Oligonucleotide modulators activating expression of coagulation factor VII and their use in treatment of hemophilia

Abstract

The present application relates to saRNA and oligonucleotide modulators for the prevention or treatment of FVII related diseases, conditions or disorders and uses thereof, such as diseases caused by or related to insufficient expression of the FVII gene or bleeding complications caused by hemophilia with inhibitors. The present application also relates to a pharmaceutical composition comprising an oligonucleotide modulator and a method of preventing or treating FVII-related diseases, conditions or disorders induced by insufficient FVII levels with the oligonucleotide modulator disclosed herein.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of nucleic acid technology, in particular to oligonucleotide modulators associated with gene expression activation and their pharmaceutical applications. Cross Reference to Related Applications

[0002] This application claims priority to the filing date of provisional patent application serial number PCT / CN2023 / 079893 filed March 06, 2023, the disclosure of which is hereby incorporated by reference into this application. SEQUENCE LISTING

[0003] The present application contains a Sequence Listing which has been submitted in computer readable form and is hereby incorporated by reference in its entirety. BACKGROUND

[0004] Coagulation factor VII (FVII, also known as proconvertin) is a coagulation factor in the coagulation system; it is a serine protease synthesized by the liver, with a concentration of 10 nM (0.5 pg / ml) in plasma and a very short half-life (4-6 hours). [Heinz S et al., 2015; Yang L et al., 2016]. Coagulation factor VII is the only coagulation factor with a small amount (1%-3%) of circulating free activated form (FVIIa) in the absence of coagulation activation. When injury occurs, the integral membrane protein tissue factor (TF) is exposed to the vascular lumen and can bind to circulating free FVIIa, triggering the coagulation reaction. Under the action of the FVIIa-TF complex, a large amount of activated factor IX (FIXa) and X (FXa) is produced, thereby forming a stable fibrin clot.

[0005] There are more than 200 mutations known for the FVII gene, including missense mutations, nonsense mutations, small insertion / deletion mutations, and splice site mutations, which can involve various sites of the gene. Mutations related to the FVII gene can affect all protein domains. Point mutations are the main cause of FVII genetic defects, among which missense mutations are the most common. Exon 8 is the largest exon of the gene and can have a large number of mutations.

[0006] Diseases, disorders, or conditions associated with FVII can include, but are not limited to, congenital coagulation factor VII deficiency (Alexander disease), acquired coagulation factor VII deficiency (AFVIID), hemophilia (such as hemophilia with inhibitors), and Glanzmann thrombasthenia (GT).

[0007] Congenital coagulation factor VII deficiency is a rare autosomal recessive hereditary bleeding disease caused by mutations in the gene encoding FVII, and severe cases can have symptoms such as joint bleeding, muscle bleeding, easy bruising, and postoperative bleeding.

[0008] Acquired Factor VII Deficiency (AFVIID) can be caused by synthesis abnormalities or reduction, accelerated catabolism, antibody neutralization or abnormal tumor mass uptake. AFVIID can involve only this factor or can be associated with other coagulation factor levels. AFVIID is more common than congenital Factor VII Deficiency.

[0009] Hemophilia is a genetic disease that results in blood failing to clot due to insufficient clotting factors, which in turn causes unexplained bleeding, joint pain, swelling or tightening, hematuria, hematochezia and epistaxis. The appearance of inhibitors (also known as antibodies) is one of the most serious complications of hemophilia treatment, and occurs when the body perceives the infused clotting factor as a threat. Recombinant human Factor VIIa (rhFVIIa) is commonly used to treat bleeding episodes on demand, but can also be used for prophylactic treatment to maintain hemostatic function in hemophilia patients.

[0010] Glanzmann Thrombasthenia (GT) is a genetic defect in the qualitative or quantitative presence of the platelet surface receptor GPIIb / IIIa (ITG αIIbβ3) that can result in impaired platelet aggregation and impaired clot retraction. There is an urgent need to develop clinical protocols that can be used to control and prevent bleeding in GT patients, but the process remains challenging. In the past, local treatment has been mainly used, including anti-fibrinolytic therapy, with or without platelet transfusion. However, in recent years, with the significant increase in the clinical use of rhFVIIa, excellent remission rates have been achieved in the treatment and prevention of bleeding in GT patients.

[0011] Fresh plasma and recombinant Factor VII (rFVII, such as rhFVIIa) are common methods for treating coagulation factor-related diseases, disorders or disturbances, but require frequent intravenous infusion, have a very short half-life (4-6 hours) and are associated with a risk of thrombosis. In addition, in hemophilia patients, the infusion of clotting factors can induce the production of inhibitors, thus causing hemophilia with inhibitors or exacerbating the condition. Therefore, there is an urgent need to develop new therapeutic drugs for the treatment and prevention of hemophilia with inhibitors and related diseases, disorders or disturbances caused by FVII gene mutations or FVII protein deficiency. SUMMARY

[0012] To address the above problems, the present application provides small activating RNA (saRNA) molecules or oligonucleotide modulators containing these molecules to activate / upregulate FVII gene transcription and increase FVII protein expression by exploiting the RNA activation (RNAa) mechanism to prevent and / or treat FVII-associated diseases, disorders or conditions (e.g., blood coagulation diseases or disorders), including diseases, disorders or conditions caused by insufficient FVII mRNA or FVII protein levels (e.g., haploinsufficiency) and diseases, disorders or conditions where FVII levels are normal but treatment or prevention can be achieved by increasing FVII levels (e.g., hemophilia, such as hemophilia with inhibitors).

[0013] In particular, the inventors found that functional saRNAs capable of activating / upregulating FVII mRNA expression are not randomly distributed across the promoter, but rather are clustered in certain specific hot spot regions. Only some regions of the FVII gene promoter are conducive to saRNA activation of the gene, such as regions -557 to -379, -346 to -298, -271 to -91, and -96 to -1 upstream of the transcription start site (TSS) of the FVII gene. A hot spot region in the present application refers to a nucleic acid region on the target gene of the saRNA (e.g., a nucleic acid region in a promoter element upstream of the TSS) that is enriched with full-length target sequences of functional saRNAs.

[0014] The inventors also found that optimal target sequences / positive strands of saRNAs within the FVII promoter region include sequences that meet the following criteria: (1) GC content between 40% and 70%; (2) no more than 5 consecutive identical nucleotides; (3) no more than 3 di- or tri-nucleotide repeats. As a beneficial result of this criteria, the target sequence (e.g., an isolated nucleic acid sequence comprising the target sequence) when interacting with the saRNA, is capable of activating / upregulating the expression of FVII mRNA at least 10% compared to the baseline level of FVII mRNA.

[0015] Based at least in part on the surprising findings, the present disclosure features saRNAs, compositions, and pharmaceutical compositions for activating / upregulating the expression of FVII mRNA at least 10% (compared to the baseline level of the FVII gene). The present application also provides methods for preventing or treating FVII-associated diseases, disorders or conditions (e.g., hemophilia with inhibitors) in a subject caused by insufficient expression of coagulation factor VII (FVII), mutations in the FVII gene, low levels of functional FVII in the blood, and / or normal FVII levels or function but where treatment or prevention can be achieved by increasing endogenous FVII levels, comprising administering one or more saRNAs, compositions, and / or pharmaceutical compositions described herein or any combination thereof.

[0016] In one aspect of the application, there is provided a saRNA molecule capable of activating / upregulating FVII gene expression in a cell or an oligonucleotide modulator comprising the same, the saRNA comprising an oligonucleotide sequence of 16 to 35 contiguous nucleotides in length, wherein the oligonucleotide sequence has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, or 100% sequence homology or complementarity to an equal length region of SEQ ID NO: 1437, whereby the expression of the gene is activated or upregulated by at least 10% compared to the baseline expression of the FVII gene. In some embodiments, the equal length region of SEQ ID NO: 1437 is located in a region selected from the group consisting of region -557 to -379, region -346 to -298, region -271 to -91, or region -96 to -1. In some embodiments, the equal length region of SEQ ID NO: 1437 is located in a region 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 the sense strand and the antisense strand, wherein each of the sense strand and the antisense strand comprises a complementary region, wherein the complementary regions of the sense strand and the antisense strand form a double-stranded nucleic acid structure. In certain embodiments, the sense strand and the antisense strand disclosed herein have at least 75% complementarity. In certain embodiments, the sense strand and the antisense strand disclosed herein are located on two different nucleic acid strands. While in certain embodiments, the sense strand and the antisense strand disclosed herein are located on a contiguous nucleic acid strand (optionally a hairpin structure single-stranded nucleic acid molecule), wherein the complementary regions of the sense strand and the antisense strand form a double-stranded nucleic acid structure. In certain embodiments, one or both ends of the double-stranded nucleic acid structure can be blunt. In certain embodiments, the sense strand and / or the antisense strand disclosed herein comprises a 3' overhang of 1 to 6 nucleotides in length (or 2 to 3 nucleotides in length). In certain embodiments, at least one of the nucleotides of the overhang is a thymine deoxyribonucleotide. In certain embodiments, the overhang is a natural overhang. In certain embodiments, the sense strand and the antisense strand disclosed herein comprise about 16 to about 35, about 17 to about 30, about 18 to about 25, or about 19 to about 22 contiguous nucleotides, respectively.

[0018] In certain embodiments, the sense strand of a saRNA disclosed herein has at least 75% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 287-858, and the antisense strand of a saRNA disclosed herein has at least 75% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 859-1430. In certain embodiments, the sense strand disclosed herein comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 287-858, and the antisense strand disclosed herein comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 859-1430.

[0019] In certain embodiments, the sense strand of a saRNA disclosed herein has at least 75% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 287-572, and the antisense strand of a saRNA disclosed herein has at least 75% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 859-1430, wherein when the SEQ ID NO of the sense strand reference nucleotide sequence is SEQ ID NO: n (where n is an integer selected from 287-572), the SEQ ID NO of the antisense strand reference nucleotide sequence is SEQ ID NO: n+572 or SEQ ID NO: n+858. For example, when the SEQ ID NO of the sense strand reference nucleotide sequence is SEQ ID NO: 287, the SEQ ID NO of the antisense strand reference nucleotide sequence is SEQ ID NO: 859 or SEQ ID NO: 1145.

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

[0021] In certain embodiments, the sense strand of the presently disclosed saRNA comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 287 to 572, and the antisense strand of the presently disclosed saRNA comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 859 to 1430, wherein when the selected nucleotide sequence comprised by the sense strand has a SEQ ID NO of SEQ ID NO: n (where n is an integer selected from 287 to 572), the selected nucleotide sequence comprised by the antisense strand has a SEQ ID NO of SEQ ID NO: n+572 or SEQ ID NO: n+858. In certain embodiments, the sense strand of the presently disclosed saRNA comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 573 to 858, and the antisense strand of the presently disclosed saRNA comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 859 to 1144, wherein when the selected nucleotide sequence comprised by the sense strand has a SEQ ID NO of SEQ ID NO: n' (where n' is an integer selected from 573 to 858), the selected nucleotide sequence comprised by the antisense strand has a SEQ ID NO of SEQ ID NO: n'+286.

[0022] In certain embodiments, the sense strand of the presently disclosed saRNA has at least 75% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1442, 1444, 1446, 1448, 1450, 1452, 1454, 1456, 1458, 1460, 1462, 1464, 1466, 1468, 1470, 1472, 1474, and 1476, and the antisense strand of the presently disclosed saRNA has at least 75% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1443, 1445, 1447, 1449, 1451, 1453, 1455, 1457, 1459, 1461, 1463, 1465, 1467, 1469, 1471, 1473, and 1475. In certain embodiments, the saRNA comprises a sense strand and an antisense strand, and the sense strand and the antisense strand form a duplex as shown in Table 11.

[0023] In certain embodiments, the oligonucleotide sequence of the presently disclosed comprises at least 75% sequence identity or complementarity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1 to 286. In certain embodiments, the sense strand of the oligonucleotide sequence of the presently disclosed comprises at least 75% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1 to 286. In certain embodiments, the antisense strand of the oligonucleotide sequence of the presently disclosed comprises at least 75% sequence complementarity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1 to 286.

[0024] In certain embodiments, the saRNA disclosed herein has at least 75% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-286, and the antisense strand has at least 75% sequence complementarity to the same reference nucleotide sequence of the sense strand selected from the group consisting of SEQ ID NOs: 1-286.

[0025] In certain embodiments, at least one nucleotide of the saRNA disclosed herein is a chemically modified nucleotide. In certain embodiments, at least one nucleotide of the antisense strand and / or the sense strand of the saRNA disclosed herein is chemically modified. In certain embodiments, the chemically modified nucleotide disclosed herein is a nucleotide having at least one of the following modifications: a) a modification to the phosphodiester linkage connecting nucleotides in the saRNA nucleotide sequence; b) a modification to the 2’-OH of ribose in the saRNA nucleotide sequence; and c) a modification to the base in the saRNA nucleotide sequence.

[0026] In certain embodiments, at least one nucleotide of the saRNA disclosed herein is a locked nucleic acid, an abasic nucleotide, a 2’-amino modified nucleotide, a 2’-alkyl modified nucleotide, a morpholino nucleotide, a phosphoramide, or a nucleotide comprising a non-natural base.

[0027] In certain embodiments, the chemical modification of the at least one chemically modified nucleotide disclosed herein is a phosphorothioate (PS) backbone modification.

[0028] In certain embodiments, the chemical modification of the at least one chemically modified nucleotide disclosed herein is a 2’-sugar modification selected from one or more of a 2’-fluoro-2’-deoxy nucleoside (2’-F) modification, a 2’-O-methyl (2’-O-Me) modification, and a 2’-O-(2-methoxyethyl) (2’-O-MOE) modification.

[0029] In certain embodiments, the chemical modification of the at least one chemically modified nucleotide disclosed herein is the addition of a 5’-phosphate moiety at the 5’ end of the nucleotide sequence. In certain embodiments, the chemical modification of the at least one chemically modified nucleotide disclosed herein is the addition of an (E)-vinylphosphonate moiety or a 5’-methylcytosine moiety at the 5’ end of the sense strand and / or the antisense strand.

[0030] In certain embodiments, the disclosure provides an oligonucleotide modulator, wherein the sense strand and / or the antisense strand of the saRNA disclosed herein is conjugated to one or more conjugating moieties selected from the group consisting of a lipid, a fatty acid, a fluorophore, a ligand, a sugar, a peptide, and an antibody.

[0031] In certain embodiments of the oligonucleotide modulator, the sense strand or the antisense strand of the saRNA disclosed herein is conjugated to one or more conjugating moieties selected from the group consisting of a cell-penetrating peptide, a polyethylene glycol, an alkaloid, a tryptamine, a benzimidazole, a quinolone, an amino acid, a cholesterol, a glucose, and an N-acetylgalactosamine. In certain embodiments, the saRNA is conjugated to a conjugating moiety selected from the group consisting of S9, tC2, tC2x6, and C5x5. 4-30 Lipid conjugation of fatty acids. In certain embodiments, the conjugating moiety is a lipid / fatty acid having a saturated or unsaturated, straight-chain or branched C 16 carbon chain.

[0032] In certain embodiments of the oligonucleotide modulator, the saRNA is conjugated to two conjugating moieties, and the two conjugating moieties are a lipid and an N-acetylgalactosamine. In certain embodiments, the two conjugating moieties are selected from the group consisting of S9, tC2, tC2x6, and C5x5, respectively. In certain embodiments, the two conjugating moieties are: 1) one derived from S9, tC2, or tC2x6; 2) one derived from C5x5. wherein, represents a carrier.

[0033] In certain embodiments, the conjugating moiety conjugated to the saRNA is S9, tC2x6, and C5x5, as shown in the present application. In certain embodiments, tC2 or tC2x6 is conjugated to the 3' end of the sense strand; C5x5 is conjugated to the 5' end of the sense strand. The conjugating moieties can be synthesized by methods known in the art, for example, the synthesis process of tC2, tC2x6, C5x5, the entire contents of WO2024002046A1 are incorporated herein by reference; S9 (HR-00214009, Anhui, China) is purchased from Wuhu Huaren Technology Co., Ltd.

[0034] In another aspect of the present application, there is provided an isolated polynucleotide of a saRNA, wherein the isolated polynucleotide is a nucleotide sequence having 16 to 35 consecutive nucleotides in SEQ ID NO: 1437. In particular, the isolated polynucleotide is a nucleic acid sequence selected from the group consisting of SEQ ID NO: 1 to 286. In another aspect of the present application, there is provided a method of using an isolated polynucleotide of a saRNA.

[0035] In another aspect of the application, there is provided an oligonucleotide complex, wherein the oligonucleotide complex comprises the antisense strand of a saRNA disclosed herein and the sense strand of a polynucleotide disclosed herein. In some embodiments, the oligonucleotide complex activates expression of the FVII gene (e.g., at least 10%) compared to baseline expression of the FVII gene.

[0036] Another aspect of the application provides a polynucleotide encoding a saRNA disclosed herein. In one embodiment, the saRNA disclosed herein is a small activating RNA (saRNA) molecule. In one embodiment, the polynucleotide is a DNA molecule. Another aspect of the application provides a vector comprising a polynucleotide disclosed herein.

[0037] In another aspect of the application, there is provided a nucleic acid complex, wherein the nucleic acid complex comprises the antisense strand of a saRNA disclosed herein and the sense strand of a polynucleotide disclosed herein. In some embodiments, the nucleic acid complex activates expression of the FVII gene (e.g., at least 10%) compared to baseline expression of the FVII gene.

[0038] Another aspect of the application provides a cell comprising a saRNA disclosed herein, a polynucleotide encoding a saRNA disclosed herein, or a vector disclosed herein. In one embodiment, the cell is a mammalian cell, optionally a human cell. In some embodiments, the cell is a host cell. The aforementioned cell can be in vitro, e.g., a cell line or cell strain, or can be present in vivo, e.g., in a human body.

[0039] Another aspect of the present application provides a composition, e.g., a pharmaceutical composition, comprising the aforementioned saRNA, a polynucleotide encoding the saRNA disclosed herein, a vector disclosed herein, or a cell disclosed herein, and optionally a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutically acceptable carrier comprises an aqueous carrier, a liposome, a polymeric molecule, or a polypeptide. In some embodiments, the pharmaceutically acceptable carrier is selected from the group consisting of an aqueous carrier, a liposome, a polymeric molecule, and a polypeptide. In some embodiments, the aqueous carrier can be, e.g., RNase-free water or RNase-free buffer. In some embodiments, the composition can comprise 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 of the aforementioned saRNA or a polynucleotide encoding the saRNA disclosed herein. In some embodiments, the composition can comprise 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 of the aforementioned saRNA or a polynucleotide encoding the saRNA disclosed herein.

[0040] Another aspect of the present application relates to the use of the aforementioned saRNA, a polynucleotide encoding the saRNA disclosed herein, or a vector or composition comprising the aforementioned saRNA or the polynucleotide disclosed herein, in the manufacture of a product for activating / upregulating FVII gene expression in a cell.

[0041] The present application also relates to a method of activating / upregulating FVII gene expression in a cell, wherein the method comprises administering to the cell the aforementioned saRNA, a polynucleotide disclosed herein, or a vector or composition comprising the aforementioned saRNA or the polynucleotide disclosed herein. Also provided is a method for increasing the level of FVII protein in a cell or the level of functional FVII protein in blood, comprising introducing into the cell a sufficient amount of the saRNA, nucleic acid, or composition disclosed herein.

[0042] The foregoing saRNA, polynucleotide disclosed herein, or composition comprising the foregoing saRNA or polynucleotide disclosed herein can be introduced directly into a cell or can be produced in a cell after introducing a nucleotide sequence encoding the saRNA into the cell. The cell is preferably a mammalian cell, more preferably a human cell. The foregoing cell can be in vitro, such as a cell line or cell strain, or can be present in a mammal, such as a human. The human is a patient having a FVII-related disease or condition, such as a disease or condition associated with a mutation in the FVII gene, a low level of FVII, a deficiency in the level of functional FVII protein in the blood of the individual, and / or no FVII deficiency but can be prevented or treated by increasing the level of FVII (e.g., hemophilia, such as hemophilia with inhibitors), and is administered a sufficient amount of the saRNA, polynucleotide disclosed herein, or composition comprising the foregoing saRNA or polynucleotide disclosed herein to treat the disease or condition. In particular, a deficiency in FVII protein, a deficiency in the expression of functional FVII protein, and / or a condition caused by hemophilia with inhibitors due to a mutation in the FVII gene. In one embodiment, the disease or condition is caused by a deficiency in the expression of FVII protein, a mutation in the FVII gene, or a deficiency in the level of functional FVII protein in the blood. In some embodiments, the disease or condition can be prevented and / or treated by increasing the level or function of FVII, such as hemophilia, e.g., hemophilia with inhibitors.

