Oligonucleotide modulators that activate complement factor H expression

Abstract

The present application relates to oligonucleotide modulators for the prevention or treatment of complement factor H deficiency (CFHD) and uses thereof. The oligonucleotide modulator 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 CFH. The present application also relates to pharmaceutical compositions comprising the oligonucleotide modulators disclosed herein and optionally a pharmaceutically acceptable carrier, and a method for upregulating expression of a target gene in a cell using the oligonucleotide modulator disclosed herein or a pharmaceutical composition comprising the oligonucleotide modulator and a method for preventing or treating a condition 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 an oligonucleotide regulator associated with gene activation and its pharmaceutical use. Background Art

[0002] Complement activation is usually controlled by regulatory factors, such as complement factor H (CFH, also known as AC3bINA, adrenomedullin binding protein-1, AMBP-1, AM binding protein-1 factor H, β1H globulin, C3b inactivation promoter, H factor, HF, H factor-1, HF1), a glycoprotein mainly produced by the liver. The mature CFH protein consists of 1213 amino acids and contains 20 short common repeat sequences (SCRs) or complement control protein (CCP) modules to form a microbead-like structure. These SCRs are called SCR1 to SCR20 from the N-terminus to the C-terminus. Each SCR consists of about 60 amino acids and is highly similar in spatial structure. The main function of the C-terminus is to recognize and bind to target cells to prevent them from being attacked by complement-activating substances. Its variation can affect the binding of CFH to the surface of target cells, making it unable to inhibit the alternative pathway of complement, leading to its overactivation and cell damage. The N-terminus is related to its complement regulatory function.

[0003] The CFH gene is a member of the regulator of complement activation (RCA) gene cluster on chromosome 1q32. CFH contains 23 exons spanning 94 kb of genomic DNA. The first exon encodes the 5' untranslated region of the mRNA and the N-terminal 18 amino acids that constitute the signal peptide. Genetic variations in the CFH gene lead to insufficient CFH expression or CFH deficiency, which is not only associated with reduced complement activation processes but also with severe inflammatory renal and ocular pathologies, such as atypical hemolytic uremic syndrome (aHUS), dense deposit disease (DDD), C3 glomerulonephritis (C3GN), CFHR5 nephropathy, lupus nephritis (LN), MPGN type I with pure complement C3 deposition (MPGN1), membranoproliferative glomerulonephritis type II (MPGN2), familial MPGN type III (MPGN3), and age-related macular degeneration (AMD). In view of the variability of the phenotype, the term complement factor H deficiency (CFHD) can be used collectively as a general term to include diseases or disorders associated with low levels of CFH.

[0004] The level of factor H in human plasma varies widely within the population (116-562 mg / mL). Although this variation is the result of a combination of genetic and environmental factors, such as age-dependent increases and smoking-related decreases, one study showed that 63% of the variation in factor H plasma levels is genetically determined [heritability (h 2)=0.63±0.07; P<0.0001]. Some reports have also described significant genotype-phenotype correlations, in which different genetic variants of CFH have specific tendencies for aHUS, AMD, or MPGN2. 30% to 50% of aHUS patients have no CFH or low concentrations in their plasma, and possible reasons include homozygotes or heterozygotes for CFH gene defects, or the presence of autoantibodies against CFH. Homozygotes usually show CFH deficiency in a familial or sporadic pattern, resulting in an earlier onset in infancy. Whereas plasma CFH levels of heterozygotes appear to be normal or slightly below a lower threshold. Most CFH gene mutations are single amino acid mutations, which result in reduced binding affinity of CFH to its ligands and endothelial cells, and thus cause clinical damage, such as CFHD, such as aHUS, AMD, and MPGN2.

[0005] Plasma infusion, as a source of CFH, has been used to treat renal disease or other CFH deficiency diseases with varying success. However, the risks of immune stimulation and protein overload limit this treatment approach. In 2004, Cheong et al. attempted liver transplantation, but the patient ultimately died 11 months after auxiliary partial orthotopic liver transplantation (APOLT). Eculizumab is a safe and effective therapy for preventing TMA recurrence and provides long-term graft function in CFH / CFHR1 heterozygous genes in aHUS. Although it was started in 2007, the annual cost is still very high, and most patients should use it for life. Gene therapy can be an alternative therapy in the future to restore CFH dysfunction instead of the widely used plasma infusion. However, as an emerging medical technology, it still has risks mainly in terms of safety, which are reflected in the vector technology itself and the off-target effects of gene editing tools. The difficulties faced by patients are not only affordability, but also the uncertainty of the long-term effects of the insertion of foreign fragments.

[0006] New therapeutic approaches are therefore needed to treat and prevent conditions and diseases caused by CFH gene mutations or CFH protein deficiency (CFHD). Summary of the invention

[0007] In order to solve the above problems, the present application provides oligonucleotide modulators, such as small activating RNA (saRNA) molecules, for treating diseases or disorders caused by lack or insufficiency of CFH protein (e.g., CFHD) levels by activating / upregulating CFH transcription and increasing the expression level of CFH protein via RNA activation (RNAa) mechanism.

[0008] Specifically, the present inventors have found that functional saRNAs capable of activating / upregulating the expression of CFH mRNA are not randomly distributed on the promoter, but are aggregated in certain specific hot spots. Only some regions on the promoter of the CFH gene are conducive to the activation / upregulation of expression by saRNA, for example, the -538 to -500 region, -468 to -396 region, -329 to -283 region, -273 to -192 region, -173 to -100 region and -64 to -14 region upstream of the transcription start site of the CFH gene. The present inventors have also found that the optimal target sequence / sense strand of saRNA in the CFH promoter region includes sequences having the following items: (1) 35% to 65% 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 CFH mRNA by at least 10% compared to the baseline level of CFH 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 CFH mRNA by at least 10% compared to the baseline level of the CFH gene. Also provided herein is a method for preventing or treating a disease or condition induced by insufficient expression of plasma complement factor F (CFH), a CFH gene mutation, and / or low functional plasma CFH levels in an individual, the method comprising administering any of the saRNA, compositions, and / or pharmaceutical compositions described herein.

[0009] In one aspect of the present application, an oligonucleotide modulator (e.g., a saRNA molecule) capable of activating / upregulating CFH gene expression in a cell is provided, wherein the oligonucleotide modulator (e.g., saRNA) comprises 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 an equal length region of SEQ ID NO: 1707, thereby activating or upregulating the expression of the gene by at least 10% compared to the baseline expression of the CFH gene. In some embodiments, the region of equal length shown in SEQ ID NO: 1707 is located in the -538 to -500 region (SEQ ID NO: 1708), -468 to -396 region (SEQ ID NO: 1709), -329 to -283 region (SEQ ID NO: 1710), -273 to -192 region (SEQ ID NO: 1711), -173 to -100 region (SEQ ID NO: 1712), or -64 to -14 region (SEQ ID NO: 1713) upstream of the transcription start site of the CFH gene.

[0010] 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 chains. And in certain embodiments, the sense strand and the antisense strand disclosed in the present application are located on a continuous nucleic acid chain, 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.

[0011] In certain embodiments, the sense strand disclosed herein has at least 75% sequence homology with a nucleotide sequence selected from SEQ ID NOs: 309-615, and the antisense strand disclosed herein has at least 75% sequence homology with a nucleotide sequence selected from SEQ ID NOs: 618-924. In certain embodiments, the sense strand disclosed herein comprises a nucleotide sequence selected from SEQ ID NOs: 309-615, and the antisense strand disclosed herein comprises a nucleotide sequence selected from SEQ ID NOs: 618-924.

[0012] 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: 1-307. 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: 1-307. 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: 1-307.

[0013] 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 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.

[0014] 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.

[0015] 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.

[0016] 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.

[0017] In certain embodiments, the sense strand or antisense strand of the saRNA disclosed herein 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.

[0018] In certain embodiments, the saRNA is conjugated to a lipid selected from a fatty acid comprising a carbon chain length of 4 to 30 carbon atoms. In certain embodiments, the conjugated group is a lipid / fatty acid having a carbon chain length of 16 carbon atoms.

[0019] In certain embodiments, the present disclosure provides a compound selected from tC2, tC2x4, and C5x5, (tC2 compound), (tC2x4 compound), and (C5x5 compound); 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 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 C5x5 has a structure as shown below: Therein 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.

[0020] In certain embodiments, saRNA is conjugated to one, two, three or more conjugated groups derived from compounds selected from tC2, tC2x4 and C5x5. 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, ether, acyl, etc.) of tC2, tC2x4 or C5x5 compounds can be detached to provide conjugated groups, so the conjugated groups can be considered to be the part obtained by subtracting the one or more atoms or terminal groups from tC2, tC2x4 and C5x5 compounds. According to one embodiment, the conjugated groups derived from tC2, tC2x4 and C5x5 are connected to the chain of asRNA through a connecting part such as –OP(O)2O- or –P(O)-O-. In certain embodiments, the saRNA is conjugated to two conjugated groups, and the two conjugated groups are lipids and N-acetylgalactosamine. In certain embodiments, the two conjugated groups are derived from: 1) a group selected from tC2 or tC2x4, and 2) C5x5, as shown in the present application, in Represents a carrier material. In certain embodiments, the conjugated group conjugated to the saRNA is derived from tC2x4 and C5x5 shown in the present application. In certain embodiments, the saRNA connected to the conjugated group has a structure represented by O1, O2 or O3:

[0021] In another aspect of the present application, an isolated polynucleotide of a saRNA is provided, wherein the isolated polynucleotide is a continuous nucleotide sequence having a length of 16 to 35 nucleotides in SEQ ID NO: 1707. Specifically, the isolated polynucleotide is a nucleic acid sequence selected from SEQ ID NO: 1-307. In another aspect of the present application, a method of using an isolated polynucleotide of a saRNA is provided.

[0022] In another aspect of the present invention, an isolated oligonucleotide complex is provided, wherein the isolated oligonucleotide complex comprises an antisense strand of a saRNA disclosed herein and a sense strand of an isolated polynucleotide disclosed herein. In some embodiments, the isolated oligonucleotide complex activates the expression of a CFH gene by at least 10%.

[0023] 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.

[0024] In another aspect of the present invention, an isolated nucleic acid complex is provided, wherein the isolated nucleic acid complex comprises an antisense strand of the saRNA disclosed herein and a sense strand of the isolated polynucleotide disclosed herein. In some embodiments, the isolated nucleic acid complex activates the expression of the CFH gene by at least 10% compared to the baseline expression of the CFH gene.

[0025] 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 exist in a mammal, such as a human body.

[0026] 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.

[0027] 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 CFH gene in a cell.

[0028] The present application also relates to a method for activating / upregulating the expression of the CFH gene in a cell, wherein the method comprises administering to the cell the above-mentioned saRNA, the isolated polynucleotide disclosed herein, or a composition comprising the above-mentioned saRNA or the isolated polynucleotide disclosed herein. At the same time, a method for increasing the level of CFH protein or the level of functional plasma CFH protein in a cell is also provided, the method comprising introducing an effective amount of saRNA, nucleic acid or composition disclosed herein into the cell.

[0029] 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 it can be present in a mammal, such as a human body. The human body is a patient with an individual disease or symptom caused by a CFH gene mutation, a low CFH level, and / or insufficient plasma levels of functional CFH 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 CFH protein deficiency and / or insufficient expression of functional CFH protein due to CFH gene mutations include, for example, aHUS, AMD, MPGN2, and other CFHD. In one embodiment, the disease caused by insufficient expression of CFH protein, or CFH gene mutation, or insufficient blood levels of functional CFH protein is CFHD. In one embodiment, the CFHD described herein includes aHUS, DDD, C3GN, CFHR5 nephropathy, LN, MPGN1, MPGN2, MPGN3, and AMD.

[0030] Another aspect of the present application relates to a method for preventing or treating a condition caused by insufficient CFH protein expression, CFH gene mutation, and / or insufficient blood levels of functional CFH 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 CFHD. The individual may be a mammal, such as a human. In one embodiment, the individual suffers from symptoms induced by insufficient CFH protein expression, CFH gene mutation, and / or low functional plasma CFH levels, which may include, for example, CFHD. In one embodiment, the disease caused by insufficient blood levels of functional CFH protein caused by CFH gene mutation is CFHD. In one embodiment, CFHD described herein includes aHUS, DDD, C3GN, CFHR5 nephropathy, LN, MPGN1, MPGN2, MPGN3, and AMD. 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 CFH gene or CFH 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 CFH protein in an individual by at least 10%.

[0031] 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 CFH protein, CFH gene mutations, and / or low functional plasma CFH levels in an individual. The individual may be a mammal, such as a human. In one embodiment, the disease or condition may include, for example, CFHD. In one embodiment, the disease caused by insufficient blood levels of functional CFH protein caused by CFH gene mutations is CFHD. In one embodiment, the CFHD described herein include aHUS, DDD, C3GN, CFHR5 nephropathy, LN, MPGN1, MPGN2, MPGN3, and AMD.

[0032] 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 package with a label indicating that the molecule or composition can be used to prevent or treat a disorder or condition induced by insufficient expression of plasma complement factor H (CFH) or for the prevention or treatment of CFHD.

[0033] 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.

[0034] Aspects of the present application include kits 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 complement factor H (CFH) or for the prevention or treatment of CFHD.

[0035] The present application also provides a kit for detecting CFH protein or CFH-regulated protein in plasma 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.

[0036] The saRNA (such as saRNA molecules) that activate / upregulate CFH gene expression provided herein can effectively and specifically upregulate the expression of the CFH gene, increase the expression level of CFH mRNA, and have low toxic side effects, and can be used to prepare drugs for preventing or treating disorders associated with insufficient CFH protein expression and diseases or conditions caused by CFH gene mutations. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1The changes in human CFH mRNA expression levels mediated by saRNA in Li-7 cells are shown. 307 saRNAs targeting human CFH promoters were transfected into human hepatoma cell lines (Li-7) at a concentration of 25nM for 3 days. The sequences and duplex compositions of 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 CFH 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 CFH mRNA expression level of each of the 307 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 CFH mRNA expression.

[0038] Figure 2 saRNAs classified by their target positions and hotspots on the human CFH promoter in Li-7 cells are shown. 307 saRNAs targeting the human CFH promoter were transfected into Li-7 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 CFH 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 CFH 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 target positions on the promoters of -544bp to -1bp upstream of the CFH transcription start site (TSS). The positions of the 6 saRNA hotspots are marked as H1 to H6 in the rectangular dotted box. The numbers above the boxes indicate the boundaries of the hotspot regions relative to the CFH TSS (site 0), spanning from the 5' end of the first saRNA target to the 3' end of the last saRNA target within each hotspot region.

[0039] Figure 3Dose response characterization of CFH saRNA in Li-7 cells is shown. The first 48 human CFH saRNAs were transfected into human Li-7 cells at concentrations of 2.5nM, 10nM, and 25nM for 3 days. Mock was transfected in the absence of oligonucleotides. The dsCon2 duplex (SEQ ID NO: 617 and 926) was used as a nonspecific duplex control. DS16A-si5 (SEQID NO: 616 and 925) is a duplex siRNA, transfected as a negative control. In a single PCR reaction, the mRNA level of CFH 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 3 The relative fold changes of CFH mRNA levels in Li-7 cells transfected with the top 48 CFH saRNAs at concentrations of 2.5 nM, 10 nM, and 25 nM, respectively, are shown. Values ​​(y-axis) are expressed as the relative fold change of CFH mRNA expression levels for each saRNA relative to Mock treatment after normalization to HPRT1 and TBP references.

