA, DNA, BNA, LNA, GNA, or peptide nucleic acid (PNA).

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

[0080] In non-limiting examples, saRNAs are 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, saRNAs are designed or selected at least in part based on criteria that enable the production of functional saRNAs. For example, in some cases, the sequence upstream of the TSS may include a sequence that, although located in a hotspot region, is detrimental to saRNA synthesis.

[0081] In some embodiments, saRNAs are designed or selected based at least in part 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 sequences that: (1) have a GC content of 35% to 70%; (2) have fewer than 5 consecutive identical nucleotides; (3) have 3 or fewer dinucleotide repeats; and (4) have 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 works such as Place et al. Molecular Therapy–Nucleic Acids (2012) 1, e15; and Li et al., PNAS These methods and principles are incorporated herein by reference in their entirety, 2006, vol.103, no. 46, 17337–17342.

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

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

[0089] In some embodiments, the double-stranded oligonucleotide agents described herein include 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, such as nucleotides modified with 2'-deoxy-2'-fluorine or 2'-deoxy.

[0090] In some embodiments, the double-stranded oligonucleotide agents described herein comprise at least one chemically modified nucleotide, which is modified at a 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 involves adding an (E)-vinylphosphonate moiety to the 5' end of the sense or antisense sequence. In some embodiments, the chemical modification of at least one of the chemically modified nucleotides involves adding 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 nucleotides modified with 2'-O-methyl groups and nucleotides containing 5'-thiophosphate groups.

[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 Currentprotocols 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' end modifications (phosphorylation, conjugation, reverse linkage, etc.), 3' end 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, (c) sugar modifications (e.g., at the 2' or 4' position) or sugar substitution, and (d) backbone modifications, including modification or substitution of phosphodiester bonds. Specific examples of double-stranded oligonucleotide agents that can be used in this disclosure include, but are not limited to, RNA comprising a modified backbone or RNA without natural 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] Modified oligonucleotide backbones include, for example, thiophosphates, chiral thiophosphates, dithiophosphates, phosphate triesters, aminoalkyl phosphate triesters, methyl and other alkylphosphonates (including 3'-alkylphosphonates and chiral phosphonates), phosphonites, 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 compounds with opposite polarities, wherein adjacent nucleoside unit pairs are 3'-5' to 5'-3' or 2'-5' 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)-Vinylphosphonates, thiophosphate backbone modifications, and combinations thereof.

[0096] Conjugating agent

[0097] Furthermore, to facilitate the entry of the double-stranded oligonucleotide agent into the cell, based on the above modifications, a chemical conjugation moiety can be introduced into the end of the sense or antisense strand of the double-stranded oligonucleotide agent to promote its 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 a conjugating agent comprising a double-stranded oligonucleotide agent and at least one conjugation moiety.

[0098] In some embodiments, the conjugating agent comprises a double-stranded oligonucleotide agent as described herein and one or more conjugated moieties covalently linked to the oligonucleotide agent. In some embodiments, the conjugated moieties alter 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 conjugated moieties impart novel properties to the linked oligonucleotide, such as the ability to detect the oligonucleotide's fluorophore or reporter group. Some conjugated moieties have been previously described, such as: helper 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., , 1993, 3, 2765-2770); Nucl.Acids Res. , 1992, 20, 533-538; aliphatic chains, such as dodecanediol or undecyl residues (Saison-Behmoaras et al., 1992, 20, 533-538); EMBO 1, 1991, 10, 1111-1118; Kabanov et al., FEBS Lett. , 1990, 259, 327-330; Svinarchuk et al., Biochimie(Manoharan et al., 1993, 75, 49-54); phospholipids, such as hexadecyl-racemic-glycerol or triethyl-ammonium 1,2-di-O-hexadecyl-racemic-glycerol-3-H-phosphonate ...). Tetrahedron Lett. , 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res., 1990, 18, 3777-3783; polyamine or polyethylene glycol chains (Manoharan et al., Nucleosides & Nucleotides (1995, 14, 969-973) or adamantaneacetic acid; palmitoyl moiety (Mishra et al., 1995, 14, 969-973); Biochim. Biophys. Acta (Crooke et al., 1995, 1264, 229-237), stearylamine or hexano-carbonyl-hydroxycholesterol moiety (Crooke et al., 1995, 1264, 229-237), J. Pharmacol. Exp. Ther. , 1996, 277, 923-937), tocopherol group (Nishina et al., Molecular Therapy Nucleic 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, antibacterial agents, or antibiotics.

[0101] In some embodiments, the double-stranded oligonucleotide agent described herein is conjugated to one or more conjugation portions selected from the following: 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 following: cell-penetrating peptides, polyethylene glycol, alkaloids, tryptophan, benzimidazole, quinolones, amino acids, cholesterol, glucose, and N-acetylgalactosamine.

[0103] In certain 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: 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 moieties selected from C 4-30 Lipids of fatty acids. In some embodiments, the conjugated moiety is a straight or branched chain with saturated or unsaturated carbon atoms. 16 Lipids / fatty acids with carbon chains.

