Small activating nucleic acid molecules and their use in treatment of hereditary angioedema

Abstract

The present application relates to small activating nucleic acid molecules for the prevention or treatment of hereditary angioedema (HAE) and uses thereof. The small activating nucleic acid molecule comprises a sense nucleic acid strand and an antisense nucleic acid strand wherein the sense nucleic acid strand and the antisense nucleic acid strand are independently oligonucleotide strands having a length of 16 to 35 nucleotides wherein one nucleotide strand has at least 75% base homology or complementarity with a target selected from the promoter region of the target gene SERPING1. The invention also relates to a pharmaceutical composition and an optional pharmaceutically acceptable carrier, a method for up-regulating expression of a target gene in a cell by using the small activating nucleic acid molecule or the pharmaceutical composition containing the small activating nucleic acid molecule disclosed by the invention, and a method for preventing or treating a disease induced by insufficient expression of the target gene.

Description

Technical Field

[0001] The present application relates to the field of nucleic acid technology, and in particular to small activating nucleic acid molecules associated with gene activation and their pharmaceutical uses. Background Art

[0002] Hereditary angioedema (HAE) is an autosomal dominant disease caused by heterozygous mutations in the gene SERPING1 encoding the plasma protein C1 inhibitory protein (C1INH). The prevalence is reported to be approximately 1 in 50,000 people. HAE is characterized by frequent and life-threatening acute attacks of skin swelling, severe abdominal pain, and upper airway swelling. Untreated individuals have an attack every 1 to 2 weeks on average, and most attacks last about 3 to 4 days. About 25% of affected individuals die in the first two decades of life, mainly due to lack of treatment.

[0003] SERPING1 is expressed primarily in the liver and encodes C1 inhibitor, which is secreted into the blood where it participates in the normal function of the contact, coagulation, and fibrinolytic systems. This gene encodes C1 esterase inhibitor (C1EI or C1INH), the largest member of the serine protease inhibitor (SERPIN) superfamily, which functions as a major inhibitor of a) C1r, C1s, mannose-binding lectin-related serine proteases MASP-1, MASP-2, factor XII, and kallikrein in the contact system, b) factor XI and thrombin in the coagulation system, and c) tissue-type plasminogen activator (tPA) and plasmin in the fibrinolytic system. Low functional C1INH levels cause the accumulation of bradykinin, which triggers episodes of increased vascular permeability. HAE type I mutations (85% of cases) result in a truncated or misfolded protein that cannot be secreted. HAE type II (15% of cases) results from mutations in amino acids at or near the protein's active site that render the enzyme inactive.

[0004] Current treatment focuses on treating acute attacks with on-demand therapy or preventing attacks with prophylactic therapy. On-demand therapy and prophylactic therapy include plasma-derived and recombinant protein C1INH. Although these therapies effectively reduce the incidence of attacks, due to the short half-life of such drugs, these therapies bring a high economic burden and require frequent dosing. There is a need for improved methods and compositions for preventing and treating conditions associated with C1INH deficiency (such as hereditary angioedema). Summary of the invention

[0005] In order to solve the above problems, the present application provides a small activating nucleic acid molecule (such as a small activating RNA (saRNA) molecule) for activating / upregulating SERPING1 transcription and increasing the expression level of C1IHN protein through RNA activation (RNAa) mechanism to treat diseases or conditions (such as HAE) caused by C1INH protein deficiency.

[0006] Specifically, the inventors have discovered that such saRNAs capable of activating / upregulating the expression of SERPING1 mRNA are not randomly distributed on the promoter, but are aggregated in certain specific hot spots. Only some regions on the promoter of the SERPING1 gene are conducive to the activation / upregulation of expression by saRNA, for example, the -466 to -399 region (SEQ ID NO: 1391), -357 to -279 region (SEQ ID NO: 1392), -256 to -215 region (SEQ ID NO: 1393), -125 to -81 region (SEQ ID NO: 1394) and -80 to -15 region (SEQ ID NO: 1395) upstream of the transcription start site of the SERPING1 gene. The inventors have also found that the optimal target sequence / sense strand of saRNA within the SERPING1 promoter region includes sequences having the following items: (1) 35% to 70% GC content; (2) less than 5 consecutive identical nucleotides; (3) 3 or fewer dinucleotide repeats; and (4) 3 or fewer trinucleotide repeats. As a beneficial result, the target sequence (e.g., an isolated nucleic acid sequence comprising the target sequence) can activate / upregulate the expression of SERPING1 mRNA by at least 10% compared to the baseline level of SERPING1 mRNA after interacting with the saRNA. Based at least in part on these findings, the present disclosure features saRNA, compositions, and pharmaceutical compositions for activating / upregulating the expression of SERPING1 mRNA by at least 10% compared to the baseline level of the SERPING1 gene. Also provided herein is a method for preventing or treating a disease or condition induced by insufficient expression of C1INH protein, C1INH gene mutation, and / or low functional C1INH level in an individual, the method comprising administering any of the saRNA, compositions, and / or pharmaceutical compositions described herein.

[0007] In one aspect of the present application, a small activating nucleic acid molecule (such as a saRNA molecule) capable of activating / upregulating the expression of a SERPING1 gene in a cell is provided, the small activating nucleic acid molecule (saRNA) comprising an oligonucleotide sequence of 16 to 35 consecutive nucleotides in length, wherein the consecutive oligonucleotide sequence has at least 75%, or at least 80%, or at least 85%, or at least 90% sequence homology or complementarity with the equal length region shown in SEQ ID NO: 1390, thereby activating or upregulating the expression of the gene by at least 10%. In some embodiments, the equal length region shown in SEQ ID NO: 1390 is located in the -466 to -399 region (SEQ ID NO: 1391), -357 to -279 region (SEQ ID NO: 1392), -256 to -215 region (SEQ ID NO: 1393), -125 to -81 region (SEQ ID NO: 1394) or -80 to -15 region (SEQ ID NO: 1395) upstream of the transcription start site of the SERPING1 gene.

[0008] In certain embodiments, the saRNA disclosed in the present application comprises a sense strand and an antisense strand, wherein the sense strand and the antisense strand each comprise a complementary region, wherein the complementary region of the sense strand and the antisense strand forms a double-stranded nucleic acid structure. In certain embodiments, the sense strand and the antisense strand disclosed in the present application have at least 90% complementarity. In certain embodiments, the sense strand and the antisense strand disclosed in the present application are located on two different nucleic acid strands. And in certain embodiments, the sense strand and the antisense strand disclosed in the present application are located on the same nucleic acid strand, optionally on a hairpin single-stranded nucleic acid molecule, wherein the complementary region of the sense strand and the antisense strand forms a double-stranded nucleic acid structure. In certain embodiments, the sense strand and the antisense strand disclosed in the present application comprise a 3' overhang with a length ranging from 0 to 6 nucleotides, or a length ranging from 2 to 3 nucleotides. In certain embodiments, at least one of the nucleotides of the overhang is a thymine deoxyribonucleotide. In certain embodiments, the sense and antisense strands disclosed herein independently comprise a length of about 16 to about 35, about 17 to about 30, about 18 to about 25, or about 19 to about 22 contiguous nucleotides.

[0009] In certain embodiments, the sense strand disclosed herein has at least 75% sequence homology with a nucleotide sequence selected from SEQ ID NOs: 274-545, and the antisense strand disclosed herein has at least 75% sequence homology with a nucleotide sequence selected from SEQ ID NOs: 548-819. In certain embodiments, the sense strand disclosed herein comprises a nucleotide sequence selected from SEQ ID NOs: 274-545, and the antisense strand disclosed herein comprises a nucleotide sequence selected from SEQ ID NOs: 548-819.

[0010] In certain embodiments, the oligonucleotide sequences disclosed herein have at least 75% sequence homology or complementarity with a nucleotide sequence selected from SEQ ID NO: 1-272. In certain embodiments, the sense strand of the oligonucleotide sequences disclosed herein has at least 75% sequence homology with a nucleotide sequence selected from SEQ ID NO: 1-272. In certain embodiments, the antisense strand of the oligonucleotide sequences disclosed herein has at least 75% sequence complementarity with a nucleotide sequence selected from SEQ ID NO: 1-272.

[0011] In certain embodiments, at least one nucleotide in the saRNA disclosed herein is a chemically modified nucleotide. In certain embodiments, at least one nucleotide in the antisense strand and / or sense strand of the saRNA disclosed herein is chemically modified. In certain embodiments, the chemically modified nucleotides disclosed herein are nucleotides having at least one of the following modifications: a) Modification of the phosphodiester bonds connecting nucleotides in the nucleotide sequence of saRNA; b) modification of the 2'-OH of ribose in the nucleotide sequence of saRNA; and c) Modification of bases in the nucleotide sequence of saRNA.

[0012] 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 phosphoramidate, or a nucleotide comprising a non-natural base.

[0013] In certain embodiments, the chemical modification of at least one chemically modified nucleotide disclosed herein is the addition of an (E)-vinylphosphonate moiety at the 5' end of the sense strand or the antisense strand.

[0014] In certain embodiments, the sense strand or antisense strand of the saRNA disclosed herein is conjugated to one or more conjugated groups selected from the group consisting of a lipid, a fatty acid, a fluorophore, a ligand, a sugar, a peptide, and an antibody.

[0015] In certain embodiments, the sense strand or antisense strand of the saRNA disclosed herein is conjugated to one or more conjugated groups selected from the following: cell penetrating peptides, polyethylene glycol, alkaloids, tryptamine, benzimidazole, quinolone, amino acids, cholesterol, glucose and N-acetylgalactosamine. In certain embodiments, the conjugated group is derived from a compound selected from tC2 and tC2x4 shown in the present application. In certain embodiments, the saRNA conjugated to one conjugated group has a structure represented by O1 and O2.

[0016] In another aspect of the present application, an isolated target site of saRNA is provided, wherein the isolated target site is a continuous nucleotide sequence having a length of 16 to 35 nucleotides in SEQ ID NO: 1390. Specifically, the isolated target site is a nucleic acid sequence selected from SEQ ID NO: 1-272. In another aspect of the present application, a method of using the isolated target site of saRNA is provided.

[0017] Surprisingly, the inventors discovered that functional saRNAs capable of activating / upregulating the expression of the SERPING1 gene in cells are not randomly distributed on the promoter, but are aggregated in certain specific hot spots. Based on such findings, the present application also provides an isolated nucleic acid sequence upstream of the transcription start site of the SERPING1 gene, wherein the isolated nucleic acid sequence is selected from SEQ ID NO: 1391-1395. Specifically, the isolated nucleic acid sequence is enriched in the isolated target site disclosed in the present application. In certain embodiments, at least 30% of the designed saRNAs targeting the isolated nucleic acid sequences disclosed in the present application can activate the expression of the SERPING1 gene by at least 10%, wherein the designed saRNA: (1) has a GC content of 35% to 65%; (2) has less than 5 consecutive identical nucleotides; (3) has 3 or fewer dinucleotide repeats; and (4) has 3 or fewer trinucleotide repeats. For example, saRNA interacts with a region of 16 to 35 nucleotides of the isolated nucleic acid sequence in a sequence-specific manner. As a beneficial result, the isolated nucleic acid sequence can activate / upregulate the expression of the SERPING1 gene by at least 10% after interacting with the saRNA.

[0018] Another aspect of the present application provides isolated polynucleotides encoding saRNA disclosed herein. In one embodiment, the saRNA disclosed herein is a small activating RNA (saRNA) molecule. In one embodiment, the nucleic acid is a DNA molecule. Another aspect of the present application provides a vector comprising isolated polynucleotides disclosed herein.

[0019] Another aspect of the present application provides a cell comprising a saRNA disclosed herein, an isolated 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 above-mentioned cell can be in vitro, such as a cell line or cell strain, or can be present in a mammal, such as a human body.

[0020] Another aspect of the present application provides a composition, such as a pharmaceutical composition, comprising the above-mentioned saRNA or an isolated polynucleotide encoding the saRNA disclosed herein, and optionally a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutically acceptable carrier includes an aqueous carrier, a liposome, a high molecular polymer or a polypeptide. In some embodiments, the pharmaceutically acceptable carrier is selected from an aqueous carrier, a liposome, a high molecular polymer and a polypeptide. In some embodiments, the aqueous carrier can be, for example, RNase-free water or RNase-free buffer. In some embodiments, the composition may comprise 0.001 nM to 150 nM (e.g., 0.001 nM to 100 nM, 0.001 nM to 50 nM, 0.001 nM to 20 nM, 10 nM to 100 nM, 10 nM to 50 nM, 20 nM to 50 nM, 20 nM to 100 nM, or 50 nM to 150 nM), or optionally 1 nM to 150 nM of the above-mentioned saRNA or an isolated polynucleotide encoding a saRNA disclosed herein.

[0021] Another aspect of the present application relates to the use of the above-mentioned saRNA, the isolated polynucleotide encoding the saRNA disclosed herein, or the composition comprising the above-mentioned saRNA or the isolated polynucleotide disclosed herein in the preparation of a formulation for activating / upregulating the expression of the SERPING1 gene in a cell.

[0022] The present application also relates to a method for activating / upregulating the expression of the SERPING1 gene in a cell, wherein the method comprises administering the above-mentioned saRNA, the isolated polynucleotide disclosed herein, or a composition comprising the above-mentioned saRNA or the isolated polynucleotide disclosed herein to the cell. At the same time, a method for increasing the level of C1IHN protein in a cell or the level of functional C1IHN protein in the blood is also provided, the method comprising introducing an effective amount of saRNA, nucleic acid or composition disclosed herein into a cell.

[0023] The above-mentioned saRNA, the isolated polynucleotides disclosed herein, or the composition comprising the above-mentioned saRNA or the isolated polynucleotides disclosed herein can be directly introduced into the cell, or can be produced in the cell after the nucleotide sequence encoding the saRNA is introduced into the cell. The cell is preferably a mammalian cell, more preferably a human cell. The above-mentioned cell can be in vitro, such as a cell line or a cell strain, or can be present in a mammal, such as a human body. The human body is a patient suffering from an individual disease or symptom caused by a SERPING1 gene mutation, low C1IHN level, and / or insufficient blood level of a functional C1IHN protein, and the saRNA, the isolated polynucleotides disclosed herein, or the composition comprising the above-mentioned saRNA or the isolated polynucleotides disclosed herein are administered in sufficient amounts to treat the disease or symptom. Specifically, symptoms caused by a lack of C1IHN protein and / or insufficient expression of a functional C1IHN protein caused by a SERPING1 gene mutation include, for example, HAE. In one embodiment, the disease caused by insufficient expression of the C1IHN protein, or a SERPING1 gene mutation, or insufficient blood level of a functional C1IHN protein is HAE. In one embodiment, the HAE described herein includes Type I and Type II HAE.

[0024] Another aspect of the present application relates to a method for preventing or treating a condition caused by insufficient expression of C1IHN protein, SERPING1 gene mutation, and / or insufficient blood levels of functional C1IHN protein in an individual, the method comprising administering to the individual a therapeutically effective amount of saRNA disclosed herein, an isolated polynucleotide encoding saRNA disclosed herein, a vector disclosed herein, or a composition comprising saRNA disclosed herein. In certain embodiments, the disease or condition is hereditary angioedema (HAE). The individual can be a mammal, such as a human. In one embodiment, the individual suffers from symptoms induced by insufficient expression of C1IHN protein, SERPING1 gene mutation, and / or low functional C1IHN levels in the blood, which may include, for example, HAE. In one embodiment, the disease caused by insufficient blood levels of functional C1IHN protein caused by SERPING1 gene mutation is HAE. In one embodiment, the HAE described herein includes type I and type II HAE. In certain embodiments, the saRNA disclosed herein, the isolated polynucleotide disclosed herein, the vector disclosed herein, or the composition disclosed herein is administered to an individual by one or more of the following routes of administration: parenteral infusion, oral administration, intranasal administration, inhalation administration, vaginal administration, and rectal administration. In certain embodiments, the route of administration is selected from one or more of the following: intrathecal, intramuscular, intravenous, intraarterial, intraperitoneal, intravesical, intraventricular, intravitreal, and subcutaneous administration. In certain embodiments, the methods disclosed herein activate / upregulate the expression of the SERPING1 gene or SERPING1 mRNA in an individual by 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%, or at least 50%). In certain embodiments, the methods disclosed herein increase the level of C1IHN protein in an individual by at least 10%.

[0025] Another aspect of the present application relates to the use of saRNA disclosed herein, isolated polynucleotides disclosed herein, or compositions comprising saRNA disclosed herein or isolated polynucleotides disclosed herein in the preparation of a medicament for preventing or treating a disorder or condition caused by insufficient blood levels of functional C1IHN protein, SERPING1 gene mutations, and / or low functional C1IHN levels in the blood in an individual. The individual may be a mammal, such as a human. In one embodiment, the disease or condition may include, for example, HAE. In one embodiment, the disease caused by insufficient blood levels of functional C1IHN protein caused by SERPING1 gene mutations is HAE. In one embodiment, the HAE described herein includes type I and type II HAE.

[0026] In addition, the present application also provides a kit for implementing the prevention or treatment methods disclosed herein, wherein the kit includes: a) saRNA, b) instructions for use, and c) optionally a means 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 a disorder or condition induced by insufficient expression of plasma protein C1 inhibitor protein (C1INH) or for the prevention or treatment of HAE.

[0027] The present application provides a kit for implementing the methods disclosed herein, wherein the kit comprises: a) saRNA disclosed herein, and b) instructions for use. In certain embodiments, the instructions for use include means or methods for administering the saRNA disclosed herein to an individual.

[0028] Aspects of the present application include a kit comprising a saRNA disclosed herein, an isolated polynucleotide disclosed herein, a vector disclosed herein, or a composition disclosed herein in a labeled package, and the label on the package indicates that the saRNA, isolated polynucleotide, vector, or composition can be used to prevent or treat a disease or condition induced by insufficient expression of plasma protein C1 inhibitory protein (C1INH) or for the prevention or treatment of HAE.

[0029] The present application also provides a kit for detecting C1INH protein or C1INH-regulated protein (e.g., bradykinin) in blood or in cells disclosed herein that have been transfected with the above-mentioned saRNA or the above-mentioned nucleic acid or the above-mentioned composition.

[0030] The saRNA (such as saRNA molecules) that activate / upregulate SERPING1 gene expression provided herein can effectively and specifically upregulate the expression of the SERPING1 gene, increase the expression level of C1IHN mRNA, and have low toxic side effects, and can be used to prepare drugs for preventing or treating disorders associated with insufficient expression of C1IHN protein and diseases or conditions caused by SERPING1 gene mutations. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1The changes in the expression level of human SERPING1 mRNA mediated by saRNA in HepG2 cells are shown. 272 ​​saRNAs targeting the human SERPING1 promoter were transfected into a human liver cancer cell line (HepG2) at a concentration of 25 nM for 3 days. The sequences and duplex compositions of the saRNA chains are shown in Table 1. Mock was transfected in the absence of oligonucleotides (not shown). In each PCR reaction of the PCR reaction, the mRNA level of SERPING1 was quantified by one-step RT-qPCR using the gene-specific primer set shown in Table 4. HPRT1 and TBP were also amplified, and their geometric mean was used as an internal reference. The value (y-axis, log2) shows the relative fold change in the SERPING1 mRNA expression level of each of the 272 saRNAs relative to the Mock treatment after normalization to HPRT1 and TBP (mean ± SEM of two replicate transfection wells). The saRNAs are classified in descending order on the x-axis by their activity (log2) in inducing SERPING1 mRNA expression.

[0032] Figure 2 saRNAs classified by their target positions and hotspots on the human SERPING1 promoter in HepG2 cells are shown. 272 ​​saRNAs targeting the human SERPING1 promoter were transfected into HepG2 at 25nM alone for 3 days. Mock was transfected in the absence of oligonucleotides (not shown). In a single PCR reaction, the mRNA level of SERPING1 was quantified by one-step RT-qPCR using the gene-specific primer sets shown in Table 4. HPRT1 and TBP were also amplified, and their geometric mean was used as an internal reference. The value (y-axis, log2) shows the relative fold change in the SERPING1 mRNA expression level of each of the saRNAs relative to the Mock treatment after normalization to HPRT1 and TBP. The saRNAs are classified on the x-axis by their positions on the promoter from -500bp to -1bp upstream of the SERPING1 transcription start site (TSS). The positions of the 5 saRNA hotspots are marked as H1 to H5 in the rectangular dashed box. The numbers above the boxes indicate the boundaries of the hotspot regions relative to the SERPING1 TSS (site 0).

[0033] FIG. 3A to FIG. 3BDose response characterization and cytotoxicity of SERPING1 saRNA in HepG2 cells are shown. The first 73 human SERPING1 saRNAs were transfected into human HepG2 cells at concentrations of 2.5nM, 10nM, and 25nM for 3 days. Mock was transfected in the absence of oligonucleotides. dsCon2 duplex (SEQ ID NO: 547 and 821) was used as a non-specific duplex control. DS12A-si2 (SEQ ID NO: 273, 546, and 820) is a duplex siRNA and was transfected as a negative control. In a single PCR reaction, the mRNA level of SERPING1 was quantified by two-step RT-qPCR using the gene-specific primer sets shown in Table 4. HPRT1 and TBP were also amplified, and their geometric mean was used as an internal reference. Figure 3A Relative fold changes of SERPING1 mRNA levels in HepG2 cells transfected with top 73 SERPING1 saRNAs at concentrations of 2.5 nM, 10 nM and 25 nM, respectively, are shown. Values ​​(y-axis) are expressed as SERPING1 mRNA expression levels relative to Mock treatment after normalization to HPRT1 and TBP references. Figure 3B The cytotoxicity of the top 73 SERPING1 saRNAs in HepG2 cells is shown. Cytotoxicity was assessed by identifying apoptotic cells with propidium iodide (PI) staining. Values ​​(y-axis) are OD values ​​of PI staining relative to Mock treatment.

[0034] FIG. 4A to FIG. 4B The activation effect of several major saRNAs on SERPING1 gene expression in HepG2 cells is shown. Cells were treated with 10 nM saRNA concentration for 5 days. Mock was transfected in the absence of oligonucleotides. dsCon2 duplex was used as a nonspecific duplex control. DS12A-si2 is a duplex siRNA and was transfected as a negative control. Figure 4A The relative mRNA levels of SERPING1 in HepG2 cells measured by two-step RT-qPCR using the gene-specific primer sets shown in Table 4 are shown. TBP was also amplified as an internal control. The values ​​(y-axis) are expressed as the SERPING1 mRNA expression levels relative to the Mock treatment after normalization to TBP. Figure 4B The SERPING1 protein level obtained by detecting the OD value using a SERPING1 ELISA kit is shown. The values ​​(y axis) are expressed as the SERPING1 protein expression level relative to the Mock treatment. Compared with the Mock treatment, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0035] Figure 5 The activation effect of several major saRNAs at low concentrations on SERPING1 mRNA expression in HepG2 cells is shown. The cells were treated with saRNA concentrations of 0.1 nM or 1 nM for 3 days. Mock was transfected in the absence of oligonucleotides. dsCon2 was used as a nonspecific duplex control. DS12A-si2 is a duplex siRNA and was transfected as a negative control. In a separate PCR reaction, the mRNA level of SERPING1 was quantified by two-step RT-qPCR using the gene-specific primer set shown in Table 4. TBP mRNA was also amplified as an internal reference. The value (y-axis) is expressed as the SERPING1 mRNA expression level relative to Mock treatment after normalization to TBP. Compared with Mock treatment, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0036] FIG. 6A to FIG. 6C The activation effects of several major saRNAs on SERPING1 expression in human liver cancer cell lines are shown. Fig. 6A )、Hep3B( Figure 6B ) and PLC / PRF / 5( Figure 6C ) cell lines were treated with 10nM saRNA for 4 days. Mock was transfected in the absence of oligonucleotides. dsCon2 was used as a nonspecific duplex control. DS12A-si2 is a duplex siRNA and was transfected as a negative control. Relative SERPING1 mRNA levels were determined by two-step RT-qPCR using a gene-specific primer set. Relative SERPING1 protein levels in HepG2, Hep3B and PLC / PRF / 5 cells were determined using an ELISA kit. As internal references, HPRT1 and TBP genes were also amplified, and their geometric means were used as internal references. The values ​​(y-axis) in these figures are expressed as SERPING1 mRNA expression levels relative to Mock treatment after normalization to HPRT1 and TBP references. The values ​​(y-axis) in these figures are also expressed as SERPING1 protein expression levels relative to Mock treatment. Compared with the Mock treatment, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0037] FIG. 7A to FIG. 7BThe time course changes of several major saRNAs on SERPING1 expression in HepG2 cells are shown. Cells were transfected with a single saRNA at a specified concentration (i.e., 0.5nM, 1.25nM, 4nM, 30nM) for 1 to 7 days. Mock is transfected in the absence of oligonucleotides. dsCon2 is used as a nonspecific duplex control. DS12A-si2 is a duplex siRNA and is transfected as a negative control. Fig. 7A The mRNA levels of SERPING1 in HepG2 cells measured by two-step RT-qPCR using the gene-specific primer sets shown in Table 4 in separate PCR reactions are shown. TBP mRNA was also amplified as an internal control. Values ​​(y-axis) are expressed as SERPING1 mRNA expression levels relative to Mock treatment after normalization to TBP. Figure 7B The SERPING1 protein level obtained by detecting the OD value using a SERPING1 ELISA kit is shown. The value (y axis) is expressed as the SERPING1 protein expression level relative to the Mock treatment.

[0038] Figure 8A to Figure 8F The activation effect of saRNA on SERPING1 mRNA expression in HepG2 cells is shown. Six saRNAs (i.e., DS12A-0043, DS12A-0072, DS12A-0187, DS12A-0182, DS12A-0240, and DS12A-0213) were transfected into HepG2 cells individually at specified concentrations (i.e., 0.0017 nM, 0.0051 nM, 0.0152 nM, 0.046 nM, 0.14 nM, 0.41 nM, 1.23 nM, 3.7 nM, 11.111 nM, 33.333 nM, and 100 nM) for 4 days. Figure 8A to Figure 8F The mRNA levels of SERPING1 were measured by two-step RT-qPCR using gene-specific primer sets. HPRT1 and TBP were also amplified and their geometric mean was used as internal reference. Values ​​(y-axis) are expressed as SERPING1 mRNA expression levels relative to Mock treatment after normalization to HPRT1 and TBP.

[0039] Fig. 9The changes in the expression level of mouse Serping1 mRNA mediated by saRNA in PMH cells are shown. 277 saRNAs targeting the mouse Serping1 promoter were transfected into primary mouse hepatocytes (PMH) at a concentration of 25 nM for 3 days. The sequence and duplex composition of the saRNA chain are shown in Table 2. Mock was transfected in the absence of oligonucleotides (not shown). In a separate PCR reaction, the mRNA level of Serping1 was quantified by one-step RT-qPCR using the gene-specific primer set shown in Table 4. Hprt1 and Tbp were also amplified, and their geometric mean was used as an internal reference. The value (y-axis) is shown as the expression level of Serping1 mRNA relative to Mock treatment after normalization to Hprt1 and Tbp. saRNAs are classified in descending order on the x-axis by their activity (log2) in inducing Serping1 mRNA expression.

