Oligonucleotide regulators that activate factor VII expression and their use in the treatment of hemophilia

Abstract

This application relates to saRNA and oligonucleotide modulators used for the prevention or treatment of FVII-related diseases, symptoms, or disorders resulting from or associated with hemorrhagic complications of hemophilia with FVII gene deficiency or inhibitors, and to the use thereof. This application also relates to pharmaceutical compositions comprising the oligonucleotide modulators disclosed herein and to methods for preventing or treating FVII-related diseases, symptoms, or disorders induced by FVII level deficiency.

Description

[Technical Field]

[0001] This application relates to the technical field of nucleic acids, and more particularly to oligonucleotide regulators involved in the activation of gene expression and their pharmaceutical applications.

[0002] Cross-reference with related applications This application claims priority to the filing date of the provisional patent application "PCT / CN2023 / 079893," filed on March 6, 2023, and its disclosures are incorporated herein by reference in their entirety.

[0003] Array List This application includes a sequence listing submitted electronically in computer-readable format, which is incorporated herein by reference in its entirety. [Background technology]

[0004] Factor VII (FVII, also known as proconvertin) is a serine protease that is one of the coagulation factors in the vasocoagulation system and is synthesized only in the liver. In plasma, it has an extremely short half-life (4-6 hours) and is present at a concentration of approximately 10 nM (0.5 μg / ml) [Heinz S et al., 2015; Yang L et al., 2016]. Factor VII is the only coagulation factor that exists at a concentration of 1-3% in the bloodstream as activated factor VIIa (FVIIa) even when coagulation is not activated. When damage occurs, tissue factor (TF), a transmembrane protein, is exposed into the vascular lumen and binds to the freely circulating FVIIa, initiating coagulation. The action of the FVIIa-TF complex leads to the rapid generation of activated factor IX (FIXa) and activated factor X (FXa), resulting in the formation of a stable fibrin clot (thrombus).

[0005] The FVII gene has over 200 different variants, including missense mutations, nonsense mutations, minor insertions and deletions, and splice site mutations, which can affect any region of the gene. Mutations identified throughout the FVII gene can potentially affect all domains of the FVII protein. Point mutations are the main cause of hereditary FVII deficiency, with missense mutations being the most frequently observed. Exon 8 is the largest exon of the FVII gene and is a concentration of numerous mutations.

[0006] FVII-related disorders include, but are not limited to, congenital FVII deficiency (Alexander's disease), acquired FVII deficiency (AFVIID), hemophilia (including hemophilia with inhibitors), and Glanzmann thrombasthenia (GT).

[0007] Congenital FVII deficiency is a rare autosomal recessive hemorrhagic disorder caused by mutations in the gene that codes for FVII. Individuals with severe FVII deficiency are prone to bleeding in the joints and muscles, easy subcutaneous bleeding (bruising), and postoperative bleeding.

[0008] Acquired FVII deficiency (AFVIID) can result from abnormal or reduced FVII synthesis, overconsumption or abnormal degradation, antibody neutralization, or abnormal resorption by tumors. AFVIID may occur alone or in conjunction with other coagulation factor deficiencies. AFVIID is more common than hereditary FVII deficiency.

[0009] Hemophilia is a genetic disorder in which the blood does not clot due to a deficiency of clotting factors. As a result, it can cause unexplained bleeding, joint pain, swelling, and stiffness, bleeding in the urine and stool, and nosebleeds. Inhibitors (also called antibodies) are one of the most serious complications of hemophilia and occur when the body recognizes administered clotting factor preparations as foreign substances. Recombinant human factor VII activated (rhFVIIa) is commonly used for on-demand treatment of bleeding and for prophylactic administration to maintain hemostasis in hemophilia patients.

[0010] Glanzmann thrombocytopenia (GT) is a hereditary platelet membrane receptor disorder caused by quantitative or qualitative abnormalities of platelet membrane glycoprotein GPIIb / IIIa (ITG αIIbβ3), which causes platelet aggregation failure and reduced thrombus contraction. Control and prevention of bleeding in GT patients is extremely important but still difficult. Once, local treatment, regardless of anti-fibrinolytic therapy or platelet transfusion, was the mainstream of treatment. However, in recent years, the use of rhFVIIa has increased significantly, and extremely high efficacy has been reported in the treatment and prevention of bleeding in GT patients.

[0011] Fresh frozen plasma and recombinant factor VII (rFVII, e.g., rhFVIIa) are treatment options for coagulation factor-related diseases, but due to their very short half-lives (4 - 6 hours), frequent intravenous administration is required, and there is also a risk of thrombosis. Furthermore, in hemophilia patients, the administered coagulation factor preparation may induce inhibitor production, which may cause or exacerbate inhibitor-bearing hemophilia. Therefore, there is a strong demand for the development of new treatment methods and pharmaceuticals for the treatment and prevention of inhibitor-bearing hemophilia and diseases caused by FVII gene mutations or FVII protein deficiencies.

Summary of the Invention

[0012] To address the above-mentioned problems, the present application provides small activating RNA (saRNA) molecules or oligonucleotide regulators containing the same. These are for the purpose of preventing and / or treating FVII-related diseases, symptoms, and disorders (e.g., blood coagulation abnormalities). Specifically, they are targeted at diseases caused by deficiencies of FVII mRNA or FVII protein (e.g., haploinsufficiency), or diseases that can be treated and prevented by increasing the amount of FVII even when the FVII level is normal (e.g., including hemophilia and inhibitor-bearing hemophilia). This is achieved by activating or upregulating FVII transcription via the RNA activation (RNAa) mechanism and increasing the FVII protein expression level.

[0013] In particular, the inventors discovered that functional saRNAs that activate or up-regulate FVII mRNA expression are not randomly distributed on the promoter, but rather concentrated in specific hotspot regions. Among the promoter regions of the FVII gene, only some regions are suitable for gene activation by saRNAs. Specifically, they are the regions of -557 to -379, -346 to -298, -271 to -91, and -96 to -1 upstream of the transcription start site (TSS). As used herein, the "hotspot region" refers to a nucleic acid region on the target gene of saRNA (such as a promoter element upstream of the transcription start point). Multiple full-length target sequences of functional saRNAs are concentrated in this region.

[0014] Furthermore, the inventors discovered that the optimal target sequence (sense strand) of saRNA within the FVII promoter region has the following characteristics: (1) The GC content is 40% to 70%. (2) No more than 5 consecutive identical nucleotides. (3) No more than 3 repeats of a 2-base or 3-base repeat sequence. As a result, the target sequence (for example, an isolated nucleic acid sequence containing the sequence) can activate or up-regulate the FVII mRNA expression level by at least 10% or more compared to the baseline by interacting with saRNA.

[0015] Based on these remarkable findings, this disclosure provides saRNAs, formulations, and pharmaceutical compositions for activating / upregulating FVII mRNA expression. Specifically, it is possible to increase FVII gene expression by at least 10% compared to baseline expression levels. Furthermore, this specification also provides methods for the prevention or treatment of FVII-related diseases, symptoms, and disorders. These include diseases resulting from FVII deficiency, FVII gene mutations, decreased functional FVII levels in the blood, or diseases that can be prevented or treated by increasing endogenous FVII levels even if FVII levels or function are normal (e.g., inhibitor-carrying hemophilia). The method involves administering one or more saRNAs, formulations, pharmaceutical compositions, or any combination thereof disclosed herein.

[0016] In one embodiment of this application, a small activated RNA (saRNA) molecule, or an oligonucleotide regulator containing the same, is provided, capable of activating or upregulating the expression of the FVII gene in a cell. The saRNA comprises a continuous oligonucleotide sequence of 16 to 35 nucleotides in length, wherein the oligonucleotide sequence has at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, 99.5%, or 100% sequence homology or complementarity with respect to the same-length region of SEQ ID NO: 1437. This enables activation or upregulation of FVII gene expression by at least 10% compared to baseline expression. In some embodiments, the same-length region of SEQ ID NO: 1437 is selected from the following region group: -557 to -379, -346 to -298, -271 to -91, or -96 to -1. In some embodiments, the same-length region of SEQ ID NO:1437 is located within one of the regions of SEQ ID NO:1438, SEQ ID NO:1439, SEQ ID NO:1440, or SEQ ID NO:1441.

[0017] In certain embodiments, the saRNA disclosed herein comprises a sense strand and an antisense strand, each having a complementary region, which together form a double-stranded nucleic acid structure. In certain embodiments, the sense strand and antisense strand disclosed herein have at least 75% complementarity. In certain embodiments, the sense strand and antisense strand disclosed herein are located on two different nucleic acid strands. In certain embodiments, the sense strand and antisense strand disclosed herein are located on a single contiguous nucleic acid strand, which may optionally be a hairpin-type single-stranded nucleic acid molecule. In this case, the complementary regions of the sense strand and antisense strand form a double-stranded nucleic acid structure. In certain embodiments, one or both ends of the double-stranded nucleic acid structure may be blunt ends. In certain embodiments, the sense strand and / or antisense strand disclosed herein includes a 3' overhang of 1 to 6 nucleotides in length, or a 3' overhang of 2 to 3 nucleotides in length. In certain embodiments, at least one nucleotide constituting the overhang is deoxythymidine. In certain embodiments, the overhang may be a natural-type overhang. In certain embodiments, the sense strand and antisense strand disclosed herein independently comprise a sequence of nucleotides of about 16–35 bases, about 17–30 bases, about 18–25 bases, or about 19–22 bases, respectively.

[0018] In certain embodiments, the sense strand of the saRNA disclosed herein has at least 75% sequence homology to a nucleotide sequence selected from SEQ ID NO: 287 to 858, and the antisense strand has at least 75% sequence homology to a nucleotide sequence selected from SEQ ID NO: 859 to 1430. In certain embodiments, the sense strand disclosed herein includes a nucleotide sequence selected from SEQ ID NO: 287 to 858, and the antisense strand includes a nucleotide sequence selected from SEQ ID NO: 859 to 1430.

[0019] In certain embodiments, the sense strand of the saRNA disclosed herein has at least 75% sequence homology to a nucleotide sequence selected from SEQ ID NO: 287 to 572, and the antisense strand disclosed herein has at least 75% sequence homology to a nucleotide sequence selected from SEQ ID NO: 859 to 1430. Furthermore, if the SEQ ID NO of the nucleotide sequence of the sense strand is SEQ ID NO: n (where n is an integer from 287 to 572), the SEQ ID NO of the reference nucleotide sequence of the corresponding antisense strand is SEQ ID NO: n+572 or n+858. For example, if the SEQ ID NO of the nucleotide sequence of the sense strand is SEQ ID NO: 287, the SEQ ID NO of the nucleotide sequence of the corresponding antisense strand will be SEQ ID NO: 859 or SEQ ID NO: 1145.

[0020] In certain embodiments, the sense strand of the saRNA disclosed herein has at least 75% sequence homology to a nucleotide sequence selected from SEQ ID NO: 573 to 858, and the antisense strand has at least 75% sequence homology to a nucleotide sequence selected from SEQ ID NO: 859 to 1144. Furthermore, if the SEQ ID NO of the reference nucleotide sequence of the sense strand is SEQ ID NO: n' (where n' is an integer from 573 to 858), the SEQ ID NO of the reference nucleotide sequence of the corresponding antisense strand is SEQ ID NO: n' + 286.

[0021] In certain embodiments, the sense strand disclosed herein includes a nucleotide sequence selected from SEQ ID NO: 287 to 572, and the antisense strand includes a nucleotide sequence selected from SEQ ID NO: 859 to 1430. Furthermore, if the SEQ ID NO of the selected nucleotide sequence included in the sense strand is SEQ ID NO: n (where n is an integer from 287 to 572), the SEQ ID NO of the corresponding selected nucleotide sequence included in the antisense strand is SEQ ID NO: n+572 or n+858. In certain embodiments, the sense strand disclosed herein includes a nucleotide sequence selected from SEQ ID NO: 573 to 858, and the antisense strand includes a nucleotide sequence selected from SEQ ID NO: 859 to 1144. Furthermore, if the SEQ ID NO of the selected nucleotide sequence included in the sense strand is SEQ ID NO: n' (where n' is an integer from 573 to 858), the SEQ ID NO of the corresponding selected nucleotide sequence included in the antisense strand is SEQ ID NO: n'+286.

[0022] In certain embodiments, the sense strand of the saRNA disclosed in this application has at least 75% sequence homology to a nucleotide sequence selected from SEQ ID NO: 1442, 1444, 1446, 1448, 1450, 1452, 1454, 1456, 1458, 1460, 1462, 1464, 1466, 1468, 1470, 1472, 1474, and 1476, and the antisense strand has at least 75% sequence homology to a nucleotide sequence selected from SEQ ID NO: 1443, 1445, 1447, 1449, 1451, 1453, 1455, 1457, 1459, 1461, 1463, 1465, 1467, 1469, 1471, 1473, and 1475. In certain embodiments, the saRNA disclosed herein comprises a sense strand and an antisense strand, forming a duplex as described in Table 11.

[0023] In certain embodiments, the oligonucleotide sequences disclosed in this application have at least 75% sequence homology or complementarity with nucleotide sequences selected from SEQ ID NO: 1 to 286. In certain embodiments, the sense strand of the oligonucleotide sequences disclosed in this application has at least 75% sequence homology with nucleotide sequences selected from SEQ ID NO: 1 to 286. In certain embodiments, the antisense strand of the oligonucleotide sequences disclosed in this application has at least 75% sequence complementarity with nucleotide sequences selected from SEQ ID NO: 1 to 286.

[0024] In certain embodiments, the sense strand of the saRNA disclosed herein has at least 75% sequence homology to a nucleotide sequence selected from SEQ ID NO: 1 to 286, and the antisense strand has at least 75% sequence complementarity to the same reference nucleotide sequence as the sense strand.

[0025] In certain embodiments, at least one nucleotide of the saRNA disclosed herein is chemically modified. In certain embodiments, at least one nucleotide of the sense strand and / or antisense strand of the saRNA disclosed herein is chemically modified. In certain embodiments, the chemically modified nucleotide disclosed herein has at least one of the following modifications: a) Modification of phosphodiester bonds that connect nucleotides within the nucleotide sequence of saRNA; b) Modification of the 2'-OH group of ribose within the nucleotide sequence of saRNA; c) Modification of base portions within the nucleotide sequence of saRNA;

[0026] In certain embodiments, at least one nucleotide of the saRNA disclosed herein may be a locked nucleotide acid (LNA), an abasic nucleotide, a 2'-amino modified nucleotide, a 2'-alkyl modified nucleotide, a morpholino nucleotide, a phosphoramidate, or a nucleotide comprising a non-natural base.

[0027] In certain embodiments, at least one of the chemically modified nucleotides disclosed herein has a phosphorothioate (PS) skeleton modification.

[0028] In certain embodiments, at least one of the chemically modified nucleotides disclosed herein has a 2' sugar site modification, selected from one or more of the following: 2'-fluoro-2'-deoxynucleoside (2'-F) modification, 2'-O-methyl (2'-O-Me) modification, or 2'-O-(2-methoxyethyl) (2'-O-MOE) modification.

[0029] In certain embodiments, at least one of the chemically modified nucleotides disclosed herein has the addition of a 5'-phosphate group to the 5' end of the nucleotide sequence. In certain embodiments, at least one of the chemically modified nucleotides disclosed herein has the addition of an (E)-vinylphosphonate group or a 5'-methylcytosine group at the 5' end of the sense strand and / or antisense strand.

[0030] In certain embodiments, the application provides oligonucleotide regulators in which the sense strand and / or antisense strand of saRNA are conjugated to one or more conjugation groups selected from lipids, fatty acids, fluorescent dyes, ligands, glycans, peptides, and antibodies.

[0031] In certain embodiments, in the oligonucleotide regulator disclosed herein, the sense or antisense strand of the saRNA is conjugated to one or more conjugation groups selected from: cell-permeable peptides, polyethylene glycol (PEG), alkaloids, tryptamine, benzimidazole, quinolone, amino acids, cholesterol, glucose, N-acetylgalactosamine. In certain embodiments, the saRNA disclosed herein is C4-C 30 It is bound to a lipid selected from fatty acids. In certain embodiments, the conjugation group is a saturated or unsaturated linear or branched C 16 It is a lipid or fatty acid that has a carbon chain.

[0032] In certain embodiments, in the oligonucleotide regulator disclosed herein, the saRNA is conjugated to two types of conjugation groups, which are lipids and N-acetylgalactosamine. In certain embodiments, the two conjugation sites are independently selected from those derived from S9, tC2, tC2x6, and C5x5. In certain embodiments, the two conjugation sites consist of: 1) one derived from S9, tC2, or tC2x6, and 2) one derived from C5x5. [ka]

[0033] In certain embodiments, the conjugation groups that bind to the saRNA disclosed herein are S9, tC2x6, and C5x5. In certain embodiments, tC2 or tC2x6 binds to the 3' end of the sense strand, and C5x5 binds to the 5' end of the sense strand. These conjugation groups can be synthesized by methods known in the art. For example, the synthesis procedures for tC2, tC2x6, and C5x5 are referred to in International Publication WO2024002046A1, the disclosure of which is incorporated herein by reference in its entirety. S9 (HR-00214009, Anhui Province, China) is purchased from Wuhu Huaren Science and Technology Co., Ltd.

[0034] In another aspect of this application, an isolated polynucleotide of saRNA is provided. This isolated polynucleotide contains a continuous nucleotide sequence consisting of 16 to 35 bases with SEQ ID NO: 1437. Specifically, this isolated polynucleotide is a nucleic acid sequence selected from SEQ ID NO: 1 to 286. In another aspect of this application, a method for utilizing the isolated polynucleotide of saRNA is provided.

[0035] In another aspect of this application, an oligonucleotide complex is provided, comprising an antisense strand of saRNA disclosed herein and a sense strand of polynucleotide disclosed herein. In some embodiments, the oligonucleotide complex activates the expression of the FVII gene by at least 10% compared to the baseline expression of the FVII gene.

[0036] In another aspect of this application, a polynucleotide encoding the saRNA disclosed herein is provided. In one embodiment, the saRNA disclosed herein is a small activated RNA (saRNA) molecule. In one embodiment, the polynucleotide is a DNA molecule. In another aspect of this application, a vector comprising the polynucleotide disclosed herein is provided.

[0037] In another aspect of this application, a nucleic acid complex is provided, comprising an antisense strand of saRNA disclosed herein and a sense strand of polynucleotide disclosed herein. In some embodiments, the nucleic acid complex activates the expression of the FVII gene by at least 10% compared to the baseline expression of the FVII gene.

[0038] In another aspect of this application, cells comprising saRNA, polynucleotides encoding saRNA, or vectors disclosed herein are provided. In one embodiment, the cells are mammalian cells, selectively human cells. In some embodiments, the cells are host cells. The cells may exist in vitro (outside the body), such as in cell lines or cell systems, or they may exist within a mammal, including a human body.

[0039] In another aspect of this application, a composition such as a pharmaceutical composition is provided. This composition comprises the aforementioned saRNA, a polynucleotide encoding the saRNA, a vector, or a cell, and may further optionally include a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutically acceptable carrier includes an aqueous carrier, a liposome, a high molecular weight polymer, or a polypeptide. In some embodiments, the pharmaceutically acceptable carrier is selected from aqueous carriers, liposomes, high molecular weight polymers, and polypeptides. In some embodiments, the aqueous carrier may be, for example, RNase-free water or RNase-free buffer. In some embodiments, a composition comprising the saRNA or polynucleotide encoding the saRNA disclosed herein may contain 0.001 to 1600 nM (e.g., 0.001 to 1000 nM, 0.01 to 500 nM, 0.1 to 400 nM, 1 to 100 nM, 10 to 50 nM, 20 to 50 nM, 20 to 100 nM, 50 to 150 nM, 50 to 400 nM, 50 to 1000 nM, or 400 to 1600 nM), or optionally 1 to 150 nM. In some embodiments, compositions comprising the saRNA or polynucleotide encoding the saRNA disclosed herein may contain 0.001 to 150 nM (e.g., 0.001 to 100 nM, 0.001 to 50 nM, 0.001 to 20 nM, 10 to 100 nM, 10 to 50 nM, 20 to 50 nM, 20 to 100 nM, or 50 to 150 nM), or optionally 1 to 150 nM.

[0040] In another aspect of this application, an application is provided for producing a product that activates or upregulates intracellular FVII gene expression using the aforementioned saRNA, polynucleotide encoding saRNA, vector, or composition containing the same.

[0041] The present application further relates to a method for activating or upregulating FVII gene expression in cells, the method comprising administering the aforementioned saRNA, polynucleotide, vector, or composition comprising them to cells. Furthermore, a method for increasing intracellular FVII protein levels or blood levels of functional FVII protein is also provided, the method comprising introducing a sufficient amount of saRNA, nucleic acid, or composition into cells.