[0104] According to another embodiment, the double-stranded oligonucleotide agent further includes at least one accessory oligonucleotide (ACO) conjugated to the oligonucleotide agent. The term "accessory oligonucleotide (ACO)" herein 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. The ACO component is not designed to specifically target any complementary nucleic acid sequence of the target. The ACO component may 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 are beneficial to improving the bioavailability and delivery of the oligonucleotide. The covalent linker portion can be a natural or non-natural nucleotide, ethylene glycol, carbohydrate, alkyl chain, or any other linker used for covalently linking any two oligonucleotides located at the 3'- or 5'-terminus of one or both chains within an oligonucleotide agent. ACOs can be prepared according to the description and preparation disclosed in WO2023280190A1, the entire text of which is incorporated herein by reference.

[0105] In some embodiments, the double-stranded oligonucleotide agent of this application relates to a sense or antisense strand of the 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) in Represents the carrier material.

[0106] In some embodiments, the conjugated portions to the double-stranded oligonucleotide agent are tC2x6 and C5x5 as 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 using 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 moiety is a lipid selected from fatty acids with a carbon chain length of 4 to 30 carbon atoms. In some embodiments, the conjugated moiety is a fatty acid with a carbon chain length of 16 carbon atoms. In some embodiments, the conjugated moiety is selected from lipophilic moieties 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 moieties via one, two, three, four, five, six, or even more linker moieties.

[0108] According to one implementation scheme, when a connecting portion is present, the connecting portion can be selected from the group consisting of: -O-, -S-, -C(O)-, -NH-, -N((Cl-C 12 )alkyl)-、-N((C1-C 12 )Alkyl)-C(O)-O-, -OC(O)-, -C(O)-O-, -OC(O)-O-, -C(O)-NH-, -OP(O)2O-, -P(O)(O - )O-, -OP(O)O-, -OP(O)(S)O-, -OS(O)2-O-, -S(O)2-O-, -S(O)-O-, -(C1-C22 )alkylene-,-(C1-C 22 )alkylene-NH-, -NH-(C1-C 22 )alkylene-,-(C1-C 22 )alkylene-NH-C(O)-、-(C1-C 22 )alkylene-C(O)-、-(C1-C 22 )alkylene-C(O)-O-, -C(O)-(C1-C 22 )alkylene-,-NH-C(O)-(C1-C 22 )alkylene-、-C(O)-NH-(C1-C 22 )alkylene-、-C(O)-(C1-C 22 )alkylene-NH-, -NH-(C1-C 22 )alkylene-C(O)-, -C(O)-(C1-C 22 )alkylene-C(O)-, -NH-(C1-C 22 )alkylene-NH-,-C(O)-(C1-C 22 )alkylene-C(O)O-, -OC(O)-(C1-C 22 )alkylene-C(O)-O-, -C(O)-O-(C1-C 22 )alkylene-OC(O)-, -C(O)-(C1-C 22 )alkylene-NH-C(O)-, -NH-C(O)-(C1-C 22 )alkylene-C(O)-, -NH-C(O)-(C1-C 22 )alkylene-C(O)-NH-, -C(O)-NH-(C1-C 22 )alkylene-NH-C(O)-、-(C1-C 22 )alkylene-OP(O)2O-、-(C1-C 22 )alkylene-OP(O)(O - )O-、-(C1-C 22 )alkylene-OP(O)(O - )O-(C1-C 22 )alkylene-,-(C1-C 22 )alkylene-OP(O)O-、-(C1-C 22 )alkylene-OP(O)(S)O-、-(C1-C 22 )alkylene-OS(O)2-O-、-(C1-C 22 )alkylene-S(O)2-O-、-(C1-C 22)alkylene-S(O)-O-、-OP(O)2-O-(C1-C 22 )alkylene-OP(O)2O-, -OP(O)-O-(C1-C 22 )alkylene-OP(O)O-, -OP(O)(S)O-(C1-C 22 )alkylene-OP(O)(S)O-、-OS(O)2-O-(C1-C 22 )alkylene-OS(O)2-O-, -S(O)2-O-(C1-C 22 )alkylene-S(O)2-O- and -OS(O)-(C1-C 22 )alkylene-S(O)-O-; wherein the connecting portion contains -(C1-C 22 The alkylene group can be an alkylene group containing 1 to 22 carbon atoms, such as 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” as used interchangeably herein may refer to a non-human (e.g., mammalian) subject or a human subject.

[0110] Double-stranded oligonucleotides activate or upregulate the expression of target genes in cells through the RNAa mechanism. The RNAa mechanism (also known as RNA activation) used in this article refers to the mechanism by which double-stranded nucleic acid structures can upregulate target genes at the transcriptional level in a sequence-specific manner.

[0111] In some implementations, the double-stranded oligonucleotide agents described herein can upregulate the expression of a target gene by at least 10% compared to the expression of an unregulated gene, 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%.

[0112] In some embodiments, the expression of the target gene 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 implementations, the double-stranded oligonucleotides described herein reduce at least the same or at least 10% of the off-target effects compared to the same but non-GNA double-stranded oligonucleotides, 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.