[0040] Figure 4 The activation effect of several major saRNAs on human CFH protein expression in Li-7 cells is shown. Cells were treated with 25nM saRNA concentration for 3 days. Mock is transfected in the absence of oligonucleotides. dsCon2 is used as a nonspecific duplex control. DS16A-si5 is a duplex siRNA, transfected as a negative control. Figure 4 The CFH protein level obtained by detecting the OD value using an ELISA kit is shown. The values ​​(y axis) are expressed as the CFH protein expression level relative to the Mock treatment. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0041] FIG. 5A to FIG. 5B The activation effects of several major saRNAs on CFH expression in human liver cancer cell lines are shown. Li-7( Figure 5A ) and Huh-7( Figure 5B) cells were treated with 10 nM saRNA for 4 days. Mock was transfected in the absence of oligonucleotides. dsCon2 was used as a nonspecific duplex control. DS16A-si5 is a duplex siRNA, transfected as a negative control. Relative CFH mRNA levels were determined using an ELISA kit. HPRT1 and TBP genes were also amplified, and their geometric mean was used as an internal reference. The values ​​(y-axis) in the figure are expressed as CFH mRNA expression levels relative to Mock treatment after normalization to HPRT1 and TBP. The values ​​(y-axis) in the figure are also expressed as CFH protein expression levels relative to Mock treatment. * / #p<0.05, ** / ##p<0.01, *** / ###p<0.001, **** / ####p<0.0001.

[0042] Figure 6A to Fig.6D The activation effect of saRNA on CFH mRNA expression in Li-7 cells is shown. Four saRNAs (i.e., DS16A-0013, DS16A-0135, DS16A-0199, and DS16A-0055) were transfected into Li-7 cells at increasing concentrations (i.e., 0.0017 nM, 0.0051 nM, 0.152 nM, 0.046 nM, 0.14 nM, 0.41 nM, 1.2 nM, 3.7 nM, 11.111 nM, 33.333 nM, and 100 nM), respectively, for 3 days. Figure 6A to Fig.6D The mRNA levels of CFH 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 CFH mRNA expression levels relative to Mock treatment after normalization to HPRT1 and TBP.

[0043] FIG. 7A to FIG. 7B The activation effect of saRNA on CFH mRNA expression in PLC / PRF / 5 and Huh-7 cells is shown. Cells were treated with saRNA at a concentration of 25 nM for 3 days. Mock is transfected in the absence of oligonucleotides. dsCon2 was used as a non-specific duplex control. DS16A-si5 is a duplex siRNA, transfected as a negative control. RD-11603 and RD-11598 are double-stranded saRNAs, used as exemplary controls. FIG. 7A to FIG. 7BThe mRNA levels of CFH were quantified by two-step RT-qPCR using gene-specific primer sets in separate PCR reactions. HMBS was also amplified and used as an internal reference. Values ​​(y-axis) are expressed as CFH mRNA expression levels relative to Mock treatment after normalization to the HMBS reference.

[0044] Figure 8 The changes of mouse Cfh expression levels in primary mouse hepatocytes (PMH) cells mediated by saRNA are shown. 383 saRNAs targeting mouse Cfh promoters were transfected into PMH cells at a concentration of 25nM for 3 days. Chain sequences and saRNA duplex compositions are shown in Table 2. Mock is transfected in the absence of oligonucleotides (not shown). In a separate PCR reaction, the mRNA levels of Cfh were quantified by one-step RT-qPCR using the gene-specific primer sets shown in Table 4. Hmbs and Tbp were also amplified, and their geometric mean was used as an internal reference. The value (y-axis) is shown as the Cfh mRNA expression level relative to Mock treatment after normalization to Hmbs and Tbp. saRNA is classified in descending order by their activity (log2) of inducing CfhmRNA expression on the x-axis.

[0045] Fig. 9 saRNAs classified by their positions and hot spots on the mouse Cfh promoter in PMH cells are shown. 383 saRNAs targeting the mouse Cfh 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 levels of Cfh were quantified by one-step RT-qPCR using the gene-specific primer sets shown in Table 4. Hmbs and Tbp were also amplified, and their geometric mean was used as an internal reference. Values ​​(y-axis, log2) show the fold changes in the Cfh mRNA expression levels of each of the saRNAs relative to Mock treatment after normalization to Hmbs and Tbp. The saRNAs are classified on the x-axis by their target positions on the promoters of -800bp to -1bp upstream of the Cfh transcription start site (TSS). The positions of the 5 saRNA hot spots are marked as H7 to H11 in the rectangular dotted frame. The numbers above the boxes indicate the boundaries of the hotspot regions relative to the Cfh TSS, spanning from the 5' end of the first saRNA target to the 3' end of the last saRNA target within each hotspot region.

[0046] Fig.10The dose response characterization of the first 64 mouse Cfh saRNAs in PMH cells is shown. The first 64 small Cfh 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. DS16B-si6 (SEQ ID NO: 1310 and 1694) is a duplex siRNA, transfected as a negative control. In a single PCR reaction, the mRNA level of Cfh was quantified by two-step RT-qPCR using the gene-specific primer sets shown in Table 4. Hmbs and Tbp were also amplified, and their geometric mean was used as an internal reference. The value (y-axis) is expressed as the fold change of Cfh mRNA levels in PMH cells relative to Mock treatment after normalization to Hmbs and Tbp references.

[0047] FIG. 11A to FIG. 11B The activation effect of several major saRNAs on Cfh gene expression in PMH cells is shown. Cells were treated with saRNA at a concentration of 25nM for 5 days. Mock was transfected in the absence of oligonucleotides. dsCon2 was used as a nonspecific duplex control. DS16B-si6 is a duplex siRNA, transfected as a negative control. Fig.11A The relative mRNA levels of Cfh in PMH cells measured by two-step RT-qPCR using the gene-specific primer sets shown in Table 4 are shown. Hmbs and Tbp were also amplified and their geometric mean was used as an internal reference. Values ​​(y-axis) are expressed as Cfh mRNA expression levels relative to Mock treatment after normalization to Hmbs and Tbp. Fig. 11B The Cfh protein level obtained by detecting the OD value using an ELISA kit is shown. The values ​​(y axis) are expressed as the Cfh protein expression level relative to the Mock treatment. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0048] Figure 12A to Fig. 12BThe time course changes of saRNA on Cfh expression in PMH cells are shown. Cells were transfected with a single saRNA at a concentration of 25nM for 3-6 days. Mock was transfected in the absence of oligonucleotides. dsCon2 was used as a nonspecific duplex control. DS16B-si6 is a duplex siRNA, transfected as a negative control. Figure 12A shows the mRNA levels of Cfh in PMH cells measured by two-step RT-qPCR using the gene-specific primer sets shown in Table 4 in a separate PCR reaction. Hmbs and Tbp were also amplified, and their geometric mean was used as an internal reference. The value (y-axis) is expressed as the Cfh mRNA expression level relative to the Mock treatment after normalization to Hmbs and Tbp. Fig. 12B The Cfh protein level obtained by detecting the OD value using an ELISA kit is shown. The value (y axis) is expressed as the Cfh protein expression level relative to the Mock treatment.

[0049] Figure 13A to Fig.13D The activation effect of saRNA on Cfh mRNA expression in PMH cells is shown. Four saRNAs (i.e., DS16B-070, DS16B-091, DS16B-098, and DS16B-0241) were transfected into PMH cells at increasing concentrations (i.e., 0.0017 nM, 0.0051 nM, 0.152 nM, 0.046 nM, 0.14 nM, 0.41 nM, 1.2 nM, 3.7 nM, 11.111 nM, 33.333 nM, and 100 nM) for 3 days. Figure 13A to Fig.13D The mRNA levels of Cfh were detected by two-step RT-qPCR using gene-specific primer sets after RNA isolation and RT reaction. Hmbs and Tbp were also amplified, and their geometric mean was used as an internal reference. The values ​​(y axis) are expressed as Cfh mRNA expression levels relative to Mock treatment after normalization to Hmbs and Tbp.

[0050] Fig.14 The activation effect of saRNA on Cfh mRNA expression in PMH cells is shown. Four saRNAs (i.e., RD-13878, RD-14662, RD-14663, and RD-14669) were transfected into PMH cells at 25 nM alone for 3 days. RD-13149 is a chemically modified siRNA used as a negative control. dsCon2 was used as a nonspecific duplex control. Fig.14The mRNA levels of Cfh were detected by two-step RT-qPCR using gene-specific primer sets after RNA isolation and RT reaction. Hmbs and Gusb were also amplified, and their geometric mean was used as an internal reference. The values ​​(y-axis) are expressed as Cfh mRNA expression levels relative to Mock treatment after normalization to Hmbs and Gusb.

[0051] Fig.15 The activation effect of saRNA on Cfh mRNA expression in PMH cells by free uptake is shown. The saRNA (i.e., RD-14660) was added to the PMH cell culture medium at specified increasing concentrations (i.e., 6.25nM, 25nM, 100nM, 400nM, 1600nM, and 6400nM) and cultured for 4 days. RD-13149 and RD-14040 were added to the PMH cell culture medium at 1000nM, respectively, and cultured for 4 days. RD-13149 is a chemically modified siRNA used as a negative control. RD-14040 was used as a single conjugated portion (i.e., tC2x4) control. Fig.15 The mRNA levels of Cfh were detected by two-step RT-qPCR using gene-specific primer sets after RNA isolation and RT reaction. Hmbs and Gusb were also amplified, and their geometric mean was used as an internal reference. The values ​​(y-axis) are expressed as Cfh mRNA expression levels relative to Mock treatment after normalization to Hmbs and Gusb.

[0052] Fig.16 The change in body weight of C57BL / 6J mice after SC injection of modified saRNA is shown. The designated saRNA (i.e., RD-13147) was administered to adult C57BL / 6J mice at 50 mg / kg via SC injection. Normal saline was injected as a vehicle control. RD-13149 is a chemically modified siRNA, injected at 3 mg / kg via SC as a negative control. Mice were sacrificed on day 10 after treatment. Fig.16 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

[0053] 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).

[0054] The embodiments of the present disclosure are based in part on the surprising discovery that oligonucleotide modulators (e.g., saRNA, also referred to herein as "CFH gene saRNA") can activate or upregulate the expression of the CFH gene in cells. After the administration of the saRNA of the present application, the increase in the production of functional CFH gene mRNA can achieve a significant increase or upregulation of CFH mRNA and CFH protein levels.

[0055] Specifically, the present inventors have found that functional saRNAs capable of activating / upregulating the expression of CFH mRNA are not randomly distributed on the promoter, but are aggregated in certain specific hot spots. Only some regions on the promoter of the CFH gene are conducive to the activation / upregulation of expression by saRNA, for example, the -538 to -500 region, -468 to -396 region, -329 to -283 region, -273 to -192 region, -173 to -100 region and -64 to -14 region upstream of the transcription start site of the CFH gene. The present inventors have also found that the optimal target sequence / sense strand of saRNA in the CFH promoter region includes sequences having the following items: (1) 35% to 65% 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 CFH mRNA by at least 10% after interacting with the saRNA compared to the baseline level of CFH mRNA. Based at least in part on these findings, the present disclosure features saRNA, compositions, and pharmaceutical compositions for activating / upregulating the expression of CFH mRNA by at least 10% compared to the baseline level of CFH mRNA. Also provided herein is a method for preventing or treating a disease or condition induced by insufficient expression of plasma complement factor F (CFH), a CFH gene mutation, and / or low functional plasma CFH levels in an individual, the method comprising administering any of the saRNA, compositions, and / or pharmaceutical compositions described herein.

[0056] 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 CFH gene in cells aggregate at specific CFH gene promoter regions, such as Figure 2 and Fig. 9As shown. The inventors have identified aggregates of these CFH gene promoter regions, which are considered to be "hotspot" promoter regions that enrich the target sites of the functional saRNA developed. These specific promoter regions identified in the present application (referred to herein as "hotspots") are optionally at least 39 nt in length, or have a length ranging from about 39 nt to about 153 nt. "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 about 40%, such as 43% 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 times or more changes in the mRNA expression of the target gene are induced. In some embodiments, at least 30%, about 40%, or more than 50% of the saRNAs designed to target the fragments in these hotspot regions are proven to be functional, i.e., 1.1 times or more changes in the mRNA level or protein expression of the target gene are induced. The inventors were surprised to find that functional saRNAs were not randomly distributed on the promoter, but gathered in specific hot spots (see, e.g., Table 9). For example, 6 hot spots of the human CFH promoter located in the -538 to -500 region (H1), -468 to -396 region (H2), -329 to -283 region (H3), -273 to -192 region (H4), -173 to -100 region (H5), and -64 to -14 region (H6), as well as 5 hot spots of the mouse Cfh promoter located in the -754 to -654 region (H7), -479 to -421 region (H8), -409 to -370 region (H9), -208 to -56 region (H10), and -52 to -4 region (H11) of the promoter TSS were detected, and they were found to be the best target sites for saRNAs that activate CFH / Cfh gene expression through RNA activation mechanisms.

[0057] This saRNA-CFH mRNA-CFH protein pathway may provide an alternative therapeutic approach to current treatments for CFH deficiency-related disorders (CFHD), such as patients with atypical hemolytic uremic syndrome.

[0058] In this application, the relevant terms are defined as follows:

[0059] 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).

[0060] 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 can be formed by hydrogen bonds in the double-stranded region, the oligonucleotide chain has 10% complementarity. In the same example, if 18 base pairs can be formed by hydrogen bonds in the double-stranded region, the oligonucleotide chain has 90% complementarity. Substantially complementary refers to at least about 75%, about 79%, about 80%, about 85%, about 90%, about 95% or 99% complementarity.

[0061] The terms "oligonucleotide" or "polynucleotide" are used interchangeably and refer to polymers of nucleotides, including single-stranded or double-stranded nucleic acid molecules of DNA, RNA, or DNA / RNA hybrids, oligonucleotide chains containing regular and irregular alternating 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 (saRNA).

[0062] 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.

[0063] 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 may 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 the target gene sequence, such as a target gene promoter, which is homologous or complementary to the sense strand or antisense strand of the saRNA. The target gene may also include one or more regulatory elements, wherein one or more saRNAs are designed to have sequence identity with the regulatory element. Non-limiting examples of one or more regulatory elements include: promoters, enhancers, silencers, insulators, TATA boxes, GC boxes, CAAT boxes, transcription start sites, DNA binding motifs for transcription factors or other proteins that regulate transcription, and 5' untranslated regions.

[0064] As used herein, in a saRNA duplex, the term "sense strand" of the saRNA refers to a strand that has sequence homology or sequence identity with a fragment of the coding strand of the target gene sequence.

[0065] 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.

[0066] 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.

[0067] 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.