[0042] The saRNA, polynucleotide, or composition containing them may be directly introduced into cells, or the nucleotide sequence encoding the saRNA may be introduced into cells and then expressed and produced within the cells. These cells are preferably mammalian cells, and more preferably human cells. These cells may exist in vitro (outside the body) as cell lines or cell systems, or they may exist within a mammal, including a human body. The human body is a patient suffering from an FVII-related disease or condition, which includes, for example, those caused by FVII gene mutations, decreased FVII levels, insufficient functional FVII protein in the blood, or conditions that are not FVII deficiency but can be prevented or treated by increased FVII levels (e.g., hemophilia, particularly inhibitor-carrying hemophilia). These patients are administered the saRNA, polynucleotide, or composition containing them disclosed in this application in amounts sufficient to treat the disease or condition. Specifically, these are conditions resulting from FVII protein deficiency due to FVII gene mutations, insufficient expression of functional FVII protein, or inhibitor-carrying hemophilia. In one embodiment, this refers to a disease or condition caused by insufficient expression of the FVII protein, a mutation in the FVII gene, or a deficiency of functional FVII protein in the blood. In some embodiments, this includes diseases or conditions that can be prevented and / or treated by increasing FVII levels or function, such as hemophilia (particularly inhibitor-carrying hemophilia).

[0043] Another aspect of this application relates to methods for preventing or treating FVII-related diseases, conditions, or disorders. The terms “FVII-related disease(s) or condition(s) or disorder(s)” and “FVII-associated disease(s) or condition(s) or disorder(s)” as used herein are synonymous and mutually interchangeable, and refer to diseases, conditions, or disorders that can be prevented or treated by increasing FVII levels. This includes, for example, diseases resulting from insufficient expression of the FVII protein, diseases associated with FVII gene mutations, diseases resulting from insufficient levels of functional FVII protein in the blood, and diseases that can be prevented or treated by increasing FVII expression even when FVII levels or function are normal. FVII-related diseases, conditions, or disorders include, but are not limited to, congenital FVII deficiency (Alexander's disease), acquired FVII deficiency (AFVIID), hemophilia (hemophilia A or B, with or without inhibitors), and Glanzmann's thrombasthenia (GT). The methods of this application involve administering to an individual in therapeutically or prophylactically effective amounts of any or more of the following: saRNA as disclosed herein, polynucleotides encoding saRNA as disclosed herein, vectors as disclosed herein, and compositions comprising saRNA as disclosed herein. In certain embodiments, the disease, condition, or disorder targeted by the methods is hemophilia. The individual may be a mammal, for example, a human. In one embodiment, the individual has symptoms resulting from or related to: insufficient expression of FVII protein, FVII gene mutation, decreased levels of functional FVII protein in the blood, and inhibitor-possessed hemophilia. In one embodiment, the disease or symptoms are caused by any of the following: insufficient expression of FVII protein, FVII gene mutation, deficiency of functional FVII protein in the blood, or inhibitor-carrying hemophilia.In certain embodiments, the saRNA, polynucleotide, vector, or composition disclosed herein is administered to an individual by one or more of the following routes of administration: parenteral administration (infusion or injection), oral administration, nasal administration, inhalation administration, vaginal administration, or rectal administration. In certain embodiments, the route of administration is selected from one or more of the following: intrathecal administration, intramuscular administration, intravenous administration, intra-arterial administration, intraperitoneal administration, intravesical administration, intracerebroventricular administration, intravitreous administration, and subcutaneous administration. In certain embodiments, the method disclosed herein activates or upregulates the expression of the FVII gene or FVII mRNA in an individual. The rate of increase may be at least 10%, for example, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or 100% or more. In certain embodiments, the method disclosed herein increases the FVII protein level in an individual. The rate of increase may be at least 10%, for example, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or 100% or more.

[0044] In another aspect of this application, uses of saRNAs, polynucleotides, or compositions comprising them disclosed herein are provided for preparing pharmaceutical formulations for the prevention or treatment of FVII-related diseases or conditions. These diseases include, for example,: deficiency of functional FVII protein levels in the blood, FVII gene mutations, decreased levels of functional FVII in the blood, and inhibitor-carrying hemophilia. The individual may be a mammal, for example, a human. In one embodiment, this disease or disorder or condition includes, for example, inhibitor-carrying hemophilia.

[0045] Furthermore, this application provides a kit for carrying out the preventive or therapeutic methods disclosed herein. The kit comprises: a) saRNA, b) instructions for use, and c) means for administering the saRNA to an individual, if necessary. Specifically, the kit is packaged in a labeled package on which the label indicates that the molecule or composition can be used for the prevention or treatment of FVII-related diseases or conditions, for example, that it is effective against FVII deficiency or inhibitor-carrying hemophilia. In some embodiments, this application provides a kit for carrying out the methods disclosed herein, which comprises: a) the saRNA disclosed herein, and b) instructions for use. In certain embodiments, the instructions for use include means or methods for administering the saRNA disclosed herein to an individual.

[0046] In another aspect of this application, a kit is provided comprising saRNAs, polynucleotides, vectors, or compositions disclosed herein enclosed in a labeled package. The label indicates that the saRNA, polynucleotide, vector, or composition is available for use in preventing or treating FVII-related diseases or conditions, including, for example, FVII deficiency or inhibitor-carrying hemophilia.

[0047] Furthermore, this application also provides a kit for detecting: FVII protein, FVII-regulated protein, the saRNA, nucleic acid, or protein levels in cells into which the composition has been introduced.

[0048] The oligonucleotide modulators (e.g., saRNA molecules) provided herein that activate or upregulate FVII gene expression efficiently and specifically increase FVII gene expression, elevate FVII mRNA expression levels, and have low toxicity and side effects. This makes them suitable for use in the manufacture of pharmaceuticals for the prevention or treatment of FVII-related diseases and conditions. Target diseases include, for example, disorders associated with FVII protein deficiency, diseases resulting from FVII gene mutations, and conditions such as inhibitor-carrying hemophilia. [Brief explanation of the drawing]

[0049] [Figure 1] Figure 1 shows the changes in human FVII mRNA expression levels mediated by saRNA in the human hepatocellular carcinoma cell line Huh-7. 858 saRNAs targeting the human FVII promoter were individually transfected into Huh-7 cells at a concentration of 25 nM for 72 hours. The strand structure and sequence (including the corresponding target site within the human FVII promoter) of each saRNA double-stranded molecule are shown in Table 1.1. Transfection was performed as a mock treatment without oligonucleotides (not shown). The dsCon2 double-stranded molecule was used as a non-targeted control (not shown). RD-13516 is a double-stranded siRNA targeting FVII mRNA and was transfected as a gene-silencing siRNA control (not shown). FVII mRNA expression levels were quantified by one-step RT-qPCR using gene-specific primer sets (see Table 3). Geometric mean values ​​of PGK1 and SDHA mRNA levels were used as internal standards. The y-axis (log2 scaling factor change) shows the relative rate of change in FVII mRNA expression levels by saRNA in response to mock treatment, after normalization with PGK1 and SDHA (mean ± standard error of 2-well replicates). The x-axis shows saRNAs sorted in descending order of activity based on FVII mRNA expression induction activity (log2 scaling factor change). [Figure 2]Figure 2 shows saRNA activity in Huh-7 cells, classified by target location on the human FVII promoter and saRNA hotspot region. 858 types of human FVII promoter-targeting saRNAs were individually transfected into Huh-7 cells at 25 nM for 72 hours. Transfection was performed as a mock treatment without oligonucleotides (not shown). The dsCon2 double-stranded molecule was used as a non-target control (not shown). RD-13516 is a double-stranded siRNA targeting FVII mRNA and was transfected as a silencing control (not shown). Geometric mean values ​​of PGK1 and SDHA mRNA levels were used as internal references. The y-axis (log2 scaling factor change) shows the relative rate of change in FVII mRNA expression levels by each saRNA compared to the mock treatment, after normalization by PGK1 and SDHA (mean ± standard error of 2-well replicates). On the x-axis, the saRNA targets are arranged in order of their positions in the region upstream of the FVII transcription start site (TSS) -558~-1. Four saRNA hotspot regions where functional saRNA (activated double-stranded RNA) targets are concentrated are shown as dotted rectangular boxes labeled H1~H4. The numbers on each box indicate the boundaries of each saRNA hotspot region, with the FVII transcription start site (TSS) as the reference point (0 coordinate on the x-axis). These regions include the 5' end of the first functional saRNA target to the 3' end of the last saRNA target. [Figure 3]Figure 3 shows the induction of FVII mRNA expression by saRNA in Huh-7 cells. Based on the screening results for FVII mRNA induction, the top 47 functional FVII saRNAs (i.e., the saRNAs with the largest relative magnification change in FVII mRNA expression levels measured in Examples 1 and 2) were selected. These saRNAs were transfected into Huh-7 cells for 72 hours at seven specified concentrations (i.e., 0.1, 0.39, 1.56, 6.25, 25, 100, and 400 nM). Transfection was performed as a mock treatment without oligonucleotides (not shown). The dsCon2 double-stranded molecule was used as a non-targeted control (not shown). RD-13516 is a double-stranded siRNA that targets FVII mRNA and was transfected as a gene-silencing siRNA control (not shown). FVII mRNA levels were quantified after each PCR reaction by one-step RT-qPCR using a gene-specific primer set. The geometric mean values ​​of PGK1 and SDHA mRNA levels were used as internal criteria. The values ​​on the y-axis represent the FVII mRNA expression levels obtained from a 7-point dose-response (concentration-dependent curve) for each saRNA. These values ​​are expressed as relative to the mock treatment after being normalized by PGK1 and SDHA (mean ± standard error of 2-well replicates). [Figure 4]Figure 4 shows the activation effect of saRNA on FVII protein expression in HepG2 cells. Nineteen designated saRNAs (e.g., DS20-027A, DS20-055A, DS20-029S, DS20-124S, DS20-086A, DS20-207B, DS20-151B, DS20-228B, DS20-069A, DS20-177A, DS20-207S, DS20-272A, DS20-252A, DS20-156S, DS20-205S, DS20-241A, DS20-082A, DS20-009A, DS20-188B) were individually transfected into HepG2 cells at 25 nM for 4 days. Oligonucleotide-free mock treatment was performed, and dsCon2 double-stranded molecules were used as a non-targeted control. FVII protein levels were detected by Western blotting using a human FVII protein-specific antibody. Furthermore, blotting with a β-tubulin antibody was performed as a control for protein loading. The values ​​on the y-axis represent the FVII protein expression level after mock treatment, normalized with β-tubulin (mean ± standard error of 2-well replicates). [Figure 5]Figures 5A-5B show the FVII mRNA expression induction activity in HepG2 and Huh-7 cells by chemically modified saRNAs (CM-saRNAs). Ten specified FVII CM-saRNAs (e.g., RD-16036, RD-16037, RD-16038, RD-16027, RD-16028, RD-16041, RD-16052, RD-16055, RD-16134, RD-16035) were transfected into HepG2 cells at 10 nM for 3 days. Eleven designated FVII CM-saRNAs (e.g., RD-16036, RD-16037, RD-16038, RD-16027, RD-16028, RD-16041, RD-16052, RD-16055, RD-16134, RD-16044, RD-16035) were transfected into Huh-7 cells at 10 nM for 3 days. Oligonucleotide-free mock treatment was performed, and dsCon2M6v double-stranded molecules were used as a non-target control. RD-15120, a chemically modified siRNA targeting the FVII gene, was transfected as a gene-silencing siRNA control. FVII mRNA levels were quantified after each PCR reaction by two-step RT-qPCR using gene-specific primer sets. Geometric mean values ​​of PGK1 and SDHA mRNA levels were used as internal standards. Figure 5A shows FVII mRNA levels in HepG2 cells. Figure 5B shows FVII mRNA levels in Huh-7 cells. The values ​​on the y-axis represent the FVII mRNA levels for each saRNA after mock treatment, normalized with PGK1 and SDHA (mean ± standard error of 4-well replicates). [Figure 6]Figures 6A-6B show the effect of chemically modified saRNAs (CM-saRNAs) on inducing FVII protein expression in HepG2 and Huh-7 cells. Seven designated FVII CM-saRNAs (e.g., RD-16012, RD-16036, RD-16013, RD-16017, RD-16029, RD-16041, RD-16120) were transfected into HepG2 cells at 25 nM for 4 days. Seven designated FVII CM-saRNAs (e.g., RD-16012, RD-16024, RD-16036, RD-16017, RD-16029, RD-16041, RD-16120) were transfected into Huh-7 cells at 25 nM for 4 days. Oligonucleotide-free mock treatment was performed, and the dsCon2M3v double-stranded molecule was used as a non-targeted control. RD-15120 is a chemically modified siRNA targeting the FVII gene and was transfected as a gene-silencing siRNA control. FVII protein levels were detected by Western blotting using a human FVII protein-specific antibody. Furthermore, blotting with a β-tubulin antibody was performed as a control for protein loading. Figure 6A shows FVII protein levels in HepG2 cells. Figure 6B shows FVII protein levels in Huh-7 cells. The values ​​on the y-axis represent the FVII protein expression level after mock treatment, normalized with β-tubulin (mean ± standard error of 2-well replicates). [Modes for carrying out the invention]

[0050] Double-stranded RNAs (dsRNAs) that target gene regulatory sequences, including promoters, have been shown to sequence-specific upregulate target genes at the transcriptional level via a mechanism called RNA activation (RNAa) (Li, LC et al., Small dsRNAs induce transcriptional activation in human cells. PNAS (2006)). Such dsRNAs are called small activating RNAs (saRNAs).

[0051] The embodiments described herein are based on the unexpected discovery that oligonucleotide modulators containing saRNA (hereinafter also referred to as "FVII gene saRNA") can activate or upregulate the expression of the FVII gene in cells. Administration of the saRNA according to this application can result in increased production of functional FVII gene mRNA and a significant increase or upregulation of FVII mRNA and FVII protein levels.

[0052] In particular, the inventors found that functional saRNAs capable of activating or upregulating FVII mRNA expression are not randomly distributed on the promoter, but are concentrated in specific hotspot regions. Only a limited portion of the promoter region of the FVII gene is suitable for gene activation by saRNA. Specifically, these are the regions of -557 to -379 (H1), -346 to -298 (H2), -271 to -91 (H3), and -96 to -1 (H4) upstream of the transcription start site (TSS). These specific promoter regions identified in this application (hereinafter referred to as "hotspots" or "hotspot regions") may have lengths of at least 25 nt, 27 nt, 30 nt, 35 nt, 40 nt, 45 nt, 48 nt, or 49 nt or more, or may be in the range of approximately 25 to 200 nt, approximately 30 to 190 nt, approximately 40 to 185 nt, or approximately 49 to 181 nt. Furthermore, it may also be a specific narrow range or concrete length within the above broad range, such as 181 nt, 179 nt, 96 nt, 49 nt, 27 nt, or 25 nt.

[0053] Furthermore, the inventors discovered that the optimal target sequence (sense strand) of saRNA within the FVII promoter region has the following characteristics: (1) a GC content of 40% to 70%; (2) no more than 5 consecutive identical nucleotides; and (3) no more than 3 repetitions of 2- or 3-base repeats. Target sequences that satisfy these conditions (e.g., isolated nucleotide sequences containing such target sequences) can interact with saRNA to upregulate FVII mRNA expression by at least 10%, or 1.1 times or more, compared to the baseline level.

[0054] In some embodiments, the hotspot region refers to the nucleotide region on the target gene of the saRNA that includes the 5' end of the first saRNA target to the 3' end of the last saRNA target. Furthermore, at least 20% (e.g., at least 30%, 40%, 45%, or about 50%) of the saRNAs designed to target this region according to the conditions (1) to (4) above are functional, meaning that FVII mRNA expression induction of 1.1 times or more compared to the baseline level of mRNA expression is possible. In some embodiments, at least 20%, about 22%, at least 30%, about 35%, about 40%, or more than 50% of the designed saRNAs are functional, meaning that mRNA levels or protein expression of the target gene can be induced or upregulated by 1.1 times or more compared to the baseline level.

[0055] Based at least in part on these findings, this disclosure features saRNAs, compositions, and pharmaceutical compositions for activating or upregulating FVII mRNA expression by at least 10% compared to a reference level. Furthermore, this specification also provides methods for preventing or treating FVII-related diseases, conditions, or disorders. For example, methods comprising administering any of the saRNAs, compositions, or pharmaceutical compositions disclosed herein for conditions such as FVII deficiency, FVII gene mutations, decreased functional FVII levels in the blood, and inhibitor-carrying hemophilia.

[0056] The embodiments described herein are based on the unexpected discovery, as shown in Figure 2, that target sequences of saRNAs capable of activating or upregulating FVII gene expression in cells are concentrated in specific regions of the FVII gene promoter. The inventors identified clusters of FVII gene promoter regions with a high density of functional saRNA target sites and designated these as "hotspot promoter regions" (see, e.g., Table 8). For example, in the human FVII promoter, the following four hotspot regions upstream of the transcription start site (TSS) were identified: —557~—379 (H1), —346~—298 (H2), —271~—91 (H3), and —96~—1 (H4). These were confirmed to be optimal target sites for inducing FVII gene expression via the RNA activation (RNAa) mechanism.

[0057] This saRNA-FVII mRNA-FVII protein pathway may offer novel therapies for FVII deficiency-related disorders and other diseases, including inhibitor-carrying hemophilia, distinct from existing treatments.

[0058] definition In this specification, the following terms are defined:

[0059] All numerical ranges provided herein include all narrow numerical ranges that fall within such broad numerical ranges, as if all such narrow numerical ranges were explicitly described herein.

[0060] The transitional terms / phrases (and their grammatical variations) “comprises,” “comprises,” and “comprise” include the phrases “consisting essentially of,” “consisting essentially of,” “consisting of,” and “consisting of,” and are interchangeable throughout this application. The open term “comprise” also optionally includes the closed term “consisting of.” As used herein, the terms “contains,” “have,” and “consist of” are used synonymously, and these terms and their variants are intended to be interpreted non-restrictively.

[0061] As used herein, the term “complementary” refers to the ability to form base pairs between two oligonucleotide chains. Base pairs are generally formed by hydrogen bonds between nucleotides in antiparallel oligonucleotide chains. The bases of complementary oligonucleotide chains can be paired by the Watson-Crick method (AT, AU, CG, etc.) or by other methods that enable the formation of double helices (such as Hoogsteen or reverse Hoogsteen base pairing).

[0062] Complementarity can be categorized into complete and incomplete complementarity. "Complete complementarity" or "100% complementarity" means that each nucleotide in the first oligonucleotide chain can form a hydrogen bond with the corresponding nucleotide in the second oligonucleotide chain within the double-stranded region of the double-stranded oligonucleotide molecule, and there are no "misspairs" in the base pairs. "Incomplete complementarity" means that not all nucleotide units in the two chains are bonded to each other by hydrogen bonds. For example, in the case of two oligonucleotide chains, each with a double-stranded region of 20 nucleotides, if only two base pairs can be formed by hydrogen bonds within this double-stranded region, the complementarity of the oligonucleotide chains is 10%. In the same example, if 18 base pairs in this double-stranded region can be formed by hydrogen bonds, the oligonucleotide chains have 90% complementarity. Substantial complementarity refers to at least approximately 75%, 79%, 80%, 85%, 90%, 95%, or 99% complementarity.

[0063] As used herein, the terms “oligonucleotide” and “polynucleotide” are interchangeable and refer to polymers of nucleotides. These include, but are not limited to, single-stranded or double-stranded nucleic acid molecules (DNA, RNA, or DNA / RNA hybrids), and oligonucleotide chains in which deoxyribose and ribose sites alternate regularly or irregularly, and oligonucleotides having modified, native, or unnatural backbones. Oligonucleotides disclosed herein for activating target gene transcription may be, or may include, small activated nucleic acid molecules (saRNAs).

[0064] As used herein, the terms “oligonucleotide chain,” “strand,” and “oligonucleotide sequence” refer to short nucleotide sequences of less than 35 base pairs (including nucleotides of deoxyribonucleic acid (DNA) or ribonucleic acid (RNA)) and can be used interchangeably. In non-limiting examples, the length of a single strand may be any length between 16 and 35 nucleotides.

[0065] As used herein, the term “target gene” can refer to a nucleic acid sequence naturally occurring in a living organism, a trans gene, a viral or bacterial sequence, a chromosomal or extrachromosomal gene, and / or a sequence transiently or stably introduced or incorporated into a cell or its chromatin. A target gene may be a protein-coding gene or a non-protein-coding gene (e.g., a microRNA gene or a long non-coding RNA gene). A target gene generally includes a promoter sequence, and positive regulation (upregulation) of target gene expression is possible by designing an saRNA identical (also called identical or homologous) to the promoter sequence. As used herein, the terms “target sequence” or “target location” are interchangeable and refer to a sequence fragment within the target gene (e.g., a target gene promoter sequence) that is homologous or complementary to the sense or antisense strand of the saRNA. A target gene may also include one or more regulatory elements, one or more of which are designed to have identical sequences to the regulatory elements. Non-limiting examples of one or more regulatory elements include: promoters, enhancers, silencers, insulators, TATA boxes, GC boxes, CAAT boxes, transcription start sites, DNA-binding motifs of transcription factors or other proteins that regulate transcription, and the 5' untranslated region.

