WO2024001172A1

WO2024001172A1 - Oligonucleotide modulators activating complement factor h expression

Info

Publication number: WO2024001172A1
Application number: PCT/CN2023/072927
Authority: WO
Inventors: Moorim KANG; Longcheng Li
Original assignee: Ractigen Therapeutics
Priority date: 2022-06-27
Filing date: 2023-01-18
Publication date: 2024-01-04
Also published as: TW202400190A; WO2024002046A1

Abstract

Provided is an oligonucleotide modulator for preventing or treating complementary factor H deficiency (CFHD) and use 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 an oligonucleotide strand of 16 to 35 nucleotides in length, in which one nucleotide strand has at least 75%base homology or complementarity to a target selected from a promoter region of a target gene CFH. Also provided is a pharmaceutical composition comprising the oligonucleotide modulator disclosed herein and optionally, a pharmaceutically acceptable carrier, and a method for up-regulating the expression of a target gene in cells and methods for preventing or treating a disorder induced by insufficient expression of a target gene with the oligonucleotide modulator or the pharmaceutical composition comprising the oligonucleotide modulator disclosed herein.

Description

OLIGONUCLEOTIDE MODULATORS ACTIVATING COMPLEMENT FACTOR H EXPRESSION

TECHNICAL FIELD

The present application relates to the technical field of nucleic acids, specifically as it relates to an oligonucleotide modulator associated with gene activation and pharmaceutical use thereof.

BACKGROUND

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-1factor H, β1H globulin, C3b inactivator accelerator, H factor, HF, H factor-1, HF1) , a glycoprotein mainly produced by the liver. Mature CFH protein consists of 1213 amino acids and is made up with 20 short consensus repeats (SCR) or complement control protein (CCP) modules to form a bead-like structure. These SCRs are termed SCR 1 to SCR 20 in order 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 C terminus is to recognize and bind target cells to prevent them from being attacked by complement activated substance. Its variation can affect the binding of CFH to the surface of target cells, disable it to inhibit the alternative pathway of complement, leading to its excessive activation and cell damage. The N terminus is related to its complement regulation function.

CFH gene is a member of the regulator of complement activation (RCA) gene cluster on chromosome 1q32. CFH comprises 23 exons and spans over 94 kb of genomic DNA. The first exon encodes the 5′untranslated region of the mRNA and the N-terminal 18 amino acids that organize the signal peptide. Genetic variations of CFH gene cause insufficient CFH expression or CFH deficiency which associates not only a decreasing of complement activating process but also 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) , type I MPGN with pure complement C3 deposition (MPGN1) , membranoproliferative glomerulonephritis type II (MPGN2) , familial type III MPGN (MPGN3) , and age-related macular degeneration (AMD) etc. In view of the variability of the phenotypes, the term complement factor H deficiency (CFHD) can be used collectively as a general term to include the diseases or disorders associated with low level of CFH.

Levels of factor H in human plasma vary widely (116-562 mg/mL) in the population. Although the variation is a combined consequence of genetic and environmental factors, such as an age-dependent increase and a smoking related decrease, one of the studies showed that 63%of the variation in plasma levels of factor H is determined genetically [heritability (h²) = 0.63±0.07; P < 0.0001] . Several reports also illustrated a remarkable genotype-phenotype correlation in which distinct genetic variations at CFH predispose specifically to aHUS, AMD or MPGN2. 30～50%of the aHUS patients has an absent or low CFH plasma concentration, the possible causes include homozygote or heterozygote of CFH gene defect, or existence of autoantibody to CFH. The homozygotes normally show lack of CFH in a familial or sporadic mode, resulted in an earlier onset in infant age. Whereas the heterozygotes’ plasma CFH levels appear to be normal or slightly under lower threshold. Most of the CFH gene mutations are single amino acid mutation which leads to a decreased binding affinity of CFH to its ligand and endothelial cells, and thus causes clinical lesions such as CFHD like aHUS, AMD and MPGN2.

Plasma infusion, as a source of CFH, has been used with variable success to treat renal disease or other CFH deficiency disorders. But the risks of immune stimulation and protein overload limit this therapeutic approach. Liver transplantation was attempted by Cheong et. al. in 2004, despite that the patient was finally died 11 months after the auxiliary partial orthotopic liver transplantation (APOLT) . Eculizumab is a safe and effective therapy for preventing TMA recurrence and provides long-term graft function in aHUS with the CFH/CFHR1 hybrid gene. Although it launched in 2007, the annual cost still remains very high and most of the patients shall use it for life. Gene therapy can be an alternative therapy in the future to restore the CFH dysfunction than the widely used plasma transfusion. However, as an emerging medical technology, it still has risks mainly in safety, which are embodied in the carrier technology itself and the off-target effects of the gene editing tools. The patients are facing difficulties not only in affordability but also the uncertainties in the long-term impact of exogenous fragment insertion.

There is a need for novel treatments for treating and preventing disorders and diseases caused by CFH gene mutation or CFH protein deficiency (CFHD) .

SUMMARY

In order to address the aforementioned problem, the present application provides an oligonucleotide modulator such as a small activating RNA (saRNA) molecule, for treating diseases or conditions caused by the lack or insufficient level of CFH protein such as CFHD by activating/upregulating CFH transcription and increasing the expression level of CFH protein via an RNA activation (RNAa) mechanism.

In particular, the inventors discovered that the functional saRNAs capable of activating/up-regulating the expression of CFH mRNA were not randomly distributed on the promoter but were clustered in certain specific hotspot regions. Only some regions on the promotor of CFH gene are in favor of the saRNAs’ function of activating/up-regulating expression, for example, the regions -538 to -500, -468 to -396, -329 to -283, -273 to -192, -173 to -100, and -64 to -14 upstream of the transcription start site of CFH gene. The inventors also discovered that optimal target sequences/sense strand of an saRNA within the CFH promoter region include sequences having: (1) a GC content between 35%and 65%; (2) less than 5 consecutive identical nucleotides; (3) 3 or less dinucleotide repeats; and (4) 3 or less trinucleotide repeats. As a beneficial consequence, a target sequence (e.g., an isolated nucleic acid sequence comprising the target sequence) , upon interacting with the saRNA, can activate/upregulate the expression of CFH mRNA by at least 10%as compared to a baseline level of CFH mRNA. Based at least in part on these discoveries, the present disclosure features saRNA, compositions, and pharmaceutical compositions for activating/up-regulating the expression of CFH mRNA by at least 10%as compared to baseline levels of CFH gene. Also provided herein are methods for preventing or treating a disease or condition induced by insufficient expression of plasma complement factor H (CFH) , a CFH gene mutation, and/or low functional CFH levels in plasma in an individual comprising administering any of the saRNA, compositions, and/or pharmaceutical compositions described herein.

In one aspect of the present application, an oligonucleotide modulator (such as saRNA molecule) capable of activating/up-regulating expression of the CFH gene in a cell is provided, the oligonucleotide modulator (e.g., the saRNA) comprising an oligonucleotide sequence of 16 to 35 consecutive nucleotides in length, wherein the continuous oligonucleotide sequence has at least 75%, or at least 80%, or at least 85%, or at least 90%sequence homology or complementary to an equal length region of SEQ ID NO: 1707, and thereby activating or up-regulating the expression of the gene by at least 10%as compared to baseline expression of the CFH gene. In some embodiments, the equal length region of SEQ ID NO: 1707 is located in the region -538 to -500 (SEQ ID NO: 1708) , region -468 to -396 (SEQ ID NO: 1709) , region -329 to -283 (SEQ ID NO: 1710) , region -273 to -192 (SEQ ID NO: 1711) , region -173 to -100 (SEQ ID NO: 1712) , or region -64 to -14 (SEQ ID NO: 1713) upstream of the transcription start site of CFH gene.

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 complementary regions, wherein the complementary regions of the sense strand and the antisense strand form a double-stranded nucleic acid structure. In certain embodiments, the sense strand and the antisense strand disclosed in the present application have a complementarity of at least 90%. In certain embodiments, the sense strand and the antisense strand disclosed in the present application are located on two different nucleic acid strands. While in certain embodiments, the sense strand and the antisense strand disclosed in the present application are located on a contiguous nucleic acid strand, optionally 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. In certain embodiments, the sense strand and the antisense strand disclosed in the present application comprises a 3′overhang ranging from 0 to 6 nucleotides in length, alternatively, from 2 to 3 nucleotides in length. In certain embodiments, at least one of the nucleotides of the overhang is a thymine deoxyribonucleotide. In certain embodiments, the sense strand and the antisense strand disclosed in the present application 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 consecutive nucleotides.

In certain embodiments, the sense strand disclosed in the present application has at least 75%sequence homology to a nucleotide sequence selected from SEQ ID NOs: 309-615, and the antisense strand disclosed in the present application has at least 75%sequence homology to a nucleotide sequence selected from SEQ ID NOs: 618-924. In certain embodiments, the sense strand disclosed in the present application comprises a nucleotide sequence selected from SEQ ID NOs: 309-615, and the antisense strand disclosed in the present application comprises a nucleotide sequence selected from SEQ ID NOs: 618-924.

In certain embodiments, the oligonucleotide sequence disclosed in the present application has at least 75%sequence homology or complementarity to a nucleotide sequence selected from SEQ ID NOs: 1-307. In certain embodiments, the sense strand of the oligonucleotide sequence disclosed in the present application has at least 75%sequence homology to a nucleotide sequence selected from SEQ ID NOs: 1-307. In certain embodiments, the antisense strand of the oligonucleotide sequence disclosed in the present application has at least 75%sequence complementarity to a nucleotide sequence selected from SEQ ID NOs: 1-307.