[0115] The exemplary target gene used in this article is the surviving motor neuron 2 (SMO2). SMN2 The surviving motor neuron (SMN) gene produces proteins that maintain the health and normal function of motor neurons. SMN1 Insufficient SMN protein due to gene mutations can lead to spinal muscular atrophy (SMA). This is known. SNM2 Higher or activated expression of the gene is associated with paralogous salvage therapy for less severe SMA symptoms.

[0116] Another exemplary target gene used in this article is SERPING1 Gene. SERPING1 Primarily expressed in the liver, it encodes 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. SERPING1 Gene expression is upregulated via RNA activation, and related diseases (especially HAE) are treated by increasing the expression levels of the C1IHN protein (i.e., the C1 repressor protein). Because... SERPING1 The gene encodes the C1IHN protein, therefore SERPING1 Increased mRNA expression raised the level of C1IHN protein.

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

[0118] Cells including oligonucleotide agents

[0119] On the other hand, this article provides a cell comprising the double-stranded oligonucleotide agent or conjugate described herein.

[0120] Upon contact with cells, the double-stranded oligonucleotide agents or conjugates described herein can effectively activate or upregulate the expression of target genes in cells, for example, by activating or upregulating the expression of target genes by at least 10% compared to the expression of unregulated genes, or by activating or upregulating the expression of target genes to a level that is the same as 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] Cells may include one or more of the double-stranded oligonucleotide agents or one or more of the 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 can be in vitro or ex vivo, such as cell lines or cell strains, or can be present in mammals, such as humans.

[0123] Compositions including oligonucleotide agents or conjugating agents

[0124] On the other hand, this document provides a composition comprising the double-stranded oligonucleotide agent or conjugating agent described herein.

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

[0126] In some embodiments, the composition is a pharmaceutical composition. In some embodiments, the composition includes 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-chain / double-chain 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, the composition includes 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 including oligonucleotides

[0129] On the other hand, this article provides a kit comprising the double-stranded oligonucleotide agent or conjugate described herein.

[0130] As used herein, “kit” is generally defined as a package, component, or container (e.g., an insulated container) that includes one or more of the components or embodiments of this disclosure and / or other components related to this disclosure, such as those described above. Any reagent or component of the kit may be provided in liquid form (e.g., a solution) or in solid form (e.g., a dried powder, frozen, etc.).

[0131] The kit may include one or more of the double-stranded oligonucleotide agents or one or more 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 the target gene in cells or a subject. In some embodiments, the kit is used to prevent or treat diseases, symptoms, or conditions 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 the 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 may be used for the prevention or treatment of a disease, symptom, or condition induced by insufficient expression of a target gene in the subject.

[0135] In another embodiment, the kit may include instructions for use of the kit in any form, or instructions from a website or other source, relating to the use of the components and / or methods described herein. 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 temperatures, 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] Uses of oligonucleotide agents or conjugators

[0137] On the other hand, this article provides a method for activating or upregulating target genes in cells or subjects, including administering the double-stranded oligonucleotide agent or conjugate described herein to the cells or subjects.

[0138] On the other hand, this article also provides a method for mitigating off-target effects caused by double-stranded oligonucleotide agents or conjugates capable of activating the expression of target genes in cells or subjects, comprising administering the double-stranded oligonucleotide agent or conjugate described herein to cells or subjects.

[0139] In some embodiments, when the double-stranded oligonucleotide agent or conjugate described herein is administered, for example, to cells or a 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 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%.

[0140] In some embodiments, when the double-stranded oligonucleotide agent or conjugate described herein is administered, for example, 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 implementations, 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 at least 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.

[0143] In some implementations, 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 can be in vitro or ex vivo, such as cell lines or cell strains, or they can be present in mammals, such as humans.

[0145] In some implementations, the subject is a mammal. In some implementations, the subject is a human. In some implementations, the subject is a person who has or is at risk of having a disease, condition, or ailment associated with insufficient expression of a 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 implementations, 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, 60 nM, 65 nM, 70 nM. nM, 75nM, 80nM, 85nM, 90nM, 95nM, 100nM, 105nM, 110nM, 115nM, 120nM, 125 nM, 130nM, 135nM, 140nM, 145nM, 150nM, 155nM, 160nM, 165nM, 170nM, 175n M, 180nM, 185nM, 190nM, 195nM, 200nM, 220nM, 240nM, 260nM, 280nM, 300nM , 320nM, 340nM, 360nM, 380nM, 400nM, 420nM, 440nM, 460nM, 480nM and 500nM.

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

[0149] In some embodiments, administering the double-stranded oligonucleotide agent or conjugate described herein to a subject includes administering a composition (e.g., a pharmaceutical composition) comprising the double-stranded oligonucleotide agent described herein in an amount effective in preventing or treating a disease, symptom, or condition.

[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 group consisting of: intrathecal, intramuscular, intravenous, intraarterial, intraperitoneal, intravesical, intraventricular, intravitreal, subcutaneous administration, 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 can range from 0.01 mg / kg to 1000 mg / kg of the subject's body weight, for example, approximately 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, etc. Single doses of g / 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, 300 mg / kg, 400 mg / kg, 500 mg / kg, 750 mg / kg, or 1000 mg / kg of the subject's body weight. The dosages described herein may include one or more of any of the double-stranded oligonucleotide agents or conjugates described herein.

