Glycerol nucleic acid-modified oligonucleotide agents and their use

Abstract

This specification 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 having a length of 15 to 35 nucleotides, wherein the sense strand and the antisense strand have at least 50% complementarity to each other and form a complementary double-stranded structure of at least 8 base pairs, and the double-stranded oligonucleotide agent comprises one or more glycerol nucleic acid (GNA) modified nucleotides located on either or both of the sense strand and the antisense strand.

Description

Technical Field

[0001] The present disclosure relates to the technical field of nucleic acids, and particularly to oligonucleotide agents capable of activating gene expression while reducing off-target effects, and their use as double-stranded RNA (dsRNA) pharmaceuticals.

Background Art

[0002] Oligonucleotides are currently a new class of therapeutics that are being actively developed for the treatment of a variety of diseases. The main classifications of therapeutic oligonucleotide agents include single-stranded antisense oligonucleotides and dsRNA. dsRNA further includes two major classifications, namely, small interfering RNA (siRNA) and small activating RNA (saRNA). Argonaute (AGO) proteins are required for both, but siRNA and saRNA have significant differences in their mechanistic frameworks.

[0003] Gene activation via saRNA provides a promising strategy for upregulating the expression of target genes by promoting endogenous transcription known as RNA activation (RNAa). SaRNA activates target genes by directly binding to the target gene promoter or interacting with antisense transcripts transcribed from overlapping promoter sequences. Despite the promise of this technology for therapeutic applications, in its development process, it faces significant challenges, particularly the undesirable "off-target" effects and the associated toxicity. Ensuring that saRNA meets strict safety requirements in both discovery and drug development remains a critically important issue in the art.

Summary of the Invention

Problems to be Solved by the Invention

[0004] To solve the above problems, the present disclosure provides oligonucleotide agents such as saRNA that reduce off-target effects while maintaining and even enhancing gene activation ability.

[0005] In one embodiment, the present 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 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, and the double-stranded oligonucleotide agent comprises one or more glycerol nucleic acid (GNA) modified nucleotides.

[0006] In another embodiment, the present disclosure provides a conjugate agent comprising a double-stranded oligonucleotide agent described herein and at least one conjugation site.

[0007] In another embodiment, the present disclosure provides cells comprising the double-stranded oligonucleotide agent or conjugate agent described herein.

[0008] In other embodiments, the Disclosure provides compositions comprising the double-stranded oligonucleotide agent or conjugate agent described herein.

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

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

[0011] In another embodiment, the Disclosure provides a method for reducing off-target effects caused by double-stranded oligonucleotide agents that can activate or upregulate the expression of a target gene in cells or subjects, the method comprising administering the double-stranded oligonucleotide agent or conjugate described herein to the cells or subjects.

[0012] In another embodiment, this disclosure provides the use of the double-stranded oligonucleotide agents or conjugate agents described herein in the manufacture of products.

[0013] The double-stranded oligonucleotide agents (such as saRNA) provided herein can efficiently 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 in the preparation of products for preventing or treating diseases, conditions, or disorders associated with insufficient expression of the aforementioned genes.

[0014] Novel features of the present invention are specifically described in the appended claims. The features and advantages of the present invention will be better understood by referring to the following detailed description illustrating exemplary embodiments in which the principles of the present invention are used, and by referring to the accompanying drawings (hereinafter also referred to as "figure" or "FIG."). [Brief explanation of the drawing]

[0015] [Figure 1]This study demonstrates the activity of glycerol-modified nucleic acid saRNAs (GNA-saRNAs) against full-length SMN2 (SMN2-FL) mRNA expression in GM03813 cells. The indicated GNA-saRNAs (i.e., RD-15977, RD-15978, RD-15979, RD-15980, RD-15981, RD-15982, and RD-15983) were transfected into GM03813 cells for 3 days at the indicated concentrations (i.e., 0.78, 1.56, 3.13, 6.25, 12.5, 25, 50, and 100 nM). RD-10994 was transfected and used as a non-GNA control. Mock treatment was performed in the absence of oligonucleotides (not shown). dsCon2 was used as a non-specific double-strand control (not shown). In each PCR reaction, SMN2-FL mRNA levels were quantified by RT-qPCR using gene-specific primer sets. The geometric mean mRNA levels of SDHA and GAPDH were used as an internal control to normalize the expression data. The data show the mean expression level of SMN2-FL relative to mock treatment after normalization for SDHA and GAPDH (mean of two replication transfection wells ± SEM). [Figure 2]This study demonstrates the activity of GNA-saRNAs on the expression of full-length (SMN2-FL) and exon 7-skipped (SMN2-Δ7) SMN2 mRNA in GM03813 cells. The indicated GNA-saRNAs (i.e., RD-15977, RD-15978, RD-15979, RD-15980, RD-15981, RD-15982, and RD-15983) were transfected into GM03813 cells for 3 days at the indicated concentrations (i.e., 0.78, 1.56, 3.13, 6.25, 12.5, 25, 50, and 100 nM). RD-10994 was transfected and used as a non-GNA control. Mock treatment was performed in the absence of oligonucleotides (not shown). dsCon2 was used as a non-specific double-strand control (not shown). Figures 2A–2H show the mRNA levels of SMN2-FL and SMN2-Δ7 quantified by RT-qPCR using gene-specific primer sets in each PCR reaction. The geometric mean mRNA levels of SDHA and GAPDH were used as an internal control to normalize the expression data. The data represent the mean expression levels of SMN2-FL or SMN2-Δ7 relative to mock treatment after normalization for SDHA and GAPDH (mean of two replication transfection wells ± SEM). [Figure 3]This study demonstrates the activity of GNA-saRNAs on the expression of SMN2-FL and SMN2-Δ7 mRNA in GM22592 cells. The indicated GNA-saRNAs (i.e., RD-15977, RD-15978, RD-15979, RD-15980, RD-15981, RD-15982, and RD-15983) were transfected into GM22592 cells for 3 days at the indicated concentrations (i.e., 0.78, 1.56, 3.13, 6.25, 12.5, 25, 50, and 100 nM). RD-10994 was transfected and used as a non-GNA control. Mock treatment was performed in the absence of oligonucleotides (not shown). dsCon2 was used as a non-specific double-strand control (not shown). Figures 3A–3H show the mRNA levels of SMN2-FL and SMN2-Δ7 quantified by RT-qPCR using gene-specific primer sets in each PCR reaction. The geometric mean of SDHA and GAPDH mRNA levels was used as an internal control to normalize the expression data. The data represent the mean expression level of SMN2-FL or SMN2-Δ7 relative to mock treatment after normalization for SDHA and GAPDH (mean of two replication transfection wells ± SEM). [Figure 4]This document describes the activity of GNA-saRNAs on the expression of SMN2-FL and SMN2-Δ7 mRNA in GM03813 cells. The indicated GNA-saRNAs (i.e., RD-15977, RD-15978, RD-15979, RD-15980, RD-15981, RD-15982, and RD-15983) were transfected at 100 nM for 3 days. RD-10994 was transfected and used as a non-GNA control. Mock treatment was performed in the absence of oligonucleotides. dsCon2 was used as a non-specific double-strand control. Figures 4A-4B show the mRNA levels of SMN2-FL and SMN2-Δ7 quantified by RT-qPCR using gene-specific primer sets in each PCR reaction. SDHA was amplified and used as an internal control to normalize the expression data. The data show the mean expression levels of SMN2-FL or SMN2-Δ7 relative to mock treatment after normalization for SDHA (mean ± SEM of four replication transfection wells). [Figure 5]This study demonstrates the off-target effects of GNA-saRNAs on the expression of the potential off-target gene P2RY2 in GM03813 cells. The indicated GNA-saRNAs (i.e., RD-15977, RD-15978, RD-15981, RD-15982, and RD-15983) were transfected into GM03813 cells at 6.25 nM and 25 nM for 3 days. RD-10994 was transfected and used as a non-GNA control. Mock treatment was performed in the absence of oligonucleotides. dsCon2 was used as a non-specific double-strand control (not shown). Figure 5A shows the "query" (antisense strand) sequence and "seed" region of RD-10994 (italicized and highlighted in gray) and its predicted complementary target sites in the P2RY2 transcript, including two mismatched nucleotides (nucleotides are italicized and in bold). Figure 5B shows the mRNA levels of P2RY2 quantified by RT-qPCR using gene-specific primer sets in each PCR reaction. SDHA was amplified and used as an internal control to normalize the expression data. The data show the mean expression level of P2RY2 relative to the mock treatment after normalization against SDHA (mean of four replication transfection wells ± SEM). [Figure 6]This study demonstrates the activity of GNA-saRNAs in the expression of SMN2-FL and SMN2-Δ7 mRNA in GM03813 cells. The indicated GNA-saRNAs (i.e., 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 at 2.5 nM for 3 days. RD-19040 was transfected and used as a non-GNA control. RD-10004 (ASO-1027) was transfected at 25 nM and used as a positive control. Mock treatment was performed in the absence of oligonucleotides. dsCon2M13v was used as a non-specific double-strand control. Figures 6A-6B show the mRNA levels of SMN2-FL and SMN2-Δ7 quantified by RT-qPCR using gene-specific primer sets in each PCR reaction. TBP was amplified and used as an internal control to normalize the expression data. The data show the mean expression levels of SMN2-FL or SMN2-Δ7 relative to mock treatment after normalization against TBP (mean of four replication transfection wells ± SEM). [Figure 7]This study demonstrates the off-target effects of GNA-saRNA on the expression of the potential off-target gene ARPIN in GM03813 cells. The identified GNA-saRNAs (i.e., RD-19650, RD-19651, RD-19652, RD-19653, RD-19654, RD-19655, RD-19657, RD-19661, RD-19662, RD-19674, and RD-19675) were transfected at 2.5 nM for 3 days. RD-19040 was transfected and used as a non-GNA control. RD-10004 (ASO-1027) was transfected at 25 nM and used as a positive control. Mock treatment was performed in the absence of oligonucleotides. dsCon2M13v was used as a non-specific double-strand control. Figure 7A shows the predicted complementary target sites in the ARPIN transcript containing the "query" (antisense strand) sequence and "seed" region of RD-19040 (italicized and highlighted in gray), as well as two mismatched nucleotides (nucleotides are italicized and in bold). Figure 7B shows the mRNA levels of ARPIN quantified by RT-qPCR using gene-specific primer sets in each PCR reaction. TBP was amplified and used as an internal control to normalize the expression data. The data show the mean expression level of ARPIN relative to mock treatment after normalization against TBP (mean of four replication transfection wells ± SEM). [Figure 8]This paper compares the activity of GNA-saRNAs and non-GNA-saRNAs in SERPING1 mRNA expression in Hep3B cells. The indicated non-GNA-saRNAs (i.e., RD-17229, RD-17235, RD-17238, RD-17241, and RD-17244) and their corresponding GNA-saRNAs (i.e., RD-17074, RD-17082, RD-17086, RD-17096, and RD-17099) were transfected into Hep3B cells for 3 days at the indicated concentrations (i.e., 0.02, 0.07, 0.21, 0.62, 1.85, 5.56, 16.67, and 50 nM). Mock treatments were performed in the absence of oligonucleotides (not shown). dsCon2 was used as a non-specific double-strand control (not shown). Figures 8A–8E show the mRNA levels of SERPING1 quantified by RT-qPCR using gene-specific primer sets in each PCR reaction. The geometric mean mRNA levels of HPRT1 and TBP were used as an internal control to normalize the expression data. The data represent the mean expression level of SERPING1 relative to mock treatment after normalization for HPRT1 and TBP (mean of four replication transfection wells ± SEM). [Figure 9]This report compares the activity of GNA-saRNAs and non-GNA-saRNAs in SERPING1 mRNA expression in HepG2 cells. The indicated non-GNA-saRNAs (i.e., RD-17229, RD-17235, and RD-17241) and their corresponding GNA-saRNAs (i.e., RD-17074, RD-17082, and RD-17096) were transfected into HepG2 cells for 3 days at the indicated concentrations (i.e., 0.02, 0.07, 0.21, 0.62, 1.85, 5.56, 16.67, and 50 nM). Mock treatment was performed in the absence of oligonucleotides (not shown). dsCon2 was used as a non-specific double-strand control (not shown). Figures 9A-9C show the SERPING1 mRNA levels quantified by RT-qPCR using gene-specific primer sets in each PCR reaction. The geometric mean mRNA levels of HPRT1 and TBP were used as an internal control to normalize the expression data. The data show the mean expression level of SERPING1 relative to mock treatment after normalization for HPRT1 and TBP (mean of four replication transfection wells ± SEM). [Modes for carrying out the invention]

[0016] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art. Furthermore, all publications, patent applications, patents, and other documents referenced herein are incorporated herein by reference. definition

[0017] As used herein, “and / or” means “and / or alternatively thereto.” Where a numerical range is provided, the range includes any number within the range (including both the upper and lower limits) and any sub-range within the range. For example, the range 1–3 may include any number 1, 2, and 3, as well as the sub-ranges 1–2 and 2–3.

[0018] As used herein, the terms “oligonucleotide,” “polynucleotide,” or “oligo” can be used interchangeably and mean polymers of nucleotides, in particular single-stranded nucleic acid molecules such as DNA, RNA, or DNA / RNA hybrids, oligonucleotide chains containing regularly and irregularly alternating deoxyribosyl and ribosyl moieties, and such oligonucleotides containing modified and naturally occurring or unnaturally occurring skeletons, such as phosphorodiamidate morpholino oligomers (PMOs). Oligonucleotides for activating the transcription of target genes as described herein may be or may contain small activated nucleic acid molecules (saRNAs).

[0019] As used herein, the term “complementary” refers to the ability to form base pairs between two oligonucleotide chains. Base pairs are typically formed by hydrogen bonds between nucleotides in antiparallel oligonucleotide chains. The bases of complementary oligonucleotide chains can form pairs in the Watson-Crick type (e.g., A and T pairs, A and U pairs, C and G pairs) or in any other form that allows for the formation of double helix (e.g., Hoogsteen type or inverse Hoogsteen type base pairing).

[0020] Complementarity includes both complete and incomplete complementarity. "Complete complementarity" or "100% complementarity" means that each nucleotide in the first oligonucleotide chain forms a hydrogen bond with a nucleotide in the second oligonucleotide chain at the corresponding position in the double-stranded region of the double-stranded oligonucleotide molecule, and no base pairs are "unpaired." "Incomplete complementarity" means that not all nucleotide units in the two chains are bonded to each other by hydrogen bonds.