[0068] 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 nucleic acid that may contain a five-membered or six-membered bridging structure with an N-O bond and a 2'-O and 4'-aminoethylene bridge. As used herein, the term "PNA" refers to a nucleic acid mimic having a pseudopeptide backbone consisting of N-(2-aminoethyl)glycine units, wherein the nucleobase is connected to the glycine nitrogen via a carbonyl methylene linker. As used herein, the term "GNA", also known as glycerol nucleic acid, is a nucleic acid similar to DNA or RNA but with a different sugar-phosphodiester backbone composition, in which propylene glycol replaces ribose or deoxyribose.

[0069] 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.

[0070] 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.

[0071] 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 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%.

[0072] 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).

[0073] 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.

[0074] As used herein, the terms "gene activation" or "activating gene expression" and "gene upregulation" or "upregulating gene expression" are used interchangeably, meaning 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.

[0075] As used herein, the terms "oligonucleotide modulator", "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, 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. 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 typically 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 typically 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 "oligonucleotide modulator," "saRNA," "small activating RNA," and "small activating nucleic acid molecule" also encompass nucleic acids other than ribonucleotides, including but not limited to modified nucleotides or analogs.

[0076] As used herein, the term "hotspot" refers to a gene promoter region of at least 39 bp in length, in which functional saRNAs are enriched, i.e., at least 40%, such as about 43%, 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 mRNA 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 a saRNA, in which the full-length target of a functional saRNA is enriched and spans from the 5' end of the first saRNA to the 3' end of the last saRNA within the 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.

[0077] 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 CFH gene by less than 10% (or less than 1.1-fold).

[0078] As used herein, the terms "isolated target site" and "isolated polynucleotide" are used interchangeably and refer herein to a target site that has complementarity or hybridization with a saRNA. For example, an isolated nucleic acid sequence of a target site may include a nucleic acid sequence that has complementarity or hybridization with a region of a saRNA. As used herein, the term "isolated polynucleotide" means a polynucleotide encoding a saRNA.

[0079] 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.

[0080] 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.

[0081] As used herein, capitalized "CFH" or "CFH gene" refers to the human gene; and capitalized "Cfh" or "Cfh gene" refers to the mouse gene.

[0082] As used herein, the term "CFH mRNA" refers to messenger RNA (mRNA) produced by the expression of a CFH gene or the transcription of a CFH gene.

[0083] As used herein, the terms "CFH" and "CFH protein" are used interchangeably to refer to the protein produced by the expression of the CFH gene or the translation of CFH mRNA. saRNA

[0084] In the present application, the expression of the CFH gene is upregulated by RNA activation, and the related diseases (particularly CFHD) are treated by increasing the expression level of the CFH protein. Since the CFH gene encodes the CFH protein, the increase in CFH mRNA expression leads to an increase in CFH protein expression, thereby treating the disease (e.g., CFHD). Therefore, in some cases, the CFH gene is the target gene in the present application.

[0085] Aspects of the present application include oligonucleotide modulators (saRNA), the small activating nucleic acid molecule 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: 1707, and wherein the saRNA activates / upregulates the expression of the CFH gene by at least 10% compared to the baseline expression of the CFH gene.

[0086] In some embodiments, the equal length portion shown in SEQ ID NO: 1707 disclosed herein is located in the -538 to -500 region (SEQ ID NO: 1708), -468 to -396 region (SEQ ID NO: 1709), -329 to -283 region (SEQ ID NO: 1710), -273 to -192 region (SEQ ID NO: 1711), -173 to -100 region (SEQ ID NO: 1712), or -64 to -14 region (SEQ ID NO: 1713) upstream of the transcription start site of the CFH gene.

[0087] In some embodiments, the contiguous oligonucleotide sequence of the saRNA has five or fewer, i.e., 5, 4, 3, 2, 1, or 0 nucleotide differences or mismatches relative to an equal length portion of SEQ ID NO: 1707. In some embodiments, these differences or mismatches are located in the middle or 3' end of the oligonucleotide sequence of the saRNA. The methods and principles of saRNA molecule design are well known to those skilled in the art and are described in detail in, for example, Place et al., Molecular Therapy-Nucleic Acids (2012) 1, e15; and Li et al., PNAS, 2006, vol. 103, no. 46, 17337-17342, which are incorporated herein by reference in their entirety.

[0088] In some embodiments, 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 CFH gene in the cell by the RNAa mechanism. The RNAa mechanism used herein (also referred to as RNA activation) refers to the mechanism by which a double-stranded nucleic acid structure can upregulate a 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 a nucleic acid chain (e.g., a continuous nucleic acid sequence). 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 thymine deoxyribonucleotides (dT). In some cases, when the sense strand and the antisense strand are located on one nucleic acid strand, the 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-mentioned saRNA have a length in the range of 16 to 35 nucleotides, respectively. 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.

[0089] 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: 1-307. 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: 309-615, 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: 618-924. More specifically, the sense strand of the saRNA disclosed herein comprises or consists of any nucleotide sequence selected from SEQ ID NOs: 309-615; and the antisense strand of the saRNA disclosed herein comprises or consists of any nucleotide sequence selected from SEQ ID NOs: 618-924, or is any nucleotide sequence selected from SEQ ID NOs: 618-924.

[0090] In certain embodiments, one strand of the saRNA has five or fewer, i.e., 5, 4, 3, 2, 1, or 0 nucleotide differences or mismatches relative to a nucleotide sequence selected from SEQ ID NOs: 1-307. Specifically, the sense strand of the saRNA disclosed herein has five or fewer, i.e., 5, 4, 3, 2, 1, or 0 nucleotide differences or mismatches relative to a nucleotide sequence selected from SEQ ID NOs: 309-615, and the antisense strand of the saRNA disclosed herein may have five or fewer, i.e., 5, 4, 3, 2, 1, or 0 nucleotide differences or mismatches relative to a nucleotide sequence selected from SEQ ID NOs: 618-924. In some embodiments, these differences or mismatches are located in the middle or 3' end of the sense or antisense strand of the saRNA.

[0091] 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: 1707, specifically, the target portion is a nucleic acid sequence selected from SEQ ID NO: 1-307.

[0092] 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.

[0093] 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.

[0094] 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.

[0095] 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.

[0096] 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.

[0097] 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.

[0098] 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, the United States, which is incorporated herein by reference. 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 an extended ligand library, 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 in the present application include, but are not limited to, RNAs containing modified backbones or containing no natural internucleoside bonds. In some embodiments, RNAs with modified backbones include RNAs that do not have phosphorus atoms in the backbone. In some embodiments, modified RNAs that do not have phosphorus atoms in their internucleoside backbones can also be considered oligonucleosides. In some embodiments, modified oligonucleotides will have phosphorus atoms in their internucleoside backbones.

[0099] 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.

[0100] 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, 534,639; 6,608,035; 6,683,167; 6,858,715; 6,867,294; 6,878,805; 7,015,315; 7,041,816; 7,273,933; 7,321,029; and U.S. Patent RE39464, which are hereby incorporated by reference in their entirety.

[0101] In certain embodiments, the small activating nucleic acid molecule is RNA, DNA, BNA, LNA, GNA, or peptide nucleic acid (PNA).

[0102] 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.

[0103] 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 oligonucleotide to which it is attached, 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 oligonucleotide to which it is attached, 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).

[0104] 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.

[0105] 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.

[0106] 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.

[0107] In some embodiments, the saRNA of the present application relates to a sense strand or antisense strand of saRNA conjugated to one or more conjugation 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, saRNA is conjugated to two conjugation groups. In certain embodiments, the two conjugation groups are lipids and N-acetylgalactosamine. In certain embodiments, the two conjugation groups are derived from: 1) a group selected from tC2 or tC2x4, and 2) C5x5, as shown in the present application, In certain embodiments, the conjugated groups conjugated to the saRNA are derived from tC2x4 and C5x5 as shown in this application.

[0108] In certain embodiments, the conjugated group is a lipid selected from a fatty acid having a carbon chain length of 4 to 30 carbon atoms. In certain embodiments, the conjugated group is a fatty acid having a carbon chain length of 16 carbon atoms. In certain embodiments, the conjugated group is selected from a lipophilic moiety as described in WO2021092371A2. In certain embodiments, saRNA may include one, two, three, four, five, six or even more oligonucleotides conjugated to one, two, three, four, five, six or even more conjugated groups respectively through one, two, three, four, five, six or even more connecting moieties.

[0109] According to one embodiment, when a linking moiety is present, the linking moiety may be selected from -O-, -S-, -C(O)-, -NH-, -N((C1-C 12 )alkyl)-, -N((C1-C 12 )Alkyl)-C(O)-O-, -OC(O)-, -C(O)-O-, -OC(O)-O-, -C(O)-NH-, -OP(O)2O-, -P(O)(O - )O-, -OP(O)O-, -OP(O)(S)O-, -OS(O)2-O-, -S(O)2-O-, -S(O)-O-, -(C1-C 22 )alkylene-, -(C1-C 22 ) alkylene-NH-, -NH-(C1-C 22 )alkylene-, -(C1-C 22 )alkylene-NH-C(O)-, -(C1-C 22 )alkylene-C(O)-, -(C1-C 22 )alkylene-C(O)-O-, -C(O)-(C1-C 22 )alkylene-, -NH-C(O)-(C1-C 22 )alkylene-, -C(O)-NH-(C1-C 22 )alkylene-, -C(O)-(C1-C 22 ) alkylene-NH-, -NH-(C1-C 22 )alkylene-C(O)-, -C(O)-(C1-C 22 )alkylene-C(O)-, -NH-(C1-C 22 )alkylene-NH-, -C(O)-(C1-C 22 )alkylene-C(O)O-, -OC(O)-(C1-C 22 )alkylene-C(O)-O-, -C(O)-O-(C1-C 22)alkylene-OC(O)-, -C(O)-(C1-C 22 ) alkylene-NH-C(O)-, -NH-C(O)-(C1-C 22 )alkylene-C(O)-, -NH-C(O)-(C1-C 22 )alkylene-C(O)-NH-, -C(O)-NH-(C1-C 22 )alkylene-NH-C(O)-, -(C1-C 22 )alkylene-OP(O)2O-, -(C1-C 22 )alkylene-OP(O)(O - )O-、-(C1-C 22 )alkylene-OP(O)(O - )O-(C1-C 22 )alkylene-, -(C1-C 22 )alkylene-OP(O)O-, -(C1-C 22 )alkylene-OP(O)(S)O-, -(C1-C 22 )alkylene-OS(O)2-O-, -(C1-C 22 )alkylene-S(O)2-O-, -(C1-C 22 )alkylene-S(O)-O-, -OP(O)2-O-(C1-C 22 )alkylene-OP(O)2O-, -OP(O)-O-(C1-C 22 )alkylene-OP(O)O-, -OP(O)(S)O-(C1-C 22 )alkylene-OP(O)(S)O-, -OS(O)2-O-(C1-C 22 )alkylene-OS(O)2-O-, -S(O)2-O-(C1-C 22 )alkylene-S(O)2-O- and -OS(O)-(C1-C 22 )alkylene-S(O)-O-; wherein the connecting part contains -(C1-C 22 ) alkylene - can be an alkylene group containing 1 to 22 carbon atoms, such as 2 to 20 carbon atoms, or 3 to 18 carbon atoms, or 4 to 16 carbon atoms, or 5 to 12 carbon atoms, or 6 to 10 carbon atoms. In one embodiment, when the linking moiety is a direct bond, the conjugated group is directly linked to the oligonucleotide.

[0110] 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.

[0111] 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 chemically modified nucleotides.

[0112] 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.

[0113] These modifications can increase the bioavailability of saRNA, improve affinity for target sequences, and increase resistance to nuclease hydrolysis in cells.

[0114] 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%).

[0115] In a non-limiting example, the saRNA is designed based at least in part on 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 saRNA is designed / selected based at least in part on the criteria of being able to produce functional saRNA. For example, in some cases, the sequence located upstream of the TSS may include a sequence that is not conducive to saRNA synthesis despite being located in a hotspot.

[0116] In some embodiments, the saRNA is designed / selected based at least in part on the following criteria: comprising a sequence having a specific GC content (e.g., a GC content of 25% to 75%) and lacking consecutive identical nucleotides, consecutive dinucleotides, or consecutive trinucleotides. In some embodiments, the saRNA sequence comprises a sequence 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.

[0117] In some embodiments, the saRNA sequence comprises a sequence having a GC content of 25% to 75%, 30% to 70%, 35% to 65%, 40% to 60%, or 45% to 55%. In some embodiments, the saRNA comprises a sequence having a GC content of 35% to 65%.

[0118] In some embodiments, the saRNA sequence comprises a sequence having less than 7 consecutive identical nucleotides, less than 6 consecutive identical nucleotides, less than 5 consecutive identical nucleotides, less than 4 consecutive identical nucleotides, or less than 3 consecutive identical nucleotides. In some embodiments, the saRNA comprises a sequence having less than 5 consecutive identical nucleotides.

[0119] In some embodiments, the saRNA sequence comprises a sequence having 5 or fewer dinucleotide repeats, 4 or fewer dinucleotide repeats, 3 or fewer dinucleotide repeats, or 2 or fewer dinucleotide repeats. In some embodiments, the saRNA comprises a sequence having 3 or fewer dinucleotide repeats.

[0120] In some embodiments, the saRNA sequence comprises a sequence having 5 or fewer trinucleotide repeats, 4 or fewer trinucleotide repeats, 3 or fewer trinucleotide repeats, or 2 or fewer trinucleotide repeats. In some embodiments, the saRNA comprises a sequence having 3 or fewer trinucleotide repeats. Target sequence

[0121] 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: 1707. In certain embodiments, the isolated target site is a nucleic acid sequence selected from SEQ ID NO: 1-307. The isolated target site is capable of interacting with the antisense strand of the saRNA disclosed in the present application, and is thus capable of activating the expression of the CFH gene (e.g., mRNA expression, protein expression, CFH expression). In some embodiments, the target site is selected at least in part based on the gene sequence. In some embodiments, the target site is selected at least in part based on a sequence close to the transcription start site (TSS) of the gene. In some embodiments, the target site is selected at least in part based on a promoter sequence upstream of the TSS. In some embodiments, the target site is selected at least in part based on a sequence from -5000bp, -4000bp, -3000bp, -2000bp, -1000bp, or -500bp upstream of the TSS. In some embodiments, the target site is selected at least in part 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.

[0122] In certain embodiments, the present application relates to an isolated oligonucleotide complex comprising a saRNA disclosed herein and an isolated target site disclosed herein. In certain embodiments, the isolated oligonucleotide complex activates the expression of the CFH gene by at least 10% (e.g., compared to the baseline expression level of the CFH gene, the expression of the CFH gene is activated). Hot Spots

[0123] In certain embodiments, the present application relates to an isolated nucleic acid sequence located upstream of the transcription start site of the CFH gene, i.e., a "hotspot". In certain embodiments, the isolated nucleic acid sequence disclosed herein is an oligonucleotide sequence having a length of at least 39 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: 1707. A "hotspot" herein is defined as a nucleic acid region on the target sequence of a saRNA, where the full-length target of a functional saRNA is enriched and spans the 5' end of the first saRNA within the hotspot to the 3' end of the last saRNA. In some embodiments, at least 40% (e.g., 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%) of the saRNA designed to target the hotspot is functional, i.e., it can induce at least a 1.1-fold change in the mRNA protein expression of the target gene. In a non-limiting example, at least 43% of the saRNAs designed for the target hotspots were functional, i.e., induced at least a 1.1-fold change in target gene mRNA expression. In a non-limiting example, the saRNAs were designed based at least in part on 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 same or similar criteria are used to select isolated nucleic acid sequences and / or target sequences. In a non-limiting example, an isolated nucleic acid sequence upstream of the TSS of the CFH gene was selected based at least in part on 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 polynucleotide sequence selected from SEQ ID NO: 1708-1713. In some embodiments, the hotspot is a nucleic acid sequence selected from the -538 to -500 region, -468 to -396 region, -329 to -283 region, -273 to -192 region, -173 to -100 region, and -64 to -14 region upstream of the transcription start site of the CFH gene. The present application also provides a method for designing saRNA, the method providing saRNA targeting the isolated nucleic acid sequence described in the present application.