[0040] Fig.10 saRNAs classified by their target positions and hotspots on the mouse Serping1 promoter in PMH cells are shown. 277 saRNAs targeting the mouse Serping1 promoter were transfected into PMH at 25nM alone for 3 days. Mock was transfected in the absence of oligonucleotides (not shown). In a separate PCR reaction, the mRNA level of Serping1 was quantified by one-step RT-qPCR using the gene-specific primer set shown in Table 4. Hprt1 and Tbp were also amplified, and their geometric mean was used as an internal reference. The value (y-axis, log2) shows the fold change of the Serping1 mRNA expression level of each of the saRNAs relative to the Mock treatment after normalization to Hprt1 and Tbp. The saRNAs are classified on the x-axis by their positions on the promoters of -500bp to -1bp upstream of the Serping1 transcription start site (TSS). The positions of the 4 saRNA hotspots are marked as H6 to H9 in the rectangular dotted box. The numbers above the boxes indicate the boundaries of the hotspot regions relative to the Serping1 TSS.

[0041] Fig.11Dose response characterization of the first 57 mouse Serping1 saRNAs in PMH cells is shown. The first 57 mouse Serping1 saRNAs were transfected into PMH cells at different concentrations of 2.5nM, 10nM and 25nM for 3 days. Mock was transfected in the absence of oligonucleotides. dsCon2 was used as a nonspecific duplex control. DS12B-si2 (SEQ ID NO: 1099 and 1377) is a duplex siRNA and was transfected as a negative control. In a single PCR reaction, the mRNA level of Serping1 was quantified by two-step RT-qPCR using a gene-specific primer set. Hprt1 and Tbp were also amplified, and their geometric mean was used as an internal reference. Values ​​(y-axis) are expressed as Serping1 mRNA expression levels relative to Mock treatment after normalization to Hprt1 and Tbp references.

[0042] FIG. 12A to FIG. 12B The dose response characterization and cytotoxicity of the first 57 mouse Serping1 saRNAs in PMH cells are shown. The first 57 mouse Serping1 saRNAs were transfected into PMH cells for 3 days at different concentrations (i.e., 0.41nM, 1.23nM, 3.7nM, 11.1nM, 33.3nM, and 100nM). Mock was transfected in the absence of oligonucleotides. dsCon2 was used as a nonspecific duplex control. DS12B-si2 is a duplex siRNA and is transfected as a negative control. In a single PCR reaction, the mRNA level of Serping1 was quantified by two-step RT-qPCR using a gene-specific primer set. Hprt1 and Tbp were also amplified, and their geometric mean was used as an internal reference. Fig. 12A Shown are changes in Serping1 mRNA levels in PMH cells after transfection with saRNA. Values ​​(y-axis) are expressed as Serping1 mRNA expression levels relative to Mock treatment after normalization to Hprt1 and Tbp references. Fig. 12B The cytotoxicity of the top 57 Serping1 saRNAs in PMH cells is shown. Cytotoxicity was assessed by identifying apoptotic cells with propidium iodide (PI) staining. The values ​​(y axis) are OD values ​​of PI staining relative to Mock treatment.

[0043] Fig.13The activation effect of several major saRNAs on Serping1 mRNA expression in PMH cells is shown. The cells were treated with saRNA at a concentration of 25 nM for 3 days. Mock was transfected in the absence of oligonucleotides. dsCon2 was used as a nonspecific duplex control. DS12B-si2 is a duplex siRNA and was transfected as a negative control. In a separate PCR reaction, the mRNA level of Serping1 was quantified by two-step RT-qPCR using a gene-specific primer set. Tbp was also amplified as an internal reference. The value (y-axis) is expressed as the Serping1 mRNA expression level relative to Mock treatment after normalization to Tbp. Compared with Mock treatment, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0044] Figure 14A to Fig. 14C The time course changes and safety of SERPING1 saRNA in HepG2 cells are shown. The specified saRNA (i.e., RD-11513, RD-11823, RD-12065, and RD-11826) were transfected into HepG2 cells at 25 nM for 7 days. Mock was transfected in the absence of oligonucleotides (not shown). Figure 14A shows that in a separate PCR reaction, SERPING1 mRNA levels were quantified by two-step RT-qPCR using gene-specific primer sets. TBP and HPRT1 were also amplified, and their geometric mean was used as an internal reference. Figure 14B shows the SERPING1 protein level obtained by detecting OD values ​​using a SERPING1 ELISA kit. Values ​​(y-axis) are expressed as SERPING1 mRNA and protein expression levels relative to Mock treatment. Fig. 14C The caspase 3 / 7 activity obtained by measuring the OD value using a caspase 3 / 7 kit (G8092, Promega) is shown. The value (y axis) is expressed as the caspase 3 / 7 activity relative to the Mock treatment.

[0045] Figure 15A to Fig. 15CThe time course changes and safety of SERPING1 saRNA in PLC / PRF / 5 cells are shown. The specified saRNA (i.e., RD-12065 and RD-11826) were transfected into PLC / PRF / 5 cells at 25nM for 7 days. Mock was transfected in the absence of oligonucleotides (not shown). Figure 15A shows that in a separate PCR reaction, SERPING1 mRNA levels were quantified by two-step RT-qPCR using gene-specific primer sets. TBP and HPRT1 were also amplified, and their geometric mean was used as an internal reference. Figure 15B shows the SERPING1 protein level obtained by detecting OD values ​​using a SERPING1 ELISA kit. Values ​​(y-axis) are expressed as SERPING1 mRNA and protein expression levels relative to Mock treatment. Fig. 15C The caspase 3 / 7 activity obtained by detecting the OD value using a caspase 3 / 7 kit is shown. The value (y axis) is expressed as the caspase 3 / 7 activity relative to the Mock treatment.

[0046] Figure 16A to Fig. 16C The activation activity and safety of SERPING1 saRNA in HepG2 cells are shown. The specified saRNA (i.e., RD-11513, RD-11823, RD-12065, and RD-11826) were transfected into HepG2 cells at 25 nM for 7 days. Mock was transfected in the absence of oligonucleotides (not shown). Figure 16A shows that in a separate PCR reaction, SERPING1 mRNA levels were quantified by two-step RT-qPCR using gene-specific primer sets. TBP and HPRT1 were also amplified, and their geometric mean was used as an internal reference. Figure 16B shows the SERPING1 protein level obtained by detecting OD values ​​using a SERPING1 ELISA kit. Values ​​(y-axis) are expressed as SERPING1 mRNA and protein expression levels relative to Mock treatment. Fig. 16C The caspase 3 / 7 activity obtained by detecting the OD value using a caspase 3 / 7 kit is shown. The value (y axis) is expressed as the caspase 3 / 7 activity relative to the Mock treatment.

[0047] FIG. 17A to FIG. 17BThe activation effect of saRNA on SERPING1 mRNA expression in HepG2 and PLC / PRF / 5 cells is shown. Cells were treated with saRNA at a concentration of 25 nM for 3 days. Mock was transfected in the absence of oligonucleotides. dsCon2 was used as a non-specific duplex control. DS12A-si2 is a duplex siRNA and was transfected as a negative control. RD-12065, RD-11513, and RD-12066 are duplex saRNAs and used as exemplary controls. FIG. 17A to FIG. 17B The mRNA levels of SERPING1 were quantified by two-step RT-qPCR using gene-specific primer sets in separate PCR reactions. HPRT1 and TBP were also amplified and their geometric mean was used as internal reference. Values ​​(y-axis) are expressed as SERPING1 mRNA expression levels relative to Mock treatment after normalization to two references, HPRT1 and TBP. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 compared to Mock treatment.

[0048] Fig.18 The time course changes and safety of Serping1saRNA in PMH cells are shown. The specified saRNA (i.e., RD-12349, RD-12350, RD-12351, and RD-12347) was transfected into PMH cells at 25nM for 7 days. Mock was transfected in the absence of oligonucleotides (not shown). In a separate PCR reaction, gene-specific primers were used to quantify Serping1 mRNA levels by two-step RT-qPCR. Tbp and Hprt1 were also amplified, and their geometric mean was used as an internal reference. The value (y-axis) is expressed as the Serping1 mRNA expression level relative to Mock treatment after normalization to Tbp and Hprt1.

[0049] Figure 19A to Fig.19C The time course changes and safety of Serping1 saRNA in PMH cells are shown. The designated saRNAs (i.e., RD-11432, RD-11931, RD-11927, and RD-12189) were injected into the cells at 25 nM by RNAiMAX ( FIG. 19A ) and at 500 nM by free uptake ( FIG. 19B to FIG. 19C ). Fig.19C ) were treated into PMH cells for 5 days. Mock was transfected in the absence of oligonucleotides (not shown). Figure 19A toFigure 19B shows quantification of Serping1 mRNA levels by two-step RT-qPCR using gene-specific primer sets in separate PCR reactions. Tbp and Hprt1 were also amplified and their geometric mean was used as an internal reference. Values ​​(y-axis) are expressed as Serping1 mRNA expression levels relative to Mock treatment after normalization to Tbp and Hprt1. Fig.19C The caspase 3 / 7 activity obtained by detecting the OD value using a caspase 3 / 7 kit is shown. The value (y axis) is expressed as the caspase 3 / 7 activity relative to the Mock treatment.

[0050] Figure 20A to Fig. 20B The activation effect of Serping1 mRNA and the change of body weight of C57BL / 6J mice after SC injection of modified saRNA are shown. The specified saRNA (ie, RD-12497) was administered to adult C57BL / 6J mice at 20 mg / kg via SC injection. Normal saline was injected as a vehicle control. RD-11432 is a chemically modified siRNA and is used as a negative control. RD-11432 was injected via SC at 3 mg / kg. Mice were sacrificed on the 7th and 14th days after treatment. Figure 20A shows that after RNA isolation and RT reaction, Serping1 mRNA levels were quantified in liver tissue using gene-specific primer sets by RT-qPCR. Tbp and Hprt1 were amplified, and their geometric mean was used as an internal reference for RNA loading. The average Serping1 mRNA levels in liver tissue of 3 animals in each group relative to the mRNA levels in the normal saline group are shown after normalization to Tbp and Hprt1. Fig. 20B The figure shows the change in body weight (g) of C57BL / 6J mice after administration. The figure shows the average body weight level of 3 animals in each group.

[0051] Figure 21A to Fig. 21BThe activation effect of Serping1 mRNA and the change of body weight of C57BL / 6J mice after SC injection of modified saRNA are shown. The specified saRNA (ie, RD-13144) was administered to adult C57BL / 6J mice at 50 mg / kg via SC injection. Normal saline was injected as a vehicle control. RD-11432 is a chemically modified siRNA and is used as a negative control. RD-11432 was injected at 3 mg / kg via SC. Mice were killed on the 10th day after treatment. Figure 21A shows that after RNA isolation and RT reaction, Serping1 mRNA levels were quantified in liver tissue using gene-specific primer sets by RT-qPCR. Tbp and Hprt1 were amplified, and their geometric mean was used as an internal reference for RNA loading. The average Serping1 mRNA level in liver tissue of 3 animals in each group relative to the mRNA level in the normal saline group after normalization to Tbp and Hprt1 is shown. Fig. 21B The figure shows the change in body weight (g) of C57BL / 6J mice after administration. The figure shows the average body weight level of 3 animals in each group. DETAILED DESCRIPTION

[0052] Double-stranded RNA (dsRNA) targeting gene regulatory sequences (including promoters) has been shown to upregulate target genes in a sequence-specific manner at the transcriptional level through a mechanism called RNA activation (RNAa) (Li, LC et al., Small dsRNAs induce transcriptional activation in human cells., PNAS (2006)). Such dsRNAs are called small activating RNAs (saRNAs).

[0053] Embodiments of the present disclosure are based in part on the surprising discovery that small activating nucleic acid molecules (saRNA, also referred to herein as "SERPING1 saRNA") can activate or upregulate the expression of the SERPING1 gene in cells. After administration of the saRNA of the present application, the increase in the production of functional SERPING1 mRNA can achieve a significant increase or upregulation of the levels of SERPING1 mRNA and SERPING1 protein (C1INH).

[0054] Embodiments of the present disclosure are also based in part on the surprising discovery that saRNAs capable of activating or upregulating the expression of the SERPING1 gene in cells are aggregated in specific SERPING1 promoter regions, such as Figure 2 and Fig.10As shown. The inventors identified aggregates of these SERPING1 promoter regions, which are considered to be "hotspot" promoter regions that enrich the target sites of the developed functional saRNA. These specific promoter regions identified in the present application (referred to herein as "hotspots") are optionally at least 42nt in length, or have a length ranging from about 42nt to about 100nt. "Hotspots" herein are defined as nucleic acid regions on the target sequence of saRNA, where the full-length targets of functional saRNAs are enriched and span the 5' end of the first saRNA target within each hotspot to the 3' end of the last saRNA target. In some embodiments, at least 30%, about 40%, or even more than 50% of the saRNAs designed to target these hotspot regions are proven to be functional, i.e., 1.1-fold or more changes in the mRNA level or protein expression of the target gene are induced. In some embodiments, at least 30%, about 40%, or even more than 50% of the saRNAs designed to target the fragments in these hotspot regions are proven to be functional, i.e., 1.1-fold or more changes in the mRNA level or protein expression of the target gene are induced. The inventors surprisingly found that functional saRNAs are not randomly distributed on the promoter, but aggregated in specific hotspots (see, e.g., Table 9). For example, 5 hotspots of the human SERPING1 promoter located in the -466 to -399 region (H1), -357 to -279 region (H2), -256 to -215 region (H3), -125 to -81 region (H4), and -80 to -15 region (H5), and 4 hotspots of the mouse Serping1 promoter located in the -424 to -325 region (H6), -257 to -182 region (H7), -170 to -117 region (H8), and -56 to -11 region (H9) of the promoter TSS were detected, and they were found to be the best target sites for saRNAs that activate SERPING1 / Serping1 gene expression through RNA activation mechanisms.

[0055] This saRNA-SERPING1 mRNA-SERPING1 protein pathway may provide an alternative therapeutic approach to current treatments for conditions associated with C1INH deficiency, such as for patients with hereditary angioedema.

[0056] In this application, the relevant terms are defined as follows:

[0057] As used herein, the term "complementarity" refers to the ability to form base pairs between two oligonucleotide chains. These base pairs are usually formed by hydrogen bonds between nucleotides in antiparallel oligonucleotide chains. The bases of complementary oligonucleotide chains can be paired in Watson-Crick mode (such as A and T, A and U and C and G) or paired in any other way that allows the formation of duplexes (such as Hoogsteen or reverse Hoogsteen base pairing).

[0058] Complementarity includes complete complementarity and incomplete complementarity. "Complete complementarity" or "100% complementarity" means that each nucleotide from the first oligonucleotide chain can form hydrogen bonds with the nucleotides at the corresponding positions in the second oligonucleotide chain in the double-stranded region of the double-stranded oligonucleotide molecule, and no base pairs are "mismatched". "Incomplete complementarity" means that not all nucleotide units of the two chains are bound to each other by hydrogen bonds. For example, for two oligonucleotide chains each having a length of 20 nucleotides in the double-stranded region, if only two base pairs in the double-stranded region can be formed by hydrogen bonds, the oligonucleotide chain has 10% complementarity. In the same example, if 18 base pairs in the double-stranded region can be formed by hydrogen bonds, the oligonucleotide chain has 90% complementarity. Basic complementarity refers to at least about 75%, about 79%, about 80%, about 85%, about 90%, about 95% or 99% complementarity.

[0059] The terms "oligonucleotide" or "polynucleotide" are used interchangeably and refer to polymers of nucleotides and include, but are not limited to, single-stranded or double-stranded nucleic acid molecules of DNA, RNA, or DNA / RNA hybrids, oligonucleotide chains containing regular and irregular alternations of deoxyribose moieties and ribose moieties, and modified and naturally or non-naturally occurring frameworks of such oligonucleotides. The oligonucleotides described herein for activating transcription of a target gene are small activating nucleic acid molecules.

[0060] As used herein, the terms "oligonucleotide chain," "chain," and "oligonucleotide sequence" are used interchangeably and are general terms referring to short nucleotide sequences having less than 35 bases, including nucleotides in deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). In a non-limiting example, the length of the chain can be any length from 16 to 35 nucleotides.

[0061] As used herein, the term "target gene" may refer to a nucleic acid sequence, transgene, viral or bacterial sequence, chromosome or extrachromosomal gene that is naturally present 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 positive regulation of the target gene can be achieved by designing a saRNA having sequence identity (also referred to as homology) with the promoter sequence, characterized in that the expression of the target gene is upregulated. "Target sequence" or "target site" are used interchangeably and refer to a sequence fragment in the sequence of a target gene sequence, such as a target gene promoter, which is homologous or complementary to the sense strand or antisense strand of the saRNA.

[0062] As used herein, in a saRNA duplex, the term "sense strand" of the saRNA refers to the strand that has sequence homology or sequence identity with a segment of the coding strand of the target gene.

[0063] As used herein, in a saRNA duplex, the term "antisense strand" of saRNA refers to a strand having sequence complementarity with a sense strand. The antisense strand can interact with a target sequence to activate or upregulate gene expression, and the target sequence can be a fragment of the coding strand of the sequence of a target gene.

[0064] As used herein, the term "coding strand" refers to a DNA strand in a target gene that cannot be used for transcription, and the nucleotide sequence of the 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 the double-stranded DNA sequence of the target gene promoter described herein refers to a promoter sequence located on the same DNA strand as the DNA coding strand of the target gene.

[0065] As used herein, the term "template strand" refers to another strand complementary to the coding strand in the double-stranded DNA of the target gene, that is, a strand that can be transcribed into RNA as a template, and the strand is complementary to the transcribed RNA (A and U and G and C). During transcription, RNA polymerase binds to the template strand, moves along the 3'→5' direction of the template strand, and catalyzes the synthesis of RNA along the 5'→3' direction. The template strand of the double-stranded DNA sequence of the target gene promoter described herein refers to a promoter sequence located on the same DNA strand as the DNA template strand of the target gene.

[0066] As used herein, the term "LNA" refers to a locked nucleic acid in which the 2'-oxygen and 4'-carbon atoms are connected by an additional bridge. As used herein, the term "BNA" refers to a 2'-O and 4'-aminoethylene bridged nucleic acid that may contain a five-membered or six-membered bridge structure with an N-O bond. As used herein, the term "PNA" refers to a nucleic acid mimetic having a pseudopeptide backbone consisting of N-(2-aminoethyl)glycine units in which the nucleobase is connected to the glycine nitrogen via a carbonyl methylene linker.

[0067] As used herein, the term "promoter" refers to a sequence that is spatially associated with a protein coding or RNA coding nucleic acid sequence and that regulates the transcription of the protein coding or RNA coding nucleic acid sequence. Typically, eukaryotic gene promoters contain 100 to 5000 base pairs, although this length range is not intended to limit the term "promoter" used herein. Although promoter sequences are typically located at the 5' end of a protein coding or RNA coding sequence, promoter sequences are also present in exon and intron sequences.

[0068] As used herein, the term "transcription start site" refers to the nucleotide that marks the start of transcription on the template strand of a gene. The transcription start site may appear on the template strand in the promoter region. A gene may have more than one transcription start site.

[0069] As used herein, the term "identity" or "homology" means that one oligonucleotide strand (sense strand or antisense strand) of saRNA has sequence similarity with the coding strand or template strand in the promoter sequence 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%.

[0070] As used herein, the term "sequence-specific manner" means a manner in which two nucleic acid fragments are bound or hybridized based on their nucleotide sequences, for example, the Watson-Crick manner (such as A and T, A and U, and C and G) or any other manner that allows the formation of a duplex (such as Hoogsteen or reverse Hoogsteen base pairing).

[0071] As used herein, the term "overhang" refers to a non-base paired nucleotide at the end (5' or 3') of an oligonucleotide chain, which is formed by one chain in a double-stranded oligonucleotide extending beyond the other chain. The single-stranded region extending beyond the 3' end and / or 5' end of the duplex is called an overhang.

[0072] As used herein, the terms "gene activation" or "activating gene expression" and "gene upregulation" or "upregulating gene expression" are used interchangeably and refer to an increase in the transcription, translation, expression or activity of a certain nucleic acid determined by measuring the level of transcription, mRNA level, protein level, enzyme activity, methylation state, chromatin state or configuration, translation level or the activity or state of a gene in a cell or biological system. These activities or states can be determined directly or indirectly. In addition, "gene activation", "activating gene expression", "gene upregulation" or "upregulating gene expression" refers to an increase in activity associated with a nucleic acid sequence, regardless of the mechanism of such activation. For example, gene activation occurs at the transcription level to increase transcription to RNA, and RNA is translated into protein, thereby increasing protein expression.

[0073] As used herein, the terms "small activating RNA", "saRNA" and "small activating nucleic acid molecule" are used interchangeably and refer to a nucleic acid molecule that can upregulate target gene expression and can be composed of a first nucleic acid fragment (sense strand) and a second nucleic acid fragment (antisense strand), the first nucleic acid fragment containing a nucleotide sequence having sequence identity with a non-coding nucleic acid sequence (e.g., a promoter or enhancer) of the target gene, and the second nucleic acid fragment containing a nucleotide sequence complementary to the first nucleic acid fragment, wherein the first nucleic acid fragment and the second nucleic acid fragment form a duplex. The saRNA can also be composed of a synthetic or vector-expressed single-stranded RNA molecule that can form a hairpin structure through two complementary regions within the molecule, wherein the first region contains a nucleotide sequence having sequence identity with the target sequence of the promoter of the gene, and the second region contains a nucleotide sequence complementary to the first region. The length of the duplex region of the saRNA molecule is generally about 10 to about 50 base pairs, about 12 to about 48 base pairs, about 14 to about 46 base pairs, about 16 to about 44 base pairs, about 18 to about 42 base pairs, about 20 to about 40 base pairs, about 22 to about 38 base pairs, about 24 to about 36 base pairs, about 26 to about 34 base pairs, and about 28 to about 32 base pairs, and generally about 10 base pairs, about 15 base pairs, about 20 base pairs, about 25 base pairs, about 30 base pairs, about 35 base pairs, about 40 base pairs, about 45 base pairs, or about 50 base pairs. In addition, the terms "saRNA", "small activating RNA" and "small activating nucleic acid molecules" also include nucleic acids other than ribonucleotides, including but not limited to modified nucleotides or analogs.

[0074] As used herein, the term "hotspot" refers to a gene promoter region of at least 42 bp in length, where functional saRNAs are enriched, i.e., at least 25%, such as about 30%, about 40%, about 50%, about 60%, or about 70%, or more than 70% of the saRNAs designed to target the region are "functional", i.e., can induce a 1.1-fold or more change in the mRNA level or protein expression of the target gene. "Hotspot" and "hotspot region" can be used interchangeably and are defined herein as a nucleic acid region on the target sequence of the saRNA where the full-length target of the functional saRNA is enriched and spans from the 5' end of the first saRNA target to the 3' end of the last saRNA target within each hotspot. In a non-limiting example, the saRNA is designed according to the following criteria: (1) having a GC content of 35% to 65%; (2) having less than 5 consecutive identical nucleotides; (3) having 3 or fewer dinucleotide repeats; and (4) having 3 or fewer trinucleotide repeats.

[0075] As used herein, the term "functional saRNA" refers to a saRNA that activates the expression of its intended target gene by at least 10% (or at least 1.1-fold). The term "non-functional saRNA" refers to a saRNA that regulates the mRNA level and protein expression of the SERPING1 gene by less than 10% (or less than 1.1-fold).

[0076] As used herein, the term "isolated target site" as used herein means a target site that has complementarity with or hybridizes to a saRNA. For example, an isolated nucleic acid sequence of a target site may include a nucleic acid sequence that has complementarity with or hybridizes to a region of a saRNA. As used herein, the term "isolated polynucleotide" as used herein means a polynucleotide encoding a saRNA.

[0077] As used herein, the term "synthetic" refers to a method of synthesizing oligonucleotides, which includes any method that allows RNA synthesis, such as chemical synthesis, in vitro transcription, and / or vector-based expression.

[0078] As used herein, the term "carrier material" refers to a solid phase starting material that is maintained in a column that allows all kit solvents to pass freely during two filtrations in an automated oligonucleotide synthesizer, and optionally produces a 3' or 5' end conjugated oligonucleotide. The carrier material can be selected from controlled pore glass (CPG), silica, silica gel, glass, ceramics, polymers, cellulose, and combinations thereof.

[0079] As used herein, capitalized "SERPING1 gene" or "SERPING1" refers to the human gene; and capitalized "Serping1 gene" or "Serping1" refers to the mouse gene orthologous to human SERPING1.

[0080] As used herein, the term "SERPING1 mRNA" refers to messenger RNA (mRNA) produced by the expression of the SERPING1 gene or the transcription of the SERPING1 gene.

[0081] As used herein, the terms "SERPING1 protein" and "C1IHN protein" are used interchangeably and refer to a protein produced by the expression of a SERPING1 gene or the translation of a SERPING1 mRNA. saRNA

[0082] In the present application, the expression of the SERPING1 gene is upregulated by RNA activation, and the related diseases (particularly HAE) are treated by increasing the expression level of the C1IHN protein (i.e., C1 inhibitory protein). Since the SERPING1 gene encodes the C1IHN protein, the increase in the expression of the SERPING1 mRNA increases the level of the C1IHN protein. In some cases, the SERPING1 gene is a target gene in the present application.

[0083] Aspects of the present application include small activating nucleic acid molecules (saRNAs) comprising an oligonucleotide sequence having a length ranging from 16 to 35 consecutive nucleotides, wherein the consecutive oligonucleotide sequence has at least 75%, or at least 80%, or at least 85%, or at least 90% sequence homology or complementarity with an equal length portion shown in SEQ ID NO: 1390, and wherein the saRNA activates / upregulates the expression of the SERPING1 gene by at least 10%.

[0084] In some embodiments, the equal length portion shown in SEQ ID NO: 1390 disclosed herein is located in the -466 to -399 region (SEQ ID NO: 1391), -357 to -279 region (SEQ ID NO: 1392), -256 to -215 region (SEQ ID NO: 1393), -125 to -81 region (SEQ ID NO: 1394), or -80 to -15 region (SEQ ID NO: 1395) upstream of the transcription start site of the SERPING1 gene.