[0066] As used herein, the term "sense strand" refers to a strand in a saRNA double-strand that has homology or identity with a fragment of the coding strand sequence of the target gene.

[0067] As used herein, the term “antisense strand” refers to a strand in a saRNA double helix that has a sequence complementary to the sense strand. The antisense strand can interact with a target sequence to activate or upregulate gene expression, the target sequence may be a fragment of a target gene coding strand sequence.

[0068] As used herein, the term "coding strand" refers to a DNA strand present in a target gene that is not used for transcription, and whose nucleotide sequence is identical to the RNA sequence obtained as a transcript (except in RNA, where T in DNA is replaced with U). The coding strand in a double-stranded DNA sequence of a target gene promoter disclosed herein refers to a promoter sequence located on the same DNA strand as the DNA coding strand of the target gene.

[0069] As used herein, the term “template strand” refers to the strand of double-stranded DNA of a target gene that is complementary to the coding strand and can be transcribed into RNA as a transcription template, and is complementary to the RNA being transcribed (A corresponds to U, G to C). During transcription, RNA polymerase binds to the template strand and catalyzes RNA synthesis in the 5'→3' direction while moving along the 3'→5' direction of the template strand. The template strand in the double-stranded DNA sequence of a target gene promoter disclosed herein refers to the promoter sequence located on the same DNA strand as the DNA template strand of the target gene.

[0070] As used herein, the term "LNA" refers to a locating nucleic acid in which a 2'-oxygen atom and a 4'-carbon atom are linked by an extra bridge. As used herein, the term "BNA" refers to a 2'-O and 4'-aminoethylene crosslinked nucleic acid that may include a 5-membered or 6-membered crosslinked structure having an NO bond. As used herein, the term "PNA" refers to a nucleic acid mimetic having a pseudopeptide backbone consisting of an N-(2-aminoethyl)glycine unit having a nucleic acid base linked to a glycine nitrogen via a carbonylmethylene linker.

[0071] 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 that protein-coding or RNA-coding nucleic acid sequence. Generally, promoters of eukaryotic genes contain 100 to 5000 base pairs, but this length range is not intended to limit the term “promoter” as used herein. Promoter sequences are usually located at the 5' end of a protein-coding or RNA-coding sequence, but may also be located within exon and intron sequences.

[0072] As used herein, the term “transcription start site (TSS)” refers to a nucleotide that indicates the start of transcription on the template strand of a gene. Transcription start sites can be located on the template strand of the promoter region. In particular, a “transcription start site (TSS)” refers to the position where transcription begins at the 5' end of the FVII gene sequence, which is a nucleotide that indicates the start of transcription on the template strand of the gene and corresponds to the first nucleotide of the RNA molecule transcribed from that gene. Different variants of the same gene may have different transcription start sites (TSSs), but their upstream sequences are often identical. TSSs within a gene can be identified using conventional methods, such as cap analysis (CAGE), oligo-capping, and robust analysis of the 5' transcription end (5'-RACE).

[0073] In some embodiments, the target site is selected at least partially based on the gene sequence. In some embodiments, the target site is selected at least partially based on the sequence near the transcription start site (TSS) of the gene. In some embodiments, the coding strand sequence of the human FVII gene promoter can be obtained from the UCSC genome database. For example, SEQ ID NO: 1437 (shown in Table 6) consists of 600 nucleotides from position -1 bp to -600 bp relative to the transcription start site (TSS).

[0074] As used herein, the terms “identity” or “homology” refer to the sequence similarity between one oligonucleotide strand (sense strand or antisense strand) of a saRNA and the coding strand or template strand in a region of the target gene. As used herein, “identity” or “homology” can be at least about 75%, about 79%, about 80%, about 85%, about 90%, about 95%, or 99%. In some embodiments, the saRNA has 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 residues that differ from the reference sequence. To determine the percentage identity between two nucleic acid sequences, both sequences are aligned for optimal comparison. For example, gaps can be introduced in one or both sequences for optimal alignment, and sequences that do not have homology for comparison purposes may be ignored. Next, the nucleotides at the corresponding nucleotide positions are compared. If a position in the first sequence is occupied by the same nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. Percentage identity between two sequences is a function based on the number of identical positions shared between the two sequences, determined by considering the number of gaps that need to be introduced to achieve optimal alignment and the length of each gap. Sequence comparison and determination of percentage identity between two sequences can be achieved using mathematical algorithms, such as the Needleman and Wunsch ((1970) J. Mol. Biol. 48:444-453) algorithm, which has been incorporated into the GAP program in the GCG software package (available at www.gcg.com). Percentage identity between two nucleotide sequences can be determined using the E. Meyers and W. Miller ((1989) CABIOS, 4:11-17) algorithm, incorporated into the ALIGN program (version 2.0), using the PAM120 weight residue table, gap length penalty 12, and gap length penalty 4.It is understood that the molecules described herein may have additional conservative or non-essential nucleic acid substitutions that do not substantially affect their function.

[0075] The term "equal-length portion" refers to a portion of a sequence that, when compared to a target sequence (e.g., a sequence of oligonucleotides from saRNA), has the same length (equal number of bases) as the target sequence.

[0076] As used herein, the term “sequence-specific mode” refers to a mode of joining or hybridizing two nucleic acid fragments according to their nucleotide sequence, such as Watson-Crick base sequences (A to T, A to U, C to G, etc.) or other modes that enable the formation of a double helix (such as Hoogsteen or reverse Hoogsteen base pairing).

[0077] As used herein, the term “overhang” refers to a non-base-paired nucleotide located at the end (5' or 3' end) of a double-stranded oligonucleotide, formed by the extension of one strand from the other. The single-stranded region extending from the 3' and / or 5' ends of a double-stranded oligonucleotide is called an “overhang.”

[0078] 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 RNA target. A natural overhang on the antisense strand consists of one or more nucleotides complementary to the corresponding position on the RNA target.

[0079] As used herein, the term “isolated” refers to a substance that has been removed from its original or natural environment (for example, if it is naturally occurring, it refers to a substance that has been removed from its natural environment). For example, a naturally occurring polynucleotide or polypeptide present in a living organism is not “isolated,” but the same polynucleotide or polypeptide that has been artificially separated from some or all of a substance coexisting in a natural system is considered “isolated.” Such polynucleotide may be part of a vector, or the polynucleotide or polypeptide may be part of a composition, but it is still considered “isolated” unless the vector or composition is part of a natural environment. Isolated molecules can be obtained, for example, by extraction from naturally occurring substances, expression of recombinant nucleic acids, or chemical synthesis of molecules. For example, the term “isolated RNA” refers to an RNA molecule that, when produced by recombinant technology, has been substantially removed from other cellular components or culture media, or, when chemically synthesized, has been substantially removed from chemical precursors or other chemicals. In some embodiments, the substances relating to this application (e.g., polynucleotides, oligonucleotides, and / or saRNAs) are isolated.

[0080] As used herein, the terms “gene activation” or “activation of gene expression,” and “upregulation of gene expression” or “upregulation of gene expression” are interchangeable and mean a decrease in the transcription, translation, expression, or activity of a particular nucleic acid, determined by measuring the transcription level, mRNA level, protein level, enzyme activity, methylation state, chromatin state or arrangement, translation level, or the activity or state of the gene in a cell or biological system. These activities or states can be determined directly or indirectly. As used herein, the terms “gene activation,” “activation of gene expression,” “upregulation of gene expression,” or “upregulation of gene expression” refer to an increase in activity related to the nucleic acid sequence, regardless of the mechanism of activation. For example, gene activation occurs at the transcription level, increasing transcription to RNA, which is then translated into protein, thereby increasing protein expression.

[0081] As used herein, the terms “small activating RNA (saRNA)” and “small activating nucleic acid molecule” are used interchangeably and refer to nucleic acid molecules capable of upregulating the expression of a target gene. The nucleic acid molecule comprises: 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. These first and second nucleic acid fragments form a double-stranded structure (duplex). saRNA may also be an artificially synthesized RNA or a single-stranded RNA molecule obtained by vector expression, which can form a hairpin structure by two complementary regions within the molecule. In this case, the first region: containing a nucleotide sequence identical to the target sequence of the gene promoter; and the second region: containing a nucleotide sequence complementary to the first region. The length of the duplex region of saRNA is typically in the range of approximately 15–35 base pairs, 16–32 base pairs, 17–30 base pairs, 18–28 base pairs, 19–26 base pairs, 20–24 base pairs, or 21–22 base pairs, and more specifically, it may be approximately 15, 16, 17, 18, 19, 20, 21, 22, or 23 base pairs. Furthermore, the terms “saRNA,” “small activating RNA,” and “small activating nucleic acid molecule” may include nucleic acids other than ribonucleotides, including, but not limited to, modified nucleotides or their analogues.

[0082] As used herein, the term “oligonucleotide modulator” means an oligonucleotide-containing substance comprising, or composed of, at least one or more saRNAs of the present invention, which has the activity to modulate target gene expression or to enhance the effect of saRNA. Furthermore, the modulator may contain other oligonucleotide moieties (e.g., ASOs) or non-oligonucleotide moieties in a form conjugated, complexed, or mixed with saRNA. In certain embodiments, the oligonucleotide modulator comprises RNA (e.g., saRNAs of the present invention), DNA, BNA, LNA, or peptide nucleic acid (PNA).

[0083] As used herein, the terms “hotspot region” and “hotspot” are interchangeable and refer to a nucleic acid region on a target gene of a functional saRNA where the full-length target sites of the functional saRNA are concentrated. For example, this region is located within a promoter upstream of the transcription start site (TSS) of the target gene and refers to the range within each hotspot that encompasses from the 5' end of the first saRNA target to the 3' end of the last saRNA target. A “hotspot region” refers to a gene promoter region of at least 25 bp (e.g., at least 49 bp) in length where the target sequences of the functional saRNA are concentrated. Specifically, of the saRNAs targeting this region, at least 20%, e.g., about 22%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more than 90% are “functional,” meaning they can induce a 1.1-fold or greater change in the mRNA or protein expression of the target gene. However, these saRNAs are designed according to the following criteria: (1) GC content is 40% to 70%. (2) No more than 5 consecutive identical nucleotides. (3) No more than 3 repeating sequences of 2 or 3 bases.

[0084] As used herein, the term "functional saRNA" refers to saRNA that activates the expression of a target gene, for example, by inducing an increase in expression of at least 10% or at least 1.1 times. As used herein, the term "non-functional saRNA" refers to saRNA that alters the mRNA level and protein expression of the FVII gene by less than 10% or less than 1.1 times.

[0085] As used herein, the terms “target site” and “oligonucleotide” are interchangeable and refer to a target site to which saRNA is complementary or hybridizes. For example, an oligonucleotide at a target site may include a nucleic acid sequence to which saRNA is complementary or hybridizes. As used herein, the term “polynucleotide” refers to a polynucleotide that codes for saRNA, including, for example, DNA.

[0086] As used herein, the term “synthesis” refers to a method of synthesizing oligonucleotides, including any method that enables RNA synthesis, such as chemical synthesis, in vitro transcription, and / or expression using a vector.

[0087] As used herein, the term “carrier material” refers to a solid-phase starting material held between filters in a column that allows all reagents and solvents to pass freely when using an automated oligonucleotide synthesizer, and which can produce oligonucleotides with 3' or 5' ends as needed. The carrier material can be selected from control porous glass (CPG), silica, silica gel, glass, ceramic, polymer, cellulose, and combinations thereof.

[0088] As used herein, the terms “FVII” or “FVII gene” refer to the human gene. As used herein, the term “FVII mRNA” refers to messenger RNA (mRNA) produced by the expression or transcription of the FVII gene.

[0089] As used herein, the terms “FVII” and “FVII protein” are used interchangeably and refer to proteins produced by the expression of the FVII gene or the translation of FVII mRNA.

[0090] As used herein, the term "reference expression of the FVII gene" refers to the expression of the FVII gene in cells or individuals that have not been treated with saRNA or are prior to treatment.

[0091] saRNA This application aims to treat related diseases by upregulating the expression of the FVII gene through RNA activation and increasing the expression level of the FVII protein. Since the FVII gene encodes the FVII protein, increased expression of FVII mRNA leads to increased expression of the FVII protein, thereby enabling the prevention or treatment of diseases (e.g., inhibitor-carrying hemophilia). Therefore, the FVII gene may be a target gene in this application.

[0092] Aspects of this application include a saRNA (or an oligonucleotide modulator containing said saRNA) comprising an oligonucleotide sequence having a length of 16 to 35 consecutive nucleotides, wherein the oligonucleotide sequence has at least 75%, 80%, 85%, or 90% sequence identity or complementarity with the equivalent length portion of SEQ ID NO:1437, and furthermore, the saRNA activates or upregulates the expression of the FVII gene by at least 10% compared to the baseline expression level of the FVII gene.

[0093] In some embodiments, the identical-length portion of SEQ ID NO:1437 disclosed herein is located in the regions -557 to -379, -346 to -298, -271 to -91, or -96 to -1 upstream of the transcription start site (TSS) of the FVII gene. In some embodiments, the identical-length region of SEQ ID NO:1437 is located within any of the regions of SEQ ID NO:1438, SEQ ID NO:1439, SEQ ID NO:1440, or SEQ ID NO:1441.

[0094] In some embodiments, the sequential oligonucleotide sequence of the saRNA has up to five nucleotide differences or mismatches relative to the equal-length portion of SEQ ID NO:1437, i.e., 5, 4, 3, 2, 1, or 0. In some embodiments, the differences or mismatches are located in the middle or at the 3' end of the oligonucleotide sequence of the saRNA. Methods and principles for designing saRNA molecules are well known to those skilled in the art, and are described in detail, for example, by Place et al. in Molecular Therapy-Nucleic Acids (2012) 1, e15, and by Li et al. in PNAS (2006) Vol.103, No.46, 17337-17342, which are incorporated herein by reference in their entirety.

[0095] In some embodiments, the saRNA disclosed herein comprises a sense strand and an antisense strand. The sense strand and antisense strand each have complementary regions and form a double-stranded nucleic acid structure that activates the expression of the FVII gene in the cell via an RNA activation (RNAa) mechanism. As used herein, the term “RNAa mechanism” refers to a mechanism by which the double-stranded nucleic acid structure sequence-specifically upregulates the target gene at the transcriptional level.

[0096] Embodiments of the present invention include a small activated RNA (saRNA) comprising a sense strand and an antisense strand. The sense strand and the antisense strand each contain complementary regions.

[0097] The sense and antisense strands of saRNA may reside on two different nucleic acid strands or on a single nucleic acid strand (e.g., a contiguous nucleic acid sequence). When the sense and antisense strands reside on two different strands, one or both ends of the saRNA may be blunt ends, or at least one strand of the saRNA may have a 3' or 5' overhang of 1 to 6 nucleotides in length (e.g., 1, 2, 3, 4, 5, or 6). In some cases, both strands may have a 3' or 5' overhang of 1 to 6 nucleotides in length (e.g., 2 or 3 nucleotides). In some cases, the nucleotides of the overhang are thymine deoxyribonucleotides (dT). In some cases, the overhang is a native overhang. When the sense and antisense strands are located on a single nucleic acid strand, in some cases the saRNA is a hairpin-type single-stranded nucleic acid molecule, where the complementary regions of the sense and antisense strands form a double-stranded nucleic acid structure with respect to each other.

[0098] In some embodiments, saRNA is a complementary double-stranded structure consisting of a sense strand and an antisense strand, with both ends forming a brant end structure. In some embodiments, saRNA is a complementary double-stranded structure consisting of a sense strand and an antisense strand, with an overhang of 1 to 6 nucleotides at the 3' end of the antisense strand. In some embodiments, saRNA is a complementary double-stranded structure consisting of a sense strand and an antisense strand, with an overhang of 1 to 6 nucleotides at the 3' end of the sense strand.

[0099] In some embodiments of the saRNA, the sense strand and antisense strand are each 16 to 35 nucleotides long. For example, in some embodiments, the sense strand and antisense strand independently have lengths of 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 nucleotides.

[0100] In certain embodiments, either strand of the saRNA has at least 75% (e.g., about 79%, about 80%, about 85%, about 90%, about 95%, or about 99%) sequence identity or complementarity with a nucleotide sequence selected from SEQ ID NO: 1-286. Specifically, the sense strand of the saRNA disclosed herein has at least 75% (e.g., about 79%, about 80%, about 85%, about 90%, about 95%, or about 99%) sequence identity with any nucleotide sequence selected from SEQ ID NO: 287-858, and the antisense strand of the saRNA disclosed herein has at least 75% (e.g., about 79%, about 80%, about 85%, about 90%, about 95%, or about 99%) sequence identity with any nucleotide sequence selected from SEQ ID NO: 859-1430. More specifically, the sense strand of the saRNA disclosed herein contains or consists of any nucleotide sequence selected from SEQ ID NO: 287 to 858, and the antisense strand contains or consists of any nucleotide sequence selected from SEQ ID NO: 859 to 1430, or is itself such sequence.

[0101] In certain embodiments, one strand of the saRNA has up to five nucleotide differences or mismatches, i.e., 5, 4, 3, 2, 1, or 0, with respect to a nucleotide sequence selected from SEQ ID NO: 1 to 286. Specifically, the sense strand of the saRNA disclosed herein has up to five nucleotide differences, i.e., 5, 4, 3, 2, 1, or 0, with respect to a nucleotide sequence selected from SEQ ID NO: 287 to 858, and the antisense strand of the saRNA disclosed herein also has up to five nucleotide differences, i.e., 5, 4, 3, 2, 1, or 0, with respect to a nucleotide sequence selected from SEQ ID NO: 859 to 1430. In some embodiments, these differences or mismatches are located in the middle or 3' end of the sense or antisense strand of the saRNA.

[0102] In certain embodiments, the antisense strands disclosed herein are capable of sequence-specific interaction with the target nucleic acid sequence of a gene promoter, meaning that the antisense strand has the ability to hybridize with the target nucleic acid via hydrogen bonds. In certain embodiments, the antisense strand, when described in the 5'-to-3' direction, has a nucleotide sequence that constitutes the reverse complement of the target portion of the target nucleic acid to which it is targeted. In certain such embodiments, the antisense strand, when described in the 5'-to-3' direction, has a nucleotide sequence containing the reverse complement to the target site at SEQ ID NO:1437, specifically, the target site is a nucleic acid sequence selected from SEQ ID NO:1-286.

[0103] In the saRNAs disclosed herein, all nucleotides may be native or unmodified nucleotides, or at least one nucleotide may be chemically modified. Non-limiting examples of chemical modifications include one or more of the following combinations: (1) Modification of phosphodiester bonds between nucleotides in the nucleotide sequence of saRNA; (2) Modification of the 2'-hydroxyl group (2'-OH) present on ribose in the nucleotide sequence of saRNA; (3) Modification of the base portion in the nucleotide of saRNA; and (4) At least one nucleotide in the nucleotide sequence of the small activated nucleic acid molecule (saRNA) is locked nucleic acid (LNA).

[0104] The chemical modifications described herein are well known to those skilled in the art, and the modification of phosphodiester bonds refers to the modification of oxygen in the phosphodiester bond, including phosphorothioate modifications and boranophosphate modifications. The modifications disclosed herein stabilize the saRNA structure and maintain high specificity and high affinity for base pairing.

[0105] In some embodiments, the saRNA of this application comprises at least one chemically modified nucleotide modified with a 2'-OH group of the pentose of the nucleotide, for example, 2'-fluoro modification, 2'-oxymethyl modification, 2'-oxyethylidene methoxy modification, 2,4'-dinitrophenol modification, loc nucleic acid (LNA), 2'-amino modification, or 2'-deoxy modification, for example, 2'-deoxy-2'-fluoro modified nucleotide, 2'-deoxy modified nucleotide.

[0106] In some embodiments, the saRNA of this application comprises at least one chemically modified nucleotide in which the base portion of the nucleotide is modified, such as 5'-bromouracil modification, 5'-iodouracil modification, N-methyluracil modification, or 2,6-diaminopurine modification.

[0107] In some embodiments, the chemical modification of saRNA is the addition of an (E)-vinylphosphonate group to the 5' end of the sense sequence or antisense sequence. In some embodiments, the chemical modification of at least one chemically modified nucleotide is the addition of a 5'-methylcytosine moiety at the 5' end of the sense sequence or antisense sequence.

[0108] In some embodiments, the saRNA of this application is a small activated nucleic acid molecule (saRNA) in which at least one nucleotide in the nucleotide sequence is a chemically modified nucleic acid, such as loct nucleotides, abasic nucleotides, 2′-amino modified nucleotides, 2′-alkyl modified nucleotides, morpholino nucleotides, phosphoramidates, and nucleotides containing non-natural bases. In some embodiments, the saRNA disclosed herein includes "endo-light" modifications with 2′-O-methyl modified nucleotides and nucleotides containing a 5′-phosphorothioate group.