[0021] As used herein, the terms “oligonucleotide chain,” “chain,” and “oligonucleotide sequence” may be used interchangeably and refer to any short nucleotide sequence having fewer than 35 bases (including nucleotides in deoxyribonucleic acid (DNA) or ribonucleic acid (RNA)). In a non-limiting example, the length of a chain may be any length from 15 to 35 nucleotides.

[0022] As used herein, the term “target gene” may refer to nucleic acid sequences, transgenes, viral or bacterial sequences, chromosomes, and / or extrachromosomal genes naturally occurring in living organisms, and / or that can be transiently or stably transfected or incorporated into cells and / or their chromatin. A target gene may be a protein-coding gene or a non-protein-coding gene (such as microRNA genes and long non-coding RNA genes). A target gene generally includes a promoter sequence, and positive regulation of the target gene may be achieved by designing an saRNA having sequence identity (also called homology) with the promoter sequence, characterized in that the expression of the target gene is upregulated. Where the terms “target sequence” or “target site” are used interchangeably, the terms refer to a sequence fragment in the target gene sequence that has homology or complementarity with the sense or antisense strand of the saRNA, such as the target gene promoter. A target gene may also include one or more regulatory elements designed such that one or more saRNAs have sequence identity with the regulatory elements. Limited 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.

[0023] As used herein, the term “guide strand” or “G strand” refers to one strand in a small RNA duplex that associates with the Argonaut (AGO) protein. The other strand, partially or completely complementary to the guide strand, is called the “passenger strand” or “P strand.” While not limited to any particular theory, the strand having a sequence complementary to the target is the antisense strand and, if properly designed, is preferentially selected as the guide strand. In this case, the passenger strand is the sense strand. However, it cannot be definitively determined that a strand is the guide strand until its 5' end is incorporated into the MID domain of AGO2. Therefore, the sense strand may be selected as the guide strand, resulting in the antisense strand becoming the passenger strand. In some embodiments, either the antisense strand or the sense strand may be selected as the guide strand. In some embodiments, both the antisense strand and the sense strand can bind to the Argonaut (AGO) protein, so that each of the sense strand and antisense strand in the RNA duplex can function as a guide strand, either alone or simultaneously.

[0024] As used herein, the term “sense strand” of saRNA in a saRNA double helix refers to a strand that has sequence homology or sequence identity with a fragment of the coding strand of the target gene sequence.

[0025] As used herein, the term “antisense strand” of saRNA in a saRNA double helix refers to a strand having a sequence complementary to the sense strand. The antisense strand may interact with a target region of a target gene to activate or upregulate gene expression, and the target region may be a segment of the coding strand of the target gene sequence.

[0026] As used herein, the term “coding strand” refers to the DNA strand in the target gene that is not available for transcription, and whose nucleotide sequence is identical to that of the RNA produced by transcription (in RNA, T in DNA is replaced with U). In the double-stranded DNA sequence of a target gene promoter as described herein, the coding strand refers to the promoter sequence on the same DNA strand as the DNA coding strand of the target gene.

[0027] As used herein, the term “template strand” refers to the other strand in the double-stranded DNA of a target gene that is complementary to the coding strand, i.e., the strand that can be transcribed into RNA as a template, and this strand is complementary to the transcribed RNA (A-U pairs and G-C pairs). 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. In the double-stranded DNA sequence of a target gene promoter as described herein, the template strand refers to the promoter sequence on the same DNA strand as the DNA template strand of the target gene.

[0028] As used herein, the term “promoter” refers to a sequence that is spatially related to a protein-coding or RNA-coding nucleic acid sequence and plays a regulatory role in the transcription of the protein-coding or RNA-coding nucleic acid sequence. Generally, eukaryotic gene promoters contain 100 to 5000 base pairs, but as used herein, the term “promoter” is not intended to be limited to this length range. Promoter sequences are generally located at the 5' end of a protein-coding or RNA-coding sequence, but can also be present in exon and intron sequences.

[0029] As used herein, the term "GNA" is also called glycerol nucleic acid and refers to a nucleic acid similar to DNA or RNA, but with a different sugar-phosphate diester backbone structure, using propylene glycol instead of ribose or deoxyribose. As used herein, the term "LNA" refers to locked nucleic acid in which the 2'-oxygen atom and 4'-carbon atom are linked by additional crosslinks. As used herein, the term "BNA" refers to nucleic acid crosslinked by 2'-O and 4'-aminoethylene and may include a five-membered or six-membered ring crosslink structure with NO bonds. As used herein, the term "PNA" refers to a nucleic acid mimetic having a pseudopeptide backbone composed of N-(2-aminoethyl)glycine units, in which nucleic acid bases are linked to the nitrogen of glycine via carbonylmethylene linkers.

[0030] As used herein, the terms “identity” or “homology” mean that one oligonucleotide strand (sense strand or antisense strand) of a saRNA has sequence similarity to the coding strand or template strand in a region of the target gene. As used herein, “identity” or “homology” may be at least about 75%, about 79%, about 80%, about 85%, about 90%, about 95%, or 99%.

[0031] As used herein, the term “overhang” refers to a nucleotide at the end (5' or 3') of a double-stranded oligonucleotide that does not form a base pair, formed by one strand extending from the other. The single-stranded region extending from the 3' and / or 5' ends of the double helix is ​​called an overhang.

[0032] As used herein, the term “natural overhang” refers to an overhang consisting of one or more nucleotides identical or complementary to the corresponding position on the target sequence. A natural overhang on the sense strand consists of one or more nucleotides identical to the corresponding position on the DNA target. A natural overhang on the antisense strand consists of one or more nucleotides complementary to the corresponding position on the DNA target.

[0033] As used herein, the term “isolated” means that a substance has been removed from its original or natural environment (e.g., its natural environment if it exists naturally). For example, a polynucleotide or polypeptide that is naturally present in a living animal is not isolated, but the same polynucleotide or polypeptide is considered isolated if it is artificially separated from some or all of the substances coexisting in a natural system. Such a polynucleotide may be part of a vector, and / or a polynucleotide or polypeptide may be part of a composition, and such a vector or composition may still be considered isolated in that it is not part of the environment in which it is found naturally. Isolated molecules can be obtained, for example, by extraction from a natural source, recombinant nucleic acid expression, or chemical synthesis of the molecule. For example, the term “isolated RNA” means an RNA molecule that, if produced by recombinant technology, substantially contains no other cellular material or culture medium, or an RNA molecule that, if chemically synthesized, substantially contains no chemical precursors or other chemical substances. In some embodiments, the materials of this application, such as the polynucleotides, oligonucleotides and / or saRNAs, are isolated.

[0034] As used herein, the terms “gene activation” or “activation of gene expression,” and “upregulation of gene” or “upregulation of gene expression” may be used interchangeably and mean an increase in the transcription, translation, expression, or activity of a particular nucleic acid, determined by measuring the activity or state of a gene at the transcriptional level, mRNA level, protein level, enzyme activity, methylation status, chromatin status or structure, translational level, or in a cell or biological system. These activities or states may be determined directly or indirectly. Furthermore, “gene activation,” “activation of gene expression,” “upregulation of gene,” or “upregulation of gene expression” refers to an increase in activity related to a nucleic acid sequence, regardless of such activation mechanisms. For example, gene activation may occur at the transcriptional level, increasing transcription to RNA, which is then translated into protein, thereby increasing protein expression.

[0035] As used herein, the terms “small activating RNA,” “saRNA,” and “small activating nucleic acid molecule” can be used interchangeably and refer to nucleic acid molecules that can upregulate the expression of a target gene, comprising a first nucleic acid fragment (sense strand) containing a nucleotide sequence having high sequence identity with respect to the non-coding nucleic acid sequence of the target gene (e.g., promoter or enhancer), and a second nucleic acid fragment (antisense strand) containing a nucleotide sequence complementary to the first nucleic acid fragment, wherein the first and second nucleic acid fragments form a double helix. saRNA can consist of a synthetic or vector-expressed single-stranded RNA molecule that can form a hairpin structure by two complementary regions within the molecule, the first region containing a nucleotide sequence having sequence identity with respect to the target region of the gene's promoter, and the second region containing a nucleotide sequence complementary to the first region. The length of the double-stranded region of saRNA is typically about 15–35, 16–32, 17–30, 18–28, 19–26, 20–24, and 21–22 base pairs, and also typically about 15, 16, 17, 18, 19, 20, 21, 22, or 23 base pairs. The terms “saRNA,” “small activated RNA,” and “small activated nucleic acid molecule” also include nucleic acids other than ribonucleotides, including but not limited to modified nucleotides or analogues.

[0036] As used herein, the term “seed region” refers to a region located near or at the 5' end of the guide strand (G strand) of a double-stranded oligonucleotide, which plays a crucial role in target recognition by the oligonucleotide. As described above, the guide strand can be the sense strand, antisense strand, or both of the sense and antisense strands of a double-stranded oligonucleotide. Typically, the length of the seed region is between 2 and 10 nucleotides.

[0037] As used herein, the term “auxiliary oligonucleotide (ACO)” refers to a non-targeted single-stranded oligonucleotide comprising at least six nucleotides, with or without having one or more linker sites conjugated to other oligonucleotides. ACO components are not designed to specifically target any complementary nucleic acid sequences present in the subject being administered the agent. The ACO component can be chemically modified at its skeleton, nucleoside, or other positions, for example, by a phosphorothioate skeleton, mesylphosphoamide skeleton, or boranophosphate skeleton, 2'-fluoro-2'-deoxynucleoside (2'-F), 2'-O-methyl (2'-O-Me), 2'-O-(2-methoxyethyl) (2'-O-MOE), locked nucleic acid (LNA), cross-linked nucleic acid (BNA), peptide nucleic acid (PNA), 5'-(E)-vinylphosphonate moiety, or 5-methylcytosine moiety, thereby conferring physicochemical properties that contribute to improving the bioavailability and delivery of oligonucleotides(s). The covalent linker moiety can be a natural or unnatural nucleotide, ethylene glycol, carbohydrate, alkyl chain, or any two oligonucleotides located at the 3' or 5' ends of one or both chains in the oligonucleotide preparation, and may be another linker used to covalently link them.

[0038] As used herein, the term “oligonucleotide agent” means an oligonucleotide-containing substance comprising or consisting of one or more saRNAs of the present invention, having activity to modulate the expression of a target gene or activity to enhance the effect of the saRNA, and may further comprise other oligonucleotide sites / components (such as ASOs) or non-oligonucleotide sites / components conjugated, bound to, or mixed with the saRNA(s). In certain embodiments, the oligonucleotide agent comprises RNA (such as the saRNAs of the present invention), DNA, BNA, LNA, GNA, or peptide nucleic acid (PNA).

[0039] As used herein, the terms “prevent,” “prevention,” and “prevention” refer to slowing the progression of a disease, disorder, or medical condition from an existing state to a more severe state.

[0040] As used herein, the terms “to treat,” “to treat,” and “treatment” refer to the prevention, alleviation, recovery, cure, and / or delay of a disease, disorder, or medical condition.

[0041] As used herein, the capital letters “SMN2-FL” or “SMN2-FL gene” and “SMN2-Δ7” or “SMN2-Δ7 gene” refer to human genes. As used herein, the terms “SMN2 mRNA,” “SMN2-FL mRNA,” or “SMN2-Δ7 mRNA” refer to messenger RNA (mRNA) produced by the expression of the SMN2 gene or the transcription of the SMN2 gene.

[0042] As used herein, the term "SERPING1 mRNA" refers to messenger RNA (mRNA) produced by the expression of the SERPING1 gene or the transcription of the SERPING1 gene.

[0043] Double-stranded oligonucleotide agents Oligonucleotides offer significant potential for the prevention or treatment of various diseases, disorders, or conditions by, for example, upregulating or downregulating the protein expression of disease-related genes and their variants. Among therapeutic oligonucleotides, saRNAs as upregulators are emerging as a new class of therapeutic agents and are being actively developed. However, undesirable side effects such as "off-target" effects are currently hindering the full realization of their therapeutic effects. There remains an unmet need in this technology for oligonucleotide agents that exhibit good efficacy in gene activation and have low off-target effects.

[0044] While sequences consisting solely of natural building blocks have been significantly utilized, oligonucleotide synthesis technology offers the potential for various nucleotide modifications. Surprisingly, specific chemical modifications of oligonucleotide agents, such as saRNA, have been found to enable both the effective reduction of undesirable off-target effects and superior gene activation by these formulations. These modified oligonucleotide agents can overcome current limitations and enable broader applications in the medical and pharmaceutical fields.

[0045] Accordingly, in one embodiment, the Specified Provisions provide 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 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, and the double-stranded oligonucleotide agent comprises one or more glycerol nucleic acid (GNA) modified nucleotides located on either or both of the sense strand and the antisense strand.

[0046] GNAs are DNA / RNA analogs in which the (deoxy)ribose portion of the sugar-phosphodiester backbone of DNA / RNA is replaced with propylene glycol. Similarly, GNA-modified nucleotides include GNA versions of non-GNA DNA / RNA nucleotides, such as the naturally occurring DNA / RNA nucleotides adenine (A), thymine (T), cytosine (C), guanine (G), and uracil (U), in which the (deoxy)ribose portion is replaced with propylene glycol.

[0047] 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).

[0048] The sense and / or antisense strands of a double-stranded oligonucleotide agent contain one or more GNA-modified nucleotides instead of the corresponding non-GNA nucleotides. In other words, one or more non-GNA nucleotides in the sense and / or antisense strands of a double-stranded oligonucleotide agent are modified or substituted with that or their GNA-modified versions.

[0049] In some embodiments, the double-chain 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 GNA-modified nucleotides (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).

[0050] In some embodiments, at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least fourteen, at least fifteen, at least sixteen, at least seventeen, at least eighteen, at least nineteen, at least twenty, at least twenty one, at least twenty two two, at least twenty three, at least twenty fourteen, at least twenty fiveteen, at least twenty sixteen, at least twenty seventeen, at least twenty eighteen, at least twenty nineteen, at least thirty, at least thirty, at least thirty one, at least thirty twoteen, at least thirty threeteen, at least thirty fourteen, at least thirty fiveteen, at least thirty sixteen, at least thirty seventeen, at least thirty nine In some embodiments, 1 to 50 (e.g., 1 to 50, 1 to 45, 1 to 40, 1 to 35, 1 to 30, 1 to 25, 1 to 20, 1 to 15, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2) GNA-modified nucleotides are located on the sense strand and / or antisense strand of the double-stranded oligonucleotide agent.