[0043] Another aspect of the present application relates to a method of preventing or treating a FVII-associated disease, disorder or condition. As used herein, the terms "FVII-associated disease, disorder or condition" and "disease, disorder or condition associated with FVII" are used interchangeably and refer to a disease, disorder or condition that can be prevented or treated by increasing FVII levels, e.g., a disease, disorder or condition caused by insufficient expression of FVII protein, FVII gene mutation, insufficient levels of functional FVII protein in the blood of an individual, and / or a disease, disorder or condition for which FVII levels or function are normal but for which prevention or treatment can be achieved by increasing FVII expression. FVII-associated diseases, conditions or disorders can include, but are not limited to, congenital FVII deficiency (Alexander's disease), acquired FVII deficiency (AFVIID), hemophilia (e.g., hemophilia A or B with or without inhibitors), and Glanzmann Thrombasthenia (GT). The methods of the present application include administering to an individual a therapeutically or prophylactically effective dose of a saRNA disclosed herein, a polynucleotide encoding a saRNA disclosed herein, a vector disclosed herein, or a composition comprising a saRNA disclosed herein. In certain embodiments, the disease, disorder or condition is hemophilia. The individual can be a mammal, e.g., a human. In one embodiment, the individual has symptoms caused by insufficient expression of FVII protein, FVII gene mutation, insufficient levels of functional FVII protein in the blood, and / or hemophilia with inhibitors. In one embodiment, the disease or symptoms are caused by insufficient expression of FVII protein, FVII gene mutation, or insufficient levels of functional FVII protein in the blood, or hemophilia with inhibitors. In certain embodiments, the saRNA disclosed herein, the polynucleotide disclosed herein, the vector disclosed herein, or the composition disclosed herein is administered to the individual by one or more routes of administration selected from the group consisting of parenteral infusion or injection, oral administration, intranasal administration, inhalation administration, vaginal administration, and rectal administration. In certain embodiments, the route of administration is selected from the group consisting of one or more of intrathecal, intramuscular, intravenous, intra-arterial, intraperitoneal, intravesical, intracerebroventricular, intravitreal, and subcutaneous administration. In certain embodiments, the methods disclosed herein activate / upregulate expression of a FVII gene or FVII mRNA in the individual (e.g., at least 10%, e.g., at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100%). In certain embodiments, the methods disclosed herein increase FVII protein levels in the individual (e.g., at least 10%, e.g., at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100%).

[0044] Another aspect of the present application relates to the use of saRNA disclosed herein, polynucleotides disclosed herein, or compositions comprising saRNA disclosed herein or polynucleotides disclosed herein in the preparation of a medicament for preventing or treating a disease, condition, or disorder caused by insufficient levels of functional FVII protein in the blood, FVII gene mutations, low levels of functional FVII in the blood, and / or hemophilia with inhibitors in an individual. The individual can be a mammal, such as a human. In one embodiment, the disease, condition, or disorder can include, for example, hemophilia with inhibitors.

[0045] In addition, the present application also provides a kit for performing the preventive or therapeutic methods disclosed herein, wherein the kit comprises: a) saRNA, b) instructions for use, and c) optionally, a method for administering the saRNA to an individual. Specifically, the kit can be packaged in a labeled package, and the label on the package indicates that the molecule or composition can be used to prevent or treat FVII-related diseases, conditions, or disorders caused by, for example, insufficient FVII expression, or for preventing or treating hemophilia with inhibitors. In some embodiments, the present application provides a kit for performing the methods disclosed herein, wherein the kit comprises: a) saRNA disclosed herein, and b) instructions for use. In certain embodiments, the instructions include an apparatus or method for administering the saRNA disclosed herein to an individual.

[0046] Aspects of the present application include a kit comprising a saRNA disclosed herein, a polynucleotide disclosed herein, a vector disclosed herein, or a composition disclosed herein in a package with a label, wherein the label on the package indicates that the saRNA, polynucleotide, vector, or composition can be used to prevent or treat FVII-related diseases, conditions, or disorders caused by, for example, insufficient FVII expression, or for preventing or treating hemophilia with inhibitors.

[0047] The present application also provides a kit for detecting FVII protein or FVII regulatory protein in blood or in cells transfected with the aforementioned saRNA, the aforementioned nucleic acid, or the aforementioned composition disclosed in the present application.

[0048] The oligonucleotide modulators (e.g., saRNA molecules) for activating / upregulating FVII gene expression provided herein can efficiently and specifically upregulate FVII gene expression, increase FVII mRNA expression levels, have low toxic side effects, and can be used to prepare drugs to prevent or treat FVII-related diseases, conditions, or disorders, such as disorders associated with insufficient FVII protein expression, diseases, conditions, or hemophilia with inhibitors caused by FVII gene mutations. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 Changes in human FVII mRNA expression levels mediated by saRNAs in human hepatocarcinoma cell line (Huh-7) are shown. 858 saRNAs targeting the human FVII promoter were transfected into Huh-7 cells at a concentration of 25 nM, and treated for 72 hours. Table 1.1 lists the strand composition and sequence of each saRNA duplex, including the homologous target site in the human FVII promoter. Cells transfected without oligonucleotides were used as mock treatment (not shown). The dsCon2 duplex was used as a non-targeting control (not shown). RD-13516 is a duplex siRNA targeting the FVII mRNA, transfected as a control for silencing double-stranded RNA (siRNA) (not shown). FVII mRNA levels were quantified by one-step RT-qPCR using a set of gene-specific primers (Table 3) in each PCR reaction. Geometric mean of mRNA levels of PGK1 and SDHA were used as internal controls. This value (y-axis, log2 fold change) shows the relative fold change in FVII mRNA expression levels induced by each saRNA relative to mock treatment (mean of two replicate transfection wells ± SEM) after normalization to PGK1 and SDHA. saRNAs are ranked in descending order according to their activity (log2 fold change) in inducing FVII mRNA expression on the x-axis.

[0050] Figure 2Figure 6 shows the activity of saRNAs in Huh-7 cells ordered by their target position on the human FVII promoter and saRNA hotspot regions. 858 saRNAs targeting the human FVII promoter were transfected into Huh-7 cells at a concentration of 25 nM, and treated for 72 hours. Cells transfected without oligonucleotides were used as mock treated (not shown). The dsCon2 duplex was used as a non-targeting control (not shown). RD-13516 is a duplex siRNA targeting the FVII mRNA and was transfected as a silencing siRNA control (not shown). FVII mRNA levels were quantified by one-step RT-qPCR using gene-specific primer sets in each PCR reaction. Geometric mean of mRNA levels of PGK1 and SDHA were used as internal controls. The values (y-axis, log2 fold change) show the relative fold change in FVII mRNA expression levels caused by each saRNA relative to mock treatment, normalized to PGK1 and SDHA, after treatment (mean of two replicate transfection wells ± SEM). The saRNAs are ordered by their target position on the promoter -558 to -1 upstream of the FVII transcription start site (TSS). Four saRNA hotspot regions with functional saRNAs (activating dsRNAs) enriched targets are marked by rectangular dashed boxes as H1 to H4, respectively. The numbers above the boxes indicate the boundaries of the hotspot regions relative to the FVII TSS (position 0 on the x-axis), which span the 5' most end of the first functional saRNA target and the 3' most end of the last saRNA target within each hotspot region.

[0051] Figure 3FVII mRNA levels in Huh-7 cells. Based on the screening results for FVII mRNA induction, the top 47 functional FVII saRNAs (i.e., saRNAs that caused the largest fold change in FVII mRNA expression levels as determined in Examples 1 and 2) were selected and transfected into Huh-7 cells at 7 indicated concentrations (i.e., 0.1, 0.39, 1.56, 6.25, 25, 100, and 400 nM) for 72 hours of treatment. Cells transfected without oligonucleotides were used as mock-treated (not shown). The dsCon2 duplex was used as a non-targeting control (not shown). RD-13516 is a duplex siRNA targeting FVII mRNA and was transfected as a silencing siRNA control (not shown). FVII mRNA levels were quantified by one-step RT-qPCR using gene-specific primers set up in a single PCR reaction. Geometric mean of mRNA levels of PGK1 and SDHA were used as internal controls. These values (y-axis) represent FVII mRNA expression levels relative to mock-treated (average of two replicate transfection wells ± SEM) after normalization to PGK1 and SDHA for the 7 dose levels of saRNAs.

[0052] Figure 4 FVII protein expression in HepG2 cells. 19 indicated 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 transfected into HepG2 cells at a transfection concentration of 25 nM for 4 days of treatment. Cells transfected without oligonucleotides were used as mock-treated. The dsCon2 duplex was used as a non-targeting control. FVII protein levels were determined by Western blotting using an antibody against human FVII protein. An antibody against β-tubulin was also blotted as a protein loading control. These values (y-axis) represent FVII protein expression levels relative to mock-treated (average of two replicate transfection wells ± SEM) after normalization to β-tubulin.

[0053] Figures 5A-5BActivation of FVII mRNA expression in HepG2 and Huh-7 cells by chemically modified saRNAs (CM-saRNAs) is shown. Ten indicated FVII CM-saRNAs (i.e. RD-16036, RD-16037, RD-16038, RD-16027, RD-16028, RD-16041, RD-16052, RD-16055, RD-16134 and RD-16035) were transfected into HepG2 cells at a transfection concentration of 10 nM for 3 days of treatment. Eleven indicated FVII CM-saRNAs (i.e. 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 a transfection concentration of 10 nM for 3 days of treatment. Cells transfected without oligonucleotide served as mock-treated. The dsCon2M6v duplex served as a non-targeting control. RD-15120, a chemically modified siRNA targeting the FVII gene, was transfected as a silencing siRNA control. FVII mRNA levels were quantified by a two-step RT-qPCR (using gene-specific primers set in individual PCR reactions). Geometric mean of mRNA levels of PGK1 and SDHA were used as internal controls. Figure 5A FVII mRNA levels in HepG2 cells are shown. Figure 5B FVII mRNA levels in Huh-7 cells are shown. These values (y-axis) represent FVII mRNA levels (mean of four replicate transfection wells ± SEM) relative to mock treatment, after normalization to PGK1 and SDHA, for each saRNA.

[0054] Figures 6A-6BActivation of FVII protein expression in HepG2 and Huh-7 cells by CM-saRNAs is shown. Seven indicated FVII CM-saRNAs (i.e. RD-16012, RD-16036, RD-16013, RD-16017, RD-16029, RD-16041 and RD-16120) were transfected into HepG2 cells at a transfection concentration of 25 nM for 4 days. Seven indicated FVII CM-saRNAs (i.e. RD-16012, RD-16024, RD-16036, RD-16017, RD-16029, RD-16041 and RD-16120) were transfected into Huh-7 cells at a transfection concentration of 25 nM for 4 days. Cells transfected without oligonucleotide served as mock treatment. dsCon2M3v duplex served as non-targeting control. RD-15120 is a chemically modified siRNA targeting the FVII gene, transfected as a silencing siRNA control. FVII protein levels were determined by Western blotting using an antibody against human FVII protein. An antibody against beta-tubulin was also blotted as a protein loading control. Figure 6A FVII protein levels in HepG2 cells are shown. Figure 6B FVII protein levels in Huh-7 cells are shown. These values (y-axis) represent FVII protein expression levels relative to mock treatment (mean of two replicate transfection wells ± SEM) after normalization to beta-tubulin. DETAILED DESCRIPTION

[0055] Double-stranded RNA (dsRNA) targeting regulatory sequences of a gene, including the promoter, has been shown to upregulate the target gene in a sequence-targeted manner at the transcriptional level via the RNA activation (RNAa) mechanism (Li, L.C. et al. Small dsRNAs induce transcriptional activation in human cells. PNAS (2006)). Such dsRNA is referred to as small activating RNA (saRNA).

[0056] Embodiments of the present disclosure are based in part on the surprising finding that oligonucleotide modulators, such as saRNAs, including those referred to herein as "FVII gene saRNAs", are capable of activating or upregulating the expression of the FVII gene in cells. Upon administration of the saRNAs described herein, an increase in the production of functional FVII gene mRNA can be achieved, resulting in a significant increase or upregulation of FVII mRNA and FVII protein levels.

[0057] In particular, the inventors of the present application found that functional saRNAs capable of activating / upregulating FVII mRNA expression are not randomly distributed across the promoter, but rather are clustered in certain specific hot spot regions. Only some regions of the FVII gene promoter are conducive to saRNAs activating the gene, for example regions -557 to -379 (H1), -346 to -298 (H2), -271 to -91 (H3), and -96 to -1 (H4) upstream of the FVII gene transcription start site (TSS). The length of these specific promoter regions (referred to herein as "hot spots" or "hot spot regions") identified by the present application is optionally at least 25 nt, at least 27 nt, at least 30 nt, at least 35 nt, at least 40 nt, at least 45 nt, at least 48 nt, or at least 49 nt; ranges from about 25 to 200 nt, about 30 to 190 nt, about 40 to 185 nt, or about 49 to about 181 nt; or is within a narrower numerical range, or at some numerical point falling within the aforementioned broader numerical ranges (e.g., 181 nt, 179 nt, 96 nt, 49 nt, 27 nt, 25 nt).

[0058] The inventors of the present application also found that optimal target sequences / sense strands for saRNAs within the FVII promoter region include sequences that meet the following criteria: (1) GC content between 40% and 70%; (2) no more than 5 consecutive identical nucleotides; (3) no more than 3 di- or tri-nucleotide repeat sequences. As a beneficial result of this criteria, the target sequences (e.g., isolated nucleic acid sequences comprising the target sequences) when interacting with a saRNA, are capable of activating / upregulating FVII mRNA expression by at least 10% or 1.1 fold compared to baseline levels of FVII mRNA.

[0059] In some embodiments, the hot spot region is a nucleic acid region on the target gene for the saRNA, spanning the 5' most end of the first saRNA target and the 3' most end of the last saRNA target in each hot spot, wherein at least 20% (e.g., at least 30%, at least 40%, at least 45%, about 50%) of the saRNAs designed to target the hot spot region meet the criteria (1), (2), (3), and (4) above are proven to be functional, i.e., capable of activating / upregulating mRNA expression of the target gene by 1.1 fold or more compared to baseline levels of mRNA expression. In some embodiments, at least 20%, about 22%, at least 30%, about 35%, about 40%, or more than 50% of the saRNAs designed meet the criteria are functional, i.e., capable of activating / upregulating mRNA levels or protein expression of the target gene by 1.1 fold or more.

[0060] Based at least in part on these findings, features of the present disclosure are saRNAs, compositions, and pharmaceutical compositions for activating / upregulating (e.g., by at least 10%) expression of FVII mRNA (as compared to baseline levels of FVII mRNA). The present application also provides methods for preventing or treating FVII-related diseases, disorders, or conditions caused by insufficient FVII expression, FVII gene mutation, low levels of functional FVII in an individual’s blood, and / or hemophilia with inhibitors, comprising administering the saRNAs, compositions, and / or pharmaceutical compositions described herein.

[0061] Embodiments of the present disclosure are also based in part on the surprising finding that the target sequence clusters of the saRNAs capable of activating or upregulating FVII gene expression in cells are clustered in specific FVII gene promoter regions, as shown in Figure 2 The inventors identified these FVII gene promoter region clusters as “hotspot” promoter regions that are enriched with target sites for the functional saRNAs developed (see, e.g., Table 8). For example, four hotspot regions of the human FVII promoter were detected, which are located at regions -557 to -379 (H1), -346 to -298 (H2), -271 to -91 (H3), and -96 to -1 (H4) of the promoter TSS, respectively, and found that these hotspot regions are the best target sites for the saRNAs to activate FVII gene expression through RNA activation mechanism.

[0062] This saRNA-FVII mRNA-FVII protein pathway can provide an alternative therapy different from the existing treatments for FVII deficiency-related diseases, disorders, or conditions, including hemophilia with inhibitors. Definitions

[0063] In the present application, the relevant terms are defined as follows:

[0064] Each numerical range given in the specification is intended to include all narrower numerical ranges falling within the broader numerical range, as if such narrower numerical ranges were explicitly written herein.

[0065] The transitional terms / phrase “comprise,” “comprises,” and “comprising” (or any grammatical variants thereof), including the transitional phrases “consist essentially of and “consisting essentially of,” are inclusive or open-ended terms (with the term “consisting of” being a closed or restrictive term) that encompassed the phrases “consisting of” and “consist of,” and permit the inclusion of additional elements notwithstanding the use of words such as “only,” “consisting of,” and “consist of’ in certain jurisdictions. The use of the terms “comprise,” “comprises,” and “comprising” also includes more restrictive terms such as “consist of” and “consisting of.” The terms “comprise,” “comprises,” and “comprising” are synonymous with the terms “include,” “includes,” and “including,” and are used in the sense of “including but not limited to.” The terms “comprise,” “comprises,” and “comprising” are synonymous with the terms “have,” “has,” and “having.”

[0066] The term "complementary" as used herein refers to the ability of two oligonucleotide strands to form base pairs between them. Base pairs are typically formed by hydrogen bonds between nucleotides in anti-parallel oligonucleotide strands. The bases of complementary oligonucleotide strands can pair in a Watson-Crick manner (e.g., A with T, A with U, and C with G), or in any other manner that allows a duplex to form (e.g., Hoogsteen or reverse Hoogsteen base pairing).

[0067] Complementarity includes perfect complementarity and imperfect complementarity. "Perfect complementarity" or "100% complementarity" means that each nucleotide from a first oligonucleotide strand is capable of forming a hydrogen bond with a nucleotide at a corresponding position in a second oligonucleotide strand in a double-stranded region of a double-stranded oligonucleotide molecule, with no base pair "mismatches." "Imperfect complementarity" means that not all nucleotide units of the two strands are bound to each other by hydrogen bonds. For example, for two oligonucleotide strands of 20 nucleotides in length in a double-stranded region, if only two base pairs in the double-stranded region are capable of forming hydrogen bonds, the oligonucleotide strands have 10% complementarity. In the same example, if 18 base pairs in the double-stranded region are capable of forming hydrogen bonds, the oligonucleotide strands have 90% complementarity. Substantial complementarity means at least about 75%, about 79%, about 80%, about 85%, about 90%, about 95%, or 99% complementarity.

[0068] The terms "oligonucleotide" or "polynucleotide" are used interchangeably to refer to polymers of nucleotides, including but not limited to single- or double-stranded nucleic acid molecules of DNA, RNA, or DNA / RNA hybrids, oligonucleotide chains containing regularly and irregularly alternating deoxyribosyl moieties and ribosyl moieties, and modifications and naturally or non-naturally occurring backbones of such oligonucleotides. The oligonucleotides described herein for activating transcription of a target gene can be or can include small activating nucleic acid molecules (saRNAs).

[0069] The terms "oligonucleotide strand," "strand," and "oligonucleotide sequence" are used interchangeably herein to refer to a short nucleotide sequence (including nucleotides in deoxyribonucleic acid (DNA) or ribonucleic acid (RNA)) having fewer than 35 bases. In one non-limiting embodiment, the length of the strand can be any length from 16 to 35 nucleotides.

[0070] The term "target gene" as used herein can refer to a nucleic acid sequence, transgene, viral or bacterial sequence, chromosome or extrachromosomal gene that naturally occurs in an organism and / or can be transiently or stably transfected or incorporated into a cell and / or its chromatin. The target gene can be a protein-coding gene or a non-protein-coding gene (such as a microRNA gene and a long non-coding RNA gene). The target gene generally contains a promoter sequence, and upregulation of the target gene can be achieved by designing a saRNA that has sequence identity (also referred to as homology) to the promoter sequence, characterized by upregulation of the target gene expression. The terms "target sequence" or "target site" as used herein can be used interchangeably and refer to a sequence fragment in the target gene sequence, for example, the target gene promoter, which is homologous or complementary to the sense strand or the antisense strand of the saRNA. The target gene can also include one or more regulatory elements, wherein one or more saRNAs are designed to have sequence identity to the regulatory elements. Non-limiting examples of one or more regulatory elements include: a promoter, an enhancer, a silencer, an insulator, a TATA box, a GC box, a CAAT box, a transcription start site, a DNA binding motif of a transcription factor or other regulatory transcriptional protein, and a 5' untranslated region.