In certain embodiments, at least one nucleotide of the saRNA disclosed in the present application is a chemically modified nucleotide. In certain embodiments, at least one nucleotide of the antisense and/or sense strand of the saRNA disclosed in the present application is chemically modified. In certain embodiments, the chemically modified nucleotide disclosed in the present application is a nucleotide with at least one the following modifications:

a) modification of a phosphodiester bond connecting nucleotides in the nucleotide sequence of the saRNA;

b) modification of 2′-OH of a ribose in the nucleotide sequence of the saRNA; and

c) modification of a base in the nucleotide sequence of the saRNA.

In certain embodiments, at least one nucleotide of the saRNA disclosed in the present application 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 non-natural base comprising nucleotide.

In certain embodiments, the chemical modification of the at least one chemically modified nucleotide disclosed in the present application is an addition of a (E) -vinylphosphonate moiety at the 5’ end of the sense strand or the antisense strand.

In certain embodiments, the sense strand or the antisense strand of the saRNA disclosed in the present application is conjugated to one or more conjugation groups selected from a lipid, a fatty acid, a fluorophore, a ligand, a saccharide, a peptide, and an antibody.

In certain embodiments, the sense strand or the antisense strand of the saRNA disclosed in the present application is conjugated to one or more conjugation groups selected from a cell-penetrating peptide, polyethylene glycol, an alkaloid, a tryptamine, a benzimidazole, a quinolone, an amino acid, a cholesterol, glucose, and N-acetylgalactosamine.

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

In certain embodiments, the present disclosure provides a compound selected from tC2, tC2x4, and C5x5,

whereinrepresents a support material. According to some embodiments, the represents a support material selected from the group consisting of silica, silica gel, glass, ceramic, polymer, cellulose, and combinations thereof. In some embodiments, the support material is in the form of a bead. The bead may be made out of any material including, without limitation, magnetic bead, paramagnetic bead, silica bead, an agarose bead, etc. According to some embodiments, conjugation group derived from tC2 has a structure as shown below:

wherein the asterisk represents the site where the conjugation group is attached to the strand, either directly or via a linking moiety, such as those indicated above. According to another embodiment, and the conjugation group derived from tC2x4 has a structure as shown below:

wherein the asterisk represents the site where the conjugation group is attached to the strand, either directly or via a linking moiety, such as those indicated above. According to another embodiment, and the conjugation group derived from C5x5 has a structure as shown below:

wherein the asterisk represents the site where the conjugation group is attached to the strand, either directly or via a linking moiety, such as those indicated above.

In certain embodiments, the saRNA is conjugated to one, two, three or more conjugation groups derived from a compound selected from tC2, tC2x4, and C5x5. It can be understood that one or more terminal atoms (such as hydrogen, halogen, nitrogen, oxygen, sulfur, phosphorus, etc. ) or terminal groups (such as hydroxyl group, amino group, ester group, ether group, acyl group, etc. ) of the tC2, tC2x4 or C5x5 compound can be detached so as to provide the conjugation group, hence the conjugation group can be considered as a moiety obtained by subtracting said one or more atoms or terminal groups from the tC2, tC2x4 and C5x5 compounds. According to an embodiment, the conjugation groups derived from tC2, tC2x4 and C5x5 are linked with the strand (s) of the asRNA via a linking moiety, such as -OP (O) ₂O-or -P (O) -O-. In certain embodiments, the saRNA is conjugated to two conjugation groups, and the two conjugation groups are a lipid and a N-acetylgalactosamine. In certain embodiments, the two conjugation groups are derived from 1) one group selected from tC2 or tC2x4, and 2) C5x5 as shown in the present application, whereinrepresents a support material. In certain embodiments, the conjugation groups conjugated to the saRNA are derived from tC2x4 and C5x5 as shown in the present application. In certain embodiments, the saRNA attached with a conjugation group has a structure represented by O1, O2 or O3:

In another aspect of the present application, an isolated polynucleotide of 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 NOs: 1-307. In another aspect of the present application, methods of using the isolated polynucleotide of saRNA is provided.

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

Another aspect of the present application provides an isolated polynucleotide encoding the 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 the isolated polynucleotide disclosed herein.

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

Another aspect of the present application provides a cell comprising the saRNA disclosed herein, the isolated polynucleotide encoding the saRNA disclosed herein, or the vector disclosed herein. In one embodiment, the cell is a mammalian cell, optionally a human cell. In some embodiments, the cell is a host cell. The aforementioned cell may be in vitro, such as a cell line or a cell strain, or may exist in a mammalian body, such as a human body.

Another aspect of the present application provides a composition, such as a pharmaceutical composition, comprising the aforementioned saRNA or 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 may be, for example, RNase-free water or RNase-free buffer. In some embodiments, the composition may comprise 0.001-150 nM (e.g., 0.001-100 nM, 0.001-50 nM, 0.001-20 nM, 10-100 nM, 10-50 nM, 20-50 nM, 20-100 nM or 50-150 nM) , or optionally1-150 nM of the aforementioned saRNA or isolated polynucleotide encoding the saRNA disclosed herein.

Another aspect of the present application relates to use of the aforementioned saRNA, isolated polynucleotide encoding the saRNA disclosed herein or the composition comprising the aforementioned saRNA or isolated polynucleotide disclosed herein in preparing a preparation for activating/up-regulating the expression of CFH gene in a cell.

The present application also relates to a method for activating/up-regulating the expression of CFH gene in a cell, wherein the method comprises administering the aforementioned saRNA, the isolated polynucleotide disclosed herein or the composition comprising the aforementioned saRNA or isolated polynucleotide disclosed herein to the cell. In the meantime, a method for increasing a level of CFH protein in a cell or a level of functional CFH protein in plasma is also provided, comprising introducing the saRNA, the nucleic acid, or the composition disclosed herein into the cell in an effective amount.

The aforementioned saRNA, the isolated polynucleotide disclosed herein or the composition comprising the aforementioned saRNA or isolated polynucleotide disclosed herein may be directly introduced into a cell or may be produced in the cell after a nucleotide sequence encoding the saRNA is introduced into the cell. The cell is preferably a mammalian cell, more preferably a human cell. The aforementioned cell may be in vitro, such as a cell line or a cell strain, or may exist in a mammalian body, such as a human body. The human body is a patient suffering from a disease or symptom caused by a CFH gene mutation, low CFH level, and/or insufficient blood levels of functional CFH protein in an individual, and the saRNA, the isolated polynucleotide disclosed herein or the composition comprising the aforementioned saRNA or the isolated polynucleotide disclosed herein is administered in a sufficient amount to treat the disease or symptom. Specifically, the symptom caused by lack of CFH protein due to CFH gene mutation, and/or insufficient expression of functional CFH protein includes, 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.

Another aspect of the present application relates to a method for preventing or treating a disorder caused by insufficient expression of CFH protein, a CFH gene mutation, and/or insufficient blood levels of functional CFH protein in an individual, which comprises administering a therapeutically effective dose of the saRNA disclosed herein, the isolated polynucleotide encoding the saRNA disclosed herein, the vector disclosed herein, or the composition comprising the saRNA disclosed to the individual. 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 a symptom caused by insufficient expression of CFH protein, a CFH gene mutation and/or low functional CFH levels in plasma may include, for example, CFHD. In one embodiment, the disease caused by insufficient blood levels of functional CFH protein due to CFH gene mutation is CFHD. In one embodiment, the 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 administrated to an individual by an administration pathway selected from one or more of: parenteral infusions, oral administration, intranasal administration, inhaled administration, vaginal administration, and rectal administration. In certain embodiments, the administration pathway is selected from one or more of intrathecal, intramuscular, intravenous, intra-arterial, intraperitoneal, intravesical, intracerebroventricular, intravitreal and subcutaneous administrations. In certain embodiments, the method disclosed herein activates/up-regulates expression of CFH gene or CFH mRNA in the individual by at least 10% (e.g. by at least 15%, by at least 20%, by at least 25%, by at least 30%, by at least 35%, by at least 40%, by at least 45%, or by at least 50%) . In certain embodiments, the method disclosed herein increases a level of CFH protein in the individual by at least 10%.

Another aspect of the present application relates to use of the saRNA disclosed herein, the isolated polynucleotide disclosed herein or the composition comprising the saRNA disclosed herein or the isolated polynucleotide disclosed herein in preparing a medicament for preventing or treating a disorder or condition caused by insufficient blood levels of functional CFH protein, a CFH gene mutation, and/or low functional CFH levels in plasma 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 due to CFH gene mutation is CFHD. In one embodiment, the CFHD described herein includes aHUS, DDD, C3GN, CFHR5 nephropathy, LN, MPGN1, MPGN2, MPGN3, and AMD.

In addition, the present application further provides kit for performing the method of prevention or treatment disclosed herein, wherein the kit comprises a) saRNA, b) instructions for use, and c) optionally, means for administering said saRNA to the individual. Specifically, a kit can be packed in a labeled package and the label on said package indicates that said molecule or composition can be used in preventing or treating a disorder or condition induced by insufficient expression of plasma complement factor H (CFH) , or against CFHD.

A kit is provided by the present application for performing the method disclosed herein, wherein the kit comprises a) saRNA disclosed herein, and b) instructions for use. In certain embodiments, the instruction for use comprising means or methods for administering the saRNA disclosed herein to an individual.

Aspects of the present application include a kit comprising the saRNA disclosed herein, the isolated polynucleotide disclosed herein, the vector disclosed herein, or the composition disclosed herein in a labeled package and the label on package indicates that the saRNA, the isolated polynucleotide, the vector or the composition can be used in preventing or treating a disease or condition induced by insufficient expression of plasma complement factor H (CFH) , or against CFHD.