[0085] In some embodiments, the saRNA disclosed herein comprises a sense strand and an antisense strand. The sense strand and the antisense strand comprise complementary regions capable of forming a double-stranded nucleic acid structure, and the double-stranded nucleic acid structure activates the expression of the SERPING1 gene in the cell through the RNAa mechanism. The RNAa mechanism used herein (also referred to as RNA activation) refers to the mechanism by which the double-stranded nucleic acid structure can upregulate the target gene in a sequence-specific manner at the transcriptional level. The sense strand and antisense strand of saRNA may be present on two different nucleic acid chains or on one nucleic acid chain. When the sense strand and the antisense strand are located on two different chains, at least one chain of saRNA has a 3' overhang of 0 to 6 nucleotides in length, so that the length of the overhang is 0, 1, 2, 3, 4, 5 or 6 nucleotides, and in some cases, both chains have a 3' overhang of 2 or 3 nucleotides in length. In some cases, the nucleotides of the overhang are dT. In some cases, when the sense strand and the antisense strand are located on one nucleic acid chain, saRNA is a hairpin single-stranded nucleic acid molecule, wherein the complementary regions of the sense strand and the antisense strand form a double-stranded nucleic acid structure with each other. In some embodiments, the sense strand and the antisense strand in the above saRNA each have a length ranging from 16 to 35 nucleotides. For example, in some embodiments, the sense strand and the antisense strand independently comprise a length of 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 nucleotides.

[0086] The present application provides that saRNA comprising the sequence of the sense chain or antisense chain of Formula I and Formula II can also cause SERPING1 gene activation. AAAUUCCUGGCCUGGCCCNNN (Formula I) CUACCAGGGNNNNNNNNNN (Formula II)

[0087] wherein N is selected from adenine (A), thymine (T), cytosine (C), guanine (G) and uracil (U).

[0088] 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 with a nucleotide sequence selected from SEQ ID NOs: 1-272. 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 with any nucleotide sequence selected from SEQ ID NOs: 274-545, 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 with any nucleotide sequence selected from SEQ ID NOs: 548-819. More specifically, the sense strand of the saRNA disclosed herein includes, comprises, or consists of any nucleotide sequence selected from SEQ ID NOs: 274-545; and the antisense strand of the saRNA disclosed herein includes, comprises, or consists of any nucleotide sequence selected from SEQ ID NOs: 548-819, or is any nucleotide sequence selected from SEQ ID NOs: 548-819. 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 with a nucleotide sequence selected from the following: AAAUUCCUGGCCUGGCCCNNN and CUACCAGGGNNNNNNNNNN. For example, the present application provides that saRNAs comprising sequences of strands in the formula AAAUUCCUGGCCUGGCCCNNN and / or CUACCAGGGNNNNNNNNNN can also lead to SERPING1 gene activation.

[0089] In certain embodiments, the antisense strand disclosed herein can interact with the target nucleic acid sequence of the promoter of a gene in a sequence-specific manner, which means that the antisense strand can hybridize with the target nucleic acid through hydrogen bonds. In certain embodiments, the antisense strand has a nucleotide sequence, and when written in a 5' to 3' direction, the nucleotide sequence comprises the reverse complementary sequence of the target portion of the target nucleic acid to which it is targeted. In certain such embodiments, the antisense strand has a nucleotide sequence, and when written in a 5' to 3' direction, the nucleotide sequence comprises the reverse complementary sequence of the target portion shown in SEQ ID NO: 1390, specifically, the target portion is a nucleic acid sequence selected from SEQ ID NO: 1-272.

[0090] In the saRNA disclosed herein, all nucleotides may be natural or non-chemically modified nucleotides, or at least one nucleotide may be a chemically modified nucleotide. Non-limiting examples of chemical modifications include one or more combinations of the following: (1) Modification of the phosphodiester bonds of nucleotides in the nucleotide sequence of saRNA; (2) modification of the 2'-OH of ribose in the nucleotide sequence of saRNA; (3) modification of bases in the nucleotides of saRNA; and (4) At least one nucleotide in the nucleotide sequence of the small activating nucleic acid molecule is a locked nucleic acid.

[0091] The chemical modifications described herein are well known to those skilled in the art, and the modification of the phosphodiester bond refers to the modification of the oxygen in the phosphodiester bond, including phosphorothioate modification and boronate modification. The modifications disclosed herein stabilize the saRNA structure, thereby maintaining high specificity and high affinity for base pairing.

[0092] In some embodiments, the saRNA of the present application comprises at least one chemically modified nucleotide, which is modified at the 2'-OH of the pentose of the nucleotide, that is, certain substituents are introduced at the hydroxyl position of the ribose, such as 2'-fluoro modification, 2'-oxymethyl modification, 2'-oxyethylenemethoxy modification, 2,4'-dinitrophenol modification, locked nucleic acid (LNA), 2'-amino modification or 2'-deoxy modification, for example, 2'-deoxy-2'-fluoro modified nucleotides, 2'-deoxy modified nucleotides.

[0093] In some embodiments, the saRNA of the present application comprises at least one chemically modified nucleotide that is modified at the base of the nucleotide, such as 5'-bromouracil modification, 5'-iodouracil modification, N-methyluracil modification, or 2,6-diaminopurine modification.

[0094] 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 antisense sequence. In some embodiments, the chemical modification of at least one chemically modified nucleotide is the addition of a 5'-methylcytosine moiety at the 5' end of the sense sequence or antisense sequence.

[0095] In some embodiments, the saRNA of the present application comprises at least one chemically modified nucleic acid nucleotide in the nucleotide sequence of the small activating nucleic acid molecule, for example, locked nucleotides, abasic nucleotides, 2'-amino modified nucleotides, 2'-alkyl modified nucleotides, morpholino nucleotides, phosphoramidates, and nucleotides containing non-natural bases. In some embodiments, the saRNA disclosed herein comprises "endo-light" modifications with 2'-O-methyl modified nucleotides and nucleotides containing 5'-phosphorothioate groups.

[0096] In some embodiments, the saRNA of the present application is chemically modified to enhance stability or other beneficial features. Specific nucleic acids in the present application can be synthesized and / or modified by conventional methods, such as described in Current protocols innucleic acid chemistry, Beaucage, SL et al. (Edrs.) John Wiley & Sons, Inc., New York, USA, incorporated herein by introduction. Modifications include, for example, (a) terminal modifications, such as 5' end modifications (phosphorylation, conjugation, reverse connection, etc.), 3' end modifications (conjugation, DNA nucleotides, reverse connection, etc.), (b) base modifications, such as replacement with stable bases, destabilized bases or bases paired with extended ligand libraries, removal of bases (absaccharide nucleotides) or conjugated bases, (c) sugar modifications (e.g., at 2' or 4' positions) or replacement of sugars, and (d) main chain modifications, including modification or replacement of phosphodiester bonds. Specific examples of saRNA molecules that can be used for the present application include, but are not limited to, RNA containing modified main chains or containing no natural internucleoside bonds. In some embodiments, the RNA with modified backbone includes RNA without phosphorus atom in the backbone. In some embodiments, the RNA with modification without phosphorus atom in its internucleoside backbone can also be considered as oligonucleoside. In some embodiments, the oligonucleotide of modification will have phosphorus atom in its internucleoside backbone.

[0097] The oligonucleotide backbone of modification comprises, for example, phosphorothioate, chiral phosphorothioate, phosphorodithioate, phosphotriester, aminoalkylphosphotriester, methyl and other alkylphosphonates (comprising 3'-alkylenephosphonates and chiral phosphonates), phosphinates, phosphoramidates (comprising 3'-phosphoramidate and aminoalkylphosphoramidate), thiophosphoramidates, thioalkylphosphonates, thioalkylphosphotriester, and boron phosphorothioate with normal 3'-5' key, the analog of 2'-5' connection of these boron phosphorothioates, and those compounds with opposite polarity, wherein adjacent nucleoside unit is to be 3'-5' connected to 5'-3' or 2'-5' connected to 5'-2'. Various salts, mixed salts and free acid forms are also included.

[0098] Non-limiting examples of preparing phosphorus-containing bonds 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; 5,58 7,361; 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; 6,5 34,639; 6,608,035; 6,683,167; 6,858,715; 6,867,294; 6,878,805; 7,015,315; 7,041,816; 7,273,933; 7,321,029; and U.S. Patent RE39464, which are hereby incorporated by reference in their entirety.

[0099] In certain embodiments, the small activating nucleic acid molecule is RNA, DNA, BNA, LNA, or peptide nucleic acid (PNA).

[0100] In addition, in order to promote the entry of saRNA into cells, on the basis of the above-mentioned modifications, a chemical conjugation group can be introduced into the end of the sense chain or antisense chain of saRNA to promote the action through the cell membrane composed of lipid bilayers and the nuclear membrane and gene promoter region in the nucleus.

[0101] In certain embodiments, the saRNA disclosed herein is covalently linked to one or more conjugated groups. In certain embodiments, the conjugated group changes one or more properties of the connected oligonucleotide, including but not limited to pharmacodynamics, pharmacokinetics, stability, binding, absorption, tissue distribution, cellular uptake, charge and clearance. In certain embodiments, the conjugated group imparts new properties to the connected oligonucleotide, such as a fluorophore or reporter group capable of detecting an oligonucleotide.Certain conjugated groups and conjugated moieties have been previously described, for example: cholesterol moieties (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556); cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4, 1053-1060); thioethers, such as hexyl-S-tritylthiol (Manoharan et al., Ann. NY Acad. Sci., 1992, 660, 306-309; Manoharan et al., Bioorg. Med. Chem. Lett., 1993, 3, 2765-2770); sulfocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20, 533-538); fatty chains, such as dodecanediol or undecyl residues (Saison-Behmoaras et al., EMBO 1, 1991, 10, 1111-1118; Kabanov et al., FEBS Lett., 1990, 259, 327-330; Svinarchuk et al., Biochimie, 1993, 75, 49-54); phospholipids, such as hexadecyl-rac-glycerol or triethyl-ammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res., 1990, 18, 3777-3783); polyamine or polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973) or adamantane acetate palmitoyl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229-237), stearylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277, 923-937), tocopherol group (Nishina et al., Molecular Therapy Nucleic Acids, 2015, 4, e220; and Nishina et al., Molecular Therapy, 2008, 16, 734-740), or GalNAc cluster (e.g. WO2014 / 179620).

[0102] In some embodiments, the saRNA of the present application relates to the sense strand or antisense strand of the saRNA conjugated to one or more conjugation groups selected from the group consisting of intercalators, reporters, polyamines, polyamides, peptides, carbohydrates, vitamin moieties, polyethylene glycols, thioethers, polyethers, cholesterol, thiocholesterol, bile acid moieties, folic acid, lipids, phospholipids, biotin, phenazine, phenanthridine, anthraquinone, adamantane, acridine, fluorescein, rhodamine, coumarin, fluorophores, and dyes.

[0103] In some embodiments, the conjugate group comprises an active drug substance, such as aspirin, warfarin, phenylbutazone, ibuprofen, suprofen, fenbufen, ketoprofen, (S)-(+)-pranoprofen, carprofen, dansylsarcosine, 2,3,5-triiodobenzoic acid, fingolimod, flufenamic acid, folinic acid, benzothiadiazide, chlorothiazide, diazepam, Indomethacin, barbiturates, cephalosporins, sulfonamides, antidiabetic medicines, antibacterials, or antibiotics.

[0104] In some embodiments, the saRNA of the present application is conjugated to one or more conjugation groups selected from the group consisting of a lipid, a fatty acid, a fluorophore, a ligand, a sugar, a peptide, and an antibody.

[0105] According to some embodiments, the present disclosure provides compounds tC2 or tC2x4, in Represents a carrier material. According to some embodiments, Represents a carrier material selected from silicon dioxide, silica gel, glass, ceramic, polymer, cellulose and combinations thereof. In some embodiments, the carrier material is in the form of microbeads. The microbeads can be made of any material, including but not limited to magnetic beads, paramagnetic beads, silica beads, agarose beads, etc. According to some embodiments, the conjugated group derived from tC2 has a structure as shown below: Wherein the asterisk indicates the site at which the conjugated group is attached to the chain directly or via a linking moiety (such as those indicated above). According to another embodiment, the conjugated group derived from tC2x4 has a structure as shown below: Wherein the asterisk represents the site that the conjugated group is connected to the chain directly or through a connecting part (such as those noted above). In some embodiments, the saRNA of the present application relates to the sense strand or antisense strand of saRNA conjugated to one or more conjugated groups selected from the following items: cell penetrating peptides, polyethylene glycol, alkaloids, tryptamine, benzimidazole, quinolone, amino acid, cholesterol, glucose and N-acetylgalactosamine. In certain embodiments, the conjugated group is derived from the compound selected from tC2 and tC2x4 shown in the present application. It is understood that one or more terminal atoms (such as hydrogen, halogen, nitrogen, oxygen, sulfur, phosphorus, etc.) or terminal groups (such as hydroxyl, amino, ester group, ether group, acyl group, etc.) of tC2 or tC2x4 compounds can be separated to provide a conjugated group, so the conjugated group can be considered to be obtained by deducting the one or more atoms or terminal groups from tC2 or tC2x4 compounds. According to one embodiment, the conjugated group derived from tC2 or tC2x4 is connected to the chain of the saRNA through a linking moiety such as -OP(O)2O- or -P(O)-O-. According to some embodiments, the saRNA connected to the conjugated group has a structure represented by O1 or O2:

[0106] In some embodiments, a saRNA conjugated to one or more conjugation groups disclosed in the embodiments is directly contacted, transferred, delivered, or administered to a cell or a patient.

[0107] In some embodiments, the sense and antisense strands of the saRNA independently 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.

[0108] In some embodiments, 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 of the saRNA are chemically modified nucleotides.

[0109] These modifications can increase the bioavailability of saRNA, improve affinity for target sequences, and increase resistance to nuclease hydrolysis in cells.

[0110] In some embodiments, the saRNA of the present application, when in contact with a cell, effectively activates or upregulates the expression of one or more genes in the cell, preferably activates or upregulates 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

[0111] In certain embodiments, the present application relates to an isolated target site of the saRNA of the present application, specifically, the isolated target site is a nucleotide sequence having a length in the range of 16 to 35 nucleotides in the nucleotide sequence shown in SEQ ID NO: 1390. In certain embodiments, the isolated target site is a nucleic acid sequence selected from SEQ ID NO: 1-272. The isolated target site is capable of interacting with the antisense strand of the saRNA disclosed in the present application, and is therefore capable of activating the expression of the SERPING1 gene (e.g., mRNA expression, protein expression, C1IHN expression). In some embodiments, the target site is selected on the gene sequence. In some embodiments, the target site is selected on a sequence close to the transcription start site (TSS) of the gene. In some embodiments, the target site is selected upstream of the promoter sequence of the TSS. In some embodiments, the target site is selected on a sequence of -5000bp, -4000bp, -3000bp, -2000bp, -1000bp, or -500bp upstream of the TSS. In some embodiments, the target site is selected by moving 1 bp toward the TSS at a time and generating a target sequence, then repeating the step and adding additional base pairs (e.g., n+1) to the TSS. 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. Hot Spots

[0112] In certain embodiments, the present application relates to an isolated nucleic acid sequence, or "hotspot", located upstream of the transcription start site of the human SERPING1 gene or the mouse Serping1 gene. In certain embodiments, the isolated nucleic acid sequence disclosed herein is an oligonucleotide sequence having a length of at least 42 consecutive nucleotides and having at least 75%, or at least 80%, or at least 85%, or at least 90% sequence homology with an equal length region within the nucleotide sequence shown in SEQ ID NO: 1390. "Hotspot" and "hotspot region" can be used interchangeably and are defined herein as a nucleic acid region on the target sequence of saRNA, where the full-length target of functional saRNA is enriched and spans the 5' end of the first saRNA target to the 3' end of the last saRNA target within each hotspot. In some embodiments, saRNAs designed to target at least 30% (e.g., 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%) of the hotspots are functional, i.e., can induce at least a 1.1-fold change in mRNA levels or protein expression of the target gene. In a non-limiting example, saRNAs are designed according to the following criteria: (1) having a GC content of 35% to 65%; (2) having less than 5 consecutive identical nucleotides; (3) having 3 or fewer dinucleotide repeats; and (4) having 3 or fewer trinucleotide repeats. In some embodiments, the isolated nucleic acid has a length of about 25 to about 250 nucleotides (e.g., about 33 to about 200 nucleotides, about 36 to about 150 nucleotides, about 39 to about 100 nucleotides, about 42 to about 75 nucleotides, about 45 to about 70 nucleotides, or about 48 to about 55 nucleotides). In some embodiments, the hotspot is a nucleic acid sequence selected from SEQ ID NO: 1391-1395. In some embodiments, the hotspot is a nucleic acid sequence selected from the -466 to -399 region, -357 to -279 region, -256 to -215 region, -125 to -81 region and -80 to -15 region upstream of the transcription start site of the SERPING1 gene. The present application also provides a method for designing saRNA, which provides a saRNA targeting the isolated nucleic acid sequence described in the present application.

[0113] The RNAa activity of each designed saRNA depends on a myriad of complex factors, such as the chromatin environment, sequence characteristics of the target itself and nearby regions, transcription factor binding, etc. The core potential determining factor may be the accessibility of the DNA target. In regions with higher accessibility, dsRNAs may show higher RNAa activity. However, dsRNAs designed to target other regions of the promoter may exhibit non-functional or even transcriptional silencing effects. This may explain the existence of hotspots where functional saRNAs cluster together.

[0114] In some aspects, methods using isolated nucleic acids upstream of the transcriptional target site of the SERPING1 gene are also provided. DNA encoding saRNA

[0115] In certain embodiments, the present application relates to nucleic acids or polynucleotides encoding saRNA, which can activate or upregulate the expression of the SERPING1 gene in the cell by at least 10%. In certain embodiments, the nucleic acid is a DNA encoding saRNA. In certain embodiments, the nucleic acid is a recombinant vector, specifically a recombinant AAV vector. The vector disclosed herein includes a DNA fragment encoding the saRNA of the present application. Cells containing saRNA

[0116] After contacting the cell, the saRNA disclosed herein can effectively activate or upregulate the expression of the SERPING1 gene in the cell, preferably upregulating the expression by at least 10%.

[0117] 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 liver cancer cell (such as a HepG2 cell) and a human liver cell, or a cell from a mouse, such as a mouse embryonic liver cell (such as an embryonic cell line BNL.CL2), a mouse liver cancer cell (such as a liver cancer cell line LPC-H12) and a primary mouse liver cell (PMH). The cells disclosed herein may be in vitro or ex vivo, such as a cell line or cell strain, or may be present in a mammal, such as a human body. The human body disclosed herein is a patient suffering from a disease or symptom caused by a SERPING1 gene mutation, a low C1IHN level and / or insufficient levels of a functional C1IHN protein in the blood. In some embodiments, the cell is from a HAE patient. Compositions comprising saRNA

[0118] In certain embodiments, the application relates to a composition or pharmaceutical composition comprising saRNA or nucleic acid of the 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, which is selected from aqueous carriers, liposomes or LNPs, polymers, micelles, colloids, metal nanoparticles, non-metallic nanoparticles, bioconjugates (e.g., GalNAc), polypeptides, and antibodies. In one embodiment, the aqueous carrier can be, for example, RNase-free water or RNase-free buffer. The composition may contain 0.001nM-150nM, such as 0.01nM-100nM, such as 0.1nM-50nM, such as 1nM-20nM, such as 0.001nM-1nM, 1nM-10nM, 10nM-100nM, 10nM-50nM, 20nM-50nM, 20nM-100nM, such as 50nM of the above-mentioned saRNA or the isolated polynucleotide according to the present application. In some embodiments, the composition comprises 1nM-150nM of the saRNA of the present application. Methods of using saRNA

[0119] Another aspect of the present application relates to a saRNA for activating / upregulating SERPING1 expression in a cell. The saRNA comprises an oligonucleotide sequence having a length of 16 to 35 consecutive nucleotides. In some embodiments, the oligonucleotide sequence has at least 75%, or at least 80%, or at least 85%, or at least 90% homology or complementarity with the equal length region shown in SEQ ID NO: 1390, specifically, the saRNA activates / upregulates the expression of the SERPING1 gene 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%). In certain embodiments, when the saRNA disclosed in the embodiments is administered, for example, to a cell or subject, the expression of the SERPING1 gene is activated / upregulated by at least 5 times (e.g., at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 12 times, at least 15 times, at least 18 times, at least 20 times, or at least 24 times). In certain embodiments, the saRNA activates / upregulates the expression of the SERPING1 gene by 24 times. In certain embodiments, the expression of the SERPING1 gene is activated / upregulated by administering to the cell a saRNA disclosed in the embodiments 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, or 150 nM. In certain embodiments, induction of SERPING1 protein (C1INH) is activated / upregulated by administering to the cells a saRNA disclosed in the embodiments 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.

[0120] Another aspect of the present application relates to a method for preventing or treating an individual's disorder or condition induced by insufficient expression of plasma protein C1 inhibitory protein (C1INH), SERPING1 gene mutations, and / or low functional C1IHN levels in the blood, the method comprising: administering an effective amount of saRNA disclosed herein, nucleic acid encoding saRNA, or isolated polynucleotides or compositions comprising saRNA to the individual. In certain embodiments, the effective amount of saRNA disclosed herein can be a concentration in the range of 0.01nM to 50nM, such as 0.01nM, 0.02nM, 0.05nM, 0.08nM, 0.1nM, 0.2nM, 0.3nM, 0.4nM, 0.5nM, 0.6nM, 0.8nM, 1nM, 5nM, 10nM, 25nM, 50nM, 75nM, 100nM or 150nM. In some embodiments, the disorder or condition is hereditary angioedema (HAE). In some embodiments, the subject is a mammal. In some embodiments, the subject is a human.

[0121] In any of the embodiments provided herein, such saRNA, nucleic acids encoding saRNA of the present application, or compositions comprising such saRNA of the present application can be directly introduced into cells, or can be produced in cells after introducing nucleotide sequences encoding saRNA into cells (e.g., mammalian cells, including but not limited to HepG2, Hep3B, PLC / PRF / 5 and PMH, or human cells). Such cells can be in vitro, such as cell lines, etc., or can be present in mammals (such as humans). In some embodiments, humans are patients or individuals with C1INH deficiency-related conditions or HAE. In certain embodiments, nucleic acids of the present application or isolated polynucleotides encoding saRNA or compositions comprising the above-mentioned saRNA are each present in an amount sufficient to achieve treatment of HAE.

[0122] Another aspect of the present application relates to the administration route of the saRNA disclosed in the embodiments to an individual. 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 other embodiments, the administration route is selected from one or more of the following: intrathecal, intramuscular, intravenous, intraarterial, intraperitoneal, intravesical, intraventricular, intravitreal, and subcutaneous administration. Dosage regimen and route of administration

[0123] Aspects of the present application relate to a pharmaceutical composition comprising the saRNA of the present invention. In some embodiments, the pharmaceutical composition comprises the saRNA of the present application and a pharmaceutically acceptable carrier, a therapeutic inert carrier, a diluent, or a pharmaceutically acceptable excipient. The pharmaceutical composition disclosed herein will be developed into a drug for preventing or treating C1INH deficiency-related conditions or HAE.

[0124] Aspects of the present application also relate to methods of preparing such compositions using the saRNA of the present application.

[0125] Another aspect of the present application relates to the use of the saRNA of the present application in the preparation of a pharmaceutical composition disclosed herein.

[0126] Another aspect of the present application relates to the use of saRNA or isolated polynucleotides according to any of the embodiments described herein or compositions according to any of the embodiments described herein in the preparation of a medicament for preventing or treating a gene or protein-related symptom induced by insufficient expression of C1IHN protein, SERPING1 gene mutation, and / or low functional C1IHN protein levels in the blood in an individual. According to the use of certain embodiments, the condition may include a SERPING1 mutation-related disorder or condition, including hereditary angioedema disease. According to the use of certain embodiments, the symptom induced by insufficient expression of C1IHN protein is hereditary angioedema. It also relates to the use according to certain embodiments, wherein the individual is a mammal, such as a human.

[0127] The dosage at which the saRNA or composition of the present application can be administered can vary within a wide range and will of course be in accordance with the individual needs in each case. In certain embodiments, the first dose of the pharmaceutical composition according to the present application is administered when the subject is less than one week, less than one month, less than 3 months, less than 6 months, less than one year old, less than 2 years old, less than 15 years old, or greater than 15 years old.

[0128] A single dose of saRNA can be 0.01 mg / kg to 1000 mg / kg, for example, a single dose in the range of about 0.01 mg / kg, 0.02 mg / kg, 0.05 mg / kg, 0.1 mg / kg, 0.2 mg / kg, 0.5 mg / kg, 1 mg / kg, 2 mg / kg, 2.5 mg / kg, 5 mg / kg, 7.5 mg / kg, 10 mg / kg, 12.5 mg / kg, 15 mg / kg, 17.5 mg / kg, 20 mg / kg, 25 mg / kg, 30 mg / kg, 40 mg / kg, 50 mg / kg, 75 mg / kg, 100 mg / kg, 120 mg / kg, 150 mg / kg, 200 mg / kg, 250 mg / kg, 300 mg / kg, 400 mg / kg, 500 mg / kg, 750 mg / kg, or 1000 mg / kg. The dosages described herein may contain two or more saRNA sequences within the saRNA of the present application.

[0129] The proposed dosing frequency is approximate, for example, in certain embodiments, if the proposed dosing frequency is dosing on day 1 and a second dosing on day 29, the HAE patient may receive the second dosing 25, 26, 27, 28, 29, 30, 31, 32, 33, or 34 days after receiving the first dosing. For example, in certain embodiments, if the proposed dosing frequency is dosing on day 1 and a second dosing on day 15, the HAE patient may receive the second dosing 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days after receiving the first dosing. For example, in certain embodiments, if the proposed dosing frequency is a dose on day 1 and a second dose on day 85, the HAE patient may receive the second dose 80 days, 81 days, 82 days, 83 days, 84 days, 85 days, 86 days, 87 days, 88 days, 89 days, or 90 days after receiving the first dose.

[0130] In certain embodiments, the dose and / or volume of injection will be adjusted based on the patient's age, the patient's weight, the patient's body surface area, the patient's symptoms, and / or the patient's sex.

[0131] In certain embodiments, the pharmaceutical composition comprises a co-solvent system. Such a co-solvent system comprises, for example, benzyl alcohol, a non-polar surfactant, a water-miscible organic polymer, and an aqueous phase. In certain embodiments, such a co-solvent system is used for hydrophobic compounds. A non-limiting example of such a co-solvent system is a VPD co-solvent system, which comprises 3% w / v benzyl alcohol, 8% w / v non-polar surfactant Polysorbate 80 TMand 65% w / v polyethylene glycol 300 in absolute ethanol. The proportions of such cosolvent systems can be varied widely without significantly changing their solubility and toxicity characteristics. In addition, the identity of the cosolvent components can be varied: for example, other surfactants can be used instead of Polysorbate 80 TM ; The fraction size of polyethylene glycol can vary; other biocompatible polymers can replace polyethylene glycol, such as polyvinyl pyrrolidone; and other sugars or polysaccharides can replace dextrose.

[0132] Examples of other compositions or components associated with the small activated nucleic acid molecules, compositions and methods described herein include, but are not limited to: diluents, salts, buffers, chelating agents, preservatives, desiccants, antimicrobial agents, needles, syringes, packaging materials, tubes, bottles, flasks, beakers, etc., such as for use, modification, assembly, storage, packaging, preparation, mixing, dilution and / or preservation of components for specific uses. In embodiments using a liquid form of any component, the liquid form may be concentrated or ready for use.