[0071] The term "sense strand" of a saRNA in a saRNA duplex as used herein refers to a strand that has sequence homology or sequence identity to a fragment of the coding strand of the target gene sequence.

[0072] The term "antisense strand" of a saRNA in a saRNA duplex as used herein refers to a strand that is complementary to the sense strand. The antisense strand can interact with a target sequence, which can be a fragment of the coding strand of the target gene sequence, to activate or upregulate gene expression.

[0073] The term "coding strand" as used herein refers to a DNA strand in a target gene that cannot be used for transcription, and the nucleotide sequence of this strand is identical to the nucleotide sequence of the RNA produced by transcription (in RNA, T in DNA is replaced by U). The coding strand of a double-stranded DNA sequence of a target gene promoter as described herein refers to the promoter sequence that is located on the same DNA strand as the coding strand of the target gene DNA.

[0074] The term "template strand" as used herein refers to the other strand in the double-stranded DNA of a target gene that is complementary to the coding strand, i.e., the strand that can be transcribed into RNA as a template, and this strand is complementary to the transcribed RNA (A to U, G to C). During transcription, the RNA polymerase binds to the template strand, moves along the 3'→5' direction of the template strand, and catalyzes the synthesis of RNA in the 5'→3' direction. The template strand of a double-stranded DNA sequence of a target gene promoter as described herein refers to the promoter sequence that is located on the same DNA strand as the template strand of the target gene DNA.

[0075] The term "LNA" as used herein refers to a locked nucleic acid in which the 2'-oxygen atom and the 4'-carbon atom are connected by an extra bridge. The term "BNA" as used herein refers to a 2'-O and 4'-aminoethylene bridged nucleic acid which can comprise a five- or six-membered bridged structure with N-O bonds. The term "PNA" as used herein refers to a nucleic acid mimic with a pseudopeptide backbone consisting of N-(2-aminoethyl)glycine units, wherein the nucleobases are connected to the glycine nitrogens via carbonylmethylene linkers.

[0076] The term "promoter" as used herein refers to a sequence spatially associated with a protein-coding or RNA-coding nucleic acid sequence and which exerts regulatory control over the transcription of the protein-coding or RNA-coding nucleic acid sequence. Typically, a eukaryotic gene promoter comprises 100 to 5000 base pairs, although this length range is not intended to limit the term "promoter" as used herein. While a promoter sequence is typically located at the 5' end of a protein-coding sequence or RNA-coding sequence, it can also be present in exon and intron sequences.

[0077] The term "transcription start site (TSS)" as used herein refers to the nucleotide on the template strand of a gene that marks the point of transcription initiation. The transcription start site can occur on the template strand of the promoter region. In particular, the "transcription start site (TSS)" can refer to the position at which transcription of the 5' end of the FVII gene sequence begins, which is the nucleotide that marks the start of transcription on the template strand of the gene and corresponds to the first nucleotide on the RNA molecule transcribed from the gene. Different variants of the same gene can have different TSS, but the same upstream sequence. The TSS in a gene can be found using traditional methods, such as Cap Analysis of Gene Expression (CAGE), oligo-capping, and 5'-Rapid Amplification of cDNA Ends (5'-RACE).

[0078] In some embodiments, the selection of the target site is based at least in part on the sequence of the gene. In some embodiments, the selection of the target site is based at least in part on the sequence proximal to the transcription start site (TSS) of the gene. In some embodiments, the coding strand sequence of the human FVII gene promoter can be retrieved from the UCSC Genome Browser database, e.g., SEQ ID NO: 1437 (as shown in Table 6) consists of 600 nucleotides ranging from -1 bp to -600 bp relative to the transcription start site (TSS).

[0079] The term "identity" or "homology" as used herein refers to sequence similarity of one oligonucleotide strand (sense or antisense strand) of a saRNA to the coding or template strand in the 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 percent identity of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., matrices can be introduced to either or both of the first and second nucleic acid sequences for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). The nucleotides at corresponding positions are then compared. When a position in the first sequence does not have a corresponding position in the second sequence, a gap can be introduced in the second sequence for alignment purposes. One molecule is considered identical to another if the two molecules share identical positions at every position considered. The percent identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be achieved using a mathematical algorithm, for example, the algorithm of Needleman and Wunsch (1970, J. Mol. Biol. 48:444-453), which has been incorporated into the GAP program in the GCG software package (available at www.gcg.com), using a NWSgapdna.CMP matrix and a 40,000,000 real value. www.gcg.com The percent identity between two nucleotide sequences can also be determined using the algorithm of E. Meyers and W. Miller (1989, CABIOS, 4: 11-17) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. It is understood that the molecules described herein can have additional conservative or non-essential nucleic acid substitutions which do not materially affect their function.

[0080] The term "isometric portion" refers to a sequence portion which is compared to a target sequence (e.g., a contiguous oligonucleotide sequence from the saRNA) and which has an equal length (equal number of bases) to the target sequence.

[0081] The term "sequence-specific pattern" as used herein refers to the manner in which two nucleic acid fragments bind or hybridize according to their nucleotide sequence, e.g., Watson-Crick manner (e.g., A with T, A with U, and C with G) or any other manner which allows for duplex formation (e.g., Hoogsteen or anti-Hoogsteen base pairing).

[0082] The term "overhang" as used herein refers to the unbasepaired nucleotides at the end (5' or 3') of an oligonucleotide strand that is formed by one strand of a duplex extending beyond the other. The single-stranded region extending from the 3' end and / or 5' end of a duplex is referred to as an overhang.

[0083] The term "natural overhang" as used herein refers to an overhang consisting of one or more nucleotides that are identical or complementary to the corresponding position on the target sequence. A natural overhang on the sense strand consists of one or more nucleotides that are identical to the corresponding position on the DNA target. A natural overhang on the sense strand consists of one or more nucleotides that are identical to the corresponding position on the DNA target. A natural overhang on the antisense strand consists of one or more nucleotides that are complementary to the corresponding position on the DNA target.

[0084] The term "isolated" as used herein refers to a material that is removed from its original or native environment, e.g., naturally-occurring environment. For example, a polynucleotide or polypeptide in its natural state in a living animal is not isolated, but the same polynucleotide or polypeptide is isolated if it is separated from other materials with which it is normally associated in nature. Such a polynucleotide can be part of a vector, and such a polynucleotide or polypeptide can be part of a composition. Inasmuch as vectors and compositions are not components of their natural environment, they are still isolated. For example, an isolated molecule can be obtained by extraction from a natural source, expression of a recombinant nucleic acid, or chemical synthesis. For example, the term "isolated RNA" refers to an RNA molecule that is produced by recombinant techniques substantially free of other cellular material, or by chemical synthesis substantially free of chemical precursors or other chemicals. In some embodiments, the materials described herein, e.g., the polynucleotides, oligonucleotides, and / or saRNAs of the application, are isolated.

[0085] The terms "gene activation" or "activating gene expression" and "gene upregulation" or "upregulating gene expression" are used interchangeably herein to refer to an increase in the transcription, translation, expression, or activity of a certain nucleic acid as determined by measuring the transcriptional level, mRNA level, protein level, enzymatic activity, methylation state, chromatin state or conformation, translational level, or activity or state of the gene in a cellular or biological system. These activities or states can be determined directly or indirectly. Furthermore, "gene activation," "activating gene expression," "gene upregulation," or "upregulating gene expression" refers to an increase in the activity associated with a nucleic acid sequence, regardless of the mechanism of activation. For example, gene activation occurs at the transcriptional level to increase transcription to RNA, which is translated into protein, thereby increasing protein expression.

[0086] The terms "small activating RNA," "saRNA," and "small activating nucleic acid molecule" are used interchangeably in the present application to refer to a nucleic acid molecule capable of upregulating target gene expression and can comprise a first nucleic acid segment (sense strand) containing a nucleotide sequence having sequence identity to a non-coding nucleic acid sequence (e.g., a promoter or enhancer) of a target gene and a second nucleic acid segment (antisense strand) containing a nucleotide sequence complementary to the first nucleic acid segment, wherein the first and second nucleic acid segments form a duplex. The saRNA can also comprise a single-stranded RNA molecule expressed synthetically or from a vector, which is capable of forming a hairpin structure through two complementary regions (first and second regions) within the molecule, wherein the first region comprises a nucleotide sequence having sequence identity to a target sequence of a gene promoter and the second region comprises a nucleotide sequence complementary to the first region. The saRNA duplex region is typically about 15 to about 35, about 16 to about 32, about 17 to about 30, about 18 to about 28, about 19 to about 26, about 20 to about 24, and about 21 to about 22 base pairs in length, and is typically about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, or about 23 base pairs in length. In addition, the terms "saRNA," "small activating RNA," and "small activating nucleic acid molecule" also encompass nucleic acids other than ribonucleotides, including but not limited to modified nucleotides or analogs.

[0087] The term "oligonucleotide modulator" refers to an oligonucleotide-containing substance comprising or consisting of one or more saRNAs of the present application, having the activity of modulating target gene expression or enhancing the action of the saRNA, and can further comprise other oligonucleotide moieties / components (such as ASOs) or non-oligonucleotide moieties / components in conjugation, combination or admixture with the saRNA. In certain embodiments, the oligonucleotide modulator comprises RNA (e.g., saRNAs described herein), DNA, BNA, LNA, or peptide nucleic acid (PNA).

[0088] The terms "hotspot region" and "hotspot" used in the present application are used interchangeably and refer to a region of nucleic acid on the saRNA target gene (e.g., a region of nucleic acid in the promoter upstream of the target gene TSS) that is enriched for full-length targets of functional saRNAs and spans the most 5' end of the first saRNA target and the most 3' end of the last saRNA target within each hotspot. A "hotspot region" refers to a region of a gene promoter that is at least 25 bp (e.g., at least 49 bp) in length that is enriched for target sequences of functional saRNAs, e.g., at least 20%, e.g., about 22%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or more than 90% of saRNAs targeting the region are "functional," i.e., can induce a 1.1-fold or greater change in mRNA or protein expression of the target gene, provided that the saRNA is designed to meet the following criteria: (1) GC content is between 40% and 70%; (2) no more than 5 consecutive identical nucleotides; (3) no more than 3 di- or tri-nucleotide repeat sequences.

[0089] The term "functional saRNA" used in the present application refers to a saRNA that causes activation (e.g., at least 10% or at least 1.1-fold) of its intended target gene expression. The term "non-functional saRNA" refers to a saRNA that modulates mRNA levels and protein expression (e.g., less than 10% or less than 1.1-fold) of the FVII gene.

[0090] The terms "target site" and "oligonucleotide" used in the present application are used interchangeably and refer to a target site that has complementarity or hybridizes to a saRNA. For example, an oligonucleotide of a target site can include a nucleic acid sequence that has complementarity or hybridizes to a region of a saRNA. The term "polynucleotide" used in the present application refers to a polynucleotide that encodes a saRNA, e.g., DNA.

[0091] The term "synthesis" used in the present application refers to a method of synthesizing an oligonucleotide, including any method that allows for the synthesis of RNA, e.g., chemical synthesis, in vitro transcription, and / or vector-based expression.

[0092] The term "support" used in the present application refers to a solid phase starting material that is immobilized between the filters of a chromatographic column, allows free passage of all reagents and solvents by use of an automated oligonucleotide synthesizer, and optionally generates 3' or 5' end conjugated oligonucleotides. The support can be selected from the group consisting of controlled pore glass beads (CPG), silica, silica gel, glass, ceramic, polymer, cellulose, and combinations thereof.

[0093] The term "FVII" or "FVII gene" as used herein refers to a human gene. The term "FVII mRNA" as used herein refers to a messenger RNA (mRNA) produced from the expression of the FVII gene or the transcription of the FVII gene.

[0094] The terms "FVII" and "FVII protein" as used herein interchangeably refer to a protein produced from the expression of the FVII gene or the translation of the FVII mRNA.

[0095] The term "baseline expression of the FVII gene" as used herein refers to the expression level of the FVII gene in a parallel control (e.g., a cell or an individual) without administration of the saRNA or before administration of the saRNA. saRNA

[0096] In the present application, the expression of the FVII gene is upregulated by RNA activation, and the associated disease is treated by increasing the expression level of the FVII protein. Since the FVII gene encodes the FVII protein, the upregulation of the FVII mRNA expression can lead to an increase in the expression of the FVII protein, thereby preventing or treating the disease (e.g., hemophilia with inhibitors). Therefore, in some cases, the FVII gene is the target gene in the present application.

[0097] Aspects of the present application include saRNAs (or oligonucleotide modulators comprising the same) comprising an oligonucleotide sequence ranging from 16 to 35 contiguous nucleotides in length, wherein the contiguous oligonucleotide sequence has at least 75%, or at least 80%, or at least 85%, or at least 90% sequence homology or complementarity to an equal length portion of SEQ ID NO: 1437, and wherein the saRNA activates / upregulates (e.g., by at least 10%) the expression of the FVII gene as compared to its baseline expression.

[0098] In some embodiments, the equal length portion of SEQ ID NO: 1437 is located in the region -557 to -379, the region -346 to -298, the region -271 to -91, or the region -96 to -1 upstream of the transcription start site of the FVII gene. In some embodiments, the equal length portion 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.

[0099] In some embodiments, the contiguous oligonucleotide sequence of the saRNA has five or fewer nucleotide differences or mismatches, i.e., 5, 4, 3, 2, 1, or 0 nucleotide differences or mismatches, relative to an equal length portion of SEQ ID NO: 1437. In some embodiments, the differences or mismatches are located in the middle or at the 3' end of the saRNA oligonucleotide sequence. Methods and principles of saRNA molecule design are well known to those skilled in the art and are described in detail in, e.g., Place et al., Molecular Therapy-Nucleic Acids (2012) 1, e15 and Li et al., PNAS, 2006, vol. 103, no. 46, 17337-17342, which are incorporated herein by reference in their entirety.

[0100] In some embodiments, the saRNA disclosed herein comprises a sense strand and an antisense strand. Each of the sense strand and the antisense strand comprises a complementary region capable of forming a double-stranded nucleic acid structure that activates expression of a FVII gene in a cell through an RNAa mechanism. The RNAa mechanism (also referred to as RNA activation) used in the present application refers to a mechanism by which a double-stranded nucleic acid structure is capable of upregulating a target gene in a sequence-specific manner at the transcriptional level.

[0101] Aspects of the present application also include a small activating RNA (saRNA) comprising a sense strand and an antisense strand. Each of the sense strand and the antisense strand comprises a complementary region.

[0102] The sense strand and the antisense strand of the saRNA can be present on two different nucleic acid strands or on the same nucleic acid strand (e.g., a contiguous nucleic acid sequence). When the sense strand and the antisense strand are on two different strands, one or both ends of the saRNA can be blunt ended, or at least one strand of the saRNA has a 3' or 5' overhang of 1 to 6 nucleotides in length, e.g., 1, 2, 3, 4, 5, or 6 nucleotides in length. In some cases, both strands have a 3' or 5' overhang of 1 to 6 nucleotides in length, e.g., 2 or 3 nucleotides in length. In some cases, the nucleotides of the overhang are thymine deoxyribonucleotides (dT). In some cases, the overhang is a natural overhang. When the sense strand and the antisense strand are on the same nucleic acid strand, in some cases, the saRNA is a hairpin single-stranded nucleic acid molecule in which the complementary regions of the sense strand and the antisense strand form a double-stranded nucleic acid structure with each other.

[0103] In some embodiments, the saRNA is a duplex consisting of a sense strand and an antisense strand, and the sense strand and the antisense strand are complementary, forming a blunt end structure at both ends. In some embodiments, the saRNA is a duplex consisting of the sense strand and the antisense strand, the sense strand and the antisense strand are complementary to each other, wherein the antisense strand has an overhang of 1 to 6 nucleotides in length at the 3 '-end of the antisense strand. In some embodiments, saRNA is a duplex consisting of a sense strand and an antisense strand, the sense strand and the antisense strand are complementary to each other, wherein the sense strand has an overhang of 1 to 6 nucleotides in length at its 3 '-end.

[0104] In the foregoing saRNAs, in some embodiments, the sense strand and the antisense strand are 16 to 35 nucleotides in length, respectively. For example, in some embodiments, the sense strand and the antisense strand comprise 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 nucleotides, respectively.

[0105] In certain embodiments, one strand of the saRNA has at least 75% (e.g., at least about 79%, about 80%, about 85%, about 90%, about 95%, or about 99%) sequence homology or complementarity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1 to 286. Specifically, the sense strand of the saRNA disclosed herein has at least 75% (e.g., at least about 79%, about 80%, about 85%, about 90%, about 95%, or about 99%) sequence homology to any of the nucleotide sequences selected from the group consisting of SEQ ID NOs: 287 to 858, and the antisense strand of the saRNA disclosed herein has at least 75% (e.g., at least about 79%, about 80%, about 85%, about 90%, about 95%, or about 99%) sequence homology to any of the nucleotide sequences selected from the group consisting of SEQ ID NOs: 859 to 1430. More specifically, the sense strand of the saRNA disclosed herein comprises or consists of any of the nucleotide sequences selected from the group consisting of SEQ ID NOs: 287 to 858; and the antisense strand of the saRNA disclosed herein comprises or consists of any of the nucleotide sequences selected from the group consisting of SEQ ID NOs: 859 to 1430.

[0106] In certain embodiments, one strand of the saRNA has five or fewer nucleotide differences or mismatches relative to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1 to 286, i.e. 5, 4, 3, 2, 1 or 0 nucleotide differences or mismatches. Specifically, the sense strand of the saRNA disclosed herein has five or fewer nucleotide differences relative to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 287 to 858, i.e. 5, 4, 3, 2, 1 or 0 nucleotide differences, and the antisense strand of the saRNA disclosed herein has five or fewer nucleotide differences relative to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 859 to 1430, i.e. 5, 4, 3, 2, 1 or 0 nucleotide differences. In some embodiments, the differences or mismatches are located in the middle or 3' end of the saRNA sense or antisense strand.

[0107] In certain embodiments, the antisense strand disclosed herein is capable of interacting with a target nucleic acid sequence on a gene promoter in a sequence-specific manner, i.e. the antisense strand is capable of hybridizing to the target nucleic acid by hydrogen bonding. In certain embodiments, the antisense strand has a nucleotide sequence comprising the reverse complement of the target portion of the target nucleic acid to which it targets when written in the 5' to 3' direction. In certain such embodiments, the antisense strand has a nucleotide sequence comprising the reverse complement of the target portion in SEQ ID NO: 1437 when written in the 5' to 3' direction; specifically, the target portion is a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1 to 286.

[0108] In the saRNA disclosed herein, all the nucleotides can be natural or non-chemically modified nucleotides, or at least one of the nucleotides is a chemically modified nucleotide. Non-limiting examples of chemical modifications include one or more combinations of: (1) modification of the phosphodiester bond of the nucleotides in the nucleotide sequence of the saRNA; (2) modification of the 2'-OH of the ribose in the nucleotide sequence of the saRNA; (3) modification of the base in the nucleotide of the saRNA; and (4) at least one of the nucleotides in the nucleotide sequence of the small activating nucleic acid molecule is a locked nucleic acid.

[0109] The chemical modifications described herein are well known to those skilled in the art, wherein the modification of the phosphodiester bond refers to the modification of the oxygen in the phosphodiester bond, including phosphorothioate modification and boranophosphate modification. The modifications disclosed herein stabilize the structure of the saRNA, maintain high specificity and high affinity for base pairing.

[0110] In some embodiments, the saRNA described herein comprises at least one chemically modified nucleotide which is modified at the 2’-OH of the pentose of the nucleotide, i.e., introducing certain substituents at the hydroxyl position of the ribose, such as 2’-fluoro modification, 2’-oxymethyl modification, 2’-oxaethylenemethoxy modification, 2,4’-dinitrophenol modification, locked nucleic acid (LNA), 2’-amino modification, or 2’-deoxy modification, such as 2’-deoxy-2’-fluoro modified nucleotide, 2’-deoxy modified nucleotide.