[0124] In some embodiments, the target sequence is designed / selected based at least in part on the criteria of being able to produce functional saRNA. For example, in some cases, the sequence located upstream of the TSS may include a sequence that is not conducive to the synthesis of the target sequence despite being located in a hotspot.

[0125] In some embodiments, target sequences within hotspots are selected based at least in part on the following criteria: including sequences having a particular GC content (e.g., a GC content of 25% to 75%) and lacking consecutive identical nucleotides, consecutive dinucleotides, or consecutive trinucleotides. In some embodiments, target sequences within hotspots include sequences that have a GC content of 35% to 65%; (2) have fewer than 5 consecutive identical nucleotides; (3) have 3 or fewer dinucleotide repeats; and (4) have 3 or fewer trinucleotide repeats.

[0126] In some embodiments, the target sequence comprises a sequence having a GC content of 25% to 75%, 30% to 70%, 35% to 65%, 40% to 60%, or 45% to 55%. In some embodiments, the saRNA comprises a sequence having a GC content of 35% to 65%.

[0127] In some embodiments, the target sequence comprises a sequence having less than 7 consecutive identical nucleotides, less than 6 consecutive identical nucleotides, less than 5 consecutive identical nucleotides, less than 4 consecutive identical nucleotides, or less than 3 consecutive identical nucleotides. In some embodiments, the saRNA comprises a sequence having less than 5 consecutive identical nucleotides.

[0128] In some embodiments, the target sequence comprises a sequence having 5 or fewer dinucleotide repeats, 4 or fewer dinucleotide repeats, 3 or fewer dinucleotide repeats, or 2 or fewer dinucleotide repeats. In some embodiments, the target sequence comprises a sequence having 3 or fewer dinucleotide repeats.

[0129] In some embodiments, the target sequence comprises a sequence having 5 or fewer trinucleotide repeats, 4 or fewer trinucleotide repeats, 3 or fewer trinucleotide repeats, or 2 or fewer trinucleotide repeats. In some embodiments, the target sequence comprises a sequence having 3 or fewer trinucleotide repeats.

[0130] The RNAa activity of each designed saRNA depends on a complex myriad of factors, such as the chromatin environment, sequence features of the target itself and nearby regions, transcription factor binding, etc. A core potential determining factor may be the accessibility of the DNA target. In regions with higher accessibility, dsRNAs may exhibit 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 are clustered together. For example, a target sequence designed at least in part based on the following criteria may not activate / upregulate the expression of the CFH gene by at least 10% compared to the baseline expression of the CFH gene, because the target sequence bound by the saRNA is not in a hotspot (e.g., any hotspot described herein): (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.

[0131] In certain embodiments, the present application relates to an isolated nucleic acid complex comprising a saRNA disclosed herein and an isolated nucleic acid sequence disclosed herein. In certain embodiments, the isolated nucleic acid complex activates the expression of the CFH gene by at least 10% compared to the baseline expression of the CFH gene.

[0132] In some aspects, methods using isolated nucleic acids upstream of a transcriptional target site of a CFH gene are also provided. DNA encoding saRNA

[0133] In certain embodiments, the present application relates to nucleic acids or polynucleotides encoding saRNAs that can activate or upregulate the expression of a CFH gene in a cell by at least 10% (e.g., compared to the baseline expression of the CFH gene). In certain embodiments, the nucleic acid is a DNA encoding a saRNA. In certain embodiments, the nucleic acid is a recombinant vector, specifically a recombinant AAV vector. The vector disclosed herein comprises a DNA fragment encoding the saRNA of the present application. Cells containing saRNA

[0134] After contacting the cell, the saRNA disclosed herein can effectively activate or upregulate the expression of the CFH gene in the cell, preferably upregulating the expression by at least 10% (e.g., compared to the baseline expression of the CFH gene).

[0135] 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 (e.g., a Li-7 cell), a human liver cancer cell (e.g., a Huh-7 cell), a human liver cancer cell (e.g., a HepG2 cell), or a cell from a mouse (e.g., a mouse embryonic liver cell or an embryonic cell line BNL.CL2), a mouse liver cancer cell (e.g., 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 CFH gene mutation, a low CFH level, and / or insufficient levels of functional plasma CFH protein. In some embodiments, the cell is from a patient with CFHD. Compositions comprising saRNA

[0136] 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. In some embodiments, the composition may include 0.001 nM to 150 nM (e.g., 0.01 nM to 100 nM, 0.1 nM to 50 nM, 1 nM to 150 nM, 1 nM to 20 nM, 0.001 nM to 1 nM, 1 nM to 10 nM, 10 nM to 100 nM, 10 nM to 50 nM, 20 nM to 50 nM, 20 nM to 100 nM) of a saRNA or an isolated polynucleotide as described herein. In some embodiments, the composition includes 50 nM of a saRNA or an isolated polynucleotide as described herein. Methods of using saRNA

[0137] Another aspect of the present application relates to saRNA for activating / upregulating CFH gene expression in cells. 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: 1707, specifically, saRNA activates / upregulates the expression of the CFH 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 CFH 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, or at least 14 times compared to the baseline expression of the CFH gene). In certain embodiments, the saRNA activates or upregulates the expression of the CFH gene by about 14 times. In certain embodiments, the expression of the CFH 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, the induction of CFH gene protein (CFH) 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.

[0138] 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 complement factor H (CFH), CFH gene mutations, and / or low functional plasma CFH levels, the method comprising: administering to the individual an effective amount of saRNA disclosed herein, nucleic acids encoding saRNA, or isolated polynucleotides, or compositions comprising saRNA. 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 CFHD. In some embodiments, the individual is a mammal. In some embodiments, the individual is a human.

[0139] 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 introduced directly 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 Li-7, Huh-7, and PMH, or human cells). Such cells can be ex vivo, such as cell lines, etc., or can be present in mammals (such as humans). In some embodiments, the human is a patient or individual with a CFH deficiency-related condition or CFHD. In certain embodiments, nucleic acids encoding saRNA or isolated polynucleotides or compositions comprising saRNA as described herein are each present in an amount sufficient to treat CFHD.

[0140] Another aspect of the present application relates to administering an effective amount of saRNA or composition to an individual using an administration route as described herein. In some embodiments, the administration route is selected from one or more of the following: parenteral infusion, oral administration, intranasal administration, inhalation administration, vaginal administration, and rectal administration. In some embodiments, the administration route is selected from one or more of the following: intrathecal, intramuscular, intravenous, intraarterial, intraperitoneal, intravesical, intraventricular, intravitreal, and subcutaneous administration. Dosage regimen and route of administration

[0141] 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 CFH deficiency-related conditions or CFHD.

[0142] Aspects of the present application also relate to methods of preparing such compositions using the saRNA of the present application.

[0143] Another aspect of the present application relates to the use of the saRNA of the present application in the preparation of the pharmaceutical composition disclosed herein.

[0144] 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 drug for preventing or treating a gene or protein-related symptom induced by insufficient expression of CFH protein, CFH gene mutation and / or low functional plasma CFH protein level in an individual. According to the use of certain embodiments, the condition may include a CFH gene mutation-related disorder or condition, including CFHD. According to the use of certain embodiments, the symptoms induced by insufficient expression of CFH protein are aHUS, DDD, C3GN, CFHR5 nephropathy, LN, MPGN1, MPGN2, MPGN3 or AMD. The present application also relates to the use according to certain embodiments, wherein the individual is a mammal, such as a human.

[0145] The dosage at which the saRNA or composition of the present application can be administered can vary over a wide range and will meet 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.

[0146] A single dose of saRNA can be a single dose in the range of 0.01 mg / kg to 1000 mg / kg, for example, 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 of any saRNA sequences described herein.

[0147] In some embodiments, 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 CFHD 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 CFHD 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 CFHD 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.

[0148] In certain embodiments, the dose and / or volume of the injection will be adjusted based on the age of the patient, the weight of the patient, and / or other factors that may require adjustment of injection parameters.

[0149] 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 TM and 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.

[0150] Examples of other compositions or components associated with the saRNA, compositions, pharmaceutical compositions, and methods described herein include, but are not limited to: diluents, salts, buffers, chelating agents, preservatives, desiccants, antimicrobial agents, needles, syringes, packaging materials, tubes, bottles, flasks, beakers, etc., such as for use, modification, assembly, storage, packaging, preparation, mixing, dilution, and / or preservation of components for a particular use. In embodiments using a liquid form of any component, the liquid form may be concentrated or ready for use.

[0151] In some embodiments, the lipid moiety used in nucleic acid therapy can be applied to the present application to deliver saRNA molecules disclosed herein. In such methods, nucleic acids (e.g., one or more saRNAs described herein) are introduced into preformed liposomes or lipid complexes made of a mixture of cationic lipids and neutral lipids. In certain 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.

[0152] 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.

[0153] 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.

[0154] 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 embodiments, a viral vector can be used. Non-limiting examples of viral 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), vaccinia virus (VV), adeno-associated virus (AAV), adenovirus, lentivirus, and retrovirus.

[0155] In some embodiments, the vector is a recombinant AAV vector (rAAV). 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 various types 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 rest of the genome is divided into two essential regions with encapsulation function: the left part of the genome, which contains the rep gene involved in viral replication and viral gene expression; and the right part of the genome, which contains the cap gene encoding viral capsid protein.

[0156] 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, JE 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 have been deleted and replaced with a gene of interest, and the use of these constructs to transfer the gene of interest in vitro (into cultured cells) or in vivo (directly into an organism).Replication-deficient recombinant AAV (rAAV) 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.

[0157] 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.

[0158] 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.

[0159] 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.

[0160] Typical formulations of oligonucleotide modulators 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

[0161] Another aspect of the present application relates to a method for detecting CFH protein or CFH-regulated protein in plasma. In certain embodiments, the method includes detecting CFH protein or CFH-regulated protein in cells transfected with saRNA disclosed herein, isolated polynucleotides, or compositions comprising saRNA. In certain embodiments, the methods disclosed herein can be used to detect a specific subgroup of patients with a disorder or condition induced by insufficient expression of plasma complement factor H (CFH) protein, CFH gene mutations, and / or low functional plasma CFH levels. 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.

[0162] In certain embodiments, the baseline measurement is obtained from a biological sample as defined herein, which is obtained from an individual prior to administering a therapy described herein. In certain embodiments, the baseline expression of the CFH gene is obtained from a biological sample prior to administering a saRNA described herein. In certain embodiments, the biological sample is peripheral blood mononuclear cells, plasma, serum, skin tissue, cerebrospinal fluid (CSF).

[0163] In some embodiments, the saRNA provided herein activates the amount of functional plasma CFH protein 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.

[0164] In some embodiments, saRNA shows a greater effect than an additive effect or a synergistic effect in the treatment, prevention, delay of progression, and / or improvement of a disease caused by a CFH gene mutation. In some embodiments, saRNA shows a greater effect than an additive effect or a synergistic effect in the protection of cells associated with the pathophysiology of the disease, particularly in the treatment, prevention, delay of progression, and / or improvement of CFHD.

[0165] Another aspect of the present application relates to a method for activating / upregulating the expression of a CFH gene in a cell, the method comprising: administering the saRNA or isolated polynucleotides or compositions of the embodiments disclosed herein. In some embodiments, the saRNA or isolated polynucleotides or compositions are introduced directly 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.

[0166] Another aspect of the present application relates to a method for increasing CFH protein levels in cells of a patient or functional plasma CFH protein levels, the method comprising introducing an effective amount of a saRNA, a nucleic acid or polynucleotide encoding the saRNA, or a composition of the embodiments disclosed herein into a cell or subject. Reagent test kit

[0167] Another aspect of the present application relates to a kit for implementing a method of increasing CFH protein levels or functional plasma CFH protein levels in a cell, the kit comprising a saRNA disclosed herein. In certain embodiments, the kit further comprises a 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 complement factor H (CFH) or directed to CFHD.

[0168] 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.).

[0169] 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.

[0170] Another aspect of the present application relates to a kit for detecting CFH protein or CFH-regulated proteins in plasma. In certain embodiments, the kit is used to detect CFH protein or CFH-regulated proteins in cells transfected with any of the saRNA disclosed herein or isolated polynucleotides or compositions disclosed herein. Also provided herein is a kit for increasing CFH protein levels in cells. saRNA synthesis

[0171] The present application provides a method for preparing an oligonucleotide modulator (saRNA), which includes sequence design and synthesis.