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

[0154] Examples of other compositions or components associated with the double-stranded oligonucleotide agents, 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 the purpose of use, modification, assembly, storage, packaging, preparation, mixing, dilution, and / or preservation. In embodiments using any component in liquid form, the liquid form may be concentrated or ready for immediate 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 containing 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 in, for example, Ansel HC et al., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems (2004), Lippincott, Williams & Wilkins, Philadelphia; Gennaro AR 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 drug (i.e., the double-stranded oligonucleotide agent or its pharmaceutical composition described herein) or to contribute to the preparation of the pharmaceutical product (i.e., the drug).

[0159] On the other hand, this article provides the use of double-stranded oligonucleotide agents or conjugators in the preparation of products.

[0160] The product can be a composition, a drug, or a kit.

[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 diseases, symptoms, or conditions in a subject that are 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 a double-stranded oligonucleotide agent or conjugate that effectively increases the level of mRNA or protein encoded by the target gene in cells or a subject. In some embodiments, the product includes 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 implementation plan

[0164] Implementation Scheme 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] Implementation Scheme 2. The double-stranded oligonucleotide agent according to Implementation Scheme 1, wherein one or both of the sense strand and the antisense strand are guide strands mediating RNA activation.

[0166] Implementation Scheme 3. The double-stranded oligonucleotide agent according to Implementation Scheme 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] Implementation Scheme 4. The double-stranded oligonucleotide agent according to Implementation Scheme 3, wherein the seed region begins no more than 3 nucleotides from the 5' end of the guide strand.

[0168] Implementation Scheme 5. The double-stranded oligonucleotide agent according to Implementation Scheme 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] Implementation Scheme 6. The double-stranded oligonucleotide agent according to Implementation Scheme 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] Implementation Scheme 7. The double-stranded oligonucleotide agent according to Implementation Scheme 1, wherein the double-stranded oligonucleotide agent comprises 1 to 50 GNA-modified nucleotides.

[0171] Implementation Scheme 8. The double-stranded oligonucleotide agent according to Implementation Scheme 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.

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

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

[0174] Implementation Scheme 11. The double-stranded oligonucleotide agent according to Implementation Scheme 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] Implementation Scheme 12. The double-stranded oligonucleotide agent according to Implementation Scheme 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) The bases mentioned herein are selected from the group consisting of: adenine nucleobases, thymine nucleobases, cytosine nucleobases, guanine nucleobases, uracil nucleobases, and analogues thereof.

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

[0177] Implementation Scheme 14. The double-stranded oligonucleotide agent according to Implementation Scheme 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] Implementation Scheme 15. The double-stranded oligonucleotide agent according to Implementation Scheme 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] Implementation Scheme 16. The double-stranded oligonucleotide agent according to Implementation Scheme 15, wherein the mismatched nucleotide is located inside or near the 3' or 5' end of the antisense strand.

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

[0181] Implementation Scheme 18. The double-stranded oligonucleotide agent according to Implementation Scheme 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] Implementation Scheme 19. The double-stranded oligonucleotide agent according to Implementation Scheme 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.

[0183] Implementation Scheme 20. A conjugating agent comprising a double-stranded oligonucleotide agent according to any one of Implementation Schemes 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] Implementation Scheme 21. The conjugating agent according to Implementation Scheme 20, wherein the conjugating moiety is selected from auxiliary oligonucleotides (ACO), lipids / fatty acids, and GalNAc clusters.

[0185] Implementation Scheme 22. The binding agent according to Implementation Scheme 21, wherein the binding portion is selected from:

[0186] (tC2x6), and

[0187] (C5x5) in Represents the carrier material.

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

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

[0190] Implementation Scheme 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 Implementation Schemes 1-19, or a conjugate agent according to any one of Implementation Schemes 20-22, or a pharmaceutical composition according to Implementation Scheme 24.

[0191] Implementation Scheme 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 Implementation Schemes 1-19 or a conjugate agent according to any one of Implementation Schemes 20-22.

[0192] Implementation Scheme 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 Implementation Schemes 1-19 or a conjugate agent according to any one of Implementation Schemes 20-22.

[0193] Implementation Scheme 28. Use of the double-stranded oligonucleotide agent according to any one of Implementation Schemes 1-19 or the conjugate agent according to any one of Implementation Schemes 20-22 in the preparation of a product for activating or upregulating target genes in cells or subjects.

[0194] Example

[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 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 provided 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 pairs, kb for kilobases, nM for nanomoles, s or sec for seconds, min for minutes, h or hr for hours, aa for amino acids, nt for nucleotides, im for intramuscular, ip for intraperitoneal, sc 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 were purchased from commercial sources and used as received. Purification of the reaction products was performed by column chromatography using silica gel (200-300 mesh) and hexane / ethyl acetate, DCM / MeOH as eluent. Thin-layer chromatography (TLC) was performed using pre-coated silica gel GF plates and developed with KMnO4 staining agent. ¹H NMR spectra were recorded using CDCl₃ with TMS at 400 or 500 MHz (Variant). High-resolution mass spectrometry (HRMS) was recorded by ESI or matrix-assisted laser desorption / ionization (MALDI) on an LC / MS (Agilent Technologies 1260 Infinity II / 6120 Quadrupole) and time-of-flight mass spectrometer.