[0111] In some embodiments, the saRNA described herein comprises at least one chemically modified nucleotide which is modified at the base of the nucleotide, such as 5’-bromouracil modification, 5’-iodouracil modification, N-methyluracil modification, or 2,6-diaminopurine modification.

[0112] In some embodiments, the chemical modification of the saRNA is the addition of an (E)-vinylphosphonate moiety at the 5’ end of the sense sequence or the antisense sequence. In some embodiments, the chemical modification of the at least one chemically modified nucleotide is the addition of a 5’-methylcytosine moiety at the 5’ end of the sense sequence or the antisense sequence.

[0113] In some embodiments, the saRNA described herein comprises at least one nucleotide in the nucleotide sequence of the small activating nucleic acid molecule, i.e., chemically modified nucleic acids, such as locked nucleotides, abasic nucleotides, 2’-amino modified nucleotides, 2’-alkyl modified nucleotides, morpholino nucleotides, phosphoramidates, and nucleotides comprising non-natural bases. In some embodiments, the saRNA disclosed herein comprises “endo-light” modifications with 2’-O-methyl modified nucleotides and nucleotides comprising 5’-thiophosphate groups.

[0114] In some embodiments, the saRNAs described herein are chemically modified to enhance stability or other beneficial properties. The nucleic acids described herein can be synthesized and / or modified by traditional methods, such as those described in Current protocols in nucleic acid chemistry, Beaucage, S. L. et al. (Edrs.), John Wiley & Sons, Inc., New York, N.Y., USA, the contents of which are incorporated herein. Modifications include, but are not limited to, (a) terminal modifications, such as 5' terminal modifications (phosphorylation, conjugation, inverted, etc.), 3' terminal modifications (conjugation, DNA nucleotides, inverted, etc.); (b) base modifications, such as substitution with a stabilizing base, destabilizing base, or base that pairs with an extended partner repertoire, removal of a base (abasic nucleotide), or conjugated base; (c) sugar modifications (e.g., at the 2' position or 4' position) or sugar substitutions; and (d) backbone modifications, including modifications or substitutions of the phosphodiester linkage. Particular embodiments of the saRNAs described herein include, but are not limited to, RNAs containing modified backbones or no natural internucleoside linkages. In some embodiments, RNAs with modified backbones include RNAs without phosphorus atoms in the backbone, and the like. In some embodiments, modified RNAs without phosphorus atoms in the internucleoside backbone can also be considered oligonucleosides. In some embodiments, the modified oligonucleotides will have phosphorus atoms in their internucleoside backbones.

[0115] Modified oligonucleotide backbones include, without limitation, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl-phosphotriesters, methyl and other alkyl phosphonates including 3'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3'-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thioalkylphosphonates, thioalkylphosphotriesters, and boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs of these, as well as others having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'. Various salts, mixed salts and free acid forms are also included.

[0116] Non-limiting examples of preparing phosphorous-containing linkages include, but are not limited to, U.S. Patent Nos. 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, the entire contents of which are incorporated herein by reference.

[0117] Furthermore, to facilitate the entry of the saRNA into the cell, a chemical conjugation moiety can be introduced at the end of the sense or antisense strand of the saRNA on the basis of the above modification to facilitate the action through the cell membrane consisting of a lipid bilayer and the nuclear membrane and the gene promoter region within the nucleus.

[0118] In certain embodiments, the saRNAs disclosed herein are covalently linked to one or more conjugate moieties. In certain embodiments, the conjugate moieties alter one or more properties of the linked oligonucleotide, including, but not limited to, pharmacodynamics, pharmacokinetics, stability, binding, absorption, tissue distribution, cellular distribution, cellular uptake, charge, and clearance. In certain embodiments, the conjugate moieties impart new properties to the linked oligonucleotide, such as a fluorophore or reporter group that enables detection of the oligonucleotide.Certain conjugation moieties and conjugate moieties have been described previously, for example: a cholesteryl moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556); a cholic acid moiety (Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4, 1053-1060); a thioether, for example, hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. 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); a lipophilic chain, for example, 10-ascndecane or undecyl residues (Saison-Behmoaras et al., EMBO 1, 1991, 10, 1111-1118; Kabanov et al., FEBS Lett., 1990, 259, 327-330; Svinarchuk et al., Biochimie, 1993, 75, 49-54); a phospholipid, for example, di-hexadecyl-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); a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973); an 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 tocopheryl group (Nishina et al., Molecular Therapy Nucleic Acids, 2015, 4, e220; Nishina et al., Molecular Therapy, 2008, 16, 734-740); a GalNAc cluster (e.g. WO2014 / 179620).

[0119] In some embodiments, the saRNA described herein involves the sense or antisense strand of the saRNA conjugated to one or more conjugated moieties selected from the group consisting of intercalators, reporter molecules, polyamines, polyamides, peptides, carbohydrates, vitamin moieties, polyethylene glycol, thioethers, polyethers, cholesterols, thiocholesterols, cholic acid moieties, folate, lipids, phospholipids, biotin, phenazines, phenanthridines, anthraquinones, adamantanes, acridines, fluoresceins, rhodamines, coumarins, fluorophores, and dyes.

[0120] In some embodiments, the conjugated moiety comprises an active drug, such as aspirin, warfarin, phenylbutazone, ibuprofen, suprofen, fenbufen, ketoprofen, (S)-(+)-pranoprofen, carprofen, dansylsarcosine, 2,3,5-triiodobenzoic acid, fingolimod, flufenamic acid, folinic acid, benzothiadiazides, chlorothiazides, diazepines, indomethacin, barbiturates, cephalosporins, sulfonamides, hypoglycemic agents, antibacterial agents, or antibiotics.

[0121] In some embodiments, the saRNA described herein is conjugated to one or more conjugated moieties selected from the group consisting of lipids, fatty acids, fluorophores, ligands, sugars, peptides, and antibodies.

[0122] In some embodiments, the saRNA of the present application involves the sense or antisense strand of the saRNA conjugated to one or more conjugated moieties selected from the group consisting of cell-penetrating peptides, polyethylene glycol, alkaloids, tryptamines, benzimidazoles, quinolones, amino acids, cholesterols, glucose, and N-acetylgalactosamine. In certain embodiments, the saRNA is conjugated to two conjugated moieties. In certain embodiments, the two conjugated moieties are a lipid and N-acetylgalactosamine. In certain embodiments, the one or more conjugated moieties are derived from S9, tC2x6, C5x5, or a combination thereof, as shown herein: wherein, represents a carrier. In certain embodiments, the conjugated moieties conjugated to the saRNA are S9, tC2x6, and C5x5, as shown herein. In certain embodiments, tC2, tC2x6, or S9 is conjugated to the 3' end of the sense strand; C5x5 is conjugated to the 5' end of the sense strand.

[0123] In certain embodiments, the conjugating moiety is a lipid selected from fatty acids having a carbon chain length of 4 to 30 carbon atoms. In certain embodiments, the conjugating moiety is a fatty acid having a carbon chain length of 16, 18, or 22 carbon atoms. In certain embodiments, the conjugating moiety is selected from the lipophilic moieties described in WO2024002046A1. In certain embodiments, the saRNA can comprise 1, 2, 3, 4, 5, 6, or even more oligonucleotides, which are conjugated to 1, 2, 3, 4, 5, 6, or even more conjugating moieties, respectively, via 1, 2, 3, 4, 5, 6, or even more linking moieties, respectively.

[0124] According to one embodiment, the linking moiety, if present, can be selected from the following groups: -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-C22 )alkylene-NH-C(O)-, -NH-C(O)-(Ci-C 22 )alkylene-C(O)-, -NH-C(O)-(Ci-C 22 )alkylene-C(O)-NH-, -C(O)-NH-(Ci-C 22 )alkylene-NH-C(O)-, -(Ci-C 22 )alkylene-OP(O)2O-, -(Ci-C 22 )alkylene-OP(O)(O-)O-, -(Ci-C 22 )alkylene-OP(O)(O-)O-(Ci-C 22 )alkylene-, -(Ci-C 22 )alkylene-OP(O)O-, -(Ci-C 22 )alkylene-OP(O)(S)O-, -(Ci-C 22 )alkylene-O-S(O)2-O-, -(Ci-C 22 )alkylene-S(O)2-O-, -(Ci-C 22 )alkylene-S(O)-O-, -O-P(O)2-O-(Ci-C 22 )alkylene-OP(O)2O-, -O-P(O)-O-(Ci-C 22 )alkylene-OP(O)O-, -OP(O)(S)O-(Ci-C 22 )alkylene-OP(O)(S)O-, -O-S(O)2-O-(Ci-C 22 )alkylene-O-S(O)2-O-, -S(O)2-O-(Ci-C 22 )alkylene-S(O)2-O-, and -O-S(O)-(Ci-C 22 )alkylene-S(O)-O-; wherein -(Ci-C 22 )alkylene- in the linking moiety can be an alkylene containing 1 to 22 carbon atoms, for example, an alkylene containing 2 to 20 carbon atoms, or 3 to 18 carbon atoms, or 4 to 16 carbon atoms, or 5 to 12 carbon atoms, or 6 to 10 carbon atoms. In one embodiment, when the linking moiety is a direct bond, the conjugating moiety is directly linked to the oligonucleotide.

[0125] In some embodiments, the saRNA conjugated to one or more conjugating moieties disclosed in embodiments is directly contacted, transferred, delivered, or administered to a cell or patient.

[0126] In some embodiments, the sense and antisense strands of the saRNA have at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or about 100% of the nucleotides that are chemically modified nucleotides.

[0127] In some embodiments, the saRNA has at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or about 100% of the nucleotides that are chemically modified nucleotides.

[0128] These modifications can improve the bioavailability of the saRNA, improve affinity for the target sequence, and enhance resistance to nuclease hydrolysis in the cell.

[0129] In non-limiting examples, the design of the saRNA is based at least in part on the following criteria: (1) GC content between 40% and 70%; (2) no more than 5 consecutive identical nucleotides; (3) no more than 3 di- or tri-nucleotide repeat sequences. In some embodiments, the design / selection of the saRNA is based at least in part on criteria that enable the production of functional saRNAs. For example, in certain cases, sequences located upstream of the TSS can include sequences that, despite being located in hot spot regions, are not conducive to saRNA synthesis.

[0130] In some embodiments, the saRNAs described herein, when contacted with a cell, can activate or upregulate the expression of one or more genes in the cell, preferably by at least 10% (e.g., at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 300%, at least 500%, at least 800%, at least 1000%, at least 2000%, or at least 5000%). Target sequence

[0131] In certain embodiments, the present application relates to a target site for a saRNA of the present application, in particular, the target site is a nucleotide sequence or oligonucleotide ranging in length from 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 the group consisting of SEQ ID NO: 1 to 286. The target site is capable of interacting with the antisense strand of a saRNA disclosed in the present application, thus the saRNA is capable of activating the expression of a FVII gene (e.g., mRNA expression, protein expression, FVII expression). In some embodiments, the selection of the target site is based at least in part on the promoter sequence upstream of the TSS. In some embodiments, the selection of the target site is based at least in part on the -5000 bp, -4000 bp, -3000 bp, -2000 bp, -1000 bp, or -500 bp sequence upstream of the TSS. In some embodiments, the target site is selected at least in part by moving 1 bp at a time towards the TSS and generating a target sequence, followed by repeating the step and adding an additional base pair to the TSS (e.g., n+1). In some embodiments, the target site has a length of about 8 to about 35 nucleotides. In some embodiments, the target site has a length of about 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 to design, screen, and / or make a corresponding saRNA.

[0132] In certain embodiments, the present application relates to an oligonucleotide complex comprising a saRNA disclosed in the present application and a target site disclosed in the present application. In certain embodiments, the oligonucleotide complex can activate the expression of a FVII gene by at least 10% (e.g., can activate the expression of a FVII gene compared to the baseline FVII gene expression level). Hot spot

[0133] In certain embodiments, the present application relates to nucleic acid sequences located upstream of the transcription start site of the FVII gene, i.e. "hotspot regions". In certain embodiments, the nucleic acid sequences disclosed herein are oligonucleotide sequences of at least 25 nt, at least 27 nt, at least 30 nt, at least 35 nt, at least 40 nt, at least 45 nt, at least 48 nt, or at least 49 contiguous nucleotides in length, and have at least 75%, at least 80%, at least 85%, or at least 90% sequence homology to an equal length region within the nucleotide sequence of SEQ ID NO: 1437. In some embodiments, the hotspot regions are isolated regions.

[0134] "Hotspot region" and "hotspot" are used interchangeably herein to refer to a nucleic acid region on a saRNA target gene (e.g. a nucleic acid region in the promoter upstream of the TSS of the target gene) that is enriched for full-length targets of functional saRNAs and spans the most 5' end of the first saRNA target and the most 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 saRNAs designed to target sequences within a hotspot are functional, i.e. induce at least 1.1-fold change in mRNA expression of the target gene. In one non-limiting example, at least 20% (e.g. at least 25%, at least 30%, at least 35%, at least 40%, at least 47%) of saRNAs designed to target a hotspot are functional, i.e. induce at least 1.1-fold change in mRNA expression of the target gene, provided that the design of the saRNAs is based at least in part on the following criteria: (1) GC content between 40% and 70%; (2) no more than 5 consecutive identical nucleotides; (3) no more than 3 di- or tri-nucleotide repeat sequences. In some embodiments, the designed functional saRNAs can be blunt-ended or overhanging, and / or unmodified or chemically modified.

[0135] In some embodiments, the same or similar criteria are used to select nucleic acid sequences and / or target sequences. In one non-limiting example, the isolated nucleic acid sequences upstream of the TSS of the FVII gene are selected based at least in part on the following criteria: (1) GC content between 40% and 70%; (2) no more than 5 consecutive identical nucleotides; (3) no more than 3 di- or tri-nucleotide repeat sequences.

[0136] In some embodiments, the nucleic acid region is about 25 to about 250 (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 length. In some embodiments, the hotspot region is a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 1438 to 1441, including any sub-region of the above-mentioned regions, as long as it is rich in target sequences of functional saRNA. In some embodiments, the hotspot region is a nucleic acid sequence selected from the group consisting of the region -557 to -379, the region -346 to -298, the region -271 to -91, and the region -96 to -1 upstream of the transcription start site of the FVII gene, including any sub-region of the above-mentioned regions, as long as it is rich in target sequences of functional saRNA. The present application also provides a method for designing saRNA, which provides saRNA targeting the nucleic acid sequences described in the present application.

[0137] In some embodiments, the design / selection of the target sequence is based at least in part on the criteria capable of generating functional saRNA. For example, in some cases, the sequence located upstream of the TSS can include sequences that are not conducive to target sequence synthesis despite being located in the hotspot region.

[0138] The RNAa activity of each designed saRNA depends on a variety of complex factors, such as chromatin environment, sequence features of the target itself and nearby regions, transcription factor binding, etc. The core determinant factor can be the accessibility of the target DNA. In regions with higher accessibility, dsRNA can exhibit higher RNAa activity. While dsRNA designed against other regions of the promoter can exhibit non-functional or even transcriptional silencing effects. This can explain the existence of hotspot regions where functional saRNA target sequences are clustered. For example, a target sequence designed based at least in part on the following criteria: “(1) GC content between 40% and 70%; (2) no more than 5 consecutive identical nucleotides; (3) no more than 3 di- or tri-nucleotide repeat sequences” can not be able to activate / upregulate the level of FVII gene expression at least 10% higher than the baseline, as the target sequence bound by saRNA is not within the hotspot region (e.g., any hotspot region described in the present application).

[0139] In certain embodiments, the present application relates to a nucleic acid complex comprising a saRNA disclosed herein and a nucleic acid sequence disclosed herein. In certain embodiments, the complex activates (e.g., at least 10%) the expression of the FVII gene compared to the baseline expression of the FVII gene.

[0140] In some aspects, methods of using nucleic acids upstream of the transcription target site of the FVII gene are also provided. DNA encoding saRNA

[0141] In certain embodiments, the present application relates to a nucleic acid or polynucleotide encoding a saRNA that can activate or upregulate the expression of a FVII gene in a cell, preferably by at least 10% (e.g., compared to the baseline expression of the FVII gene). In certain embodiments, the nucleic acid is a DNA encoding a saRNA. In certain embodiments, the nucleic acid is a recombinant vector, in particular, a recombinant AAV vector. The vectors disclosed herein comprise a DNA segment encoding a saRNA of the present application. saRNA-containing cell

[0142] Upon contacting a cell, the saRNA disclosed herein can effectively activate or upregulate the expression of a FVII gene in a cell, preferably by at least 10% (e.g., compared to the baseline expression of the FVII gene).

[0143] In certain embodiments, the present application relates to a cell comprising a saRNA disclosed herein. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a human cell, such as a human embryonic liver cell, a human hepatoma cell (e.g., Huh-7 cell), a human hepatoma cell (e.g., PLC / PRF / 5 cell). The cell disclosed herein can be in vitro or ex vivo, such as a cell line or a cell strain, or can be present in a mammal, such as a human. The human disclosed herein is a patient suffering from a FVII-related disease or condition, such as a disease or condition caused by a mutation in the FVII gene, low FVII levels, insufficient levels of functional FVII protein in the blood, and / or hemophilia with inhibitors. In some embodiments, the cell is from a hemophilia patient. saRNA-containing composition

[0144] In certain embodiments, the present application relates to a composition or pharmaceutical composition comprising a saRNA or nucleic acid of the present application. In some embodiments, the composition comprises at least one pharmaceutically acceptable carrier. In some embodiments, the composition comprises at least one pharmaceutically acceptable carrier selected from an aqueous carrier, a liposome or LNP, a polymer, a micelle, a colloid, a metal nanoparticle, a non-metal nanoparticle, a bioconjugate (e.g., GalNAc), a polypeptide, and an antibody. In one embodiment, the aqueous carrier can be, for example, RNase-free water or RNase-free buffer. In some embodiments, the composition can contain 0.001-1600 nM (e.g., 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) of a saRNA or polynucleotide described herein. In some embodiments, the composition includes 25 nM of a saRNA or polynucleotide described herein. In some embodiments, the composition can contain 0.001-150 nM (e.g., 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) of a saRNA or polynucleotide described herein. In some embodiments, the composition includes 25 nM of a saRNA or polynucleotide described herein. Methods of using saRNA

[0145] Another aspect of the application relates to methods of activating / upregulating expression of a FVII gene in a cell using a saRNA. The saRNA comprises an oligonucleotide sequence of 16 to 35 contiguous nucleotides in length. In some embodiments, the oligonucleotide sequence has at least 75%, at least 80%, at least 85%, or at least 90% homology or complementarity to an equal length region of SEQ ID NO: 1437, in particular, the saRNA can activate / upregulate expression of a FVII gene, e.g., at least 10% (e.g., at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 300%, at least 500%, at least 800%, at least 1000%, at least 2000%, or at least 5000% compared to baseline expression of the FVII gene). In certain embodiments, the expression of a FVII gene is activated / upregulated at least 2-fold (e.g., at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, or at least 7-fold compared to baseline expression of the FVII gene) when a saRNA disclosed in the embodiments is administered to a cell or subject. In certain embodiments, the saRNA activates or upregulates expression of a FVII gene by about 6.5-fold. In certain embodiments, the FVII gene expression of a cell is activated / upregulated by administering a saRNA disclosed in the embodiments to the cell at a concentration of at least 0.01 nM, e.g., 0.02 nM, 0.05 nM, 0.08 nM, 0.1 nM, 0.2 nM, 0.3 nM, 0.4 nM, 0.5 nM, 0.6 nM, 0.8 nM, 1 nM, 5 nM, 10 nM, 25 nM, 50 nM, 75 nM, 100 nM, 150 nM, 200 nM, 400 nM, 800 nM, 1000 nM, or 1600 nM. In certain embodiments, the induction of a FVII protein (FVII) is activated / upregulated by administering a saRNA disclosed in the embodiments to the cell at a concentration of at least 0.01 nM, e.g., 0.02 nM, 0.05 nM, 0.08 nM, 0.1 nM, 0.2 nM, 0.3 nM, 0.4 nM, 0.5 nM, 0.6 nM, 0.8 nM, 1 nM, 2 nM, 3 nM, 4 nM, 5 nM, 10 nM, 25 nM, 50 nM, 75 nM, 100 nM, or 150 nM.