[0133] The lipid moieties used in nucleic acid therapy can be applied to the present application to deliver saRNA molecules disclosed herein. In some such methods, nucleic acids, such as oligomeric compounds, are introduced into preformed liposomes or lipid complexes made of a mixture of cationic lipids and neutral lipids. In some methods, saRNA complexes with monocationic or polycationic lipids are formed in the absence of neutral lipids. In certain embodiments, the lipid moiety is selected to increase the distribution of the agent to specific cells or tissues. In certain embodiments, the lipid moiety is selected to increase the distribution of the agent to adipose tissue. In certain embodiments, the lipid moiety is selected to increase the distribution of the agent to muscle tissue.

[0134] In certain embodiments, the pharmaceutical composition comprises a delivery system. Examples of delivery systems include, but are not limited to, liposomes and emulsions. Certain delivery systems can be used to prepare certain pharmaceutical compositions, including those comprising hydrophobic compounds. In certain embodiments, certain organic solvents are used, such as dimethyl sulfoxide.

[0135] In certain embodiments, the pharmaceutical composition comprises one or more tissue-specific delivery molecules designed to deliver one or more agents of the invention to specific tissues or cell types. For example, in certain embodiments, the pharmaceutical composition comprises liposomes coated with tissue-specific antibodies.

[0136] In some embodiments, saRNA can be delivered or administered via a vector. Any vector that can be used for gene delivery can be used. In some variations, viral vectors (such as AAV, adenovirus, lentivirus, retrovirus) can be used. Non-limiting examples of vectors that can be used in this 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) and vaccinia virus (VV).

[0137] In some embodiments, the vector is a recombinant AAV vector. AAV vectors are relatively small DNA viruses that can be integrated into the genome of the cells they infect in a stable and site-specific manner. They can infect a wide range of cells without inducing any effects on cell growth, morphology or differentiation, and they are not shown to be involved in human pathology. The AAV genome has been cloned, sequenced and characterized. It contains approximately 4700 bases and contains an inverted terminal repeat (ITR) region of approximately 145 bases at each end, which serves as the origin of viral replication. The remainder of the genome is divided into two essential regions with encapsulation function: 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 viral capsid protein.

[0138] AAV vectors can be prepared using standard methods in the art. Adeno-associated virus of any serotype is suitable (see, e.g., Blacklow, pp. 165-174 of "Parvoviruses and Human Disease", edited by JR Pattison (1988); Rose, Comprehensive Virology 3:1, 1974; P. Tattersall, "The Evolution of Parvovirus Taxonomy" In Parvoviruses (edited by JR Kerr, SF Cotmore., ME Bloom, RM Linden, CR Parrish) pp. 5-14, Hudder Arnold, London, England (2006); and DE Bowles, J E Rabinowitz, RJ Samulski, "The Genus Dependovirus" (edited by JR Kerr, SF Cotmore., ME Bloom, RM Linden, CR Parrish) pp. 15-23, Hudder Arnold, London, England (2006), the disclosures of which are incorporated herein by reference in their entirety). Methods for purifying vectors can be found, for example, in U.S. Pat. Nos. 6,566,118, 6,989,264, and 6,995,006 and WO / 1999 / 011764, entitled "Methods for Generating High Titer Helper-free Preparation of Recombinant AAV Vectors," the disclosures of which are incorporated herein by reference in their entirety. Preparation of hybrid vectors is described, for example, in PCT Application No. PCT / US2005 / 027091, the disclosures of which are incorporated herein by reference in their entirety. Gene transfer in vitro and in vivo using vectors derived from AAV has been described (see, for example, International Patent Application Publication Nos. 91 / 18088 and WO 93 / 09239; U.S. Pat. Nos. 4,797,368, 6,596,535, and 5,139,941; and European Patent No. 0488528, all of which are incorporated herein by reference in their entirety). These publications describe various AAV-derived constructs in which the rep and / or cap genes are deleted and replaced with a gene of interest, and the use of these constructs to transfer a gene of interest in vitro (into cultured cells) or in vivo (directly into an organism).The replication-deficient recombinant AAV according to the present application can be prepared by co-transfecting a plasmid containing a nucleic acid sequence of interest flanked by two AAV inverted terminal repeat (ITR) regions and a plasmid carrying AAV encapsulation genes (rep and cap genes) into a cell line infected with a human helper virus (e.g., adenovirus). The resulting AAV recombinant is then purified by standard techniques.

[0139] In some embodiments, the vectors used in the methods of the present application are encapsulated into viral particles (e.g., AAV viral particles, including but not limited to AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, and AAV16). Thus, the present application may include recombinant viral particles (recombinant because they contain recombinant polynucleotides) comprising any of the vectors described herein. Methods for preparing such particles are known in the art and are described in U.S. Pat. No. 6,596,535.

[0140] The formulations, pharmaceutical compositions or medicaments of the present application are formulated, dosed and administered in a manner consistent with "good medical practice". Factors considered in this context include the specific disorder being treated, the specific mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the delivery site of the agent, the method of administration, the timing of administration, and other factors known to physicians.

[0141] For the formulations, pharmaceutical compositions or medicaments of the present application, delivery can optionally be by parenteral infusion, including intrathecal, intramuscular, intravenous, intraarterial, intraperitoneal, intravesical, intracerebroventricular, intravitreal or subcutaneous administration; or by oral administration, intranasal administration, inhalation administration, vaginal administration or rectal administration.

[0142] Typical formulations of small activated nucleic acid molecules in the present application are prepared by mixing the saRNA of the present application with a carrier or excipient. Suitable carriers and excipients are well known to those skilled in the art and are described in detail in, for example, Ansel HC et al., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems (2004) Lippincott, Williams & Wilkins, Philadelphia; Gennaro AR et al., Remington: The Science and Practice of Pharmacy (2000) Lippincott, Williams & Wilkins, Philadelphia; and Rowe RC, Handbook of Pharmaceutical Excipients (2005) Pharmaceutical Press, Chicago. The formulation may also include one or more buffers, stabilizers, surfactants, wetting agents, lubricants, emulsifiers, suspending agents, preservatives, antioxidants, opacifiers, glidants, processing aids, colorants, sweeteners, fragrances, flavorings, diluents and other known additives to provide an elegant presentation of the drug (i.e., the saRNA of the present application or its pharmaceutical composition) or facilitate the preparation of the drug product (i.e., the drug). Diagnostic Methods

[0143] Another aspect of the present application relates to a method for detecting C1INH protein or C1INH-regulated protein (e.g., bradykinin) in blood. In certain embodiments, the method is used to detect C1INH protein or C1INH-regulated protein (e.g., bradykinin) in cells transfected with saRNA disclosed herein, isolated polynucleotides, or compositions comprising saRNA. As certain embodiments, the methods disclosed herein can be used to detect patients in a specific subgroup who suffer from disorders or conditions induced by insufficient expression of plasma protein C1 inhibitory protein (C1INH), SERPING1 gene mutations, and / or low functional C1IHN levels in the blood. As an alternative embodiment of the method disclosed herein, the method can be used for the efficacy or safety monitoring of patients treated with saRNA, nucleic acids encoding saRNA, or isolated polynucleotides, compositions, or drugs described above.

[0144] In certain embodiments, the baseline measurement is obtained from a biological sample as defined herein, obtained from an individual prior to administering a therapy described herein. In certain embodiments, the biological sample is peripheral blood mononuclear cells, plasma, serum, skin tissue, cerebrospinal fluid (CSF).

[0145] In some embodiments, the saRNA provided herein activates the amount of functional C1INH protein in the 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 above baseline measurements.

[0146] In a specific embodiment, saRNA shows an effect greater than an additive effect or a synergistic effect in the treatment, prevention, delayed progression and / or improvement of a disease caused by a SPERING1 gene mutation, and additionally in the protection of cells associated with the pathophysiology of the disease, in particular for the treatment, prevention, delayed progression and / or improvement of HAE.

[0147] Another aspect of the present application relates to a method for activating / upregulating the expression of the SERPING1 gene in a cell, the method comprising: administering the saRNA or isolated polynucleotide or composition of the embodiments disclosed herein. In some embodiments, the saRNA or isolated polynucleotide or composition is directly introduced into the cell. In some embodiments, the saRNA of the embodiments disclosed herein is produced in the cell after the nucleotide sequence encoding the saRNA is introduced into the cell. In some embodiments, the cells disclosed herein are mammalian cells, preferably human cells.

[0148] Another aspect of the present application relates to a method for increasing the level of C1IHN protein in a patient's cells or the level of functional C1IHN protein in the blood, the method comprising introducing an effective amount of saRNA, a nucleic acid or polynucleotide encoding saRNA, or a composition of the embodiments disclosed herein into a cell or subject. Reagent test kit

[0149] Another aspect of the present application relates to a kit for implementing a method for increasing C1IHN protein levels in cells or functional C1IHN protein levels in blood, the kit comprising saRNA and instructions for use disclosed in the embodiments. In certain embodiments, the kit also includes means for administering the saRNA to an individual. In certain embodiments, the kit is in a labeled package, and the label on the package indicates that the saRNA or composition can be used to prevent or treat a disease or condition induced by insufficient expression of plasma protein C1 inhibitory protein (C1INH) or directed to HAE.

[0150] As used herein, "kit" is generally defined as a package, assembly or container (e.g., an insulated container) comprising one or more of the components or embodiments of the present application and / or other components related to the present application, such as those described above. Any agent or component of the kit can be provided in liquid form (e.g., solution) or in solid form (e.g., dry powder, frozen, etc.).

[0151] In other embodiments, the kit may include instructions for use of any form of kit related to components and / or methods as described herein or instructions from websites or other sources. For example, instructions may include instructions for use, modification, mixing, dilution, preservation, assembly, storage, packaging and / or preparation of components and / or other components related to the kit. In some cases, instructions may also include instructions for delivery (e.g., transport or storage under conditions such as room temperature, subzero temperature, low temperature) of components. Instructions may be provided in any form (such as written or oral (e.g., telephone), digital, optical, visual (e.g., videotape, DVD, etc.) and / or electronic communication (including Internet or network-based communication) forms) available to the user of the kit.

[0152] Another aspect of the present application relates to a kit for detecting C1INH protein or C1INH-regulated protein (e.g., bradykinin) in blood. In certain embodiments, the kit is used to detect C1INH protein or C1INH-regulated protein (e.g., bradykinin) in cells transfected with saRNA or isolated polynucleotides of any embodiment of the embodiments disclosed herein or compositions disclosed herein. saRNA synthesis

[0153] The present application provides a method for preparing small activating nucleic acid molecules, which includes sequence design and synthesis.

[0154] Small activating nucleic acid molecules can be chemically synthesized or obtained from biotechnology companies specializing in nucleic acid synthesis. In general, the chemical synthesis of nucleic acids includes the following four steps: a) synthesis of oligoribonucleotides; b) deprotection; c) purification and separation; d) desalting and annealing. For example, the specific steps for chemically synthesizing saRNA are as follows: Synthetic oligoribonucleotides:

[0155] 1 μM RNA was synthesized in an automatic DNA / RNA synthesizer (e.g., Applied Biosystems EXPEDITE8909), and the coupling time for each cycle was set to 10 to 15 minutes. Using a solid-phase-bound 5'-Op-dimethoxytrityl-thymidine matrix as an initiator, one base was bound to the solid phase matrix in the first cycle, and then one base was bound to the base bound in the n-1 cycle in the nth (19≥n≥2) cycle. This process was repeated until the synthesis of the entire nucleic acid sequence was completed. Deprotection

[0156] The solid phase matrix bound to saRNA is placed in a test tube, and a mixture solution of 1 mL of ethanol and ammonium hydroxide (volume ratio: 1:3) is added to the test tube. The test tube is then sealed and placed in an incubator, the mixture is incubated at 25°C to 70°C for 2 to 30 hours, the solution containing the solid phase matrix bound to saRNA is filtered, and the filtrate is collected. The solid phase matrix is ​​rinsed twice with double distilled water (1 mL each time), and the filtrate is collected. The collected eluents are combined, vacuum dried for 1 to 12 hours, and then 1 mL of tetrabutylammonium fluoride in tetrahydrofuran (1M) solution is added to the solution, left to stand at room temperature for 4 to 12 hours, and then 2 mL of n-butanol is added. The precipitate is collected by high-speed centrifugation to obtain a crude single-stranded saRNA product. Purification and isolation

[0157] The obtained saRNA crude product was dissolved in 2 mL of an aqueous solution of ammonium acetate having a concentration of 1 mol / mL, and the solution was separated using a reverse phase C18 high pressure liquid chromatography column to obtain a purified saRNA single-stranded product. Desalting and annealing

[0158] Salts were removed by gel filtration (size exclusion chromatography). A single sense oligoribonucleic acid chain and a single antisense oligoribonucleic acid chain were mixed in a 1:1 molar ratio into 1 mL to 2 mL of buffer (10 mM Tris, pH = 7.5-8.0, 50 mM NaCl). The solution was heated to 95 ° C and then slowly cooled to room temperature to obtain a solution containing saRNA. Cell culture and treatment

[0159] Human hepatoma HepG2 (SCSP-510, National Certified Cell Culture Collection, China) cells were cultured in modified DMEM medium (Gibco, Thermo Fisher Scientific, Carlsbad, CA) supplemented with 10% calf serum (Sigma-Aldrich) and 1% penicillin / streptomycin (Gibco) at 37°C and 5% CO2. PMH were isolated from the liver of C57BL / 6J mice (Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd.) and cultured in modified Willian's Medium E (WME) medium (A12176-01, Gibco, Thermo Fisher Scientific, Carlsbad, CA) supplemented with 1% insulin (S6955, Selleck, USA) and 1% penicillin / streptomycin (Gibco) at 5% CO2 and 37°C. HepG2 and PMH cells were seeded into 96-well plates at 5000 cells / well, respectively. Following the reverse transfection protocol, saRNAs were transfected individually into HepG2 cells in each well with 0.3 μL of RNAiMAX (Invitrogen, Carlsbad, CA) at a final concentration of 2.5 nM, 10 nM, or 25 nM or any other concentration, respectively, for a transfection duration of 72 hours. Mock (blank control) was transfected in the absence of oligonucleotides. dsCon2 (SEQ ID NO: 547 and 821) was used as a non-specific duplex control. DS12A-si2 (SEQ ID NO: 273, 546, and 820) is a duplex siRNA of the SERPING1 gene and was transfected as a negative control. RT-qPCR, PI staining and ELISA assays (1) One-step reverse transcription-quantitative polymerase chain reaction (one-step RT-qPCR)

[0160] At the end of transfection, the medium was discarded and the cells were washed once with 150 μL PBS per well. After removing the PBS, 100 μL cell lysis buffer (Power Green Cells to-Ct TM Kit, Life Technologies) was added to each well and incubated at room temperature for 5 minutes. 0.5 μL of cell lysate was taken from each well and PCR was performed using One Step TB Green in a Roche Lightcycler 480 real-time PCR instrument. TM PrimeScript TMRT-PCR Kit II (Takara, RR086A) was used for analysis by RT-qPCR. PCR reactions were prepared using the Bravo automated liquid handling platform (Agilent). Each transfected sample was amplified in 3 replicate wells. PCR reaction conditions are shown in Table 3 below. Table 3. PCR reaction preparation

[0161] The reaction conditions are as follows: reverse transcription reaction (stage 1): 42°C for 5 minutes, 95°C for 10 seconds; PCR reaction (stage 2): 95°C for 5 seconds, 59°C for 20 seconds, 72°C for 10 seconds; 40 amplification cycles; and melting curve (stage 3). Human SERPING1 gene and mouse Serping1 gene were amplified as target genes. Human reference genes (HPRT1 and TBP) and mouse reference genes (Hprt1 and Tbp) were also amplified as internal controls for RNA loading. All primer sequences are listed in Table 4. Table 4. Primer sequences used for RT-qPCR assays (2) Two-step RT-qPCR

[0162] To quantify the expression of mRNA in cells, total cellular RNA was isolated from treated cells using the RNeasy Plus Mini kit (Qiagen, Hilden, Germany) according to its instructions. The resulting RNA (1 μg) was reverse transcribed into cDNA using a PrimeScript RT kit containing gDNA Eraser (Takara, Shiga, Japan). The resulting cDNA was amplified using SYBR Premix Ex Taq II (Takara, Shiga, Japan) reagent and primers that specifically amplify the target gene of interest in Roche Light Cycler 480 Multiwell Plate 384 (Roche, reference: 4729749001, USA). The reaction conditions were as follows: reverse transcription reaction (stage 1): 42°C for 5 minutes, 95°C for 10 seconds; PCR reaction (stage 2): 95°C for 5 seconds, 60°C for 30 seconds, 72°C for 10 seconds; 40 amplification cycles; melting curve (stage 3). The PCR reaction conditions are shown in Tables 5 and 6. Table 5. RT reaction Table 6. RT-qPCR reaction

[0163] To calculate the expression level of SERPING1 or Serping1 (target gene) in saRNA transfected samples relative to control treatment (Mock) (E rel ), substitute the Ct values ​​of the target gene and the two internal reference genes into formula III, E rel =2 (CtTm-CtTs) / ((2 (CtR1m-CtR1s) *2 (CtR2m-CtR2s) ) (1 / 2) ) (Formula III) Among them, CtT m is the Ct value of the target gene from the mock treated sample; CtT s is the Ct value of the target gene from saRNA treated samples; CtR1 m is the Ct value of the internal reference gene 1 from the mock treated sample; CtR1 s is the Ct value of reference gene 1 from saRNA treated samples; CtR2 m is the Ct value of the reference gene 2 from the mock treated sample; and CtR2 s is the Ct value of internal reference gene 2 from saRNA treated samples. (3) Propidium iodide (PI) staining

[0164] After saRNA treatment, HepG2 or PMH cells were placed in 96-well plates and cultured for 72 hours. The cells were washed with cold PBS and lysed with 40 μL / well of cell lysis buffer (0.25% Igeal CA-630, 140 mM NaCl, 2 mM DTT, 10 mM Tris, pH 7.4) containing 1.5 μM PI. The plate was incubated on ice for 5 minutes, and then the optical density (OD) was measured at 535 nm excitation and 615 nm emission wavelength on a microplate reader system (Infinite M2000Pro). (4) ELISA assay

[0165] To quantify the expression level of human SERPING1 protein, the supernatant of cultured cells (e.g., HepG2, Hep3B or PLC / PRF / 5) was collected and detected by OD value using an ELISA kit (R&D Systems, catalog number: DY008, DY2488-05, USA). The expression level of human SERPING1 protein was obtained according to the following ELISA kit instructions provided by the kit manufacturer. (5) Statistical analysis

[0166] One-way analysis of variance (ANOVA) followed by Tukey's multiple comparisons was used to compare the differences between groups of continuous variables. P values ​​less than 0.05 were considered significantly different. * / # indicates p < 0.05, ** / ## indicates p < 0.01, *** / ### indicates p < 0.001, and **** / #### indicates p < 0.0001. Animal surgery

[0167] All animal surgical procedures were performed by certified laboratory personnel using protocols that complied with local and state regulations and were approved by the Institutional Animal Care and Use Committee. C57BL / 6J mice (B204, Beijing, China) purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd. were dosed by subcutaneous (SC) injection. Primary mouse hepatocyte (PMH) isolation and free uptake assay

[0168] C57BL / 6J mice (Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.) were anesthetized with isoflurane, and the initial flushing reagent and digestion reagent were perfused in sequence, and then the liver was placed in a 10 cm culture dish containing culture medium. The liver lobe was torn open with two tweezers to obtain a suspension, which was filtered through a 70-75 micron membrane, and the cell suspension was collected in a 50 mL conical tube. Then, centrifuged in a rocker centrifuge at 100 × g for 2 minutes at 4 ° C, the supernatant was removed and 20 mL of cold PBS was drawn with a pipette to wash the cells (repeat the wash twice). Cell viability was tested using 0.4% trypan blue, and the cells were inoculated into cell culture plates coated with type I collagen 4 to 12 hours before cell inoculation to ensure that at least 60% of the cells in the culture medium were confluent. Ensure that all cells grow to their full size after inoculation of cells, resulting in a final confluence of 90% to 95% for experiments.

[0169] It was found in the present application that the above-mentioned saRNA, after being introduced into cells, can effectively increase the expression levels of SERPING1 mRNA and protein.

[0170] The present application will be further described below with reference to specific examples and accompanying drawings. It should be understood that these examples are only used 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 specifying specific conditions are usually based on conventional conditions, such as the conditions described in Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Example Example 1: Design and synthesis of saRNA targeting the human SERPING1 promoter

[0171] The coding chain of the promoter sequence of the human SERPING1 gene (from the transcription start site (TSS) to the upstream -500bp fragment) (SEQ ID NO: 1390) was retrieved from the ENSEMBL genome database. In order to identify functional small activating RNA (saRNA) capable of activating the expression of the SERPING1 gene, a series of 19nt saRNA targets were selected on the 500bp SERPING1 promoter sequence (Table 7), starting from the upstream -500bp of the TSS, moving 1bp to the TSS each time, and generating target sequences. Table 7. Deduced human SERPING1 promoter sequence (5'-3') (SEQ ID NO: 1390)

[0172] The target sequences were then filtered to retain those that met the following criteria: (1) had a GC content of 35% to 65%; (2) had less than 5 consecutive identical nucleotides; (3) had 3 or fewer dinucleotide repeats; and (4) had 3 or fewer trinucleotide repeats. After filtering, 272 target sequences were retained for determining the sense strand sequence of candidate saRNAs.

[0173] The saRNA is designed so that the sense strand sequence and the antisense strand sequence form a saRNA duplex, wherein each of the sense strand and the antisense strand is 21 nt in length. The 19 nucleotides in the 5' region of the first ribonucleic acid chain (sense strand) of the saRNA have 100% sequence identity with the target sequence of the promoter, and the 3' end of the first ribonucleic acid chain includes an additional 2nt dTdT sequence. It is possible that the 19 nucleotides in the 5' region of the second ribonucleic acid chain (antisense strand) are complementary to the first ribonucleic acid chain sequence, and the 3' end of the second ribonucleic acid chain includes an additional 2nt dTdT sequence. The two chains of the above saRNA are chemically synthesized, mixed and annealed at a molar ratio of 1: 1 to obtain a duplex saRNA. Table 1 lists the saRNA target sequences, their sense strands and antisense strands designed in the embodiment. Example 2: High-throughput screening of saRNA targeting the human SERPING1 promoter

[0174] To identify saRNAs that can activate SERPING1 transcription, HepG2 cells were transfected with each of the above 272 saRNAs at a transfection concentration of 25 nM. After 72 hours, the transfected cells were lysed and the lysates were analyzed by one-step RT-qPCR to obtain the relative (compared to Mock treatment) expression level of the SERPING1 gene for each saRNA-treated sample. The results of the relative expression of SERPING1 mRNA (fold change) are listed in Table 1. Figure 1 The activity distribution (log2) of SERPING1 saRNA sorted from the strongest activating protein to the strongest inhibitory protein is shown. Table 8 further shows that 101 (37.1%), 27 (9.9%) and 11 (4.0%) saRNAs showed high activation (≥1.5 times), moderate activation (1.2 times-1.5 times) and mild activation (1.1 times-1.2 times) on SERPING1 mRNA levels, respectively. 133 (49.0%) saRNAs had no significant effect on the expression of SERPING1 mRNA. The highest activation observed for SERPING1 mRNA expression was 24.69 times. SaRNAs with activation activity are referred to as "functional saRNAs" in this application. Table 8. Summary of the activities of all screened SERPING1 saRNAs

[0175] When functional saRNAs were classified by their location on the human SERPING1 promoter ( Figure 2 ), it can be clearly seen that functional saRNAs are distributed on the promoter in an aggregated manner, that is, in certain promoter regions, there are "hot spots" where functional saRNAs are enriched. Among the tested promoter sequences, there are 5 hot spots located at -466 to -399 region (H1), -357 to -279 region (H2), -256 to -215 region (H3), -125 to -81 region (H4) and -80 to -15 region (H5) relative to TSS, and contain highly enriched functional saRNAs. By following the design criteria: (1) having a GC content of 35% to 65%; (2) having less than 5 consecutive identical nucleotides; (3) having 3 or fewer dinucleotide repeats; and (4) having 3 or fewer trinucleotide repeats, at least 30% of the designed saRNAs targeting the provided hot spot sequences can activate the expression of the SERPING1 gene by at least 10%.

[0176] The functional saRNA sequences that were able to upregulate SERPING1 expression by at least 1.1-fold and located on hotspot H1 (-466 to -399) were as follows: DS12A-0224, DS12A-0156, DS12A-0126, DS12A-0193, DS12A-0201, DS12A-0137, DS 12A-0141, DS12A-0168, DS12A-0113, DS12A-0079, DS12A-0094, DS12A-0063, DS12A -0022, DS12A-0007, DS12A-0037, DS12A-0020, DS12A-0096, DS12A-0116, DS12A-01 75. DS12A-0042, DS12A-0089, DS12A-0111, DS12A-0112, DS12A-0133, DS12A-0090.

[0177] The functional saRNA sequences located at hotspot H2 (-357 to -279) are as follows: DS12A-0011, DS12A-0012, DS12A-0026, DS12A-0027, DS12A-0051, DS12A-0100, DS12A-0147, DS12A-0148, DS12A-0101, DS12A-0119, DS12A-0144, DS12A -0066, DS12A-0085, DS12A-0067, DS12A-0220, DS12A-0221, DS12A-0209, DS12A-0271, DS12A-0261, DS12A-0115, DS12A-0138, DS12A-0062, DS12A-0139, DS12A-0080, DS12A-0231, DS12A-0217, DS12A-0140, DS12A-0173, DS12A-0169, DS12A-0114, DS12A-0092, DS12A-0023, DS12A-0021, DS12A-0043, DS12A -0177, DS12A-0182, DS12A-0178, DS12A-0084, DS12A-0102, DS12A-0104, DS12A-0056, DS12A-0004, DS12A-0013, DS12A-0014, DS12A-0070, DS12A-0028.

[0178] Possibly, the functional saRNA sequence located at hotspot H3 (-256 to -215) is as follows: DS12A-0048, DS12A-0045, DS12A-0097, DS12A-0117, DS12A-0082, DS12A-0129, DS12A-0194, DS12A-0215, DS12A-0132, DS12A-0134, DS12A-0025, DS12A-0136, DS12A-0093, DS12A-0077, DS12A-0130, DS12A-0058.

[0179] Hotspot H4 (-125 to -81) contains the following functional saRNAs: DS12A-0030, DS12A-0029, DS12A-0099, DS12A-0145, DS12A-0206, DS12A-0149, DS 12A-0118, DS12A-0151, DS12A-0187, DS12A-0121, DS12A-0017, DS12A-0123, DS12A -0190, DS12A-0210, DS12A-0071, DS12A-0184, DS12A-0272, DS12A-0256, DS12A-02 62. DS12A-0265, DS12A-0170, DS12A-0171, DS12A-0200, DS12A-0172, DS12A-0266.