[0198] Example 1 Preparation of the compound GNA-U disclosed herein

[0199] In this embodiment, compound GNA-U was prepared using the following method.

[0200]

[0201] (1) Preparation of compound 2

[0202] DMTrCl (64.5 g, 190 mmol) was added to a solution of compound 1 (10 mL, 151 mmol, 1.0 eq) and Et3N (42 mL, 302 mmol, 2.0 eq) in CH2Cl2 (340 mL). After 12 hours, the reaction mixture was poured into a saturated aqueous solution of NaHCO3 (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 used... 1 H NMR characterization. 1 H NMR (400 MHz, CDCl3) δ7.48 – 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] To 45 mL of anhydrous DMF solution of compound 11 (2.5 g, 22.3 mmol, 1.2 eq), NaH (148 mg, 3.7 mmol, 60% mineral oil solution, 0.2 eq) was added, 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 residue was purified by rapid chromatography (silica gel, gradient eluent: 50%-100% ethyl acetate / hexane, 1% Et₃N) to give compound 3 (6.14 g, 67% yield). The product was analyzed by mass spectrometry and... 1 1H NMR characterization. Calculated MW: 488.19; Measured MW: 487.63 [MH] + .1 H NMR (400 MHz, CDCl3) δ 7.42 – 7.38 (m, 2H), 7.33 – 7.24 (m, 7H), 7.16 (d, J = 7.9Hz, 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, 2-cyanoethyl N,N-diisopropylphosphonamide (2.7 mL, 12.3 mmol, 3.0 eq) was added to an anhydrous DCM (40 mL) solution of compound 3 (2.0 g, 4.1 mmol, 1.0 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... 1 H NMR characterization. 1 H 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 2Preparation of the compound GNA-T disclosed herein

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

[0209]

[0210] (1) Preparation of compound 4

[0211] To 122.0 mL of anhydrous DMF solution of compound 12 (4.8 g, 38.3 mmol, 1.2 eq), NaH (256 mg, 6.4 mmol, 60% mineral oil solution, 0.2 eq) was added, and the mixture was stirred for 1 hour under nitrogen protection. To the same solution, 10.0 mL of anhydrous DMF solution of compound 2 (12.0 g, 31.9 mmol, 1.0 eq) was added, 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 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 analyzed by mass spectrometry and... 1 1H NMR characterization. Calculated MW: 502.21; Measured MW: 500.74 [MH] + . 1 HNMR (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, 2-cyanoethyl N,N-diisopropylphosphonamide (11.3 mL, 50.7 mmol, 3.0 eq) was added to an anhydrous DCM solution of compound 4 (8.5 g, 16.9 mmol, 1.0 eq) and DIPEA (8.4 mL, 50.7 mmol, 3.0 eq) (169.0 mL). 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-T (10.0 g, 84% yield). The product was analyzed by mass spectrometry and... 1 ¹H NMR characterization. Calculated MW: 702.32; Measured MW: 617.66 [M-diisopropyl-H] + . 1 H NMR (400 MHz, CDCl3) δ 7.48 – 7.41 (m, 2H), 7.36 – 7.26 (m, 7H), 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 the compound GNA-C disclosed herein

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

[0216]

[0217] (1) Preparation of compound 5

[0218] To 55.0 mL of anhydrous DMF solution of compound 13 (2.68 g, 17.5 mmol, 1.1 eq), NaH (136 mg, 3.7 mmol, 60% mineral oil solution, 0.2 eq) was added, and the mixture was stirred for 1 hour under nitrogen protection. 5.0 mL of anhydrous DMF solution of compound 2 (6.0 g, 15.9 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 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... 1 H NMR characterization. 1 H 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– 3.06 (m, 1H), 2.21 (s, 3H).

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

[0220] Under a nitrogen atmosphere, at room temperature, 2-cyanoethyl N,N-diisopropylphosphonamide (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). The reaction mixture was stirred for 1.0 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-C (2.2 g, 94% yield). The product was... 1 H NMR characterization. 1 H 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 disclosed herein

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

[0223]

[0224] (1) Preparation of compound 6

[0225] To 65.0 mL of anhydrous DMF solution of compound 14 (2.28 g, 16.9 mmol, 1.1 eq), NaH (140 mg, 3.51 mmol, 60% mineral oil solution, 0.22 eq) was added, and the mixture was stirred for 2 hours under nitrogen protection. 5.0 mL of anhydrous DMF solution of compound 2 (6.0 g, 15.9 mmol, 1.0 eq) was added to the above solution, and the reaction mixture was 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 analyzed by mass spectrometry and... 1 1H NMR characterization. Calculated MW: 511.58; Measured MW: 512.37 [M+H] + . 1H NMR (400MHz, 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 analyzed by mass spectrometry and... 1 1H NMR characterization. Calculated MW: 566.66; Measured MW: 567.62 [M+H] + . 1 H 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 of compound GNA-A