[0146] In another aspect of the application relates to a method of preventing or treating a FVII related disease or disorder, such as a disease or disorder induced by insufficient expression of coagulation factor VII (FVII), FVII gene mutation, low functional FVII levels in the blood of an individual, and / or having normal FVII levels but can be treated by increasing FVII levels, such as hemophilia with inhibitors, comprising: administering to the individual an effective amount of a saRNA, a nucleic acid or polynucleotide encoding a saRNA, or a composition comprising a saRNA disclosed herein. In certain embodiments, the effective amount of a saRNA disclosed herein can be a concentration in the range of 0.01 nM to 1600 nM, such as 0.01 nM, 0.02 nM, 0.05 nM, 0.08 nM, 0.1 nM, 0.2 nM, 0.3 nM, 0.4 nM, 0.5 nM, 0.6 nM, 0.8 nM, 1 nM, 5 nM, 10 nM, 25 nM, 50 nM, 75 nM, 100 nM, 150 nM, 200 nM, 400 nM, 800 nM, 1000 nM, or 1600 nM. In certain embodiments, the effective amount of a saRNA disclosed herein can be a concentration in the range of 0.01 nM to 150 nM, such as 0.01 nM, 0.02 nM, 0.05 nM, 0.08 nM, 0.1 nM, 0.2 nM, 0.3 nM, 0.4 nM, 0.5 nM, 0.6 nM, 0.8 nM, 1 nM, 5 nM, 10 nM, 25 nM, 50 nM, 75 nM, 100 nM, or 150 nM. In some embodiments, the disease or disorder is hemophilia, such as hemophilia with inhibitors. In some embodiments, the individual is a mammal. In some embodiments, the individual is a human.

[0147] In any of the embodiments provided herein, such saRNAs, nucleic acids encoding saRNAs of the application, or compositions comprising such saRNAs of the application can be introduced directly into a cell, or can be produced within a cell after introduction of a nucleotide sequence encoding a saRNA into a cell, such as a mammalian cell, including but not limited to PLC / PRF / 5 and Huh-7, or a human cell. Such cells can be ex vivo cells, such as cell lines, etc., or can be present in a mammal, such as a human. In some embodiments, human refers to a patient or individual having a FVII deficiency related disease or hemophilia with inhibitors. In certain embodiments, the nucleic acids or polynucleotides encoding saRNAs or compositions of saRNAs described herein are each in an amount sufficient to treat hemophilia.

[0148] Another aspect of the application relates to administering an effective amount of a saRNA or a composition thereof to an individual using an administration route described herein. In some embodiments, the administration route is selected from one or more of the following: parenteral infusion, oral administration, intranasal administration, inhalation administration, vaginal administration, and rectal administration. In some embodiments, the administration route is selected from one or more of the following: intrathecal, intramuscular, intravenous, intra-arterial, intraperitoneal, intravesical, intracerebroventricular, intravitreal, and subcutaneous administration. Dosing regimen and routes of administration

[0149] Aspects of the application relate to pharmaceutical compositions comprising a saRNA of the application. In some embodiments, the pharmaceutical composition comprises a saRNA of the application and a pharmaceutically acceptable carrier, a therapeutically inert carrier, a diluent, or a pharmaceutically acceptable adjuvant. The pharmaceutical compositions disclosed herein will be developed into a medicament for preventing or treating a FVII deficiency related condition or a hemophilia (e.g., hemophilia with inhibitors).

[0150] Aspects of the application also relate to methods of using a saRNA of the application to prepare such a composition.

[0151] Another aspect of the application relates to the use of a saRNA of the application in the manufacture of a pharmaceutical composition disclosed herein.

[0152] Another aspect of the application relates to the use of a saRNA or polynucleotide according to any embodiment described herein, or a composition according to any embodiment described herein, in the manufacture of a medicament for preventing or treating a condition related to the FVII gene or FVII protein, such as a condition induced by insufficient expression of the FVII protein in an individual, a mutation in the FVII gene, low functional FVII levels in the blood, and / or hemophilia (e.g., hemophilia with inhibitors). According to certain embodiments of use, the condition can include a FVII gene mutation related disease or disorder, or hemophilia with inhibitors. According to certain embodiments of use, the condition is induced by insufficient expression of the FVII protein or hemophilia with inhibitors. Also contemplated are certain embodiments of use, wherein the individual is a mammal, such as a human.

[0153] The dosage ranges of the various saRNAs or compositions of the application can vary widely from one to another and will be determined by the requirements of the individual case. In certain embodiments, the pharmaceutical composition of the application is administered for the first time when the subject is less than 1 week old, less than 1 month old, less than 3 months old, less than 6 months old, less than 1 year old, less than 2 years old, less than 15 years old, or more than 15 years old.

[0154] The single dose of the saRNA ranges from 0.01 mg / kg to 1000 mg / kg, such as about 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 can comprise two or more of any of the saRNA sequences described herein.

[0155] In some embodiments, the intended frequency of administration is an approximation. For example, in certain embodiments, if the intended frequency of administration is first administration on day 1 and second administration on day 29, the patient can receive the second administration 25, 26, 27, 28, 29, 30, 31, 32, 33, or 34 days after receiving the first administration. In certain embodiments, if the intended frequency of administration is first administration on day 1 and second administration on day 15, the patient can receive the second administration 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days after receiving the first administration. In certain embodiments, if the intended frequency of administration is first administration on day 1 and second administration on day 85, the patient can receive the second administration 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, or 90 days after receiving the first administration.

[0156] In certain embodiments, the injection dose and / or volume will be adjusted based on patient age, patient weight, and / or other factors that can require adjustment of injection parameters.

[0157] In certain embodiments, the pharmaceutical composition comprises a cosolvent system. For example, such cosolvent systems include benzyl alcohol, a nonpolar surfactant, a water-miscible organic polymer, and an aqueous phase. In certain embodiments, such cosolvent systems are used for hydrophobic compounds. A non-limiting example of such a cosolvent system is the VPD cosolvent system, which is an anhydrous ethanol solution comprising 3% w / v benzyl alcohol, 8% w / v nonpolar surfactant polysorbate 80 TM , and 65% w / v polyethylene glycol 300. The proportions of such a cosolvent system can vary considerably without significantly altering its solubility and toxicity properties. In addition, the identity of the cosolvent components can vary: for example, other surfactants can be used instead of polysorbate 80 TM ; the fraction size of the polyethylene glycol can vary; other biocompatible polymers can be used instead of polyethylene glycol, such as polyvinylpyrrolidone; other sugars or polysaccharides can be used instead of glucose.

[0158] Examples of other compositions or components associated with the saRNAs, compositions, pharmaceutical compositions, and methods described herein include, but are not limited to, diluents, salts, buffers, chelating agents, preservatives, desiccants, antibacterial agents, needles, syringes, packaging materials, tubes, bottles, flasks, beakers, and the like, for example, for use in using, modifying, assembling, storing, packaging, preparing, mixing, diluting, and / or preserving components for a particular use. In embodiments in which any component is in a liquid form, the liquid can be in a concentrated or ready-to-use form.

[0159] In some embodiments, lipid moieties for nucleic acid therapy can be applied in the present application to deliver the saRNA molecules disclosed herein. In these methods, the nucleic acid (e.g., one or more saRNAs described herein) is introduced into preformed liposomes or lipoplexes made from a mixture of cationic lipids and neutral lipids. In certain methods, saRNA complexes are formed with single or multi-cationic lipids in the absence of neutral lipids. In certain embodiments, the lipid moieties are selected to increase the distribution of the agent into a particular cell or tissue. In certain embodiments, the lipid moieties are selected to increase the distribution of the agent in adipose tissue. In certain embodiments, the lipid moieties are selected to increase the distribution of the agent in muscle tissue.

[0160] In certain embodiments, the pharmaceutical compositions include a delivery system. Examples of delivery systems include, but are not limited to, liposomes and emulsions. Certain delivery systems can be used to prepare certain pharmaceutical compositions, including compositions comprising a hydrophobic compound. In certain embodiments, certain organic solvents, such as dimethylsulfoxide, are used.

[0161] In certain embodiments, the pharmaceutical compositions comprise one or more tissue-specific delivery molecules designed to deliver one or more agents of the present application to a particular tissue or cell type. For example, in certain embodiments, the pharmaceutical compositions include liposomes and / or liposomes coated with tissue-specific antibodies.

[0162] In some embodiments, the saRNAs can be delivered or administered by a vector. Any vector useful for gene delivery can be used. In some embodiments, a viral vector can be used. Non-limiting examples of viral vectors useful in the present application include, but are not limited to, human immunodeficiency virus, herpes simplex virus (HSV), Moloney murine sarcoma virus (MMSV), mouse stem cell virus (MSCV), Semliki Forest virus (SFV), Sindbis virus (SIN), Venezuelan equine encephalitis virus (VEE), vesicular stomatitis virus (VSV), vaccinia virus (VV), adeno-associated virus (AAV), adenovirus, lentivirus, and retrovirus.

[0163] In some embodiments, the vector is a recombinant AAV vector (rAAV). AAV vectors are relatively small DNA viruses that can integrate into the genome of the cell they infect in a stable and site-specific manner. They are able to infect multiple types of cells without any impact on cell growth, morphology, or differentiation, and do not appear to be associated with human pathologies. The AAV genome has been cloned, sequenced, and characterized. It comprises about 4700 bases, with inverted terminal repeat (ITR) regions of about 145 bases at each end, which serve as the origin of viral replication. The remainder of the genome is divided into two essential regions with encapsidation functions: the left portion of the genome, which contains the rep gene involved in viral replication and viral gene expression; and the right portion of the genome, which contains the cap gene encoding the viral capsid proteins.

[0164] The formulations, pharmaceutical compositions, or medicaments of the present application are formulated, dosed, and administered in a fashion consistent with good medical practice. Factors for consideration in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the part of the body to be treated, the method of administration, the scheduling of the administration, and other factors known to medical practitioners. The formulations, pharmaceutical compositions, or medicaments of the present application can be administered in either single or multiple doses.

[0165] The formulations, pharmaceutical compositions, or medicaments of the present application can be delivered by parenteral infusion, including intrathecal, intramuscular, intravenous, intraarterial, intraperitoneal, intravesical, intracerebroventricular, intravitreal, or subcutaneous administration; or by oral, intranasal, inhalation, vaginal, or rectal administration.

[0166] Typical formulations of the oligonucleotide modulators of the application are prepared by mixing a saRNA of the application with carriers or excipients. Suitable carriers and excipients are well known to those skilled in the art and are described in, for example, Ansel H.C. et al., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems (2004; Lippincott, Williams & Wilkins, Philadelphia); Gennaro A.R. et al., Remington: The Science and Practice of Pharmacy (2000; Lippincott, Williams & Wilkins, Philadelphia); and Rowe R.C, Handbook of Pharmaceutical Excipients (2005; Pharmaceutical Press, Chicago). The formulations can also include one or more buffers, stabilizing agents, surfactants, wetting agents, lubricating agents, emulsifiers, suspending agents, preservatives, antioxidants, opaquing agents, glidants, processing aids, colorants, sweeteners, perfuming agents, flavoring agents, diluents, and other known additives to render the drug product less unpalatable or to provide additional pharmacological benefits. Diagnostic methods

[0167] Another aspect of the application relates to a method for detecting FVII protein or FVII modulating protein in blood. In certain embodiments, the method comprises detecting FVII protein or FVII modulating protein in a cell transfected with a saRNA, a polynucleotide or a composition comprising a saRNA as disclosed herein. In certain embodiments, the method disclosed herein can be used to detect a specific subset of patients suffering from a disease or disorder induced by Factor VII (FVII) protein under-expression, FVII gene mutation, low functional FVII levels in blood and / or hemophilia with inhibitors. As an alternative embodiment to the method disclosed herein, the method can be used for therapeutic efficacy or safety monitoring in patients receiving a saRNA, a nucleic acid encoding a saRNA, a polynucleotide, a composition or a medicament of the application.

[0168] In certain embodiments, a baseline measurement is obtained from a biological sample (as defined herein) obtained from the individual prior to administration of the treatment described herein. In certain embodiments, a baseline expression of the FVII gene is obtained from the biological sample prior to administration of a saRNA described herein. In certain embodiments, the biological sample is a peripheral blood mononuclear cell, plasma, serum, a portion of skin tissue or an organ.

[0169] In some embodiments, the saRNA provided herein can activate the amount of functional FVII protein in blood by at least 10% (e.g., at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 300%, at least 500%, at least 800%, at least 1000%, at least 2000%, or at least 5000%) compared to the baseline measurement described above.

[0170] In some embodiments, the saRNA exhibits a greater additive effect or synergy in treating, preventing, delaying progression, and / or ameliorating a disease caused by a mutation in the FVII gene. In some embodiments, the saRNA exhibits a greater additive effect or synergy in protecting cells involved in the pathophysiology of the disease, particularly in treating, preventing, delaying progression, and / or ameliorating hemophilia.

[0171] Another aspect of the present application relates to a method of activating / upregulating FVII gene expression in a cell, comprising administering a saRNA, a polynucleotide, or a composition of the embodiments disclosed herein. In some embodiments, the saRNA or polynucleotide or composition is introduced into a cell. In some embodiments, the saRNA of the embodiments disclosed herein is produced in a cell after introducing a nucleotide sequence encoding the saRNA into the cell. In some embodiments, the cell disclosed herein is a mammalian cell, preferably a human cell.

[0172] Another aspect of the present application relates to a method for increasing the level of FVII protein in a cell or the level of functional FVII protein in blood of a patient, comprising introducing into the cell or subject an effective amount of a saRNA, a nucleic acid encoding a saRNA, or a polynucleotide or a composition of the embodiments disclosed herein. Kits

[0173] Another aspect of the present application relates to a kit for increasing the level of FVII protein in a cell or the level of functional FVII protein in blood, comprising a saRNA disclosed herein. In certain embodiments, the kit further comprises a method for administering the saRNA to an individual. In certain embodiments, the kit is packaged in labeled packaging, and the label on the packaging shows that the saRNA or composition can be used for preventing or treating a FVII-related disease, disorder, or condition, such as a disease or condition induced by insufficient expression of coagulation factor VII (FVII), or for preventing or treating hemophilia (e.g., hemophilia with inhibitors).

[0174] As used herein, a "kit" generally refers to a package, assembly, or container (e.g., insulated container) comprising one or more components or embodiments of the present application, and / or other components related to the present application, e.g., as previously described. Any agent or component of the kit can be provided in liquid form (e.g., solution) or solid form (e.g., dry powder, frozen, etc.).

[0175] In other embodiments, the kit can provide instructions for using the kit and components and / or methods described herein, or include a website or any form of other resource indicating such information. For example, the instructions can include instructions for use, modification, mixing, dilution, preservation, assembly, storage, packaging, and / or preparation of the components and / or other components associated with the kit. In some cases, the instructions can also include instructions for delivery of the components, e.g., instructions for shipping or storage at room temperature, subzero temperature, cryogenic temperature, etc.

[0176] Another aspect of the present application relates to a kit for detecting a FVII protein or a FVII modulating protein in blood. In certain embodiments, the kit is used to detect a FVII protein or a FVII modulating protein in a cell transfected with any one or more of the saRNAs, polynucleotides, or compositions disclosed herein. The present application also provides a kit for increasing the level of a FVII protein in a cell. Particular embodiments