[0180] Hotspot H5 (-80 to -15) contains the following functional saRNAs: DS12A-0160, DS12A-0191, DS12A-0240, DS12A-0229, DS12A-0225, DS12A-001 8. DS12A-0006, DS12A-0019, DS12A-0049, DS12A-0044, DS12A-0050, DS12A-00 86. DS12A-0219, DS12A-0146, DS12A-0203, DS12A-0207, DS12A-0188, DS12A- 0222, DS12A-0189, DS12A-0212, DS12A-0257, DS12A-0241, DS12A-0242, DS12A -0213, DS12A-0192, DS12A-0214, DS12A-0076, DS12A-0088, DS12A-0110, DS1 2A-0108, DS12A-0128, DS12A-0125, DS12A-0124, DS12A-0258, DS12A-0075, DS 12A-0055, DS12A-0072, DS12A-0106, DS12A-0196, DS12A-0197, DS12A-0195, D S12A-0135, DS12A-0163, DS12A-0164, DS12A-0216, DS12A-0232, DS12A-0247.

[0181] Similar results were observed in the distribution of functional saRNA on the mouse Serping1 promoter. Fig.10 As shown, there are 4 hot spots -424 to -325 region (H6), -257 to -182 region (H7), -170 to -117 region (H8) and -56 to -11 region (H9), where functional saRNA is enriched.

[0182] The functional saRNA sequences that can upregulate Serping1 expression by at least 1.1-fold and are located on the hotspot H6 (-424 to -325) are as follows: DS12B-0170, DS12B-0171, DS12B-0034, DS12B-0035, DS12B-0036, DS12B-0056, DS12B-0085, DS12B-0086, DS12B-0087, DS12B-0269, DS12B-0270, DS 12B-0252, DS12B-0253, DS12B-0254, DS12B-0224, DS12B-0172, DS12B-0173, DS12B-0174, DS12B-0175, DS12B-0097, DS12B-0098, DS12B-0099, DS12B -0100, DS12B-0101, DS12B-0102, DS12B-0176, DS12B-0103, DS12B-0104, DS12B-0105, DS12B-0106, DS12B-0177, DS12B-0225, DS12B-0226, DS12B-02 27. DS12B-0178, DS12B-0179, DS12B-0107, DS12B-0108, DS12B-0109, DS12B-0110, DS12B-0111, DS12B-0112, DS12B-0180, DS12B-0228, DS12B-0181.

[0183] The functional saRNA sequences located at hotspot H7 (-257 to -182) are as follows: DS12B-0188, DS12B-0137, DS12B-0138, DS12B-0139, DS12B-0140, DS12B-0141, DS12B-0142, DS12B-0143, DS12B-0144, DS12B-0145, DS12B-0275, DS12B-0276, DS12B-0277, DS12B-0001, DS12B-0015, DS12B-0016, DS12B-0017, DS12B-0018, DS12B-0019, DS12B-0020, DS12B-0037, DS12B-0021, DS12B-0022, DS12B-0038, DS12B-0057, DS12B-0039, DS12B-0040, DS12B-0023, DS12B-0146, DS12B-0147, DS12B-0189, DS12B-0190.

[0184] It is possible that the functional saRNA sequence located at hotspot H8 (-170 to -117) is as follows: DS12B-0234, DS12B-0255, DS12B-0235, DS12B-0196, DS12B-0236, DS12B-0237, DS12B-0238, DS12B-02 39. DS12B-0047, DS12B-0048, DS12B-0067, DS12B-0090, DS12B-0091, DS12B-0068, DS12B-0049, DS12B -0069, DS12B-0070, DS12B-0071, DS12B-0092, DS12B-0093, DS12B-0094, DS12B-0271, DS12B-0256, DS 12B-0257, DS12B-0072, DS12B-0050, DS12B-0073, DS12B-0074, DS12B-0095, DS12B-0075, DS12B-0096.

[0185] Hotspot H9 (-56 to -11) contains the following functional saRNAs: DS12B-0258, DS12B-0241, DS12B-0259, DS12B-0260, DS12B-0261, DS12B-0242, DS12B-0206, DS12B-0207, DS12B -0156, DS12B-0208, DS12B-0209, DS12B-0210, DS12B-0211, DS12B-0157, DS12B-0212, DS12B-0213, DS12B-0243.

[0186] These results indicate that functional saRNAs are not randomly distributed on the promoter, but are aggregated in specific hotspot regions. The hotspot sequences obtained from human SERPING1 promoter and mouse Serping1 promoter are listed in Table 9. Table 9. Human SERPING1 and mouse Serping1 saRNA hotspot regions and their sequences Example 3: Dose response characterization and cytotoxicity assessment of saRNA in HepG2 cells

[0187] In order to verify the functional saRNA that can activate the SERPING1 gene and its cytotoxicity, based on the high-throughput screening results, the top 73 SERPING1 saRNAs were selected from 139 functional saRNAs (see Table 1) and transfected HepG2 cells with 2.5nM, 10nM and 25nM single saRNAs for 3 days. The SERPING1 mRNA expression of transfected cells was analyzed by RT-qPCR.

[0188] like Figure 3A As shown, all saRNAs induced more than 1.1-fold SERPING1 mRNA at 3 tested concentrations (2.5nM, 10nM and 25nM), of which DS12A-0156 showed the highest activity of 43.3-fold at 10nM. When treated at 2.5nM, 5 (6.8%) and 17 (23.3%) saRNAs induced SERPING1 mRNA levels more than 10-fold and 5-fold, respectively. When treated at 10nM, 9 (12.3%) and 28 (38.4%) saRNAs induced SERPING1 mRNA levels more than 10-fold and 5-fold, respectively. When treated at 25nM, 10 (13.7%) and 29 (39.7%) saRNAs induced SERPING1 mRNA levels more than 10-fold and 5-fold, respectively. Cytotoxicity analysis was subsequently performed via PI staining to further identify duplexes with acceptable in vitro safety characteristics. As Figure 3B As shown, HepG2 cells tolerated all 73 saRNAs at all three doses with a relative cell proliferation threshold of 0.5. Overall, the human target sequence selection, saRNA design, and screening criteria identified a set of strong and safe candidates for all dose ranges for further drug development. Example 4: Evaluation of the activation effect of saRNA on SERPING1 mRNA and protein expression in HepG2 cells

[0189] In order to analyze the activation effect of saRNA on SERPING1 mRNA and protein expression in HepG2 cells, the specified saRNA (see Table 1) was selected to transfect HepG2 cells at 10 nM for 5 days. Figure 4A As shown in Figure 2, 4 (about 26.7%) and 9 (about 60%) saRNAs induced more than 2.0-fold and 1.5-fold activity on SERPING1 mRNA levels, respectively, among which DS12A-0184 showed the highest activity (4.7-fold). Figure 4B As shown, 5 (about 20.8%) and 18 (about 75.8%) saRNAs induced more than 2.0-fold and 1.5-fold activity on SERPING1 protein levels, respectively, of which DS12A-0184 and DS12A-0072 showed the highest activity (2.4-fold). The results showed that the activation effect of saRNA was highly correlated with the expression of SERPING1 mRNA and protein in HepG2 cells. Example 5: Activation effect of low concentration saRNA on the expression of SERPING1 mRNA in HepG2 cells

[0190] To confirm the activation effect of saRNA on SERPING1 mRNA expression in HepG2 cells, 29 saRNAs (see Table 1) were transfected at low concentrations (0.1 nM and 1 nM) for 3 days. Figure 5As shown in A, when treated at 0.1 nM, 11 (37.9%) and 26 (89.7%) saRNAs induced more than 1.5-fold and 1.1-fold increases in SERPING1 mRNA levels, respectively, of which DS12A-0184 showed the highest activity (2.2-fold). When treated at 1 nM, 22 (75.9%) and 28 (96.6%) saRNAs induced more than 1.5-fold and 1.1-fold increases in SERPING1 mRNA levels, respectively, of which DS12A-0184 showed the highest activity (4.7-fold). The results show that saRNA is able to activate the expression of SERPING1 mRNA at low concentrations, which will enable them to be further developed as drugs. Example 6: Activation of SERPING1 mRNA and protein expression in different human liver cancer cells by saRNA Evaluate

[0191] The selected saRNAs (see Table 1) were transfected into human liver cancer cell lines HepG2, Hep3B and PLC / PRF / 5 at 10 nM for 4 days and the activation effects in various cells were verified. Table 10 shows the activation effects of the saRNAs derived from Fig. 6A (HepG2), Figure 6B (Hep3B) and Figure 6CRelative fold changes of SERPING1 mRNA and protein of (PLC / PRF / 5). In HepG2 cells, all saRNAs induced significant activation of SERPING1 compared to Mock, ranging from 1.9-fold to 9.3-fold and 1.5-fold to 6.9-fold in SERPING1 mRNA and protein, respectively, with DS12A-0213 showing the highest activity of mRNA (9.3-fold) and protein (3.6-fold). In Hep3B cells, all saRNAs induced variable activation of SERPING1 compared to Mock, ranging from 1.2-fold to 2.4-fold, except DS12A-240, which caused a 0.8-fold increase in SERPING1 mRNA and a 2.1-fold increase in SERPING1 protein. For protein, all saRNAs induced variable activation of SERPING1 compared to Mock, ranging from 1.1 to 3.2 times, with DS12A-0077 showing the highest activity of protein (3.2 times). In PLC / PRF / 5 cells, all saRNAs induced variable activation of SERPING1 compared to Mock, ranging from 1.1 to 1.9 times and 1.3 to 2.6 times in SERPING1 mRNA and protein, respectively, with DS12A-0225 showing the highest activity of mRNA (1.9 times) and protein (2.6 times). Data statistics show that saRNA has a moderate correlation with the expression of SERPING1 mRNA and protein in different cell lines. Table 10. Relative expression of SERPING1 mRNA and protein (fold change) Note: / means not tested. Example 7: Time course changes in the expression of SERPING1 mRNA and protein in saRNA-induced HepG2 cells change

[0192] Six saRNAs (i.e., DS12A-0043, DS12A-0072, DS12A-0187, DS12A-0182, DS12A-0240, and DS12A-0213) were selected (see Table 1) and transfected in HepG2 cells at the specified concentrations (i.e., 0.5 nM, 1.25 nM, 4 nM, 30 nM) for a duration ranging from 1 day to 7 days. The SERPING1 mRNA level was detected at each time point (Table 11, Fig. 7A ) and SERPING1 protein levels (Table 12, Figure 7B) Relative fold change. Overall, a single transfection of saRNA induced the relevant expression of SERPING1 mRNA and protein for at least 7 days, with DS12A-0213 showing the highest effect among all tested saRNAs. Table 11. Relative expression of SERPING1 mRNA (fold change) Table 12. Relative expression of SERPING1 protein (fold change) Example 8: saRNA induces EC expression of SERPING1 mRNA in HepG2 cells 50

[0193] To further test the EC 50 For the effect on SERPING1 mRNA expression, 6 saRNAs (i.e., DS12A-0043, DS12A-0072, DS12A-0187, DS12A-0182, DS12A-0240, and DS12A-0213) (see Table 1) were selected to transfect HepG2 cells at increasing doses (i.e., 0.0017 nM, 0.0051 nM, 0.0152 nM, 0.046 nM, 0.14 nM, 0.41 nM, 1.23 nM, 3.7 nM, 11.111 nM, 33.333 nM, and 100 nM) for 4 days. The levels of SERPING1 mRNA of the 6 saRNAs are shown in Figure 8A to Figure 8F As summarized in Table 13, DS12A-0072 and DS12A-0240 showed the highest EC in inducing SERPING1 mRNA, respectively. 50 (2.78nM) and the lowest EC 50 (0.98 nM). The data provide a clearer comparison of the potency of candidate saRNAs in future development. Table 13. EC of SERPING1 mRNA in HepG2 cells 50 value Example 9: Design and synthesis of saRNA targeting the mouse Serping1 promoter

[0194] The coding strand of the promoter sequence (SEQ ID NO: 1400) (Table 14) of the mouse Serping1 gene from TSS to upstream -500bp was retrieved from the ENSEMBL genome database. In order to identify functional small activating RNAs (saRNAs) capable of activating the expression of the Serping1 gene, a series of 19nt saRNA targets were selected on the 500bp Serping1 promoter sequence, starting from upstream -500bp of the TSS and moving 1bp to the TSS each time, thereby generating target sequences. Table 14. Deduced mouse Serping1 promoter sequence (5'-3') (SEQ ID NO: 1400)

[0195] The target sequences were then filtered to retain those that met the following criteria: (1) GC content of 35% to 65%; (2) less than 5 consecutive identical nucleotides; (3) 3 or fewer dinucleotide repeats; and (4) 3 or fewer trinucleotide repeats. After filtering, 277 target sequences remained, and their corresponding double-stranded saRNAs were chemically synthesized. The length of each of the sense and antisense strands of the saRNA used in this study was 21 nt. The 19 nucleotides in the 5' region of the first RNA strand (sense strand) of the saRNA had 100% sequence identity with the target sequence of the promoter, and the 3' end of the first RNA strand was an additional 2nt dTdT sequence. The 19 nucleotides in the 5' region of the second RNA strand were complementary to the first RNA strand sequence, and the 3' end of the second RNA strand was an additional 2nt dTdT sequence. The two strands of the above-mentioned single saRNA were mixed and annealed at a molar ratio of 1:1 to obtain a duplex saRNA. Table 2 lists the saRNA target sequences designed in the Examples, their sense strands and antisense strands. Example 10: High-throughput screening of saRNA targeting the mouse Serping1 promoter

[0196] To identify saRNAs capable of activating Serping1 transcription, PMH cells were transfected with each of the above 277 saRNAs at a transfection concentration of 25 nM. Fig. 9The activity distribution (log2) of Serping1 saRNA sorted from the strongest activating protein to the strongest inhibitory protein is presented. Table 15 further shows that 8 (2.9%), 49 (17.7%) and 33 (11.9%) saRNAs showed high activation (≥1.5 times), moderate activation (1.2 times-1.5 times) and mild activation (1.1 times-1.2 times) on Serping1 mRNA levels, respectively. 187 (67.5%) saRNAs had no obvious effect on the expression of Serping1 mRNA. The highest activation observed for Serping1 mRNA expression was 1.65 times. Table 15. Summary of the activities of all screened Serping1 saRNAs

[0197] Fig.10 Further showing the activity distribution of Serping1saRNA classified by their position on the Serping1 promoter, it can be clearly seen that the functional saRNA is distributed on the promoter in an aggregated manner, that is, in certain promoter regions, there are "hot spots" enriched with functional saRNA. From the TSS of the promoter, there are 4 hot spots located in the -424 to -325 region (H6), -257 to -182 region (H7), -170 to -117 region (H8) and -56 to -11 region (H9), and contain highly enriched functional saRNA. This result (i.e., functional saRNA is not randomly distributed on the promoter) indicates that specific hotspot regions are essential for regulating Serping1 gene expression by RNA activation mechanisms. All hotspot sequences are listed in Table 9. Example 11: Dose response of saRNA in activating the Serping1 gene in PMH cells

[0198] The top 57 Serping1 saRNAs were selected from the 90 functional saRNAs derived from the screening (see Table 2) and PMH cells were transfected at 2.5 nM, 10 nM and 25 nM for 3 days. Serping1 mRNA expression of transfected cells was analyzed by RT-qPCR.

[0199] like Fig.11As shown. When treated with 2.5nM, 4 (7.0%) and 12 (21.1%) saRNAs induced more than 2-fold and 1.5-fold activity on Serping1 levels, respectively, of which DS12B-0206 and DS12B-0130 showed the highest activity (2.6 times). When treated with 10nM, 13 (22.8%) and 30 (52.6%) saRNAs induced more than 2-fold and 1.5-fold on Serping1 levels, respectively, of which DS12B-0188 showed the highest activity (3.7 times). When treated with 25nM, 22 (38.6%) and 37 (64.9%) saRNAs induced more than 2-fold and 1.5-fold on Serping1 levels, respectively, of which DS12B-0188 showed the highest activity (5.7 times).

[0200] To evaluate the dose-effect relationship between saRNA treatment and Serping1 activation, the first 57 Serping1 saRNAs were transfected in PMH cells at 6 increasing concentrations (i.e., 0.41 nM, 1.23 nM, 3.7 nM, 11.1 nM, 33.3 nM, and 100 nM). Fig. 12A Serping1 mRNA was quantified by two-step RT-qPCR using gene-specific primers in a single PCR reaction. Table 16 shows the percentage of positive saRNAs that activated at least a 1.5-fold change in Serping1 mRNA levels. Cytotoxicity analysis was then performed via PI staining to further identify duplexes with possible safety issues. Fig. 12B As shown, PMH cells tolerated all 57 saRNAs at all six doses with a >50% cell proliferation threshold relative to mock treatment. Table 16. Summary of saRNA activity against mouse Serping1 mRNA Example 12: Evaluation of the activation effect of saRNA on Serping1 mRNA expression in PMH cells

[0201] The top 24 saRNAs (see Table 2) were selected to transfect PMH cells at 25 nM for 3 days to confirm the activation effect of saRNA on Serping1 mRNA expression in PMH cells. Fig.13As shown, 22 of the 24 saRNAs (91.7%) induced significant activation of Serping1 mRNA, showing more than 2-fold activation, of which DS12B-0110 showed the highest activity (5.5-fold). Only the remaining two DS12B-0276 and DS12B-0274 induced 1.8-fold and 1.2-fold activation of Serping1 mRNA. The data show that the selected saRNA can activate the expression of Serping1 mRNA in vitro. Example 13: Design and Synthesis of Mutant SERPING1 saRNA

[0202] To evaluate the sequence requirements of the sense strand by mutational ‘walk’ of SERPING1 saRNA, a series of 3nt mutant saRNAs were designed and synthesized.

[0203] RD-12118 is a duplex saRNA, which is replaced by any 3nt nucleotide of the sense strand, starting from the 5' end of the sense strand and moving 3nt to the 3' end each time, thereby generating the new sequence of RD-12226 shown in Table 17.

[0204] RD-12126 is a duplex saRNA, which is replaced by any 3nt nucleotide of the sense strand, starting from the 5' end of the sense strand, and moving 3nt to the 3' end each time, thereby sequentially generating new sequences (ie, RD-12232, RD-12233, and RD-12234) shown in Table 17. It is worth noting that RD-12234 was synthesized by replacing the last 4nt nucleotides of RD-12126. Example 14: Assessing the sequence requirements of the sense strand by mutational “walk” of SERPING1 saRNA

[0205] To evaluate the sequence requirements of the positive strand by mutational "walk" of SERPING1 mRNA, the designated SERPING1 saRNA was transfected in HepG2 cells at 25 nM for 3 days. The SERPING1 mRNA levels of saRNA were quantified by RT-qPCR using gene-specific primers. RD-11633 is a chemically modified siRNA and was used as a negative control. RD-12118 and RD-12126 were used as known effective saRNAs.

[0206] As shown in Table 18, RD-12118 induced a 3.95-fold increase in SERPING1 mRNA, and RD-12226 (a mutant saRNA in which the last 3nt nucleotides were replaced) induced a 2.36-fold increase in SERPING1 mRNA, indicating that the last 3nt nucleotide mutation does not lose activation activity. Similarly, RD-12126 induced high activation activity for SERPING1 mRNA, and RD-12232, RD-12233, and RD-12234 all induced high activation activity for SERPING1 mRNA by about 4.0 times, indicating that the last 10nt continuous nucleotide mutation does not lose activation activity. The present application provides saRNAs comprising the sequences of the positive chains of Formula I and Formula II that can also cause SERPING1 gene activation. Both formulas are summarized in Table 19.

[0207] The results showed that mutagenesis at specific sites of the target gene still maintained activation activity. Table 18. Summary of SERPING1 mRNA expression in HepG2 cells Table 19. Sequence formula of SERPING1 saRNA Mode SEQ ID NO Sense strand sequence (5'-3') Length (nt) Formula I 1443 AAAUUCCUGGCCUGGCCCNNN 21 Formula II 1444 CUACCAGGGNNNNNNNNNN 19 Note: N is selected from adenine (A), thymine (T), cytosine (C), guanine (G) and uracil (U). Example 15: Time course treatment of SERPING1 saRNA in HepG2 and PLC / PRF / 5 cells

[0208] To detect the time course changes of SERPING1 mRNA induced by SERPING1 saRNA, the designated saRNAs (i.e., RD-11513, RD-11823, RD-12065 and RD-11826) (see Table 17) were transfected at 25 nM in HepG2 and PLC / PRF / 5 cells for 1 to 7 days. RD-11823 and RD-11826 are fully modified saRNAs derived from duplex saRNAs of RD-11513 and RD-12065, respectively. The SERPING1 mRNA levels of saRNAs were quantified by RT-qPCR using gene-specific primer sets.

[0209] As shown in FIG14A , RD-11513, RD-11823, RD-12065, and RD-11826 all induced an increase in SERPING1 mRNA levels and sustained activation activity within 7 days, with increases ranging from about 1.1 to 27.8 times, about 1.3 to 20.7 times, about 0.8 to 4.9 times, and about 0.8 to 8.8 times, respectively. RD-11513 showed the greatest and most sustained effect in HepG2 cells (Tmax ≥6 days). RD-11826 (chemically modified saRNA) improved the activation activity compared to RD-12065 without modification, while RD-11823 could not further improve the activation of SERPING1 mRNA. As shown in Figure 14B, RD-11513, RD-11823, RD-12065 and RD-11826 all induced an increase in SERPING1 protein levels and persistent activation activity within 7 days, inducing about 1.1 to 24.4 times, about 1.0 to 24.9 times, about 0.9 to 5.8 times and about 1.1 to 13.3 times, respectively, of which RD-11823 showed the largest and most persistent (T max ≥6 days). RD-11826 showed increased activation activity compared to RD-12065 lacking the modification, and RD-11823 and RD-11513 showed similar activation activity of the SERPING1 protein. Caspase 3 / 7 activity was also quantified to determine the induction of undesired cytotoxicity as a safety marker. Fig. 14C As shown, the caspase 3 / 7 activities of RD-11513 and RD-12065 showed slight cytotoxicity on days 6 and 7, while RD-11823 and RD-11826 were not detected at any of the test points up to day 7, indicating that chemical modification can attenuate cytotoxicity.

[0210] FIG. 15A depicts that RD-12065 and RD-11826 induced increasing activation activity of SERPING1 mRNA level from day 1 to day 4, and decreased from day 5 to day 7, wherein the peak of SERPING1 mRNA induction occurred on day 4. RD-12065 and RD-11826 showed similar trends in the expression of SERPING1 mRNA level, wherein RD-12065 showed the highest induction (3.0-fold) on day 4. As shown in FIG. 15B , RD-12065 and RD-11826 induced increasing activation activity of SERPING1 protein level from day 1 to day 5, and decreased from day 6 to day 7, wherein the peak of SERPING1 protein induction occurred on day 5. RD-12065 and RD-11826 showed similar trends in the expression of SERPING1 protein level, wherein RD-12065 showed the highest induction (1.6-fold) on day 5. Fig. 15C As shown, no caspase 3 / 7 activity was detected for RD-12065 and RD-11826 at any of the test points up to 7 days.

[0211] The data showed that SERPING1 saRNA induced maximal and sustained activation of SERPING1 mRNA and protein, and chemical modification enhanced the activation effect and attenuated cytotoxicity. Example 16: Evaluation of the activation activity and safety of SERPING1 saRNA in HepG2 cells

[0212] To detect the activation activity and safety of SERPING1 saRNA, the designated saRNAs (i.e., RD-11513, RD-11823, RD-12065, and RD-11826) were transfected in HepG2 cells at increasing concentrations (i.e., 0.098 nM, 0.195 nM, 0.39 nM, 0.78 nM, 1.56 nM, 3.13 nM, 6.25 nM, 12.5 nM, 25 nM, 50 nM, and 100 nM) for 5 days.

[0213] Dose-response curves of activation activity were plotted, where the EC values ​​of SERPING1 mRNA ( FIG. 16A ) and protein ( FIG. 16B ) were 50 and E max The values ​​were extrapolated to define the activation activity of each of the saRNAs tested. FIG16A shows that for each saRNA, activation of SERPING1 mRNA was dose-dependent, with EC 50 and E max The values ​​were approximately 0.21 nM to 13.35 nM and approximately 4.78-fold to 14.23-fold in the lower nanomolar range, respectively. Figure 16B shows that for each saRNA, the activation of SERPING1 protein was dose-dependent, with EC 50 and E max The values ​​were approximately 0.24 nM to 9.86 nM and approximately 3.36-fold to 10.09-fold in the lower nanomolar range, respectively. As summarized in Table 20, RD-12065 was the most potent saRNA for SERPING1 mRNA and protein, with the lowest EC values ​​at 0.21 nM and 0.24 nM, respectively. 50 In RD-11553, the E of SERPING1 mRNA appeared at 100 nM. max Activation activity (14.23 times). In RD-11823, the E of SERPING1 protein appeared at 25 nM. max Activation activity (10.09 times). Fig. 16C As shown, no caspase 3 / 7 activity was detected at any of the tested dose concentrations up to 100 nM. The results indicate that SERPING1 saRNA can induce activation of SERPING1 mRNA and protein in vitro. Table 20. EC of SERPING1 saRNA in HepG2 cells50 and E max Summary of Values Example 17: Design and synthesis of SERPING1 saRNAs of different lengths, overhangs, and mutations

[0214] To evaluate the effects of homology, overhangs, and mutations on saRNA activity, a series of saRNA variants were designed and synthesized in-house.

[0215] RD-12065 is an exemplary duplex saRNA of 21nt, which contains a sense strand and an antisense strand. The 19nt nucleotides in the 5' end of the sense strand have 100% sequence identity with the target sequence of the promoter, the 19 nucleotides in the 5' end of the antisense strand are complementary to the sense strand, and the 3' ends of the sense strand and the antisense strand respectively include an additional 2nt TT sequence. Based on RD-12065, 8 compounds (i.e., RD-13950, RD-13951, RD-13952, RD-13953, RD-13955, RD-13956, RD-13957, and RD-13958) as shown in Table 21 were designed.

[0216] RD-13950 was synthesized with 16 nt nucleotides obtained by removing the last 3 nt GCU nucleotides at the 3' end of the sense strand of RD-12065 without a 2 nt TT sequence, the antisense strand was complementary to the sense strand, and the 3' ends of the sense and antisense strands protruded with an additional 2 nt TT sequence.

[0217] RD-13951 was synthesized with 18 nt nucleotides obtained by removing the last 1 nt U nucleotide at the 3' end of the sense strand of RD-12065 without a 2 nt TT sequence, the antisense strand was complementary to the sense strand, and the 3' ends of the sense strand and the antisense strand were blunt ends.

[0218] RD-13952 was synthesized with 22 nt nucleotides obtained by adding an extra 3 nt AAA sequence at the 3' end of the sense strand of RD-12065 without the 2 nt TT sequence, and an antisense strand with 20 nt nucleotides complementary to the sense strand without the 2 nt AA sequence overhang.