[0051] In some embodiments, either or both of the sense strand and / or antisense strand of a double-stranded oligonucleotide agent can serve as guide strands that mediate RNA activation.

[0052] In some embodiments, the guide chain of the double-stranded oligonucleotide agent contains 1 to 50 GNA-modified nucleotides (for example, 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).

[0053] 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 (or more) from the 5' end of the guide strand, which can be either the sense strand or the antisense strand or both. In some embodiments, one or more GNA-modified nucleotides are located at positions 1 to 25 (e.g., positions 2 to 25, 2 to 24, 2 to 23, or 2 to 22) of the guide strand, which can be either the sense strand or the antisense strand or both. Typically, the guide chain of a double-stranded oligonucleotide agent includes a seed region that plays a role in the recognition of the target gene. In some embodiments, the length of the seed region is 2 to 10 nucleotides (e.g., 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, or 2 to 4).

[0054] The seed region is located at or near the 5' end of the guide strand. In some embodiments, the seed region begins within three nucleotides (e.g., 3, 2, 1, or 0) from the 5' end of the guide strand. In some embodiments, the 5' terminal nucleotide of the seed region is located within three nucleotides (e.g., 3, 2, 1, or 0) from the 5' end of the guide strand. In some embodiments, the 3' terminal nucleotide of the seed region is six or more nucleotides (e.g., 6, 7, 8, 9, or 10) from the 5' end of the guide strand. In some embodiments, the seed region includes consecutive nucleotides from the 5' end of the guide strand, starting from positions 2-10, or 2-9, or 2-8, or 2-7, or 2-6, or 3-6, or 2-5, or 3-6, or 3-5, or 4-6.

[0055] In some embodiments, the seed region has at least 80%, at least 85%, at least 90%, at least 95%, or 100% complementarity to a comparable segment of the sense strand of the double-stranded oligonucleotide agent. In certain embodiments, the seed region has 100% complementarity to a comparable segment of the sense strand of the double-stranded oligonucleotide agent.

[0056] One or more GNA-modified nucleotides may be located within and / or outside the seed region. In some embodiments, the seed region contains 1 to 8 GNA-modified nucleotides (or any number thereof). In some embodiments, the seed region contains 1 to 6 GNA-modified nucleotides (or any number thereof). In some embodiments, the seed region contains 1 to 5 GNA-modified nucleotides (or any number thereof). In some embodiments, the seed region contains 1 to 4 GNA-modified nucleotides (or any number thereof). In some embodiments, the seed region contains 1 to 3 GNA-modified nucleotides (or any number thereof). In some embodiments, the seed region contains 1 or 2 GNA-modified nucleotides (or any number thereof). 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.

[0057] In some embodiments, all or some of the GNA-modified nucleotides in the double-stranded oligonucleotide preparation are located in the seed region. In some embodiments, all or some of the GNA-modified nucleotides of the guide strand in the double-stranded oligonucleotide preparation are located in the seed region.

[0058] The double-stranded oligonucleotide preparation may include one or more GNA-modified nucleotides located at or near either the 5' end and / or the 3' end of the seed region. In an exemplary embodiment, the double-stranded oligonucleotide preparation includes one or more GNA-modified nucleotides located at the first three nucleotides (e.g., the first two or one) counting from the 5' end and 3' end of the seed region.

[0059] In some embodiments, the double-stranded oligonucleotide agent further comprises one or more GNA-modified nucleotides located outside the seed region. In some embodiments, the double-stranded oligonucleotide agent further comprises one or more GNA-modified nucleotides located in the passenger strand (P strand). In some embodiments, the double-stranded oligonucleotide agent comprises one or more GNA-modified nucleotides in either the sense strand or the antisense strand, or both.

[0060] The sense and antisense strands of a double-stranded oligonucleotide agent can be obtained by chemical synthesis, for example, by a phosphoramidite method (e.g., solid-phase phosphoramidite method). In some embodiments, either or both of the sense and antisense strands are synthesized to contain one or more GNA-modified nucleotides. In some embodiments, the antisense strand is synthesized to contain one or more GNA-modified nucleotides. The synthesized sense and antisense strands can be annealed to form a double-stranded structure.

[0061] In certain embodiments, the GNA-modified nucleotide monomer of formula (1) can be used in synthesis. [ka]

[0062] In the formula, the base can be selected from the group consisting of adenine nucleic acid bases, thymine nucleic acid bases, cytosine nucleic acid bases, guanine nucleic acid bases, uracil nucleic acid bases, and their analogues.

[0063] In a particular embodiment, the base of formula (1) can be selected from the following structures. [ka]

[0064] The sense strand of a 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 a segment of equivalent length of the coding strand of the target gene. In some embodiments, the sense strand has five or fewer nucleotide differences, i.e., 5, 4, 3, 2, 1, or 0, with respect to a segment of equivalent length of the coding strand of the target gene.

[0065] Generally, the antisense strand of a double-stranded oligonucleotide agent is complementary to a segment of the target gene's coding strand, particularly a segment in the promoter region of the target gene, and can therefore interact with the gene (or the aforementioned 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 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 complementary nucleotides with respect to a segment of the target gene's coding strand of equivalent length. In some embodiments, the antisense strand has 5 or fewer nucleotide differences or mismatches with respect to a segment of the target gene's coding strand of equivalent length, i.e., 5, 4, 3, 2, 1, or 0. In some embodiments, the above-mentioned differences or mismatches are located within the antisense chain or at or near its 3' end.

[0066] The sense and antisense strands of a double-stranded oligonucleotide agent include a complementary region, which forms a double-stranded structure consisting of at least eight base pairs, for example, at least 10, at least 12, at least 15, at least 18, or at least 20 base pairs. In some embodiments, the sense and antisense strands are at least 50%, for example, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% complementary to each other. In some embodiments, the number of mismatched nucleotides in the complementary region between the sense and antisense strands is five or less, i.e., 5, 4, 3, 2, 1, or 0. In some embodiments, the complementary double-stranded structure consisting of at least eight base pairs has five or fewer mismatched nucleotides between the sense and antisense strands, i.e., 5, 4, 3, 2, 1, or 0. In some embodiments, the mismatched nucleotide(s) are located within the antisense strand, or at or near the 3' or 5' end. In some embodiments, the mismatched nucleotide(s) are located at the GNA(s) position(s) in the sense strand and / or antisense strand.

[0067] The sense and antisense strands of the double-stranded oligonucleotide agents 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 located on two different strands, at least one strand of the double-stranded oligonucleotide agent has a 3' overhang consisting of 0 to 6 nucleotides in length, the overhang having a length of 0, 1, 2, 3, 4, 5, or 6 nucleotides, and optionally both strands having a 3' overhang consisting of 2 or 3 nucleotides in length. In some embodiments, the nucleotides of the overhang may be selected from corresponding positions on the DNA target or may be complementary nucleotides to those positions, i.e., a natural overhang. When the sense and antisense strands are located on a single nucleic acid strand, optionally the double-stranded oligonucleotide agent may be a hairpin-type single-stranded nucleic acid molecule, where the complementary regions of the sense and antisense strands form a double-stranded structure with respect to each other.

[0068] The sense and antisense strands of the double-stranded oligonucleotide agents described herein each have a length of 15 to 35 nucleotides. For example, in some embodiments, the sense and antisense strands each have a length of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 nucleotides, or any length within these ranges.

[0069] In one exemplary embodiment, the sense strand of the double-stranded oligonucleotide agent may contain a nucleotide sequence shown in any of SEQ ID NOs: 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 contain a nucleotide sequence shown in any of SEQ ID NOs: 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.

[0070] In some embodiments, the double-stranded oligonucleotide agent may include RNA, DNA, BNA, LNA, GNA, or peptide nucleic acid (PNA).

[0071] In some embodiments, the double-stranded oligonucleotide agent is a saRNA that upregulates the expression of a target gene.

[0072] In non-limiting examples, saRNAs are designed at least in part to criteria including (1) a GC content of 35% to 70%, (2) fewer than 5 identical nucleotide sequences, (3) three or fewer dinucleotide repeats, and (4) three or fewer trinucleotide repeats. In some embodiments, saRNAs are designed or selected at least in part to criteria that enable the production of functional saRNAs. For example, in some cases, sequences located upstream of the TSS may be located within a hotspot region but still contain sequences that are not favorable for saRNA synthesis.

[0073] In some embodiments, saRNAs are designed or selected at least in part on the basis of having a specific GC content (e.g., 25% to 75% GC content) and a sequence lacking consecutive identical nucleotides, consecutive dinucleotides, or consecutive trinucleotides. In some embodiments, the saRNA sequence includes a sequence having (1) a GC content of 35% to 70%, (2) fewer than 5 consecutive identical nucleotides, (3) 3 or fewer dinucleotide repeats, and (4) 3 or fewer trinucleotide repeats.

[0074] In some embodiments, the saRNA sequence includes sequences having a GC content of 25%-75%, 30%-70%, 35%-70%, 40%-60%, or 45%-55%. In some embodiments, the saRNA includes sequences having a GC content of 35%-70%.

[0075] In some embodiments, the saRNA sequence includes 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 includes a sequence having fewer than 5 consecutive identical nucleotides.

[0076] In some embodiments, the saRNA sequence includes 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 includes a sequence having 3 or fewer dinucleotide repeats.

[0077] In some embodiments, the saRNA sequence includes 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 includes a sequence having 3 or fewer trinucleotide repeats.

[0078] Methods and principles for designing saRNA molecules are well known to those skilled in the art, and are described in detail, for example, in Place et al., Molecular Therapy-Nucleic Acids (2012)1, e15, and Li et al., PNAS, 2006, vol.103, no.46, 17337-17342, which are incorporated herein by reference.

[0079] In the double-stranded oligonucleotide preparations described herein, all nucleotides may be naturally occurring or chemically unmodified nucleotides, or at least one nucleotide may be a chemically modified nucleotide. Non-limiting examples of chemical modification include: (1) Modification of phosphodiester bonds in the nucleotide sequence of double-stranded oligonucleotide agents; (2) Modification of the 2'-OH group of ribose in the nucleotide sequence of double-stranded oligonucleotide agents; (3) Modification of bases in the nucleotides of double-stranded oligonucleotide agents; and (4) One or more combinations of the following: at least one nucleotide in the nucleotide sequence of the double-stranded oligonucleotide agent is locked nucleic acid, cross-linked nucleic acid, DNA, GNA, or peptide nucleic acid (PNA).

[0080] The chemical modifications described herein are well known to those skilled in the art. The modifications described herein can stabilize the structure of the above-mentioned formulations and maintain high specificity and high affinity for base pairing.

[0081] In some embodiments, the double-stranded oligonucleotide agents described herein include at least one chemically modified nucleotide in which the 2'-OH of the pentose of the nucleotide is modified, i.e., modifications by introducing a specific substituent at the hydroxyl position of ribose, such as 2'-fluoro modification, 2'-oxymethyl modification, 2'-oxyethylidenemethoxy modification, 2,4'-dinitrophenol modification, locked nucleic acid (LNA), 2'-amino modification, or 2'-deoxy modification (e.g., 2'-deoxy-2'-fluoro modified nucleotide, 2'-deoxy modified nucleotide).

[0082] In some embodiments, the double-stranded oligonucleotide agents described herein include at least one chemically modified nucleotide in which the bases of the nucleotide are modified, for example, 5'-bromouracil modification, 5'-iodouracil modification, N-methyluracil modification, or 2,6-diaminopurine modification.

[0083] In some embodiments, the chemical modification of the double-stranded oligonucleotide agent is the addition of an (E)-vinylphosphonate moiety to the 5' end of the sense sequence and / or antisense sequence. In some embodiments, the chemical modification of at least one chemically modified nucleotide is the addition of a 5-methylcytosine moiety or 5-methyluracil to the 5' end of the sense sequence and / or antisense sequence.

[0084] In some embodiments, the double-stranded oligonucleotide preparations described herein include a chemically modified nucleic acid in which at least one nucleotide in the nucleotide sequence of the preparation is, for example, a locked nucleotide, an abasic nucleotide, a 2'-amino modified nucleotide, a 2'-alkyl modified nucleotide, a morpholino nucleotide, a phosphoramidate, and a non-natural base-containing nucleotide. In some embodiments, the double-stranded oligonucleotide preparations described herein include "endo-light" modifications, which include a 2'-O-methyl modified nucleotide and a nucleotide containing a 5'-phosphorothioate group.

[0085] In some embodiments, the double-stranded oligonucleotide agents described herein are chemically modified to improve stability or other advantageous properties. The nucleic acids characterized in this disclosure may be synthesized and / or modified by conventionally known methods, such as the methods described in “Current protocols in nucleic acid chemistry,” Beaucage, SL et al. (Edrs.), John Wiley & Sons, Inc., New York, NY, USA (this document is incorporated herein by reference). Modifications include, for example, (a) terminal modifications, e.g., 5' terminal modifications (phosphorylation, conjugation, reverse bond, etc.), 3' terminal modifications (conjugation, DNA nucleotide, reverse bond, etc.), (b) base modifications, e.g., substitution with a stabilizing base, destabilizing base, or base forming a base pair with an extended partner repertoire, removal of a base (debased nucleotide), or conjugate base, (c) sugar modifications (e.g., at the 2' or 4' position) or sugar substitution, and (d) skeletal modifications, including modification or substitution of phosphodiester bonds. Specific examples of double-stranded oligonucleotide agents usable in this disclosure include, but are not limited to, RNA having a modified skeleton or RNA lacking natural internucleoside bonds. In some embodiments, RNA having a modified skeleton includes, in addition to others, RNA lacking a phosphorus atom in its skeleton. In some embodiments, modified RNA lacking a phosphorus atom in its internucleoside skeleton can be considered an oligonucleoside. In some embodiments, the modified oligonucleotide will have a phosphorus atom in its internucleoside skeleton.

[0086] Modified oligonucleotide skeletons include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl phosphotriesters, methyl and other alkyl phosphonates including 3'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramides including 3'-aminophosphoamides and aminoalkylphosphoamides, thionophosphoamides, thionoalkyl phosphonates, thionoalkyl phosphotriesters, and boranophosphates having the usual 3'-5' bond, their 2'-5' bond analogues, and those with inverted polarity where adjacent nucleoside unit pairs are bonded from 3'-5' to 5'-3' or 2'-5' to 5'-2'. Various salts, mixed salts, and free acid forms are also included.