Further to provide by the present application is a kit for detecting CFH protein, or CFH regulated protein in plasma, or in a cell disclosed herein having been transfected with the saRNA aforementioned, or the nucleic acid aforementioned, or the composition aforementioned.

The saRNA activating/upregulating the expression of CFH gene provided herein (such as an saRNA molecule) can efficiently and specifically upregulate the expression of CFH gene and increase the expression level of CFH mRNA with low toxic and adverse effects, and can be used in preparing a drug for preventing or treating disorders associated with insufficient expression of CFH protein and diseases or conditions caused by a CFH gene mutation.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows changes in expression level of human CFH mRNA mediated by saRNAs in Li-7 cells. 307 human CFH promotor-targeting saRNAs were individually transfected at a concentration of 25 nM for 3 days into human hepatocarcinoma cell line (Li-7) . Sequences of saRNA strands and duplex composition are shown in Table 1. Mock was transfected in the absence of an oligonucleotide (not shown) . mRNA levels of CFH were quantified by one-step RT-qPCR using a gene specific primer set shown in Table 4 in each of PCR reactions. HPRT1 and TBP were also amplified and their geometric means was used as an internal reference. The value (y-axis, log2) shows the relative fold changes on CFH mRNA expression levels by each of the 307 saRNAs relative to Mock treatment after normalized to HPRT1 and TBP (mean ± SEM of two replicate transfection wells) . saRNAs are sorted on x-axis by their activity of inducing CFH mRNA expression (log2) in a descending order.

FIG. 2 shows saRNA sorted by their target location and hotspot regions on human CFH promoter in Li-7 cells. 307 human CFH promoter-targeting saRNAs were individually transfected at 25 nM into Li-7 for 3 days. Mock was transfected in the absence of an oligonucleotide (not shown) . mRNA levels of CFH were quantified by one-step RT-qPCR using a gene specific primer set shown in Table 4 in individual PCR reactions. HPRT1 and TBP were also amplified and their geometric means was used as an internal reference. The value (y-axis, log2) shows the relative fold changes on CFH mRNA expression levels by each of the saRNAs relative to Mock treatment after normalized to HPRT1 and TBP. saRNAs are sorted on x-axis by their target location on the promoter -544 bp to -1 bp upstream of CFH transcription start site (TSS) . Locations of the 6 saRNA hotspot regions were marked as H1 to H6 in rectangular dotted boxes. The numbers above the boxes indicate the boundaries of the hotspot regions relative to the CFH TSS (0 site) which span the very 5’ end of the first saRNA′s target and the very 3’ end of the last saRNA’s target within each hotspot region.

FIG. 3 shows the dose-response characterization of CFH saRNAs in Li-7 cells. Top 48 human CFH saRNAs transfecting into human Li-7 cells at concentrations of 2.5 nM, 10 nM and 25 nM for 3 days. Mock was transfected in the absence of an oligonucleotide. dsCon2 duplex (SEQ ID NO: 617, 926) was served as a non-specific duplex control. DS16A-si5 (SEQ ID NO: 616, 925) was a duplex siRNA and transfected as a negative control. mRNA levels of CFH were quantified by two step RT-qPCR using a gene specific primer set shown in Table 4 in individual PCR reactions. HPRT1 and TBP were also amplified and their geometric means was used as an internal reference. FIG. 3 shows the relative fold changes of CFH mRNA level in Li-7 cells transfected by top 48 CFH saRNAs at concentrations of 2.5nM, 10nM and 25nM respectively. The values (y-axis) show relative fold changes on CFH mRNA expression levels by each of saRNA relative to Mock treatment after normalized to the reference of HPRT1 and TBP.

FIG. 4 shows the activating effects of several lead saRNAs on the expression of human CFH protein in Li-7 cells. Cells were treated at a saRNA concentration of 25 nM for 3 days. Mock was transfected in the absence of an oligonucleotide. dsCon2 was served as a non-specific duplex control. DS16A-si5 was a duplex siRNA and transfected as a negative control. FIG. 4 shows the CFH protein level by detecting the OD value using an ELISA kit. The values (y-axis) are presented as CFH protein expression levels relative to Mock treatment. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

FIGs. 5A-5B show the activating effects of several lead saRNAs on the expression of CFH in human hepatocellular carcinoma cell lines. The Li-7 (FIG. 5A) and Huh-7 (FIG. 5B) cells were treated by the saRNA at 10 nM for 4 days. Mock was transfected in the absence of an oligonucleotide. dsCon2 was served as a non-specific duplex control. DS16A-si5 was a duplex siRNA and transfected as a negative control. Relative CFH mRNA levels were determined by using an ELISA kit. HPRT1 and TBP genes were also amplvified and their geometric means was used as an internal reference. The values (y-axis) in the figures are presented as CFH mRNA expression levels relative to Mock treatment after normalized to HPRT1 and TBP. The values (y-axis) in the figures are also presented as CFH protein expression levels relative to Mock treatment. */#p < 0.05, **/##p < 0.01, ***/###p < 0.001, ****/####p < 0.0001.

FIGs. 6A-6D show the activating effect of saRNAs on the expression of CFH mRNA in Li-7 cells. Four saRNAs (i.e., DS16A-0013, DS16A-0135, DS16A-0199 and DS16A-0055) were individually transfected into Li-7 cells at the ascending concentrations (i.e., 0.0017, 0.0051, 0.152, 0.046, 0.14, 0.41, 1.2, 3.7, 11.111, 33.333 and 100 nM) for 3 days. FIG. 6A-6D show the mRNA levels of CFH were determined by two step RT-qPCR using a gene specific primer set. HPRT1 and TBP were also amplified and their geometric means was used as an internal reference. The values (y-axis) are presented as CFH mRNA expression level relative to Mock treatment after normalized to HPRT1 and TBP.

FIGs. 7A-7B show the activating effects of saRNAs on the expression of CFH mRNA in PLC/PRF/5 and Huh-7 cells. Cells were treated by the saRNAs at a concentration of 25 nM for 3 days. Mock was transfected in the absence of an oligonucleotide. dsCon2 was served as a non-specific duplex control. DS16A-si5 was a duplex siRNA and transfected as a negative control. RD-11603 and RD-11598 were duplex saRNAs and served exemplary controls. FIG. 7A-7B show mRNA levels of CFH were quantified by two step RT-qPCR using a gene specific primer set in separate PCR reactions. HMBS was also amplified and served as an internal reference. The values (y-axis) are presented as CFH mRNA expression levels relative to Mock treatment after normalized to the reference of HMBS.

FIG. 8 shows changes in expression levels of mouse Cfh in primary mouse hepatocytes (PMH) cells mediated by saRNAs. 383 mouse Cfh promoter-targeting saRNAs were individually transfected at a concentration of 25 nM for 3 days in PMH cells. Strand sequence and saRNA duplex composition are shown in Table 2. Mock was transfected in the absence of an oligonucleotide (not shown) . mRNA levels of Cfh were quantified by one-step RT-qPCR using a gene specific primer set shown in Table 4 in separate PCR reactions. Hmbs and Tbp were also amplified and their geometric means was used as an internal reference. The values (y-axis) are shown as Cfh mRNA expression levels relative to Mock treatment after normalized to Hmbs and Tbp. saRNAs are sorted on x-axis by their activity of inducing Cfh mRNA expression (log2) in a descending order.

FIG. 9 shows saRNA sorted by their location and hotspot regions on mouse Cfh promoter in PMH cells. 383 mouse Cfh promoter-targeting saRNAs were individually transfected at 25 nM into PMH for 3 days. Mock was transfected in the absence of an oligonucleotide (not shown) . mRNA levels of Cfh were quantified by one-step RT-qPCR using a gene specific primer set shown in Table 4 in separate PCR reactions. Hmbs and Tbp were also amplified and their geometric means was used as an internal reference. The value (y-axis, log2) shows the fold changes on Cfh mRNA expression levels by each of saRNAs relative to Mock treatment after normalized to Hmbs and Tbp. saRNAs are sorted on x-axis by their target location on the promoter -800 bp to -1 bp upstream of Cfh transcription start site (TSS) . Locations of the 5 saRNA hotspot regions were marked as H7 to H11 in rectangular dotted boxes. The numbers above the boxes indicate the boundaries of the hotspot regions relative to the Cfh TSS which span the very 5’ end of the first saRNA′s target and the very 3’ end of the last saRNA’s target within each hotspot region.

FIG. 10 shows the dose-response characterization of top 64 mouse Cfh saRNAs in PMH cells. Top 64 mouse Cfh saRNAs were transfected at different concentrations of 2.5 nM, 10 nM and 25 nM into PMH cells for 3 days. Mock was transfected in the absence of an oligonucleotide. dsCon2 was served as a non-specific duplex control. DS16B-si6 (SEQ ID: 1310, 1694) was a duplex siRNA and transfected as a negative control. mRNA levels of Cfh were quantified by two step RT-qPCR using a gene specific primer set shown in Table 4 in individual PCR reactions. Hmbs and Tbp were also amplified and their geometric means was used as an internal reference. The values (y-axis) were presented as fold changes of Cfh mRNA level in PMH cells relative to Mock treatment after normalizing to the reference of Hmbs and Tbp.