[0229] Under a nitrogen atmosphere, at room temperature, 2-cyanoethyl N,N-diisopropylphosphonamide (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 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: 1%–5% MeOH / DCM, 1% Et3N) to give compound GNA-A (2.4 g, 89% yield). The product was... 1 H NMR characterization. 1 H 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 the compound GNA-G disclosed herein

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

[0232]

[0233] (1) Preparation of compound 8

[0234] To 55.0 mL of anhydrous DMF solution of 6-(benzyloxy)-9H-purine-2-amine compound 15 (4.20 g, 17.5 mmol, 1.05 eq), NaH (140 mg, 3.50 mmol, 60% mineral oil solution, 0.22 eq) was added, and the mixture was stirred for 1 hour under nitrogen protection. 5.0 mL of anhydrous DMF solution of compound 2 (6.0 g, 15.9 mmol, 1.0 eq) was added to the above solution, and the reaction mixture was heated to 90 °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 8 (5.0 g, 51% yield). The product was analyzed by mass spectrometry and... 1 1H NMR characterization. Calculated MW: 617.26; Measured MW: 618.75 [M+H] + . 1 H 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] To 30.0 mL of anhydrous DMF solution of compound 9 (3.8 g, 7.2 mmol, 1.0 eq), dimethylformamide dimethyl acetal (3.4 mL, 25.1 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 (2.8 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, at room temperature, 2-cyanoethyl N,N-diisopropylphosphonamide (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). The reaction mixture was stirred for 0.5 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: 1%–5% MeOH / DCM, 1% Et3N) to give compound GNA-G (1.2 g, 59% yield). The product was... 1 H NMR characterization. 1 H 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 was designed as a targeted weapon. SMN2 The promoter of a gene is a sequence that activates the transcription of its alleles via an RNA activation (RNAa) mechanism. This can be regulated by transfecting cell lines with chemically modified saRNA derived from RD-12318. SMN2-FL (i.e., the full length including exon 7) SMN2 )and SMN2-Δ7 (i.e., excluding exon 7) SMN2 The expression level of mRNA of ) can be increased, thereby enhancing the expression level of ) by SMN2-FL The level of SMN protein encoded by mRNA. "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 and has chemical modifications, namely 2'-fluorine, 2'-O-methyl (2'-OMe), and 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 was designed as a target SMN2 The promoter of a gene is a sequence that activates the transcription of alleles 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'-fluorine, 2'-O-methyl (2'-OMe), 5'-( E )-Vinylphosphonate and thiophosphate (PS) main chain modification. 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 they have GNA modification at positions 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, and 22, counting from the 5' end of their antisense strand. The synthesis of RD-19672 is the same as that of RD-10994, except that it has two GNA modifications at positions 8 and 17 (counting from the 5' end) on its antisense strand. The synthesis of RD-19674 is the same as that of RD-10994, except that it has a GNA modification at position 9 (counting from the 5' end) on its antisense strand and at position 13 (counting from the 5' end) on its sense strand. The synthesis of RD-19675 is the same as that of RD-10994, except that it has a GNA modification at position 10 (counting from the 5' end) on its antisense strand and at position 12 (counting from the 5' end) on its sense strand. "nG", "nU", "nA", and "nC" represent GNA-modified nucleotides.

[0246] Non-GNA-saRNAs (namely RD-17229, RD-17235, RD-17238, RD-17241, and RD-17244) were designed to target SERPING1The promoters of these genes activate the transcription of alleles via the RNAa mechanism. The synthesis of their corresponding GNA-saRNAs (RD-17074, RD-17082, RD-17086, RD-17096, and RD-17099) is the same as that of 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 through 8, counting from the 5' end of their sense strand. The "seed" region of RD-17099 is located at positions 2 through 8, counting from the 5' end of its antisense strand. "nG", "nA", and "nC" represent GNA-modified nucleotides.

[0247] Table 1. Oligonucleotide sequences and compositions

[0248] Note: Uppercase letters indicate RNA; Indicates main chain modification of thiophosphate (PS); f represents 2'-fluorine; m represents 2'-O-methyl (2'-OMe); Vp represents 5'-( E )-Vinylphosphonate; me represents 2'-O-methoxyethyl (2'MOE); meC represents 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, in italics, bold, or gray highlighted. GUUGCUU and GCAGGCC Indicates the "seed" region; n / a: not applicable.

[0249] Example 7 GNA-saRNA in regulating GM03813 cells SMN2-Δ7 Transform into SMN2-FL in vitro activity of mRNA

[0250] To assess the in vitro activity of GNA-saRNA at various nucleotide positions, designated 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 designated 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 and used as a non-GNA control. In each PCR reaction, gene-specific primer sets were used for RT-qPCR. SMN2-FL The mRNA level was quantified and plotted on [the graph / plot]. Figure 1 Table 2 summarizes... SMN2-FL The maximum efficacy of expression (i.e., E) max The area under the curve (AUC) and the area under the curve were calculated using GraphPad Prism software, as described in the Materials and Methods section. SERPING1 The area under the curve (AUC) of mRNA expression was measured. The results showed that, compared to non-GNA-saRNA RD-10994, GNA-saRNA enhanced the expression of mRNA. SMN2-FL mRNA expression activity.