[0087] In certain embodiments, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or about 100% of the non-GNA modified nucleotides (non-GNA nucleotides) of the double-stranded oligonucleotide agent include modifications selected from the group consisting of 2'-fluoro(2'-F), 2'-O-methyl(2'-OMe), 2'-O-methoxyethyl(2'-MOE), 5'-(E)-vinylphosphonate, phosphorothioate skeleton modifications, and combinations thereof.

[0088] Conjugate Furthermore, to facilitate the introduction of double-stranded oligonucleotide agents into cells, a chemical conjugation site may be introduced to the end of the sense or antisense strand of the double-stranded oligonucleotide agent based on the above modifications, thereby facilitating action via the cell membrane, which consists of a lipid bilayer, as well as the nuclear membrane and gene promoter regions within the nucleus. Accordingly, this disclosure also provides a conjugate agent comprising a double-stranded oligonucleotide agent and at least one conjugation site.

[0089] In certain embodiments, the conjugate agent comprises a double-stranded oligonucleotide agent described herein and one or more conjugation sites covalently bound to the oligonucleotide agent. In certain embodiments, the conjugation site modifies one or more properties of the conjugated oligonucleotide, including but not limited to pharmacodynamics, pharmacokinetics, stability, binding, absorption, tissue distribution, intracellular distribution, intracellular uptake, charge, and clearance. In certain embodiments, the conjugation site imparts a novel property to the conjugated oligonucleotide, such as a fluorescent labeling group or reporter group that enables detection of the oligonucleotide. Specific conjugation sites have been previously described, for example, accessory oligonucleotides (ACO, WO2023280190A1 and PCT / CN2024 / 084814), lipids / fatty acids (WO2024002046A1), cholesterol sites (Letsinger et al., Proc.Natl.Acad.Sci.USA, 1989, 86, 6553-6556), cholic acid (Manoharan et al., Bioorg.Med.Chem.Lett., 1994, 4, 1053-1060), thioethers, for example, hexyl-S-tritylthiol (Manoharan et al., Ann.NYAcad.Sci., 1992, 660, 306-309; Manoharan et al. (al., Bioorg. Med. Chem. Lett., 1993, 3, 2765-2770), thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20, 533-538), aliphatic chains, e.g., dodecane-diol or undecyl residues (Saison-Behmoaras et al., EMBO 1, 1991, 10, 1111-1118; Kabanov et al., FEBS Lett., 1990, 259, 327-330; Svinarchuk et al., Biochimie, 1993, 75, 49-54), phospholipids, e.g., dihexadecyl-rac-glycerol or triethyl-ammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al.Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucleosides & Nucleotides, 1990, 18, 3777-3783), polyamine or polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), adamantane acetate, palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229-237), octadecylamine or hexylaminocarbonyloxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277, 923-937), tocopherol group (Nishina et al., Molecular Therapy Nucleic Examples include Acids, 2015, 4, e220; and Nishina et al., Molecular Therapy, 2008, 16, 734-740), or the GalNAc cluster (e.g., WO2024002046A1).

[0090] In some embodiments, the sense or antisense strand of a double-stranded oligonucleotide agent is conjugated with one or more conjugation sites selected from intercalators, reporter molecules, polyamines, polyamides, peptides, carbohydrates, vitamin moieties, polyethylene glycol, thioethers, polyethers, cholesterol, thiocholesterol, cholic acid moieties, folic acid, lipids, phospholipids, biotin, phenazine, phenanthridine, anthraquinone, adamantane, acridine, fluorescein, rhodamine, coumarin, fluorophores, and pigments.

[0091] In some embodiments, the conjugation site contains an active drug component, such as aspirin, warfarin, phenylbutazone, ibuprofen, suprofen, fenbufen, ketoprofen, (S)-(+)-pranoprofen, carprofen, dansylsarcosine, 2,3,5-triiodobenzoic acid, fingolimod, flufenamic acid, holinic acid, benzothiadiazide, chlorothiazide, diazepine, indomethacin, barbiturates, cephalosporins, sulfonamides, antidiabetic drugs, antibacterial agents, or antibiotics.

[0092] In some embodiments, the double-stranded oligonucleotide agents described herein conjugate with one or more conjugation sites selected from lipids, fatty acids, fluorophores, ligands, sugars, peptides, and antibodies.

[0093] In some embodiments, the sense or antisense strand of a double-stranded oligonucleotide agent is conjugated to one or more conjugation sites selected from cell-permeable peptides, polyethylene glycol, alkaloids, tryptamine, benzimidazole, quinolone, amino acids, cholesterol, glucose, and N-acetylgalactosamine.

[0094] In certain embodiments of the double-stranded oligonucleotide agent, the sense or antisense strand of the formulation disclosed herein is conjugated to one or more conjugation sites selected from cell-permeable peptides, polyethylene glycol, alkaloids, tryptamine, benzimidazole, quinolone, amino acids, cholesterol, glucose, and N-acetylgalactosamine. In certain embodiments, the double-stranded oligonucleotide agent is C 4-30 Conjugate to lipids selected from fatty acids. In certain embodiments, the conjugation site is saturated or unsaturated and linear or branched C 16 It is a lipid / fatty acid that has a carbon chain.

[0095] According to other embodiments, the double-stranded oligonucleotide agent further comprises at least one accessory oligonucleotide (ACO) conjugated with the oligonucleotide agent. The term “accessory oligonucleotide (ACO)” as used herein refers to a non-targeted single-stranded oligonucleotide comprising at least six nucleotides, having or not having one or more linker sites conjugated to other oligonucleotides. The ACO component is not designed to specifically target any complementary nucleic acid sequence present in the subject being administered the agent. The ACO component can be chemically modified at its skeleton, nucleoside, or other positions, for example, by a phosphorothioate skeleton, mesylphosphoamide skeleton, or boranophosphate skeleton, 2'-fluoro-2'-deoxynucleoside (2'-F), 2'-O-methyl (2'-O-Me), 2'-O-(2-methoxyethyl) (2'-O-MOE), locked nucleic acid (LNA), cross-linked nucleic acid (BNA), peptide nucleic acid (PNA), 5'-(E)-vinylphosphonate moiety, or 5-methylcytosine moiety, thereby conferring physicochemical properties that contribute to improving the bioavailability and delivery of oligonucleotides(s). The covalent linker moiety can be a natural or unnatural nucleotide, ethylene glycol, carbohydrate, alkyl chain, or any two oligonucleotides located at the 3' or 5' ends of one or both chains in the oligonucleotide preparation, and may be another linker used to covalently link them. The ACO may be described and prepared in accordance with what is disclosed in WO2023280190A1, which is incorporated herein by reference in its entirety.

[0096] In some embodiments, the double-stranded oligonucleotide agents of this specification relate to a sense or antisense strand of the double-stranded oligonucleotide agent that is conjugated to one or more conjugation sites selected from cell-permeable peptides, polyethylene glycol, alkaloids, tryptamine, benzimidazole, quinolone, amino acids, cholesterol, glucose, and N-acetylgalactosamine. In certain embodiments, the double-stranded oligonucleotide agent is conjugated to two conjugation sites. In certain embodiments, the two conjugation sites are a lipid and N-acetylgalactosamine. In certain embodiments, one or more conjugation sites are derived from tC2×6, C5×5, or a combination thereof, as shown in this application. [ka]

[0097] In certain embodiments, the conjugation sites conjugated to the double-stranded oligonucleotide agent are tC2×6 and C5×5, as shown in this application. In certain embodiments, tC2×6 is conjugated to the 3' end of the sense strand, and C5×5 is conjugated to the 5' end of the sense strand. The conjugation sites can be synthesized by methods well known to those skilled in the art, for example, the synthesis steps for tC2×6 and C5×5 are described in WO2024002046A1, which is incorporated herein by reference in whole.

[0098] In certain embodiments, the conjugation site is a lipid selected from fatty acids having a carbon chain length of 4 to 30 carbon atoms. In certain embodiments, the conjugation site is a fatty acid having a carbon chain length of 16 carbon atoms. In certain embodiments, the conjugation site is selected from the lipophilic sites described in WO2021092371A2. In certain embodiments, the double-stranded oligonucleotide agent comprises 1, 2, 3, 4, 5, 6, or more oligonucleotides, each of which may be individually conjugated to 1, 2, 3, 4, 5, 6, or more conjugation sites via 1, 2, 3, 4, 5, 6, or more linking sites.

[0099] According to one embodiment, the linking site, when present, is -O-, -S-, -C(O)-, -NH-, -N((C1-C 12 )alkyl)-, -N((C1-C 12 )alkyl)-C(O)-O-, -O-C(O)-, -C(O)-O-, -O-C(O)-O-, -C(O)-NH-, -OP(O)2O-, -P(O)(O - )O-, -OP(O)O-, -OP(O)(S)O-, -O-S(O)2-O-, -S(O)2-O-, -S(O)-O-, -(C1-C 22 )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 )Alkilen-OP(O)(O - )O-,-(C1-C 22 )Alkilen-OP(O)(O - )O-(C1-C 22 )Alkylene-,-(C1-C 22 )Alkilen-OP(O)O-,-(C1-C 22 )Alkilen-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 )Alkilen-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 ) can be selected from the group consisting of alkylene-S(O)-O-, in which the above linking part is -(C1-C 22The alkylene group may be an alkylene group having 1 to 22 carbon atoms, for example, an alkylene group having 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 site is a direct bond, the conjugation site is directly bonded to the oligonucleotide.

[0100] In some embodiments, the double-stranded oligonucleotide agent conjugates with one or more conjugation sites disclosed in the embodiments to be directly contacted, transferred, delivered, or administered to cells or subjects. The terms “patient,” “individual,” or “subject,” as used interchangeably herein, may refer to a non-human (e.g., mammalian) subject or a human subject.

[0101] Double-stranded oligonucleotide agents activate or upregulate the expression of target genes within cells via the RNAa mechanism. As used herein, the RNAa mechanism (also known as RNA activation) refers to a mechanism by which double-stranded nucleic acid structures can sequence-specifically upregulate target genes at the transcriptional level.

[0102] In some embodiments, the double-stranded oligonucleotide agents described herein can upregulate target gene expression by at least 10%, for example, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 300%, at least 500%, at least 800%, at least 1000%, at least 2000%, or at least 5000%, compared to unregulated gene expression.

[0103] In some embodiments, the expression of a target gene upregulated by the double-stranded oligonucleotide agent described herein is at least equivalent to, or at least 10%, for example, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 300%, or at least 500% higher than the expression regulated by the same but GNA-free double-stranded oligonucleotide agent.

[0104] In some embodiments, the double-stranded oligonucleotide agents described herein reduce off-target effects by at least equivalent amounts, or by at least 10%, for example, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100%, compared to identical double-stranded oligonucleotide agents that do not contain GNA.

[0105] In some embodiments, the double-stranded oligonucleotide agents described herein interact with and activate target genes associated with a disease, disorder, or medical condition. In some embodiments, the disease, disorder, or medical condition is caused by insufficient expression of the target gene. In some embodiments, upregulated expression of the target gene promotes the prevention of the disease, disorder, or medical condition. In some embodiments, upregulated expression of the target gene alleviates the symptoms of the disease, disorder, or medical condition, or promotes the treatment of the disease, disorder, or medical condition.

[0106] An exemplary target gene used herein is the Survival Motor Neuron 2 (SMN2) gene. The Survival Motor Neuron (SMN) protein produced by this gene can maintain the health and normal function of motor neurons, while a deficiency of SMN protein resulting from mutations in the SMN1 gene can cause spinal muscular atrophy (SMA). High or activated expression of the SNM2 gene is known as a paralog rescue therapy to alleviate the symptoms of SMA.

[0107] Another representative target gene used herein is the SERPING1 gene. SERPING1 is primarily expressed in the liver, secreted into the bloodstream, and encodes a C1 inhibitor involved in the normal functioning 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 by RNA activation, thereby treating related diseases (particularly HAE) by increasing the expression of the C1IHN protein (i.e., the C1 inhibitor). Since the SERPING1 gene encodes the C1IHN protein, increased SERPING1 mRNA expression leads to increased levels of the C1IHN protein.

[0108] However, it should be understood that the SMN2 gene, SERPING1 gene, and double-stranded oligonucleotide agents (e.g., saRNA) for these genes were selected and used solely for illustrative purposes and do not limit the scope of the present invention in any way.

[0109] Cells containing oligonucleotide agents In another embodiment, the Specified herein provides cells comprising a double-stranded oligonucleotide agent or a conjugate agent.

[0110] The double-stranded oligonucleotide agents or conjugates described herein can effectively activate or upregulate the expression of target genes within cells after contact with cells. For example, they activate or upregulate the expression of target genes by at least 10% compared to the expression when the gene is not regulated, or they activate or upregulate the expression of target genes to a level equivalent to or at least 10% higher than the expression level obtained by the same but GNA-free double-stranded oligonucleotide agent or conjugate, while reducing off-target effects compared to the GNA-free double-stranded oligonucleotide agent.

[0111] The cells described above may contain one or more double-stranded oligonucleotide agents or one or more conjugate agents as described herein.

[0112] In certain embodiments, this disclosure relates to cells comprising the double-stranded oligonucleotide agent or conjugate agent described herein. In some embodiments, the cells are mammalian cells. In some embodiments, the cells are human cells. The cells described herein may exist in vitro or outside the body, such as in cell lines or cell lineages, or they may exist in mammalian bodies, such as in the human body.

[0113] Composition containing an oligonucleotide agent or a conjugate agent In another embodiment, this specification provides compositions comprising the double-stranded oligonucleotide agent or conjugate agent described herein.

[0114] The above composition may contain one or more double-stranded oligonucleotide agents or one or more conjugate agents as described herein.

[0115] In some embodiments, the composition is a pharmaceutical composition. In some embodiments, the composition comprises at least one pharmaceutically acceptable carrier. In some embodiments, the composition comprises at least one pharmaceutically acceptable carrier selected from an aqueous carrier, liposomes or LNPs, polymers, micelles, colloids, metal nanoparticles, nonmetal nanoparticles, bioconjugates (e.g., GalNAc, lipids, antibodies, peptides, or single- / double-stranded oligonucleotides), polypeptides, antibodies, and any combination thereof. In one embodiment, the aqueous carrier may be, for example, RNase-free water or RNase-free buffer.