FIGs. 11A-11B show the activating effects of several lead saRNAs on the expression of Cfh gene in PMH cells. Cells were treated by the saRNA at a concentration of 25 nM for 5 days. Mock was transfected in the absence of an oligonucleotide. dsCon2 was served as a non-specific duplex control. DS16B-si6 was a duplex siRNA and transfected as a negative control. FIG. 11A shows the relative mRNA levels of Cfh in PMH cells determined by two step RT-qPCR using a gene specific primer set shown in Table 4. Hmbs and Tbp were also amplified and their geometric means was used as an internal reference. The values (y-axis) are presented as Cfh mRNA expression levels relative to Mock treatment after normalized to Hmbs and Tbp. FIG. 11B shows the Cfh protein level by detecting the OD value using an ELISA kit. The values (y-axis) are presented as Cfh protein expression levels relative to Mock treatment. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

FIGs. 12A-12B shows the time course change of saRNAs on the expression of Cfh in PMH cells. Cells were transfected by the individual saRNAs at a concentration of 25 nM for 3-6 days. Mock was transfected in the absence of an oligonucleotide. dsCon2 was served as a non-specific duplex control. DS16B-si6 was a duplex siRNA and transfected as a negative control. FIG. 12A shows the mRNA levels of Cfh in PMH cells determined by two step RT-qPCR using a gene specific primer set shown in Table 4 in separate PCR reactions. Hmbs and Tbp were also amplified and their geometric means was used as an internal reference. The values (y-axis) are presented as Cfh mRNA expression levels relative to Mock treatment after normalized to Hmbs and Tbp. FIG. 12B shows the Cfh protein levels by detecting the OD values using an ELISA kit. The values (y-axis) are presented as Cfh protein expression levels relative to Mock treatment.

FIGs. 13A-13D shows the activating effect of saRNAs on the expression of Cfh mRNA in PMH cells. Four saRNAs (i.e., DS16B-070, DS16B-091, DS16B-098 and DS16B-0241) were individually transfected into PMH cells at the ascending concentrations (i.e., 0.0017, 0.0051, 0.152, 0.046, 0.14, 0.41, 1.2, 3.7, 11.111, 33.333 and 100 nM) for 3 days. FIG. 13A-13D show mRNA level of Cfh were detected by two-step RT-qPCR using a gene specific primer set after RNA isolation and RT reaction. Hmbs and Tbp were also amplified and their geometric means was used as an internal reference. The values (y-axis) are presented as Cfh mRNA expression levels relative to Mock treatment after normalized to Hmbs and Tbp.

FIG. 14 shows the activating effect of saRNAs on the expression of Cfh mRNA in PMH cells. Four saRNAs (i.e., RD-13878, RD-14662, RD-14663 and RD-14669) were individually transfected into PMH cells at 25 nM for 3 days. RD-13149 was chemical modified siRNA and served as a negative control. dsCon2 was served as a non-specific duplex control. FIG. 14 shows mRNA level of Cfh were detected by two-step RT-qPCR using a gene specific primer set after RNA isolation and RT reaction. Hmbs and Gusb were also amplified and their geometric means was used as an internal reference. The values (y-axis) are presented as Cfh mRNA expression levels relative to Mock treatment after normalized to Hmbs and Gusb.

FIG. 15 shows the activating effect of saRNAs on the expression of Cfh mRNA by free uptake in PMH cells. saRNAs (i.e., RD-14660) were added into PMH cell culture medium at the indicated escalating concentrations (i.e., 6.25, 25, 100, 400, 1600 and 6400 nM) for 4 days. RD-13149 and RD-14040 were individually added into PMH cell culture medium at 1000 nM for 4 days. RD-13149 was chemical modified siRNA and served as a negative control. RD-14040 was served as a conjugation moiety (i.e., tC2x4) alone control. FIG. 15 shows mRNA level of Cfh were detected by two-step RT-qPCR using a gene specific primer set after RNA isolation and RT reaction. Hmbs and Gusb were also amplified and their geometric means was used as an internal reference. The values (y-axis) are presented as Cfh mRNA expression levels relative to Mock treatment after normalized to Hmbs and Gusb.

FIG. 16 show the body weight change of C57BL/6J mice after SC injection of modified saRNAs. The indicated saRNA (i.e., RD-13147) was administrated into adult C57BL/6J mice via SC injection at 50 mg/kg. Saline was injected as a vehicle control. RD-13149 was chemical modified siRNA and injected at 3 mg/kg via SC as a negative control. Mice were sacrificed at day 10 following treatment. FIG. 16 shows the body weight (g) change of C57BL/6J mice post dosing. Mean body weight levels of 3 animals per group are shown in each of group.

DETAILED DESCRIPTION

Double-stranded RNAs (dsRNAs) targeting gene regulatory sequences, including promoters, have been shown to upregulate target genes in a sequence-specific manner at the transcriptional level via a mechanism known as RNA activation (RNAa) (Li, L. C., et al.Small dsRNAs induce transcriptional activation in human cells. PNAS (2006) ) . Such dsRNAs are termed small activating RNAs (saRNAs) .

Embodiments of the present disclosure are based in part on the surprising discovery that an oligonucleotide modulator (for example, saRNA, also referred to as “CFH gene saRNA” herein) can activate or upregulate the expression of a CFH gene in a cell. The increase in production of functional CFH gene mRNA following administration with an saRNA of the present application can achieve a significant increase or upregulation in the level of CFH mRNA and CFH protein.

In particular, the inventors discovered that the functional saRNAs capable of activating/up-regulating the expression of CFH mRNA were not randomly distributed on the promoter but were clustered in certain specific hotspot regions. Only some regions on the promotor of CFH gene are in favor of the saRNAs’ function of activating/up-regulating expression, for example, the regions -538 to -500, -468 to -396, -329 to -283, -273 to -192, -173 to -100, and -64 to -14 upstream of the transcription start site of CFH gene. The inventors also discovered that optimal target sequences/sense strand of an saRNA within the CFH promoter region include sequences having: (1) a GC content between 35%and 65%; (2) less than 5 consecutive identical nucleotides; (3) 3 or less dinucleotide repeats; and (4) 3 or less trinucleotide repeats. As a beneficial consequence, a target sequence (e.g., an isolated nucleic acid sequence comprising the target sequence) , upon interacting with the saRNA, can activate/upregulate the expression of CFH mRNA by at least 10%as compared to a baseline level of CFH mRNA. Based at least in part on these discoveries, the present disclosure features saRNA, compositions, and pharmaceutical compositions for activating/up-regulating the expression of CFH mRNA by at least 10%as compared to baseline levels of CFH mRNA. Also provided herein are methods for preventing or treating a disease or condition induced by insufficient expression of plasma complement factor H (CFH) , a CFH gene mutation, and/or low functional CFH levels in plasma in an individual comprising administering any of the saRNA, compositions, and/or pharmaceutical compositions described herein.

Embodiments of the present disclosure are also based in part on the surprising discovery that the saRNAs capable of activating or upregulating the expression of CFH gene in a cell are clustered in particular CFH gene promoter regions, as shown in FIGs. 2 and 9. The present inventors identified these clusters of CFH gene promoter regions that were considered “hotspot” promoter regions that enrich target sites for the functional saRNAs developed. These specific promoter regions (referred to as “hotspot” herein) identified by the present application are optionally at least 39 nt in length, or alternatively have a length ranging from about 39 to about 153 nt. A “hotspot” herein is defined by a nucleic acid region on the target sequence of the saRNAs, where full length targets of functional saRNAs are enriched and spanned the very 5’ end of the first saRNA′s target and the 3’ end of the last saRNA’s target within each hotspot. In some embodiments, at least about 40%, e.g., 43%or even over 50%of the saRNAs designed to target a fragment in these hotspot regions are turned out to be functional, i.e., can induce a 1.1-fold or more change in the mRNA expression of the target gene. In some embodiments, at least 30%, about 40%, or over 50%of the saRNAs designed to target a fragment in these hotspot regions are turned out to be functional, i.e., can induce a 1.1-fold or more change in mRNA level or protein expression of the target gene. The present inventors surprisingly found that functional saRNAs were not randomly distributed on the promoter but were clustered in the specific hotspot regions (see e.g., Table 9) . For example, the 6 hotspots of the human CFH promoter located in 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) ; and the 5 hotspots of the mouse Cfh promoter located in regions -754 to -654 (H7) , -479 to -421 (H8) , -409 to -370 (H9) , -208 to -56 (H10) and -52 to -4 (H11) from the TSS of the promoter were detected, and were found to be optimal target sites for saRNAs in activating CFH/Cfh gene expression by the RNA activation mechanism.

This saRNA-CFH mRNA-CFH protein pathway can provide an alternative therapeutic method different from the current treatment of CFH-deficiency-related disorders (CFHD) , e.g., for atypical hemolytic uremic syndrome patients.

In the present application, the related terms are defined as follows:

The term ″complementary″ as used herein refers to the capability of forming base pairs between two oligonucleotide strands. The base pairs are generally formed through hydrogen bonds between nucleotides in the antiparallel oligonucleotide strands. The bases of the complementary oligonucleotide strands can be paired in the Watson-Crick manner (such as A to T, A to U, and C to G) or in any other manner allowing the formation of a duplex (such as Hoogsteen or reverse Hoogsteen base pairing) .

Complementarity includes complete complementarity and incomplete complementarity. ″Complete complementarity″ or ″100%complementarity″ means that each nucleotide from the first oligonucleotide strand can form a hydrogen bond with a nucleotide at a corresponding position in the second oligonucleotide strand in the double-stranded region of the double-stranded oligonucleotide molecule, with no base pair being ″mispaired″ . ″Incomplete complementarity″ means that not all the nucleotide units of the two strands are bound with each other by hydrogen bonds. For example, for two oligonucleotide strands each of 20 nucleotides in length in the double stranded region, if only two base pairs in this double-stranded region can be formed through hydrogen bonds, the oligonucleotide strands have a complementarity of 10%. In the same example, if 18 base pairs in this double-stranded region can be formed through hydrogen bonds, the oligonucleotide strands have a complementarity of 90%. Substantial complementarity refers to at least about 75%, about 79%, about 80%, about 85%, about 90%, about 95%or 99%complementarity.