[0172] saRNA can be chemically synthesized or obtained from biotechnology companies specializing in nucleic acid synthesis. Generally speaking, 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:

[0173] 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 a starter, 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

[0174] 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

[0175] 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

[0176] 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

[0177] Human hepatoma Li-7 (TCHu183, Shanghai Institutes for Biological Sciences, China) cells were cultured in modified RPMI1640 medium (Gibco) supplemented with 10% calf serum (Sigma-Aldrich) and 1% penicillin / streptomycin (Gibco, Thermo Fisher Scientific, Carlsbad, CA) at 37°C and 5% CO2. Human hepatoma Huh-7 (JCRB0403, Nanjing Kebai Biotechnology Co., Ltd., China) cells were cultured in modified DMEM medium (Gibco) supplemented with 10% calf serum (Sigma-Aldrich) and 1% penicillin / streptomycin (Gibco, Thermo Fisher Scientific, Carlsbad, CA) at 37°C and 5% CO2. Primary mouse hepatocytes (PMH) were isolated from the liver of C57BL / 6 mice (Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd., China). PMH were cultured in a modified Willian's Medium E (WME) medium (A12176-01, Gibco) supplemented with 1% insulin (S6955, Selleck, USA) and 1% penicillin / streptomycin (Gibco, Thermo Fisher Scientific, Carl Pasteur, California) at 5% CO2 and 37°C. Li-7 and PMH cells were seeded into 96-well plates at 4000 cells / well, respectively. According to the reverse transfection protocol, saRNA was transfected into Li-7 cells in each well with 0.3 μL of RNAiMAX (Invitrogen, Carl Pasteur, California) at a final concentration of 2.5 nM, 10 nM or 25 nM or any other concentration, respectively, for 72 hours. Mock (blank control) was transfected in the absence of oligonucleotides. dsCon2 (SEQ ID NO: 617 and 926) was used as a non-specific duplex control. DS16A-si5 (SEQ ID NO: 616 and 925) is a duplex siRNA of CFH gene, and was transfected as a negative control. DS16B-si6 (SEQ ID NO: 1310 and 1694) is a duplex siRNA of Cfh gene, and was transfected as a negative control. RT-qPCR and ELISA assays (1) One-step reverse transcription-quantitative polymerase chain reaction (one-step RT-qPCR)

[0178] At the end of transfection, the medium was discarded and the cells were washed once with 150 μL PBS per well. After discarding PBS, 100 μL cell lysis buffer (Power Green Cells to-Ct TMKit, 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 TBGreen in a Roche Lightcycler 480 real-time PCR instrument (Roche, reference number: 4729749001, USA). TM PrimeScript TM RT-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

[0179] 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 CFH gene and mouse Cfh gene were amplified as target genes. Human reference genes (HPRT1 and TBP) were also amplified, and their geometric mean was used as an internal reference for RNA loading. Mouse reference genes (Hmbs and Tbp) were also amplified, and their geometric mean was used as an internal reference 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

[0180] 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 LightCycler 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

[0181] To calculate the expression level of CFH or Cfh (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 1, E rel =2 (CtTm-CtTs) / ((2 (CtR1m-CtR1s) *2 (CtR2m-CtR2s) ) (1 / 2) ) (Formula 1) 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 internal 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) ELISA assay

[0182] In order to quantify the expression level of human CFH protein, the supernatant of cultured cells (e.g., Li-7 or Huh-7) was collected and detected by OD value using an ELISA kit (Signalway Antibody, catalog number: EK2066, USA). According to the following ELISA kit instructions provided by the kit manufacturer, the expression level of human CFH protein was obtained. (4) Statistical analysis

[0183] One-way analysis of variance (ANOVA) followed by Tukey's multiple comparisons was used to compare the differences between groups of continuous variables. Differences were considered significant when the P value was less than 0.05. * / #p<0.05, ** / ##p<0.01, *** / ###p<0.001, **** / ####p<0.0001. Animal surgery

[0184] 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 hepatocytes (PMH) isolation

[0185] C57BL / 6J mice (Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.) were anesthetized with isoflurane and perfused with initial flushing reagent and digestion reagent in sequence. The liver was placed in a 10 cm culture dish and torn open with tweezers in the culture medium. The cell suspension was collected by 70-75 micron membrane filtration into a 50 mL conical tube, and then centrifuged at 100 × g in a swing arm centrifuge for 2 minutes at 4 ° C. After removing the supernatant, 20 mL of cold PBS was drawn to wash the cells (repeat this step twice). Cells with at least 80% viability tested by 0.4% trypan blue can be measured. The cells were inoculated onto a cell culture plate coated with collagen I for 4 to 12 hours in advance, and the final confluence was 90% to 95%, and the measurement was started.

[0186] It was found in the present application that the above-mentioned saRNA, after being introduced into cells, can effectively increase the expression levels of CFH and Cfh mRNA and protein.

[0187] 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 CFH promoter

[0188] The coding sequence (SEQ ID NO: 1707) from the human CFH gene promoter was retrieved from the UCSC genome database. The sequence consists of 580 nucleotides, ranging from -1 bp relative to the transcription start site (TSS) to -580 bp upstream (Table 7). Table 7. Deduced human CFH promoter sequence (5'-3') (SEQ ID NO: 1707)

[0189] A total of 307 small activating RNA (saRNA) duplexes of 19 bp in length targeting promoter sequences were synthesized according to the following criteria: (i) 35% to -65% GC content; (ii) less than 5 consecutive identical nucleotides; (iii) 3 or fewer total dinucleotide repeats; and (iv) 3 or fewer total trinucleotide repeats. A double dTdT overhang was also added to the 3' end of each strand, resulting in a total length of 21 nt. The strand composition and sequence of each saRNA duplex, including the homologous target site in the CFH promoter, are listed in Table 1. Example 2: High-throughput screening of saRNA targeting the human CFH promoter

[0190] To identify saRNAs capable of upregulating CFH, Li-7 cells were transfected with 25 nM concentrations of each of the above saRNAs for 72 hours, followed by gene expression analysis by one-step RT-qPCR. A nonspecific duplex (i.e., dsCon2) was used as a nonspecific duplex control, while siRNA targeting human CFH transcripts (i.e., DS16A-si5) was used as a negative control for transfection efficiency. Of the 307 saRNAs, 44 (14.3%), 53 (17.3%), and 35 (11.4%) saRNAs showed high activation (≥1.5-fold), moderate activation (1.2 to 1.5-fold), and Table 8. Summary of saRNA screening for CFH expression

[0191] 175 (57.0%) saRNAs reduced CFH levels or had no significant effect. The relative changes in CFH expression caused by saRNA treatment are also summarized in Table 1, and the expression data obtained by gene induction are plotted in Figure 1 Moderate. Mild activation (1.1 to 1.2-fold) (Table 8). Sorting the expression data by target site location within the human CFH promoter revealed six “hot spots” where saRNA activity was concentrated, including regions -538 to -500 (H1), -468 to -396 (H2), -329 to -283 (H3), -273 to -192 (H4), -173 to -100 (H5), and -64 to -14 (H6) relative to the TSS ( Figure 2 ). Nearly 53% of all functional saRNAs target sequences within the indicated "hotspots". The promoter sequences corresponding to each "hotspot" are listed in Table 7.

[0192] At least 30% of the designed saRNAs targeting the provided hotspot sequences can activate the expression of the CFH gene by at least 10% following the following design criteria: (i) 35% to 65% GC content; (ii) less than 5 consecutive identical nucleotides; (iii) 3 or fewer total dinucleotide repeats; and (iv) 3 or fewer trinucleotide repeats.

[0193] The saRNA duplexes capable of upregulating human CFH expression by 1.1-fold or more in the “hotspot region” H1 (-538 to -500) are as follows: DS16A-011, DS16A-012, DS16A-013, DS16A-014, DS16A-015, DS16A-016, DS16A-017, DS16A-018, DS16A-019, DS16A-020, DS16A-021, DS16A-022, DS16A-023, DS16A-024.

[0194] The saRNA duplexes capable of upregulating human CFH expression by 1.1-fold or more in the “hotspot region” H2 (-468 to -396) are as follows: DS16A-025, DS16A-026, DS16A-027, DS16A-028, DS16A-029, DS16A-030, DS16A-031, DS16A-032, DS16A-033, DS16A-034, DS16A-035, DS16A-036, DS16A-037, DS16A-038, DS16A-039, DS16A-040, DS16A-041, DS16A-042, DS16A-043, DS16A-044, DS16A-045, DS16A-046, DS16A-047, DS16A-048, DS16A-049, DS16A-050, DS16A-051, DS16A-052, DS16A-053, DS16A-054, DS16A-055, DS16A-056, DS16A-057, DS16A-058, DS16A-059, DS16A-060.

[0195] The saRNA duplexes capable of upregulating human CFH expression by 1.1-fold or more in the “hotspot region” H3 (-329 to -283) are as follows: DS16A-099, DS16A-100, DS16A-101, DS16A-102, DS16A-103, DS16A-104, DS16A-105, DS16A-106, DS16A-107, DS16A-108, DS16A-109, DS16A -110, DS16A-111, DS16A-112, DS16A-113, DS16A-114, DS16A-115, DS16A-116, DS16A-117, DS16A-118, DS16A-119, DS16A-120, DS16A-121.

[0196] The saRNA duplexes capable of upregulating human CFH expression by 1.1-fold or more in the “hotspot region” H4 (-273 to -192) are as follows: DS16A-129, DS16A-130, DS16A-131, DS16A-132, DS16A-133, DS16A-134, DS16A-135, DS16A-136, DS16A-137, DS16A-138, DS16A-139, DS16A-140, DS16A-1 41. DS16A-142, DS16A-143, DS16A-144, DS16A-145, DS16A-146, DS16A-147, DS16A-148, DS16A-149, DS16A-150, DS16A-151, DS16A-152, DS16A-153, DS16A -154, DS16A-155, DS16A-156, DS16A-157, DS16A-158, DS16A-159, DS16A-160, DS16A-161, DS16A-162, DS16A-163, DS16A-164, DS16A-165, DS16A-166, DS 16A-167, DS16A-168, DS16A-169, DS16A-170, DS16A-171, DS16A-172, DS16A- 173, DS16A-174, DS16A-175, DS16A-176, DS16A-177, DS16A-178, DS16A-179.

[0197] The saRNA duplexes capable of upregulating human CFH expression by 1.1-fold or more in the “hotspot region” H5 (-173 to -100) are as follows: DS16A-193, DS16A-194, DS16A-195, DS16A-196, DS16A-197, DS16A-198, DS16A-199, DS16A-200, DS16A-201, DS16A-202, DS16A-203, DS16A-204, DS16A-205, DS16A-206, DS16A-207, DS16A-208, DS16A-209, DS16A-210, DS16A-211, DS16A-212, DS16A-213, DS16A-214, DS16A-215, DS16A-216, DS16A-217, DS16A-218, DS16A-219, DS16A-220, DS16A-221, DS16A-222, DS16A-223, DS16A-224, DS16A-225, DS16A-226, DS16A-227, DS16A-228, DS16A-229, DS16A-230, DS16A-231, DS16A-232, DS16A-233, DS16A-234, DS16A-235, DS16A-236, DS16A-237, DS16A-238, DS16A-239, DS16A-240.

[0198] The saRNA duplexes capable of upregulating human CFH expression by 1.1-fold or more in the “hotspot region” H6 (-64 to -14) are as follows: DS16A-266, DS16A-267, DS16A-268, DS16A-269, DS16A-270, DS16A-271, DS16A-272, DS 16A-273, DS16A-274, DS16A-275, DS16A-276, DS16A-277, DS16A-278, DS16A-279, DS16A -280, DS16A-281, DS16A-282, DS16A-283, DS16A-284, DS16A-285, DS16A-286, DS16A-2 87. DS16A-288, DS16A-289, DS16A-290, DS16A-291, DS16A-292, DS16A-293, DS16A-294. Table 9. Human CFH and mouse Cfh saRNA hotspot regions and their sequences Example 3: Dose-dependent activation of human CFH expression by saRNA treatment in Li-7 cells

[0199] Based on the CFH-induced screening results, the top 48 saRNAs with good performance (see Table 1) were transfected into Li-7 cells at 3 concentrations (i.e., 2.5 nM, 10 nM, and 25 nM) for 72 hours to quantify dose-dependent gene induction by one-step RT-qPCR. dsCon2 and DS16A-si5 were used as nonspecific duplex controls for gene activation and negative controls for transfection efficiency, respectively. Figure 3 As shown, most saRNAs exhibited dose-dependent induction of CFH, with DS16A-274 showing the highest level of activity at 25 nM treatment concentration, up to 9.5-fold. The most effective duplexes included 42 saRNAs (87.5%), which showed a ≥1.5-fold increase in CFH levels at the lowest concentration (i.e., 2.5 nM), of which 13 (27.1%) activated CFH more than 3-fold. Example 4: saRNA treatment increases secreted CFH protein levels in Li-7 cells

[0200] The best performing subset (23 saRNAs in total) was transfected into Li-7 cells at a concentration of 25 nM to quantify the secretion of CFH protein in the culture medium after 72 hours by ELISA. dsCon2 and DS16A-si5 were used as nonspecific duplex controls for gene activation and negative controls for transfection efficiency, respectively. Figure 4 As shown, an increase in CFH protein levels was measured for each saRNA after 72 hours of treatment compared to baseline (dashed line), with 9 duplexes showing statistical significance. Example 5: Correlation between CFH protein and mRNA induction after saRNA treatment of human hepatocellular carcinoma cell lines

[0201] CFH mRNA upregulation and protein secretion were evaluated in two liver cancer cell lines (i.e., Li-7 and Huh-7) using 10 saRNAs (i.e., DS16A-011, DS16A-013, DS16A-038, DS16A-055, DS16A-144, DS16A-135, DS16A-153, DS16A-170, DS16A-178, and DS16A-199). Each saRNA was transfected at 10 nM for 4 days, and mRNA expression and CFH protein levels were quantified by two-step RT-qPCR and ELISA in the same samples, respectively. dsCon2 treatment served as a nonspecific duplex control for gene activation, while DS16A-si5 served as a negative control for transfection efficiency and comparison of CFH mRNA and protein knockdown. Li-7 ( Figure 5A ) and Huh-7( Figure 5B) cells all had the same general pattern, i.e., mRNA induction by saRNA treatment was associated with a measurable increase in secreted protein. The relative fold changes in protein levels were generally not as high as those for mRNA induction, which may be due to the kinetic delay in protein translation and secretion compared to transcriptional activation. In support, knockdown of CFH mRNA by DS16A-si5 treatment was also associated with a delay in maximal inhibition of protein levels ( FIG. 5A to FIG. 5B The relative changes in CFH mRNA and protein levels in Li-7 and Huh-7 cells are also summarized in Table 10. Table 10. Relative expression (fold change) of CFH mRNA and protein levels in Li-7 and Huh-7 cells Note: / means not tested. Example 6: CFH gene activation efficacy of saRNA in Li-7 cells Dose response curves were generated by two-step RT-qPCR for the 4 best performing saRNAs (i.e., DS16A-013, DS16A-135, DS16A-199, and DS16A-055) after 72 hours of treatment using 11 increasing concentrations (i.e., 0.0017, 0.0051, 0.0152, 0.046, 0.14, 0.41, 1.23, 3.7, 11.111, 33.333, and 100 nM) to characterize their potency ( Figure 6A to Fig.6D As summarized in Table 11, each saRNA exhibited a dose-dependent activation of CFH expression, EC 50 Values ​​were in the low nanomolar range (ie, approximately 1.18 nM to 2.8 nM). Table 11. ECs that activate CFH expression in vitro 50 value Example 7: Effect of saRNA structure and sequence on saRNA activity in vitro

[0202] In order to evaluate the effect of duplex structure and sequence specificity on saRNA activity, two of the best performing saRNAs (i.e., DS16A-135 and DS16A-013) were selected to synthesize a series of variants for activating the expression of human CFH. Table 12 lists the sequence composition and design of each saRNA variant. Each duplex was transfected into PLC / PRF / 5 or Huh-7 cells at a concentration of 25nM for 3 days, and mRNA expression was analyzed by two-step RT-qPCR. dsCon2 and DS16A-si5 treatments were used as nonspecific duplex controls for gene activation and negative controls for transfection efficiency, respectively. FIG. 7A to FIG. 7BAs shown in Figure 1, shortening the duplex length of DS16A-135 to 16 bp (i.e., RD-14694) abolished the induction effect of saRNA on CFH mRNA, while extending the duplex length to 20 bp with asymmetric overhangs (i.e., RD-14696) or removing the overhangs on the 18 bp duplex (i.e., RD-14695) did not significantly affect saRNA activity. However, further extending the duplex length to 28 bp and adding asymmetric overhangs (i.e., RD-14697) significantly reduced CFH induction, at least in Huh-7 cells ( Figure 7B ). Mutation of the "seed" region on the antisense strand in DS16A-135 (i.e., RD-14698) interfered with maximal gene induction only in PLC / PRF / 5 cells, while preserving its function in Huh-7 cells ( FIG. 7A to FIG. 7B ). However, mutations in the "seed" region on the sense strand (i.e., RD-14699) reduced maximal activity in Huh-7 cells, suggesting that the sense strand is preferentially responsible for gene activation in this cell line ( Figure 7B The overhang length of the sense strand in the asymmetric duplex was modified to 1, 3, or 5 nucleotides (i.e., RD-14700, RD-14701, and RD-14702, respectively), which performed similarly to the dual-nucleotide overhang saRNA (i.e., RD-14696) ( Figure 7B ).