[0251] Table 2. GNA-saRNA treatment results in GM03813 cells SMN2-FL E expressed max and AUC

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

[0253] like Figure 2 A to Figure 2 As shown in Figure H, GNA-saRNA exhibited higher induction rates in GM03813 cells. SMN2-FL The activity was reduced at the same time. SMN2-Δ7 mRNA. Figure 3 A to Figure 3 As shown in Figure H, GNA-saRNA exhibited higher induction rates in GM22592 cells. SMN2-FL The activity was reduced at the same time. SMN2-Δ7 mRNA levels were increased in all GNA-saRNAs. SMN2-FL mRNA levels indicate that GNA-saRNA can regulate SMN2-Δ7 Transform into SMN2-FL mRNA to enhance the effect SMN2-FL mRNA expression activity.

[0254] Example 8 GNA-saRNA in regulating GM03813 cells SMN2-Δ7 Transform into SMN2-FL in vitro activity of mRNA

[0255] To confirm the in vitro activity of GNA modifications at various nucleotide sites, designated GNA-saRNAs (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, gene-specific primer sets were used for RT-qPCR. SMN2-FL and SMN2-Δ7 Quantification of mRNA levels. For example... Figure 4A and Figure 4B As shown, compared to RD-10994 (a non-GNA saRNA), all GNA-saRNAs exhibited higher induction rates at 100 nM. SMN2-FL The activity was reduced at the same time. SMN2-Δ7 mRNA. SMN2-FL and SMN2-Δ7 The expression of mRNA is summarized in Table 3. The results indicate that GNA-saRNA can regulate... SMN2-Δ7 Transform into SMN2-FL mRNA to enhance the effect SMN2-FL mRNA expression activity.

[0256] Table 3. GM03813 cells SMN2-FL and SMN2-Δ7 mRNA expression

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

[0258] Example 9 GNA-saRNA reduces potential off-target genes in GM03813 cells. P2RY2 Off-target effects of expression.

[0259] To assess the off-target mitigation effects of GNA-saRNA, the antisense strand of RD-10994 was used as a "search" sequence to search for potential off-target genes via computer analysis and to predict them. P2RY2 These are potential off-target genes. Figure 5A The image shows the "retrieval" sequence (antense strand) and "seed" region (highlighted in gray) of RD-10994, as well as its position within a region containing two mismatched nucleotides (italicized and bolded nucleotides). P2RY2Predicted complementary sequences for target sites in transcripts. GM03813 cells were transfected with designated GNA-saRNAs (RD-15977, RD-15978, RD-15981, RD-15982, and RD-15983) 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, gene-specific primer sets were used for RT-qPCR to target the target sites. P2RY2 Quantification of mRNA levels. For example... Figure 5B As shown, all GNA-saRNAs reduced off-target effects compared to the non-GNA control RD-10994, except for RD-15981 when treated with 25 nM. P2RY2 The expression of mRNA is summarized in Table 4. The results show that GNA modification at different locations can mitigate off-target effects to varying degrees.

[0260] Table 4. GM03813 cells P2RY2 mRNA expression

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

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

[0263] To assess the in vitro activity of GNA modifications at various nucleotide sites, 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) 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. Figure 6A and Figure 6B As shown, compared to RD-19040 (a non-GNA saRNA), all GNA-saRNAs exhibited higher induction rates at 2.5 nM. SMN2-FL and SMN2-Δ7 mRNA activity. SMN2-FL and SMN2-Δ7The expression of mRNAs is summarized in Table 5. The results indicate that GNA modification of SMN2 saRNAs enhances their ability to induce... SMN2-FL and SMN2-Δ7 Targeting activity in mRNA expression.

[0264] Table 5. GM03813 cells SMN2-FL and SMN2-Δ7 mRNA expression

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

[0266] Example 11 GNA-saRNA reduces potential off-target genes in GM03813 cells. ARPIN Off-target effects of expression.

[0267] To assess the off-target mitigation effects of GNA-saRNA, the antisense strand of RD-19040 was used as a "search" sequence to search for potential off-target genes via computer analysis and to predict them. ARPIN These are potential off-target genes. Figure 7A The image shows the "retrieval" sequence (antense strand) and "seed" region (highlighted in gray) of RD-19040, as well as its region containing two mismatched nucleotides (italicized and bolded nucleotides). ARPIN Predicted complementary sequences for target sites in transcripts. Specific 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 and used as a non-GNA control. In each PCR reaction, gene-specific primer sets were used for RT-qPCR to target the target sites. ARPIN Quantification of mRNA levels. For example... Figure 7B As shown, all GNA-saRNAs reduced off-target effects compared to RD-19040, which is a non-GNA saRNA. ARPIN The expression of mRNA is summarized in Table 6. The results show that GNA modification at different positions can mitigate off-target effects to varying degrees.