[0116] In some embodiments, the composition may contain 0.001 to 200 nM of the double-stranded oligonucleotide agent or conjugate described herein (e.g., 0.01 to 100 nM, 0.1 to 50 nM, 1 to 150 nM, 1 to 200 nM, 1 to 20 nM, 0.001 to 1 nM, 1 to 10 nM, 10 to 100 nM, 10 to 50 nM, 20 to 50 nM, 20 to 100 nM, 25 to 100 nM, or 30 to 100 nM). In a particular embodiment, the composition may contain 20 nM of the double-stranded oligonucleotide agent or conjugate described herein. In a particular embodiment, the composition may contain 25 nM of the double-stranded oligonucleotide agent or conjugate described herein. In a particular embodiment, the composition may contain 30 nM of the double-stranded oligonucleotide agent or conjugate described herein. In certain embodiments, the composition contains 50 nM of the double-stranded oligonucleotide agent or conjugate agent described herein. In certain embodiments, the composition contains 100 nM of the double-stranded oligonucleotide agent or conjugate agent described herein.

[0117] kit containing an oligonucleotide agent In another embodiment, this specification provides a kit comprising a double-stranded oligonucleotide agent or conjugate agent as described herein.

[0118] As used herein, “Kit” typically means a package, assembly, or container (e.g., an insulated container) that includes one or more components or embodiments of the Disclosure and / or other components relating to the Disclosure as described above. Any formulation or component of a Kit may be provided in liquid form (e.g., in solution) or solid form (e.g., dry powder, frozen, etc.).

[0119] The above kit may contain one or more double-stranded oligonucleotide agents or one or more conjugate agents as described herein.

[0120] In certain embodiments, the kit is intended to activate or upregulate the expression of a target gene in cells or subjects. In certain embodiments, the kit is intended to increase the level of mRNA or protein encoded by the target gene in cells or subjects. In certain embodiments, the kit is intended to prevent or treat diseases, disorders, or conditions associated with insufficient expression of a target gene.

[0121] In some embodiments, the kit includes a composition (e.g., a pharmaceutical composition) comprising a double-stranded oligonucleotide agent or conjugate agent as described herein.

[0122] In some embodiments, the kit further includes means for administering a double-stranded oligonucleotide agent or conjugate agent to a subject. In certain embodiments, the kit is housed in a labeled package, the label of which indicates that the double-stranded oligonucleotide agent, or conjugate agent, or composition can be used to prevent or treat diseases, disorders, or conditions resulting from insufficient expression of a target gene in a subject.

[0123] In additional embodiments, the kit may include instructions for use of the kit in relation to the components and / or methods described herein, or a link to a website or other information source, which may be in any form. For example, the instructions for use may include instructions for the use, modification, mixing, dilution, storage, assembly, storage, packaging, and / or preparation of the components and / or other components related to the kit. In some cases, the instructions for use may include instructions for the transport of the components, such as instructions for transport or storage at room temperature, below freezing temperatures, cryogenic temperatures, etc. The instructions for use may be provided in any form useful to the user of the kit, for example, in writing or orally (e.g., by telephone), digital, optical, visual (e.g., videotape, DVD, etc.), and / or electronic communication (including the Internet or web-based communication).

[0124] Application of oligonucleotide agents or conjugate agents In another embodiment, this specification provides a method for activating or upregulating a target gene in a cell or subject, comprising administering a double-stranded oligonucleotide agent or conjugate agent described herein to the cell or subject.

[0125] In another embodiment, the Specified herein also provides a method for reducing off-target effects caused by double-stranded oligonucleotide agents or conjugates that can activate the expression of a target gene in cells or subjects, the method comprising administering the double-stranded oligonucleotide agent or conjugate described herein to the cells or subjects.

[0126] In certain embodiments, when a 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%, for example, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 300%, at least 500%, at least 800%, at least 1000%, at least 2000%, or at least 5000% compared to the expression of the gene in an unregulated state.

[0127] In certain embodiments, when a 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 equivalent to, or at least 10%, for example, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 300%, or at least 500% higher than the expression regulated by the same but GNA-free double-stranded oligonucleotide agent.

[0128] In some embodiments, when a double-stranded oligonucleotide agent or conjugate described herein is administered, for example, to cells or a subject, it reduces off-target effects by at least the same amount, or at least 10%, for example, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100% compared to an identical double-stranded oligonucleotide agent that does not contain GNA.

[0129] In certain embodiments, the expression of a target gene is activated / upregulated by administering a double-stranded oligonucleotide agent or conjugate agent described herein 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.

[0130] In some embodiments, activation of a target gene promotes the prevention and / or treatment of diseases, disorders, or conditions associated with or resulting from insufficient expression of the target gene.

[0131] In some embodiments, the cells are mammalian cells. In some embodiments, the cells are human cells. The cells described herein may exist outside the body or in vitro, such as in cell lines or cell lineages, or they may exist within mammalian bodies, such as in the human body.

[0132] In some embodiments, the subject is a mammal. In some embodiments, the subject is a human. In some embodiments, the subject is a human having a disease, disorder, or condition that is associated with or at risk of developing insufficient expression of a gene (i.e., a target gene).

[0133] In some embodiments, the double-chain oligonucleotide agent or conjugate agent is applied 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.

[0134] In some embodiments, the double-chain oligonucleotide agent or conjugate agent is available in concentrations ranging from 0.01 nM to 500 nM, for example, 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, 75 nM, 80 nM, 85 nM, 90 nM, 95 nM, 100 nM, 105 nM, 110 nM It is applied to subjects or transfected into cells at concentrations within the range obtained by combining any two of the following endpoints: nM, 115 nM, 120 nM, 125 nM, 130 nM, 135 nM, 140 nM, 145 nM, 150 nM, 155 nM, 160 nM, 165 nM, 170 nM, 175 nM, 180 nM, 185 nM, 190 nM, 195 nM, 200 nM, 220 nM, 240 nM, 260 nM, 280 nM, 300 nM, 320 nM, 340 nM, 360 nM, 380 nM, 400 nM, 420 nM, 440 nM, 460 nM, 480 nM, and 500 nM.

[0135] In some embodiments, administration of the double-stranded oligonucleotide agents or conjugates described herein to a subject activates / upregulates the expression of a target gene, thereby promoting the prevention and / or treatment of a disease, disorder, or medical condition in the subject.

[0136] In some embodiments, administering a double-stranded oligonucleotide agent or conjugate described herein to a subject involves administering a composition (e.g., a pharmaceutical composition) containing the double-stranded oligonucleotide agent described herein in an amount effective for the prevention or treatment of the disease, disorder, or condition described herein.

[0137] In some embodiments, the route of administration is selected from one or more of intravenous administration, oral administration, nasal administration, inhalation administration, vaginal administration, and rectal administration. In some embodiments, the route of administration is selected from the group consisting of intrathecal administration, intramuscular administration, intravenous administration, intraarterial administration, intraperitoneal administration, intravesical administration, intraventricular administration, intravitreous administration, subcutaneous administration, and combinations thereof.

[0138] The dosage of the double-stranded oligonucleotide agent or composition disclosed herein can be varied over a wide range and is determined to suit the individual requirements of each case.

[0139] The single dose of a double-stranded oligonucleotide agent can be in the range of 0.01 mg to 1000 mg per kg of body weight of the subject, for example, approximately 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1, 2, 2.5, 5, 7.5, 10, 12.5, 15, 17.5, 20, 25, 30, 40, 50, 75, 100, 120, 150, 200, 250, 300, 400, 500, 750, or 1000 mg / kg per kg of body weight of the subject. The doses described herein may include one or more of the double-stranded oligonucleotide agents or one or more conjugate agents described herein.

[0140] In certain embodiments, the dose will be adjusted based on the subject's age, body weight, and / or other factors requiring adjustment of the injection parameters.

[0141] Examples of other compositions or components related to 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., used for the use, modification, assembly, storage, packaging, preparation, mixing, dilution, and / or preservation of the components for specific applications. In embodiments in which a liquid form of any component is used, the liquid form may be a concentrated form or a usable form.

[0142] In certain embodiments, the pharmaceutical composition includes a delivery system. Examples of delivery systems include, but are not limited to, liposomes and emulsions. Certain delivery systems are useful for preparing pharmaceutical compositions containing hydrophobic compounds. In certain embodiments, certain organic solvents, such as dimethyl sulfoxide, are used.

[0143] In certain embodiments, the pharmaceutical composition comprises one or more tissue-specific delivery molecules designed to deliver one or more pharmaceutical formulations of the present disclosure to a specific tissue or cell type. For example, in certain embodiments, the pharmaceutical composition comprises liposomes coated with a tissue-specific antibody.

[0144] The preparations, pharmaceutical compositions, or medicinal products of this disclosure are formulated, dosage-determined, and administered in accordance with best medical practices. Factors to be considered in this regard include the specific disorder being treated, the specific mammal being treated, the clinical condition of the individual subject, the cause of the disorder, the site of drug delivery, the method of administration, the administration schedule, and other factors known to healthcare professionals.

[0145] Typical formulations of the double-stranded oligonucleotide agents or conjugates of this disclosure are prepared by mixing the formulations described herein with a carrier or excipient. Suitable carriers and excipients are widely known to those skilled in the art and are described in detail, for example, Ansel HC et al., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems (2004), Lippincott, Williams & Wilkins, Philadelphia; Gennaro AR et al., Remington: The Science and Practice of Pharmacy (2000), Lippincott, Williams & Wilkins, Philadelphia; and Rowe RC, Handbook of Pharmaceutical Excipients (2005), Pharmaceutical Press, Chicago. The formulation may contain one or more known additives, such as buffers, stabilizers, surfactants, wetting agents, smoothing agents, emulsifiers, suspending agents, preservatives, antioxidants, opacifiers, lubricants, processing aids, colorants, sweeteners, fragrances, flavorings, diluents, and other known additives, to impart a refined appearance to the drug (i.e., the double-stranded oligonucleotide preparation or its pharmaceutical composition as described herein) or to assist in the manufacture of the pharmaceutical product (i.e., the drug).

[0146] In another embodiment, the use of the double-stranded oligonucleotide agent or conjugate agent described herein in the manufacture of products is provided.

[0147] The product may be a composition, a drug, or a kit.

[0148] In some embodiments, the product is intended to activate or upregulate the expression of a target gene in cells or subjects. In some embodiments, the product is intended to increase the level of mRNA or protein encoded by the target gene in cells or subjects. In some embodiments, the product is intended to prevent or treat diseases, disorders, or conditions associated with or resulting from insufficient expression of a target gene in subjects.

[0149] In some embodiments, the product comprises an amount effective to activate or upregulate the expression of a target gene in cells or subjects. In some embodiments, the product comprises an amount effective to increase the level of mRNA or protein encoded by the target gene in cells or subjects. In some embodiments, the product comprises an amount effective to prevent or treat a disease, disorder, or condition associated with or resulting from insufficient expression of a target gene in a subject.

[0150] Specific Embodiments Embodiment 1. A double-stranded oligonucleotide agent capable of activating or upregulating the expression of a target gene, comprising a sense strand and an antisense strand, each 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, and the double-stranded oligonucleotide agent comprises one or more glycerol nucleic acid (GNA) modified nucleotides located on either or both of the sense strand and the antisense strand.

[0151] Embodiment 2. The double-stranded oligonucleotide agent according to Embodiment 1, wherein either or both of the sense strand and the antisense strand are guide strands (or multiple guide strands) that mediate RNA activation.

[0152] Embodiment 3. The double-stranded oligonucleotide agent according to Embodiment 2, wherein the guide chain has a seed region of 2 to 10 nucleotides in length located at or near the 5' end of the guide chain.

[0153] Embodiment 4. The double-stranded oligonucleotide agent according to Embodiment 3, wherein the seed region starts within three or fewer nucleotides from the 5' end of the guide strand.

[0154] Embodiment 5. The double-stranded oligonucleotide agent according to Embodiment 3, wherein the seed region comprises nucleotides from the 2nd to 10th position, or from the 2nd to 9th position, or from the 2nd to 8th position, or from the 2nd to 7th position, or from the 2nd to 6th position, or from the 3rd to 6th position, or from the 2nd to 5th position, or from the 3rd to 6th position, or from the 3rd to 5th position, or from the 4th to 6th position, from the 5' end of the guide chain.

[0155] Embodiment 6. The double-stranded oligonucleotide agent according to Embodiment 1, wherein the 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).

[0156] Embodiment 7. The double-stranded oligonucleotide agent described in Embodiment 1, wherein the double-stranded oligonucleotide agent contains 1 to 50 GNA-modified nucleotides.

[0157] Embodiment 8. A double-stranded oligonucleotide agent according to Embodiment 2, comprising one or more GNA-modified nucleotides located at positions 1 and / or 2 and / or 3 and / or 4 and / or 5 and / or 6 and / or 7 and / or 8 and / or 9 and / or 10 and / or 11 and / or 12 and / or 13 and / or 14 and / or 15 and / or 16 and / or 17 and / or 18 and / or 19 and / or 20 and / or 21 and / or 22 and / or 23 and / or 24 and / or 25 (multiple positions possible) from the 5' end of the guide chain.

[0158] Embodiment 9. The double-stranded oligonucleotide agent according to Embodiment 3, wherein one or more GNA-modified nucleotides are located within and / or outside the seed region of the guide strand.

[0159] Embodiment 10. The double-stranded oligonucleotide agent according to Embodiment 3, wherein one or more GNA-modified nucleotides are located on the passenger strand.

[0160] Embodiment 11. The double-stranded oligonucleotide agent according to Embodiment 1, wherein the sense strand comprises a nucleotide sequence shown in any of SEQ ID NOs: 1, 3, 12, 14, 15, 17, 18, 20, 21, 23, 24, 27, 29, 52, 54, and the antisense strand comprises a nucleotide sequence shown in any of SEQ ID NOs: 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. Embodiment 12. The guide chain is synthesized using the GNA-modified nucleotide monomer of formula (1) to include one or more GNA-modified nucleotides. [ka] The double-stranded oligonucleotide agent according to Embodiment 1, wherein the base is selected from the group consisting of adenine nucleic acid bases, thymine nucleic acid bases, cytosine nucleic acid bases, guanine nucleic acid bases, uracil nucleic acid bases, and analogs thereof.

[0161] Embodiment 13. The base in formula (1) is selected from the following structures: [ka] A double-stranded oligonucleotide agent according to Embodiment 12.

[0162] Embodiment 14. The double-stranded oligonucleotide agent according to Embodiment 1, wherein the sense strand or 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 complementary nucleotides with respect to a segment of the coding strand of the target gene of equivalent length.

[0163] Embodiment 15. The double-stranded oligonucleotide agent according to Embodiment 1, wherein the complementary double-stranded structure, composed of at least eight base pairs, has five or fewer mismatched nucleotides between the sense strand and the antisense strand, i.e., 5, 4, 3, 2, 1, or 0.