[0219] RD-13953 was synthesized with 30 nt nucleotides obtained by adding an additional 11 nt AAAUUCCUGGC sequence at the 3' end of the sense strand of RD-12065 without the 2 nt TT sequence, and an antisense strand with 28 nt nucleotides complementary to the sense strand without the 2 nt GC sequence overhang.

[0220] RD-13955 was synthesized with the same sense strand of RD-12065 and 5 mismatched nucleotides that were mismatched every other nucleotide at the 3' end of the antisense strand and included an additional 2nt TT sequence at the 3' end of both the sense and antisense strands.

[0221] RD-13956 was synthesized with 20 nt nucleotides obtained by protruding an extra 1 nt A nucleotide at the 3' end of the sense strand of RD-12065 without the 2 nt TT sequence, and the antisense strand was identical to RD-12065 without the 2 nt TT sequence.

[0222] RD-13957 was synthesized with 22 nt nucleotides obtained by protruding an extra 3 nt AAA nucleotide at the 3' end of the sense strand of RD-12065 without the 2 nt TT sequence, and the antisense strand was identical to RD-12065 without the 2 nt TT sequence.

[0223] RD-13958 was synthesized with 24 nt nucleotides obtained by overhanging an extra 5 nt AAAUU nucleotide at the 3' end of the sense strand of RD-12065 without the 2 nt TT sequence, and the antisense strand was identical to RD-12065 without the 2 nt TT sequence.

[0224] RD-11513 is an exemplary duplex saRNA of 21nt, which contains a sense strand and an antisense strand. The 19nt nucleotides in the 5' end of the sense strand have 100% sequence identity with the target sequence of the promoter, the 19 nucleotides in the 5' end of the antisense strand are complementary to the sense strand, and the 3' ends of the sense strand and the antisense strand respectively include an additional 2nt TT sequence. Based on RD-11513, 5 compounds (i.e., RD-13959, RD-13960, RD-13962, RD-13965, and RD-13966) as shown in Table 21 were designed.

[0225] RD-13959 was synthesized with 17 nt nucleotides obtained by removing the last 2 nt CA nucleotides at the 3' end of the sense strand of RD-11513 without a 2 nt TT sequence, the antisense strand was complementary to the sense strand, and the 3' ends of the sense and antisense strands protruded with an additional 2 nt TT sequence. Rd-13960 was synthesized with a 19 nt sense strand that was identical to RD-11513 without the 2 nt TT sequence, an antisense strand that was complementary to the sense strand, and 3' ends of the sense and antisense strands were blunt ends.

[0226] RD-13962 was synthesized with 35 nt nucleotides obtained by adding an additional 16 nt CAAAGAAGACCAAGCG sequence at the 3' end of the sense strand of RD-11513 without the 2 nt TT sequence, and the antisense strand with 33 nt nucleotides was complementary to the sense strand without the 2 nt CG sequence overhang.

[0227] RD-13965 was synthesized with 23 nt nucleotides obtained by overhanging an extra 4 nt CAAA nucleotide at the 3' end of the sense strand of RD-11513 without the 2 nt TT sequence, and the antisense strand was identical to RD-11513 without the 2 nt TT sequence.

[0228] RD-13966 was synthesized with 25 nt nucleotides obtained by protruding an extra 6 nt CAAAGA nucleotide at the 3' end of the sense strand of RD-11513 without the 2 nt TT sequence, and the antisense strand was identical to RD-11513 without the 2 nt TT sequence.

[0229] RD-12066 is an exemplary duplex saRNA of 21nt, which contains a sense strand and an antisense strand. The 19nt nucleotides in the 5' end of the sense strand have 100% sequence identity with the target sequence of the promoter, the 19 nucleotides in the 5' end of the antisense strand are complementary to the sense strand, and the 3' ends of the sense strand and the antisense strand respectively include an additional 2nt TT sequence. Based on RD-12066, 4 compounds (ie, RD-13967, RD-13968, RD-13969, and RD-13970) as shown in Table 21 were designed.

[0230] RD-13967 was synthesized with 16 nt nucleotides obtained by removing the last 3 nt CAA nucleotides at the 3' end of the sense strand of RD-12066 without a 2 nt TT sequence, the antisense strand was complementary to the sense strand, and the 3' ends of the sense and antisense strands protruded with an additional 2 nt TT sequence.

[0231] RD-13968 was synthesized with 35 nt nucleotides obtained by adding an additional 16 nt UUUCCAAAAAGUUCAU sequence at the 3' end of the sense strand of RD-12066 without the 2 nt TT sequence, and an antisense strand with 29 nt nucleotides complementary to the sense strand without the 6 nt GUUCAU sequence overhang.

[0232] RD-13969 was synthesized with 16 nt nucleotides obtained by removing the last 3 nt UUC nucleotides at the 3' end of the antisense strand of RD-12066 without a 2 nt TT sequence, the sense strand was complementary to the antisense strand, and the 3' ends of the sense and antisense strands protruded with an additional 2 nt TT sequence.

[0233] RD-13970 was synthesized with 35 nt nucleotides obtained by adding an additional 16 nt CUCAAAGUAACCCUUU sequence at the 3' end of the antisense strand of RD-12066 without the 2 nt TT sequence, and a sense strand with 29 nt nucleotides complementary to the antisense strand without the 6 nt CCCUUU sequence overhang. Example 18: Evaluation of the effects of homology, overhangs and mutations on saRNA activity in HepG2 and PLC / PRF / 5 cells ring

[0234] To evaluate the effects of homology, overhangs, and mutations on SERPING1 saRNA activity, the designated saRNAs were transfected at 25 nM in HepG2 and PLC / PRF / 5 cells for 3 days. Mock was transfected in the absence of oligonucleotides. dsCon2 was used as a nonspecific duplex control. DS12A-si2 is a duplex siRNA and was transfected as a negative control. RD-12065, RD-11513, and RD-12066 are duplex saRNAs and serve as exemplary controls. SERPING1 mRNA levels of saRNAs were quantified by RT-qPCR using gene-specific primer sets.

[0235] Group A includes 7 compounds (i.e., RD-12065, RD-13950, RD-13951, RD-13952, RD-13956, RD-13957, and RD-13958) (see Table 21). All compounds in this group except RD-13950 and RD-13952 have better activation activity than the exemplary saRNA control (RD-12065) by more than 4.0 to 5.4 times, indicating that saRNA nucleotide sequences can tolerate different overhang lengths, with certain variants providing greater potency ( Fig.17A). For example, RD-13956, RD-13957, and RD-13958 containing 1, 3, or 5 nucleotide overhangs at the 3' end of the sense strand showed better activation activity than the exemplary saRNA control, especially the 3 nucleotide overhang (i.e., RD-13957), indicating that the overhang can provide benefits for inducing SERPING1 mRNA levels and can be optimized independently. The shorter size RD-13951 with a blunt-ended 18nt nucleotide had an excellent induction effect on SERPING1 mRNA, indicating that shorter nucleotide sequences are feasible and provide benefits. The shortest size RD-13950 with a 16nt nucleotide with a 2nt TT at the 3' end had little induction effect on SERPING1 mRNA, indicating that the shortest nucleotide sequence is also feasible. The longer size RD-13952 with a sense strand and antisense strand length of 22nt and 20nt, respectively, had a moderate induction effect, indicating that longer nucleotide sequences are feasible. Overall, the data suggest that saRNA nucleotide sequences ranging in length from 16 to 30 contiguous nucleotides, with overhangs of 1 to 5 nucleotides and at least 80% homology or complementarity to their target sequence are functional.

[0236] Group B includes 6 compounds (i.e., RD-11513, RD-13959, RD-13960, RD-13962, RD-13965, and RD-13966) (see Table 21). Fig.17A As shown, RD-13959 and RD-13960, which are shorter in size, with 17nt nucleotides of 2nt TT and 19nt nucleotides with blunt ends, respectively, caused excellent induction of SERPING1 mRNA. RD-13962, which is longer in size with a sense strand length of 35nt and an antisense strand length of 33nt, had a slight induction of SERPING1 mRNA. Sense strand 3' end lengths of 4nt overhangs (i.e., RD-13965) and 6nt overhangs (i.e., RD-13966) showed high activation activity, especially for the 4nt overhangs. Overall, the data indicate that saRNA nucleotide sequences ranging from 17 to 35 consecutive nucleotides in length are functional, and their overhangs range from 4 to 6 nucleotides, with at least 76% homology or complementarity to their target sequences.

[0237] Group C includes compounds (i.e., RD-12066, RD-13968, and RD-13970) (see Table 21). Fig.17AAs shown, RD-13968 with a longer size (35nt sense strand and 29nt antisense strand) has a slight induction effect on SERPING1 mRNA. RD-13970 with a longer size (29nt sense strand and 35nt antisense strand) has a better induction effect on SERPING1 mRNA than the exemplary saRNA control (RD-12066). Overall, the data indicate that saRNAs ranging in size from 29nt to 35nt and having at least 83% homology or complementarity to their target sequences are functional.

[0238] Group D includes 9 compounds (i.e., RD-12065, RD-13950, RD-13951, RD-13952, RD-13953, RD-13955, RD-13956, RD-13957, and RD-13958) (see Table 21). All compounds in this group except RD-13950 and RD-13955 have activation activities equal to or better than the exemplary saRNA control (RD-12065) by 2.4 to 3.1 times, indicating that saRNA nucleotide sequences can tolerate different overhang lengths, with certain variants providing greater potency ( Fig. 17B). For example, RD-13956, RD-13957, and RD-13958 containing 1, 3, or 5 nucleotide overhangs at the 3' end of the sense strand showed high activation activity equal to or better than the exemplary saRNA control, especially the 3 nucleotide overhang (i.e., RD-13957), indicating that the overhang can provide benefits for inducing SERPING1 mRNA levels and can be optimized independently. The shorter size RD-13951 with a blunt-ended 18nt nucleotide had a high induction effect on SERPING1 mRNA, indicating that shorter nucleotide sequences are feasible and provide benefits. The shortest size RD-13950 with a 16nt nucleotide with a 2nt TT at the 3' end had little induction effect on SERPING1 mRNA, indicating that the shortest nucleotide sequence is also feasible. The longer size RD-13952 with a sense strand and antisense strand length of 22nt and 20nt, respectively, had a high induction effect, indicating that longer nucleotide sequences are feasible. The longest size of RD-13953, with sense and antisense strands of 30 nt and 28 nt, respectively, caused high induction, indicating that the longest nucleotide sequence is feasible. RD-13955 with a 5 nt mismatch in the antisense strand of 19 nt in total length caused a 1.3-fold induction of SERPING1 mRNA levels, indicating that saRNA nucleotide sequences with at least 26% mutations do not lose activation activity (data not shown). Overall, the data indicate that saRNAs with sizes of 16 to 30 nt, with overhangs of 1 to 5 nucleotides, containing up to 26% sequence mutations, and having at least 80% homology or complementarity to their target sequences are functional.

[0239] Group E includes 5 compounds (i.e., RD-12066, RD-13967, RD-13968, RD-13969, and RD-13970) (see Table 21). Fig. 17B As shown, RD-13967 and RD-13969, both of the shorter sizes of 16nt nucleotides with 2nt TT at the 3' end, have little induction effect on SERPING1 mRNA, indicating that shorter nucleotide sequences are usable. RD-13968, of the longer size of 35nt sense strand length and 29nt antisense strand length, has a moderate induction effect on SERPING1 mRNA, indicating that longer nucleotide sequences are feasible. RD-13970, of the longer size (29nt sense strand and 35nt antisense strand), has a stronger induction effect on SERPING1 mRNA than the exemplary saRNA control (RD-12066). Overall, the data indicate that saRNAs ranging in size from 16nt to 35nt and having at least 83% homology or complementarity to their target sequences are functional.

[0240] In summary, these results indicate that saRNA nucleotide sequences ranging in length from 16 to 35 contiguous nucleotides with 1 to 6 overhangs and 26% mutations are viable and have at least 76% homology or complementarity. Example 19: Time course changes in Serping1 mRNA induced by Serping1 saRNA in PMH cells

[0241] To detect the kinetic activity of Serping1saRNA, chemically modified saRNAs (ie, RD-12349, RD-12350, RD-12351, and RD-12347) (see Table 17) were transfected at 25 nM in PMH cells for 1 to 7 days. RD-12349, RD-12350, RD-12351, and RD-12347 are all fully modified saRNAs derived from duplex saRNAs RD-12369, RD-12370, RD-12371, and RD-12367, respectively. Serping1mRNA levels of saRNAs were quantified by RT-qPCR using gene-specific primer sets.

[0242] like Fig.18 As shown, RD-12349 and RD-12350 induced an increase in Serping1 mRNA levels and persistent activation activity within 7 days, by about 1.1 to 3.3 times and about 1.1 to 3.2 times, respectively, among which RD-12349 showed the greatest and most persistent effect (T max ≥7 days). RD-12351 induced increasing activation activity of Serping1 mRNA levels from day 1 to day 5 and decreased from day 6 to day 7, with a peak of Serping1 mRNA induction occurring on day 5 (2.6-fold). RD-12347 induced increasing activation activity of Serping1 mRNA levels from day 1 to day 4 and decreased from day 5 to day 7, with a peak of Serping1 mRNA induction occurring on day 4 (2.5-fold). The data indicate that chemically modified saRNAs exhibit various kinetic activities in PMH cells.

[0243] In addition, the RNAiMAX reagent was then used at 25 nM ( FIG. 19A ) and 500 nM ( FIG. 19B and Fig.19C) were tested for free uptake of additional Serping1 saRNAs (i.e., RD-11931, RD-11927, and RD-12189) (see Table 17) for 1 to 5 days. RD-11432 is a chemically modified siRNA and was used as a negative control. RD-11931, RD-11927, and RD-12189 were designed based on RD-12351, RD-12350, and RD-12349 by conjugating a conjugation group derived from tC2 to the 3' end of the sense strand.

[0244] Specifically, the structure of tC2 is as follows: And the conjugated group derived from tC2 has the structure shown below: The asterisk indicates the site at which the conjugated group is attached to the chain, either directly or through a linking moiety such as those noted above.

[0245] As shown in FIG19A , RD-11927 and RD-12189 induced activation activity at the Serping1 mRNA level within 5 days, about 1.0-fold to 2.8-fold and about 0.9-fold to 1.8-fold, respectively, wherein activation decreased on day 4 and increased on day 5. RD-11931 induced increasing activation activity at the Serping1 mRNA level from day 1 to day 4 and decreased on day 5, wherein a peak value (2.4-fold) of Serping1 mRNA induction occurred on day 4. As shown in FIG19B , RD-11931 and RD-11927 induced increasing activation activity at the Serping1 mRNA level from day 1 to day 4 and reached a plateau on day 5, wherein RD-11931 exhibited the highest Serping1 mRNA induction (1.6-fold). RD-12189 induced a slight activation activity of Serping1 mRNA levels from day 2 to day 4 and recovered on day 5, with the highest Serping1 mRNA induction occurring on day 4 (1.2-fold). RD-11432 showed substantial knockdown (approximately 90%) of Serping1 mRNA expression by RNAiMAX reagent or free uptake within 5 days. Fig.19C As shown, no caspase 3 / 7 activity was detected for any of the compounds tested at any of the test points up to day 5.

[0246] Taken together, these results indicate that chemically modified saRNAs display various kinetic activities and are well tolerated in PMH cells. Example 20: Persistent Activation of Serping1 saRNA in the Liver of C57BL / 6J Mice

[0247] To evaluate the activation persistence of Serping1saRNA, RD-12497 was administered to adult female C57BL / 6J mice at 20 mg / kg by SC injection. RD-12497, which was conjugated with an auxiliary single-stranded oligonucleotide (ACO) sequence via an S9 linker at the 3' end of the sense chain, was designed based on RD-12350. Normal saline was used as a vehicle control to establish the baseline expression level of Serping1 mRNA. RD-11432 is a chemically modified siRNA and is used as a negative control. Under the same administration, RD-11432 was injected at 3 mg / kg. Mice were sacrificed on days 7 and 14 after treatment, and Serping1mRNA induction was quantified in liver tissue by RT-qPCR. As shown in Figure 20A, RD-12497 induced 1.3-fold and 1.4-fold expression of Serping1 mRNA on days 7 and 14, respectively. RD-11432 caused 92% and 90% knockdown of Serping1 mRNA on days 7 and 14, respectively. Fig. 20B As shown, there was no significant weight loss in animals treated with RD-12497 up to day 14 compared to the saline group, indicating that the modified saRNA was well tolerated in C57BL / 6J mice.

[0248] To further evaluate the activation persistence of Serping1saRNA, RD-13144 was administered to adult female C57BL / 6J mice at 50 mg / kg by SC injection. RD-13144 (fully modified Serping1 saRNA) is based on RD-12350 by connecting a conjugated group derived from a delivery enhancement compound (DEC; i.e., tC2x4) to the 3' end of the sense strand and conjugating the vinylphosphonate (VP) modification to the 5' position (5'VP) of the antisense strand. Physiological saline was used as a vehicle control to establish the baseline expression level of Serping1 mRNA. The structure of the tC2 compound or tC2x4 compound is shown below:

[0249] RD-11432 is a chemically modified siRNA and was used as a negative control. Under the same administration, RD-11432 was injected at 3 mg / kg. Mice were sacrificed on day 10 after treatment, and Serping1 mRNA induction was quantified in liver tissue by RT-qPCR. As shown in Figure 21A, RD-13114 induced 1.3-fold expression of Serping1 mRNA on day 10. RD-11432 caused a 91% knockdown of Serping1 mRNA on day 10. Fig. 21BAs shown, there was no significant weight loss in animals treated with RD-12497 and RD-11432 up to day 10 compared to the saline group, indicating that the modified saRNA was well tolerated in C57BL / 6J mice.

[0250] Overall, these results indicate that chemically modified saRNA has persistent Serping1 mRNA activation activity without obvious toxicity. Example 21: Preparation of Compound tC2 of the Present Disclosure 1. Part 1: Synthesis of compound 3. (1) Preparation of Compound 2:

[0251] Dicarboxylic acid (20 g, 86.8 mmol) was dissolved / suspended in anhydrous CH2Cl2 (100 mL). Oxalyl chloride (16.2 mL, 190.96 mmol) and DMF (5 drops) were then added to the solution. The reaction mixture was stirred at room temperature for 3 hours, then concentrated under reduced pressure to give a crude compound 2, which was used directly in the next step without further purification. (2) Preparation of compound 3:

[0252] A solution of benzyl alcohol (9.39 g, 86.8 mmol) and Et3N (12 mL, 86.8 mmol) in THF (200 mL) was added dropwise to an ice-cold solution of compound 2 (86.8 mmol) in THF (100 mL) over 2 hours. The solution was then warmed to room temperature and stirred overnight. A mixture of H2O (80 mL), Et3N (12 mL, 86.8 mmol) and THF (80 mL) was slowly added to the solution over 1 hour, and stirring continued for 2 hours. THF was then removed, and 20 mL of H2O was added to the residue. The mixture was extracted with ether (3×100 mL), the organic phases were combined, dried over Na2SO4, and concentrated. Ethyl acetate (20 mL) was added to the residue, and the suspension was filtered to remove dodecanedioic acid. The filtrate was concentrated and chromatographically analyzed (hexane: ethyl acetate = 4:1 to 2:1) to obtain compound 3 (12.5 g, 45% yield) as a white solid. MW calculated value: 320.20; MW measured value: 319.02 [MH] - . 2. Part 2: Synthesis of compound 6. (1) Preparation of compound 5:

[0253] Borane-methyl sulfide complex (6.16 g, 80 mmol) was slowly added to a solution of Fmoc-L-hydroxyproline (compound 4, 13.3 g, 37.6 mmol) in anhydrous THF (250 mL) at room temperature. The reaction mixture was stirred at room temperature for 5 minutes and then heated to reflux for about 1 hour. Methanol (15 mL) was carefully added to the reaction mixture and refluxed for 15 minutes. The reaction mixture was then concentrated under reduced pressure. The crude product was then evaporated three times with methanol (100 mL each time) to remove boron-related impurities. The resulting crude product of compound 5 was used directly in the next step without further purification. (2) Preparation of Compound 6:

[0254] Under ice bath, DMTrCl (14 g, 41.4 mmol) was slowly added to a solution of compound 5 (37.6 mmol) in anhydrous pyridine (200 mL). The reaction system was stirred overnight under nitrogen protection and then concentrated under reduced pressure. The crude product was dissolved in anhydrous MeCN (300 mL), and Et3N (112.8 mmol, 15.6 mL) was then added to the mixture and heated to 60 ° C for 4 hours. After concentration under reduced pressure, the resulting residue was purified by flash chromatography (silica gel, gradient eluent: 1%-8% MeOH / DCM) to obtain compound 6 (7.57 g, 48% yield) as a yellow solid. 1 H NMR(400MHz, CDCl3)δ7.41(d,J=7.4Hz,2H),7.30(d,J=8.8Hz,4H),7.28–7.2 2(m,2H),7.18(t,J=7.2Hz,1H),6.80(d,J=8.8Hz,4H),4.34(s,1H),3.75(d, J=11.1Hz,6H),3.60(dd,J=12.7,6.7Hz,1H),3.10–2.92(m,5H),2.86(d,J=1 1.5Hz, 1H), 1.85 (dd, J=13.5, 7.1Hz, 1H), 1.63 (ddd, J=13.7, 7.9, 5.9Hz, 1H). 3. Overall synthesis pathway. (1) Preparation of compound 9:

[0255] Sodium hydroxide solution (0.83 g NaOH dissolved in 4 mL H2O) was slowly added to a solution of 2-amino-2-(hydroxymethyl)propane-1,3-diol (compound 7, 25 g, 206 mmol) in DMSO (50 mL). Then 3-(tert-butoxy)-3-oxoprop-1-en-1-ylammonium (90 g, 700 mmol) was slowly added under nitrogen protection. The reaction system was stirred at room temperature for 2 days. Then 100 mL H2O was added to the reaction system, the mixture was extracted with ethyl acetate (3×150 mL), the organic phases were combined, dried with Na2SO4, and concentrated. The resulting residue was purified by flash chromatography (silica gel, gradient eluent: 1%-5% MeOH / DCM) to give compound 9 (42 g, 40% yield) as a colorless oil. (2) Preparation of compound 10:

[0256] Under nitrogen protection, HBTU (7.81 g, 20.6 mmol) and DIPEA (5.1 mL, 30.9 mmol) were added to a DCM (50 mL) solution of 12-(benzyloxy)-12-oxododecanoic acid (compound 3, 3.3 g, 10.3 mmol). After 5 minutes, compound 9 (7.8 g, 15.45 mmol) was added to the reaction system. The reaction system was then stirred at room temperature for 4 hours, and then 50 mL of H2O was added to the reaction system, the mixture was extracted with DCM (3×100 mL), and the organic phases were combined, dried over Na2SO4, and concentrated. The resulting residue was purified by flash chromatography (silica gel, gradient eluent: 1%-30% EA / HX) to obtain compound 10 (7.16 g, 86% yield) as a colorless oil. 1 H NMR (400MHz, CDCl3) δ7.42–7.27(m,5H),6.00(s,1H),5.11(s,2H),3.70(s,6H),3.64(t,J=6.3Hz,6H),2.44(t ,J=6.3Hz,6H),2.34(t,J=7.5Hz,2H),2.17–2.09(m,2H),1.59(d,J=11.6Hz,4H),1.45(s,27H),1.26(s,12H). (3) Preparation of compound 12:

[0257] Under nitrogen protection, CF3COOH (50mL) was added to a DCM (100mL) solution of compound 10 (7.0g, 8.67mmol). The reaction mixture was stirred at room temperature overnight, then concentrated under reduced pressure to obtain a crude product as a colorless oil. Afterwards, the colorless oil was dissolved in 100mL DCM, and then HBTU (19.73g, 52.02mmol) and DIPEA (13mL, 78.03mmol) were added to the reaction system under nitrogen protection. Five minutes later, tert-butyl (3-aminopropyl) carbamate (compound 11, 6.8g, 39mmol) was added to the reaction system. The reaction mixture was stirred at room temperature overnight. Then 50mL H2O was added to the reaction solution, the mixture was extracted with DCM (3×100mL), and the organic phase was combined, dried over Na2SO4, and concentrated. The resulting residue was purified by flash chromatography (silica gel, gradient eluent: 1%-8% MeOH / DCM) to afford compound 12 (6.7 g, 70% yield) as a colorless oil. (4) Preparation of compound 14:

[0258] HCl / dioxane (4M, 8mL) was added to a DCM (8mL) solution of compound 12 (1.0g, 0.9mmol, 1.0eq). The reaction mixture was stirred at room temperature for 3 hours, then concentrated under reduced pressure to obtain a crude product as a yellow solid. Afterwards, the crude product was dissolved in 15mL DCM, and compound 13 (1.81g, 4.05mmol, 4.5eq), HBTU (1.53g, 4.05mmol, 4.5eq) and DIPEA (1.79mL, 10.8mmol, 12.0eq) were added to the reaction system under nitrogen protection. The reaction mixture was stirred at room temperature overnight. Then 10mL H2O was added to the reaction mixture. The mixture was extracted with DCM (3×20mL). The organic phases were combined, dried over Na2SO4, and concentrated. The resulting residue was purified by flash chromatography (silica gel, gradient eluent: 1%-8% MeOH / DCM) to afford compound 14 (0.99 g, 52% yield) as a white solid. 1 H NMR characterization. 1 HNMR (400MHz,CDCl3) 1H NMR (400MHz, CDCl3) δ7.37–7.32(m,5H),5.34(d,J=2.9Hz,3H),5.29(s,2H),5.19(dd,J=11.2,3.2Hz,3H),5. 10(s,2H),4.59(d,J=8.4Hz,3H),4.12(dd,J=13.4,5.8Hz,6H),3.90(d,J=6.2Hz,4H),3.67(s,12H),3.47(s,6 H),3.26(d,J=5.3Hz,10H),3.06(q,J=7.4Hz,4H),2.42(t,J=5.4Hz,6H),2.34(t,J=7.5Hz,3H),2.22(dd,J=1 6.2,7.7Hz,5H),2.13(s,9H),2.03(s,9H),1.98(s,9H),1.94(s,9H),1.40-1.35(m,22H),1.27-1.22(m,12H). (5) Preparation of Compound 15:

[0259] Under nitrogen protection, Pd / C (98 mg) was slowly added to a MeOH (10 mL) solution of compound 14 (0.98 g, 0.47 mmol). The reaction atmosphere was then replaced with hydrogen three times. Afterwards, the reaction mixture was stirred overnight by purging with a hydrogen balloon at room temperature. The reaction mixture was then filtered and concentrated under reduced pressure to obtain a crude product.