[0109] In some embodiments, the saRNAs of this application are chemically modified to enhance stability or other beneficial properties. The nucleic acids characterized in this application can be synthesized and / or modified by well-known methods, such as those described in "Current Protocols in Nucleic Acid Chemistry" (Beaucage, SL et al., John Wiley & Sons, Inc., New York, NY, USA). The references herein are incorporated herein by reference. (Edrs.), John Wiley & Sons, Inc., New York, NY, USA, which is hereby incorporated herein by reference. Modifications include, for example, (a) terminal modifications, e.g., 5' terminal modifications (phosphorylation, conjugation, reverse bond, etc.) and 3' terminal modifications (conjugation, DNA nucleotide, reverse bond, etc.), (b) base modifications, e.g., substitution of a stabilizing base, a destabilizing base, or a base that forms a base pair with an expanded repertoire of partners, removal of a base (abasic nucleotide), or a conjugate base, (c) sugar modifications (e.g., at the 2' or 4' position) or sugar substitutions, and (d) skeletal modifications, including modification or substitution of phosphodiester bonds. Specific examples of saRNA molecules that can be used in this application include, but are not limited to, RNA containing a modified skeleton or RNA that does not contain natural nucleoside bonds. In some embodiments, RNA having a modified skeleton includes, among other things, RNA that does not have a phosphorus atom in the skeleton. In some embodiments, modified RNA that does not contain a phosphorus atom in its internucleoside backbone can also be considered an oligonucleoside. In some embodiments, the modified oligonucleotide will have a phosphorus atom in its internucleoside backbone.

[0110] 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, phosphoramidates including 3'-aminophosphoramides and aminoalkyl phosphoramidates, thionophosphoramides, thioalkyl phosphonates, thioalkyl phosphotriesters, and boranophosphates having the usual 3'-5' bond, their 2'-5' bonded analogues, and those having reverse polarity where adjacent pairs of nucleoside units 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.

[0111] Non-limiting examples of the preparation of the phosphorus-containing linkages include, but are not limited to, the following. US 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,195; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,316; 5,550,111; 5,563,253; 5,571,799; 5,587,361; 5,625,050; 6,028,188; 6,124,445; 6,160,109; 6,169,170; 6,172,209; 6,239,265; 6,277,603; 6,326,199; 6,346,614; 6,444,423; 6,531,590, 6,534,639, 6,608,035, 6,683,167, 6,858,715, 6,867,294, 6,878,805, 7,015,315, 7,041,816, 7,273,933, 7,321,029, and U.S. Patent RE39464, which are incorporated in their entirety by reference, respectively, in particular herein.

[0112] Furthermore, to facilitate the introduction of saRNA into cells, chemical conjugation sites may be introduced to the ends of the sense or antisense strands of saRNA based on the above modifications, thereby promoting their action through gene promoter regions within the cell membrane, nuclear membrane, and nucleus, which are composed of lipid bilayers.

[0113] In certain embodiments, the saRNA disclosed herein is covalently bound to one or more conjugate sites. In certain embodiments, the conjugation sites modify one or more properties of the bound oligonucleotide. These properties include, but are not limited to, pharmacodynamics, pharmacokinetics, stability, binding, absorption, tissue distribution, intracellular distribution, intracellular uptake, charge, and clearance. In certain embodiments, the conjugation sites impart new properties to the bound oligonucleotide. Examples include fluorescent dyes or reporter groups that enable the detection of the oligonucleotide. Specific conjugation sites and conjugate sites have already been described in the following literature. For example: cholesterol group (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), thioether (e.g., hexyl-S-tritylthiol) (Manoharan et al., Ann. NY Acad. Sci., 1992, 660, 306-309; Manoharan et al., Bioorg. Med. Chem. Lett., 1993, 3, 2765-2770), thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20, 533-538), aliphatic chains (e.g., dodecanediol or undecyl residues) (Saison-Behmoaras et al., EMBO J., 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 triethylammonium-1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate) Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res., 1990, 18, 3777-3783), polyamine or polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), adamantane acetate, palmityl group (Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229-237), octadecylamine or hexylaminocarbonyloxycholesterol group (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277, 923-937), tocopherol group (Nishina et al., Molecular Therapy Nucleic Acids, 2015, 4, e220; Nishina et al., Molecular Therapy, 2008, 16, 734-740), GalNAc cluster (e.g., WO2014 / 179620). Natl. Acad. Sci. USA, 1989, 86, 6553-6556), cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4, 1053-1060), a thioether, eg, hexyl-S-tritylthiol (Manoharan et al., Ann. NY Acad. Sci., 1992, 660, 306-309; Manoharan et al., Bioorg. Med. Chem. Lett., 1993, 3, 2765-2770), a thiocholesterol (Oberhauser et al., Nucl. Acids Res.,1992, 20, 533-538), an aliphatic chain, eg, do-decan-diol or undecyl residues (Saison-Behmoaras et al., EMBO1, 1991, 10, 1111-1118; Kabanov et al., FEBS Lett., 1990, 259, 327-330; Svinarchuk et al., Biochimie,1993, 75, 49-54), a phospholipid, eg, di-hexadecyl-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., Nucl. Acids Res.,1990, 18, 3777-3783), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides&Nucleotides, 1995, 14, 969-973), or adamantane acetic acid, a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229-237), an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277, 923-937), a tocopherol group (Nishina et al., Molecular Therapy Nucleic Acids, 2015, 4, e220; and Nishina et al., Molecular Therapy, 2008, 16, 734-740), or a GalNAc cluster (e.g., WO2014 / 179620).

[0114] In some embodiments, the saRNA of this application has a sense strand or antisense strand attached to one or more conjugation sites selected from intercalators, reporter molecules, polyamines, polyamides, peptides, carbohydrates, vitamin groups, polyethylene glycol, thioethers, polyethers, cholesterol, thiocholesterol, cholic acid groups, folic acid, lipids, phospholipids, biotin, phenazine, phenanthridine, anthraquinone, adamantane, acridine, fluorescein, rhodamine, coumarin, fluorescent dyes, and dyes.

[0115] In some embodiments, the conjugation site may contain a pharmaceutically active ingredient. Examples include aspirin, warfarin, phenylbutazone, ibuprofen, suprofen, fenbufen, ketoprofen, (S)-(+)-pranoprofen, carprofen, dansyl sarcosine, 2,3,5-triiodobenzoic acid, fingolimod, fluphenamic acid, folinic acid, benzothiadiazide, chlorothiazide, diazepine, indomethacin, barbiturates, cephalosporin antibiotics, sulfonamides, antidiabetic drugs, antibacterial agents, or antibiotics.

[0116] In some embodiments, the saRNA of this application is bound to one or more conjugation sites selected from lipids, fatty acids, fluorescent dyes, ligands, sugars, peptides, and antibodies.

[0117] In some embodiments, the saRNA of this application is a sense or antisense strand conjugated to one or more conjugation sites selected from cell-permeable peptides, polyethylene glycol, alkaloids, tryptamine, benzimidazole, quinolone, amino acid, cholesterol, glucose, and N-acetylgalactosamine. In certain embodiments, the saRNA is conjugated to two conjugation sites. In certain embodiments, these two conjugation sites are a lipid and N-acetylgalactosamine. In certain embodiments, one or more conjugation sites are derived from S9, tC2, tC2x6, C5x5, or a combination thereof, and include (S9), (tC2), (tC2x6), or (C5x5) as shown herein. [ka] In certain embodiments, the conjugation sites that bind to saRNA are S9, tC2x6, and C5x5, as shown herein. In certain embodiments, tC2, tC2x6, or S9 is bound to the 3' end of the sense strand, and C5x5 is bound to the 5' end of the sense strand.

[0118] In certain embodiments, the conjugation site is a lipid selected from fatty acids having carbon chains with 4 to 30 carbon atoms. In certain embodiments, the conjugation site is a fatty acid having carbon chains with 16, 18, or 22 carbon atoms. In certain embodiments, the conjugation site is selected from the lipid-soluble sites described in International Publication WO2024002046A1. In certain embodiments, the saRNA 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 an additional 1, 2, 3, 4, 5, 6, or more linker sites.

[0119] In one embodiment, the linker moiety, if present, may be selected from the group consisting of: -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-, -O-C(O)-(C1-C 22 )alkylene-C(O)-O-, -C(O)-O-(C1-C 22 )alkylene-O-C(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 )alkylene-S(O)-O-; where the -(C1~C22)alkylene- included in the linker moiety may be an alkylene group having 1 to 22 carbon atoms, for example, 2 to 20, 3 to 18, 4 to 16, 5 to 12, or 6 to 10 carbon atoms. In one embodiment, when the linker moiety is directly bonded, the conjugation moiety is directly bonded to the oligonucleotide.

[0120] In some embodiments, saRNAs conjugated to one or more conjugation sites disclosed in the embodiment are directly contacted, transported, delivered, or administered to cells or patients.

[0121] In some embodiments, the sense strand and antisense strand of the saRNA are, independently, nucleotides in which at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or about 100% of the nucleotides are chemically modified.

[0122] In some embodiments, at least about 50%, 60%, 70%, 80%, 90%, 95%, or about 100% of the nucleotides of the saRNA are chemically modified nucleotides.

[0123] These modifications can improve the bioavailability of saRNA, enhance affinity to target sites, and increase resistance to hydrolysis by nucleases within cells.

[0124] In non-limiting examples, saRNAs are designed based on at least the following criteria: (1) a GC content of 40% to 70%, (2) no more than 5 consecutive identical nucleotides, and (3) no more than 3 repetitions of 2- or 3-base repeats. In some embodiments, saRNAs are designed or selected, at least in part, based on criteria that enable the generation of functional saRNAs. For example, in some cases, sequences located upstream of the TSS may contain sequences that are unsuitable for saRNA synthesis, even if they are located within the hotspot region.

[0125] In some embodiments, the saRNA of this application, upon contact with a cell, has the effect of activating or upregulating the expression of one or more genes within the cell, preferably at least 10% (e.g., at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 300%, 500%, 800%, 1000%, 2000%, or 5000%).

[0126] target sequence In certain embodiments, this application relates to a target site of saRNA, specifically, a nucleotide or oligonucleotide having a length of 16 to 35 nucleotides in the nucleotide sequence of SEQ ID NO: 1437. In certain embodiments, the target site or oligonucleotide is a nucleic acid sequence selected from SEQ ID NO: 1 to 286. The target site is capable of interacting with the antisense strand of the saRNA disclosed in this application, resulting in a saRNA that activates the expression of the FVII gene (e.g., mRNA expression, protein expression, FVII expression). In some embodiments, the target site is selected based at least in part on a promoter sequence upstream of the TSS. In some embodiments, the target site is selected based at least in part on a sequence upstream of the TSS at -5000 bp, -4000 bp, -3000 bp, -2000 bp, -1000 bp, or -500 bp. In some embodiments, the target site is selected by moving 1 bp increments in the TSS direction, thereby determining the target sequence, and this process is repeated to extend it by 1 base pair (e.g., n+1) in the TSS direction. In some embodiments, the target site has a length of approximately 8 to approximately 35 nucleotides. In some embodiments, the target site has a length of approximately 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, or 35 nucleotides. In some embodiments, the target site or oligonucleotide is an isolated molecule. In some embodiments, the isolated target site or isolated oligonucleotide is used for the design, screening, and / or preparation of the corresponding saRNA.

[0127] In certain embodiments, this application relates to an oligonucleotide complex comprising a saRNA and a target site disclosed herein. In certain embodiments, the oligonucleotide complex activates the expression of the FVII gene by at least 10% (e.g., activates expression compared to a baseline FVII gene expression level).

[0128] hotspot In certain embodiments, this application relates to nucleic acid sequences located upstream of the transcription start site (TSS) of the FVII gene, i.e., “hotspot regions”. In certain embodiments, the nucleic acid sequences disclosed herein are oligonucleotide sequences having a length of at least 25, 27, 30, 35, 40, 45, 48, or 49 consecutive nucleotides and having at least 75%, 80%, 85%, or 90% sequence homology to the co-length region in SEQ ID NO: 1437. In some embodiments, the hotspot region is an isolated region.

[0129] The terms "hotspot region" and "hotspot" are synonymous and are defined herein as a nucleic acid region (e.g., a promoter region upstream of the TSS) on the target gene of a saRNA in which the full-length target sites of functional saRNAs are concentrated, and which includes the region from the 5' end of the first saRNA target to the 3' end of the last saRNA target within each hotspot. In some embodiments, at least 20% (e.g., 22%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%) of the saRNAs designed to target sequences within a hotspot are functional, i.e., capable of altering the mRNA expression of the target gene by at least 1.1 times. As a non-limiting example, at least 20% (e.g., 25%, 30%, 35%, 40%, or 47%) of the saRNAs designed to target a hotspot are functional and capable of altering the mRNA expression of the target gene by at least 1.1 times. However, the saRNA is designed based on at least the following criteria: (1) the GC content is 40% to 70%, (2) the same nucleotide does not exceed 5 consecutive times, and (3) the 2-base or 3-base repeat sequence does not exceed 3 times. In some embodiments, the designed functional saRNA may be blunt-ended or have overhangs, and may be unmodified or modified.

[0130] In some embodiments, nucleic acid sequences and / or target sites are selected using the same or similar criteria. As a non-limiting example, isolated nucleic acid sequences upstream of the TSS of the FVII gene are selected based on at least the following criteria: (1) GC content is 40% to 70%; (2) No more than 5 consecutive identical nucleotides; (3) No more than 3 repetitions of 2- or 3-base repeats.

[0131] In some embodiments, the nucleic acid region has a length of about 25 to about 250 nucleotides (e.g., about 33 to about 200, about 36 to about 150, about 39 to about 100, about 42 to about 75, about 45 to about 70, or about 48 to about 55 nucleotides). In some embodiments, the hotspot region is a polynucleotide sequence selected from SEQ ID NO: 1438 to 1441, and includes a subregion within the region where the target sequence of the functional saRNA accumulates. In some embodiments, the hotspot region is a nucleic acid sequence selected from the -557 to -379, -346 to -298, -271 to -91, and -96 to -1 regions located upstream of the TSS of the FVII gene, and includes a subregion within the region where the target sequence of the functional saRNA accumulates. This application also provides a method for designing saRNAs that target the nucleic acid sequences described herein.

[0132] In some embodiments, the target site is designed or selected based at least in part on criteria that enable the generation of a functional saRNA. For example, in some cases, a sequence located upstream of the TSS may not be suitable for target site synthesis, even if it is located within the hotspot region.

[0133] The RNAa activity of each designed saRNA depends on a complex multi-factor system, including the chromatin environment, the target sequence and its surrounding sequence characteristics, and transcription factor binding. A major determinant may be the accessibility of the DNA target. In highly accessible regions, double-stranded RNA (dsRNA) may exhibit higher RNAa activity. Conversely, dsRNAs designed to target other regions of the promoter may be non-functional or exhibit transcriptional silencing effects. This is thought to explain the existence of hotspot regions where functional saRNA target sites accumulate. For example, even if a target site is designed based at least partially on the following criteria: (1) GC content of 40% to 70%, (2) no more than 5 consecutive identical nucleotides, and (3) no more than 3 repetitions of 2- or 3-base repeats, if the target site is not located within a hotspot region (e.g., any of the hotspot regions described herein), it may not be possible to activate or upregulate FVII gene expression by at least 10% compared to the baseline expression level.

[0134] In certain embodiments, this application relates to a nucleic acid complex comprising a saRNA and nucleic acid sequence disclosed herein. In certain embodiments, the complex activates the expression of the FVII gene by at least 10% compared to a baseline expression level.

[0135] In some embodiments, methods are also provided that use nucleic acids upstream of the transcription target site of the FVII gene.

[0136] DNA that encodes saRNA In certain embodiments, this application relates to a nucleic acid or polynucleotide encoding a saRNA that can activate or upregulate the expression of the FVII gene in a cell. Preferably, the expression is increased by at least 10% compared to the baseline expression level of the FVII gene. In certain embodiments, the nucleic acid is DNA encoding the saRNA. In certain embodiments, the nucleic acid is a recombinant vector, specifically a recombinant adeno-associated virus (AAV) vector. The vectors disclosed herein include a DNA fragment encoding the saRNA of this application.

[0137] Cells containing saRNA The saRNAs disclosed herein, after contact with cells, effectively activate or upregulate the expression of the FVII gene within the cell. Preferably, they increase the expression of the FVII gene by at least 10% compared to the baseline expression level.

[0138] In certain embodiments, this application relates to cells containing the saRNA disclosed herein. In some embodiments, the cells are mammalian cells. In some embodiments, the cells are human cells, including, for example, human fetal hepatocytes, human hepatocellular carcinoma cells (e.g., Huh-7 cells), and human hepatocellular carcinoma cells (e.g., PLC / PRF / 5 cells). The cells disclosed herein may be present in vitro or ex vivo (e.g., cell lines or cell lines), or in a mammalian body (e.g., in a human body). The human body disclosed herein is a patient suffering from an FVII-related disease or condition, caused, for example, by an FVII gene mutation, low FVII levels, insufficient levels of functional FVII protein in the blood, or hemophilia with inhibitors. In some embodiments, the cells are derived from a hemophilia patient.

[0139] Composition containing saRNA In certain embodiments, this application relates to compositions or pharmaceutical compositions comprising saRNA or nucleic acids disclosed herein. In some embodiments, the composition comprises at least one pharmaceutically acceptable carrier. In some embodiments, the composition comprises at least one pharmaceutically acceptable carrier, which is selected from aqueous carriers, liposomes or LNPs (lipid nanoparticles), polymers, micelles, colloids, metal nanoparticles, nonmetal nanoparticles, bioconjugates (e.g., GalNAc), polypeptides, and antibodies. In one embodiment, the aqueous carrier may be RNase-free water or RNase-free buffer. In some embodiments, the composition may contain 0.001 to 1600 nM of the saRNA or polynucleotide described herein. (Examples: 0.001-1000 nM, 0.001-500 nM, 0.001-400 nM, 10-100 nM, 10-50 nM, 20-50 nM, 20-100 nM, 50-150 nM, 50-400 nM, 50-1000 nM, or 400-1600 nM) In some embodiments, the composition contains 25 nM of the saRNA or polynucleotide described herein. In some embodiments, the composition may contain 0.001-150 nM of the saRNA or polynucleotide described herein. (Examples: 0.01-100 nM, 0.1-50 nM, 1-150 nM, 1-20 nM, 0.001-1 nM, 1-10 nM, 10-100 nM, 10-50 nM, 20-50 nM, 20-100 nM) In some embodiments, the composition contains 25 nM of the saRNA or polynucleotide described herein.

[0140] How to use saRNA Another aspect of this application relates to a method for using saRNA to activate / upregulate FVII gene expression in cells. The saRNA comprises an oligonucleotide sequence consisting of 16 to 35 consecutive nucleotides. In some embodiments, the oligonucleotide sequence has at least 75%, 80%, 85%, or 90% homology or complementarity to the co-length region of SEQ ID NO: 1437, and activates / upregulates FVII gene expression (e.g., at least 10%, and further 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 300%, 500%, 800%, 1000%, 2000%, or 5000%) compared to the baseline expression level. In certain embodiments, administration of the saRNA disclosed in the embodiments to cells or subjects activates / upregulates FVII gene expression by at least twofold (e.g., threefold, fourfold, fivefold, sixfold, or sevenfold). In certain embodiments, the saRNA activates or upregulates FVII gene expression by approximately 6.5fold. In certain embodiments, administration of the saRNA disclosed in the embodiments to cells at a concentration of at least 0.01 nM (e.g., 0.02, 0.05, 0.08, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.8, 1, 5, 10, 25, 50, 75, 100, 150, 200, 400, 800, 1000, 1600 nM) activates / upregulates FVII gene expression. In certain embodiments, administration of the saRNA disclosed in the embodiments to cells at a concentration of at least 0.01 nM (e.g., 0.02, 0.05, 0.08, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.8, 1, 2, 3, 4, 5, 10, 25, 50, 75, 100, 150 nM) activates / upregulates the production of FVII protein (FVII).

[0141] Another aspect of this application relates to a method for preventing or treating FVII-related disorders or conditions, such as insufficient FVII expression, FVII gene mutations, low functional FVII levels in the blood of an individual, or diseases treatable by increased FVII levels even if FVII levels are normal (e.g., hemophilia with inhibitors), comprising administering to the individual an effective amount of saRNA, a nucleic acid or polynucleotide encoding said saRNA, or a composition containing said saRNA. In certain embodiments, the effective amount of saRNA disclosed herein may be in the concentration range of 0.01 to 1600 nM (e.g., 0.01, 0.02, 0.05, 0.08, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.8, 1, 5, 10, 25, 50, 75, 100, 150, 200, 400, 800, 1000, 1600 nM). In certain embodiments, the effective amount of saRNA disclosed herein may be in the concentration range of 0.01 to 1600 nM (e.g., 0.01, 0.02, 0.05, 0.08, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.8, 1, 5, 10, 25, 50, 75, 100, 150, 200, 400, 800, 1000, 1600 nM). In some embodiments, the disorder or condition is hemophilia (e.g., hemophilia with inhibitors). In some embodiments, the individual is a mammal. In some embodiments, the individual is a human.