[0164] Embodiment 16. The double-stranded oligonucleotide agent according to Embodiment 15, wherein the mismatched nucleotide(s) are located inside the antisense strand, or at or near the 3' or 5' end.

[0165] Embodiment 17. The double-stranded oligonucleotide agent according to Embodiment 15, wherein the mismatched nucleotide(s) are located at the position(s) of the GNA in the sense strand and / or the antisense strand.

[0166] Embodiment 18. The double-stranded oligonucleotide agent according to Embodiment 1, wherein the double-stranded oligonucleotide agent is a small molecule activated RNA (saRNA) that upregulates the expression of the target gene by at least 10%.

[0167] Embodiment 19. The double-stranded oligonucleotide agent according to Embodiment 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 comprises a modification selected from the group consisting of 2'-fluoro(2'-F), 2'-O-methyl(2'-OMe), 2'-O-methoxyethyl(2'-MOE), 5'-(E)-vinylphosphonate, phosphorothioate skeleton modification, and combinations thereof.

[0168] Embodiment 20. A conjugate agent comprising a double-stranded oligonucleotide agent according to any one of Embodiments 1 to 19 and at least one conjugation site, wherein the conjugate agent is selected from lipids, fatty acids, fluorophores, ligands, sugars, peptides, and antibodies.

[0169] Embodiment 21. The conjugate agent according to Embodiment 20, wherein the conjugation site is selected from auxiliary oligonucleotides (ACOs), lipids / fatty acids, and GalNAc clusters. Embodiment 22. The above conjugation area is, [ka] Selected from, During the ceremony, [ka] The conjugate agent according to Embodiment 21, which shows a support material.

[0170] Embodiment 23. Cells comprising the double-stranded oligonucleotide agent described in Embodiment 1 or the conjugate agent described in Embodiment 20.

[0171] Embodiment 24. A pharmaceutical composition comprising a double-stranded oligonucleotide agent described in Embodiment 1 or a conjugate agent described in Embodiment 20, and at least one pharmaceutically acceptable carrier.

[0172] Embodiment 25. A kit for activating or upregulating a target gene in cells or a subject, comprising a double-stranded oligonucleotide agent according to any of Embodiments 1 to 19, a conjugate agent according to any of Embodiments 20 to 22, or a pharmaceutical composition according to Embodiment 24.

[0173] Embodiment 26. A method for activating or upregulating a target gene in cells or a subject, comprising administering a double-stranded oligonucleotide agent according to any one of Embodiments 1 to 19, or a conjugate agent according to any one of Embodiments 20 to 22, to the cells or subject.

[0174] Embodiment 27. A method for reducing off-target effects caused by double-stranded oligonucleotide agents that can activate or upregulate the expression of a target gene in cells or subjects, comprising administering a double-stranded oligonucleotide agent according to any of Embodiments 1 to 19, or a conjugate agent according to any of Embodiments 20 to 22, to the cells or subjects.

[0175] Embodiment 28. Use of a double-stranded oligonucleotide agent according to any of Embodiments 1 to 19, or a conjugate agent according to any of Embodiments 20 to 22, in the manufacture of a product for activating or upregulating a target gene in cells or subjects. [Examples]

[0176] The present application will be further described below with reference to specific examples and drawings. These examples should be understood not as limiting the scope of the present application, but merely as illustrative examples. In the following examples, test methods where specific conditions are not explicitly stated were generally carried out in accordance with conventionally known conditions, such as those described in Sambrook, et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or conditions recommended by the manufacturer.

[0177] The following examples are provided to those skilled in the art to provide a complete disclosure and description of how the present invention may be prepared and used, and are not intended to limit the scope of what the inventors consider to be the invention, nor are they intended to imply that the following experiments are all, or that only these experiments have been performed. Efforts have been made to ensure accuracy in the numbers used (e.g., quantity, temperature, etc.), but some experimental errors and deviations exist. Unless otherwise specified, parts are parts by weight, molecular weight is weight-average molecular weight, temperature is Celsius, and pressure is atmospheric pressure or near it. Standard abbreviations, such as bp, base pair(s); kb, kilobase(s); nM, nanomoles(s); s or sec, seconds; min, minutes; h or hr, hours; aa, amino acids(s); nt, nucleotide(s); im, intramuscular; ip, intraperitoneal; sc, subcutaneous; ivt or IVT, intravitreous; iv or IV, tail vein, intravenous; icv or icv or ICV, intracerebral, etc. may be used.

[0178] Unless otherwise specified, all starting materials, reagents, and solvents used below were purchased from commercially available suppliers and used as is. Reaction products were purified by column chromatography using silica gel (200-300 mesh), with hexane / ethyl acetate and DCM / MeOH as eluents. Thin-layer chromatography (TLC) was performed using plates pre-coated with silica gel GF, and visualization was performed by KMnO4 staining. ¹H NMR spectra were recorded at 400 or 500 MHz (Varian) using CDCl3 in conjunction with TMS. High-resolution mass spectrometry (HRMS) was recorded using LC / MS (Agilent Technologies 1260 Infinity II / 6120 Quadrupole) and time-of-flight mass spectrometry with ESI or matrix-assisted laser desorption / ionization (MALDI). Example 1 Preparation of the compound GNA-U of this disclosure

[0179] In this example, compound GNA-U was prepared using the following procedure. [ka]

[0180] (1) Preparation of compound 2 [ka]

[0181] 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), DMTrCl (64.5 g, 190 mmol) was added. After 12 h, the reaction mixture was poured into saturated NaHCO3 aqueous solution (500 mL). The organic layer was extracted with ethyl acetate, washed with brine, dried over anhydrous Na2SO4, and concentrated under reduced pressure. The resulting residue was purified by flash chromatography (silica gel, gradient elution: 1-10% ethyl acetate / hexane, 1% Et3N) to obtain compound 2 (52.2 g, 90% yield) as a yellow oil. The product was, 1 Characterized by 1H NMR. 1 H NMR (400 MHz, CDCl3) δ 7.48 - 7.42 (m, 2H), 7.39 - 7.23 (m, 7H), 6.85 - 6.80 (m, 4H), 3.78 (s, 6H), 3.36 - 3.27 (m, 1H), 3.17 - 3.08 (m, 2H), 2.81 - 2.59 (m, 2H).

[0182] (2) Preparation of compound 3 [ka]

[0183] To a solution of compound 11 (2.5 g, 22.3 mmol, 1.2 eq) in 45 mL of anhydrous DMF, NaH (148 mg, 3.7 mmol, 60% in mineral oil, 0.2 eq) was added, and the mixture was stirred under a nitrogen atmosphere for 1 hour. A solution of compound 2 (7.0 g, 18.6 mmol, 1.0 eq) in 5.0 mL of anhydrous DMF was added to the above solution, and the reaction was heated overnight at 110°C. The solution was cooled in an ice bath and quenched with saturated ammonium chloride. The reaction was extracted twice with ethyl acetate, and the organic phase was washed three times with saturated LiCl aqueous solution and once with brine. Then, it was dried over anhydrous Na2SO4 and concentrated under reduced pressure. The resulting residue was purified by flash chromatography (silica gel, gradient elution: 50-100% ethyl acetate / hexane, 1% Et3N) to obtain compound 3 (6.14 g, yield 67%). The product was analyzed by mass spectrometry and 1 Characterized by 1H NMR. Calculated MW value: 488.19; Measured MW value: 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 = 7.9 Hz, 1H), 6.87 - 6.79 (m, 4H), 5.55 (d, J = 7.9 Hz, 1H), 4.08 -4.02 (m, 2H), 3.78 (s, 6H), 3.68 - 3.59 (m, 1H), 3.23 - 3.14 (m, 2H).

[0184] (3) Preparation of compound GNA-U [ka]

[0185] To a solution of compound 3 (2.0 g, 4.1 mmol, 1.0 eq) and DIPEA (2.2 mL, 12.3 mmol, 3.0 eq) in anhydrous DCM (40 mL), 2-cyanoethyl N,N-diisopropyl chlorophosphoramidite (2.7 mL, 12.3 mmol, 3.0 eq) was added at room temperature under a nitrogen atmosphere. The reaction mixture was stirred for 1.5 hours. The mixture was extracted twice with DCM, then washed with saturated NaHCO3 and brine, and dried over anhydrous Na2SO4. The organic layer was concentrated under reduced pressure, and the resulting residue was purified by flash chromatography (silica gel, gradient elution: 20-50% ethyl acetate / hexane, 1% Et3N) to obtain compound GNA-U (3.4 g, yield 72%). The product was, 1 Characterized by 1H NMR. 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).

[0186] Example 2 Preparation of the compound GNA-T of this disclosure

[0187] In this example, compound GNA-T was prepared using the following procedure. [ka]

[0188] (1) Preparation of compound 4 [ka]

[0189] To a solution of compound 12 (4.8 g, 38.3 mmol, 1.2 eq) in 122.0 mL of anhydrous DMF, NaH (256 mg, 6.4 mmol, 60% in mineral oil, 0.2 eq) was added, and the mixture was stirred under a nitrogen atmosphere for 1 hour. A solution of compound 2 (12.0 g, 31.9 mmol, 1.0 eq) in 10.0 mL of anhydrous DMF was added to the above solution, and the reaction was heated overnight at 110°C. The solution was cooled in an ice bath and quenched with saturated ammonium chloride. The reaction was extracted twice with ethyl acetate, and the organic phase was washed three times with saturated LiCl aqueous solution and once with brine. Then, it was dried over anhydrous Na2SO4 and concentrated under reduced pressure. The resulting residue was purified by flash chromatography (silica gel, gradient elution: 50-100% ethyl acetate / hexane, 1% Et3N) to obtain compound 4 (8.5 g, yield 53%). The product was analyzed by mass spectrometry and 1 Characterized by 1H NMR. Calculated MW value: 502.21; Measured MW value: 500.74 [MH] + . 1 H NMR (400 MHz, CDCl3) δ 7.43 - 7.38 (m, 2H), 7.33 - 7.26 (m, 7H), 7.04 (s, 1H), 6.87 - 6.80 (m, 4H), 4.10 - 3.98 (m, 2H), 3.79 (s, 6H), 3.69 - 3.62 (m, 1H), 3.18 (d, J = 5.4 Hz, 2H), 1.84 (s, 3H).

[0190] (2) Preparation of compound GNA-T [ka]

[0191] To a solution of compound 4 (8.5 g, 16.9 mmol, 1.0 eq) and DIPEA (8.4 mL, 50.7 mmol, 3.0 eq) in anhydrous DCM (169.0 mL), 2-cyanoethyl N,N-diisopropyl chlorophosphoramidite (11.3 mL, 50.7 mmol, 3.0 eq) was added at room temperature under a nitrogen atmosphere. The reaction mixture was stirred for 1.5 hours. The mixture was extracted twice with DCM, then washed with saturated NaHCO3 and brine, and dried over anhydrous Na2SO4. The organic layer was concentrated under reduced pressure, and the resulting residue was purified by flash chromatography (silica gel, gradient elution: 20-50% ethyl acetate / hexane, 1% Et3N) to obtain compound GNA-T (10.0 g, yield 84%). The product was analyzed by mass spectrometry and 1 Characterized by 1H NMR. Calculated MW value: 702.32; Measured MW value: 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).

[0192] Example 3 Preparation of compound GNA-C of this disclosure

[0193] In this example, compound GNA-C was prepared using the following procedure. [ka]

[0194] (1) Preparation of compound 5 [ka]

[0195] To a solution of compound 13 (2.68 g, 17.5 mmol, 1.1 eq) in 55.0 mL of anhydrous DMF, NaH (136 mg, 3.7 mmol, 60% in mineral oil, 0.2 eq) was added, and the mixture was stirred under a nitrogen atmosphere for 1 hour. A solution of compound 2 (6.0 g, 15.9 mmol, 1.0 eq) in 5.0 mL of anhydrous DMF was added to the above solution, and the reaction was heated overnight at 110°C. The solution was cooled in an ice bath and quenched with saturated ammonium chloride. The reaction was extracted twice with ethyl acetate, and the organic phase was washed three times with saturated LiCl aqueous solution and once with brine. Then, it was dried over anhydrous Na2SO4 and concentrated under reduced pressure. The resulting residue was purified by flash chromatography (silica gel, gradient elution: 50-100% ethyl acetate / hexane, 1% Et3N) to obtain compound 5 (2.5 g, yield 30%). The product was, 1 Characterized by 1H NMR. 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).

[0196] (2) Preparation of compound GNA-C [ka]

[0197] To a solution of compound 5 (1.7 g, 3.21 mmol, 1.0 eq) and DIPEA (1.4 mL, 8.03 mmol, 2.5 eq) in anhydrous DCM (38 mL), 2-cyanoethyl N,N-diisopropyl chlorophosphoramidite (1.8 mL, 8.03 mmol, 2.5 eq) was added at room temperature under a nitrogen atmosphere. The reaction mixture was stirred for 1.0 h. The mixture was extracted twice with DCM, then washed with saturated NaHCO3 and brine, and dried over anhydrous Na2SO4. The organic layer was concentrated under reduced pressure, and the resulting residue was purified by flash chromatography (silica gel, gradient elution: 20-50% ethyl acetate / hexane, 1% Et3N) to obtain compound GNA-C (2.2 g, 94% yield). The product was, 1 Characterized by 1H NMR. 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).