[0177] The present application provides the following specific embodiments: In some aspects, the present application provides a plurality of saRNAs, in particular: 1. A small activating RNA (saRNA) comprising a sense strand and an antisense strand, wherein: each of the sense or antisense strands of the saRNA comprises a contiguous oligonucleotide sequence ranging in length from 16 to 35 contiguous nucleotides, wherein each of the contiguous oligonucleotide sequences has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% homology or complementarity to an equal-length contiguous fragment of SEQ ID NO: 1437; wherein the saRNA increases expression of a FVII gene by at least 10% compared to baseline expression of the FVII gene. 2. The saRNA according to embodiment 1, wherein the contiguous stretch of SEQ ID NO: 1437 is located in a region upstream of the transcription start site (TSS) of the FVII gene, and wherein the contiguous stretch of SEQ ID NO: 1437 is located within a region selected from the group of: region -557 to -379; region -346 to -298; region -271 to -91; and region -96 to -1; and / or the contiguous stretch of SEQ ID NO: 1437 is located in a region of SEQ ID NO: 1438, SEQ ID NO: 1439, SEQ ID NO: 1440, or SEQ ID NO: 1441. 3. The saRNA according to embodiment 1 or 2, wherein the contiguous stretch of SEQ ID NO: 1437 is selected from SEQ ID NOs: 1 to 286. 4. The saRNA according to any one of embodiments 1 to 3, wherein the contiguous stretch of SEQ ID NO: 1437 is selected from SEQ ID NOs: 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 embodiments 1 to 4, wherein the contiguous oligonucleotide sequence has (1) a GC content between 40% and 70%; (2) no more than 5 consecutive identical nucleotides; (3) no more than 3 di- or tri-nucleotide repeat sequences; and / or the contiguous oligonucleotide sequence is complementary to the sense strand or the antisense strand of the saRNA. 6. The saRNA according to any one of embodiments 1 to 5, wherein the sense strand and the antisense strand each have a length of about 16 to about 35, about 17 to about 30, about 18 to about 25, or about 19 to about 22 nucleotides. 7. The saRNA according to any one of embodiments 1 to 6, wherein the sense strand and the antisense strand have at least 90% complementarity; and / or wherein the sense strand and antisense strand are located on two different nucleic acid strands or adjacent nucleic acid strands; and / or wherein the contiguous oligonucleotide sequence comprises 0, 1, 2, or 3 mismatches to the complementary region of the sense strand or the complementary region of the antisense strand. 8. The saRNA according to any one of embodiments 1 to 7, wherein the sense strand and antisense strand each comprise a complementary region, and wherein the complementary regions of the sense strand and antisense strand form a double-stranded nucleic acid structure. 9. The saRNA according to embodiment 8, wherein the sense strand or the antisense strand comprises a 3’ overhang of 1-6, 1-5, or 2-3 nucleotides in length, respectively; or the double stranded nucleic acid structure is blunt ended. 10. The saRNA according to embodiment 9, wherein at least one of the nucleotides of the overhang is a thymine deoxyribonucleotide (dT). 11. The saRNA according to any one of embodiments 1-10, wherein the contiguous oligonucleotide sequence of the sense strand has at least 75% sequence homology to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 287-858, and / or the contiguous oligonucleotide sequence of the antisense strand has at least 75% sequence homology to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 859-1430. 12. The saRNA according to any one of embodiments 1-11, wherein the contiguous oligonucleotide sequence of the sense strand is selected from the group consisting of SEQ ID NOs: 287-858, and the contiguous oligonucleotide sequence of the antisense strand is selected from the group consisting of SEQ ID NOs: 859-1430. 13. The saRNA according to any one of embodiments 1-12, wherein when the contiguous oligonucleotide sequence of the sense strand is set forth as SEQ ID NO: n, the contiguous oligonucleotide sequence of the antisense strand is set forth as SEQ ID NO: n+572 or n+858, wherein n is an integer selected from 287-572; and / or, wherein when the contiguous oligonucleotide sequence of the sense strand is set forth as SEQ ID NO: n’, the contiguous oligonucleotide sequence of the antisense strand is set forth as SEQ ID NO: n’+286, wherein n’ is an integer selected from 573-858. 14. The saRNA according to any one of embodiments 1-13, wherein the sense strand comprises a contiguous nucleotide sequence selected from the group consisting of 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 comprises a contiguous nucleotide sequence selected from the group consisting of 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 saRNA according to any one of embodiments 1-14, wherein the sense strand comprises the nucleotide sequence of SEQ ID NO: m, the antisense strand comprises the nucleotide sequence of SEQ ID NO: m+572, and wherein m is selected from the group consisting of 323, 341, 363, 372, 402, 403, 406, 410, 431, 432, 437, 463, 474, 491, 493, 504, 514, 527, 528; and / or wherein the sense strand comprises the nucleotide sequence of SEQ ID NO: m', the antisense strand comprises the nucleotide sequence of SEQ ID NO: m+858, and wherein m is selected from the group consisting of 296, 297, 315, 374, 407, 410, 442, 491, 493, 504, 514, 527, 545, 547, 560; and / or wherein the sense strand comprises the nucleotide sequence of SEQ ID NO: m", the antisense strand comprises the nucleotide sequence of SEQ ID NO: m" +286, and wherein m is selected from the group consisting of 581, 582, 599, 627, 641, 653, 654, 658, 679, 749, 812, 813, and 824. 16. The saRNA of any one of embodiments 1 to 15, wherein the sense strand comprises a contiguous nucleotide sequence selected from the group consisting of SEQ ID NOs: 1442, 1444, 1446, 1448, 1450, 1452, 1454, 1456, 1458, 1460, 1462, 1464, 1466, 1468, 1470, 1472, 1474, and 1476; and / or the antisense strand comprises a contiguous nucleotide sequence selected from the group consisting of SEQ ID NOs: 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 strand, as shown in Table 11. 17. The saRNA of any one 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 nucleotides are nucleotides having at least one of the following modifications: a) modification of the phosphodiester bonds connecting nucleotides in the saRNA nucleotide sequence; b) modification of the 2'-OH of ribose in the saRNA nucleotide sequence; and c) modification of the bases in the saRNA nucleotide sequence. 19. The saRNA according to embodiment 18, wherein the modification of the phosphodiester bond linking the nucleotides is selected from phosphorothioate modification and boranophosphate modification; and / or the modification of 2'-OH is selected from 2'-fluoro modification, 2'-oxymethyl modification, 2'-oxyethylenemethoxy modification, 2,4'-dinitrophenol modification, 2'-amino modification and 2'-deoxy modification; and / or the modification of the base is selected from 5'-bromouracil modification, 5'-iodouracil modification, N-methyluracil modification and 2,6-diaminopurine modification. 20. The saRNA of embodiment 17, wherein at least one nucleotide of the saRNA is a locked nucleic acid, an abasic nucleotide, a 2'-amino modified nucleotide, a 2'-alkyl modified nucleotide, a morpholino nucleotide, a phosphoramide, or a nucleotide containing a non-natural base; and / or wherein the chemical modification of the at least one chemically modified nucleotide is the addition of an (E)-vinylphosphonate moiety to the 5' end of the sense strand or the antisense strand. In some aspects, the present application provides oligonucleotide modulators, particularly: 21. An oligonucleotide modulator comprising one or more saRNAs according to any one of embodiments 1 to 20. 22. The oligonucleotide modulator of embodiment 21, further comprising one or more moieties or components conjugated, combined or mixed with the one or more saRNAs. 23. The oligonucleotide modulator of embodiment 21, wherein the sense and / or antisense strand of the saRNA is conjugated to one or more conjugation moieties selected from the group consisting of a lipid, a fatty acid (e.g. comprising a carbon chain having 4-30, 12-24, 16-22 carbon atoms), a fluorophore, a ligand, a sugar, a peptide and an antibody. 24. The oligonucleotide modulator of embodiment 22, wherein the conjugation moieties are each respectively selected from the group consisting of a lipid, a cell penetrating peptide, a polyethylene glycol, an alkaloid, a tryptamine, a benzimidazole, a quinolone, an amino acid, a cholesterol, a glucose, a N-acetylgalactosamine and any combination thereof. 25. The oligonucleotide modulator of embodiment 22, wherein the conjugation moieties are respectively selected from the group consisting of S9, tC2, tC2x6, C5x5 and any combination thereof, wherein, represents a carrier. 26. The oligonucleotide modulator of embodiment 21, wherein the oligonucleotide modulator further comprises a saRNA conjugated or bound to one or more other active moieties for the treatment of a FVII related disease, disorder or condition, wherein the one or more other active moieties are respectively selected from the group consisting of a small molecule chemical moiety, a polypeptide and an antibody. In some aspects, the present application provides a plurality of target sites, in particular: 27. An isolated oligonucleotide, wherein (a) the isolated oligonucleotide is an oligonucleotide sequence of 16 to 35 contiguous nucleotides having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% homology to an equal length contiguous fragment of SEQ ID NO: 1437; or wherein (b) the isolated oligonucleotide is an oligonucleotide sequence of 16 to 35 contiguous nucleotides having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% complementarity to an equal length contiguous fragment of SEQ ID NO: 1437. 28. The isolated oligonucleotide of embodiment 27, wherein an equal length contiguous fragment of SEQ ID NO: 1437 is in a region upstream of the transcription start site (TSS) of the FVII gene, and wherein an equal length contiguous fragment of SEQ ID NO: 1437 is in a region selected from the group of: Region -557 to -379; Region -346 to -298; Region -271 to -91 ; and Region -96 to -1 ; and / or an equal length contiguous fragment of SEQ ID NO: 1437 is in a region of SEQ ID NO: 1438, SEQ ID NO: 1439, SEQ ID NO: 1440, or SEQ ID NO: 1441. 29. The isolated oligonucleotide of embodiment 27, wherein the isolated oligonucleotide (a) is a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1 to 286, such as selected from the group consisting of SEQ ID NOs: 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 the isolated oligonucleotide (b) is a nucleic acid sequence complementary to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1 to 286, such as complementary to SEQ ID NOs: 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. An oligonucleotide complex comprising: the antisense strand of the saRNA of any one of embodiments 1 to 20 and the sense strand of the isolated oligonucleotide (a) of any one of embodiments 27 to 29; or the sense strand of the saRNA of any one of embodiments 1 to 20 and the antisense strand of the isolated oligonucleotide (b) of any one of embodiments 27 to 29. 31. The oligonucleotide complex of embodiment 30, wherein the oligonucleotide complex activates expression of the FVII gene at least 10% above baseline expression of the FVII gene. In some aspects, the present application provides a plurality of hotspots, in particular: 32. An isolated nucleic acid molecule, wherein at least 20% (such as at least 30%, at least 40%, at least 45%, at least 50%) of saRNAs designed to target the sequence of the isolated nucleic acid molecule are designed to activate FVII gene expression by at least 10%, wherein the designed saRNAs have (1) a GC content between 40% and 70%; (2) no more than 5 consecutive identical nucleotides; (3) no more than 3 di- or tri-nucleotide repeat sequences. 33. An isolated nucleic acid molecule, the sequence of which corresponds to a region upstream of the transcription start site of a FVII gene, wherein the sequence is located upstream of the transcription start site of a FVII gene in a region selected from the group consisting of region -557 to -379, region -346 to -298, region -271 to -91 and region -96 to -1, or any sub-region within any of the aforementioned regions. 34. An isolated nucleic acid molecule, the sequence of which is selected from the group consisting of SEQ ID NOs: 1438 to 1441. 35. The isolated nucleic acid molecule according to any one of embodiments 32 to 34, comprising the isolated oligonucleotide according to embodiment 27(a) or 29(a). 36. The isolated nucleic acid molecule according to any one of embodiments 32 to 34, wherein the saRNAs designed to target the nucleic acid molecule are as defined in any one of embodiments 1 to 20; and / or the desired saRNAs are blunt-ended or have overhanging ends, and / or are unmodified or chemically modified. In some aspects, the present application provides a plurality of DNA, in particular: 37. An isolated polynucleotide encoding the saRNA according to any one of embodiments 1 to 20 or the oligonucleotide modulator according to any one of embodiments 21 to 26. 38. The isolated polynucleotide according to embodiment 37, wherein the isolated polynucleotide is DNA. 39. A vector comprising the isolated polynucleotide according to any one of embodiments 37 to 38. In some aspects, the present application provides a plurality of cells, in particular: 40. A host cell comprising the saRNA according to any one of embodiments 1 to 20, the oligonucleotide modulator according to any one of embodiments 21 to 26, the isolated polynucleotide according to any one of embodiments 37 to 38 or the vector according to embodiment 39. In some aspects, the present application provides a plurality of products, in particular: 41. A product comprising the saRNA of any one of embodiments 1-20, the oligonucleotide modulator of any one of embodiments 21-26, the isolated oligonucleotide of any one of embodiments 27-29, the oligonucleotide complex of any one of embodiments 30-31, the isolated nucleic acid molecule of any one of embodiments 32-36, the isolated polynucleotide of any one of embodiments 37-38, the vector of embodiment 39, or the host cell of embodiment 40. 42. The product according to embodiment 41, wherein the product is used for preventing, treating or diagnosing a FVII-associated disease, disorder or condition (such as a FVII deficiency-associated disease or haemophilia), for designing and / or screening saRNAs. In some aspects, the present application provides a pharmaceutical composition, in particular: 43. A composition comprising the saRNA of any one of embodiments 1-20, the oligonucleotide modulator of any one of embodiments 21-26, or the isolated polynucleotide of embodiments 37-38, and optionally a pharmaceutically acceptable carrier. 44. The composition according to embodiment 43, wherein the composition comprises 0.001-1600 nM, such as 1-150 nM, of the saRNA. In some aspects, the present application provides products for and / or for use in the manufacture of a medicament for activating / upregulating FVII gene expression, in particular: In some aspects, the present application provides products for and / or for use in the manufacture of a medicament for activating / upregulating FVII gene expression, in particular: 45. A product for activating / upregulating expression of a FVII gene in a cell, wherein the product can activate expression of the FVII gene by at least 10% compared to baseline expression of the FVII gene, and wherein the product comprises an active substance selected from one or more of the saRNA of any one of embodiments 1-20 or the oligonucleotide modulator of any one of embodiments 21-26, the isolated polynucleotide of any one of embodiments 37-38, the vector of embodiment 39, or the composition of any one of embodiments 43-44. 46. Use of an active substance for the manufacture of a product for activating / upregulating expression of a FVII gene in a cell, wherein the product can activate expression of the FVII gene by at least 10% compared to baseline expression of the FVII gene, and wherein the active substance is selected from one or more of the saRNA of any one of embodiments 1-20 or the oligonucleotide modulator of any one of embodiments 21-26, the isolated polynucleotide of any one of embodiments 37-38, the vector of embodiment 39, or the composition of any one of embodiments 43-44. 47. A method of activating / upregulating expression of a FVII gene in a cell, wherein the product can activate expression of the FVII gene by at least 10% compared to baseline expression of the FVII gene, and wherein the method comprises administering to the cell an effective amount of an active substance, wherein the active substance is selected from one or more of the saRNAs of any one of embodiments 1-20 or the oligonucleotide modulators of any one of embodiments 21-26, the isolated polynucleotide of any one of embodiments 37-38, the vector of embodiment 39, or the composition of any one of embodiments 43-44. 48. The product for activating / upregulating expression of a FVII gene in a cell according to embodiment 45, the use according to embodiment 46, or the method of embodiment 47, wherein the active substance is introduced into a cell; and / or wherein the cell is an in vitro, ex vivo, or in vivo cell; and / or wherein the cell is a mammalian cell. 49. The product, use, or method according to embodiment 48, wherein the active substance is introduced into the cell by: 1) composing the active substance with a physiologically acceptable or pharmaceutically acceptable carrier, such as one or more selected from the group consisting of an aqueous carrier, a liposome, a polymeric molecule, a polypeptide, and an antibody, and / or 2) conjugating the active substance to one or more conjugating moieties, such as one or more (e.g. two conjugating moieties, wherein one is a lipid and the other is an N-acetylgalactosamine) selected from the group consisting of a lipid, a cell penetrating peptide, a polyethylene glycol, a biogenic alkaloid, a tryptamine, a benzimidazole, a quinolone, an amino acid, a cholesterol, a glucose, and an N-acetylgalactosamine, and any combination thereof. 50. The product, use, or method according to embodiment 49, wherein the conjugating moiety is one or more of S9, tC2, tC2x6, and C5x5, or any combination thereof (such as C5x5 and tC2x6): wherein, represents a carrier. 51. The product, use, or method according to embodiment 49, wherein the conjugating moiety is a lipid selected from a fatty acid having a carbon chain length of 4-30, 12-24, 16-22 carbon atoms; and / or wherein the conjugating moiety is a fatty acid having a carbon chain length of 4-30, 12-24, 16-22, or 16 carbon atoms; and / or wherein the conjugating moiety is derived from a fluorophore, a ligand, a sugar, a peptide, and an antibody, respectively. 52. The product for activating / upregulating FVII gene expression in a cell according to embodiment 45, the use according to embodiment 46 or the method according to embodiment 47, wherein the cell is from a patient suffering from or at risk of suffering from a disease, disorder or condition that can be prevented or treated by activating / upregulating FVII gene expression, e.g. a haemophilia (such as haemophilia with inhibitors) or Glanzmann Thrombasthenia (GT), wherein a sufficient amount of the active substance is administered to prevent or treat the disease, disorder or condition. In some aspects, the present application provides products for and / or for use in the manufacture of a medicament for activating / upregulating FVII gene expression, in particular: In some aspects, the present application provides diagnostic methods, in particular: 53. A product for preventing or treating a FVII related disease, disorder or condition, wherein the product comprises an active substance selected from one or more of the saRNAs according to any one of embodiments 1-20 or the oligonucleotide modulators according to any one of embodiments 21-26, the isolated polynucleotide according to any one of embodiments 37-38, the vector according to embodiment 39 or the composition according to any one of embodiments 43-44. 54. Use of an active substance in the manufacture of a product for preventing or treating a FVII related disease or disorder or condition, wherein the active substance is selected from one or more of the saRNAs according to any one of embodiments 1-20 or the oligonucleotide modulators according to any one of embodiments 21-26, the isolated polynucleotide according to any one of embodiments 37-38, the vector according to embodiment 39 or the composition according to any one of embodiments 43-44. 55. A method for preventing or treating a FVII related disease, disorder or condition, wherein the method comprises administering to a subject an effective amount of an active substance, wherein the active substance is selected from one or more of the saRNAs according to any one of embodiments 1-20 or the oligonucleotide modulators according to any one of embodiments 21-26, the isolated polynucleotide according to any one of embodiments 37-38, the vector according to embodiment 39 or the composition according to any one of embodiments 43-44. 56. The product according to embodiment 53, the use according to embodiment 54 or the method according to embodiment 55, wherein the subject is a mammal (such as a human), preferably a mammal (such as a human) suffering from or at risk of suffering from a disease, disorder or condition that can be prevented or treated by activating / upregulating Factor VII (FVII) protein expression, FVII gene mutation, low functional FVII levels in blood and / or other disease, disorder or condition that can be prevented or treated by activating / upregulating FVII levels (such as haemophilia with inhibitors). 57. The product of embodiment 53, use of embodiment 54, or method of embodiment 55, wherein the active ingredient is administered to the individual by one or more of the following routes of administration: parenteral infusion, oral administration, intranasal administration, inhalation administration, vaginal administration, and rectal administration. 58. The product of embodiment 53, use of embodiment 54, or method of embodiment 55, wherein the active ingredient is administered to the individual by one or more of the following routes of administration: intrathecal, intramuscular, intravenous, intraarterial, intraperitoneal, intravesical, intracerebroventricular, intravitreal, and subcutaneous administration. 59. The product of embodiment 53, use of embodiment 54, or method of embodiment 55, wherein the expression of FVII gene mRNA in the individual is activated / upregulated by at least 10% as compared to baseline expression of the FVII gene; and / or, wherein the level of FVII protein in the individual is increased by at least 10% as compared to baseline level of FVII protein; and / or, wherein the FVII-related disease, disorder, or condition comprises congenital FVII deficiency (Alexander disease), acquired FVII deficiency (AFVIID), hemophilia (such as hemophilia with inhibitors), and Glanzmann Thrombasthenia (GT). In some aspects, the present application provides kits, in particular: 60. A method of diagnosing a FVII-related disease or disorder by detecting FVII protein or FVII modulated protein in the cell of embodiment 40. In some aspects, the present application provides methods for obtaining saRNA, in particular: 61. A kit for use in practicing the method of embodiment 60, comprising the saRNA of any one of embodiments 1-20 or the oligonucleotide modulator of any one of embodiments 21-26. 62. The kit of embodiment 61, wherein the instructions for use comprise a method of administering the saRNA of any one of embodiments 1-20 or the oligonucleotide modulator of any one of embodiments 21-26 to an individual. 63. A kit comprising the saRNA of any one of embodiments 1-20 or the oligonucleotide modulator of any one of embodiments 21-26, the isolated polynucleotide of any one of embodiments 37-38, the vector of embodiment 39, or the composition of any one of embodiments 43-44, packaged in labeled packaging, the label on the packaging indicating that the saRNA, the isolated polynucleotide, the vector, or the composition can be used to prevent or treat a disease, disorder, or condition induced by insufficient expression of coagulation factor VII (FVII), or to prevent or treat hemophilia. 64. A kit for detecting a FVII protein or a FVII modulating protein in the cell of embodiment 40. In some aspects, the present application provides a plurality of isolated double-stranded functional RNAs having blunt-end structure, in particular: 65. A method for obtaining a saRNA capable of upregulating FVII gene expression at least 10% compared to baseline expression of the FVII gene, wherein the method comprises: (a) synthesizing a saRNA comprising a sense strand and an antisense strand, wherein each of the sense strand or the antisense strand comprises a contiguous oligonucleotide sequence of 16 to 35 contiguous nucleotides, wherein the contiguous oligonucleotide sequence has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% homology or complementarity to an equal-length contiguous fragment of SEQ ID NO: 1437, respectively, and wherein the contiguous oligonucleotide sequence has (1) a GC content between 40% to 70%; (2) no more than 5 consecutive identical nucleotides; (3) no more than 3 di- or trinucleotide repeats; and (b) determining the ability of the saRNA to upregulate FVII gene expression. 66. A method for obtaining a saRNA capable of upregulating FVII gene expression at least 10% compared to baseline expression of the FVII gene, wherein the method comprises: (a) synthesizing a saRNA targeting a contiguous fragment of SEQ ID NO: 1437, wherein the saRNA has (1) a GC content between 40% to 70%; (2) no more than 5 consecutive identical nucleotides; (3) no more than 3 di- or trinucleotide repeats; and (b) determining the ability of the saRNA to upregulate FVII gene expression. 67. The method of embodiment 65 or 66, wherein the contiguous fragment of SEQ ID NO: 1437 is located in a region upstream of the transcription start site (TSS) of the FVII gene, and wherein the contiguous fragment of SEQ ID NO: 1437 is located within a region selected from the group of: Region -557 to -379; Region -346 to -298; Region -271 to -91; and Region -96 to -1; and / or the contiguous fragment of SEQ ID NO: 1437 is located within a region of SEQ ID NO: 1438, SEQ ID NO: 1439, SEQ ID NO: 1440, or SEQ ID NO: 1441. 68. The method of embodiment 65 or 66, wherein the contiguous fragment of SEQ ID NO: 1437 is selected from SEQ ID NO: 1 to 286. Examples 69. A double-stranded functional saRNA molecule, wherein both ends of the functional RNA molecule are blunt ends; and / or wherein each strand of the functional RNA molecule is about 16 to about 35, about 17 to about 30, about 18 to about 25, or about 19 to about 22 nucleotides in length. 70. The double-stranded functional RNA molecule of embodiment 69, wherein the functional RNA molecule targets and modulates FVII gene expression. 71. An oligonucleotide agent comprising one or more saRNAs of any of embodiments 69-70. 72. The oligonucleotide agent of embodiment 71, further comprising one or more moieties or components conjugated to the one or more agents. 73. The oligonucleotide agent of embodiment 72, wherein the sense and / or antisense strand of the functional saRNA is conjugated to one or more conjugating moieties selected from a lipid, a fatty acid (e.g., comprising a carbon chain having 4-30, 12-24, 16-22 carbon atoms), a fluorophore, a ligand, a sugar, a peptide, and an antibody. 74. The oligonucleotide agent of embodiment 73, wherein the conjugating moieties are selected from a lipid, a cell-penetrating peptide, a polyethylene glycol, an alkaloid, a tryptamine, a benzimidazole, a quinolone, an amino acid, a cholesterol, a glucose, a N-acetylgalactosamine, and any combination thereof, respectively. 75. The oligonucleotide agent of embodiment 74, wherein the conjugating moieties are selected from S9, tC2, tC2x6, C5x5, and any combination thereof, respectively. wherein, denotes a carrier.