[0142] In any embodiment provided herein, the saRNA, the nucleic acid encoding the saRNA, or a composition containing the saRNA may be introduced directly into cells, or administered by introducing the nucleotide sequence encoding the saRNA into cells to induce intracellular production (e.g., mammalian cells including, but not limited to, PLC / PRF / 5 and Huh-7, or human cells). The cells may be ex vivo (e.g., cell lines) or present in a mammal such as a human. In some embodiments, the human is a patient or individual suffering from hemophilia with FVII deficiency-related conditions or inhibitors. In certain embodiments, the nucleic acid encoding the saRNA or polynucleotide, or the composition containing the saRNA, is present in an amount sufficient to treat hemophilia.

[0143] Another aspect of this application relates to administering an effective amount of saRNA or a composition to an individual using the administration routes described herein. In some embodiments, the administration route is selected from one or more of parenteral infusion, oral administration, nasal administration, inhalation administration, vaginal administration, and rectal administration. In some embodiments, the administration route is selected from one or more of intrathecal administration, intramuscular administration, intravenous administration, intra-arterial administration, intraperitoneal administration, intravesical administration, intraventricular administration, intravitreous administration, and subcutaneous administration.

[0144] Administration regimen and route of administration Aspects of this application relate to pharmaceutical compositions comprising the saRNA of this application. In some embodiments, the pharmaceutical composition comprises the saRNA of this application and a pharmaceutically acceptable carrier, a therapeutically inert carrier, a diluent, or a pharmaceutically acceptable excipient. Aspects of this application also relate to methods of using the saRNA of this application to prepare such pharmaceutical compositions.

[0145] Aspects of this application also relate to methods of using the saRNA of this application to prepare such compositions.

[0146] Another aspect of this application relates to the use of the saRNA of this application in the manufacture of the pharmaceutical compositions disclosed herein.

[0147] Another aspect of this application relates to the use of saRNA or polynucleotides, or compositions, according to any embodiment described herein, in the manufacture of pharmaceuticals for the prevention or treatment of conditions associated with the FVII gene or FVII protein. These conditions include those caused by insufficient expression of the FVII protein, mutations in the FVII gene, decreased levels of functional FVII in the blood, or hemophilia (e.g., hemophilia with inhibitors). Uses in specific embodiments may include diseases or conditions associated with mutations in the FVII gene, or hemophilia with inhibitors. Uses in specific embodiments may target conditions induced by insufficient expression of the FVII protein or hemophilia with inhibitors. Uses in specific embodiments may also include cases where the subject is a mammal, such as human.

[0148] The dosages for administering the saRNA or composition of this application can be obtained over a wide range and can be adapted to the individual requirements of each case. In certain embodiments, the initial dose of the pharmaceutical composition of this application is administered when the subject is less than one week, less than one month, less than three months, less than six months, less than one year, less than two years, less than fifteen years, or 15 years or older.

[0149] The single dose of saRNA ranges from 0.01 mg / kg to 1000 mg / kg, 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. The doses described herein may contain two or more of the saRNA sequences described herein.

[0150] In some embodiments, the proposed dosing frequencies are approximate. For example, in a particular embodiment, if the proposed dosing frequency is the first dose on day 1 and the second dose on day 29, the patient may receive the second dose 25, 26, 27, 28, 29, 30, 31, 32, 33, or 34 days after the first dose. In a particular embodiment, if the proposed dosing frequency is the first dose on day 1 and the second dose on day 15, the patient may receive the second dose 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days after the first dose. In a particular embodiment, if the proposed dosing frequency is the first dose on day 1 and the second dose on day 85, the patient may receive the second dose 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, or 90 days after the first dose.

[0151] In certain embodiments, the dosage and / or volume of injection are adjusted based on the patient's age, weight, and / or other factors requiring adjustment of injection parameters.

[0152] In certain embodiments, the pharmaceutical composition includes a cosolvent system. Some such cosolvent systems include, for example, benzyl alcohol, a nonpolar surfactant, a water-miscible organic polymer, and an aqueous phase. In certain embodiments, such a cosolvent system is used for hydrophobic compounds. A non-limiting example of such a cosolvent system is the VPD cosolvent system, which consists of 3% w / v benzyl alcohol and 8% w / v nonpolar surfactant polysorbate 80 TM and an absolute ethanol solution containing 65% w / v polyethylene glycol 300. The proportions of such cosolvent systems can be changed considerably without significantly altering solubility or toxic properties. Furthermore, the identity of the cosolvent components can be changed: for example, Polysorbate 80 TM Other surfactants may be used instead; the fractional size of polyethylene glycol may be changed; other biocompatible polymers, such as polyvinylpyrrolidone, may be used instead of polyethylene glycol; and other sugars or polysaccharides may be used instead of dextrose.

[0153] Examples of other compositions or components related to the saRNA, compositions, pharmaceutical compositions, and methods described herein include, but are not limited to, diluents, salts, buffers, chelating agents, preservatives, desiccants, antimicrobial agents, needles, syringes, packaging materials, tubes, bottles, flasks, beakers, etc., for example, those for using, modifying, assembling, preserving, packaging, preparing, mixing, diluting, and / or storing the components for specific uses. In embodiments in which a liquid form of any of the components is used, the liquid form may be concentrated or ready for immediate use.

[0154] In some embodiments, the lipid moiety used in nucleic acid therapy may be applied in this application for the delivery of saRNA molecules disclosed herein. In such a method, nucleic acids (e.g., one or more saRNAs described herein) are introduced into a pre-formed liposome or lipoplex consisting of a mixture of cationic and neutral lipids. In certain methods, the complex of saRNA with mono- or polycationic lipids is formed without the presence of neutral lipids. In certain embodiments, the lipid moiety is selected to increase the distribution of a pharmaceutical agent to specific cells or tissues. In certain embodiments, the lipid moiety is selected to increase the distribution of a pharmaceutical agent to adipose tissue. In certain embodiments, the lipid moiety is selected to increase the distribution of a pharmaceutical agent to muscle tissue.

[0155] 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 certain pharmaceutical compositions, including those containing hydrophobic compounds. In certain embodiments, certain organic solvents, such as dimethyl sulfoxide, are used.

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

[0157] In some embodiments, saRNA may be delivered or administered via a vector. Any vector that can be used for gene delivery may be used. In some embodiments, viral vectors may be used. Non-limiting examples of viral vectors that may be used in this application include, but are not limited to, human immunodeficiency virus;HSV, herpes simplex virus;MMSV, Moloney's mouse sarcoma virus;MSCV, mouse stem cell virus;SFV, Semryki forest virus;SIN, Sindbis virus;VEE, Venezuelan encephalitis virus;VSV, vesicular stomatitis virus;VV, vaccinia virus;AAV, adeno-associated virus; adenovirus; lentivirus; and retrovirus.

[0158] In some embodiments, the vector is a recombinant AAV vector (rAAV). AAV vectors are relatively small DNA viruses that can be incorporated into the genome of infected cells in a stable and site-specific manner. AAV can infect a variety of cells without affecting cell growth, morphology, or differentiation, and does not appear to be involved in human pathogenesis. The AAV genome has been cloned, sequenced, and characterized. The genome consists of approximately 4,700 base pairs, with approximately 145 base pairs of reverse terminal repeat (ITR) regions at both ends, which serve as the origin of viral replication. The remainder of the genome is divided into two important regions responsible for encapsulation: the left side of the genome, which contains the rep gene involved in viral replication and viral gene expression, and the right side of the genome, which contains the cap gene encoding the viral capsid protein.

[0159] The formulations, pharmaceutical compositions, or medicinal products of this application are formulated, administered, and given in a manner consistent with good medical practice. Factors to be considered in this context include the specific disorder being treated, the specific mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of drug delivery, the method of administration, the administration schedule, and other factors known to healthcare professionals.

[0160] In the case of the formulations, pharmaceutical compositions, or pharmaceuticals of this application, delivery may be selectively parenteral infusion, including intrathecal, intramuscular, intravenous, intra-arterial, intraperitoneal, intravesical, intraventricular, intravitreous, or subcutaneous administration; or by oral, intranasal, inhalation, vaginal, or rectal administration.

[0161] Typical formulations of the oligonucleotide modulator of this application are prepared by mixing the saRNA of this application with a carrier or excipient. Suitable carriers and excipients are well known to those skilled in the art and are described in detail, for example, Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems by Ansel HC et al. (2004, Lippincott, Williams & Wilkins, Philadelphia), Remington: The Science and Practice of Pharmacy by Gennaro AR et al. (2000, Lippincott, Williams & Wilkins, Philadelphia), and Handbook of Pharmaceutical Excipients by Rowe RC (2005, Pharmaceutical Press, Chicago). The formulation may also include one or more buffers, stabilizers, surfactants, wetting agents, lubricants, emulsifiers, suspending agents, preservatives, antioxidants, opacifiers, lubricants, processing aids, colorants, sweeteners, flavorings, diluents, and other known additives to provide a sophisticated presentation of a pharmaceutical (i.e., saRNA or its pharmaceutical composition) or to assist in the manufacture of a pharmaceutical (i.e., a drug).

[0162] Diagnostic methods Another aspect of this application relates to a method for detecting FVII protein or FVII regulatory protein in the blood. In certain embodiments, the method includes detecting FVII protein or FVII regulatory protein in cells into which the saRNA, polynucleotide, or saRNA-containing composition disclosed herein has been introduced. In certain embodiments, the method disclosed herein can be applied to detect a specific subgroup of patients suffering from diseases or conditions induced by insufficient FVII protein expression, FVII gene mutations, low levels of functional FVII in the blood, and / or inhibitor-carrying hemophilia. As another embodiment of the method disclosed herein, the method can be used to monitor the efficacy or safety of the above patients receiving treatment with saRNA, nucleic acids or polynucleotides encoding saRNA, compositions, or pharmaceuticals.

[0163] In certain embodiments, baseline measurements are obtained from a biological sample as defined herein before administering the treatment described herein. In certain embodiments, baseline expression levels of the FVII gene are obtained from a biological sample before administering the saRNA described herein. In certain embodiments, the biological sample may be peripheral blood mononuclear cells, plasma, serum, skin tissue, or a portion of an organ.

[0164] In some embodiments, the saRNAs provided herein increase the amount of functional FVII protein in the blood by at least 10% (e.g., at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 300%, 500%, 800%, 1000%, 2000%, or 5000%) compared to the baseline measurements described above.

[0165] In some embodiments, saRNA exhibits effects beyond additive or synergistic in the treatment, prevention, progression delay, and / or improvement of diseases caused by FVII gene mutations. In some embodiments, saRNA exhibits effects beyond additive or synergistic in the protection of cells involved in the pathophysiology of diseases, particularly in the treatment, prevention, progression delay, and / or improvement of hemophilia.

[0166] Another aspect of this application relates to a method for activating / upregulating FVII gene expression in cells, comprising administering saRNA, polynucleotides, or compositions disclosed herein. In some embodiments, saRNA, polynucleotides, or compositions are introduced into cells. In some embodiments, the saRNAs disclosed herein are produced in cells after the nucleotide sequence encoding the saRNA has been introduced into the cells. In some embodiments, the cells disclosed herein are mammalian cells, preferably human cells.

[0167] Another aspect of this application relates to a method for increasing the amount of FVII protein in cells or the amount of functional FVII protein in a patient's blood, comprising introducing an effective amount of saRNA, a nucleic acid or polynucleotide encoding saRNA, or a composition disclosed herein into a cell or subject.

[0168] kit Another aspect of this application relates to a kit for carrying out a method for increasing intracellular FVII protein levels or functional FVII protein levels in the blood, the kit comprising saRNA disclosed herein. In certain embodiments, the kit further comprises means for administering the saRNA to an individual. In certain embodiments, the kit is housed in a labeled package, the label of which states that the saRNA or the composition can be used to prevent or treat diseases, conditions or disorders induced by insufficient expression of factor VII (FVII), or hemophilia (e.g., inhibitor-carrying hemophilia).

[0169] As used herein, the term “kit” generally means a package, assembly, or container (e.g., an insulated container) containing one or more components or embodiments of this application, or other components related to this application (e.g., those described above). Any reagent or component of the kit may be provided in liquid form (e.g., in solution) or solid form (e.g., dry powder, frozen product, etc.).

[0170] In further embodiments, the kit may include instructions in any form or to a website or other source provided for using the kit in relation to the components and / or methods described herein. For example, the instructions may include instructions for the use, modification, mixing, dilution, storage, assembly, preservation, packaging, and / or preparation of the components and / or other components related to the kit. In some cases, the instructions may also include instructions for the delivery of components for transport or storage at room temperature, sub-zero temperatures, cryogenic temperatures, etc.

[0171] Another aspect of this application relates to a kit for detecting FVII proteins or FVII regulatory proteins in blood. In certain embodiments, the kit is for detecting FVII proteins or FVII regulatory proteins in cells transfected with one or more saRNAs, polynucleotides, or compositions disclosed herein. Furthermore, kits for increasing intracellular FVII protein levels are also provided herein.

[0172] Specific Embodiments This application provides the following specific embodiments. In one aspect of this specification, saRNA is provided in particular: 1. A small activated RNA (saRNA) comprising a sense strand and an antisense strand, characterized by the following: Each sense strand or antisense strand of the saRNA contains a continuous oligonucleotide sequence consisting of 16 to 35 consecutive nucleotides, and the continuous oligonucleotide sequence has at least 75%, 80%, 85%, 90%, 95%, 98%, or 100% homology or complementarity with respect to a continuous fragment of equal length of SEQ ID NO:1437, respectively. The saRNA increases the expression of the FVII gene by at least 10% compared to the baseline expression of the FVII gene. 2. The saRNA of Embodiment 1, wherein a continuous fragment of equal length with SEQ ID NO:1437 is located in the region upstream of the transcription start site (TSS) of the FVII gene, and further Consecutive segments of equal length with SEQ ID NO:1437 are located in regions selected from the following groups: regions -557 to -379, regions -346 to -298, regions -271 to -91, or regions -96 to -1; and / or Consecutive segments of equal length with SEQ ID NO:1437 are located in the region of SEQ ID NO:1438, SEQ ID NO:1439, SEQ ID NO:1440, or SEQ ID NO:1441. 3. A sequence of equal-length fragments of saRNA according to Embodiment 1 or 2, with SEQ ID NO: 1437, is selected from SEQ ID NO: 1 to 286. 4. A saRNA of any of Embodiments 1 to 3, wherein a continuous fragment of equal length with SEQ ID NO: 1437 is selected from SEQ ID NO: 37, 55, 77, 86, 116, 117, 120, 124, 145, 146, 151, 177, 188, 205, 207, 218, 228, 241, 242, 10, 11, 29, 88, 121, 156, 259, 261, 274, 9, 27, 69, 81, 82, 107, 240, and 252. 5. A saRNA according to any of Embodiments 1 to 4, wherein the continuous oligonucleotide sequence satisfies the following: (1) the GC content is 40% to 70%; (2) the same nucleotide does not exceed 5 consecutive times; (3) the 2-base or 3-base repeat sequence does not exceed 3 times; and / or, the continuous oligonucleotide sequence is complementary to the sense strand or antisense strand of the saRNA.

[0173] 6. A saRNA according to any of Embodiments 1 to 5, wherein the sense strand and antisense strand each independently have a length of about 16 to 35, about 17 to 30, about 18 to 25, or about 19 to 22 nucleotides. 7. A saRNA according to any of Embodiments 1 to 6, wherein the sense strand and antisense strand have at least 90% complementarity; and / or, The sense strand and antisense strand are located on two different nucleic acid strands or on one contiguous nucleic acid strand; and / or, The continuous oligonucleotide sequence contains 0, 1, 2, or 3 mismatches with respect to the complementary region of the sense or antisense strand. 8. A saRNA according to any of Embodiments 1 to 7, wherein the sense strand and the antisense strand each include complementary regions, and the complementary regions of the sense strand and the antisense strand form a double-stranded nucleic acid structure. 9. The saRNA of Embodiment 8, wherein the sense strand or antisense strand independently contains a 3' overhang of 1-6, 1-5, or 2-3 nucleotides in length, or The double-stranded nucleic acid structure has blunt ends. 10. The saRNA of Embodiment 9, wherein at least one of the nucleotides in the overhang is a thymine deoxyribonucleotide (dT).

[0174] 11. A saRNA of any of Embodiments 1 to 10, wherein the continuous oligonucleotide sequence of the sense strand has at least 75% sequence homology to a nucleotide sequence selected from SEQ ID NO: 287 to 858, and / or The sequence of oligonucleotides in the antisense strand has at least 75% sequence homology to a nucleotide sequence selected from SEQ ID NO: 859~1430. 12. A saRNA according to any of Embodiments 1 to 11, wherein the sense strand sequence is selected from SEQ ID NO: 287 to 858, and the antisense strand sequence is selected from SEQ ID NO: 859 to 1430. 13. A saRNA of any of Embodiments 1 to 12, wherein the sequential oligonucleotide sequence of the sense strand is SEQ ID NO: n, and the sequential oligonucleotide sequence of the antisense strand is SEQ ID NO: n+572 or n+858, where n is an integer selected from SEQ ID NO: 287 to 572; and / or, If the sequence of oligonucleotides on the sense strand is SEQ ID NO: n' and the sequence of oligonucleotides on the antisense strand is SEQ ID NO: n'+286, then n' is an integer selected from SEQ ID NO: 573 to 858. 14. A saRNA of any of Embodiments 1 to 13, wherein the sense strand comprises a sequence of nucleotides selected from SEQ ID NO: 323, 341, 363, 372, 402, 403, 406, 410, 431, 432, 437, 463, 474, 491, 493, 504, 514, 527, 528, 296, 297, 315, 374, 407, 442, 545, 547, 560, 581, 582, 599, 627, 641, 653, 654, 658, 679, 749, 812, 813, and 824, and / or The antisense strand contains a sequence of nucleotides selected from SEQ ID NO: 859, 913, 935, 944, 974, 975, 978, 982, 1003, 1004, 1009, 1035, 1046, 1063, 1065, 1076, 1086, 1099, 1100, 1154, 1155, 1173, 1232, 1265, 1268, 1300, 1349, 1351, 1362, 1372, 1385, 1403, 1405, 1418, 867, 868, 885, 927, 939, 940, 965, 1098, and 1110.

[0175] 15. A saRNA according to any of Embodiments 1 to 14, wherein the sense strand comprises the nucleotide sequence of SEQ ID NO: m, and the antisense strand comprises the nucleotide sequence of SEQ ID NO: m+572, where m is selected from 323, 341, 363, 372, 402, 403, 406, 410, 431, 432, 437, 463, 474, 491, 493, 504, 514, 527, 528; and / or, The sense strand contains the nucleotide sequence of SEQ ID NO: m', and the antisense strand contains the nucleotide sequence of SEQ ID NO: m+858, where m is selected from 296, 297, 315, 374, 407, 410, 442, 491, 493, 504, 514, 527, 545, 547, 560; and / or, The sense strand contains the nucleotide sequence with SEQ ID NO: m" and the antisense strand contains the nucleotide sequence with SEQ ID NO: m"+286, where m is selected from 581, 582, 599, 627, 641, 653, 654, 658, 679, 749, 812, 813, and 824. 16. A saRNA of any of Embodiments 1 to 15, wherein the sense strand comprises a sequence of nucleotides selected from SEQ ID NO: 1442, 1444, 1446, 1448, 1450, 1452, 1454, 1456, 1458, 1460, 1462, 1464, 1466, 1468, 1470, 1472, 1474, and 1476, and / or The antisense strand contains a sequence of nucleotides selected from SEQ ID NO: 1443, 1445, 1447, 1449, 1451, 1453, 1455, 1457, 1459, 1461, 1463, 1465, 1467, 1469, 1471, 1473, and 1475, and / or The saRNA contains a sense strand and an antisense strand, which form a double-stranded structure, and this double-stranded structure is selected from any of the double-stranded structures listed in Table 11. 17. A saRNA according to any of Embodiments 1 to 16, wherein at least one nucleotide of the saRNA is a chemically modified nucleotide. 18. The saRNA of Embodiment 17, wherein the chemically modified nucleotide has at least one of the following modifications: a) Modification of phosphodiester bonds that connect nucleotides within the nucleotide sequence of saRNA; b) Modification of the 2'-OH group of ribose within the nucleotide sequence of saRNA; c) Modification of base portions within the nucleotide sequence of saRNA; 19. The saRNA of Embodiment 18, wherein the modification of the phosphodiester bond linking the nucleotides is selected from phosphorothioate modification or boranophosphate modification; and / or, The modification of the 2'-OH group is selected from the group consisting of 2'-fluoro modification, 2'-oxymethyl modification, 2'-oxyethylidene methoxy modification, 2,4'-dinitrophenol modification, 2'-amino modification, and 2'-deoxy modification; and / or, The base modification is selected from the group consisting of 5'-bromouracil modification, 5'-iodouracil modification, N-methyluracil modification, and 2,6-diaminopurine modification.