[0203] In PLC / PRF / 5 cells, DS16A-013 (i.e., RD-11598) did not significantly activate CFH expression; however, extending the duplex length to 23 bp with an asymmetric overhang on the sense strand (i.e., RD-14705) did measure an approximately 1.75-fold increase in mRNA levels ( Fig. 7A DS16A-013 (i.e., RD-11598) treatment in Huh-7 increased the expression level by nearly 2-fold ( Figure 7B Only shortening the duplex length to 16 bp (i.e., RD-14703), extending the duplex length to 33 bp (i.e., RD-14708), or mutation of the “seed” region on the antisense strand (i.e., RD-14707) reduced saRNA activity in Huh-7 cells.

[0204] In summary, the results show that saRNA duplex length can affect gene induction activity, and nucleotide sequences with lengths of ≤16bp or ≥33bp are significantly weakened. In addition, mutations in the "seed" region of the positive strand (S) reduce saRNA activity, indicating that there is sequence specificity in target gene activation. Finally, saRNA activity is generally resilient to changes in overhang length, although the best performing variants preferably have dinucleotide overhangs or blunt-end structures (no overhangs). Example 8: Design and synthesis of saRNA targeting the mouse Cfh promoter

[0205] The coding sequence (SEQ ID NO: 1719) from the mouse Cfh gene promoter was retrieved from the UCSC genome database. The sequence consists of 800 nucleotides ranging from -1 bp to -800 bp upstream relative to the transcription start site (TSS) (Table 13). Table 13. Deduced mouse Cfh promoter sequence (5'-3') (SEQ ID NO: 1719)

[0206] A total of 383 saRNA duplexes with a length of 19 bp targeting mouse promoter sequences were synthesized according to the following criteria: (i) 35% to -65% GC content; (ii) less than 5 consecutive identical nucleotides; (iii) 3 or less total dinucleotide repeats; and (iv) 3 or less total trinucleotide repeats. A dinucleotide overhang was also synthesized at the 3' end of each chain, resulting in a total length of 21 nt. The chain composition and sequence of each saRNA duplex targeting the mouse Cfh promoter are listed in Table 2. Example 9: High-throughput screening of saRNA targeting the mouse Cfh promoter

[0207] In order to identify saRNAs capable of activating Cfh expression, primary mouse hepatocytes (PMH) grown in culture medium were transfected with each of the above saRNAs at a concentration of 25 nM for 72 hours, and then gene expression analysis was performed by one-step RT-qPCR. siRNA targeting mouse Cfh transcripts (i.e., DS16B-si6) was also used as a negative control for transfection efficiency. Among the 383 mouse saRNAs, 89 (23.2%), 58 (15.1%), and 41 (10.7%) saRNAs showed high activation (≥1.5 times), moderate activation (1.2 to 1.5 times), and mild activation (1.1 to 1.2 times) of Cfh expression (Table 14). Table 14. Summary of the activities of all screened Cfh saRNAs

[0208] 195 (51.0%) saRNAs reduced the expression level of Cfh mRNA or had no significant effect. The relative changes in mouse Cfh expression caused by saRNA treatment are also summarized in Table 2, and the expression data obtained by gene induction are plotted in Figure 8Table 15 also compares the results of mouse and human saRNA high-throughput screening grouped by target gene expression fold change. Table 15. Summary of the activities of all screened human CFH and mouse Cfh saRNAs

[0209] Sorting the expression data by target site location within the mouse Cfh promoter revealed five “hotspots” where saRNA activity was concentrated, including regions −754 to −654 (H7), −479 to −421 (H8), −409 to −370 (H9), −208 to −56 (H10), and −52 to −4 (H11) ( Fig. 9 ). Nearly 68% of all functional saRNAs target sequences within the indicated "hotspots". The promoter sequences corresponding to each "hotspot" are listed in Table 9.

[0210] The saRNA duplexes capable of upregulating mouse Cfh expression by at least 1.1-fold or more in the “hotspot region” H7 (-754 to -654) are as follows: DS16B-069, DS16B-070, DS16B-071, DS16B-072, DS16B-073, DS16B-074, DS16B- 075, DS16B-076, DS16B-077, DS16B-078, DS16B-079, DS16B-080, DS16B-081, DS 16B-082, DS16B-083, DS16B-084, DS16B-085, DS16B-086, DS16B-087, DS16B-08 8. DS16B-089, DS16B-090, DS16B-091, DS16B-092, DS16B-093, DS16B-094, DS16B -095, DS16B-096, DS16B-097, DS16B-098, DS16B-099, DS16B-100, DS16B-101, D S16B-102, DS16B-103, DS16B-104, DS16B-105, DS16B-106, DS16B-107, DS16B-1 08. DS16B-109, DS16B-110, DS16B-111, DS16B-112, DS16B-113, DS16B-114, DS1 6B-115, DS16B-116, DS16B-117, DS16B-118, DS16B-119, DS16B-120, DS16B-121.

[0211] The saRNA duplexes capable of upregulating mouse Cfh expression by at least 1.1-fold or more in the “hotspot region” H8 (-479 to -421) are as follows: DS16B-157, DS16B-158, DS16B-159, DS16B-160, DS16B-161, DS16B-162, DS16B-163, DS16B-164, DS16B-165, DS16B-166, DS16B-167, DS16B-168, DS16B-169, DS16B-170, DS16B-171, DS16B-172, DS16B-173, DS16B-174, DS16B-175, DS16B-176, DS16B-177, DS16B-178, DS16B-179, DS16B-180, DS16B-181, DS16B-182, DS16B-183, DS16B-184, DS16B-185, DS16B-186, DS16B-187, DS16B-188, DS16B-189, DS16B-190, DS16B-191, DS16B-192.

[0212] The saRNA duplexes capable of upregulating mouse Cfh expression by at least 1.1-fold or more in the “hotspot region” H9 (-409 to -370) are as follows: DS16B-217, DS16B-218, DS16B-219, DS16B-220, DS16B-221, DS16B-222, DS16B-223, DS16B-224, DS16B-225, DS16B-226, DS16B-227, DS16B-228, DS16B-229, DS16B-230, DS16B-231, DS16B-232, DS16B-233, DS16B-234, DS16B-235, DS16B-236, DS16B-237, DS16B-238.

[0213] The saRNA duplexes capable of upregulating mouse Cfh expression by at least 1.1-fold or more in the “hotspot region” H10 (-208 to -56) are as follows: DS16B-279, DS16B-280, DS16B-281, DS16B-282, DS16B-283, DS16B-284, DS16B-285, D S16B-286, DS16B-287, DS16B-288, DS16B-289, DS16B-290, DS16B-291, DS16B-292, DS 16B-293, DS16B-294, DS16B-295, DS16B-296, DS16B-297, DS16B-298, DS16B-299, DS1 6B-300, DS16B-301, DS16B-302, DS16B-303, DS16B-304, DS16B-305, DS16B-306, DS16B -307, DS16B-308, DS16B-309, DS16B-310, DS16B-311, DS16B-312, DS16B-313, DS16B- 314, DS16B-315, DS16B-316, DS16B-317, DS16B-318, DS16B-319, DS16B-320, DS16B-3 21. DS16B-322, DS16B-323, DS16B-324, DS16B-325, DS16B-326, DS16B-327, DS16B-32 8. DS16B-329, DS16B-330, DS16B-331, DS16B-332, DS16B-333, DS16B-334, DS16B-335.

[0214] The saRNA duplexes capable of upregulating mouse Cfh expression by at least 1.1-fold or more in the “hotspot region” H11 (-52 to -4) are as follows: DS16B-357, DS16B-358, DS16B-359, DS16B-360, DS16B-361, DS16B-362, DS16B-363, DS16B-364, DS16B-365, DS16B-366, DS16B-367, DS16B-368, DS16B-369, DS16B-370, DS16B-371, DS16B-372, DS16B-373, DS16B-374, DS16B-375, DS16B-376, DS16B-377, DS16B-378, DS16B-379, DS16B-380. Example 10: Dose-dependent activation of mouse Cfh expression by saRNA treatment in PMH cells

[0215] Based on the screening results of mouse Cfh induction, the top 64 saRNAs with good performance (see Table 2) were transfected into PMH cells at 3 concentrations (i.e., 2.5 nM, 10 nM, and 25 nM) for 72 hours to quantify dose-dependent gene induction by one-step RT-qPCR. dsCon2 and DS16B-si6 were used as nonspecific duplex controls for gene activation and negative controls for transfection efficiency, respectively. Fig.10 As shown, most saRNAs exhibited dose-dependent induction of Cfh, with DS16B-070 having the highest level of activity at a treatment concentration of 25 nM, reaching approximately 4.0-fold. The most effective duplexes included 41 saRNAs (64.1%), which showed a ≥1.1-fold increase in mouse Cfh levels at the lowest concentration (i.e., 2.5 nM), but at the same concentration, only 4 (6.3%) activated Cfh expression by more than 1.5-fold. Example 11: saRNA treatment increases Cfh mRNA and protein levels in PMH cells over time

[0216] The best performing subset (a total of 19 saRNAs) was transfected into PMH cells at a concentration of 25 nM and further evaluated for Cfh induction five days later. dsCon2 and DS16B-si6 were used as nonspecific duplex controls for gene activation and negative controls for transfection efficiency, respectively. Fig.11A As shown, greater fold changes in mRNA expression were detected on day 5, including 8 saRNAs (42.1%), which induced more than 5-fold changes in Cfh levels, of which DS16B-098 had the highest gene expression measurement, an increase of 14.8-fold. On day 5 after saRNA treatment, Cfh protein levels secreted into the culture medium were also detected by ELISA (i.e., DS16B-085, DS16B-091, DS16B-098, and DS16B-237). Three saRNAs (i.e., DS16B-091, DS16B-098, and DS16B-237) measured Cfh protein levels increased by more than 1.5-fold relative to baseline (dashed line) ( Fig. 11B ).

[0217] To quantify the accumulation of Cfh over time, two saRNAs (i.e., DS16B-089 and DS16B-091) were transfected into PMH cells at a concentration of 25 nM, and mRNA and protein levels were measured daily by two-step RT-qPCR and ELISA, respectively, from day 3 to day 6 after treatment. The increase in Cfh mRNA levels for both DS16B-089 and DS16B-091 increased and plateaued from day 3 to day 6 (Figure 12A). Although protein levels did not exceed 1.25-fold compared to the Mock and dsCon2 control treatments, Cfh protein did correlate with the rise and plateau of Cfh mRNA after saRNA treatment ( Fig. 12B The relative fold changes of Cfh mRNA and protein levels are also listed in Table 16 and Table 17, respectively. Table 16. Relative levels of Cfh mRNA over time (fold change) Table 17. Relative levels of Cfh protein over time (fold change) Example 12: Cfh gene activation efficacy of saRNA in PMH cells

[0218] Dose response curves were generated by two-step RT-qPCR for the 4 best performing saRNAs (i.e., DS16B-070, DS16B-091, DS16B-098, and DS16B-241) after 5 days of treatment using 11 increasing concentrations (i.e., 0.0017, 0.0051, 0.0152, 0.046, 0.14, 0.41, 1.23, 3.7, 11.111, 33.333, and 100 nM) to characterize their potency ( Figure 13A to Fig.13D As summarized in Table 18, each saRNA exhibited a dose-dependent activation of mouse Cfh expression, EC 50 Values ​​were in the low nanomolar range (ie, approximately 1.58 nM to 15.3 nM). Table 18. ECs that activate Cfh expression in vitro 50 value Example 13: Development of DEC-conjugated saRNA for inducing Cfh expression in mice in vivo

[0219] Variants of DS16B-098 and DS16B-091 (i.e., 2'-F, 2'-Ome, and PS modifications) were synthesized by medicinal chemistry methods, including conjugation of a delivery enhancing compound (DEC) such as GalNac (i.e., tC2 or tC2x4) at the 3' end of the antisense strand and / or conjugation of a lipid C5x5 at the 5' end of the sense strand. DS16B-098 variants (i.e., RD-13878, RD-14662, RD-14663, and RD-14669) were transfected into PMH cells at a concentration of 25 nM for 72 hours by Lipofectamine RNAiMax to confirm the retention of saRNA activity after chemical modification. Chemically modified siRNAs (i.e., RD-13149) targeting Cfh transcripts with tC2x4 coupling were used as negative controls for cell transfection. All DEC-conjugated variants increased Cfh expression in mice, similar to or better than RD-13878, compared to unconjugated saRNA (i.e., RD-13878). Fig.14 ). This data suggests that both conjugation and medicinal chemistry approaches are well tolerated by saRNA.

[0220] A chemically modified variant of DS16B-091 (i.e., RD-14660) with two DEC conjugates (i.e., tC2x4 and C5x5) was applied at 5 increasing concentrations (i.e., 6.25, 25, 100, 1600, and 6400 nM) to determine whether DEC conjugation can deliver saRNA to PMH cells in the absence of a transfection agent (i.e., free uptake). Nonspecific DEC-saRNA (i.e., RD-14040) served as a negative control for saRNA activity. Fig.15 As shown, RD-14660 showed a dose-dependent induction of Cfh up to approximately 3.0-fold, indicating that DEC-conjugation can deliver saRNA to target cells in vitro.

[0221] To determine the activation of Cfh expression in vivo, tC2x4 DEC-saRNA (ie, RD-13147) containing a 5'-VP modification on the antisense strand was administered to adult female C57BL / 6J mice by SC injection at a dose of 50 mg / kg. Saline was used as a vehicle control, while DEC-siRNA (ie, RD-13149) treatment was used as a negative control for in vivo liver-targeted delivery. Mice were sacrificed on day 10 after treatment, and Cfh mRNA levels in liver tissue were quantified by two-step RT-qPCR. As summarized in Table 20, RD-13147 increased the average Cfh expression level in mouse liver by approximately 1.22 times compared to animals in the saline control group. In addition, the knockdown of Cfh by RD-13149 also supports the in vivo delivery of DEC to liver tissue. No adverse results were observed in monitoring animal weight after treatment, indicating that DEC-saRNA treatment is generally safe in C57BL / 6J mice ( Fig.16 ).

[0222] The chemical compositions and strand sequences of all DEC-conjugated saRNA duplexes are listed in Table 19. The structures of tC2, tC2x4, and C5x5 compounds are shown below:

[0223]

[0224]

[0225] Table 19. Oligonucleotide sequences and duplex compositions Note: Capital letters indicate RNA; * indicates phosphorothioate (PS) backbone modification; f indicates 2'-fluoro; m indicates 2'-O-methyl (2'-OMe); VP indicates 5'-(E)-vinylphosphonate. Table 20. Relative expression of Cfh mRNA in mouse liver tissue (SC, day 10) Note: #1, #2, and #3 represent 3 animals in each group. Example 14: Preparation of Compound tC2 of the Present Disclosure 1. Part 1: Synthesis of compound 3. (1) Preparation of Compound 2:

[0226] 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:

[0227] 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:

[0228] 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:

[0229] 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:

[0230] 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:

[0231] 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:

[0232] 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:

[0233] 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) 1 H 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:

[0234] 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.