[0260] The crude product was dissolved in DCM (20 mL), and HBTU (267 mg, 0.705 mmol, 1.5 eq) and DIPEA (265 μL, 1.598 mmol, 3.4 eq) were added to the reaction under nitrogen protection. Five minutes later, compound 6 (236 mg, 0.564 mmol, 1.2 eq) was added to the reaction mixture. The reaction mixture was stirred at room temperature overnight. H2O (10 mL) was then added to the reaction mixture. The mixture was extracted with DCM (3×20 mL). The organic phases were combined, dried over Na2SO4, and concentrated. The resulting residue was purified by flash chromatography (silica gel, gradient eluent: 1%-10% MeOH / DCM) to give compound 15 (0.79 g, 69% yield) as a yellow solid. The product was purified by 1 H NMR characterization. 1H NMR (400MHz, CDCl3) δ7.34(d,J=8.5Hz,2H),7.25–7.20(m,5H),6.97–6.89(m,2H),6.80(t,J=8.6Hz,4H),5.33(s,3 H),5.17(ddd,J=11.0,7.7,3.2Hz,3H),4.60(t,J=9.1Hz,3H),4.19–4.06(m,9H),3.95–3.85(m,6H),3.78(d,J=3.5H z,6H),3.67(s,12H),3.48(t,J=10.8Hz,4H),3.26(s,12H),2.42(t,J=5.2Hz,6H),2.24(dd,J=26.4,11.2Hz,11H), 2.14(s,9H),2.03(s,9H),1.98(s,9H),1.93(s,9H),1.72–1.51(m,22H),1.38–1.31(m,6H),1.27(d,J=8.6Hz,12H). (6) Preparation of Compound 16:

[0261] Under nitrogen protection, DMAP (138 mg, 1.13 mmol, 3.5 eq) and succinic anhydride (97 mg, 0.97 mmol, 3.0 eq) were added to a solution of compound 15 (780 mg, 0.324 mmol, 1.0 eq) in anhydrous DCM (6 mL). The reaction mixture was stirred at room temperature overnight, and then H2O (10 mL) was added to the reaction system. The mixture was extracted with DCM (3×20 mL). The organic phases were combined, dried over Na2SO4, and concentrated. The resulting residue was purified by flash chromatography (silica gel, gradient eluent: 1%-15% MeOH / DCM) to give compound 16 (690 mg, 85% yield) as a white solid. The product was purified by 1 H NMR characterization. 1H NMR (400MHz, CDCl3) δ7.38–7.31(m,4H),7.21–7.12(m,5H),6.80(dd,J=13.7,5.2Hz,4H),5.33(d,J=2.7Hz,3H), 5.19(d,J=11.2Hz,3H),4.63(dd,J=8.2,4.9Hz,3H),4.16–4.07(m,9H),3.78(s,6H),3.77(s,6H),3.67(s,12H),3 .51–3.45(m,4H),3.25(s,12H),2.58(d,J=7.7Hz,4H),2.43(t,J=5.2Hz,6H),2.25(td,J=15.1,7.7Hz,11H),2.1 3(s,9H),2.03(s,9H),1.97(s,9H),1.94(s,9H),1.83–1.51(m,24H),1.46–1.29(m,6H),1.26(d,J=12.0Hz,12H). (7) Preparation of compound tC2:

[0262] Under nitrogen protection, HBTU (208 mg, 0.55 mmol, 2.0 eq) was added to a solution of compound 16 (690 mg, 0.275 mmol, 1.0 eq), controlled pore glass (CPG; code: C3006-1000, Hebei Dinaxingke Biotechnology Co., Ltd., China; 4.8 g), N,N-diisopropylethylamine (DIPEA; 137 μL, 0.825 mmol, 3.0 eq) in acetonitrile (40 mL). The reaction mixture was shaken at 25 ° C overnight and then washed with DCM and ether to obtain a crude support material.

[0263] Under nitrogen protection, the crude support material was added to a solution of acetic anhydride (15 mL), pyridine (34 mL), NEt3 (500 μL) in acetonitrile (22 mL). The reaction mixture was shaken at 25° C. for 1 h, then washed with DCM and ether to obtain compound tC2 (4.7 g) of the present disclosure. Example 22: Preparation of an oligonucleotide linked to a conjugated group derived from compound tC2 of the present disclosure General methods for synthesizing oligonucleotides (1) Single-strand synthesis method

[0264] Single-stranded oligonucleotides were synthesized by solid phase synthesis on a K&A DNA synthesizer (K&ALaborgeraete GbR, Schaffheim, Germany). Starting material is general carrier material or special carrier material, it is commercially available or as disclosed synthesis in the foregoing text.Usually, in DNA synthesizer, phosphoramidite monomer (0.1M, acetonitrile or dichloromethane solution) including various joints and conjugates is added to carrier material successively, to generate desired full-length oligonucleotide.Each phosphoramidite adds circulation and is made up of four chemical reactions, including trityl removal, coupling, oxidation / thiolation and end-capping.In the first step, the trityl removal reaction of 45 seconds is carried out using the DCM of 3% dichloroacetic acid (TCA).In the second step, all amidites are carried out to the phosphoramidite coupling of 6 minutes by 12eq. In the third step, oxidation was performed for 1 minute using 0.02M iodine in THF: pyridine: water (70: 20: 10, v / v / v); if phosphorothioate modification is required, oxidation was replaced by thiolation for 3 minutes using 0.1M hydrogenated xanthocyanin in pyridine: ACN (50: 50, v / v) solution. In the fourth step, end-capping was performed for 20 seconds using THF: acetic anhydride: pyridine (80: 10: 10, v / v / v) (CAP A) and N-methylimidazole: THF (10: 90, v / v) (CAP B). The cycle of the four chemical reactions will depend on the length of the individual oligonucleotides.

[0265] Deprotection I (Nucleobase Deprotection): After the synthesis is complete, the support material is transferred to a screw-cap microcentrifuge tube. For a 1 μmol synthesis scale, 1 mL of a mixture of methylamine and ammonium hydroxide is added. The tube containing the support material is then heated in an oven at 60°C to 65°C for 15 minutes and then allowed to cool to room temperature. The cleavage solution is collected and evaporated to dryness in a SpeedVac to obtain a crude single-stranded oligonucleotide.

[0266] 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 DMSO. After adding 1 mL triethylamine trihydrofluoride, the tube cap is covered and the mixture is vigorously shaken to ensure complete dissolution, and then heated in an oven at 65°C for 15 minutes. The tube is removed from the oven and cooled to room temperature. The solution containing the fully desilylated oligonucleotide is cooled on dry ice. 2 mL of ice-cold n-butanol (-20°C) is carefully added in 0.5 mL portions 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

[0267] Purification of oligonucleotides was performed on an AKTA explorer 10 equipped with a Source 15Q 4.6 / 100PE 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 min, Flow rate: 1 mL / min. Pure oligonucleotides were collected and desalted through a HiPrep 26 / 10 desalting column. (3) Annealing to form a duplex

[0268] For duplexes, after generating a desalted purified single-stranded solution, the sense and antisense strands were mixed in equal volumes at equimolar concentrations in a tube. The tube was placed in a heating block at 95°C for 5 minutes, then cooled to room temperature and subsequently lyophilized to powder.

[0269] O1 was generated according to the general synthesis method of oligonucleotides described above, using the conjugated group derived from compound tC2 as the starting carrier material. An exemplary structure of a DEC-conjugated oligonucleotide is O1 as shown below: O1(Compound tC2 application)

[0270] It can be seen that in the structure of O1, the conjugated group derived from the delivery enhancing compound tC2 is connected to a double-stranded RNA (dsRNA) duplex (including but not limited to saRNA or siRNA) at the 3' end of the sense strand (S) through a linking portion, such as -OP(O)2O- or -P(O)-O-, wherein (S) is the sense strand and (AS) is the antisense strand. Example 23: Preparation of Compound tC2x4 of the Present Disclosure (1) Compound 18 was prepared by using (2S,3R,4R,5R,6R)-3-acetamido-6-(acetoxymethyl)tetrahydro-2H-pyran-2,4,5-triyl triacetate (Compound 17) as a starting material.

[0271] Under nitrogen protection, TMSOTf (15.3 mL, 84.7 mmol, 1.1 eq) was added to a DCM (300 mL) solution of (2S, 3R, 4R, 5R, 6R)-3-acetamido-6-(acetoxymethyl)tetrahydro-2H-pyran-2,4,5-triyl triacetate (30 g, 77 mmol, 1.0 eq). The reaction mixture was stirred at room temperature for 6 hours. Then NaHCO3 solution (21 g NaHCO3 dissolved in 200 mL water) was added in an ice bath, and the reactants were transferred to room temperature. After stirring for 30 minutes, the mixture was extracted 3 times with DCM, the organic phases were combined, washed with brine, dried over Na2SO4, and concentrated. The resulting residue was purified by flash chromatography (silica gel, gradient eluent: 1%-50% EA / hexane) to give compound 18 (19.8 g, 78% yield). The product was analyzed by mass spectrometry and 1 H NMR characterization. MW calculated value: 329.11; MW found value: 330.33 [M+H] + . 1 H NMR (400MHz, CDCl3) δ5.97(d,J=9.5Hz,1H),5.39(s,1H),5.26(dd,J=11.4,3.2Hz,1H),4.53(td,J=11.4,3.4H z,1H),4.43(t,J=6.5Hz,1H),4.27(s,1H),4.11–4.08(m,1H),2.17(s,3H),2.05(s,3H),2.00(d,J=7.3Hz,6H). (2) Preparation of Compound 20

[0272] Under nitrogen protection, hex-5-en-1-ol (compound 19; 7.2 g, 71.8 mmol, 1.2 eq) and TMSOTf (2.2 mL, 12 mmol, 0.2 eq) were added to a solution of compound 18 (19.7 g, 59.8 mmol, 1.0 eq) in DCE (260 mL). The reaction mixture was stirred at room temperature for 6 hours, then a saturated NaHCO3 solution (100 mL) was added, and the mixture was extracted 3 times with DCM. The organic phases were combined, washed with brine, dried over Na2SO4, and concentrated. The resulting residue was purified by flash chromatography (silica gel, gradient eluent: 1%-5% MeOH / DCM) to give compound 20 (18.7 g, 73% yield). (3) Preparation of Compound 13

[0273] In an ice bath, NaIO4 (37.3 g, 174.4 mol, 4.0 eq) and RuCl3·3H2O (1.8 g, 8.72 mol, 0.2 eq) were added to a solution of compound 20 (18.7 g, 43.6 mmol, 1.0 eq) in ACN / DCM / H2O (140 mL / 140 mL / 210 mL). After 10 minutes, the reaction mixture was transferred to room temperature and stirred overnight. The reaction mixture was then filtered and concentrated under reduced pressure. Water (100 mL) was then added to the mixture, and the mixture was then extracted 3 times with DCM and the organic phase was washed once with brine. After drying over anhydrous Na2SO4 and concentrating under reduced pressure, the resulting residue was purified by flash chromatography (silica gel, gradient elution: 1%-8% MeOH / DCM) to give compound 13 (19 g, 97% yield). The product was analyzed by mass spectrometry and 1 H NMR characterization. MW calculated value: 447.17; MW found value: 448.36 [M+H] + . 1 H NMR (400MHz, CDCl3) δ6.15(d,J=8.7Hz,1H),5.33(d,J=3.0Hz,1H),5.29–5.26(m,1H),4.66(d,J=8.4Hz,1H),4.13(dd,J=9.3,6.8Hz,2H),3.92(dd,J =8.4,5.4Hz,2H),3.50(dd,J=5.7,4.0Hz,1H),2.35(dd,J=14.3,7.0Hz,2H ),2.13(s,3H),2.03(s,3H),1.98(s,3H),1.95(s,3H),1.69–1.61(m,4H). (4) This step involves preparing compound 23 from methyl 4-fluoro-3-nitrobenzoate.

[0274] Under nitrogen protection, to a solution of methyl 4-fluoro-3-nitrobenzoate (compound 21; 8.0 g, 40.2 mmol, 1.0 eq) and K2CO3 (5.5 g, 40.2 mmol) in anhydrous DMF (100 mL) was added (3-aminopropyl)benzyl carbamate (compound 22; 8.3 g, 40.2 mmol, 1.0 eq). The reaction mixture was stirred at 25°C for 6 hours, and then cold water (100 mL) was added. The mixture was extracted 3 times with ethyl acetate, and the organic phase was washed 3 times with saturated LiCl solution and 1 time with brine. It was then dried over anhydrous Na2SO4 and concentrated under reduced pressure to form a yellow solid compound 23, which was used directly in the next step without further purification. (5) Preparation of Compound 24

[0275] In an ice bath, HCOONH4 (9.78 g, 155.04 mmol, 6.0 eq) and Zn powder (10.14 g, 155.04 mmol, 6.0 eq) were added to a solution of compound 23 (10 g, 25.84 mmol, 1.0 eq) in THF / H2O (9:1, 100 mL). After 10 minutes, the reaction mixture was moved to room temperature and stirred overnight. The reaction mixture was then filtered and concentrated under reduced pressure. Water (100 mL) was then added, extracted 3 times with ethyl acetate, and the organic phase was washed once with brine. After drying with anhydrous Na2SO4 and concentrating under reduced pressure, the light red solid compound 24 formed was used directly in the next step without further purification. (6) Preparation of Compound 26

[0276] Under nitrogen protection, 5-((tert-butyldimethylsilyl)oxy)pentanal (compound 25; 5.4 g, 24.84 mmol, 1.0 eq) and AcOH (5.73 mL, 99.36 mmol, 4.0 eq) were added to a solution of compound 24 (8.87 g, 24.84 mmol, 1.0 eq) in EtOH (220 mL). The reaction mixture was stirred at 80°C overnight and then concentrated under reduced pressure. Saturated NaHCO3 solution (100 mL) was then added, and the mixture was extracted three times with ethyl acetate. The organic phases were combined, washed with brine, dried over Na2SO4, and concentrated. The resulting residue was purified by flash chromatography (silica gel, gradient eluent: 1%-5% MeOH / DCM) to give compound 26 (6.3 g, 46% yield) as a red solid. The product was analyzed by mass spectrometry, 1 H NMR and 13 C NMR characterization. MW calculated: 553.30; MW found: 554.29 [M+H] + . 1 H NMR (400MHz, CDCl3) δ8.42 (d, J=0.8Hz, 1H), 7.95 (dd, J=8.5, 1.1Hz, 1H), 7.44 –7.28(m,5H),7.26(d,J=6.8Hz,1H),5.12(s,2H),4.16(dd,J=12.6,5.3Hz,2H) ,3.93(s,3H),3.67(t,J=6.3Hz,2H),3.28(d,J=6.2Hz,2H),2.87(t,J=7.6Hz,2 H), 2.04–1.88 (m, 4H), 1.67 (dd, J = 14.7, 6.6Hz, 2H), 0.88 (s, 9H), 0.04 (s, 6H).13 C NMR (100MHz, CDCl3) δ167.72,156.63,142.35,138.19,136.30,128.60,128.24,124.10,123.87,121.63,1 08.66,66.96,62.71,60.41,52.06,41.31,38.57,32.46,30.43,27.30,25.97,24.15,21.07,18.34,14.21. (7) Preparation of Compound 27

[0277] Under nitrogen protection, 1M TBAF THF solution (17.1 mL, 17.1 mmol, 1.5 eq) was added to a solution of compound 26 (6.3 g, 11.4 mmol, 1.0 eq) in anhydrous THF (50 mL). The reaction mixture was stirred at room temperature for 1 hour and then concentrated under reduced pressure. Water (100 mL) was then added, the mixture was extracted 3 times with DCM, the organic phases were combined, washed with brine, dried over Na2SO4, and concentrated. The resulting residue was dissolved in 50 mL of pyridine, and DMTrCl (4.6 g, 13.68 mmol, 1.2 eq) was added thereto. The reaction mixture was stirred at room temperature for 6 hours and then concentrated under reduced pressure. The resulting residue was purified by flash chromatography (silica gel, gradient eluent: 1%-5% MeOH / DCM) to give compound 27 (6.23 g, 74% yield) as a yellow solid. The product was purified by mass spectrometry, 1 H NMR and 13 C NMR characterization. MW calculated: 741.34; MW found: 303.11 [DMT]-, 440.14 [removed DMT+H] + . 1H NMR (400MHz, CDCl3) δ8.63(dd,J=5.7,1.5Hz,1H),8.43(d,J=1.0Hz,1H),7.97(dd,J=8.5,1.1Hz ,1H),7.45(d,J=7.4Hz,2H),7.37–7.28(m,11H),7.21(dd,J=8.1,6.1Hz,1H),6.83(t,J=5.9Hz,4 H),5.13(s,2H),4.12(t,J=7.2Hz,2H),3.95(s,3H),3.79(s,6H),3.24(d,J=6.2Hz,2H),3.14(t, J=6.2Hz, 2H), 2.83 (t, J=7.4Hz, 2H), 2.01 (dd, J=16.0, 8.5Hz, 4H), 1.79 (dd, J=14.1, 6.6Hz, 2H). 13 C NMR (100MHz, CDCl3) δ167.81,158.43,156.64,149.95,145.34,142.43,138.26,136.57,136.05,130.11,128.69,128.39,128.26,128.23 ,127.84,126.75,124.17,123.89,121.70,113.11,108.75,85.93,67 .02,62.90,55.31,52.16,41.37,38.64,30.50,29.76,27.30,24.53. (8) The carbonyloxy group contained in compound 27 is reduced to a methyleneoxy group to obtain compound 28.

[0278] Under nitrogen protection and ice bath, LiAlH4 (384mg, 10.11mmol, 1.5eq) was added to anhydrous THF (60mL) solution of compound 27 (5.0g, 6.74mmol, 1.0eq). After 10 minutes, the mixture was transferred to room temperature and stirred for about 1 hour. The reactant was then transferred to an ice bath, and saturated potassium sodium tartrate solution (30mL) was slowly added to the mixture. After 30 minutes of reaction, the reaction system was extracted 3 times with Et2O, and then the organic phase was combined, washed with salt water, dried with Na2SO4, and concentrated. The obtained residue was dissolved in 20mL DMF, and imidazole (688mg, 10.11mmol, 1.5eq) and TBSCl (1.524g, 10.11mmol, 1.5eq) were added. The reaction mixture was stirred at room temperature for 1 hour. After concentration under reduced pressure, the resulting residue was purified by flash chromatography (silica gel, gradient eluent: 1%-5% MeOH / DCM) to give compound 28 (4.78 g, 86% yield) as a yellow solid. The product was analyzed by mass spectrometry, 1 H NMR and 13 C NMR characterization. MW calculated: 827.43; MW found: 303.16 [DMT]-, 526.50 [remove DMT+H] + . 1 H NMR(400MHz, CDCl3)δ7.69(s,1H),7.47–7.42(m,2H),7.40–7.28(m,11H),7.22 (t,J=3.5Hz,3H),6.86–6.80(m,4H),5.13(s,2H),4.86(s,2H),4.15(dd,J=14.3 ,7.2Hz,2H),3.79(s,6H),3.13(t,J=6.3Hz,2H),2.82(t,J=7.5Hz,2H),2.05–1 .94(m,4H),1.81–1.74(m,2H),1.29(t,J=7.1Hz,2H),0.97(s,9H),0.13(s,6H). 13C NMR (100MHz, CDCl3) δ171.27,158.45,156.60,154.95,145.40,142.93,136 .66,135.52,134.14,130.14,128.70,128.37,128.28,127.85,126.74,120 .93,117.18,113.13,108.73,85.93,67.00,65.53,63.00,60.51,55.32,53.55,41.19,38.74,30.51,29.85,27.33,26.14,24.78,21.17,18.57,14.33. (9) Compound 30 was prepared from the starting material 4-fluoro-3-nitrobenzoic acid (Compound 29).

[0279] Under nitrogen protection, benzyl bromide (35.6 g, 207.98 mmol, 1.1 eq) was slowly added to a solution of 4-fluoro-3-nitrobenzoic acid (compound 29; 35 g, 189.1 mmol, 1.0 eq) and Na2CO3 (30.6 g, 283.6 mmol, 1.5 eq) in anhydrous DMF (500 mL). The reaction mixture was stirred at 45 ° C for 3 hours, and then cold water (200 mL) was added. The mixture was extracted 3 times with ethyl acetate, and the organic phase was washed 3 times with saturated LiCl solution and 1 time with brine. The organic phase was dried over anhydrous Na2SO4 and concentrated under reduced pressure to form compound 30 as a yellow oil, which was used directly in the next step without further purification. (10) Preparation of Compound 31

[0280] Under nitrogen protection, to compound 30 (52g, 189.1mmol, 1.0eq) and K2CO3 (26.13g, 189.1mmol, 1.0eq) in anhydrous DMF (500mL) solution, add (3-aminopropyl) tert-butyl carbamate (compound 11; 32.9g, 189.1mmol, 1.0eq). The reaction mixture was stirred at room temperature for 3 hours, and then cold water (200mL) was added. The mixture was extracted with ethyl acetate 3 times, and the organic phase was washed 3 times with saturated LiCl solution and 1 time with brine. After drying with anhydrous Na2SO4 and concentrating under reduced pressure, yellow solid compound 31 was formed, which was directly used in the next step without further purification. (11) Reduction of compound 31 to generate compound 32.

[0281] In an ice bath, HCOONH4 (71.58 g, 1.134 mol, 6.0 eq) and Zn powder (74.19 g, 1.134 mol, 6.0 eq) were added to a solution of compound 31 (81.16 g, 189.1 mmol, 1.0 eq) in THF / H2O (9:1, 666 mL). After 10 minutes, the reaction mixture was moved to room temperature and stirred overnight. The reaction mixture was then filtered and concentrated under reduced pressure. Water (200 mL) was then added to the mixture, and the mixture was then extracted 3 times with ethyl acetate and the organic phase was washed once with brine. The organic brine was dried over anhydrous NaSO4 and concentrated under reduced pressure to form a light red solid compound 32, which was used directly in the next step without further purification. (12) Compound 34 was generated by acylation of compound 32 with N2,N6-bis(tert-butoxycarbonyl)-L-lysine.

[0282] Under nitrogen protection, N2,N6-bis(tert-butoxycarbonyl)-L-lysine (compound 33; 21.82 g, 63 mmol, 1.5 eq), EDCI (12.08 g, 63 mmol, 1.5 eq) and DMAP (2.565 g, 21 mmol, 0.5 eq) were added to a solution of compound 32 (16.8 g, 42 mmol, 1.0 eq) in DCM (300 mL). The reaction mixture was stirred at room temperature overnight. Then 200 mL of H2O was added to the reaction mixture, and the mixture was extracted with DCM (3×100 mL). The organic phases were combined, dried over Na2SO4, and concentrated. The resulting residue was purified by flash chromatography (silica gel, gradient eluent: 1%-50% EA / hexane) to give compound 34 (27.5 g, 90% yield) as a yellow solid. The product was analyzed by mass spectrometry and 1 H NMR characterization. MW calculated value: 727.42; MW found value: 728.47 [M+H] + . 1 H NMR (400MHz, CDCl3) δ7.88(dd,J=8.6,1.9Hz,1H),7.79(s,1H),7.35(ddt,J=9.8,7.1,5.5Hz,5H),6.62(d,J=8.6Hz,1H), 5.29(s,2H),4.16(s,1H),3.37–2.97(m,6H),1.78(d,J=25.4Hz,4H),1.51(s,2H),1.48–1.35(m,27H),1.36–1.13(m,2H). (13) Compound 35 containing a benzimidazole structure was prepared from compound 34.

[0283] Under nitrogen protection, compound 34 (20 g, 27.5 mmol, 1.0 eq) was added to AcOH (100 mL). The reaction mixture was stirred at 95 ° C for 4 hours, then saturated NaHCO3 solution (100 mL) was added, and the mixture was extracted 3 times with ethyl acetate, and then the organic phases were combined, washed with saturated NaHCO3 solution (3×100 mL), dried with Na2SO4, and concentrated. The resulting residue was purified by flash chromatography (silica gel, gradient eluent: 1%-50% EA / hexane) to obtain compound 35 (8.78 g, 45% yield) as a white solid. The product was purified by mass spectrometry and 1 H NMR characterization. MW calculated: 709.41; MW found: 710.57 [M+H] + . 1 H NMR (400MHz, CDCl3) δ8.50(s,1H),8.04(dd,J=8.5,1.4Hz,1H),7.46(d,J=7.0Hz,2H),7.41(s,1H),7.38–7.34(m,3H),5. 39(s,2H),5.02(dd,J=15.8,7.7Hz,1H),3.20-3.10(m,6H),2.08-1.96(m,4H),1.57–1.48(m,4H),1.41(d,J=8.6Hz,27H). (14) Preparation of Compound 36

[0284] HCl / dioxane (4M, 20mL) was added to a DCM (10mL) solution of compound 35 (1.8g, 2.54mmol, 1.0eq). The reaction mixture was stirred at room temperature for 3 hours, then concentrated under reduced pressure to obtain a crude product as a yellow solid. Afterwards, the crude product was dissolved in 25mL DCM, and compound 13 (3.4g, 7.62mmol, 3.0eq), HBTU (3.37g, 8.89mmol, 3.5eq) and DIPEA (5.05mL, 30.48mmol, 12.0eq) were added to the reaction system under nitrogen protection. The reaction mixture was stirred at room temperature overnight. Then 20mL H2O was added to the reaction mixture. The mixture was extracted with DCM (3×30mL). The organic phases were combined, dried over Na2SO4, and concentrated. The resulting residue was purified by flash chromatography (silica gel, gradient eluent: 1%-8% MeOH / DCM) to afford compound 36 (3.67 g, 85% yield) as a yellow solid. The product was analyzed by mass spectrometry and 1H NMR characterization. MW calculated value: 1697.80; MW found value: 1698.53 [M+H] + . 1 H NMR (400MHz, CDCl3) δ8.41(d,J=22.5Hz,1H),8.04(d,J=8.5Hz,1H),7.44(d,J=7.0Hz,2H),7.40–7.31(m,4H),5.42–5. 26(m,7H),5.19(td,J=11.0,3.3Hz,2H),4.64–4.55(m,2H),4.24–3.99(m,10H),3.94–3.75(m,6H),3.68–3.62(m,1H),3 .35–3.16(m,4H),3.11–3.06(m,1H),2.35–2.26(m,3H),2.23–2.15(m,6H),2.12(d,J=6.9Hz,9H),2.03(s,9H),1.99(s, 9H), 1.92 (dd, J=11.6, 5.2Hz, 9H), 1.78 (s, 2H), 1.71–1.61 (m, 6H), 1.51 (dd, J=12.6, 6.5Hz, 8H), 1.42 (d, J=6.6Hz, 4H). (15) Connecting compound 36 to compound 37 derived from compound 28

[0285] Under nitrogen protection, Pd / C (185 mg) was slowly added to a MeOH (20 mL) solution of compound 36 (1.85 g, 1.09 mmol, 1.0 eq). The reaction atmosphere was then replaced with hydrogen three times. Afterwards, the reaction mixture was stirred overnight by purging with a hydrogen balloon at room temperature. The reaction mixture was then filtered and concentrated under reduced pressure to obtain a crude product.