[0176] 20. saRNA of Embodiment 17, wherein at least one nucleotide of the saRNA is a locked nucleic acid (LNA), an abasic nucleotide, a 2'-amino modified nucleotide, a 2'-alkyl modified nucleotide, a morpholino nucleotide, a phosphoramidate, or a nucleotide comprising a non-natural base; and / or, The chemical modification of at least one chemically modified nucleotide is the addition of an (E)-vinylphosphonate group to the 5' end of the sense strand or antisense strand. In one embodiment of this specification, in particular, an oligonucleotide modulator is provided: 21. An oligonucleotide modulator comprising one or more saRNAs according to any of Embodiments 1 to 20. 22. An oligonucleotide modulator of Embodiment 21, further comprising one or more portions or components bound, complexed, or mixed with the saRNA. 23. An oligonucleotide modulator of Embodiment 21, wherein the sense strand and / or antisense strand of saRNA are bound to one or more binding sites (conjugation sites) selected from the group consisting of lipids, fatty acids (e.g., fatty acids having carbon chains with 4-30, 12-24, or 16-22 carbon atoms), fluorescent dyes, ligands, sugars, peptides, and antibodies. 24. An oligonucleotide modulator of Embodiment 22, wherein the conjugation site is independently selected from lipids, cell-permeable peptides, polyethylene glycol, alkaloids, tryptamine, benzimidazole, quinolone, amino acids, cholesterol, glucose, N-acetylgalactosamine, and any combination thereof.

[0177] 25. An oligonucleotide modulator of Embodiment 22, wherein the conjugation site is independently selected from S9, tC2, tC2x6, C5x5, and any combination thereof. [ka]

[0178] 26. In the oligonucleotide modulator of Embodiment 21, the oligonucleotide modulator further comprises saRNA conjugated or combined with one or more other active sites for the treatment of FVII-related disease or condition or disorder, wherein the one or more other active sites are independently selected from small molecular weight chemical sites, polypeptides, and antibodies. In one embodiment of this specification, a target site is provided in particular: 27. An isolated oligonucleotide, (a) the isolated oligonucleotide is a sequence of 16-35 bases having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% homology to a sequence of equal-length fragments of SEQ ID NO: 1437, Alternatively, (b) the isolated oligonucleotide is a sequence of 16–35 bases of oligonucleotides having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% complementarity to the equal-length sequence fragment of SEQ ID NO: 1437. 28. In the isolated oligonucleotide of Embodiment 27, the contiguous fragment of equal length with SEQ ID NO: 1437 is located in the region upstream of the transcription start site (TSS) of the FVII gene, and further, Consecutive segments of equal length with SEQ ID NO:1437 are located in regions selected from the following groups: regions -557 to -379, regions -346 to -298, regions -271 to -91, or regions -96 to -1; and / or Consecutive segments of equal length with SEQ ID NO:1437 are located in the region of SEQ ID NO:1438, SEQ ID NO:1439, SEQ ID NO:1440, or SEQ ID NO:1441. 29. In the isolated oligonucleotide of Embodiment 27, (a) the isolated oligonucleotide is a nucleic acid sequence selected from SEQ ID NO: 1 to 286, for example, selected from SEQ ID NO: 37, 55, 77, 86, 116, 117, 120, 124, 145, 146, 151, 177, 188, 205, 207, 218, 228, 241, 242, 10, 11, 29, 88, 121, 156, 259, 261, 274, 9, 27, 69, 81, 82, 107, 240, and 252, or (b) The isolated oligonucleotide is a nucleic acid sequence complementary to a nucleic acid sequence selected from SEQ ID NO: 1 to 286, for example, complementary to SEQ ID NO: 37, 55, 77, 86, 116, 117, 120, 124, 145, 146, 151, 177, 188, 205, 207, 218, 228, 241, 242, 10, 11, 29, 88, 121, 156, 259, 261, 274, 9, 27, 69, 81, 82, 107, 240, and 252.

[0179] 30. Oligonucleotide complexes containing the following: An antisense strand of saRNA from any of Embodiments 1 to 20 and a sense strand of (a) isolated oligonucleotide from any of Embodiments 27 to 29, or A sense strand of saRNA from any of Embodiments 1 to 20 and an antisense strand of (b) isolated oligonucleotide from any of Embodiments 27 to 29. 31. In the oligonucleotide complex of Embodiment 30, the oligonucleotide complex activates the expression of the FVII gene by at least 10% compared to baseline expression. In one embodiment of this specification, a hotspot is provided in particular: 32. At least 20% (e.g., at least 30%, at least 40%, at least 45%, or at least 50%) of an isolated nucleic acid molecule, which is a saRNA designed to target the sequence of the isolated nucleic acid molecule, is a nucleic acid molecule that activates the expression of the FVII gene by at least 10%, wherein the designed saRNA satisfies the following conditions: (1) GC content is 40% to 70%; (2) No more than 5 consecutive identical nucleotides; (3) No more than 3 repetitions of 2-base or 3-base repeat sequences. 33. An isolated nucleic acid molecule having a sequence corresponding to the region upstream of the transcription start site of the FVII gene, wherein the sequence is located in a region selected from the following regions located upstream of the transcription start site, or in any sub-region within these regions: regions -557 to -379, regions -346 to -298, regions -271 to -91, and regions -96 to -1. 34. Isolated nucleic acid molecules having a sequence selected from SEQ ID NO: 1438~1441.

[0180] 35. An isolated nucleic acid molecule according to any of Embodiments 32 to 34, comprising an isolated oligonucleotide according to Embodiment 27(a) or Embodiment 29(a). 36. An isolated nucleic acid molecule according to any of embodiments 32 to 34, wherein the saRNA designed to target the nucleic acid molecule is a saRNA as defined in any of embodiments 1 to 20; and / or The desired saRNA is either a brand-end type, has an overhang, has no chemical modification, or has chemical modification. In one embodiment of this specification, DNA is provided in particular: 37. Isolated polynucleotides encoding saRNA according to any of Embodiments 1 to 20, or oligonucleotide modulators according to any of Embodiments 21 to 26. 38. An isolated polynucleotide according to Embodiment 37, wherein the isolated polynucleotide is DNA. 39. A vector comprising an isolated polynucleotide as described in any of embodiments 37 to 38. In one embodiment of this specification, cells are provided in particular:

[0181] 40. A host cell comprising saRNA according to any of Embodiments 1 to 20, an oligonucleotide modulator according to any of Embodiments 21 to 26, an isolated polynucleotide according to any of Embodiments 37 to 38, or a vector according to Embodiment 39. In one embodiment of this specification, in particular, products are provided: 41. A product comprising saRNA according to any of Embodiments 1 to 20, an oligonucleotide modulator according to any of Embodiments 21 to 26, an isolated oligonucleotide according to any of Embodiments 27 to 29, an oligonucleotide complex according to any of Embodiments 30 to 31, an isolated nucleic acid molecule according to any of Embodiments 32 to 36, an isolated polynucleotide according to any of Embodiments 37 to 38, a vector according to Embodiment 39, or a host cell according to Embodiment 40. 42. The product described in Embodiment 41 is a product for the prevention, treatment or diagnosis of FVII-related diseases or disorders or symptoms (e.g., diseases associated with FVII deficiency or hemophilia), and for the design and / or screening of saRNAs. In one embodiment of this specification, a pharmaceutical composition is provided in particular: 43. A composition comprising saRNA according to any of Embodiments 1 to 20, an oligonucleotide modulator according to any of Embodiments 21 to 26, or an isolated polynucleotide according to any of Embodiments 37 to 38, and optionally a pharmaceutically acceptable carrier. 44. The composition according to Embodiment 43, wherein the composition contains 0.001 to 1600 nM, for example, 1 to 150 nM of saRNA.

[0182] In one aspect of this specification, a product is provided that is used in the manufacture of a pharmaceutical product for activating or upregulating FVII gene expression: 45. A product for activating / upregulating FVII gene expression in cells, wherein the product activates FVII gene expression by at least 10% compared to a reference expression level of the FVII gene, and the product comprises one or more active ingredients selected from any of the saRNAs described in Embodiments 1 to 20, the oligonucleotide modulators described in any of Embodiments 21 to 26, the isolated polynucleotides described in any of Embodiments 37 to 38, the vectors described in Embodiment 39, or the compositions described in any of Embodiments 43 to 44. 46. ​​Use of an active ingredient in the manufacture of a product for activating / upregulating FVII gene expression in cells, wherein the product activates FVII gene expression by at least 10% compared to a reference expression level of the FVII gene, and the active ingredient is one or more selected from the saRNA described in any of Embodiments 1 to 20, the oligonucleotide modulator described in any of Embodiments 21 to 26, the isolated polynucleotide described in any of Embodiments 37 to 38, the vector described in Embodiment 39, or the composition described in any of Embodiments 43 to 44.

[0183] 47. A method for activating / upregulating FVII gene expression in cells, wherein the method activates FVII gene expression by at least 10% compared to a reference expression level of the FVII gene, and the method comprises administering an effective amount of an active ingredient to cells, the active ingredient being one or more selected from saRNA as described in any of Embodiments 1 to 20, oligonucleotide modulators as described in any of Embodiments 21 to 26, isolated polynucleotides as described in any of Embodiments 37 to 38, vectors as described in Embodiment 39, or compositions as described in any of Embodiments 43 to 44. 48. Products, uses, or methods for activating / upregulating FVII gene expression in cells as described in Embodiment 45, the use described in Embodiment 46, or the method described in Embodiment 47, wherein the active ingredient is introduced into the cells; and / or Products, uses, or methods in which the cells are in vitro (in a test tube), ex vivo (outside the body), or in vivo (in the body); and / or Products, uses, or methods in which the cells in question are mammalian cells. 49. A product, use, or method described in Embodiment 48, wherein the active ingredient is introduced into cells by the following method: 1) Composition of the active ingredient with a physiologically or pharmaceutically acceptable carrier, wherein the carrier comprises one or more selected from the group consisting of aqueous carriers, liposomes, polymers, polypeptides, and antibodies; and / or 2) Conjugating the active ingredient to one or more conjugation sites, where the conjugation sites are one or more selected from the group consisting of lipids, cell-permeable peptides, polyethylene glycol, alkaloids, tryptamine, benzimidazole, quinolone, amino acids, cholesterol, glucose, and N-acetylgalactosamine, and further include any combination thereof (for example, two conjugation sites, one of which is a lipid and the other is N-acetylgalactosamine).

[0184] 50. A product, use, or method according to Embodiment 49, wherein the conjugation site is one or more selected from S9, tC2, tC2x6, and C5x5, or any combination thereof (e.g., C5x5 and tC2x6). [ka]

[0185] 51. A product, use, or method according to Embodiment 49, wherein the conjugation site is a lipid selected from fatty acids having carbon chains with 4 to 30, 12 to 24, and 16 to 22 carbon atoms. and / or A product, use, or method wherein the conjugation site is a fatty acid having a carbon chain with 4 to 30, 12 to 24, 16 to 22, or 16 carbon atoms. and / or a product, use, or method in which the conjugation site is independently selected from fluorescent dyes, ligands, sugars, peptides, and antibodies. 52. A product, use, or method for activating / upregulating FVII gene expression in cells as described in Embodiment 45, the use as described in Embodiment 46, or the method as described in Embodiment 47, wherein the cells are derived from a patient who has or is at risk of having a disease, condition, or disorder induced by FVII protein deficiency, FVII gene mutation, decreased blood functional FVII protein levels, and / or activation / upregulation of FVII gene expression (e.g., hemophilia (e.g., inhibitor-carrying hemophilia) or Glanzmann thrombasthenia (GT)), and the active ingredient is administered in an amount sufficient to prevent or treat the disease, condition, or disorder.

[0186] In certain embodiments of this specification, products are provided that are used in methods for treating or preventing diseases, or in the manufacture of pharmaceuticals: 53. A product for preventing or treating an FVII-related disease, condition or disorder, the product comprising an active ingredient, the active ingredient being one or more selected from: saRNA as described in any of Embodiments 1 to 20; oligonucleotide modulator as described in any of Embodiments 21 to 26; isolated polynucleotide as described in any of Embodiments 37 to 38; vector as described in Embodiment 39; or composition as described in any of Embodiments 43 to 44. 54. Use of an active ingredient in the manufacture of a product for preventing or treating an FVII-related disease, condition or disorder, wherein the active ingredient is one or more selected from: saRNA as described in any of Embodiments 1 to 20, oligonucleotide modulator as described in any of Embodiments 21 to 26, isolated polynucleotide as described in any of Embodiments 37 to 38, vector as described in Embodiment 39, or composition as described in any of Embodiments 43 to 44. 55. A method for preventing or treating an FVII-related disease, condition, or disorder, the method comprising the step of administering an effective amount of an active ingredient to a subject, the active ingredient being one or more selected from: a saRNA as described in any of Embodiments 1 to 20, an oligonucleotide modulator as described in any of Embodiments 21 to 26, an isolated polynucleotide as described in any of Embodiments 37 to 38, a vector as described in Embodiment 39, or a composition as described in any of Embodiments 43 to 44. 56. The product described in Embodiment 53, the use described in Embodiment 54, or the method described in Embodiment 55, wherein the subject is a mammal (e.g., human), preferably a mammal suffering from or at risk of having a disease, condition, or disorder (e.g., hemophilia with inhibitors) that can be prevented or treated by deficiency of factor VII (FVII) protein expression, FVII gene mutation, decreased functional FVII levels in the blood, and / or activation / upregulation of FVII levels. 57. The product described in Embodiment 53, the use described in Embodiment 54, or the method described in Embodiment 55, wherein the active ingredient is administered to an individual by one or more routes of administration selected from the following: parenteral administration (intravenous infusion), oral administration, nasal administration, inhalation administration, vaginal administration, and rectal administration. 58. The product described in Embodiment 53, the use described in Embodiment 54, or the method described in Embodiment 55, wherein the active ingredient is administered to an individual by one or more routes of administration selected from the following: intrathecal, intramuscular, intravenous, intra-arterial, intraperitoneal, intravesical, intraventricular, intracerebral, intravitreous, and subcutaneous. 59. A product described in Embodiment 53, a use described in Embodiment 54, or a method described in Embodiment 55, wherein the expression of FVII gene mRNA in an individual is activated / upregulated by at least 10% compared to a baseline expression level of the FVII gene, and / or the FVII protein level in an individual is increased by at least 10% compared to a baseline level of the FVII protein, and / or FVII-related diseases, conditions, or disorders are selected from the following: congenital FVII deficiency (Alexander disease), acquired FVII deficiency (AFVIID), hemophilia (e.g., hemophilia with inhibitors), and Glanzmann thrombasthenia (GT).

[0187] In one embodiment of this specification, a diagnostic method is provided: 60. A method for diagnosing an FVII-related disease or disorder by detecting an FVII protein or a protein controlled by FVII in the cells described in Embodiment 40.

[0188] In one embodiment of this specification, a kit is provided: 61. A kit for carrying out the method of Embodiment 60, comprising saRNA as described in any of Embodiments 1 to 20, or oligonucleotide modulator as described in any of Embodiments 21 to 26. 62. A kit according to Embodiment 61, wherein the instructions for use include means for administering to an individual the saRNA described in any of Embodiments 1 to 20 or the oligonucleotide modulator described in any of Embodiments 21 to 26. 63. A kit comprising saRNA according to any of Embodiments 1 to 20, or oligonucleotide modulator according to any of Embodiments 21 to 26, isolated polynucleotide according to any of Embodiments 37 to 38, vector according to Embodiment 39, or composition according to any of Embodiments 43 to 44, wherein the kit is housed in a labeled package, the label on the package stating that the saRNA, isolated polynucleotide, vector, or composition may be used for the prevention or treatment of diseases, conditions or disorders induced by deficiency of factor VII (FVII), or for use in relation to hemophilia. 64. A kit for detecting FVII protein or FVII-controlled protein in the cells described in Embodiment 40.

[0189] In one embodiment of this specification, a method for obtaining saRNA is provided: 65. A method for obtaining saRNA capable of increasing the expression of the FVII gene by at least 10% compared to a reference expression level, the method comprising: (a) A step of synthesizing saRNA including a sense strand and an antisense strand, wherein each sense strand or antisense strand includes a continuous oligonucleotide sequence consisting of 16 to 35 consecutive nucleotides, and each of these continuous oligonucleotide sequences independently has at least 75%, 80%, 85%, 90%, 95%, 98%, or 100% homology or complementarity with the isolength continuous fragment of SEQ ID NO:1437, and furthermore, the continuous oligonucleotide sequence satisfies the following conditions: (1) the GC content is 40% to 70%; (2) the same nucleotide does not exceed 5 consecutive times; and (3) the 2-base or 3-base repeat sequence does not exceed 3 times. (b) A step to determine the ability of the saRNA to increase the expression of the FVII gene. 66. A method for obtaining saRNA capable of increasing the expression of the FVII gene by at least 10% compared to a reference expression level, the method comprising: (a) A step of synthesizing saRNA targeting a continuous fragment of SEQ ID NO:1437, wherein the saRNA satisfies the following conditions: (1) The GC content is 40% to 70%. (2) The same nucleotide does not exceed 5 consecutive occurrences. (3) The 2-base or 3-base repeat sequence does not exceed 3 occurrences. (b) A step to determine the ability of the saRNA to increase the expression of the FVII gene. 67. The sequence fragment with SEQ ID NO:1437 is located upstream of the transcription start site (TSS) of the FVII gene, and furthermore: The sequence of SEQ ID NO:1437 is located in a region selected from the following groups: region -557~-379, region -346~-298, region -271~-91, or region -96~-1; and / or The sequence fragment in question is located in the region of SEQ ID NO:1438, SEQ ID NO:1439, SEQ ID NO:1440, or SEQ ID NO:1441. 68. A method according to Embodiment 65 or 66, wherein the continuous segment of SEQ ID NO:1437 is selected from SEQ ID NO:1 to 286.

[0190] In one aspect of this specification, isolated double-stranded functional RNA having a brant end structure is provided: 69. A double-stranded functional saRNA molecule, wherein both ends of the functional RNA molecule are blunt-ended; and / or, The length of each strand in the functional RNA molecule is approximately 16-35 nucleotides, 17-30 nucleotides, 18-25 nucleotides, or 19-22 nucleotides. 70. A double-stranded functional RNA molecule according to Embodiment 69, wherein the functional RNA molecule targets and controls the expression of the FVII gene. 71. An oligonucleotide agent comprising one or more saRNAs described in any of embodiments 69 to 70. 72. An oligonucleotide agent according to Embodiment 71, wherein one or more sites or components are conjugated (bound) to the agent. 73. Oligonucleotide agents according to Embodiment 72, wherein the sense strand and / or antisense strand of the functional saRNA are conjugated to one or more conjugation sites selected from: lipids, fatty acids having a carbon chain length of 4-30, 12-24, or 16-22 carbon atoms, fluorescent dyes, ligands, polysaccharides, peptides, and antibodies. 74. Oligonucleotide agents according to Embodiment 73, wherein the conjugation site is independently selected from: lipids, cell-permeable peptides, polyethylene glycol (PEG), alkaloids, tryptamine, benzimidazole, quinolone, amino acids, cholesterol, glucose, N-acetylgalactosamine, and any combination thereof.

[0191] 75. An oligonucleotide agent according to Embodiment 74, wherein the conjugation site is independently selected from S9, tC2, tC2x6, and C5x5, or any combination thereof ((S9), (tC2), (tC2x6), and (C5x5)). [ka]

[0192] While specific embodiments of active substances (e.g., saRNA), products, compositions, and methods have been described herein, many variations of the invention will become apparent to those skilled in the art by examining this specification and the claims. The scope of the invention should be determined by referring to the claims and this specification, which encompass the entire range of its equivalents, as well as the above-described variations. [Examples]

[0193] The present invention will be further described below with reference to specific examples and drawings. It should be understood that these examples are not intended to limit the scope of this application, but merely to illustrate it. In the following examples, research methods where no specific conditions are described were generally carried out according to conventional conditions, such as those described in Sambrook et al.'s "Molecular Cloning: Laboratory Manual" (New York: Cold Spring Harbor Laboratory Press, 1989), or conditions recommended by the manufacturer.

[0194] Materials and methods saRNA synthesis (1) Single-strand synthesis Single-stranded oligonucleotides were synthesized using solid-phase synthesis with the K&A DNA synthesizer (K&A Laboratories GbR, Chaafheim, Germany).

[0195] The starting materials were commercially available universal or specialized solid supports, or synthesized as disclosed in the previous context. Generally, phosphoramidite monomers (0.1 M in acetonitrile or dichloromethane) containing various linkers and conjugates were sequentially added to a solid support in a DNA synthesizer to produce the desired full-length oligonucleotides.

[0196] Amidite addition: Each cycle consists of four chemical reactions including detritylation, coupling, oxidation / thiolation, and capping. In the first step, detritylation was performed for 45 seconds using a DCM solution containing 3% dichloroacetic acid (DCA). In the second step, phosphoramidite coupling was performed on all amidites with 12 equivalents for 6 minutes. In the third step, oxidation was performed for 1 minute using a 0.02 M iodine solution in THF:pyridine:water (70:20:10, v / v / v). However, if phosphorothioate modification was required, oxidation was replaced with thiolation, and the mixture was treated 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 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 these four chemical reactions was determined by the length of the single-stranded oligonucleotide.