[0235] 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. 1 H 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:

[0236] 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. 1 H 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:

[0237] 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.

[0238] 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 15: 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

[0239] Single-stranded oligonucleotides are synthesized on K&A DNA synthesizer (K&A Laborgeraete GbR, Schaffheim, Germany) by carrier material synthesis technology. Starting material is a general carrier material or a special carrier material, which is commercially available or as disclosed in the foregoing synthesis. Usually, in a DNA synthesizer, phosphoramidite monomers (0.1M, acetonitrile or dichloromethane solution) including various joints and conjugates are added to the carrier material in sequence to generate the desired full-length oligonucleotide. Each phosphoramidite adds a cycle consisting of four chemical reactions, including detritylation, coupling, oxidation / thiolation and end-capping. In the first step, the detritylation reaction is carried out for 45 seconds using the DCM of 3% dichloroacetic acid (TCA). In the second step, all amidites are subjected to 6 minutes of phosphoramidite coupling 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.

[0240] 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.

[0241] 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

[0242] 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 minutes, flow rate: 1 mL / min. Pure oligonucleotides were collected and desalted by HiPrep 26 / 10 desalting column. (3) Annealing to form a duplex

[0243] 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.

[0244] 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:

[0245] 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 16: 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.

[0246] Under nitrogen protection, TMSOTf (15.3 mL, 84.7 mmol, 1.1 eq) was added to a 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) in DCM (300 mL). 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 purified 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

[0247] 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

[0248] 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 eluent: 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 HNMR (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,2 H),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.

[0249] 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

[0250] 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

[0251] 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 3 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 HNMR and 13C 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

[0252] 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]+ . 1 H 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.

[0253] 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).

[0254] 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

[0255] 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.

[0256] 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.

[0257] 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.

[0258] 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, 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 value: 709.41; MW found value: 710.57 [M+H] + . 1 HNMR(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

[0259] 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 1HNMR characterization. MW calculated value: 1697.80; MW measured 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

[0260] 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.

[0261] 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 analyzed 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.

[0262] 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.

[0263] 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.

[0264] 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.

[0265] 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 17: Preparation of conjugated oligonucleotides linked to conjugated groups derived from compounds tC2x4 of the present disclosure

[0266] O2 was generated according to the general synthesis method of oligonucleotides shown in Example 15 above, using the conjugated group derived from compound tC2x4 as the starting support material. An exemplary structure of a DEC-conjugated oligonucleotide is O2 as shown below:

[0267] It can be seen that in the structure of O2, the conjugate group derived from the delivery enhancing compound is linked 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 moiety, such as -OP(O)2O- or -P(O)-O-, wherein (S) is the sense strand and (AS) is the antisense strand. Example 18: Preparation of Compound C5x5 of the Present Disclosure

[0268] In this example, compound C5x5 was prepared by using the following method. (1) Preparation of Compound 42

[0269] Under nitrogen protection, compound 40 (19.56 g, 81 mmol, 1.0 eq) was added to a solution of 2-(4-fluoro-3-nitrophenyl) methyl acetate (compound 41; 17.3 g, 81 mmol, 1.0 eq) and K2CO3 (11.2 g, 81 mmol) in anhydrous DMF (200 mL). The reaction mixture was stirred at 55 ° C for 6 hours, and then cold water (100 mL) was added. The mixture was extracted three times with ethyl acetate, and the organic phase was washed three times with saturated LiCl solution and once with brine. It was then dried over anhydrous Na2SO4 and concentrated under reduced pressure to form a yellow oily compound 42, which was used directly in the next step without further purification. (2) Preparation of Compound 43

[0270] In an ice bath, HCOONH4 (30.67 g, 486 mmol, 6.0 eq) and Zn powder (31.78 g, 486 mmol, 6.0 eq) were added to a solution of compound 42 (35.18 g, 81 mmol, 1.0 eq) in THF / H2O (9:1, 280 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, extracted three times with ethyl acetate, and the organic phase was washed once with brine. After drying with anhydrous Na2SO4 and concentrating under reduced pressure, the formed compound 43 was used directly in the next step without further purification. Compound 43 was characterized by mass spectrometry. MW calculated value: 404.34. MW measured value: 405.3 [M+H] + . (3) Preparation of Compound 45

[0271] Under nitrogen protection, 3-((tert-butyldimethylsilyl)oxy)propanal (compound 44; 9.0 g, 48 mmol, 1.0 eq) and AcOH (11 mL, 192 mmol, 4.0 eq) were added to a solution of compound 43 (19.36 g, 48 mmol, 1.0 eq) in EtOH (200 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 residual compound 45 was used directly in the next step without further purification. (4) Preparation of Compound 46

[0272] Under nitrogen protection, 1M TBAF THF solution (26.3mL, 26.3mmol, 1.5eq) was added to a solution of compound 45 (10g, 17.5mmol, 1.0eq) in anhydrous THF (50mL). The reaction mixture was stirred at room temperature for 1 hour and then concentrated under reduced pressure. Water (100mL) was then added, the mixture was extracted three times with DCM, the organic phases were combined, washed with brine, dried over Na2SO4, and concentrated. The resulting residue was dissolved in 50mL pyridine, and DMTrCl (7.12g, 21mmol, 1.2eq) 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%-3% MeOH / DCM) to give compound 46 (8.1g, 61% yield) as a yellow solid. The product was purified by mass spectrometry and 1 H NMR characterization. MW calculated: 760.48. MW found: 761.8 [M+H] + . 1 H NMR (400MHz, CDCl3) δ7.59–7.53(m,1H),7.38–7.31(m,2H),7.23-7.19(m,6H),7.18–7.14(m,3H),6.76(dd,J=7.8,5.6Hz,4H),4.17–4. 02(m,2H),3.76(s,6H),3.73(s,2H),3.67(s,3H),3.59(t,J=7.0Hz,2H),3.19–3.05(m,2H),1.29–1.25(m,28H),0.88(t,J=6.5Hz,3H). (5) Preparation of compound C5x5

[0273] Under nitrogen protection and ice bath, LiAlH4(202mg, 5.33mmol, 1.5eq) was added to anhydrous THF(20mL) solution of compound 46(2.7g, 3.55mmol, 1.0eq). After 10 minutes, the mixture was transferred to room temperature and stirred for 1 hour. The reaction system was then transferred to an ice bath, and saturated potassium sodium tartrate solution(20mL) was slowly added to the mixture. After 30 minutes, the reaction system was extracted with Et2O three times, and then the organic phase was combined, washed with salt water, dried with Na2SO4, and concentrated. The crude product (300 mg, 0.41 mmol, 1.0 eq) was dissolved in anhydrous DCM (5 mL), and DIPEA (204 μL, 1.23 mmol, 3.0 eq) and 3-((chloro(diisopropylamino)phosphinoyl)oxy)propionitrile (compound 47; 274 μL, 1.23 mmol, 3.0 eq) were added at 25 ° C under nitrogen protection. The reaction mixture was stirred for 1 hour. The mixture was extracted twice with DCM, then washed with brine and dried over anhydrous Na2SO4. The organic layer was concentrated under reduced pressure and the resulting residue was purified by flash chromatography (silica gel, gradient eluent: 1%-5% MeOH / DCM, 1% Et3N) to give compound C5x5 (299 mg, 78% yield) as a colorless oil. The product was purified by mass spectrometry and 1 HNMR characterization. MW calculated: 946.61; MW found: 303.2 [DMT] - . 1 H NMR (400MHz, CDCl3) δ7.62 (s, 1H), 7.32 (dd, J = 7.6, 4.1Hz, 2H), 7.27–7.11 (m, 9H), 6. 73(dd,J=7.9,5.6Hz,4H),4.34–4.31(m,2H),4.10–4.06(m,2H),3.75(s,6H),3.64–3. 61(m,2H),3.58(dd,J=11.8,5.1Hz,2H),3.24–3.18(m,2H),3.09–2.88(m,4H),2.65– 2.55(m,4H),1.37–1.28(m,28H),1.22(dd,J=6.8,3.2Hz,12H),0.88(t,J=6.5Hz,3H). Example 19: Preparation of conjugated oligonucleotides linked to conjugated groups derived from compound C5x5 of the present disclosure

[0274] O3 was generated using the conjugated group derived from compound C5x5 as the terminal imine according to the general synthesis method of oligonucleotides shown in Example 15 above. An exemplary structure of a DEC-conjugated oligonucleotide is O3 shown below:

[0275] It can be seen that in the structure of O3, the conjugated group derived from the delivery enhancing compound C5x5 is connected to a double-stranded RNA (dsRNA) duplex (including but not limited to saRNA or siRNA) at the 5' 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.

[0276] In summary, the high-throughput screening data revealed multiple "hot spots" of saRNA activity in the promoters of human CFH and mouse Cfh genes. Exemplary saRNAs increased expression at both mRNA and protein levels, while exhibiting dose-dependent gene induction. Moreover, saRNA activity was easily tolerated by medicinal chemistry and DEC conjugation, enabling in vivo delivery. These results provide evidence that targeted activation of CFH expression in the liver through saRNA administration is a promising strategy for treating CFHD.

[0001] References 1. (López-Larrea C, Dieguez MA, Enguix A, Dominguez O, Marin B, Gomez E. A familial deficiency of complement factor H. Biochem Soc Trans 1987;15:648–9. 2. Caprioli, J., F. Castelletti, S. Bucchioni, P. Bettinaglio, E. Bresin, G. Pianetti, S. Gamba, S. Brioschi, E. Daina, G. Remuzzi, M. Noris, Recurrent International Registry of, and Hus Ttp Familial. 2003. 'Complement factor H mutations and gene polymorphisms in haemolytic uraemic syndrome: the C-257T, the A2089G and the G2881T polymorphisms are strongly associated with the disease', Human molecular genetics, 12:3385-95. 3. Edwards, A.O., R. Ritter, 3rd, K.J. Abel, A. Manning, C. Panhuysen, and L.A. Farrer. 2005. 'Complement factor H polymorphism and age-related macular degeneration', Science, 308:421-4. 4. Esparza-Gordillo, J., J.M. Soria, A. Buil, L. Almasy, J. Blangero, J. Fontcuberta, and S. Rodriguez de Cordoba. 2004. 'Genetic and environmental factors influencing the human factor H plasma levels', Immunogenetics, 56:77-82. 5.Haines,J.L.,M.A.Hauser,S.Schmidt,W.K.Scott,L.M.Olson,P.Gallins,K.L.Spencer,S.Y.Kwan,M.Noureddine,J.R.Gilbert,N.Schnetz-Boutaud,A.Agarwal,E.A.Postel,and M.A.Pericak-Vance.2005.'Complement factor H variant increasesthe risk of age-related macular degeneration',Science,308:419-21. 6.Klein,R.J.,C.Zeiss,E.Y.Chew,J.Y.Tsai,R.S.Sackler,C.Haynes,A.K.Henning,J.P.SanGiovanni,S.M.Mane,S.T.Mayne,M.B.Bracken,F.L.Ferris,J.Ott,C.Barnstable,and J.Hoh.2005.'Complement factor H polymorphismin age-relatedmacular degeneration',Science,308:385-9. 7.Richards,A.,M.R.Buddles,R.L.Donne,B.S.Kaplan,E.Kirk,M.C.Venning,C.L.Tielemans,J.A.Goodship,and T.H.Goodship.2001.'Factor H mutations inhemolytic uremic syndrome cluster in exons 18-20,a domain important for hostcell recognition',American journal of human genetics,68:485-90. 8.Warwicker,P.,T.H.Goodship,R.L.Donne,Y.Pirson,A.Nicholls,R.M.Ward,P.Turnpenny,and J.A.Goodship.1998.'Genetic studies into inherited andsporadic hemolytic uremic syndrome',Kidney Int,53:836-44. 9.Ault,B.H.,B.Z.Schmidt,N.L.Fowler,C.E.Kashtan,A.E.Ahmed,B.A.Vogt,andH.R.Colten.1997.'Human factor H deficiency.Mutations in framework cysteineresidues and block in H protein secretion and intracellular catabolism',JBiol Chem,272:25168-75. 10.Boyer,O.,L.H.Noel,E.Balzamo,G.Guest,N.Biebuyck,M.Charbit,R.Salomon,V.Fremeaux-Bacchi,and P.Niaudet.2008.'Complement factor Hdeficiency and posttransplantation glomerulonephritis with isolated C3deposits',Am J Kidney Dis,51:671-7. 11.Cheong,H.I.,B.S.Lee,H.G.Kang,H.Hahn,K.S.Suh,I.S.Ha,andY.Choi.2004.'Attempted treatment of factor H deficiency by livertransplantation',Pediatr Nephrol,19:454-8. 12.Fortin,M.C.,W.Schurch,H.Cardinal,and M.J.Hebert.2004.'Complementfactor H deficiency in acute allograft glomerulopathy and post-transplanthemolytic uremic syndrome',Am J Transplant,4:270-3. 13.Gonzales,E.,T.Ulinski,D.Habes,G.Deschenes,V.Fremeaux-Bacchi,andA.Bensman.2016.'Long-term successful liver-kidney transplantation in a childwith atypical hemolytic uremic syndrome caused by homozygous factor Hdeficiency',Pediatr Nephrol,31:2375-78. 14.Kim,S.,E.Park,S.I.Min,N.J.Yi,J.Ha,I.S.Ha,H.I.Cheong,andH.G.Kang.2018.'Kidney Transplantation in Patients with Atypical HemolyticUremic Syndrome due to Complement Factor H Deficiency:Impact of LiverTransplantation',J Korean Med Sci,33:e4. 15.Larakeb,A.,S.Leroy,V.Fremeaux-Bacchi,M.Montchilova,B.Pelosse,O.Dunand,G.Deschenes,A.Bensman,and T.Ulinski.2007.'Ocular involvement inhemolytic uremic syndrome due to factor H deficiency--are there therapeuticconsequences?',Pediatr Nephrol,22:1967-70. 16.Licht, C., A. Weyersberg, S. Heinen, L. Stapenhorst, J. Devenge, B. Beck, R. Waldherr, M. Kirschfink, PFZipfel, and B. Hoppe cofactor protein domain 15′,Am JKidney Dis,45:415–21. 17.Michaux,K.,J.Bacchetta,E.Javouhey,P.Cochat,V.Fremaux-Bacchi,andAL.Sellier-Leclerc. 18. Nathanson, S., T. Ulinski, V. Fremeaux-Bacchi, and G. Deschenes. 19.Rougier,N.,MDKazatchkine,JPRougier,V.Fremeaux-Bacchi,J.Blouin,G.Deschenes,B.Soto,V.Baudouin,B.Pautard,W.Proesmans,E.Weiss,andL.Weiss syndrome',J Am Soc Nephrol,9:2318-26. 20.Ruseva,M.M.,K.A.Vernon,A.M.Lesher,W.J.Schwaeble,Y.M.Ali,M.Botto,T.Cook,W.Song,C.M.Stover,and M.C.Pickering.2013.'Loss of properdinexacerbates C3 glomerulopathy resulting from factor H deficiency',J Am SocNephrol,24:43-52. 21.Schejbel,L.,I.M.Schmidt,M.Kirchhoff,C.B.Andersen,H.V.Marquart,P.Zipfel,and P.Garred.2011.'Complement factor H deficiency and endocapillaryglomerulonephritis due to paternal isodisomy and a novel factor H mutation',Genes Immun,12:90-9. 22.Servais,A.,L.H.Noel,M.A.Dragon-Durey,M.C.Gubler,P.Remy,D.Buob,C.Cordonnier,R.Makdassi,W.Jaber,E.Boulanger,P.Lesavre,and V.Fremeaux-Bacchi.2011.'Heterogeneous pattern of renal disease associated withhomozygous factor H deficiency',Hum Pathol,42:1305-11. 23.Suga,K.,S.Kondo,S.Matsuura,Y.Kinoshita,E.Kitano,M.Hatanaka,H.Kitamura,Y.Hidaka,T.Oda,and S.Kagami.2010.'A case of dense deposit diseaseassociated with a group A streptococcal infection without the involvement ofC3NeF or complement factor H deficiency',Pediatr Nephrol,25:1547-50. 24.Taylor,C.M.2001.'Hemolytic-uremic syndrome and complement factor Hdeficiency:clinical aspects',Semin Thromb Hemost,27:185-90. 25.Wilson,V.,R.Darlay,W.Wong,K.M.Wood,J.McFarlane,L.Schejbel,I.M.Schmidt,C.L.Harris,J.Tellez,E.M.Hunze,K.Marchbank,J.A.Goodship,andT.H.Goodship.2013.'Genotype / phenotype correlations in complement factor Hdeficiency arising from uniparental isodisomy',Am J Kidney Dis,62:978-83.