The term ″oligonucleotide″ or “polynucleotide” can be used interchangeably, and refers to polymers of nucleotides, and includes, but is not limited to, single-stranded or double-stranded nucleic acid molecules of DNA, RNA, or DNA/RNA hybrid, oligonucleotide strands containing regularly and irregularly alternating deoxyribosyl portions and ribosyl portions, as well as modified and naturally or unnaturally existing frameworks for such oligonucleotides. The oligonucleotide for activating target gene transcription described herein is a small activating nucleic acid molecule (saRNA) .

The terms ″oligonucleotide strand″ , “strand” and ″oligonucleotide sequence″ as used herein can be used interchangeably, referring to a generic term for 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 a strand can be any length from 16 to 35 nucleotides.

The term ″target gene″ as used herein can refer to nucleic acid sequences, transgenes, viral or bacterial sequences, chromosomes or extrachromosomal genes that are naturally present in organisms, and/or can be transiently or stably transfected or incorporated into cells and/or chromatins thereof. The target gene can be a protein-coding gene or a non-protein-coding gene (such as a microRNA gene and a long non-coding RNA gene) . The target gene generally contains a promoter sequence, and the positive regulation for the target gene can be achieved by designing a saRNA having sequence identity (also called homology) to the promoter sequence, characterized as the up-regulation of expression of the target gene. ″Target sequence″ or “target site” used interchangeably refers to a sequence fragment in the sequence of a target gene sequence, such as, a target gene promoter, which is homologous or complementary with a sense strand or an antisense strand of a saRNA. The target gene can also include one or more regulatory elements where one or more saRNA are designed to have sequence identity to a regulatory element. Non-limiting examples of one or more regulatory elements include: a promoter, an enhancer, a silencer, an insulator, a TATA box, a GC box, a CAAT box, a transcriptional start site, a DNA binding motif of a transcription factor or other protein that regulates transcription, and a 5’ untranslated region.

As used herein, the terms ″sense strand″ of a saRNA in the saRNA duplex refers to the strand having sequence homology or sequence identity with a fragment of the coding strand of the sequence of a target gene.

As used herein, the terms ″antisense strand″ of a saRNA in the saRNA duplex refers to the strand having sequence complementary with the sense strand. Said antisense strand may interact with a target sequence to active or up-regulate gene expression, said target sequence may be a fragment of the coding strand of the sequence of a target gene.

The term ″coding strand″ as used herein refers to a DNA strand in the target gene which cannot be used for transcription, and the nucleotide sequence of this strand is the same as that of a RNA produced from transcription (in the 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 on the same DNA strand as the DNA coding strand of the target gene.

The term ″template strand ″ as used herein refers to the other strand complementary with the coding strand in the double-stranded DNA of the target gene, i.e., the strand that, as a template, can be transcribed into RNA, and this strand is complementary with the transcribed RNA (A to U and G to C) . In the process of transcription, RNA polymerase binds to the template strand, moves along the 3′→5′direction of the template strand, and catalyzes the synthesis of the 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 on the same DNA strand as the DNA template strand of the target gene.

As used herein, the term “LNA” refers to a locked nucleic acid in which the 2′-oxygen and 4′-carbon atoms are joined by an extra bridge. As used herein, the term “BNA” refers to a 2′-O and 4′-aminoethylene bridged nucleic acid that can contain a five-membered or six-membered bridged structure with an N-O linkage. As used herein, the term “PNA” refers to a nucleic acid mimic with a pseudopeptide backbone composed of N- (2-aminoethyl) glycine units with the nucleobases attached to the glycine nitrogen via carbonyl methylene linkers. As used herein, the term “GNA” also referred to as glycerol nucleic acid, is a nucleic acid similar to DNA or RNA but differing in the composition of its sugar-phosphodiester backbone, using propylene glycol in place of ribose or deoxyribose.

The term ″promoter″ as used herein refers to a sequence which is spatially associated with a protein-coding or RNA-coding nucleic acid sequence and plays a regulatory role for the transcription of the protein-coding or RNA-coding nucleic acid sequence. Generally, a eukaryotic gene promoter contains 100 to 5000 base pairs, although this length range is not intended to limit the term ″promoter″ as used herein. Although the promoter sequence is generally located at the 5′terminus of a protein-coding or RNA-coding sequence, it may also exist in exon and intron sequences.

The term ″transcription start site″ as used herein refers to a nucleotide marking the transcription start on the template strand of a gene. The transcription start site can appear on the template strand of the promoter region. A gene can have more than one transcription start site.

The term ″identity″ or ″homology″ as used herein means that one oligonucleotide strand (sense or antisense strand) of an saRNA has sequence similarity with a coding strand or template strand in a region of a target gene. As used herein, the ″identity″ or ″homology″ may be at least about 75%, about 79%, about 80%, about 85%, about 90%, about 95%or 99%.

The term “sequence specific mode” as used herein means a binding or hybridization way of two nucleic acid fragments according to their nucleotide sequence, e.g., a Watson-Crick manner (such as A to T, A to U, and C to G) or any other manner allowing the formation of a duplex (such as Hoogsteen or reverse Hoogsteen base pairing) .

The term ″overhang″ as used herein refers to non-base-paired nucleotides at the terminus (5′or 3′) of an oligonucleotide strand, which is formed by one strand extending out of the other strand in a double-stranded oligonucleotide. A single-stranded region extending out of the 3′terminus and/or 5′terminus of a duplex is referred to as an overhang.

As used herein, the terms ″gene activation″ or ″activating gene expression″ and ″gene upregulation″ or ″up-regulating gene expression″ can be used interchangeably, and mean an increase in transcription, translation, expression or activity of a certain nucleic acid as determined by measuring the transcriptional level, mRNA level, protein level, enzymatic activity, methylation state, chromatin state or configuration, translation level or the activity or state in a cell or biological system of a gene. These activities or states can be determined directly or indirectly. In addition, ″gene activation″ , ″activating gene expression″ , ″gene up-regulation″ or ″up-regulating 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 transcriptional level to increase transcription into RNA and the RNA is translated into a protein, thereby increasing the expression of the protein.

As used herein, the terms “oligonucleotide modulator” , ″small activating RNA″ , ″saRNA″ , and ″small activating nucleic acid molecule″ can be 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) containing a nucleotide sequence having sequence identity to the non-coding nucleic acid sequence (e.g., a promoter or an enhancer) of a target gene and a second nucleic acid fragment (antisense strand) containing a nucleotide sequence complementary with the first nucleic acid fragment, wherein the first nucleic acid fragment and the second nucleic acid fragment form a duplex. The saRNA can also be comprised of a synthesized or vector-expressed single-stranded RNA molecule that can form a hairpin structure by two complementary regions within the molecule, wherein the first region contains a nucleotide sequence having sequence identity to the target sequence of a promoter of a gene, and the second region contains a nucleotide sequence which is complementary with the first region. The length of the duplex region of the saRNA is typically about 10 to about 50, about 12 to about 48, about 14 to about 46, about 16 to about 44, about 18 to about 42, about 20 to about 40, about 22 to about 38, about 24 to about 36, about 26 to about 34, and about 28 to about 32 base pairs, and typically about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45 or about 50 base pairs. In addition, the terms “oligonucleotide modulator” , ″saRNA″ , ″small activating RNA″ , and ″small activating nucleic acid molecule″ also contain nucleic acids other than the ribonucleotide, including, but not limited to, modified nucleotides or analogues.

As used herein, the terms ″hotspot″ refers to a gene promoter region of at least 39 bp in length where functional saRNAs are enriched, i.e., at least 40%, e.g., about 43%, about 50%, about 60%, or about 70%, or more than 70%of the saRNAs designed to target this region is “functional” , i.e., can induce a 1.1-fold or more change in the mRNA or protein expression of the target gene. A “hotspot” and “hotspot region” can be used interchangeably and herein is defined by a nucleic acid region on the target sequence of the saRNAs , where full length targets of functional saRNAs are enriched and spanned the very 5’ end of the first saRNA and the 3’ end of the last saRNA within the hotspot. In a non-limiting example, a saRNA is designed according to the following criteria: (1) having a GC content between 35%and 65%; (2) with less than 5 consecutive identical nucleotides; (3) with 3 or less dinucleotide repeats; and (4) with 3 or less trinucleotide repeats.

As used herein, the term “functional saRNA” refers to a saRNA which 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 an saRNA which modulates both mRNA level and protein expression of CFH gene by less than 10% (or less than 1.1 fold) .

As used herein, the term “an isolated target site” and “an isolated polynucleotide″ can be used interchangeably, and herein means a target site which a saRNA has complementarity or hybridizes to. For example, an isolated nucleic acid sequence of a target site can include a nucleic acid sequence which a region of saRNAs have complementarity or hybridize to. As used herein, the term “an isolated polynucleotide” used herein means a polynucleotide which encodes an saRNA.

As used herein, the term ″synthesis″ refers to a method for synthesis of an oligonucleotide, including any method allowing RNA synthesis, such as chemical synthesis, in vitro transcription, and/or vector-based expression.

As used herein, the term ″support material″ refers to a solid phase starting material held between filters, in columns that enable all reagents and solvents to pass through freely using an automated oligonucleotide synthesizer, and optionally, generate 3′or 5′end conjugated oligonucleotide. A support material can be selected from the group consisting of control pore glass (CPG) , silica, silica gel, glass, ceramic, polymer, cellulose, and combinations thereof.

As used herein, the upper cased “CFH” or “CFH gene” refers to a human gene; whereas the first letter capitalized “Cfh” or “Cfh gene” refers to a mouse gene.