[0197] Deprotection step I (base deprotection): After the synthesis was complete, the solid support was transferred to a screw-capped microcentrifuge tube. For 1 μmol scale synthesis, 1 ml of a mixture of methylamine and ammonium hydroxide was added. The tube containing the solid support was 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 dried using a speed vacuum to obtain crudely purified single-chain oligonucleotides.

[0198] Deprotection step II (removal of 2'-TBDMS group): If the crude RNA oligonucleotide still contained a 2'-TBDMS group, it was dissolved in 0.1 ml of DMSO. After adding 1 ml of triethylamine trifluoroacetate, the tube was capped and vigorously stirred to ensure complete dissolution, then 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. 2 ml of ice-cold n-butanol (-20°C) was carefully added in 0.5 ml increments to precipitate the oligonucleotide. The precipitate was filtered, washed with 1 ml of ice-cold n-butanol, and then dissolved in 0.01 M tris(hydroxymethyl)aminomethane buffer.

[0199] (2) Single-strand purification 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, 2M NaCl, pH 7.5), Gradient: The concentration of Buffer B was changed from 10% to 60% over 25 minutes at a flow rate of 1 ml / min. Pure oligonucleotides were recovered and desalted using a HiPrep 26 / 10 desalting column.

[0200] (3) Annealing for double helix formation For double-chain compounds, a desalted and purified single-chain solution was prepared, and then equal volumes of the sense and antisense chains were mixed in a tube at equimolar concentrations. The tube was placed on a heat block set to 95°C for 5 minutes, and then cooled to room temperature. The resulting double-chain compound was then freeze-dried to obtain a powder.

[0201] Conjugation sites The conjugation sites can be synthesized by methods well known to those skilled in the art. For example, the synthesis processes for tC2, tC2x6, and C5x5 are described in WO2024002046A1, which is incorporated herein by reference.

[0202] Cell culture and processing Human hepatocellular carcinoma-derived Huh-7 cells (JCRB0403, Cobioer Biosciences CO.LTD, China) and HepG2 cells (SCSP-510, National Collection of Authenticated Cell Cultures, China) were cultured at 37 °C and 5% CO2 in modified DMEM medium (Gibco, Thermo Fisher Scientific, Carlsbad, CA) supplemented with 10% calf serum (Sigma-Aldrich) and 1% penicillin / streptomycin (Gibco). saRNAs were transfected individually into each well of Huh-7 and HepG2 cells using RNAiMAX (Invitrogen, Carlsbad, CA) according to a reverse transcription protocol, at final concentrations of 10 nM, 25 nM, or specified concentrations (0.1, 0.39, 1.56, 6.25, 25, 100, 400 nM). Transfection was performed under conditions that did not include oligonucleotides, and mock treatment was performed. dsCon2, dsCon2M6v, and dsCon2M3v were used as non-targeted double-stranded RNA controls, respectively. RD-13516 is a double-stranded siRNA targeting the FVII gene and was transfected as a silencing siRNA control. RD-15120 is a chemically modified siRNA targeting the FVII gene and was transfected as a silencing siRNA control.

[0203] RNA extraction and reverse transcription quantitative polymerase chain reaction (RT-qPCR) (1) RNA extraction and one-step RT-qPCR At the end of transfection, the culture medium was removed and each well was washed once with 150 μL of PBS. After removing the PBS, 100 μL of cell lysis buffer (Power SYBR(R) Green Cells-to-Ct) was added to each well. TM The kit (Life Technologies) was added and incubated at room temperature for 5 minutes. 0.5 μL of cell lysate was taken from each well and One Step TB Green(R) PrimeScript was added. TM RT-qPCR analysis was performed using the RT-PCR kit II (Takara, RR086A, Shiga, Japan) on a Roche LightCycler 480 real-time PCR instrument (Roche, product number: 4729749001, USA). The PCR reaction was prepared using the Bravo automated liquid handling platform (Agilent, USA). Each transfection sample was amplified using triple assay. Details of the PCR reaction mixture are shown in Table 2. [Table 2]

[0204] The reaction conditions were as follows: Reverse transcription reaction (stage 1): 5 minutes at 42°C, 10 seconds at 95°C. PCR reaction (stage 2): 5 seconds at 95°C, 20 seconds at 59°C, 10 seconds at 72°C, repeated for 40 cycles. Furthermore, melting curve analysis (stage 3) was performed. The human FVII gene was amplified as the target gene. Human reference genes (PGK1 and SDHA) were also amplified in the same way, and the geometric mean of these genes was used as an internal control of RNA loading. The primer sequences are shown in Table 3. [Table 3]

[0205] (2) RNA extraction and two-step RT-qPCR To quantify intracellular mRNA expression levels, total cellular RNA was extracted from treated cells using the RNeasy Plus Mini kit (Qiagen, Hilden, Germany) according to the instructions. The obtained RNA (approximately 1 μg) was then processed using PrimeScript. TM cDNA was prepared by reverse transcription using an RT reagent kit (with gDNA Eraser, Takara Bio Inc., RR047A, Shiga, Japan). The obtained cDNA was then processed on a Roche LightCycler 480 Multiwell Plate 384 (Roche, 4729749001, USA) using TB Green(R) Premix Ex Taq. TM Amplification was performed using reagent II (Takara Bio Inc., RR820A, Shiga, Japan) and specific primers for target gene amplification.

[0206] The reaction conditions were as follows: Reverse transcription reaction (Stage 1): 5 minutes at 42°C, 10 seconds at 95°C. PCR reaction (Stage 2): 5 seconds at 95°C, 30 seconds at 60°C, 10 seconds at 72°C, repeated for 40 cycles. Furthermore, melting curve analysis (Stage 3) was performed. The PCR reaction conditions are shown in Tables 4 and 5. [Table 4]

[0207] [Table 5]

[0208] To calculate the relative expression level (Erel) of FVII (target gene) mRNA in samples into which saRNA was introduced, compared with the control treatment (Mock), the Ct values ​​of the target gene and two endogenous reference genes were substituted into Equation 1.

number

[0209] Animal experiments Cynomolgus monkeys (male and female) were purchased from Kunming Biomed International (KBI). All animal experimental procedures were carried out by certified laboratory staff following protocols compliant with regional and state regulations and approved by the Institutional Animal Care and Use Committee (IACUC). Formulations for animal administration were freshly prepared immediately before use by dissolving lyophilized oligonucleotides in physiological saline to prepare a stock solution and then diluting to a predetermined administration concentration. Animals were randomly assigned to test groups based on body weight and sex.

[0210] Coagulation function test To evaluate the coagulation function in cynomolgus monkeys, which are non-human primates, CM-saRNA was administered. One group of cynomolgus monkeys was administered CM-saRNA by subcutaneous injection (SC) on day 0 (10 mg / kg) and day 7 (10 mg / kg) of administration. Another group of cynomolgus monkeys was administered siRNA control by subcutaneous injection (SC) on day 0 (5 mg / kg) of administration. The group administered only physiological saline was used as the vehicle control group (vehicle control). Plasma samples were collected from cynomolgus monkeys on day 21 and day 28 after the first administration. Plasma samples were prepared by collecting 1.8 mL of blood from each cynomolgus monkey into a plastic blood collection tube with sodium citrate as an anticoagulant, mixing, and centrifuging at 2500 g for 10 minutes within 30 minutes after collection. The samples were immediately measured using an automatic coagulation analyzer (BCA-700, GeteinBiotech).

[0211] Example 1: Design and synthesis of saRNA targeting the human FVII promoter The coding strand sequence of the human FVII gene promoter was obtained from the UCSC Genome Database (SEQ ID NO: 1437, shown in Table 6). This sequence consists of 600 bases from -1 bp to -600 bp based on the transcription start site (TSS). [Table 6]

[0212] Using the 1-bp walk method of shifting one base at a time in the 600-bp promoter region, 579 candidate target positions with a length of 22 bases (nt) were identified, and 286 of them were selected as saRNA target sites based on the following criteria: (i) GC content of 40 - 70%. (ii) Not containing 5 or more consecutive identical bases. (iii) The number of 2-base or 3-base repeat sequences not exceeding 3 times. For the 286 selected target sequences, a total of 858 types of double-stranded saRNAs were designed (listed in Table 1.1).

[0213] Example 2: High-throughput screening of saRNAs targeting the human FVII promoter To identify saRNAs that can up-regulate FVII mRNA expression, each of the aforementioned saRNAs was transfected into Huh-7 cells at 25 nM for 72 hours, and then gene expression analysis was performed by one-step RT-qPCR. Non-targeting double-stranded (dsCon2) was used as a non-targeting control, while siRNA (RD-13516) targeting human FVII transcripts was used as a transfection control, and knockdown by RNA interference (RNAi) was monitored.

[0214] As a result, among the 858 types of saRNAs, 133 (15.5%) showed high activity (1.5-fold or more), 187 (21.8%) showed medium activity (1.2 - 1.5-fold), and 84 (9.8%) showed low activity (1.1 - 1.2-fold) in activating FVII expression. The results for each group of high, medium, and low activities are summarized in Table 7. [Table 7]

[0215] The relative changes in FVII expression due to saRNA treatment are summarized in Table 1.2 and plotted in Figure 1.

[0216] When all saRNAs were sorted by their target site locations on the human FVII promoter, regions where the target sequences of functional saRNAs were concentrated, i.e., "saRNA hotspot regions," were identified (Figure 2). These regions, relative to the transcription start site (TSS), are -557 to -379 (H1), -346 to -298 (H2), -271 to -91 (H3), and -96 to -1 (H4) (Figure 2). The DNA sequences corresponding to each hotspot region are shown in Table 8. [Table 8]

[0217] Example 3: Dose-dependent induction of FVII mRNA by saRNA in Huh-7 cells and confirmation of screening results. To confirm the screening results, 47 functional FVII saRNAs were transfected into Huh-7 cells for 72 hours at seven different concentrations (0.1, 0.39, 1.56, 6.25, 25, 100, and 400 nM), and dose-response curves were created by RT-qPCR (Figure 3). For each saRNA that showed dose-dependent FVII mRNA induction, the efficacy relative to maximal activity could be defined by extrapolating the EC50 value and Emax level (see Table 9). Table 9. FVII mRNA levels mediated by saRNA in Huh-7 cells [Table 9] JPEG2026509436000015.jpg208170JPEG2026509436000016.jpg158170

[0218] Example 4: In vitro activation of FVII saRNA and chemically modified saRNA (CM-saRNA) in HepG2 cells and Huh-7 cells To evaluate the activating effect of FVII saRNAs on FVII protein expression, 19 designated saRNAs (i.e., DS20-027A, DS20-055A, DS20-029S, DS20-124S, DS20-086A, DS20-207B, DS20-151B, DS20-228B, DS20-069A, DS20-177A, DS20-207S, DS20-272A, DS20-252A, DS20-156S, DS20-205S, DS20-241A, DS20-082A, DS20-009A, and DS20-188B) were individually transfected into HepG2 cells for 4 days at a final concentration of 25 nM. FVII protein levels were detected by Western blotting using a human FVII protein-specific antibody. Figure 4 shows the FVII protein levels in HepG2 cells after saRNA treatment. Table 10 summarizes the FVII protein levels in HepG2 cells after saRNA treatment. [Table 10]

[0219] To evaluate the activation of FVII mRNA expression by FVII CM-saRNA, ten types of FVII CM-saRNA (RD-16036, RD-16037, RD-16038, RD-16027, RD-16028, RD-16041, RD-16052, RD-16055, RD-16134, and RD-16035) shown in Table 11 were transfected into HepG2 cells at 10 nM for 3 days. Eleven types of FVII CM-saRNA (RD-16036, RD-16037, RD-16038, RD-16027, RD-16028, RD-16041, RD-16052, RD-16055, RD-16134, RD-16044, and RD-16035) were transfected into Huh-7 cells at 25 nM for 3 days. RD-15120 is a chemically modified siRNA targeting the FVII gene and was transfected as a gene silencing siRNA control. The FVII mRNA level was quantified by a two-step RT-qPCR method. Figures 5A and 5B show the FVII mRNA levels after CM-saRNA treatment in HepG2 cells and Huh-7 cells. Table 12 summarizes the FVII mRNA levels after CM-saRNA treatment in HepG2 cells and Huh-7 cells. Table 11. Oligonucleotide Sequences and Compositions

Table 11

[0220]

Table 12

[0221] To further evaluate the effect of FVII CM-saRNAs on activating FVII protein expression, seven specified FVII CM-saRNAs (i.e., RD-16012, RD-16036, RD-16013, RD-16017, RD-16029, RD-16041, RD-16120) were transfected into HepG2 cells at a concentration of 25 nM for 4 days. The same seven FVII CM-saRNAs (i.e., RD-16012, RD-16024, RD-16036, RD-16017, RD-16029, RD-16041, RD-16120) were transfected into Huh-7 cells at a concentration of 25 nM for 4 days. RD-15120, a chemically modified siRNA targeting the FVII gene, was transfected as a gene-silencing siRNA control. FVII protein levels were detected by Western blotting using human FVII protein-specific antibodies. As shown in Figures 6A-6B, all CM-saRNAs showed expression induction of 1.3 times or more. Table 13 summarizes the FVII protein levels after CM-saRNA treatment in HepG2 and Huh-7 cells. [Table 13]

[0222] Example 5: Improvement of coagulation function in non-human primates treated with FVII CM-saRNA. To evaluate coagulation function in cynomolgus monkeys, a non-human primate, CM-saRNA was administered. One group of monkeys (one male: age approximately 4.1–4.9 years, weight 3.33–4.68 kg; one female: age approximately 4.2–4.8 years, weight 2.54–3.09 kg) received a specified CM-saRNA (e.g., RD-17272) subcutaneously at a dose of 10 mg / kg on days 0 and 7. Another group of monkeys (one male: age approximately 4.1–4.9 years, weight 3.33–4.68 kg; one female: age approximately 4.2–4.8 years, weight 2.54–3.09 kg) received RD-16985 subcutaneously at a dose of 5 mg / kg on day 0 as a silencing siRNA control. A group receiving only saline was used as the vehicle control. Plasma samples were collected from cynomolgus monkeys on days 21 and 28 after the initial dose. The prothrombin time (seconds) of the monkey plasma was measured using an automated coagulation analyzer on days 21 and 28 after the initial dose. The thrombin time (seconds) of the monkey plasma was also measured using an automated coagulation analyzer on day 28 after the initial dose. The results for prothrombin time and thrombin time are summarized in Table 14. [Table 14]

[0223] In summary, high-throughput screening data revealed multiple "hotspot regions" of saRNA activity in the human FVII gene promoter. Representative saRNAs increased the expression of both FVII mRNA and FVII protein, exhibiting dose-dependent behavior. These results suggest that targeted activation of FVII expression via saRNAs is a promising strategy for treating FVII-related diseases or conditions, including congenital FVII deficiency (Alexander disease), acquired FVII deficiency (AFVIID), hemophilia (e.g., hemophilia with inhibitors), and Glanzmann thrombasthenia (GT).

[0224] [Table 1-1] JPEG2026509436000024.jpg207170JPEG2026509436000025.jpg209170JPEG2026509436000026.jpg206170JPEG2026509436000027.jpg210170JPEG2026509436000028.jpg211170JPEG2026509436000029.jpg213170JPEG2026509436000030.jpg211170JPEG2026509436000031.jpg213170JPEG2026509436000032.jpg210170JPEG2026509436000033.jpg208170JPEG2026509436000034.jpg213170JPEG2026509436000035.jpg208170JPEG2026509436000036.jpg214170JPEG2026509436000037.jpg212170JPEG2026509436000038.jpg214170JPEG2026509436000039.jpg211170JPEG2026509436000040.jpg212170JPEG2026509436000041.jpg212170JPEG2026509436000042.jpg214170JPEG2026509436000043.jpg214170JPEG2026509436000044.jpg211170JPEG2026509436000045.jpg213170JPEG2026509436000046.jpg212170JPEG2026509436000047.jpg212170JPEG2026509436000048.jpg212170JPEG2026509436000049.jpg214170

[0225]

Table 1-2

[0226] Equal portions Specific embodiments of the composition and method have been described herein, but the above description is illustrative and not limiting. A variety of modifications of the invention will become apparent to those skilled in the art by examining this specification and the following claims. The scope of the invention should be determined by referring to the claims and this specification, including the entire range of its equivalents, and the above modifications.

[0227] Reference All publications, patents, and accession numbers referenced herein are incorporated herein by reference in whole, as if each individual publication or patent were specifically and individually indicated to be incorporated by reference. [Table 15]

Claims

1. Small activated RNA (saRNA) containing sense and antisense strands, characterized by the following: Each sense strand or antisense strand of saRNA contains a sequence of oligonucleotides consisting of 16 to 35 consecutive nucleotides, and the sequence of oligonucleotides has at least 75%, 80%, 85%, 90%, 95%, 98%, or 100% homology or complementarity with respect to a sequence of equal lengths of SEQ ID NO:1437. This saRNA increases the expression of the FVII gene by at least 10% compared to the baseline expression of the FVII gene.

2. The sequence of equal-length fragments with SEQ ID NO:1437 is located in the region upstream of the transcription start site (TSS) of the FVII gene, and further The sequence of equal-length consecutive segments with SEQ ID NO:1437 lies in a region selected from the following groups: Regions -557 to -379, -346 to -298, -271 to -91, or -96 to -1; and / or The saRNA according to claim 1, wherein a contiguous fragment of equal length of SEQ ID NO:1437 is located in the region of SEQ ID NO:1438, SEQ ID NO:1439, SEQ ID NO:1440, or SEQ ID NO:1441.

3. The saRNA according to claim 1 or 2, wherein a sequence of equal-length fragments of SEQ ID NO:1437 is selected from SEQ ID NO:1 to 286.

4. The saRNA according to any one of claims 1 to 3, wherein the continuous fragments of equal length of SEQ ID NO:1437 are selected from SEQ ID NO:37, 55, 77, 86, 116, 117, 120, 124, 145, 146, 151, 177, 188, 205, 207, 218, 228, 241, 242, 10, 11, 29, 88, 121, 156, 259, 261, 274, 9, 27, 69, 81, 82, 107, 240, and 252.

5. The saRNA according to any one of claims 1 to 4, wherein the continuous oligonucleotide sequence satisfies the following conditions: (1) The GC content is 40% to 70%. (2) No more than 5 consecutive identical nucleotides. (3) The sequence of two or three bases does not exceed three repetitions, and / or the sequence of oligonucleotides is complementary to the sense strand or antisense strand of the saRNA.

6. The saRNA according to any one of claims 1 to 5, wherein the sense strand and the antisense strand each independently have a length of about 16–35, about 17–30, about 18–25, or about 19–22 nucleotides.

7. The sense strand and antisense strand have at least 90% complementarity; and / or, The sense strand and antisense strand are located on two different nucleic acid strands or on one contiguous nucleic acid strand; and / or, The saRNA according to any one of claims 1 to 6, wherein the continuous oligonucleotide sequence contains 0, 1, 2, or 3 mismatches with respect to the complementary region of the sense strand or antisense strand.

8. The saRNA according to any one of claims 1 to 7, wherein the sense strand and the antisense strand each include a complementary region, and the complementary regions of the sense strand and the antisense strand form a double-stranded nucleic acid structure.

9. The sense strand or antisense strand independently contains a 3' overhang of 1–6, 1–5, or 2–3 nucleotides in length, or The saRNA according to claim 8, wherein the double-stranded nucleic acid structure has blunt ends.

10. The saRNA according to claim 9, wherein at least one of the nucleotides in the overhang is a thymine deoxyribonucleotide (dT).

11. The sense strand sequence of oligonucleotides has at least 75% sequence homology to a nucleotide sequence selected from SEQ ID NO: 287–858, and / or The saRNA according to any one of claims 1 to 10, wherein the sequence oligonucleotide sequence of the antisense strand has at least 75% sequence homology to a nucleotide sequence selected from SEQ ID NO: 859 to 1430.

12. The saRNA according to any one of claims 1 to 11, wherein the sense strand sequence is selected from SEQ ID NO: 287 to 858, and the antisense strand sequence is selected from SEQ ID NO: 859 to 1430.

13. If the sequence of oligonucleotides on the sense strand is SEQ ID NO: n, and the sequence of oligonucleotides on the antisense strand is SEQ ID NO: n+572 or n+858, where n is an integer selected from SEQ ID NO: 287 to 572; and / or, The saRNA according to any one of claims 1 to 12, wherein the sequence of oligonucleotides of the sense strand is SEQ ID NO: n', and the sequence of oligonucleotides of the antisense strand is SEQ ID NO: n'+286, where n' is an integer selected from SEQ ID NO: 573 to 858.