[0198] Example 4 Preparation of compound GNA-A of this disclosure

[0199] In this example, compound GNA-A was prepared using the following procedure. [ka]

[0200] (1) Preparation of compound 6 [ka]

[0201] To a solution of compound 14 (2.28 g, 16.9 mmol, 1.1 eq) in 65.0 mL of anhydrous DMF, NaH (140 mg, 3.51 mmol, 60% in mineral oil, 0.22 eq) was added, and the mixture was stirred under a nitrogen atmosphere for 2 hours. A solution of compound 2 (6.0 g, 15.9 mmol, 1.0 eq) in 5.0 mL of anhydrous DMF was added to the above solution, and the reaction was heated overnight at 105°C. The solution was cooled in an ice bath and quenched with saturated ammonium chloride. The reaction was extracted twice with ethyl acetate, and the organic phase was washed three times with saturated LiCl aqueous solution and once with brine. Then, it was dried over anhydrous Na2SO4 and concentrated under reduced pressure. The resulting residue was purified by flash chromatography (silica gel, gradient elution: 1-5% MeOH / DCM, 1% Et3N) to obtain compound 6 (5.4 g, yield 66%). The product was analyzed by mass spectrometry and 1 Characterized by 1H NMR. Calculated MW value: 511.58; Measured MW value: 512.37 [M+H] + . 1 H NMR (400 MHz, CDCl3) δ 8.26 (s, 1H), 7.73 (s, 1H), 7.45 - 7.39 (m, 2H), 7.34 - 7.20 (m, 7H), 6.87 - 6.80 (m, 4H), 5.99 (s, 2H), 4.46 - 4.38 (m, 1H), 4.33 - 4.17 (m, 2H), 3.80 (s, 6H), 3.32 - 3.24 (m, 1H), 3.12 - 3.04 (m, 1H). (2) Preparation of compound 7 [ka]

[0202] To a solution of compound 6 (4.4 g, 8.6 mmol, 1.0 eq) in 44.0 mL of anhydrous DMF, dimethylformamide dimethyl acetal (4.1 mL, 30.1 mmol, 3.5 eq) was added, and the mixture was heated at 60°C for 1 hour. The solution was cooled in an ice bath, extracted twice with ethyl acetate, and the organic phase was washed three times with saturated LiCl aqueous solution and once with brine. The mixture was then dried over anhydrous Na2SO4 and concentrated under reduced pressure. The resulting residue was purified by flash chromatography (silica gel, gradient elution: 1-5% MeOH / DCM, 1% Et3N) to obtain compound 7 (4.0 g, yield 82%). The product was analyzed by mass spectrometry and 1 Characterized by 1H NMR. Calculated MW value: 566.66; Measured MW value: 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).

[0203] (3) Preparation of compound GNA-A [ka]

[0204] To a solution of compound 7 (2.0 g, 3.53 mmol, 1.0 eq) and DIPEA (1.54 mL, 8.82 mmol, 2.5 eq) in anhydrous DCM (40 mL), 2-cyanoethyl N,N-diisopropyl chlorophosphoramidite (1.97 mL, 8.82 mmol, 2.5 eq) was added at room temperature under a nitrogen atmosphere. The reaction mixture was stirred for 0.5 hours. The mixture was extracted twice with DCM, then washed with saturated NaHCO3 and brine, and dried over anhydrous Na2SO4. The organic layer was concentrated under reduced pressure, and the resulting residue was purified by flash chromatography (silica gel, gradient elution: 1-5% MeOH / DCM, 1% Et3N) to obtain compound GNA-A (2.4 g, yield 89%). The product was, 1 Characterized by 1H NMR. 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).

[0205] Example 5 Preparation of the compound GNA-G of this disclosure

[0206] In this example, compound GNA-G was prepared using the following procedure. [ka]

[0207] (1) Preparation of compound 8 [ka]

[0208] To a solution of 6-(benzyloxy)-9H-purine-2-amine compound 15 (4.20 g, 17.5 mmol, 1.05 eq) in 55.0 mL of anhydrous DMF, NaH (140 mg, 3.50 mmol, 60% in mineral oil, 0.22 eq) was added, and the mixture was stirred under a nitrogen atmosphere for 1 hour. A solution of compound 2 (6.0 g, 15.9 mmol, 1.0 eq) in 5.0 mL of anhydrous DMF was added to the above solution, and the reaction was heated overnight at 90°C. The solution was cooled in an ice bath and quenched with saturated ammonium chloride. The reaction was extracted twice with ethyl acetate, and the organic phase was washed three times with saturated LiCl aqueous solution and once with brine. The solution was then dried over anhydrous Na2SO4 and concentrated under reduced pressure. The resulting residue was purified by flash chromatography (silica gel, gradient elution: 1-5% MeOH / DCM, 1% Et3N) to obtain compound 8 (5.0 g, yield 51%). The product was analyzed by mass spectrometry and 1 Characterized by 1H NMR. Calculated MW value: 617.26; Measured MW value: 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).

[0209] (2) Preparation of compound 9 [ka]

[0210] Compound 8 (4.80 g, 7.80 mmol) and Pd / C (2.40 g, 10% on carbon) were suspended in EtOAc (192.0 mL). The solution was purged with nitrogen, then purged with hydrogen, and stirred under a hydrogen atmosphere. After 3 h, when the completion of the reaction was confirmed by TLC, the mixture was filtered through celite and washed with 5% MeOH / DCM to obtain crude compound 9. The product was characterized by mass spectrometry. Calculated MW: 527.22; Measured MW: 528.60 [M+H] + .

[0211] (3) Preparation of Compound 10 [Chemical formula]

[0212] Dimethylformamide dimethylacetal (3.4 mL, 25.1 mmol, 3.5 eq) was added to a solution of 9 (3.8 g, 7.2 mmol, 1.0 eq) in 30.0 mL of anhydrous DMF, and the mixture was heated at 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 a saturated aqueous LiCl solution and once with brine. Then, it was dried over anhydrous Na2SO4 and concentrated under reduced pressure. The obtained residue was purified by flash chromatography (silica gel, gradient elution: 1 - 5% MeOH / DCM, 1% Et3N) to obtain compound 7 (2.8 g, yield 67%). The product was characterized by mass spectrometry. Calculated MW: 582.26; Measured MW: 583.45 [M+H] + .

[0213] (4) Preparation of Compound GNA - G [Chemical formula]

[0214] To a solution of compound 10 (1.5 g, 2.58 mmol, 1.0 eq) and DIPEA (0.97 mL, 6.44 mmol, 2.5 eq) in anhydrous DCM (15.0 mL), 2-cyanoethyl N,N-diisopropyl chlorophosphoramidite (1.83 mL, 6.44 mmol, 2.5 eq) was added at room temperature under a nitrogen atmosphere. The reaction mixture was stirred for 0.5 hours. The mixture was extracted twice with DCM, then washed with saturated NaHCO3 and brine, and dried over anhydrous Na2SO4. The organic layer was concentrated under reduced pressure, and the resulting residue was purified by flash chromatography (silica gel, gradient elution: 1-5% MeOH / DCM, 1% Et3N) to obtain compound GNA-G (1.2 g, yield 59%). The product was, 1 Characterized by 1H NMR. 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).

[0215] Example 6 Synthesis and design of GNA-modified oligonucleotides

[0216] Table 1 shows the oligonucleotides tested in the following examples. RD-12318 is a sequence designed to target the promoter of the SMN2 gene and to activate the transcription of the gene allele via an RNA activation (RNAa) mechanism. Transfecting cell lines with chemically modified saRNA derived from RD-12318 can regulate the expression levels of SMN2-FL (i.e., full-length SMN2 including exon 7) and SMN2-Δ7 (i.e., SMN2 lacking exon 7) mRNA, thereby boosting the level of SMN protein encoded by SMN2-FL mRNA. The "seed" region (GUUGCUU) is part of the antisense sequence (SEQ ID NO: 2) of RD-12318.

[0217] RD-10994 was synthesized based on RD-12318, with chemical modifications including 2'-fluoro, 2'-O-methyl (2'-OMe), 5'-(E)-vinylphosphonate, and phosphorothioate (PS) skeleton modifications. RD-15977, RD-15978, RD-15979, RD-15980, RD-15981, RD-15982, and RD-15983 were synthesized in the same manner as RD-10994, except that glycerol nucleic acid (GNA) modifications were introduced at positions 2, 3, 4, 5, 6, 7, and 8 from the 5' end of the antisense strand. "nG", "nU", and "nC" represent GNA-modified nucleotides.

[0218] RD-19588 is a sequence designed to target the promoter of the SMN2 gene and activate the transcription of the gene allele via the RNAa mechanism. The "seed" region (GCAGGCC) is part of the antisense sequence (SEQ ID NO: 28) of RD-19588. RD-19040 was synthesized based on RD-19588, with chemical modifications including 2'-fluoro, 2'-O-methyl (2'-OMe), 5'-(E)-vinylphosphonate, and phosphorothioate (PS) skeleton modifications. 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-196 RD-19668, RD-19669, and RD-19670 were synthesized in the same manner as RD-10994, except that glycerol nucleic acid (GNA) modifications were introduced 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 the antisense strand. RD-19672 was synthesized in the same manner as RD-10994, except that two GNA modifications were introduced at positions 8 and 17, counting from the 5' end of the antisense strand. RD-19674 was synthesized in the same manner as RD-10994, except that a GNA modification was introduced at position 9, counting from the 5' end of the antisense strand, and at position 13, counting from the 5' end of the sense strand. RD-19675 was synthesized in the same manner as RD-10994, except that a GNA modification was introduced at the 10th position from the 5' end of the antisense strand and at the 12th position from the 5' end of the sense strand. "nG", "nU", "nA", and "nC" represent GNA-modified nucleotides.

[0219] The non-GNA-saRNAs (i.e., RD-17229, RD-17235, RD-17238, RD-17241, and RD-17244) were designed to target the SERPING1 gene promoter and aim to activate the transcription of the gene allele via the RNAa mechanism. The corresponding GNA-saRNAs (i.e., RD-17074, RD-17082, RD-17086, RD-17096, and RD-17099) were synthesized in the same manner as the non-GNA-saRNAs, except that a GNA modification was introduced at position 7 from the 5' end of the sense or antisense strand. The "seed" regions of RD-17074, RD-17082, RD-17086, and RD-17096 are located consecutively from position 2 to 8 from the 5' end of the sense strand. The "seed" region of RD-17099 is located consecutively from position 2 to 8, counting from the 5' end of the antisense strand. "nG", "nA", and "nC" represent GNA-modified nucleotides. [Table 1] JPEG2026528711000028.jpg250170JPEG2026528711000029.jpg250170JPEG20265287110 00030.jpg250170JPEG2026528711000031.jpg250170JPEG2026528711000032.jpg250170

[0220] Example 7 In vitro activity of GNA-saRNA in the regulation of SMN2-Δ7 to SMN2-FL mRNA in .GM03813 cells To evaluate the in vitro activity of GNA-saRNAs at various nucleotide positions, the indicated GNA-saRNAs (i.e., RD-15977, RD-15978, RD-15979, RD-15980, RD-15981, RD-15982, and RD-15983) were transfected into GM03813 cells for 3 days at the indicated concentrations (i.e., 0.78, 1.56, 3.13, 6.25, 12.5, 25, 50, and 100 nM). RD-10994 was transfected and used as a non-GNA control. SMN2-FL mRNA levels were quantified by RT-qPCR using gene-specific primer sets in each PCR reaction and plotted in Figure 1. Table 2 shows the maximum efficacy of SMN2-FL expression (i.e., E max This summarizes the area under the curve (AUC) and the expression area under the curve (ANA). The area under the curve (AUC) for SERPING1 mRNA expression was calculated using GraphPad Prism software, as described in the Materials and Methods section. The results suggest that GNA-saRNA can enhance the activity of SMN2-FL mRNA expression compared to the non-GNA-saRNA RD-10994.

[0221] [Table 2]

[0222] As shown in Figures 2A-2H, GNA-saRNA showed high SMN2-FL induction activity and simultaneous decrease in SMN2-Δ7 mRNA expression in GM03813 cells. As shown in Figures 3A-3H, GNA-saRNA showed high SMN2-FL induction activity and simultaneous decrease in SMN2-Δ7 mRNA expression in GM22592 cells. All GNA-saRNAs increased SMN2-FL mRNA levels, suggesting that GNA-saRNAs can enhance SMN2-FL mRNA expression by regulating SMN2-Δ7 to SMN2-FL mRNA.

[0223] Example 8In vitro activity of GNA-saRNA in the regulation of SMN2-Δ7 to SMN2-FL mRNA in .GM03813 cells To verify the in vitro activity of GNA modifications at various nucleotide positions, the indicated GNA-saRNAs (i.e., RD-15977, RD-15978, RD-15979, RD-15980, RD-15981, RD-15982, and RD-15983) were transfected into GM03813 cells at 100 nM for 3 days. RD-10994 was transfected and used as a non-GNA control. SMN2-FL and SMN2-Δ7 mRNA levels were quantified by RT-qPCR using gene-specific primer sets in each PCR reaction. As shown in Figures 4A and 4B, all GNA-saRNAs showed higher SMN2-FL inducing activity and simultaneous decreased SMN2-Δ7 mRNA expression compared to the non-GNA saRNA RD-10994 at 100 nM treatment. The expression levels of SMN2-FL and SMN2-Δ7 mRNA are summarized in Table 3. The results suggest that GNA-saRNA can enhance the activity of SMN2-FL mRNA expression by regulating SMN2-Δ7 to SMN2-FL mRNA.

[0224] [Table 3]

[0225] Example 9 .GNA-saRNA reduces off-target effects on the expression of the off-target latent gene P2RY2 in GM03813 cells. To evaluate the reduction of off-target effects by GNA-saRNA, the antisense strand of RD-10994 was used as the "query" sequence, and potential off-target genes were searched by in silico analysis. As a result, P2RY2 was predicted as a potential off-target gene. Figure 5A shows the "query" sequence (antisense strand) of RD-10994 and the "seed" region (highlighted in gray), and the predicted complementary sequences to the target sites in the P2RY2 transcript (nucleotides shown in italics and bold) containing two mismatched nucleotides. The indicated GNA-saRNAs (i.e., RD-15977, RD-15978, RD-15981, RD-15982, and RD-15983) were transfected into GM03813 cells at 6.25 nM and 25 nM for 3 days. RD-10994 was transfected and used as a non-GNA control. The mRNA level of P2RY2 was quantified by RT-qPCR using gene-specific primer sets in each PCR reaction. As shown in Figure 5B, all GNA-saRNAs except RD-15981 at the 25 nM treatment reduced the off-target effect compared to the non-GNA control RD-10994. The expression levels of P2RY2 mRNA are summarized in Table 4. The results suggest that the degree of reduction of off-target effects may vary depending on the position of GNA modification.

[0226]

Table 4

[0227] Example 10 . In vitro activity of GNA-saRNAs regarding the induction of SMN2-FL and SMN2-Δ7 mRNA expression in GM03813 cells To evaluate the in vitro activity of GNA modifications at various nucleotide positions, the listed GNA-saRNAs (i.e., 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 at 2.5 nM for 3 days. RD-19040 was transfected and used as a non-GNA control. RD-10004 (ASO-1027) was transfected at 25 nM and used as a positive control. As shown in Figures 6A and 6B, all GNA-saRNAs showed higher inductive activity towards SMN2-FL and SMN2-Δ7 mRNA compared to the non-GNA saRNA RD-19040 at 2.5 nM treatment. The expression levels of SMN2-FL and SMN2-Δ7 mRNA are summarized in Table 5. The results suggest that GNA modification of SMN2 saRNA can enhance on-target activity that induces the expression of SMN2-FL and SMN2-Δ7 mRNA.