[0286] The crude product was dissolved in DCM (20 mL), and then HBTU (826 mg, 2.18 mmol, 2.0 eq) and DIPEA (614 μL, 3.7 mmol, 3.4 eq) were added to the reaction under nitrogen protection. Five minutes later, compound 37 (849 mg, 1.2 mmol, 1.1 eq) produced by reducing compound 28 was added to the reaction mixture. The reaction mixture was stirred at room temperature overnight. H2O (20 mL) was then added to the reaction system, and the mixture was extracted with DCM (3×50 mL). The organic phases were combined, dried over Na2SO4, and concentrated. The resulting residue was purified by flash chromatography (silica gel, gradient eluent: 1%-8% MeOH / DCM) to give compound 38 (1.8 g, 72% yield) as a white solid. The product was purified by mass spectrometry and1 H NMR characterization: MW calculated: 2282.08; MW found: 1142.33 [M+2H] + / 2. 1 H NMR (400MHz, CDCl3) δ8.08 (s, 1H), 7.64 (s, 1H), 7.38 (dd, J = 12.7, 8.1Hz, 4H), 7.28 (d, J=8.8Hz,5H),7.23(d,J=7.8Hz,2H),7.20–7.15(m,2H),6.78(d,J=8.8Hz,4H),5.33(s ,3H),5.23–5.11(m,2H),4.81(s,2H),4.58(dd,J=8.4,2.1Hz,2H),4.18(d,J=10.6Hz, 3H),4.17–4.04(m,8H),3.96–3.80(m,6H),3.75(s,6H),3.63(dd,J=13.3,6.6Hz,1H), 3.57–3.40(m,5H),3.22(s,3H),3.07(dd,J=10.9,4.7Hz,2H),2.85(t,J=7.5Hz,2H),2 .42(s,6H),2.20(dd,J=34.7,6.1Hz,8H),2.10(d,J=7.6Hz,9H),2.06(d,J=9.1Hz,2H) ,2.01(d,J=3.3Hz,9H),1.97(dd,J=7.5,2.4Hz,9H),1.89(dd,J=28.9,13.9Hz,9H),1. 80–1.64(m,6H),1.62–1.46(m,12H),1.40(d,J=6.0Hz,3H),0.92(s,9H),0.08(s,6H). (16) Compound 39 was generated by deprotecting the TBS group of compound 38 and then acylating it with succinic anhydride.

[0287] Under nitrogen protection, 1M TBAF THF solution (2.06 mL, 2.06 mmol, 3.0 eq) was added to a solution of compound 38 (1.57 g, 0.687 mmol, 1.0 eq) in anhydrous THF (10 mL). The reaction mixture was stirred at room temperature for 1 hour and then concentrated under reduced pressure to obtain a crude product.

[0288] The crude product was dissolved in DCM (15 mL), and DMAP (587 mg, 4.8 mmol, 3.5 eq) and succinic anhydride (137 mg, 1.374 mmol, 2.0 eq) were added under nitrogen protection. The reaction mixture was stirred at room temperature overnight, and then H2O (20 mL) was added to the reaction mixture. The mixture was extracted with DCM (3×50 mL). The organic phases were combined, dried over Na2SO4, and concentrated. The resulting residue was purified by flash chromatography. The resulting residue was purified by flash chromatography (silica gel, gradient eluent: 1%-10% MeOH / DCM) to give compound 39 (1.0 g, 64% yield) as a yellow oil. The product was analyzed by mass spectrometry and 1 H NMR characterization: MW calculated: 2268.01; MW found: 1135.22 [M+2H]+ / 2. 1 H NMR (400MHz, CDCl3) δ8.17(d,J=6.6Hz,2H),7.40(d,J=7.6Hz,2H),7.30–7. 26(m,5H),7.24(s,2H),7.18(d,J=6.8Hz,2H),6.79(d,J=8.8Hz,4H),6.50( s,2H),5.32(s,2H),5.29(s,5H),5.25–5.12(m,4H),4.60(dd,J=15.7,8.3H z,2H),4.17(d,J=10.0Hz,2H),4.14–3.96(m,8H),3.87(dd,J=15.1,6.5Hz, 5H),3.76(s,6H),3.45(s,6H),3.19(d,J=8.7Hz,2H),3.08(t,J=6.0Hz,2H) ,3.01(s,10H),2.90–2.77(m,2H),2.63(d,J=17.4Hz,4H),2.19(dd,J=24.8 ,17.2Hz,9H),2.12–2.08(m,9H),2.01(d,J=3.7Hz,9H),1.98–1.94(m,9H), 1.87(dd,J=24.8,13.3Hz,9H), 1.76–1.62(m,6H), 1.55(d,J=15.4Hz,12H). (17) Compound 39 was connected to the carrier material controlled pore glass (CPG) to prepare the compound tC2x4 of the present invention.

[0289] Under nitrogen protection, HBTU (78 mg, 0.206 mmol, 2.0 eq) was added to a solution of compound 39 (233 mg, 0.103 mmol, 1.0 eq), controlled pore glass (CPG; 1.8 g) and N,N-diisopropylethylamine (DIPEA; 51 μL, 0.309 mmol, 3.0 eq) in acetonitrile (14 mL). The reaction mixture was shaken at 25 ° C overnight and then washed with DCM and diethyl ether to obtain a crude support material.

[0290] Under nitrogen protection, the crude support material was added to a solution of acetic anhydride (6.2 mL), pyridine (12 mL), NEt3 (186 μL) in acetonitrile (7.9 mL). The reaction mixture was shaken at 25° C. for 1 h, then washed with DCM and ether to obtain compound tC2x4 of the present disclosure (1.84 g). Example 24: Preparation of conjugated oligonucleotides linked to conjugated groups derived from compounds tC2x4 of the present disclosure Preparation

[0291] O2 was generated according to the general synthesis method of oligonucleotides shown in Example 22 above, using the conjugated group derived from compound tC2x4 as the starting support material. An exemplary structure of a DEC-conjugated oligonucleotide is O2 shown below:

[0292] It can be seen that in the structure of O2, the conjugated group derived from the delivery enhancing compound tC2x4 is connected to a double-stranded RNA (dsRNA) duplex (including but not limited to saRNA or siRNA) at the 3' end of the sense strand (S) through a linking portion, such as -OP(O)2O- or -P(O)-O-, wherein (S) is the sense strand and (AS) is the antisense strand.

[0293] In summary, based on high-throughput screening of saRNA targeting the SERPING1 promoter, multiple hotspots and saRNA targeting the hotspots that can significantly activate SERPING1 gene expression were identified. These saRNAs can not only upregulate the expression of the SERPING1 gene in a dose-dependent manner, but also significantly increase the proportion of functional proteins in cells. In addition, in vivo efficacy studies have demonstrated that the saRNA disclosed herein can significantly improve the blood levels of functional C1INH protein. These results clearly show that activating SERPING1 expression at the transcriptional level with saRNA targeting the SERPING1 promoter to increase C1INH protein expression is a promising strategy for treating HAE. References 1.Chen,M.,and M.A.Riedl.2017.'Emerging Therapies in HereditaryAngioedema',Immunol Allergy Clin North Am,37:585-95. 2.Cohn,D.M.,N.J.Viney,L.M.Fijen,E.Schneider,V.J.Alexander,S.Xia,G.E.Kaeser,C.Nanavati,B.F.Baker,R.S.Geary,M.Levi,J.C.M.Meijers,andE.S.G.Stroes.2020.'Antisense Inhibition of Prekallikrein to ControlHereditary Angioedema',The New England journal of medicine,383:1242-47. 3.Craig,T.,P.Busse,R.G.Gower,D.T.Johnston,J.M.Kashkin,H.H.Li,W.R.Lumry,M.A.Riedl,and D.Soteres.2018.'Long-term prophylaxis therapy inpatients with hereditary angioedema with C1 inhibitor deficiency',Ann AllergyAsthma Immunol,121:673-79. 4.Farfara,Dorit,Emily Feierman,Allison Richards,Alexey S.Revenko,Robert A.MacLeod,Erin H.Norris,and Sidney Strickland.2018. 5.Farkas,H.,M.L.Debreczeni,and K.V.Kohalmi.2018.'Investigationaldrugs in phase I and phase II clinical trials for hereditary angioedema',Expert Opin Investig Drugs,27:87-103. 6.Farkas,Henriette,Nora Veszeli,Kinga V. and LilianVarga.2018.'Real-life experience with recombinant human C1-inhibitor intreatment of hereditary angioedema with C1-inhibitor deficiency',Journal ofAllergy and Clinical Immunology,141:AB49. 7.Ferrone,J.D.,G.Bhattacharjee,A.S.Revenko,T.A.Zanardi,M.S.Warren,F.J.Derosier,N.J.Viney,N.C.Pham,G.E.Kaeser,B.F.Baker,E.Schneider,S.G.Hughes,B.P.Monia,and A.R.MacLeod.2019.'IONIS-PKKRx a Novel Antisense Inhibitor ofPrekallikrein and Bradykinin Production',Nucleic acid therapeutics,29:82-91. 8.Germenis,A.E.,and M.Speletas.2016.'Genetics of HereditaryAngioedema Revisited',Clin Rev Allergy Immunol,51:170-82. 9.Hahn,J.,M.Nordmann-Kleiner,C.Bonner,G.Kojda,T.K.Hoffmann,andJ.Greve.2019.'The Influence of ACE Inhibition on C1-Inhibitor:A Biomarker forACE Inhibitor-Induced Angioedema?',Biomed Hub,4:1-9. 10.Han,Eun D.,Ryan C.MacFarlane,Aideen N.Mulligan,Jennifer Scafidi,and Alvin E.Davis.2002.'Increased vascular permeability in C1inhibitor–deficient mice mediated by the bradykinin type 2receptor',Journal of ClinicalInvestigation,109:1057-63. 11.Liu,J.,J.Qin,A.Borodovsky,T.Racie,A.Castoreno,M.Schlegel,M.A.Maier,T.Zimmerman,K.Fitzgerald,J.Butler,and A.Akinc.2019.'Aninvestigational RNAi therapeutic targeting Factor XII(ALN-F12)for thetreatment of hereditary angioedema',RNA,25:255-63. 12.Liu,S.,Y.Xu,Y.Liu,and Y.Zhi.2019.'Hereditary angioedema:a Chineseperspective',Eur J Dermatol,29:14-20. 13.Liu,Yang,Thomas L.Saunders,Thomas Sisson,Robert Blackburn,DavidS.Ginsberg,and Duane Day.2018. 14.Lumry,W.R.2018.'Hereditary Angioedema:The Economics of Treatmentof an Orphan Disease',Front Med(Lausanne),5:22. 15.Prada,Anne E.,Kamyar Zahedi,and Alvin E.Davis.1998.'Regulation ofC1Inhibitor Synthesis',Immunobiology,199:377-88. 16.Qiu,T.,M.J.Chiuchiolo,A.S.Whaley,A.R.Russo,D.Sondhi,S.M.Kaminsky,R.G.Crystal,and O.E.Pagovich.2019.'Gene therapy for C1 esterase inhibitordeficiency in a Murine Model of Hereditary angioedema',Allergy,74:1081-89. 17.Valerieva,A.,S.Caccia,and M.Cicardi.2018.'Recombinant human C1esterase inhibitor(Conestat alfa)for prophylaxis to prevent attacks in adultand adolescent patients with hereditary angioedema',Expert Rev Clin Immunol,14:707-18. 18.Zahedi,K.,A.E.Prada,and A.E.Davis,3rd.1994.'Transcriptionalregulation of the C1 inhibitor gene by gamma-interferon',J Biol Chem,269:9669-74. 19.Zeerleder,S.,and M.Levi.2016.'Hereditary and acquired C1-inhibitor-dependent angioedema:from pathophysiology to treatment',Ann Med,48:256-67. 20.Zuraw,B.L.2010.'The pathophysiology of hereditary angioedema',World Allergy Organ J,3:S25-8.

Claims

1. A small activating nucleic acid molecule (saRNA), comprising an oligonucleotide sequence having a length ranging from 16 to 35 consecutive nucleotides, wherein the consecutive oligonucleotide sequence comprises a nucleotide sequence having at least 75%, at least 80%, at least 85% or at least 90% homology or complementarity to an equal length portion as shown in SEQ ID NO: 1390, wherein the saRNA activates the expression of the SERPING1 gene by at least 10%.

2. The saRNA according to claim 1, wherein the equal length portion shown in SEQ ID NO: 1390 is located in the -466 to -399 region (SEQ ID NO: 1391), -357 to -279 region (SEQ ID NO: 1392), -256 to -215 region (SEQ ID NO: 1393), -125 to -81 region (SEQ ID NO: 1394) or -80 to -15 region (SEQ ID NO: 1395) upstream of the transcription start site of the SERPING1 gene.

3. The saRNA of any one of claims 1 to 2, wherein the saRNA comprises a sense strand and an antisense strand, wherein the sense strand and the antisense strand each comprise a complementary region, wherein the complementary regions of the sense strand and the antisense strand form a double-stranded nucleic acid structure.

4. The saRNA of any one of claims 1 to 2, wherein the sense strand and the antisense strand have at least 90% complementarity.

5. The saRNA of claim 3, wherein the sense strand and the antisense strand are located on two different nucleic acid strands.

6. The saRNA of claim 3, wherein the sense strand and the antisense strand are located on the same nucleic acid strand, optionally on a hairpin single-stranded nucleic acid molecule, wherein the complementary regions of the sense strand and the antisense strand form a double-stranded nucleic acid structure.

7. The saRNA of claim 3, wherein at least one of the sense strand and the antisense strand comprises a 3' overhang ranging from 0 to 6 nucleotides in length.

8. The saRNA of claim 7, wherein the sense strand and the antisense strand comprise 3' overhangs ranging from 2 to 3 nucleotides in length.

9. The saRNA of claim 7, wherein at least one of the nucleotides of the overhang is a thymidine deoxyribonucleotide.

10. The saRNA of any one of claims 3 to 9, wherein the sense strand and the antisense strand independently comprise a length of about 16 to about 35, about 17 to about 30, about 18 to about 25, or about 19 to about 22 contiguous nucleotides.

11. The saRNA of any one of claims 3 to 10, wherein the sense strand has at least 75% sequence homology to a nucleotide sequence selected from SEQ ID NOs: 274-545, and the antisense strand has at least 75% sequence homology to a nucleotide sequence selected from SEQ ID NOs: 548-819; or wherein the sense strand or the antisense strand has at least 75% sequence homology to a nucleotide sequence of AAAUUCCUGGCCUGGCCCNNN and / or CUACCAGGGNNNNNNNNNN.

12. The saRNA of claim 11, wherein the sense strand comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 274-545, and the antisense strand comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 548-819.

13. The saRNA of claim 1, wherein the oligonucleotide sequence has at least 75% sequence homology or complementarity with a nucleotide sequence selected from SEQ ID NOs: 1-272.

14. The saRNA of claim 1, wherein the sense strand of the oligonucleotide sequence has at least 75% sequence homology to a nucleotide sequence selected from SEQ ID NOs: 1-272.

15. The saRNA of claim 1, wherein the antisense strand of the oligonucleotide sequence has at least 75% sequence complementarity with a nucleotide sequence selected from SEQ ID NOs: 1-272.

16. The saRNA of any one of claims 1 to 15, wherein at least one nucleotide of the saRNA is a chemically modified nucleotide.

17. The saRNA of claim 16, wherein at least one nucleotide in the antisense strand and / or the sense strand of the saRNA is chemically modified.

18. The saRNA of claim 16, wherein the chemically modified nucleotide is a nucleotide having at least one of the following modifications: a) modification of the phosphodiester bonds connecting nucleotides in the nucleotide sequence of the saRNA; b) modification of the 2'-OH of ribose in the nucleotide sequence of the saRNA; and c) modification of bases in the nucleotide sequence of the saRNA.

19. The saRNA of claim 16, 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 phosphoramidate, or a nucleotide containing a non-natural base.

20. The saRNA of claim 16, wherein the chemical modification of the at least one chemically modified nucleotide is the addition of an (E)-vinylphosphonate moiety at the 5' end of the sense strand or the antisense strand.

21. The saRNA of any one of claims 1 to 20, wherein the sense strand or the antisense strand of the saRNA is conjugated to one or more conjugated groups selected from the group consisting of a lipid, a fatty acid, a fluorophore, a ligand, a sugar, a peptide, and an antibody.

22. The saRNA of claim 21, wherein the sense strand or the antisense strand of the saRNA is conjugated to one or more conjugation groups selected from the group consisting of cell penetrating peptides, polyethylene glycol, alkaloids, tryptamines, benzimidazoles, quinolones, amino acids, cholesterol, glucose, and N-acetylgalactosamine.

23. The saRNA of claim 22, wherein the conjugated group is selected from tC2 and tC2x4, in Represents carrier material.

24. An isolated target site for the saRNA according to claim 1, wherein the isolated target site is a nucleotide sequence shown in SEQ ID NO: 1390 having a length ranging from 16 to 35 consecutive nucleotides.

25. The isolated target site of claim 24, wherein the isolated target site is a nucleic acid sequence selected from SEQ ID NO: 1-272.

26. An isolated nucleic acid sequence upstream of the transcription start site of the SERPING1 gene, wherein the isolated nucleic acid sequence is selected from SEQ ID NOs: 1391-1395.

27. An isolated nucleic acid sequence selected from the group consisting of -466 to -399 region, -357 to -279 region, -256 to -215 region, -125 to -81 region and -80 to -15 region upstream of the transcription start site of the SERPING1 gene.

28. The isolated nucleic acid sequence of claim 26 or 27, wherein the isolated nucleic acid sequence is enriched for the isolated target site of claim 24 or 25.

29. The isolated nucleic acid sequence of claim 26 or 27, wherein at least 30% of the designed saRNAs targeting the isolated nucleic acid sequence can activate the expression of the SERPING1 gene by at least 10%, wherein the designed saRNAs: (1) have a GC content of 35% to 65%; (2) have less than 5 consecutive identical nucleotides; (3) have 3 or fewer dinucleotide repeats; and (4) have 3 or fewer trinucleotide repeats.

30. The isolated nucleic acid sequence of claim 26 or 27, wherein a nucleotide region of the isolated nucleic acid sequence having a length ranging from 16 to 35 nucleotides binds to the saRNA of claim 1 in a sequence-specific manner.

31. A method of designing a saRNA, wherein the method provides a saRNA targeting the isolated nucleic acid sequence of any one of claims 26 to 30.

32. An isolated polynucleotide encoding the saRNA of any one of claims 1 to 23.

33. The isolated polynucleotide of claim 32, wherein the isolated polynucleotide is DNA.

34. A vector comprising the isolated polynucleotide according to claim 32 or 33.

35. A host cell comprising the saRNA of any one of claims 1 to 23, the isolated polynucleotide of claim 32 or 33, or the vector of claim 34.

36. A composition comprising the saRNA of any one of claims 1 to 23 or the isolated polynucleotide of claims 32 or 33, and optionally, a pharmaceutically acceptable carrier.

37. The composition of claim 36, wherein the pharmaceutically acceptable carrier is selected from aqueous carriers, liposomes, high molecular polymers, polypeptides and antibodies.

38. The composition of claim 36 or 37, wherein the composition comprises 0.001 nM to 150 nM of the saRNA.

39. The composition of claim 38, wherein the composition comprises 1 nM to 150 nM of the saRNA.

40. A saRNA comprising an oligonucleotide sequence having a length ranging from 16 to 35 consecutive nucleotides for activating / upregulating SERPING1 expression in a cell, wherein the oligonucleotide sequence has at least 75%, at least 80%, at least 85%, or at least 90% sequence homology or complementarity to an equal length portion shown as SEQ ID NO; 1390, wherein the saRNA activates the expression of the SERPING1 gene by at least 10%.

41. The saRNA of claim 40, wherein the equal length region shown in SEQ ID NO: 1390 is located in the -466 to -399 region (SEQ ID NO: 1391), -357 to -279 region (SEQ ID NO: 1392), -256 to -215 region (SEQ ID NO: 1393), -125 to -81 region (SEQ ID NO: 1394), or -80 to -15 region (SEQ ID NO: 1395) upstream of the transcription start site of the SERPING1 gene.

42. The saRNA of claim 41, wherein the saRNA comprises a sense strand and an antisense strand, wherein the sense strand comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 274-545, and the antisense strand comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 548-819.

43. A method for preventing or treating a disease or condition induced by insufficient expression of plasma protein C1 inhibitory protein (C1INH), SERPING1 gene mutation and / or low functional C1IHN levels in the blood in an individual, the method comprising: An effective amount of the saRNA of any one of claims 1 to 23 and 40 to 42, the isolated polynucleotide of claim 32 or 33, the vector of claim 34, or the composition of any one of claims 36 to 39 is administered to the individual.

44. The method of claim 43, wherein the disease or condition is hereditary angioedema (HAE).

45. The method of claim 43, wherein the individual is a mammal.

46. ​​The method of claim 45, wherein the individual is a human.

47. The method of claim 43, wherein the individual suffers from symptoms induced by insufficient expression of C1IHN protein, mutations in the SERPING1 gene, and / or low functional C1IHN levels in the blood.

48. The method of claim 43, wherein the saRNA of any one of claims 1 to 23 and 40 to 42, the isolated polynucleotide of claim 32 or 33, the vector of claim 34, or the composition of any one of claims 36 to 39 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.

49. The method of claim 48, wherein the route of administration is selected from one or more of the following: intrathecal, intramuscular, intravenous, intraarterial, intraperitoneal, intravesical, intracerebroventricular, intravitreal, and subcutaneous administration.

50. The method of claim 43, wherein the method activates / upregulates expression of the SERPING1 gene or SERPING1 mRNA in the individual by at least 10%.

51. The method of claim 43, wherein the method increases C1IHN protein levels in the individual by at least 10%.

52. A method for detecting C1INH protein or C1INH-regulated protein (bradykinin) in the cell according to claim 35.

53. A kit for carrying out the method according to claim 43, the kit comprising: a) the saRNA according to claims 1 to 23 and 40 to 42, and b) instructions for use.

54. The kit of claim 53, wherein the instructions for use comprise means for administering the saRNA of claims 1 to 23 and 40 to 42 to an individual.

55. A kit comprising the saRNA of any one of claims 1 to 23 and 40 to 42, the isolated polynucleotide of claim 32 or 33, the vector of claim 34, or the composition of any one of claims 36 to 39 in a labeled package, the label on the package indicating that the saRNA, the isolated polynucleotide, the vector, or the composition can be used to prevent or treat a disease or condition induced by insufficient expression of plasma protein C1 inhibitory protein (C1INH) or for prevention or treatment of HAE.

56. A kit for detecting the C1INH protein or the C1INH-regulated protein (bradykinin) in the cell according to claim 35.

57. Use of the saRNA of any one of claims 1 to 23 and 40 to 42, the isolated polynucleotide of claim 32 or 33, the vector of claim 34, or the composition of any one of claims 36 to 39 in the preparation of a medicament for preventing or treating a disease or condition induced by insufficient expression of C1IHN protein, a SERPING1 gene mutation, and / or low functional C1IHN levels in the blood in an individual.

58. The use according to claim 57, wherein the disease or condition is hereditary angioedema.

59. The use according to claim 57, wherein the individual is a mammal, optionally wherein the mammal is a human.

60. Use of the saRNA of any one of claims 1 to 23 and 40 to 42, the isolated polynucleotide of claim 32 or 33, the vector of claim 34, or the composition of any one of claims 36 to 39 in the preparation of a formulation for activating / upregulating expression of the SERPING1 gene in a cell.

61. The use of claim 60, wherein the saRNA of any one of claims 1 to 23 and 40 to 42, the isolated polynucleotide of claim 32 or 33, the vector of claim 34, or the composition of any one of claims 36 to 39 is directly introduced into the cell.

62. The use of claim 61, wherein the saRNA is produced in the cell after introducing a nucleotide sequence encoding the saRNA into the cell.

63. The use according to any one of claims 60 to 62, wherein the cell is a mammalian cell, optionally wherein the mammalian cell is a human cell.

64. The use according to claim 63, wherein the cell is in a human.

65. The use of claim 64, wherein the human is a patient suffering from symptoms induced by insufficient expression of C1IHN protein, mutation of the SERPING1 gene, and / or low functional C1IHN levels in the blood, wherein the saRNA, the isolated polynucleotide, or the composition is administered in an amount sufficient to treat the symptoms.

66. The use according to claim 65, wherein the symptom induced by insufficient expression of the C1IHN protein is hereditary angioedema.

67. A method for activating / upregulating the expression of a SERPING1 gene in a cell, the method comprising: An effective amount of the saRNA of any one of claims 1 to 23 and 40 to 42, the isolated polynucleotide of claim 32 or 33, the vector of claim 34, or the composition of any one of claims 36 to 39 is administered to the cell.

68. The method of claim 67, wherein the saRNA of any one of claims 1 to 23 and 40 to 42, the isolated polynucleotide of claim 32 or 33, the vector of claim 34, or the composition of any one of claims 36 to 39 is introduced directly into the cell.

69. The method of claim 68, wherein the saRNA of any one of claims 1 to 23 and 40 to 42, the isolated polynucleotide of claim 32 or 33, the vector of claim 34, or the composition of any one of claims 36 to 39 is introduced into the cell by one or more of the means consisting of: 1) combining the saRNA according to any one of claims 1 to 23 and 40 to 42, the isolated polynucleotide according to claim 32 or 33, the vector according to claim 34, or the saRNA in the composition according to any one of claims 36 to 39 with a pharmaceutically acceptable carrier selected from the group consisting of an aqueous carrier, a liposome, a high molecular weight polymer, a polypeptide, and an antibody; and 2) covalently conjugating the saRNA according to any one of claims 1 to 23 and 40 to 42, the isolated polynucleotide according to claim 32 or 33, the vector according to claim 34, or the composition according to any one of claims 36 to 39 to one or more conjugating groups selected from the group consisting of a cell penetrating peptide, a polyethylene glycol, an alkaloid, a tryptamine, a benzimidazole, a quinolone, an amino acid, cholesterol, glucose, and N-acetylgalactosamine.

70. The method of claim 69, wherein the conjugated group is derived from a compound selected from tC2 and tC2x4, in Represents carrier material.

71. The method according to any one of claims 67 to 70, wherein the cell is a mammalian cell, preferably a cell from the human body.

72. The method of claim 71, wherein the human is a patient suffering from symptoms induced by insufficient expression of C1IHN protein, mutation of the SERPING1 gene, and / or low functional C1IHN levels in the blood, wherein the saRNA, the isolated polynucleotide, or the composition is administered in an amount sufficient to treat the symptoms.

73. The method of claim 72, wherein the symptom caused by insufficient expression of the C11HN protein is hereditary angioedema.

74. A method for increasing the level of C1IHN protein in a cell, the method comprising: An effective amount of the saRNA according to any one of claims 1 to 23 and 40 to 42, the isolated polynucleotide according to claim 32 or 33, the vector according to claim 34, or the saRNA in the composition of any one of claims 36 to 39 is introduced into the cell.

75. Compound tC2 or tC2x4, in Represents carrier material.

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