Claims

1. An oligonucleotide modulator (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: 1707, wherein the saRNA activates the expression of the CFH gene by at least 10% compared to the baseline expression of the CFH gene.

2. The saRNA according to claim 1, wherein the equal length region shown in SEQ ID NO: 1707 is located in the -538 to -500 region (SEQ ID NO: 1708), -468 to -396 region (SEQ ID NO: 1709), -329 to -283 region (SEQ ID NO: 1710), -273 to -192 region (SEQ ID NO: 1711), -173 to -100 region (SEQ ID NO: 1712), or -64 to -14 region (SEQ ID NO: 1713) upstream of the transcription start site of the CFH gene.

3. The saRNA of any one of claims 1-2, wherein the 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.

4. The saRNA of any one of claims 1-3, 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.

5. The saRNA of any one of claims 1-3, wherein the sense strand and the antisense strand have at least 90% complementarity.

6. The saRNA of claim 4, wherein the sense strand and the antisense strand are located on two different nucleic acid strands.

7. The saRNA of claim 4, wherein the sense strand and the antisense strand are located on a continuous 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.

8. The saRNA of claim 4, wherein at least one of the sense strand and the antisense strand comprises a 3' overhang ranging from 0 to 6 nucleotides in length.

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

10. The saRNA of claim 8, wherein at least one of the nucleotides of the overhang is a thymidine deoxyribonucleotide.

11. The saRNA of any one of claims 4-10, 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.

12. The saRNA of any one of claims 4-10, wherein the sense strand has at least 75% sequence homology to a nucleotide sequence selected from SEQ ID NOs: 309-615, and the antisense strand has at least 75% sequence homology to a nucleotide sequence selected from SEQ ID NOs: 618-924.

13. The saRNA of claim 12, wherein the sense strand comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 309-615, and the antisense strand comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 618-924.

14. The saRNA of claim 1, wherein the oligonucleotide sequence has at least 75% sequence homology or complementarity to a nucleotide sequence selected from SEQ ID NOs: 1-307.

15. 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: 1-307.

16. 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: 1-307.

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

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

19. The saRNA of claim 17, 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.

20. The saRNA of claim 17, wherein at least one nucleotide of the saRNA is a locked nucleic acid, an abasic nucleotide, a 2'-amino modified nucleotide, a 2'-alkyl modified nucleotide, a morpholino nucleotide, a phosphoramidate, or a nucleotide containing a non-natural base.

21. The saRNA of claim 17, 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.

22. The saRNA of any one of claims 1-21, 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.

23. The saRNA of claim 22, 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.

24. The saRNA of claim 23, wherein the conjugated group is derived from a compound selected from tC2, tC2x4, and C5x5, (tC2 compound), (tC2x4 compound), and (C5x5 compound); in Represents the carrier material.

25. The saRNA of claim 22, wherein the conjugated group is a lipid selected from fatty acids comprising a carbon chain length of 4 to 30 carbon atoms.

26. The saRNA of claim 25, wherein the conjugated group is a fatty acid comprising a carbon chain length of 16 carbon atoms.

27. The saRNA of any one of claims 22 and 23, wherein the sense strand or the antisense strand of the saRNA is conjugated to two conjugating groups, wherein the two conjugating groups are a lipid and N-acetylgalactosamine.

28. The saRNA of claim 27, wherein the two conjugated groups are C5x5 and tC2x4.

29. An isolated polynucleotide of the saRNA according to claim 1, wherein the isolated polynucleotide is a nucleotide sequence shown in SEQ ID NO: 1707 having a length ranging from 16 to 35 consecutive nucleotides.

30. The isolated polynucleotide of claim 29, wherein the isolated polynucleotide is a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-307.

31. An isolated oligonucleotide complex comprising the antisense strand of the saRNA according to any one of claims 1-28 and the sense strand of the isolated polynucleotide according to any one of claims 29-30.

32. The isolated oligonucleotide complex of claim 31, wherein the isolated oligonucleotide complex activates expression of the CFH gene by at least 10% compared to baseline expression of the CFH gene.

33. An isolated nucleic acid sequence upstream of the transcription start site of the CFH gene, wherein the isolated nucleic acid sequence is selected from SEQ ID NO: 1708-1713.

34. An isolated nucleic acid sequence selected from the group consisting of -538 to -500 region, -468 to -396 region, -329 to -283 region, -273 to -192 region, -173 to -100 region and -64 to -14 region upstream of the transcription start site of the CFH gene.

35. An isolated nucleic acid sequence according to claim 33 or 34, wherein the isolated nucleic acid sequence comprises an isolated polynucleotide according to any one of claims 29-30.

36. The isolated nucleic acid sequence of claim 33 or 34, wherein at least 30% of the designed saRNAs targeting the isolated nucleic acid sequence can activate the expression of the CFH 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.

37. An isolated nucleic acid complex comprising the antisense strand of the saRNA of any one of claims 1-28 and the sense strand of the isolated nucleic acid sequence of any one of claims 33-36.

38. The isolated nucleic acid complex of claim 37, wherein the isolated nucleic acid complex activates expression of the CFH gene by at least 10% compared to baseline expression of the CFH gene.

39. An isolated polynucleotide encoding the saRNA of any one of claims 1-28.

40. The isolated polynucleotide of claim 39, wherein the isolated polynucleotide is DNA.

41. A vector comprising the isolated polynucleotide according to any one of claims 39-40.

42. A host cell comprising the saRNA of any one of claims 1-28, the isolated polynucleotide of any one of claims 39-40, or the vector of claim 41.

43. A composition comprising the saRNA of any one of claims 1-28 or the isolated polynucleotide of claims 39 or 40, and optionally, a pharmaceutically acceptable carrier.

44. The composition of claim 43, wherein the pharmaceutically acceptable carrier is selected from aqueous carriers, liposomes, high molecular polymers, polypeptides and antibodies.

45. The composition of claim 43 or 44, wherein the composition comprises 0.001 nM-150 nM of the saRNA.

46. ​​The composition of claim 45, wherein the composition comprises 1 nM-150 nM of the saRNA.

47. A saRNA comprising an oligonucleotide sequence having a length ranging from 16 to 35 consecutive nucleotides for activating / upregulating CFH gene expression in a cell, wherein the oligonucleotide sequence has at least 75%, or at least 80%, or at least 85%, or at least 90% sequence homology or complementarity to an equal length portion shown as SEQ ID NO; 1707, wherein the saRNA activates the expression of the CFH gene by at least 10% compared to the baseline expression of the CFH gene.

48. The saRNA of claim 47, wherein the equal length region shown in SEQ ID NO: 1707 is located in the -538 to -500 region (SEQ ID NO: 1708), -468 to -396 region (SEQ ID NO: 1709), -329 to -283 region (SEQ ID NO: 1710), -273 to -192 region (SEQ ID NO: 1711), -173 to -100 region (SEQ ID NO: 1712), or -64 to -14 region (SEQ ID NO: 1713) upstream of the transcription start site of the CFH gene.

49. The saRNA of claim 48, 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: 309-615, and the antisense strand comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 618-924.

50. A method for preventing or treating a disease or condition induced by insufficient expression of plasma complement factor F (CFH) protein, CFH gene mutation and / or low functional plasma CFH levels in an individual, the method comprising: An effective amount of the saRNA of any one of claims 1-28, the isolated polynucleotide of any one of claims 39-40, the vector of claim 41, or the composition of any one of claims 43-46 is administered to the individual.

51. The method of claim 50, wherein the disease or disorder is complement factor H deficiency (CFHD).

52. The method of claim 50, wherein the individual is a mammal.

53. The method of claim 52, wherein the individual is a human.

54. The method of claim 50, wherein the individual suffers from symptoms induced by insufficient expression of CFH protein, mutations in the CFH gene, and / or low functional plasma CFH levels.

55. The method of claim 50, wherein the saRNA of any one of claims 1-28, the isolated polynucleotide of any one of claims 39-40, the vector of claim 41, or the composition of any one of claims 43-46 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.

56. The method of claim 55, 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.

57. The method of claim 50, wherein the method activates / upregulates expression of the CFH gene or Cfh gene mRNA in the individual by at least 10% compared to a baseline expression of the CFH gene or Cfh gene.

58. The method of claim 50, wherein the method increases CFH protein levels in the individual by at least 10% compared to baseline expression of the CFH gene.

59. A method for detecting CFH protein or a CFH-regulated protein in the cell of claim 42.

60. A kit for performing the method of claim 59, comprising the saRNA of any one of claims 1-28.

61. The kit of claim 60, wherein the instructions for use comprise means for administering the saRNA of any one of claims 1-28 to an individual.

62. A kit comprising the saRNA of any one of claims 1-28, the isolated polynucleotide of any one of claims 39-40, the vector of claim 41, or the composition of any one of claims 43-46 in a labeled package, wherein the label on the package indicates 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 complement factor H (CFH) or for the prevention or treatment of CFHD.

63. A kit for detecting CFH protein or CFH-regulated protein in the cell according to claim 42.

64. Use of the saRNA of any one of claims 1-28, the isolated polynucleotide of any one of claims 39-40, the vector of claim 41, or the composition of any one of claims 43-46 in the preparation of a medicament for preventing or treating a disease or condition induced by insufficient CFH protein expression, CFH gene mutation, and / or low functional plasma CFH levels in an individual.

65. The use according to claim 64, wherein the disease or disorder is CFHD.

66. The use according to claim 64, wherein the individual is a mammal, optionally wherein the mammal is a human.

67. Use of the saRNA of any one of claims 1-28, the isolated polynucleotide of any one of claims 39-40, the vector of claim 41, or the composition of any one of claims 43-46 in the preparation of a formulation for activating / upregulating the expression of a CFH gene in a cell.

68. The use of claim 67, wherein the saRNA of any one of claims 1-28, the isolated polynucleotide of any one of claims 39-40, the vector of claim 41, or the composition of any one of claims 43-46 is introduced directly into the cell.

69. The use of claim 68, wherein the saRNA is produced in the cell after introducing a nucleotide sequence encoding the saRNA into the cell.

70. The use according to any one of claims 67-69, wherein the cell is a mammalian cell, optionally wherein the mammalian cell is a human cell.

71. The use of claim 70, wherein the cell is in a human.

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

73. The use according to claim 72, wherein the symptom induced by insufficient expression of CFH protein is CFHD.

74. A method for activating / upregulating the expression of a CFH gene in a cell, the method comprising: An effective amount of the saRNA of any one of claims 1-28, the isolated polynucleotide of any one of claims 39-40, the vector of claim 41, or the composition of any one of claims 43-46 is administered to the cell.

75. The method of claim 74, wherein the saRNA of any one of claims 1-28, the isolated polynucleotide of any one of claims 39-40, the vector of claim 41, or the composition of any one of claims 43-46 is directly introduced into the cell.

76. The method of claim 75, wherein the method for directly introducing into the cell comprises: 1) combining the saRNA according to any one of claims 1-28, the isolated polynucleotide according to any one of claims 39-40, the vector according to claim 41, or the saRNA in the composition according to any one of claims 43-46 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) conjugating the saRNA according to any one of claims 1-28, the isolated polynucleotide according to any one of claims 39-40, the vector according to claim 41, or the saRNA in the composition according to any one of claims 43-46 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.

77. The method of claim 76, wherein the conjugated group is derived from a compound selected from tC2, tC2x4, and C5x5, (tC2 compound), (tC2x4 compound), and (C5x5 compound); in Represents the carrier material.

78. The method of claim 76, wherein the conjugated group is a lipid selected from fatty acids comprising a carbon chain length of 4 to 30 carbon atoms.

79. The method of claim 78, wherein the conjugated group is a fatty acid comprising a carbon chain length of 16 carbon atoms.

80. The method of claim 76, wherein the sense strand or the antisense strand of the saRNA is conjugated to two conjugating groups, wherein the two conjugating groups are a lipid and N-acetylgalactosamine.

81. The method of claim 80, wherein the two conjugated groups are derived from C5x5 and tC2x4.

82. The method of any one of claims 74-81, wherein the cell is a mammalian cell, preferably a cell from a human.

83. The method of claim 82, wherein the human is a patient suffering from symptoms induced by insufficient expression of CFH protein, mutations in the CFH gene, and / or low functional plasma CFH levels, wherein the saRNA, the isolated polynucleotide, or the composition is administered in an amount sufficient to treat the symptoms.

84. The method of claim 83, wherein the symptoms caused by insufficient expression of CFH protein are atypical hemolytic uremic syndrome (aHUS), dense deposit disease (DDD), C3 glomerulonephritis (C3GN), CFHR5 nephropathy, lupus nephritis (LN), MPGN type I with pure complement C3 deposition (MPGN1), membranoproliferative glomerulonephritis type II (MPGN2), familial MPGN type III (MPGN3), and age-related macular degeneration (AMD).

85. A method for increasing the level of CFH protein in a cell, the method comprising: An effective amount of the saRNA according to any one of claims 1-28, the isolated polynucleotide according to any one of claims 39-40, the vector according to claim 41, or the composition according to any one of claims 43-46 is introduced into the cell, wherein the saRNA, the isolated polynucleotide, or the composition activates expression of the CFH gene by at least 10% compared to baseline expression of the CFH gene.

86. A compound selected from tC2, tC2x4 and C5x5, (tC2 compound), (tC2x4 compound), and in Represents the carrier material.

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