As used herein, the term ″CFH mRNA″ refers to a message RNA (mRNA) generated from the expression of CFH gene, or the transcription of CFH gene.

As used herein, the term “CFH” and “CFH protein” can be used interchangeably, and refers to a protein generated from the expression of CFH gene, or translation of the CFH mRNA.

saRNA

In the present application, expression of the CFH gene is upregulated by RNA activation, and a related disease (particularly CFHD) is treated by increasing the expression level of CFH protein. As the CFH gene encodes the CFH protein, an increase in CFH mRNA expression results in an increase in expression of the CFH protein, thereby treating the disease (e.g., CFHD) . Therefore, the CFH gene, in some cases, is a target gene in the present application.

Aspects of the present application include an oligonucleotide modulator (saRNA) comprising an oligonucleotide sequence having a length ranging from 16 to 35 consecutive nucleotides, wherein the continuous oligonucleotide sequence has at least 75%, or at least 80%, or at least 85%, or at least 90%sequence homology or complementary to an equal length portion of SEQ ID NO: 1707, and wherein the saRNA activates/upregulates the expression of CFH gene by at least 10%as compared to baseline expression of the CFH gene.

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

In some embodiments, the continuous oligonucleotide sequence of the saRNA has five or less, i.e., 5, 4, 3, 2, 1, or 0 nucleotide differences or mismatches relative to the equal length portion of SEQ ID NO: 1707. In some embodiments, the differences or mismatches locate in the middle or 3’ terminus of the oligonucleotide sequence of the saRNA. 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 herein incorporated by reference in their entireties.

In some embodiments, the saRNA disclosed herein comprises a sense strand and an antisense strand. The sense strand and the antisense strand comprise complementary regions capable of forming a double-stranded nucleic acid structure that activates the expression of the CFH gene in a cell via the RNAa mechanism. The RNAa mechanism (also known as RNA activation) used herein refers to a mechanism that a double-strand nucleic acid structure is capable of upregulating target genes in a sequence-specific manner at the transcriptional level. The sense strand and the antisense strand of the saRNA can exist either on two different nucleic acid strands or on one nucleic acid strand (e.g., a contiguous nucleic acid sequence) . When the sense strand and the antisense strand are located on two different strands, at least one strand of the saRNA has a 3′overhang of 0 to 6 nucleotides in length, such that the overhangs of 0, 1, 2, 3, 4, 5 or 6 nucleotides in length, and in some cases, both strands have a 3′overhang of 2 or 3 nucleotides in length. The nucleotide of the overhang is, in some cases thymine deoxyribonucleotide (dT) . When the sense strand and the antisense strand are located on one nucleic acid strand, in some cases, the saRNA is a hairpin single-stranded nucleic acid molecule, where the complementary regions of the sense strand and the antisense strand form a double stranded nucleic acid structure with each other. In the aforementioned saRNA, in some embodiments, the sense strand and the antisense strand have a length ranging from16 to 35 nucleotides, respectively. For example, in some embodiments, the sense strand and the antisense strand, independently comprises 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.

In certain embodiments, one strand of the saRNA has at least 75% (e.g., at least about 79%, about 80%, about 85%, about 90%, about 95%or about 99%) sequence homology or complementarity to a nucleotide sequence selected from 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 to 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 to 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 or is any nucleotide sequence selected from SEQ ID NOs: 618-924.

In certain embodiments, one strand of the saRNA has five or less, i.e., 5, 4, 3, 2, 1, or 0 nucleotide differences or mismatches relative to the nucleotide sequence selected from SEQ ID NOs: 1-307. Specifically, the sense strand of the saRNA disclosed herein has five or less, i.e., 5, 4, 3, 2, 1, or 0 nucleotide differences relative to the nucleotide sequence selected from SEQ ID NOs: 309-615, and the antisense strand of the saRNA disclosed herein has five or less, i.e., 5, 4, 3, 2, 1, or 0 nucleotide differences relative to the nucleotide sequence selected from SEQ ID NOs: 618-924. In some embodiments, the differences or mismatches locate in the middle or 3’ terminus of the sense or antisense strand of the saRNA.

In certain embodiments, the antisense strand disclosed herein is capable of interact with a target nucleic acid sequence of a promoter of a gene in a sequence specific manner, meaning that the antisense strand is capable of undergoing hybridization to a target nucleic acid through hydrogen bonding. In certain embodiments, an antisense strand has a nucleotide sequence that, when written in the 5′to 3′direction, comprises the reverse complement of the target portion of a target nucleic acid to which it is targeted. In certain such embodiments, an antisense strand has a nucleotide sequence that, when written in the 5′to 3′direction, comprises the reverse complement of the target portion in SEQ ID NO: 1707, specifically, the target portion is a nucleic acid sequence selected from SEQ ID NO: 1-307.

In the saRNAs disclosed herein, all nucleotides may be natural or non-chemically modified nucleotides, or at least one nucleotide is a chemically modified nucleotide. Non-limiting examples of the chemical modification include one or more of a combination of the following:

(1) modification of a phosphodiester bond of nucleotides in the nucleotide sequence of the saRNA;

(2) modification of 2′-OH of the ribose in the nucleotide sequence of the saRNA;

(3) modification of a base in the nucleotide of the saRNA; and

(4) at least one nucleotide in the nucleotide sequence of a small activating nucleic acid molecule being a locked nucleic acid.

The chemical modification described herein is well-known to those skilled in the art, and the modification of the phosphodiester bond refers to the modification of oxygen in the phosphodiester bond, including phosphorothioate modification and boranophosphate modification. The modifications disclosed herein stabilize a saRNA structure, maintaining high specificity and high affinity for base pairing.

In some embodiments, the saRNA of the present application includes at least one chemically modified nucleotide which is modified at 2′-OH in pentose of a nucleotide, i.e., the introduction of certain substituents at the hydroxyl position of the ribose, such as 2′-fluoro modification, 2′-oxymethyl modification, 2′-oxyethylidene methoxy modification, 2, 4′-dinitrophenol modification, locked nucleic acid (LNA) , 2′-amino modification or 2′-deoxy modification, e.g., a 2′-deoxy-2′-fluoro modified nucleotide, a 2′-deoxy-modified nucleotide.

In some embodiments, the saRNA of the present application includes at least one chemically modified nucleotide which is modified at the base of the nucleotide, e.g., 5 ′-bromouracil modification, 5′-iodouracil modification, N-methyluracil modification, or 2, 6-diaminopurine modification.

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

In some embodiments, the saRNA of the present application includes at least one nucleotide in the nucleotide sequence of the small activating nucleic acid molecule being a chemically modified nucleic acid, e.g., a locked nucleotide, an abasic nucleotide, a 2′-amino-modified nucleotide, a 2′-alkyl-modified nucleotide, a morpholino nucleotide, a phosphoramidate, and a non-natural base comprising nucleotide. In some embodiments, the saRNA disclosed herein includes an “endo-light” modification with 2′-O-methyl modified nucleotides and nucleotides comprising a 5′-phosphorothioate group.

In some embodiments, the saRNA of the present application is chemically modified to enhance stability or other beneficial characteristics. The nucleic acids featured in the present application may be synthesized and/or modified by conventional methods, such as those described in “Current protocols in nucleic acid chemistry, ” Beaucage, S. L. et al. (Edrs. ) , John Wiley &Sons, Inc., New York, N.Y., USA, which is hereby incorporated herein by reference. Modifications include, for example, (a) end modifications, e.g., 5′end modifications (phosphorylation, conjugation, inverted linkages, etc. ) 3′end modifications (conjugation, DNA nucleotides, inverted linkages, etc. ) , (b) base modifications, e.g., replacement with stabilizing bases, destabilizing bases, or bases that base pair with an expanded repertoire of partners, removal of bases (abasic nucleotides) , or conjugated bases, (c) sugar modifications (e.g., at the 2′position or 4′position) or replacement of the sugar, as well as (d) backbone modifications, including modification or replacement of the phosphodiester linkages. Specific examples of saRNA molecules that can be used in this present application include, but are not limited to RNAs containing modified backbones or no natural internucleoside linkages. In some embodiments, RNAs having modified backbones include, among others, those that do not have a phosphorus atom in the backbone. In some embodiments, modified RNAs that do not have a phosphorus atom in their internucleoside backbone can also be considered to be oligonucleosides. In some embodiments, the modified oligonucleotide will have a phosphorus atom in its internucleoside backbone.

Modified oligonucleotide backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3′-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3′-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3′-5′linkages, 2′-5′linked analogs of these, and those) having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3′-5′to 5′-3′or 2′-5′to 5′-2′. Various salts, mixed salts and free acid forms are also included.

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

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

In addition, to facilitate entry of the saRNA into a cell, chemical conjugation groups may be introduced at the ends of the sense or antisense strands of the saRNA on the basis of the above modifications to facilitate action through a cell membrane composed of lipid bilayers and gene promoter regions within the nuclear membrane and nucleus.

In certain embodiments, saRNAs disclosed in the present application are covalently attached to one or more conjugate groups. In certain embodiments, conjugate groups modify one or more properties of the attached oligonucleotide, including but not limited to pharmacodynamics, pharmacokinetics, stability, binding, absorption, tissue distribution, cellular distribution, cellular uptake, charge and clearance. In certain embodiments, conjugate groups impart a new property on the attached oligonucleotide, e.g., fluorophores or reporter groups that enable detection of the oligonucleotide. Certain conjugate groups and conjugate moieties have been described previously, for example: cholesterol moiety (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) , a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660, 306-309; Manoharan et al., Bioorg. Med. Chem. Lett., 1993, 3, 2765-2770) , a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20, 533-538) , an aliphatic chain, e.g., do-decan-diol or undecyl residues (Saison-Behmoaras et al., EMBO1, 1991, 10, 1111-1118; Kabanov et al., FEBS Lett., 1990, 259, 327-330; Svinarchuk et al., Biochimie, 1993, 75, 49-54) , a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethyl-ammonium 1, 2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res., 1990, 18, 3777-3783) , a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides &Nucleotides, 1995, 14, 969-973) , or adamantane acetic acid, a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229-237) , an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277, 923-937) , a tocopherol group (Nishina et al., Molecular Therapy Nucleic Acids, 2015, 4, e220; and Nishina et al., Molecular Therapy, 2008, 16, 734-740) , or a GalNAc cluster (e.g., WO2014/179620) .