[0178] While specific embodiments of active substances (e.g., saRNAs), products, compositions, and methods have been discussed herein, many variations of the present application will become apparent to those of ordinary skill in the art after a study of the specifications and claims. The entire scope of the application should be determined by reference to the entire scope of the claims and their equivalents, along with the specification and these variations. Materials and Methods

[0179] The present application will be further described with reference to specific examples and the accompanying drawings. It is to be understood that these examples are only intended to illustrate the present application and are not intended to limit the scope of the present application. In the following examples, the research methods without specific conditions generally conform to the conventional conditions, such as those described in Sambrook et al. in Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or the conditions recommended by the manufacturer. Example 1: Design and synthesis of saRNAs targeting the human FVII promoter saRNA synthesis (1) Single-strand synthesis

[0180] Single-strand oligonucleotides were synthesized by solid-phase synthesis technique on a K&A DNA synthesizer (K&A Laborgeraete GbR, chaafheim, Germany).

[0181] The starting material is a commercially available general solid-phase carrier or a special solid-phase carrier, or a synthetic material as disclosed hereinbefore. Generally, phosphoramidite monomers (0.1 M acetonitrile or dichloromethane solution) including various linkers and conjugates are added to the solid carrier in the DNA synthesizer in turn to generate the desired full-length oligonucleotide.

[0182] Amide addition: Each amide addition cycle includes four chemical reactions, namely, detritylation, coupling, oxidation / thiolation, and capping. In the first step, detritylation is performed using 3% dichloroacetic acid (DCA) in DCM for 45 seconds. In the second step, all the phosphoramidites are subjected to phosphoramidite coupling at 12 equivalents for 6 minutes. In the third step, 0.02 M iodonium oxidation is added in THF:pyridine:water (70:20:10, v / v / v) for 1 minute; if a phosphorothioate modification is required, thiolation with 0.1 M hydroxylamine in pyridine:ACN (50:50, v / v) is used instead of oxidation for 3 minutes. In the fourth step, capping is performed using THF:acetic anhydride:pyridine (80:10:10, v / v / v) (CAP A) and N-methylimidazole:THF (10:90, v / v) (CAP B) for 20 seconds. The cycle of the four chemical reactions depends on the length of the individual oligonucleotide.

[0183] Deprotection I (nucleobase deprotection): After completion of synthesis, the solid-phase carrier is transferred to a screw-capped microcentrifuge tube. For a 1 pmol synthesis scale, a mixture of 1 ml methylamine and ammonium hydroxide is added. The tube containing the solid-phase carrier is heated in an oven at 60-65 °C for 15 minutes, and then cooled to room temperature. The lysis solution is collected and evaporated to dryness in a speedvac to obtain the single-strand oligonucleotide crude product.

[0184] Deprotection II (removal of 2'-TBDMS group): If the crude RNA oligonucleotide still carries the 2'-TBDMS group, it is dissolved in 0.1 ml of DMSO. After addition of 1 ml of triethylamine trifluoride, the tube is capped, the mixture is shaken vigorously to ensure complete dissolution, and then heated in an oven at 65°C for 15 minutes. The test tube is removed from the oven and cooled to room temperature. The solution containing the completely desilylated oligonucleotide is cooled on dry ice. 2 ml of ice-cold n-butanol (-20°C) are carefully added in portions of 0.5 ml to precipitate the oligonucleotide. The precipitate is filtered, washed with 1 ml of ice-cold n-butanol, and then dissolved in 0.01 M tris(hydroxymethyl)aminomethanol hydrochloride buffer. (2) Single strand purification

[0185] Oligonucleotide purification was performed on an AKTA Explorer 10 equipped with a Source 15Q 4.6 / 100 PE column using the following conditions: Buffer A: (10 mM Tris-HCl, 1 mM EDTA, pH 7.5), B: (10 mM Tris-HCl, 1 mM EDTA, 2 M NaCl, pH 7.5), gradient: 10% B to 60% B in 25 minutes, and flow rate: 1 ml / min. The pure oligonucleotide was collected and desalted by a HiPrep 26 / 10 desalting column. (3) Annealing to form double strand

[0186] For double strand, after generating the desalted purified single strand solution, the sense strand and the antisense strand were mixed in equal molar concentration in equal volume in a tube. The test tube was placed in a heating block at 95°C for 5 minutes and cooled to room temperature. Subsequently, the double strand thus obtained was lyophilized into a powder. Conjugation moiety

[0187] The conjugation moiety can be synthesized by methods known in the art, for example, the synthesis process of tC2, tC2x6 and C5x5, the entire contents of WO2024002046A1 are incorporated herein. Cell culture and treatment

[0188] Human hepatocellular carcinoma Huh-7 (JCRB0403, Nanjing Kebai Biotechnology Co., Ltd., China) and HepG2 cells (SCSP-510, National Model and Specialty Experimental Cell Resource Bank, China) were cultured at 37 ° C, 5% CO2 in modified DMEM medium (Gibco, ThermoFisher Scientific, Carlsbad, California) supplemented with 10% calf serum (Sigma-Aldrich) and 1% penicillin / streptomycin (Gibco). Following the reverse transfection protocol, saRNA was transfected into Huh-7 cells and HepG2 cells at a final concentration of 10, 25 nM or the specified concentrations (i.e., 0.1, 0.39, 1.56, 6.25, 25, 100, and 400 nM) using RNAiMAX (Invitrogen, Carlsbad, California). Cells were transfected in the absence of oligonucleotides as mock treatment. dsCon2, dsCon2M6v, and dsCon2M3v were used as non-targeting duplex controls. RD-13516 is a duplex 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. RNA isolation and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) (1) RNA isolation and one-step RT-qPCR

[0189] After transfection, the culture medium was discarded and the cells were washed once with 150 μL PBS per well. After discarding PBS, 100 μL cell lysis buffer (Power Green Cells-to-Ct TM The mixture was incubated at room temperature for 5 minutes. 0.5 μl of cell lysate was taken from each well and PCR was performed on a Roche Lightcycler 480 real-time PCR instrument (Roche, reference number: 4729749001, USA) using the One Step TB PrimeScript TM RT-qPCR analysis was performed using the RT-PCR Kit II (Takara, RR086A, Shlga, Japan). PCR pre-processing was performed using the Bravo automated liquid handling platform (Agilent, USA). Each transfected sample was amplified in triplicate. The PCR reaction mixture is shown in Table 2. Table 2. Composition of PCR reaction mixture

[0190] The reaction conditions were as follows: reverse transcription reaction (1st stage): 42°C for 5 min, 95°C for 10 sec; PCR reaction (2nd stage): 95°C for 5 sec, 59°C for 20 sec, 72°C for 10 sec, 40 amplification cycles; melting curve (3rd stage). Human FVII gene amplification was performed as a target gene. Human reference genes (PGK1 and SDHA) were also amplified and their geometric mean was used as an internal control for RNA load. Primer sequences are shown in Table 3. Table 3. Primer sequences for RT-qPCR detection (2) RNA isolation and two-step RT-qPCR

[0191] For quantification of mRNA expression in cells, total cellular RNA was isolated from treated cells using the RNeasy Plus Mini Kit (Qiagen, Hilden, Germany) according to its manual. The PrimeScript TM RT-qPCR detection RT kit (Takara, RR047A, Shlga, Japan) was used to reverse transcribe the obtained RNA (about 1 pg) into cDNA. The obtained cDNA was amplified using TB Premix Ex Taq™ II (Takara, RR820A, Shlga, Japan) reagents and primers specific for amplification of the target gene of interest in a Roche LightCycler 480 Multiwell Plate 384 (Roche, ref: 4729749001, USA).

[0192] The reaction conditions were as follows: reverse transcription reaction (1st stage): 42°C for 5 min, 95°C for 10 sec; PCR reaction (2nd stage): 95°C for 5 sec, 60°C for 30 sec, 72°C for 10 sec; 40 amplification cycles; melting curve (3rd stage). PCR reaction conditions are shown in Tables 4 and 5. Table 4: RT reaction Table 5: RT-qPCR reaction

[0193] To calculate the relative expression level of FVII (target gene) mRNA in saRNA transfected samples relative to control treatment (mock) (E rel ), the Ct values of the target gene and two internal control genes were inserted into equation 1, where CtT mCtT is the Ct value of the target gene in the mock-treated sample; CtT s CtR1 is the Ct value of the target gene from the saRNA-treated sample; CtR1 m CtR1 is the Ct value of the internal control gene 1 in the mock-treated sample; CtR1 s CtR2 is the Ct value of the internal control gene 1 from the saRNA-treated sample; CtR2 m CtR2 is the Ct value of the internal control gene 2 from the mock-treated sample; CtR2 s CtR2 is the Ct value of the internal control gene 2 from the saRNA-treated sample. Animal experiments

[0194] Cynomolgus monkeys (male and female) were purchased from Kunming KBI Biotechnology Co., Ltd. (KBI). All animal procedures were performed by certified laboratory personnel in accordance with protocols approved by the Institutional Animal Care and Use Committee in compliance with local and national regulations. Animal dosing formulations were prepared fresh prior to use by dissolving lyophilized oligonucleotides in normal saline to prepare stock solutions, which were diluted to the intended dosing concentration. Animals were randomly assigned to each study group according to body weight and gender. Coagulation function test

[0195] To assess coagulation function in non-human primates, cynomolgus monkeys were administered CM-saRNA. One group of cynomolgus monkeys was given the specified CM-saRNA by subcutaneous injection at day 0 (10 mg / kg) and day 7 (10 mg / kg). Another group of cynomolgus monkeys was given siRNA control (5 mg / kg) by subcutaneous injection at day 0. Cynomolgus monkeys injected with normal saline only served as vehicle controls. Monkey plasma samples were collected at day 21 and day 28 after the first dose. 1.8 ml of blood was collected from each cynomolgus monkey into plastic blood collection tubes containing sodium citrate as an anticoagulant, and the plasma samples were prepared by mixing and centrifuging at 2500 g for 10 minutes within 30 minutes after collection. The samples were immediately detected using a fully automatic coagulation analyzer (BCA-700, Getein Biotech). Example 2: High-throughput screening of saRNAs targeting the human FVII promoter

[0196] The coding strand sequence of the human FVII gene promoter was retrieved from the UCSC Genome Browser database (SEQ ID NO: 1437, as shown in Table 6). It consists of 600 nucleotides, ranging from -1 bp to -600 bp relative to the transcription start site (TSS). Table 6. Putative human FVII promoter sequence (5'-3') (SEQ ID NO: 1437)

[0197] By simple 1 bp stepping within the 600 bp promoter region, 579 possible target sites of 22 nucleotides (nt) in length were identified in the promoter sequence and 286 of them were selected as targets for saRNAs according to the following criteria: (i) GC content between 40% and 70%, (ii) no more than 5 consecutive identical nucleotides, and (iii) less than 3 di- or trinucleotide repeats. For the 286 selected target sequences, a total of 858 duplexes were designed (see Table 1.1). Figure 1

[0198] To identify saRNAs capable of up-regulating FVII mRNA expression, Huh-7 cells were transfected with each of the above saRNAs at a concentration of 25 nM, treated for 72 hours, and then subjected to gene expression analysis by one-step RT-qPCR. A non-targeting duplex (dsCon2) was used as a non-targeting control, while an siRNA targeting the human FVII transcript (i.e., RD-13516) was used as a transfection control to monitor knockdown by RNA interference (RNAi).

[0199] The results show that among the 858 saRNAs tested, 133 (15.5%), 187 (21.8%), and 84 (9.8%) exhibited high (≥ 1.5-fold), moderate (1.2-1.5-fold), and low (1.1-1.2-fold) activation of FVII expression, respectively. The results grouped by high, moderate, and low activation are summarized in Table 7. Table 7. Summary of saRNAs activities inducing FVII mRNA expression screened in Huh-7 cells

[0200] The summary of the relative changes in FVII expression caused by saRNA treatment is shown in Table 1.2 and plotted in Figure 2 .

[0201] Upon sorting all saRNAs according to their target site positions on the human FVII promoter, it is clearly visible that functional saRNAs are clustered within discrete regions or saRNA "hotspot regions" where the target sequences of functional saRNAs are enriched Figure 2 ). They are the regions -557 to -379 (H1), -346 to -298 (H2), -271 to -91 (H3), and -96 to -1 (H4) Example 3: Confirmation of screening results and dose-dependent induction of FVII mRNA in Huh-7 cells by saRNAs ) relative to the TSS. The corresponding DNA sequences of the hotspot regions are shown in Table 8. Table 8. Human FVII saRNA hotspot regions and their sequences Figure 3

[0202] To confirm the screening results, 47 functional FVII saRNAs were transfected into Huh-7 cells at 7 designated concentrations (i.e. 0.1, 0.39, 1.56, 6.25, 25, 100 and 400 nM) for 72 hours and dose response curves were generated by RT-qPCR (Figure 9). Example 4: In vitro activation of FVII saRNAs and chemically modified saRNAs (CM-saRNAs) in HepG2 and Huh-7 cells For the saRNAs that showed dose-dependent induction of FVII mRNA, the EC 50 values and E max levels could be extrapolated to determine the induction capacity of each of the saRNAs in the context of maximum activity (see Table 9). Table 9. FVII mRNA levels in Huh-7 cells after saRNA induction Note: SEM represents standard error of the mean. Figure 4 Figures 5A-5B

[0203] To assess the activation of FVII protein expression by FVII saRNAs, 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 transfected into HepG2 cells at a concentration of 25 nM for 4 days. FVII protein levels were determined by Western blotting using an antibody against human FVII protein. Figures 6A-6B FVII protein levels in HepG2 cells after saRNA treatment are shown. Table 10 summarizes FVII protein levels in HepG2 cells after saRNA treatment. Table 10. FVII protein levels in HepG2 cells after FVII saRNA treatment Note: SEM represents standard error of the mean.

[0204] To assess the activation of FVII mRNA expression by FVIICM-saRNAs, ten designated FVIICM-saRNAs (i.e. RD-16036, RD-16037, RD-16038, RD-16027, RD-16028, RD-16041, RD-16052, RD-16055, RD-16134 and RD-16035) (see Table 11) were transfected at a level of 10 nM into HepG2 cells for 3 days. Eleven designated FVIICM-saRNAs (i.e. RD-16036, RD-16037, RD-16038, RD-16027, RD-16028, RD-16041, RD-16052, RD-16055, RD-16134, RD-16044 and RD-16035) (see Table 11) were transfected at a level of 25 nM into Huh-7 cells for 3 days. RD-15120 is a chemically modified siRNA targeting FVII gene, transfected as a silent siRNA control. FVII mRNA levels were quantitatively determined by two-step RT-qPCR. Example 5: FVII CM-saRNA treatment improves coagulation function in non-human primates FVII mRNA levels in HepG2 and Huh-7 cells after CM-saRNA treatment are shown. Table 12 summarizes FVII mRNA levels in HepG2 and Huh-7 cells after CM-saRNA treatment. Table 11. Oligonucleotide sequences and compositions Note: Capital, RNA; *, phosphorothioate (PS) backbone modification; f, 2’-fluoro; m, 2’-O-methyl (2’-OMe); Vp, 5’-(E)-vinylphosphonate; dC, cytosine deoxyribonucleic acid; dA, adenosine deoxyribonucleic acid; tC2x6 compound see WO2024002046A1 application. Table 12. FVII mRNA levels in HepG2 and Huh-7 cells after FVIICM-saRNA treatment Note: SEM represents standard error of the mean. / indicates not detected.

[0205] To further assess the activation of FVII protein expression by FVIICM-saRNAs, seven designated FVIICM-saRNAs (i.e., RD-16012, RD-16036, RD-16013, RD-16017, RD-16029, RD-16041, and RD-16120) were transfected into HepG2 cells at a transfection concentration of 25 nM for 4 days. Seven designated FVIICM-saRNAs (i.e., RD-16012, RD-16024, RD-16036, RD-16017, RD-16029, RD-16041, and RD-16120) were transfected into Huh-7 cells at a transfection concentration of 25 nM for 4 days. RD-15120, a chemically modified siRNA targeting FVII gene, was transfected as a silent siRNA control. FVII protein levels were determined by Western blotting using an antibody against human FVII protein. As shown in Table 13, all CM-saRNAs induced more than 1.3-fold. The FVII protein levels in HepG2 and Huh-7 cells after CM-saRNA treatment are summarized in Table 13. Doctrine of Equivalents Table 13. FVII protein levels in HepG2 and Huh-7 cells after FVIICM-saRNA treatment Table 13. FVII protein levels in HepG2 and Huh-7 cells after FVIICM-saRNA treatment Note: SEM represents standard error of the mean. / indicates not detected. Incorporation by Reference

[0206] To assess the coagulation function in non-human primates cynomolgus monkeys, CM-saRNAs were administered to cynomolgus monkeys. One group of monkeys (1 male, about 4.1-4.9 years old, weighing 3.33-4.68 kg; 1 female, about 4.2-4.8 years old, weighing 2.54-3.09 kg) was administered a designated CM-saRNA (i.e., RD-17272) by subcutaneous injection at day 0 (10 mg / kg) and day 7 (10 mg / kg), respectively. Another group of monkeys (1 male, about 4.1-4.9 years old, weighing 3.33-4.68 kg; 1 female, about 4.2-4.8 years old, weighing 2.54-3.09 kg) was administered RD-16985 (5 mg / kg) by subcutaneous injection at day 0 as a silent siRNA control. Cynomolgus monkeys injected with normal saline only served as vehicle controls. Monkey plasma samples were collected at day 21 and day 28 after the first administration. At day 21 and day 28 after the first administration, the prothrombin time (seconds, s) of monkey plasma was determined by a fully automatic coagulation analyzer. At day 28 after the first administration, the thrombin time (seconds, s) of monkey plasma was determined by a fully automatic coagulation analyzer. The prothrombin time and thrombin time are summarized in Table 14. Table 14. Prothrombin time and thrombin time of monkey plasma. Note: SEM represents standard error of the mean.

[0207] In summary, high-throughput screening data revealed multiple "hot spot regions" of saRNA activity in the human FVII gene promoter. Exemplary saRNAs increased expression of FVII mRNA and FVII protein levels in a dose-dependent manner. These results demonstrate that targeting activation of FVII expression by saRNAs is a promising strategy for the treatment of FVII-related diseases, disorders, or conditions, such as congenital FVII deficiency (Alexander disease), acquired FVII deficiency (AFVIID), hemophilia (e.g., hemophilia with inhibitors), and Glanzmann thrombasthenia (GT). References

[0208] While specific embodiments of the present application compositions and methods have been discussed, the above specification is illustrative and not restrictive. Many variations of the application will become apparent to those of skill in the art upon review of this specification and the claims below. The entire scope of the application should be determined by reference to the entire scope of the claims, and the specification and drawings, along with the variations thereof. ​

[0209] All publications, patents, and accession numbers mentioned in this application are hereby incorporated by reference in their entirety into this application, as if each individual publication or patent was specifically and individually incorporated by reference herein. ​ 1. Heinz S, Braspenning J. Measurement of blood coagulation factor synthesis in cultures of human hepatocytes. Methods in molecular biology. 2015; 1250: 309-316. 2. Yang L, Li Y, Bhattacharya A, Zhang Y. A plasma proteolysis pathway comprising blood coagulation proteases. Oncotarget. 2016; 7(27): 40919-40938. 3. O'Hara PJ, Grant FJ, Haldeman BA, Gray CL, Insley MY, Hagen FS, Murray MJ. Nucleotide sequence of the gene coding for human factor VII, a vitamin K-dependent protein participating in blood coagulation. Proceedings of the national academy of sciences of the United States of America. 1987; 84(15): 5158-5162. 4. Barbon E, Pignani S, Branchini A, Bernardi F, Pinotti M, Bovolenta M. An engineered tale-transcription factor rescues transcription of factor VII impaired by promoter mutations and enhances its endogenous expression in hepatocytes. Scientific reports. 2016; 6: 28304. 5. Long-Cheng Li, Steven T Okino, Hong Zhao, Deepa Pookot, Robert F Place, Shinji Urakami, Hideki Enokida, Rajvir Dahiya. Small dsRNAs induce transcriptional activation in human cells. PNAS. 2006 Nov. 14; 103(46): 17337-42. 6. Robert F Place, Ji Wang, Emily J Noonan, Rachel Meyers, Muthiah Manoharan, Klaus Charisse, Rick Duncan, Vera Huang, Xiaoling Wang, Long-Cheng Li. Formulation of small activating RNA into lipidoid nanoparticles inhibits xenograft prostate tumor growth by inducing p21 expression. Mol Ther Nucleic Acids. 2012 Mar. 27; 1(3): e15.