14. The sense strand contains a sequence of nucleotides selected from SEQ ID NO: 323, 341, 363, 372, 402, 403, 406, 410, 431, 432, 437, 463, 474, 491, 493, 504, 514, 527, 528, 296, 297, 315, 374, 407, 442, 545, 547, 560, 581, 582, 599, 627, 641, 653, 654, 658, 679, 749, 812, 813, and 824, and / or The saRNA according to any one of claims 1 to 13, wherein the antisense strand comprises a sequence of nucleotides selected from SEQ ID NO: 859, 913, 935, 944, 974, 975, 978, 982, 1003, 1004, 1009, 1035, 1046, 1063, 1065, 1076, 1086, 1099, 1100, 1154, 1155, 1173, 1232, 1265, 1268, 1300, 1349, 1351, 1362, 1372, 1385, 1403, 1405, 1418, 867, 868, 885, 927, 939, 940, 965, 1098, and 1110.

15. The sense strand contains the nucleotide sequence of SEQ ID NO: m, and the antisense strand contains the nucleotide sequence of SEQ ID NO: m+572, where m is selected from 323, 341, 363, 372, 402, 403, 406, 410, 431, 432, 437, 463, 474, 491, 493, 504, 514, 527, 528; and / or, The sense strand contains the nucleotide sequence of SEQ ID NO: m', and the antisense strand contains the nucleotide sequence of SEQ ID NO: m+858, where m is selected from 296, 297, 315, 374, 407, 410, 442, 491, 493, 504, 514, 527, 545, 547, 560; and / or, The saRNA according to any one of claims 1 to 14, wherein the sense strand comprises a nucleotide sequence of SEQ ID NO: m" and the antisense strand comprises a nucleotide sequence of SEQ ID NO: m"+286, where m is selected from 581, 582, 599, 627, 641, 653, 654, 658, 679, 749, 812, 813, and 824.

16. The sense strand contains a sequence of nucleotides selected from SEQ ID NO: 1442, 1444, 1446, 1448, 1450, 1452, 1454, 1456, 1458, 1460, 1462, 1464, 1466, 1468, 1470, 1472, 1474, and 1476, and / or The saRNA according to any one of claims 1 to 15, wherein the antisense strand comprises a sequence of nucleotides selected from SEQ ID NO: 1443, 1445, 1447, 1449, 1451, 1453, 1455, 1457, 1459, 1461, 1463, 1465, 1467, 1469, 1471, 1473, and 1475, and / or the saRNA comprises a sense strand and an antisense strand to form a double-stranded (duplex) as described in Table 11.

17. The saRNA according to any one of claims 1 to 16, wherein at least one nucleotide of the saRNA is a chemically modified nucleotide.

18. The saRNA according to claim 17, wherein the chemically modified nucleotide is a nucleotide having at least one of the following modifications: a) Phosphodiester bonds that connect nucleotides within the nucleotide sequence of saRNA. Modification of; b) Modification of the 2'-OH group of ribose within the nucleotide sequence of saRNA; c) Modification of the base portion within the nucleotide sequence of saRNA.

19. The modification of the phosphodiester bond linking the nucleotides is selected from phosphorothioate modification or boranophosphate modification; and / or, The modification of the 2'-OH group is selected from the group consisting of 2'-fluoro modification, 2'-oxymethyl modification, 2'-oxyethylidene methoxy modification, 2,4'-dinitrophenol modification, 2'-amino modification, and 2'-deoxy modification; and / or, The saRNA according to claim 18, wherein the base modification is selected from the group consisting of 5'-bromouracil modification, 5'-iodouracil modification, N-methyluracil modification, and 2,6-diaminopurine modification.

20. At least one nucleotide of the saRNA is a locked nucleic acid (LNA), an abasic nucleotide, a 2'-amino modified nucleotide, a 2'-alkyl modified nucleotide, a morpholino nucleotide, a phosphoramidate, or a nucleotide containing a non-natural base; and / or, The saRNA according to claim 17, wherein the chemical modification of the at least one chemically modified nucleotide is the addition of an (E)-vinylphosphonate group to the 5' end of the sense strand or antisense strand.

21. An oligonucleotide modulator comprising one or more saRNAs according to any one of claims 1 to 20.

22. The oligonucleotide modulator according to claim 21, further comprising one or more portions or components bound, complexed, or mixed with the saRNA.

23. The oligonucleotide modulator according to claim 21, wherein the sense strand and / or antisense strand of saRNA are bound to one or more binding sites (conjugation sites) selected from the group consisting of lipids, fatty acids (e.g., fatty acids having carbon chains with 4 to 30, 12 to 24, or 16 to 22 carbon atoms), fluorescent dyes, ligands, sugars, peptides, and antibodies.

24. The oligonucleotide modulator according to claim 22, wherein the conjugation site is independently selected from lipids, cell-permeable peptides, polyethylene glycol, alkaloids, tryptamine, benzimidazole, quinolone, amino acids, cholesterol, glucose, N-acetylgalactosamine, and any combination thereof.

25. The oligonucleotide modulator according to claim 22, wherein the conjugation site is independently selected from S9, tC2, tC2x6, C5x5, and any combination thereof. 【Chemistry 1】

26. The oligonucleotide modulator according to claim 21, further comprising saRNA conjugated or combined with one or more other active sites for the treatment of FVII-related disease or condition or disorder, wherein the one or more other active sites are independently selected from small molecular weight chemical sites, polypeptides, and antibodies.

27. Isolated oligonucleotides, (a) The isolated oligonucleotide is a sequence of 16 to 35 bases having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% homology to a continuous fragment of equal length of SEQ ID NO: 1437, or (b) The isolated oligonucleotide is a sequence of 16 to 35 bases having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% complementarity to the continuous fragment of equal length of SEQ ID NO: 1437.

28. The contiguous fragments of equal length with SEQ ID NO: 1437 are located in the region upstream of the transcription start site (TSS) of the FVII gene, and furthermore, The sequence of equal-length consecutive segments with SEQ ID NO:1437 lies in a region selected from the following groups: Regions -557 to -379, -346 to -298, -271 to -91, or -96 to -1; and / or The isolated oligonucleotide according to claim 27, wherein a continuous fragment of equal length of SEQ ID NO:1437 is located in the region of SEQ ID NO:1438, SEQ ID NO:1439, SEQ ID NO:1440, or SEQ ID NO:1441.

29. (a) The isolated oligonucleotide is a nucleic acid sequence selected from SEQ ID NO: 1 to 286, for example, selected from SEQ ID NO: 37, 55, 77, 86, 116, 117, 120, 124, 145, 146, 151, 177, 188, 205, 207, 218, 228, 241, 242, 10, 11, 29, 88, 121, 156, 259, 261, 274, 9, 27, 69, 81, 82, 107, 240, and 252, or (b) The isolated oligonucleotide according to claim 27, wherein the isolated oligonucleotide is a nucleic acid sequence complementary to a nucleic acid sequence selected from SEQ ID NO: 1 to 286, for example, complementary to SEQ ID NO: 37, 55, 77, 86, 116, 117, 120, 124, 145, 146, 151, 177, 188, 205, 207, 218, 228, 241, 242, 10, 11, 29, 88, 121, 156, 259, 261, 274, 9, 27, 69, 81, 82, 107, 240, and 252.

30. Oligonucleotide complexes including the following: An antisense strand of saRNA according to any one of claims 1 to 20, and a sense strand of (a) isolated oligonucleotide according to any one of claims 27 to 29, or A sense strand of saRNA according to any one of claims 1 to 20, and an antisense strand of (b) isolated oligonucleotide according to any one of claims 27 to 29.

31. The oligonucleotide complex according to claim 30, wherein the oligonucleotide complex activates the expression of the FVII gene by at least 10% compared to baseline expression.

32. An isolated nucleic acid molecule in which at least 20% (e.g., at least 30%, at least 40%, at least 45%, or at least 50%) of a saRNA designed to target the sequence of the isolated nucleic acid molecule activates the expression of the FVII gene by at least 10%, wherein the designed saRNA satisfies the following conditions: (1) GC content is between 40% and 70%, (2) no more than 5 consecutive identical nucleotides, and (3) no more than 3 repetitions of 2-base or 3-base repeat sequences.

33. An isolated nucleic acid molecule having a sequence corresponding to the region upstream of the transcription start site of the FVII gene, wherein the sequence is located in a region selected from the following regions located upstream of the transcription start site, or in any sub-region within these regions: regions -557 to -379, regions -346 to -298, regions -271 to -91, and regions -96 to -1.

34. An isolated nucleic acid molecule having a sequence selected from SEQ ID NO: 1438–1441.

35. An isolated nucleic acid molecule according to any one of claims 32 to 34, comprising an isolated oligonucleotide according to claim 27(a) or claim 29(a).

36. An isolated nucleic acid molecule according to any one of claims 32 to 34, wherein the saRNA designed to target the nucleic acid molecule is a saRNA as defined in any one of claims 1 to 20; and / or, An isolated nucleic acid molecule according to any one of claims 32 to 34, wherein the desired saRNA is of the brand end type, has an overhang, is without chemical modification, or has chemical modification.

37. An isolated polynucleotide encoding an saRNA according to any one of claims 1 to 20, or an oligonucleotide modulator according to any one of claims 21 to 26.

38. The isolated polynucleotide according to claim 37, wherein the isolated polynucleotide is DNA.

39. A vector comprising an isolated polynucleotide according to any one of claims 37 to 38.

40. A host cell comprising saRNA according to any one of claims 1 to 20, an oligonucleotide modulator according to any one of claims 21 to 26, an isolated polynucleotide according to any one of claims 37 to 38, or a vector according to claim 39.

41. A product comprising saRNA according to any one of claims 1 to 20, an oligonucleotide modulator according to any one of claims 21 to 26, an isolated oligonucleotide according to any one of claims 27 to 29, an oligonucleotide complex according to any one of claims 30 to 31, an isolated nucleic acid molecule according to any one of claims 32 to 36, an isolated polynucleotide according to any one of claims 37 to 38, a vector according to claim 39, or a host cell according to claim 40.

42. The product according to claim 41, the product for the prevention, treatment or diagnosis of FVII-related diseases or disorders or symptoms (e.g., diseases associated with FVII deficiency or hemophilia), and for the design and / or screening of saRNAs.

43. A composition comprising saRNA according to any one of claims 1 to 20, an oligonucleotide modulator according to any one of claims 21 to 26, or an isolated polynucleotide according to any one of claims 37 to 38, and optionally a pharmaceutically acceptable carrier.

44. The composition according to claim 43, wherein the composition comprises 0.001 to 1600 nM, for example, 1 to 150 nM of saRNA.

45. A product for activating / upregulating FVII gene expression in cells, wherein the product activates FVII gene expression by at least 10% compared to a reference expression level of the FVII gene, and the product comprises one or more active ingredients selected from saRNA according to any one of claims 1 to 20, oligonucleotide modulators according to any one of claims 21 to 26, isolated polynucleotides according to any one of claims 37 to 38, vectors according to claim 39, or compositions according to any one of claims 43 to 44.

46. Use of an active ingredient in the manufacture of a product for activating / upregulating FVII gene expression in cells, wherein the product activates FVII gene expression by at least 10% compared to a reference expression level of the FVII gene, and the active ingredient is one or more selected from the saRNA described in any of claims 1 to 20, the oligonucleotide modulator described in any of claims 21 to 26, the isolated polynucleotide described in any of claims 37 to 38, the vector described in claim 39, or the composition described in any of claims 43 to 44.

47. A method for activating / upregulating FVII gene expression in cells, wherein the method activates FVII gene expression by at least 10% compared to a reference expression level of the FVII gene, and the method comprises administering an effective amount of an active ingredient to cells, the active ingredient being one or more selected from saRNA according to any one of claims 1 to 20, oligonucleotide modulators according to any one of claims 21 to 26, isolated polynucleotides according to any one of claims 37 to 38, vectors according to claim 39, or compositions according to any one of claims 41 to 44.

48. A product for activating / upregulating FVII gene expression in cells according to claim 45, a use according to claim 46, or a method according to claim 47, wherein the active ingredient is introduced into the cell; and / or Products, uses, or methods in which the cells are in vitro, ex vivo, or in vivo; and / or Products, uses, or methods in which the cells in question are mammalian cells.

49. A product, use, or method according to claim 48, wherein the active ingredient is introduced into cells by the following method: 1) Composition of the active ingredient with a physiologically or pharmaceutically acceptable carrier, wherein the carrier comprises one or more selected from the group consisting of aqueous carriers, liposomes, polymers, polypeptides, and antibodies; and / or 2) Conjugating the active ingredient to one or more conjugation sites, where the conjugation sites are one or more selected from the group consisting of lipids, cell-permeable peptides, polyethylene glycol, alkaloids, tryptamine, benzimidazole, quinolone, amino acids, cholesterol, glucose, and N-acetylgalactosamine, and further include any combination thereof (for example, two conjugation sites, one of which is a lipid and the other is N-acetylgalactosamine).

50. A product, use, or method according to claim 49, wherein the conjugation site is one or more selected from S9, tC2, tC2x6, and C5x5, or any combination thereof (e.g., C5x5 and tC2x6). 【Chemistry 2】

51. A product, use, or method according to claim 49, wherein the conjugation site is a lipid selected from fatty acids having carbon chains with 4 to -30, 12 to 24, and 16 to 22 carbon atoms. and / or A product, use, or method wherein the conjugation site is a fatty acid having a carbon chain with 4 to 30, 12 to 24, 16 to 22, or 16 carbon atoms. and / or a product, use, or method in which the conjugation site is independently selected from fluorescent dyes, ligands, sugars, peptides, and antibodies.

52. A product for activating / upregulating FVII gene expression in cells according to claim 45, a use according to claim 46, or a method according to claim 47, wherein the cells are derived from a patient who has or is at risk of having a disease, condition, or disorder that is induced by FVII protein deficiency, FVII gene mutation, decreased blood functional FVII protein levels, and / or activation / upregulation of FVII gene expression (e.g., hemophilia (e.g., inhibitor-carrying hemophilia) or Glanzmann thrombasthenia (GT)), and the active ingredient is administered in an amount sufficient to prevent or treat the disease, condition, or disorder.

53. Products for the prevention or treatment of FVII-related diseases, conditions, or disorders, wherein the product contains an active ingredient, and the active ingredient is one or more selected from the following: A saRNA according to any one of claims 1 to 20, an oligonucleotide modulator according to any one of claims 21 to 26, an isolated polynucleotide according to any one of claims 37 to 38, a vector according to claim 39, or a composition according to any one of claims 43 to 44.

54. Use of an active ingredient in the manufacture of a product for the prevention or treatment of FVII-related disease, condition, or disorder, wherein the active ingredient is one or more selected from the following: A saRNA according to any one of claims 1 to 20, an oligonucleotide modulator according to any one of claims 21 to 26, an isolated polynucleotide according to any one of claims 37 to 38, a vector according to claim 39, or a composition according to any one of claims 43 to 44.

55. A method for preventing or treating an FVII-related disease, condition, or disorder, the method comprising the step of administering an effective amount of an active ingredient to a subject, wherein the active ingredient is one or more selected from the following: A saRNA according to any one of claims 1 to 20, an oligonucleotide modulator according to any one of claims 21 to 26, an isolated polynucleotide according to any one of claims 37 to 38, a vector according to claim 39, or a composition according to any one of claims 43 to 44.

56. The product according to claim 53, the use according to claim 54, or the method according to claim 55, wherein the subject is a mammal (e.g., human) and preferably a mammal suffering from or at risk of having another disease, condition, or disorder (e.g., hemophilia with inhibitors) that can be prevented or treated by deficiency of factor VII (FVII) protein expression, FVII gene mutation, decreased functional FVII levels in the blood, and / or activation / upregulation of FVII levels.

57. The active ingredient is administered to an individual by one or more routes of administration selected from the following: the product according to claim 53, the use according to claim 54, or the method according to claim 55: Parenteral administration (intravenous infusion), oral administration, nasal administration, inhalation administration, vaginal administration, and rectal administration.

58. The active ingredient is administered to an individual by one or more routes of administration selected from the following: the product according to claim 53, the use according to claim 54, or the method according to claim 55: Intrathecal administration, intramuscular administration, intravenous administration, intra-arterial administration, intraperitoneal administration, intravesical administration, intraventricular administration, intravitreous administration, and subcutaneous administration.

59. A product according to claim 53, a use according to claim 54, or a method according to claim 55, wherein the expression of FVII gene mRNA in an individual is activated / upregulated by at least 10% compared to the baseline expression level of the FVII gene, and / or FVII protein levels in an individual are elevated by at least 10% compared to the reference level of FVII protein, and / or FVII-related diseases, conditions, or disorders are selected from the following: congenital FVII deficiency (Alexander disease), acquired FVII deficiency (AFVIID), hemophilia (e.g., hemophilia with inhibitors), and Glanzmann thrombasthenia (GT). The product according to claim 53, the use according to claim 54, or the method according to claim 55.

60. A method for diagnosing an FVII-related disease or disorder by detecting an FVII protein or a protein controlled by FVII in the cells described in claim 40.

61. A kit for carrying out the method of claim 60, comprising a saRNA according to any one of claims 1 to 20, or an oligonucleotide modulator according to any one of claims 21 to 26.

62. A kit according to claim 61, wherein the instructions for use include means for administering to an individual the saRNA according to any one of claims 1 to 20 or the oligonucleotide modulator according to any one of claims 21 to 26.

63. A kit comprising saRNA according to any one of claims 1 to 20, or oligonucleotide modulator according to any one of claims 21 to 26, isolated polynucleotide according to any one of claims 37 to 38, vector according to claim 39, or composition according to any one of claims 43 to 44, the kit being housed in a labeled package, the label on the package stating that the saRNA, isolated polynucleotide, vector, or composition may be used for the prevention or treatment of diseases, conditions or disorders induced by deficiency of factor VII (FVII), or for use in relation to hemophilia.

64. A kit for detecting FVII protein or FVII-regulated protein in the cells described in claim 40.

65. A method for obtaining saRNA capable of increasing the expression of the FVII gene by at least 10% compared to a reference expression level, the method comprising: (a) A step of synthesizing saRNA comprising a sense strand and an antisense strand, wherein each sense strand or antisense strand comprises a continuous oligonucleotide sequence consisting of 16 to 35 consecutive nucleotides, and each of these continuous oligonucleotide sequences independently has at least 75%, 80%, 85%, 90%, 95%, 98%, or 100% homology or complementarity with the isolength continuous fragment of SEQ ID NO:1437, and furthermore, the continuous oligonucleotide sequence satisfies the following conditions: (1) the GC content is 40% to 70%, (2) the same nucleotide does not exceed 5 consecutive times, and (3) the 2-base or 3-base repeat sequence does not exceed 3 times. (b) A step to determine the ability of the saRNA to increase the expression of the FVII gene.

66. A method for obtaining saRNA capable of increasing the expression of the FVII gene by at least 10% compared to a baseline expression level, the method comprising: (a) A step of synthesizing saRNA targeting a continuous fragment of SEQ ID NO:1437, wherein the saRNA satisfies the following conditions: (1) the GC content is 40% to 70%, (2) the same nucleotide does not exceed 5 consecutive times, and (3) the 2-base or 3-base repeat sequence does not exceed 3 times. (b) A step to determine the ability of the saRNA to increase the expression of the FVII gene.

67. The method according to claim 65 or 66, wherein the sequence fragment of SEQ ID NO:1437 is located upstream of the transcription start site (TSS) of the FVII gene, and further: The sequence of segments with SEQ ID NO:1437 is located in a region selected from the following groups: regions -557 to -379, regions -346 to -298, regions -271 to -91, or regions -96 to -1; and / or The method according to claim 65 or 66, wherein the contiguous segment is located in the region of SEQ ID NO:1438, SEQ ID NO:1439, SEQ ID NO:1440, or SEQ ID NO:1441.

68. The method according to claim 65 or 66, wherein the consecutive segment of SEQ ID NO:1437 is selected from SEQ ID NO:1 to 286.

69. A double-stranded functional saRNA molecule, wherein both ends of the functional RNA molecule are blunt-ended; and / or, A double-stranded functional saRNA molecule in which the length of each strand is approximately 16–35 nucleotides, approximately 17–30 nucleotides, approximately 18–25 nucleotides, or approximately 19–22 nucleotides.

70. A double-stranded functional RNA molecule according to claim 69, wherein the functional RNA molecule targets and regulates the expression of the FVII gene.

71. An oligonucleotide agent comprising one or more saRNAs according to any one of claims 69 to 70.

72. An oligonucleotide agent according to claim 71, wherein one or more sites or components are conjugated (bound) to the agent.

73. The oligonucleotide agent according to claim 72, wherein the sense strand and / or antisense strand of the functional saRNA are bound to one or more conjugation sites selected from the following: Lipids, fatty acids with carbon chain lengths of 4–30, 12–24, or 16–22 carbon atoms, fluorescent dyes, ligands, polysaccharides, peptides, and antibodies.

74. The conjugation site is independently selected from the following, according to claim 73: Lipids, cell-permeable peptides, polyethylene glycol (PEG), alkaloids, tryptamine, benzimidazole, quinolones, amino acids, cholesterol, glucose, N-acetylgalactosamine, and any combination thereof.

75. The oligonucleotide agent according to claim 74, wherein the conjugation site is independently selected from S9, tC2, tC2x6, and C5x5, or any combination thereof ((S9), (tC2), (tC2x6), and (C5x5)). 【Transformation 3】

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