In some embodiments, the saRNA of the present application relates to the sense strand or the antisense strand of the saRNA that is conjugated to one or more conjugation groups selected from: intercalators, reporter molecules, polyamines, polyamides, peptides, carbohydrates, vitamin moieties, polyethylene glycols, thioethers, polyethers, cholesterols, thiocholesterols, cholic acid moieties, folate, lipids, phospholipids, biotin, phenazine, phenanthridine, anthraquinone, adamantane, acridine, fluoresceins, rhodamines, coumarins, fluorophores, and dyes.

In some embodiments, a conjugate group comprises an active drug substance, for example, aspirin, warfarin, phenylbutazone, ibuprofen, suprofen, fen-bufen, ketoprofen, (S) - (+) -pranoprofen, carprofen, dansylsarcosine, 2, 3, 5-triiodobenzoic acid, fingolimod, flufenamic acid, folinic acid, a benzothiadiazide, chlorothiazide, a diazepine, indo- methicin, a barbiturate, a cephalosporin, a sulfa drug, an antidiabetic, an antibacterial or an antibiotic.

In some embodiments, the saRNA of the present application is conjugated to one or more conjugation groups selected from: a lipid, a fatty acid, a fluorophore, a ligand, a saccharide, a peptide, and an antibody.

In some embodiments, the saRNA of the present application relates to the sense strand or the antisense strand of the saRNA that is conjugated to one or more conjugation groups selected from a cell-penetrating peptide, polyethylene glycol, an alkaloid, a tryptamine, a benzimidazole, a quinolone, an amino acid, a cholesterol, glucose and N-acetylgalactosamine. In certain embodiments, the saRNA is conjugated to two conjugation groups. In certain embodiments, the two conjugation groups are a lipid and a N-acetylgalactosamine. In certain embodiments, the two conjugation groups are derived from 1) one group selected from tC2 or tC2x4, and 2) C5x5, as shown in the present application, In certain embodiments, the conjugation groups conjugated to the saRNA are derived from tC2x4 and C5x5, as shown in the present application.

In certain embodiments, said conjugation group is a lipid selected from fatty acid comprising a carbon chain length of from 4 to 30 carbon atoms. In certain embodiments, said conjugation group is fatty acid comprising a carbon chain length of 16 carbon atoms. In certain embodiments, the conjugation group is selected from lipophilic moieties as described in WO2021092371A2. In certain embodiments, the saRNA may comprise one, two, three, four, five, six or even more oligonucleotides separately conjugated to one, two, three, four, five, six or even more of the conjugation groups via one, two, three, four, five, six or even more linking moieties.

According to an embodiment, the linking moieties, when present, can be selected from the group consisting of -O-, -S-, -C (O) -, -NH-, -N ( (C₁-C₁₂) alkyl) -, -N ( (C₁-C₁₂) alkyl) -C (O) -O-, -O-C (O) -, -C (O) -O-, -O-C (O) -O-, -C (O) -NH-, -OP (O) ₂O-, -P (O) (O^-) O-, -OP (O) O-, -OP (O) (S) O-, -O-S (O) ₂-O-, -S (O) ₂-O-, -S (O) -O-, - (C₁-C₂₂) alkylene-, - (C₁-C₂₂) alkylene-NH-, -NH- (C₁-C₂₂) alkylene-, - (C₁-C₂₂) alkylene-NH-C (O) -, - (C₁-C₂₂) alkylene-C (O) -, - (C₁-C₂₂) alkylene-C (O) -O-, -C (O) - (C₁-C₂₂) alkylene-, -NH-C (O) - (C₁-C₂₂) alkylene-, -C (O) -NH- (C₁-C₂₂) alkylene-, -C (O) - (C₁-C₂₂) alkylene-NH-, -NH- (C₁-C₂₂) alkylene-C (O) -, -C (O) - (C₁-C₂₂) alkylene-C (O) -, -NH- (C₁-C₂₂) alkylene-NH-, -C (O) - (C₁-C₂₂) alkylene-C (O) O-, -O-C (O) - (C₁-C₂₂) alkylene-C (O) -O-, -C (O) -O- (C₁-C₂₂) alkylene-O-C (O) -, -C (O) - (C₁-C₂₂) alkylene-NH-C (O) -, -NH-C (O) - (C₁-C₂₂) alkylene-C (O) -, -NH-C (O) - (C₁-C₂₂) alkylene-C (O) -NH-, -C (O) -NH- (C₁-C₂₂) alkylene-NH-C (O) -, - (C₁-C₂₂) alkylene-OP (O) ₂O-, - (C₁-C₂₂) alkylene-OP (O) (O^-) O-, - (C₁-C₂₂) alkylene-OP (O) (O^-) O- (C₁-C₂₂) alkylene-, - (C₁-C₂₂) alkylene-OP (O) O-, - (C₁-C₂₂) alkylene-OP (O) (S) O-, - (C₁-C₂₂) alkylene-O-S (O) ₂-O-, - (C₁-C₂₂) alkylene-S (O) ₂-O-, - (C₁-C₂₂) alkylene-S (O) -O-, -O-P (O) ₂-O- (C₁-C₂₂) alkylene-OP (O) ₂O-, -O-P (O) -O- (C₁-C₂₂) alkylene-OP (O) O-, -OP (O) (S) O- (C₁-C₂₂) alkylene-OP (O) (S) O-, -O-S (O) ₂-O- (C₁-C₂₂) alkylene-O-S (O) ₂-O-, -S (O) ₂-O- (C₁-C₂₂) alkylene-S (O) ₂-O-and -O-S (O) - (C₁-C₂₂) alkylene-S (O) -O-; wherein the - (C₁-C₂₂) alkylene-contained in the linking moiety can be an alkylene group comprising from 1 to 22 carbon atoms, such as from 2 to 20 carbon atoms, or from 3 to 18 carbon atoms, or from 4 to 16 carbon atoms, or from 5 to 12 carbon atoms, or from 6 to 10 carbon atoms. In one embodiment, the conjugation group is directly linked with the oligonucleotide when the linking moiety is a direct bond.

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

In some embodiments, the sense strand and the antisense strand 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%nucleotides which are chemically modified nucleotides.

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%nucleotides of the saRNA are chemically modified nucleotides.

These modifications can increase the bioavailability of the saRNA, improve affinity to a target sequence, and enhance resistance to nuclease hydrolysis in a cell.

In some embodiments, the saRNA of the present application which, upon contact with a cell, are effective in activating or up-regulating the expression of one or more genes in the cell, preferably by at least 10% (e.g., at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 300%, at least 500%, at least 800%, at least 1000%, at least 2000%, or at least 5000%) .

In a non-limiting example, a saRNA is designed based at least in part on the following criteria: (1) having a GC content between 35%and 65%; (2) with less than 5 consecutive identical nucleotides; (3) with 3 or less dinucleotide repeats; and (4) with 3 or less trinucleotide repeats. In some embodiments, a saRNA is design/selected based, at least in part, on criteria that enables production of functional saRNA. For example, in some cases, a sequence located upstream of a TSS may include a sequence that does not favor synthesis of a saRNA despite being located in a hotspot.

In some embodiments, a saRNA is designed/selected based, at least in part, on criteria that includes a sequence having a particular GC content (e.g., a GC content between 25%and 75%) and lacking consecutive identical nucleotides, consecutive dinucleotides, or consecutive trinucleotides. In some embodiments, a saRNA sequence comprises a sequence having a GC content between 35%and 65%; (2) with less than 5 consecutive identical nucleotides; (3) with 3 or less dinucleotide repeats; and (4) with 3 or less trinucleotide repeats.

In some embodiments, a saRNA sequence comprises a sequence having a GC content between 25%and 75%, between 30%and 70%, between 35%and 65%, between 40%and 60%, or between 45%and 55%. In some embodiments, the saRNA comprises a sequence having a GC context between 35%and 65%.

In some embodiments, a 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.

In some embodiments, a saRNA sequence comprises a sequence having 5 or less dinucleotide repeats, 4 or less dinucleotide repeats, 3 or less dinucleotide repeats, or 2 or less dinucleotide repeats. In some embodiments, the saRNA comprises a sequence having 3 or less dinucleotide repeats.

In some embodiments, a saRNA sequence comprises a sequence having 5 or less trinucleotide repeats, 4 or less trinucleotide repeats, 3 or less trinucleotide repeats, or 2 or less trinucleotide repeats. In some embodiments, the saRNA comprises a sequence having 3 or less trinucleotide repeats.

Target sequence

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 ranging from 16 to 35 nucleotides in the nucleotide sequence of 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 an antisense strand of the saRNA disclosed in the present application, and thus capable of activating the expression of CFH gene (e.g., mRNA expression, protein expression, CFH expression) . In some embodiments, the target site is selected based at least in part on a gene sequence. In some embodiments, the target site is selected based at least in part on a sequence close to a transcription starting site (TSS) of the gene. In some embodiments, the target site is selected based at least in part on a promoter sequence upstream of the TSS. In some embodiments, the target site is selected based at least in part on a sequence from -5000 bp, -4000bp, -3000 bp, -2000bp, -1000 bp or -500 bp upstream of the TSS. In some embodiments, the target site is selected at least in part by moving toward the TSS by 1 bp each time, and resulting in a target sequence, followed by repeating this step and increasing towards the TSS by an additional base pair (e.g., n +1) . In some embodiments, the target site has a length of about 8 to about 35 nucleotides. In some embodiments, the target site has a length of about 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 nucleotides.