[0228] [Table 5]

[0229] Example 11 .GNA-saRNA reduces off-target effects on the expression of the off-target latent gene ARPIN in GM03813 cells. To evaluate the reduction of off-target effects by GNA-saRNA, the antisense strand of RD-19040 was used as a "query" sequence, and potential off-target genes were searched for by in silico analysis. As a result, ARPIN was predicted as a potential off-target gene. Figure 7A shows the "query" sequence (antisense strand) and "seed" region (highlighted in gray) of RD-19040, and the predicted complementary sequences for the target site in the ARPIN transcript containing two mismatched nucleotides (nucleotides shown in italics and bold). The indicated GNA-saRNAs (i.e., RD-19650, RD-19651, RD-19652, RD-19653, RD-19654, RD-19655, RD-19657, RD-19661, RD-19662, RD-19674, and RD-19675) were transfected at 2.5 nM for 3 days. RD-19040 was transfected and used as a non-GNA control. ARPIN mRNA levels were quantified by RT-qPCR using gene-specific primer sets in each PCR reaction. As shown in Figure 7B, all GNA-saRNAs reduced off-target effects compared to the non-GNA-saRNA RD-19040. ARPIN mRNA expression levels are summarized in Table 6. The results suggest that the degree of off-target effect reduction may differ depending on the location of the GNA modification.

[0230] [Table 6]

[0231] Example 12 In vitro activity of GNA-saRNA in inducing SERPING1 mRNA levels in Hep3B and HepG2 cells To verify the in vitro activity of GNA-saRNAs, Hep3B cells were transfected for 3 days with the indicated non-GNA-saRNAs (i.e., RD-17229, RD-17235, RD-17238, RD-17241, and RD-17244) and their corresponding GNA-saRNAs (i.e., RD-17074, RD-17082, RD-17086, RD-17096, and RD-17099) at the indicated concentrations (i.e., 0.02, 0.07, 0.21, 0.62, 1.85, 5.56, 16.67, and 50 nM). The mRNA levels of SERPING1 induced by non-GNA-saRNAs and GNA-saRNAs are shown in Figures 8A–8E.

[0232] Similarly, the indicated non-GNA-saRNAs (i.e., RD-17229, RD-17235, and RD-17241) and their corresponding GNA-saRNAs (i.e., RD-17074, RD-17082, and RD-17096) were transfected into HepG2 cells for 3 days at the indicated concentrations (i.e., 0.02, 0.07, 0.21, 0.62, 1.85, 5.56, 16.67, and 50 nM). The mRNA levels of SERPING1 induced by non-GNA-saRNAs and GNA-saRNAs are shown in Figures 9A–9C.

[0233] Table 7 shows E max The AUC of SERPING1 mRNA expression is summarized. The AUC of SERPING1 mRNA expression was calculated using GraphPad Prism software as described in the Materials and Methods section. The results indicate that GNA-saRNA can enhance on-target activity in inducing SERPING1 mRNA expression by non-GNA-saRNA.

[0234] [Table 7]

[0235] Materials and methods saRNA synthesis (1) Single-strand synthesis Single-stranded oligonucleotides were synthesized by solid-phase synthesis using the K&A DNA synthesizer (K&A Laboratories GbR, Schaafheim, Germany).

[0236] The starting materials were either commercially available universal solid supports or special solid supports, or the synthesis disclosed in the above description. Generally, phosphoramidite monomers (0.1 M in acetonitrile or dichloromethane) containing various linkers and conjugates were sequentially added to the solid support in a DNA synthesizer to produce the desired full-length oligonucleotide.

[0237] Amidite addition: Each cycle of amidite addition consisted of four chemical reactions: detritylation, coupling, oxidation / thiolation, and capping. In the first step, detritylation was performed for 45 seconds using 3% dichloroacetic acid (DCA) in DCM. In the second step, phosphoramidite coupling was performed on all amidites at 12 eq for 6 minutes. In the third step, as an oxidation reaction, treatment was performed for 1 minute with a 0.02 M iodine solution in THF:pyridine:water (70:20:10, v / v / v). If phosphorothioate modification was required, thiolation was performed instead of oxidation, and treatment was performed for 3 minutes with a 0.1 M xanthan hydride solution in pyridine:ACN (50:50, v / v). In the fourth step, capping was performed for 20 seconds using mixtures of THF:acetic anhydride:pyridine (80:10:10, v / v / v) (CAP A) and N-methylimidazole:THF (10:90, v / v) (CAP B). The number of cycles for the four chemical reactions depended on the length of the single oligonucleotide.

[0238] Deprotection I (nucleoside base deprotection): After synthesis was complete, the solid support was transferred to a microcentrifuge tube with a screw cap. For synthesis on a 1 μmol scale, 1 mL of a mixture of methylamine and ammonium hydroxide was added. The tube containing the solid support was then heated in an oven at 60°C to 65°C for 15 minutes, and then cooled to room temperature. The cleavage solution was collected and evaporated to dryness by speed vacuum to obtain crude single-stranded oligonucleotides.

[0239] Deprotection II (Removal of 2'-TBDMS group): If the crude RNA oligonucleotide retained a 2'-TBDMS group, it was dissolved in 0.1 mL of DMSO. After adding 1 mL of triethylamine hydrofluoric acid, the tube was capped, and the mixture was shaken vigorously to completely dissolve it. Then, it was heated in a 65°C oven for 15 minutes. The tube was removed from the oven and allowed to cool to room temperature. The solution containing the completely desilylated oligonucleotide was cooled on dry ice. To precipitate the oligonucleotide, 2 mL of ice-cold n-butanol (-20°C) was carefully added in 0.5 mL increments. The precipitate was filtered, washed with 1 mL of ice-cold n-butanol, and then dissolved in 0.01 M tris(hydroxymethyl)aminomethane hydrochloride buffer.

[0240] (2) Purification of single strands Oligonucleotide purification was performed using 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 for 25 minutes, flow rate: 1 mL / min or less. The purified oligonucleotides were collected and desalted using a HiPrep 26 / 10 Desalting column.

[0241] (3) Formation of double helix by annealing To obtain the double chain, a desalted, purified single-chain solution was prepared, and then the sense and antisense chains were mixed in equimolar concentrations and volumes in a tube. The tube was placed on a 95°C heat block for 5 minutes, and then cooled to room temperature. The resulting double chain was then freeze-dried to obtain a powder. Cell culture and processing

[0242] Fibroblasts derived from SMA patients were obtained from the Coriell 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 5% CO2 and 37°C in modified MEM medium (Gibco, Thermo Fisher Scientific, Carlsbad, CA) supplemented with 15% fetal bovine serum (Sigma-Aldrich), 1% NEAA (Gibco), and 1% penicillin / streptomycin (Gibco). HepG2 cells (SCSP-510, National collection of authenticated cell cultures, 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, Cobioer, China) were cultured at 37°C and 5% CO2 in modified MEM medium supplemented with 10% fetal bovine serum, 1% NEAA, and 1% penicillin / streptomycin. Transfection was performed using Lipofectamine RNAiMax (ThermoFisher, Waltham, MA, USA) in growth medium according to the manufacturer's protocol.

[0243] RNA isolation and reverse transcription quantification by polymerase chain reaction (RT-qPCR) (1) RNA isolation and two-step RT-qPCR To quantify intracellular mRNA expression levels, total RNA from treated cells was isolated using the RNeasy Plus Mini kit (Qiagen, Hilden, Germany) according to the manual. The obtained RNA (approximately 1 μg) was analyzed using PrimeScript. TM cDNA was reverse transcribed using an RT reagent kit with gDNA Eraser (Takara, RR047A, Shiga, Japan). The resulting cDNA was then processed with primers that specifically amplify the target gene and TB Green. (R) Premix Ex Chapter TM The sample was amplified using reagent II (Takara, RR820A, Shiga, Japan) on a Roche LightCycler 480 Multiwell Plate 384 (Roche, catalog number: 4729749001, US).

[0244] The reaction conditions include: reverse transcription (first stage): 5 min at 42°C, 10 sec at 95°C; PCR reaction (second stage): 5 sec at 95°C, 30 sec at 60°C, 10 sec at 72°C, repeating the above cycle 40 times for amplification; and a melting curve (third stage). The PCR reaction conditions are shown in Tables 8 and 9. The primer sequences are listed in Table 10. [Table 8]

[0245] [Table 9]

[0246] [Table 10]

[0247] One type of standard gene Expression level of target mRNA in saRNA-treated samples (E rel To calculate the result by comparing it with the control treatment, the Ct values ​​of the target gene and the internal standard gene were substituted into Equation I.

number

[0248] In the formula, CtTm represents the Ct value of the target gene in the control treatment sample; CtTs represents the Ct value of the target gene in the saRNA treatment sample; CtRm represents the Ct value of the internal standard gene in the control treatment sample; and CtRs represents the Ct value of the internal standard gene in the saRNA treatment sample. Two types of standard genes

[0249] Expression level of target gene mRNA in saRNA-treated samples (E rel To calculate the result by comparing it with the control treatment, the Ct values ​​of the target gene and two internal standard genes were substituted into the following equation II.

number

[0250] In the formula, CtTm represents the Ct value of the target gene in the control treatment sample; CtTs represents the Ct value of the target gene in the saRNA treatment sample; CtR1m represents the Ct value of internal standard gene 1 in the control treatment sample; CtR1s represents the Ct value of internal standard gene 1 in the saRNA treatment sample; CtR2m represents the Ct value of internal standard gene 2 in the control treatment sample; and CtR2s represents the Ct value of internal standard gene 2 in the saRNA treatment sample.

[0251] Calculation of Area Under the Curve (AUC) The area under the curve (AUC) for a particular test substance is calculated based on a curve obtained by plotting dose-response data across eight dose levels. This curve reflects the cumulative effect of the test substance across all test doses. Essentially, AUC is a metric that quantifies the overall response to treatment, integrating the responses at each dose to express them as a single metric, such as mean efficacy. To calculate AUC, first plot the response curve to agonist concentration based on dose-response data. Then, evaluate the curve using the "Area under the Curve" plugin in GraphPad Prism software. Although AUC values ​​are unitless, they can be used as a comparative index to evaluate the relative response in comparison to other test substances.

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 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, and the double-stranded oligonucleotide agent comprises one or more glycerol nucleic acid (GNA) modified nucleotides located on either or both of the sense strand and the antisense strand.

2. The double-stranded oligonucleotide agent according to claim 1, wherein either or both of the sense strand and the antisense strand are guide strands (or multiple guide strands) that mediate RNA activation.

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

4. The double-stranded oligonucleotide agent according to claim 3, wherein the seed region starts within three or fewer nucleotides from the 5' end of the guide strand.

5. The double-stranded oligonucleotide agent according to claim 3, wherein the seed region includes nucleotides from the 2nd to 10th position, or from the 2nd to 9th position, or from the 2nd to 8th position, or from the 2nd to 7th position, or from the 2nd to 6th position, or from the 3rd to 6th position, or from the 2nd to 5th position, or from the 3rd to 6th position, or from the 3rd to 5th position, or from the 4th to 6th position, from the 5' end of the guide chain.

6. The double-stranded oligonucleotide agent according to claim 1, wherein the 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).

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, comprising one or more GNA-modified nucleotides located at the 1st and / or 2nd and / or 3rd and / or 4th and / or 5th and / or 6th and / or 7th and / or 8th and / or 9th and / or 10th and / or 11th and / or 12th and / or 13th and / or 14th and / or 15th and / or 16th and / or 17th and / or 18th and / or 19th and / or 20th and / or 21st and / or 22nd and / or 23rd and / or 24th and / or 25th positions (or more) from the 5' end of the guide chain.

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 guide strand.

10. The double-stranded oligonucleotide agent according to claim 3, wherein one or more GNA-modified nucleotides are located on the passenger strand.

11. The double-stranded oligonucleotide agent according to claim 1, wherein the sense strand comprises a nucleotide sequence shown in any of SEQ ID NOs: 1, 3, 12, 14, 15, 17, 18, 20, 21, 23, 24, 27, 29, 52, 54, and the antisense strand comprises a nucleotide sequence shown in any of SEQ ID NOs: 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 guide chain is synthesized using the GNA-modified nucleotide monomer of formula (1) to include one or more GNA-modified nucleotides. 【Chemistry 1】 The double-stranded oligonucleotide agent according to claim 1, wherein the base is selected from the group consisting of adenine nucleic acid bases, thymine nucleic acid bases, cytosine nucleic acid bases, guanine nucleic acid bases, uracil nucleic acid bases, and analogs thereof.

13. The base in formula (1) is selected from the following structures: 【Chemistry 2】 The double-stranded oligonucleotide agent according to claim 12.

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 complementary nucleotides with respect to a segment of the coding strand of the target gene of equivalent length.

15. The double-stranded oligonucleotide agent according to claim 1, wherein the complementary double-stranded structure, composed of at least eight base pairs, has five or fewer mismatched nucleotides between the sense strand and the antisense strand, i.e., 5, 4, 3, 2, 1, or 0.

16. The double-stranded oligonucleotide agent according to claim 15, wherein the mismatched nucleotide(s) are located inside the antisense strand, or at or near the 3' or 5' end.

17. The double-stranded oligonucleotide agent according to claim 15, wherein the mismatched nucleotide(s) are located at the position(s) of the GNA 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 molecule activated RNA (saRNA) that upregulates the expression of the target gene by at least 10%.

19. The double-stranded oligonucleotide agent according to 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 comprises a modification selected from the group consisting of 2'-fluoro(2'-F), 2'-O-methyl(2'-OMe), 2'-O-methoxyethyl(2'-MOE), 5'-(E)-vinylphosphonate, phosphorothioate skeleton modification, and combinations thereof.

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

21. The conjugation site is selected from auxiliary oligonucleotides (ACOs), lipids / fatty acids, and GalNAc clusters, as described in claim 20.

22. The aforementioned conjugation site is, 【Transformation 3】 Selected from, During the ceremony, 【Chemistry 4】 The conjugate agent according to claim 21, wherein the support material is shown.

23. A cell comprising the double-stranded oligonucleotide agent described in claim 1 or the conjugate agent described in claim 20.

24. A pharmaceutical composition comprising a double-stranded oligonucleotide agent according to claim 1 or a conjugate 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 to 19, a conjugate agent according to any one of claims 20 to 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 a double-stranded oligonucleotide agent according to any one of claims 1 to 19 or a conjugate agent according to any one of claims 20 to 22 to the cell or subject.

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

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

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