Claims

1. A small activating RNA (saRNA) comprising a sense strand and an antisense strand, wherein each sense strand or antisense strand of the saRNA comprises an oligonucleotide sequence ranging in length from 16 to 35 contiguous nucleotides, wherein each of the oligonucleotide sequences has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% homology or complementarity to an equal-length contiguous fragment of SEQ ID NO: 1437; wherein the saRNA increases expression of a FVII gene by at least 10% compared to baseline expression of the FVII gene.

2. The saRNA of claim 1, wherein the equal-length contiguous fragment of SEQ ID NO: 1437 is located in an upstream region of a transcription start site (TSS) of the FVII gene, and wherein the equal-length contiguous fragment of SEQ ID NO: 1437 is located within a region selected from the group consisting of: Region -557 to -379; Region -346 to -298; Region -271 to -91; and Region -96 to -1; and / or the equal-length contiguous fragment of SEQ ID NO: 1437 is located in a region of SEQ ID NO: 1438, SEQ ID NO: 1439, SEQ ID NO: 1440, or SEQ ID NO: 1441.

3. The saRNA of claim 1 or 2, wherein the equal-length contiguous fragment of SEQ ID NO: 1437 is selected from the group consisting of SEQ ID NOs: 1 to 286.

4. The saRNA of any one of claims 1 to 3, wherein the equal-length contiguous fragment of SEQ ID NO: 1437 is selected from the group consisting of SEQ ID NOs: 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 of any one of claims 1 to 4, wherein the contiguous oligonucleotide sequence has (1) a GC content between 40% and 70%; (2) no more than 5 consecutive identical nucleotides; (3) no more than 3 di- or tri-nucleotide repeat sequences; and / or the contiguous oligonucleotide sequence is complementary to the sense strand or antisense strand of the saRNA.

6. The saRNA of any one of claims 1 to 5, wherein the sense strand and the antisense strand each have a length of about 16 to about 35, about 17 to about 30, about 18 to about 25, or about 19 to about 22 nucleotides. ​ 7. The saRNA according to any one of claims 1 to 6, wherein the sense strand and the antisense strand have at least 90% complementarity; and / or wherein the sense strand and antisense strand are located on two different nucleic acid strands or adjacent nucleic acid strands; and / or wherein the contiguous oligonucleotide sequence comprises 0, 1, 2 or 3 mismatches to the complementary region of the sense strand or the complementary region of the antisense strand.

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

9. The saRNA according to claim 8, wherein the sense strand or the antisense strand comprises a 3’ overhang of 1 to 6, 1 to 5 or 2 to 3 nucleotides in length, respectively; or the double stranded nucleic acid structure is blunt ended.

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

11. The saRNA according to any one of claims 1 to 10, wherein the contiguous oligonucleotide sequence of the sense strand has at least 75% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 287 to 858, and / or the contiguous oligonucleotide sequence of the antisense strand has at least 75% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 859 to 1430.

12. The saRNA according to any one of claims 1 to 11, wherein the contiguous oligonucleotide sequence of the sense strand is selected from the group consisting of SEQ ID NOs: 287 to 858, and the contiguous oligonucleotide sequence of the antisense strand is selected from the group consisting of SEQ ID NOs: 859 to 1430.

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

14. The saRNA of any one of claims 1 to 13, wherein the sense strand comprises a contiguous nucleotide sequence selected from the group consisting of SEQ ID NOs: 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 comprises a contiguous nucleotide sequence selected from the group consisting of SEQ ID NOs: The consecutive nucleotide sequence of 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 saRNA of any one of claims 1 to 14, wherein the sense strand comprises the nucleotide sequence of SEQ ID NO: m, the antisense strand comprises the nucleotide sequence of SEQ ID NO: m+572, and wherein m is selected from the group consisting of 323, 341, 363, 372, 402, 403, 406, 410, 431, 432, 437, 463, 474, 491, 493, 504, 514, 527, 528; and / or wherein the sense strand comprises the nucleotide sequence of SEQ ID NO: m', the antisense strand comprises the nucleotide sequence of SEQ ID NO: m+858, and wherein m is selected from the group consisting of 296, 297, 315, 374, 407, 410, 442, 491, 493, 504, 514, 527, 545, 547, 560; and / or wherein the sense strand comprises the nucleotide sequence of SEQ ID NO: m' NO: m" nucleotide sequence, the antisense strand comprises SEQ ID NO:m"+286 nucleotide sequence, and wherein m is selected from 581, 582, 599, 627, 641, 653, 654, 658, 679, 749, 812, 813 and 824.

16. The saRNA of any one of claims 1-16, wherein the sense strand comprises a contiguous nucleotide sequence selected from the group consisting of SEQ ID NOs: 1442, 1444, 1446, 1448, 1450, 1452, 1454, 1456, 1458, 1460, 1462, 1464, 1466, 1468, 1470, 1472, 1474, and 1476; and / or the antisense strand comprises a contiguous nucleotide sequence selected from the group consisting of SEQ ID NOs: 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 duplex, as shown in Table 11.

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

18. The saRNA of claim 17, wherein the chemically modified nucleotide is a nucleotide having at least one of the following modifications: a) a modification to a phosphodiester linkage connecting nucleotides in the saRNA nucleotide sequence; b) a modification to a 2’-OH of a ribose in the saRNA nucleotide sequence; and c) a modification to a base in the saRNA nucleotide sequence.

19. The saRNA of claim 18, wherein the modification to the phosphodiester linkage connecting nucleotides is selected from the group consisting of a phosphorothioate modification and a boranophosphate modification; and / or the modification to the 2’-OH is selected from the group consisting of a 2’-fluoro modification, a 2’-oxymethyl modification, a 2’-oxyethylenemethoxy modification, a 2,4’-dinitrophenol modification, a 2’-amino modification, and a 2’-deoxy modification; and / or the modification to the base is selected from the group consisting of a 5’-bromouracil modification, a 5’-iodouracil modification, an N-methyluracil modification, and a 2,6-diaminopurine modification.

20. The saRNA of claim 17, wherein at least one nucleotide of the saRNA is a locked nucleic acid, an abasic nucleotide, a 2’-amino modified nucleotide, a 2’-alkyl modified nucleotide, a morpholino nucleotide, a phosphoramide, or a nucleotide comprising a non-natural base; and / or wherein the chemical modification of the at least one chemically modified nucleotide is an addition of an (E)-vinylphosphonate moiety at the 5’ end of the sense strand or the antisense strand.

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

22. The oligonucleotide modulator of claim 21, further comprising one or more moieties or components conjugated to, bound to, or mixed with the one or more saRNAs.

23. The oligonucleotide modulator of claim 21, wherein the sense strand and / or the antisense strand of the saRNA is conjugated to one or more conjugating moieties selected from a lipid, a fatty acid (e.g., a fatty acid comprising a carbon chain having 4-30, 12-24, or 16-22 carbon atoms), a fluorophore, a ligand, a sugar, a peptide, and an antibody.

24. The oligonucleotide modulator of claim 22, wherein the conjugating moieties are each, respectively, selected from a lipid, a cell-penetrating peptide, a polyethylene glycol, an alkaloid, a tryptamine, a benzimidazole, a quinolone, an amino acid, a cholesterol, a glucose, an N-acetylgalactosamine, and any combination thereof.

25. The oligonucleotide modulator of claim 22, wherein the conjugating moieties are each, respectively, selected from S9, tC2, tC2x6, C5x5, and any combination thereof. wherein, represents a carrier.

26. The oligonucleotide modulator of claim 21, wherein the oligonucleotide modulator further comprises a saRNA conjugated or bound to one or more other active moieties for treatment of a FVII-related disease, disorder, or condition, wherein the one or more other active moieties are each, respectively, selected from a small molecule chemical moiety, a polypeptide, and an antibody.

27. An isolated oligonucleotide, wherein (a) the isolated oligonucleotide is an oligonucleotide sequence having 16 to 35 contiguous nucleotides, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% homologous to an equal-length contiguous fragment of SEQ ID NO: 1437; or wherein (b) the isolated oligonucleotide is an oligonucleotide sequence having 16 to 35 contiguous nucleotides, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% complementary to an equal-length contiguous fragment of SEQ ID NO: 1437.

28. The isolated oligonucleotide of claim 27, wherein the equal-length contiguous fragment of SEQ ID NO: 1437 is located in a region upstream of a transcription start site (TSS) of a FVII gene, and wherein the equal-length contiguous fragment of SEQ ID NO: 1437 is located within a region selected from the group consisting of: Region -557 to -379; Region -346 to -298; Region -271 to -91; and Region -96 to -1; and / or the equal-length contiguous fragment of SEQ ID NO: 1437 is located within a region of SEQ ID NO: 1438, SEQ ID NO: 1439, SEQ ID NO: 1440, or SEQ ID NO: 1441. ​ ​ 29. The isolated oligonucleotide of claim 27, wherein the isolated oligonucleotide (a) is a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1 to 286, such as selected from the group consisting of SEQ ID NOs: 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 the isolated oligonucleotide (b) is a nucleic acid sequence complementary to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1 to 286, such as complementary to SEQ ID NOs: 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. An oligonucleotide complex comprising: an antisense strand of the saRNA of any one of claims 1 to 20 and a sense strand of the isolated oligonucleotide (a) of any one of claims 27 to 29; or a sense strand of the saRNA of any one of claims 1 to 20 and an antisense strand of the isolated oligonucleotide (b) of any one of claims 27 to 29.

31. The oligonucleotide complex of claim 30, wherein the oligonucleotide complex can activate expression of the FVII gene at least 10% up-regulation compared to baseline expression of the FVII gene.

32. An isolated nucleic acid molecule, wherein at least 20% (such as at least 30%, at least 40%, at least 45%, at least 50%) of saRNAs designed to target the sequence of the isolated nucleic acid molecule can activate FVII gene expression by at least 10%, wherein the designed saRNAs have (1) a GC content between 40% and 70%; (2) no more than 5 consecutive identical nucleotides; (3) no more than 3 di- or tri-nucleotide repeat sequences.

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

34. An isolated nucleic acid molecule, the sequence of which is selected from the group consisting of SEQ ID NOs: 1438 to 1441.

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

36. The isolated nucleic acid molecule according to any one of claims 32 to 34, wherein the saRNA targeted by the nucleic acid molecule design is as defined in any one of claims 1 to 20; and / or the desired saRNA is blunt-ended or has overhanging ends, and / or is unmodified or chemically modified.

37. An isolated polynucleotide encoding the saRNA of any one of claims 1 to 20 or the oligonucleotide modulator of 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 the isolated polynucleotide of any one of claims 37 to 38.

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

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

42. The product according to claim 41, wherein the product is used for the prevention, treatment or diagnosis of a FVII-related disease, disorder or condition (such as a FVII deficiency-related disease or haemophilia), for the design and / or screening of saRNAs.

43. A composition comprising the saRNA of any one of claims 1 to 20, the oligonucleotide modulator of any one of claims 21 to 26, or the isolated polynucleotide 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, such as 1 to 150 nM, of the saRNA.

45. A product for activating / upregulating the expression of a FVII gene in a cell, wherein the product can activate the expression of the FVII gene by at least 10% compared to the baseline expression of the FVII gene, and wherein the product comprises an active substance selected from one or more of the saRNA of any one of claims 1 to 20 or the oligonucleotide modulator of any one of claims 21 to 26, the isolated polynucleotide of any one of claims 37 to 38, the vector of claim 39, or the composition of any one of claims 43 to 44.

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

47. A method of activating / upregulating expression of a FVII gene in a cell, wherein the product can activate expression of the FVII gene at least 10% compared to baseline expression of the FVII gene, and wherein the method comprises administering to the cell an effective amount of an active substance, wherein the active substance is selected from one or more of a saRNA of any one of claims 1-20 or an oligonucleotide modulator of any one of claims 21-26, an isolated polynucleotide of any one of claims 37-38, a vector of claim 39, or a composition of any one of claims 41-44.

48. The product for activating / upregulating expression of a FVII gene in a cell according to claim 45, the use according to claim 46, or the method according to claim 47, wherein the active substance is introduced into a cell; and / or wherein the cell is an in vitro, ex vivo, or in vivo cell; and / or wherein the cell is a mammalian cell.

49. The product, use, or method according to claim 48, wherein the active substance is introduced into the cell by: 1) composing the active substance with a physiologically acceptable or pharmaceutically acceptable carrier, such as one or more selected from the group consisting of an aqueous carrier, a liposome, a polymeric molecule, a polypeptide, and an antibody, and / or 2) conjugating the active substance to one or more conjugating moieties, such as one or more (e.g. two conjugating moieties, wherein one is a lipid and the other is an N-acetylgalactosamine) selected from the group consisting of a lipid, a cell penetrating peptide, a polyethylene glycol, a biogenic alkaloid, a tryptamine, a benzimidazole, a quinolone, an amino acid, a cholesterol, a glucose, and an N-acetylgalactosamine, and any combination thereof.

50. The product, use, or method according to claim 49, wherein the conjugating moiety is one or more of S9, tC2, tC2x6, and C5x5, or any combination thereof (such as C5x5 and tC2x6): wherein, represents a carrier.

51. The product, use, or method according to claim 49, wherein the conjugating moiety is a lipid selected from the group consisting of a fatty acid having a carbon chain length of 4-30, 12-24, 16-22 carbon atoms; and / or wherein the conjugating moiety is a fatty acid having a carbon chain length of 4-30, 12-24, 16-22, or 16 carbon atoms; and / or wherein the conjugating moiety is derived from a fluorophore, a ligand, a sugar, a peptide, and an antibody, respectively.

52. The product for activating / upregulating FVII gene expression in a cell according to claim 45, the use according to claim 46 or the method according to claim 47, wherein the cell is from a patient suffering from or at risk of suffering from a disease, disorder or condition that can be prevented or treated by activating / upregulating FVII gene expression, e.g. a haemophilia (e.g. haemophilia with inhibitors) or Glanzmann thrombasthenia (GT), wherein a sufficient amount of the active substance is administered to prevent or treat the disease, disorder or condition.

53. A product for preventing or treating a FVII-associated disease, disorder or condition, wherein the product comprises an active substance selected from one or more of the saRNAs according to any one of claims 1-20 or the oligonucleotide modulators according to any one of claims 21-26, the isolated polynucleotide according to any one of claims 37-38, the vector according to claim 39 or the composition according to any one of claims 43-44.

54. Use of an active substance in the manufacture of a product for preventing or treating a FVII-associated disease or disorder or condition, wherein the active substance is selected from one or more of the saRNAs according to any one of claims 1-20 or the oligonucleotide modulators according to any one of claims 21-26, the isolated polynucleotide according to any one of claims 37-38, the vector according to claim 39 or the composition according to any one of claims 43-44.

55. A method for preventing or treating a FVII-associated disease, disorder or condition, wherein the method comprises administering to a subject an effective amount of an active substance, wherein the active substance is selected from one or more of the saRNAs according to any one of claims 1-20 or the oligonucleotide modulators according to any one of claims 21-26, the isolated polynucleotide according to any one of claims 37-38, the vector according to claim 39 or the composition according to any one of claims 43-44.

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. a human), preferably a mammal (e.g. a human) suffering from or at risk of suffering from a disease, disorder or condition that can be prevented or treated by activating / upregulating Factor VII (FVII) protein expression, e.g. a haemophilia with inhibitors.

57. The product according to claim 53, the use according to claim 54 or the method according to claim 55, wherein the active ingredient is administered to the individual by one or more of the following administration routes: parenteral infusion, oral administration, intranasal administration, inhalation administration, vaginal administration and rectal administration.

58. The product of claim 53, the use of claim 54, or the method of claim 55, wherein the active ingredient is administered to the individual by one or more of the following routes of administration: intrathecally, intramuscularly, intravenously, intra-arterially, intraperitoneally, intravesically, intracerebroventricularly, intravitreally, and subcutaneously.

59. The product of claim 53, the use of claim 54, or the method of claim 55, wherein the expression of FVII gene mRNA in the individual is activated / upregulated by at least 10% as compared to baseline expression of the FVII gene; and / or, wherein the level of FVII protein in the individual is increased by at least 10% as compared to baseline level of FVII protein; and / or, wherein the FVII-related disease, disorder, or condition comprises congenital FVII deficiency (Alexander disease), acquired FVII deficiency (AFVIID), hemophilia (such as hemophilia with inhibitors), and Glanzmann thrombasthenia (GT).

60. A method of diagnosing a FVII-related disease or disorder by detecting FVII protein or FVII modulating protein in the cell of claim 40.

61. A kit for performing the method of claim 60, comprising the saRNA of any one of claims 1-20 or the oligonucleotide modulator of any one of claims 21-26.

62. The kit of claim 61, wherein the instructions for use comprise a method of administering the saRNA of any one of claims 1-20 or the oligonucleotide modulator of any one of claims 21-26 to an individual.

63. A kit comprising the saRNA of any one of claims 1-20 or the oligonucleotide modulator of any one of claims 21-26, the isolated polynucleotide of any one of claims 37-38, the vector of claim 39, or the composition of any one of claims 43-44, packaged in labeled packaging, the label on the packaging indicating that the saRNA, the isolated polynucleotide, the vector, or the composition can be used to prevent or treat a disease, disorder, or condition induced by insufficient expression of coagulation factor VII (FVII), or to prevent or treat hemophilia.

64. A kit for detecting FVII protein or FVII modulating protein in the cell of claim 40.

65. A method for obtaining a saRNA capable of upregulating FVII gene expression by at least 10% as compared to baseline expression of the FVII gene, wherein the method comprises: (a) synthesizing a saRNA comprising a sense strand and an antisense strand, wherein each of the sense strand or antisense strand comprises a contiguous oligonucleotide sequence of 16 to 35 contiguous nucleotides, wherein the contiguous oligonucleotide sequence has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% homology or complementarity to an equal-length contiguous fragment of SEQ ID NO: 1437, and wherein the contiguous oligonucleotide sequence has (1) a GC content between 40% and 70%; (2) no more than 5 consecutive identical nucleotides; (3) no more than 3 dinucleotide or trinucleotide repeat sequences; and (b) determining the ability of the saRNA to upregulate FVII gene expression.

66. A method for obtaining a saRNA capable of upregulating FVII gene expression by at least 10% compared to baseline expression of the FVII gene, wherein the method comprises: (a) synthesizing a saRNA targeting a contiguous fragment of SEQ ID NO: 1437, wherein the saRNA has (1) a GC content between 40% and 70%; (2) no more than 5 consecutive identical nucleotides; (3) no more than 3 dinucleotide or trinucleotide repeat sequences; and (b) determining the ability of the saRNA to upregulate FVII gene expression.

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

68. The method of claim 65 or 66, wherein the contiguous fragment of SEQ ID NO: 1437 is selected from the group consisting of SEQ ID NOs: 1 to 286.

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

70. The double-stranded functional RNA molecule of claim 69, wherein the functional RNA molecule targets and modulates FVII gene expression.

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

72. The oligonucleotide agent of claim 71, further comprising one or more moieties or components conjugated to the one or more agents.

73. The oligonucleotide agent of claim 72, wherein the sense strand and / or the antisense strand of the functional saRNA is conjugated to one or more conjugating moieties selected from a lipid, a fatty acid (e.g., a fatty acid comprising a carbon chain having 4-30, 12-24, 16-22 carbon atoms), a fluorophore, a ligand, a sugar, a peptide, and an antibody.

74. The oligonucleotide agent of claim 73, wherein the conjugating moieties are selected from a lipid, a cell-penetrating peptide, a polyethylene glycol, an alkaloid, a tryptamine, a benzimidazole, a quinolone, an amino acid, a cholesterol, a glucose, a N-acetylgalactosamine, and any combination thereof, respectively.

75. The oligonucleotide agent of claim 74, wherein the conjugating moieties are selected from S9, tC2, tC2x6, C5x5, and any combination thereof, respectively. wherein represents a carrier.

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