[0268] Table 6. GM03813 cells ARPIN mRNA expression

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

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

[0271] To verify the in vitro activity of GNA-saRNA, 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. The activity of non-GNA-saRNAs and GNA-saRNAs was... SERPING1 mRNA levels such as Figure 8 A to Figure 8 As shown in E.

[0272] Similarly, specified non-GNA-saRNAs (RD-17229, RD-17235, and RD-17241) and their corresponding GNA-saRNAs (RD-17074, RD-17082, and RD-17096) were transfected into HepG2 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. The non-GNA-saRNA and GNA-saRNA... SERPING1 mRNA levels such as Figures 9A to 9C As shown.

[0273] Table 7 summarizes... SERPING1 E expressed max And AUC. As described in the Materials and Methods section, calculated using GraphPadPrism software. SERPING1 The AUC of mRNA expression was calculated. The results indicate that GNA-saRNA can enhance the effect of non-GNA-saRNA in inducing mRNA expression. SERPING1 Targeting activity in mRNA expression.

[0274] Table 7. GNA-saRNA treatment results in Hep3B and HepG2 cells SERPING1 E expressed max and AUC

[0275] Note: "n / a" indicates not applicable, and " / " indicates not detected.

[0276] Materials and methods

[0277] saRNA synthesis

[0278] (1) Single-chain synthesis

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

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

[0281] Phosphamide addition: Each phosphoramide addition cycle consists of four chemical reactions, including detriphenylmethylation, coupling, oxidation / thiolization, and capping. In the first step, detriphenylmethylation is performed for 45 seconds using DCM in 3% dichloroacetic acid (DCA). In the second step, all phosphoramides are coupled at 12 eq for 6 minutes. In the third step, oxidation is performed for 1 minute using a solution of 0.02 M iodine in THF:pyridine:water (70:20:10, v / v / v); if thiophosphate modification is required, thiolization is performed for 3 minutes instead of oxidation using a solution of 0.1 M hydroflavin in pyridine:ACN (50:50, v / v). In the fourth step, capping is 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 depends on the length of the individual oligonucleotide.

[0282] Deprotection I (nucleobase deprotection): After synthesis, the solid support was transferred to a screw-cap 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 60-65 °C for 15 minutes, followed by cooling to room temperature. The lysis solution was collected and evaporated to dryness in a SpeedVac to obtain crude single-chain oligonucleotides.

[0283] Deprotection II (removal of 2'-TBDMS groups): If the crude RNA oligonucleotide still carries 2'-TBDMS groups, dissolve it in 0.1 mL DMSO. Add 1 mL triethylamine trihydrofluoride, cap the tube, and vigorously shake the mixture to ensure complete dissolution. Then heat in an oven at 65 °C for 15 minutes. Remove the tube from the oven and cool to room temperature. Cool the solution containing the fully desilylated oligonucleotide on dry ice. Carefully add 2 mL of ice-cold n-butanol (-20 °C) in 0.5 mL increments to precipitate the oligonucleotide. Filter the precipitate, wash with 1 mL of ice-cold n-butanol, and then dissolve in 0.01 M tris(hydroxymethyl)aminomethanol hydrochloride buffer.

[0284] (2) Single-chain purification

[0285] Oligonucleotide purification was performed 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 forms a double chain

[0287] For the duplex, after generating a desalted purified single-stranded solution, the sense and antisense strands were mixed in an equimolar volume in a tube. The tube was placed in a heating block at 95°C for 5 minutes, and then cooled to room temperature. The resulting duplex was then lyophilized into a 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 with Lipofectamine RNAiMax (Thermo Fisher Scientific, Waltham, Massachusetts, USA) was performed in the growth medium 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. This was achieved using PrimeScript containing a gDNA Eraser. TM The resulting RNA (1 μg) was reverse transcribed into cDNA using an RT kit (Takara, RR047A, Shiga, Japan). TBGreen was used in a Roche LightCycler 480 Multiwell Plate 384 (Roche, Reference: 4729749001, USA). ® Premix Ex Taq TM The obtained cDNA was amplified using the Takara (RR820A, Shiga, Japan) reagent and primers specifically designed for amplifying the target gene.

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

[0294] Table 8. RT reaction

[0295] Table 9. RT-qPCR reactions

[0296] Table 10. Primer sequences used for RT-qPCR assay

[0297] A reference gene

[0298] To calculate the expression level of target mRNA in saRNA-treated samples relative to control samples ( E rel Substitute the Ct values ​​of the target gene and the internal reference gene into Formula I.

[0299] (Formula I)

[0300] Where CtTm is the Ct value of the target gene from the control 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 sample; and 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 of target gene mRNA in saRNA-treated samples relative to control samples ( E rel Substitute the Ct values ​​of the target gene and the two internal reference genes into the following formula II.

[0303] (Formula II)

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

[0305] Calculate the area under the curve (AUC).

[0306] The area under the curve (AUC) of a specific test product is derived from a curve plotted using dose-response data at eight doses. This curve reflects the cumulative effect of the test product across 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 the AUC, we first plotted an agonist versus response curve based on the dose-response data. The curve was then evaluated using the "Area Under the Curve" 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 articles.

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.

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