In certain embodiments, the present application relates to an isolated oligonucleotide complex comprising the saRNA disclosed herein and the isolated target site disclosed in the present application. In certain embodiments, the isolated oligonucleotide complex activates the expression of CFH gene by at least 10% (e.g., activates expression of the CFH gene as compared to baseline CFH gene expression levels) .

Hotspot

In certain embodiments, the present application relates to an isolated nucleic acid sequence, or namely “hotspot” , located upstream of the transcription start site of CFH gene. In certain embodiments, isolated nucleic acid sequence disclosed herein is an oligonucleotide sequence having least 39 consecutive nucleotides in length and has at least 75%, or at least 80%, or at least 85%, or at least 90%sequence homology to an equal length region within the nucleotide sequence of SEQ ID NO: 1707. A “hotspot” herein is defined by a nucleic acid region on the target sequence of the saRNAs, where full length targets of functional saRNAs are enriched and spanned the very 5’ end of the first saRNA and the 3’ end of the last saRNA within the hotspot. In some embodiments, at least 40%(e.g., 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%or 100%) of the saRNAs designed to target hotspot is functional, i.e., can induce an at least 1.1-fold change in the mRNA expression of the target gene. In a non-limiting example, at least 43%of the saRNAs designed to the targeted hotspots is functional, i.e., can induce an at least 1.1-fold change in the mRNA expression of the target gene. In a non-limiting example, a saRNA is designed based at least in part on the following criteria: (1) having a GC content between 35%and 65%; (2) with less than 5 consecutive identical nucleotides; (3) with 3 or less dinucleotide repeats; and (4) with 3 or less trinucleotide repeats. In some embodiments, the same or similar criteria is used to select an isolated nucleic acid sequence and/or a target sequence. In a non-limiting example, an isolated nucleic acid sequence upstream of the CFH gene’s TSS is selected based at least in part on the following criteria: (1) having a GC content between 35%and 65%; (2) with less than 5 consecutive identical nucleotides; (3) with 3 or less dinucleotide repeats; and (4) with 3 or less trinucleotide repeats. In some embodiments, the isolated nucleic acid has about 25 to about 250 (e.g., about 33 to about 200, about 36 to about 150, about 39 to about 100, about 42 to about 75, about 45 to about 70, or about 48 to about 55) nucleotides in length. In some embodiments, a hotspot is a polynucleotide sequence selected from the group consisting of SEQ ID NO: 1708-1713. In some embodiments, a hotspot is a nucleic acid sequence selected from the group consisting of region -538 to -500, region -468 to -396, region -329 to -283, region -273 to -192, region -173 to -100 and region -64 to -14 upstream of the transcription start site of the CFH gene. The present application also provides a method of designing saRNA, said method provide saRNA targeting said isolated nucleic acid sequence of the present application.

In some embodiments, a target sequence is design/selected based, at least in part, on criteria that enables production of functional saRNA. For example, in some cases, a sequence located upstream of a TSS may include a sequence that does not favor synthesis of a target sequence despite being located in a hotspot.

In some embodiments, a target sequence within a hotspot is selected based, at least in part, on criteria that includes a sequence having a particular GC content (e.g., a GC content between 25%and 75%) and lacking consecutive identical nucleotides, consecutive dinucleotides, or consecutive trinucleotides. In some embodiments, a target sequence within a hotspot comprises a sequence having a GC content between 35%and 65%; (2) with less than 5 consecutive identical nucleotides; (3) with 3 or less dinucleotide repeats; and (4) with 3 or less trinucleotide repeats.

In some embodiments, a target sequence comprises a sequence having a GC content between 25%and 75%, between 30%and 70%, between 35%and 65%, between 40%and 60%, or between 45%and 55%. In some embodiments, the saRNA comprises a sequence having a GC context between 35%and 65%.

In some embodiments, a 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.

In some embodiments, a target sequence comprises a sequence having 5 or less dinucleotide repeats, 4 or less dinucleotide repeats, 3 or less dinucleotide repeats, or 2 or less dinucleotide repeats. In some embodiments, the target sequence comprises a sequence having 3 or less dinucleotide repeats.

In some embodiments, a target sequence comprises a sequence having 5 or less trinucleotide repeats, 4 or less trinucleotide repeats, 3 or less trinucleotide repeats, or 2 or less trinucleotide repeats. In some embodiments, the target sequence comprises a sequence having 3 or less trinucleotide repeats.

RNAa activity of each designed saRNA is depended on a complex myriad of factors, such as chromatin environments, sequence features of the target per se and nearby regions, transcriptional factor binding etc. The core underlying determinant may be accessibility of the DNA target. In the regions with higher accessibility, dsRNAs may show a higher activity of RNAa. While dsRNAs designed targeting other regions of the promotor may exhibit non-functional or even transcriptional silencing effect. This may explain the existing of hotspot regions where functional saRNAs are clustered together. For example, a target sequence designed based at least in part on the following criteria: (1) having a GC content between 35%and 65%; (2) with less than 5 consecutive identical nucleotides; (3) with 3 or less dinucleotide repeats; and (4) with 3 or less trinucleotide repeats may not activate/upregulate the expression of CFH gene by at least 10%as compared to baseline expression of the CFH gene because the target sequence that the saRNA binds to is not within a hotspot (e.g., any of hotspots described herein) .

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

In some aspects, methods of using the isolated nucleic acid upstream of the transcription target site of CFH gene is also provided.

DNA encoding saRNA

In certain embodiments, the present application relates to a nucleic acid or polynucleotide encoding the saRNA which can activate or upregulate the expression of CFH gene in a cell by at least 10% (e.g., as compared to 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 vectors disclosed herein comprise a fragment of DNA that encodes a saRNA of the present application.

Cell comprising saRNA

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

In certain embodiments, the present application relates to a cell comprising the 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 embryo liver cell, a human hepatoma cell (e.g., a Li-7 cell) , a human hepatoma cell (e.g., a Huh-7 cell) , a human hepatoma cell (e.g., an HepG2 cell) , or a cell from a mouse (e.g., mouse embryonic liver cell or embryonic cell line BNL. CL2) , a mouse liver cancer cell (e.g., LPC-H12) , and a primary mouse hepatocyte (PMH) . The cell disclosed herein may be in vitro, or ex vivo, such as a cell line or a cell strain, or may exist in a mammalian body, 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, low CFH level, and/or insufficient levels of functional CFH protein in plasma. In some embodiments, the cell is from a patient of suffering CFHD.

Composition comprising saRNA

In certain embodiments, the present application relates to a composition or pharmaceutical composition comprising the saRNA or the nucleic acid of the present application. In some embodiments, the composition comprises at least one pharmaceutically acceptable carrier. In some embodiments, the composition comprising at least one pharmaceutically acceptable carrier selected from an aqueous carrier, liposome or LNP, polymer, micelle, colloid, metal nanoparticle, non-metallic nanoparticle, bioconjugate (e.g., GalNAc) , polypeptide and antibody. In one embodiment, the aqueous carrier may be, for example, RNase-free water, or RNase-free buffer. In some embodiments, the composition may contain 0.001-150 nM (e.g., 0.01-100 nM, 0.1-50 nM, 1-150 nM, 1-20 nM, 0.001-1 nM, 1-10 nM, 10-100 nM, 10-50 nM, 20-50 nM, 20-100 nM) of the saRNA or isolated polynucleotide as described herein. In some embodiments, the composition includes 50 nM of the saRNA or isolated polynucleotide as described herein.

Methods of using saRNA

Another aspect of the present application relates to a saRNA for activating/upregulating the CFH gene expression in a cell. The saRNA comprises an oligonucleotide sequence having a length of 16 to 35 consecutive nucleotides. In some embodiments, the oligonucleotide sequence has at least 75%, or at least 80%, or at least 85%, or at least 90%homology or complementary to an equal length region of SEQ ID NO: 1707, specifically, the saRNA activates/up-regulates 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%as compared to baseline expression of the CFH gene) . In certain embodiments, upon administering the saRNA disclosed in the embodiments, e.g., to a cell or a subject, the expression of the CFH gene is activated/up-regulated by at least 5 fold (e.g., at least 6 fold, at least 7 fold, at least 8 fold, at least 9 fold, at least 10 fold, at least 12 fold, or at least 14 folds compared to baseline expression of the CFH gene) . In certain embodiments, an saRNA activates or upregulates the expression of the CFH gene by about 14-fold. In certain embodiments, the expression of CFH gene is activated/up-regulated by administering the saRNA disclosed in the embodiments to a cell at a concentration of at least 0.01 nM, e.g., 0.02 nM, 0.05 nM, 0.08 nM, 0.1 nM, 0.2 nM, 0.3 nM, 0.4 nM, 0.5 nM, 0.6 nM, 0.8 nM, 1 nM, 5 nM, 10 nM, 25 nM, 50 nM, 75 nM, 100 nM, or 150 nM. In certain embodiments, the induction of CFH gene protein (CFH) is activated/up-regulated by administering the saRNA disclosed in the embodiments to a cell at a concentration of at least 0.01 nM, e.g., 0.02 nM, 0.05 nM, 0.08 nM, 0.1 nM, 0.2 nM, 0.3 nM, 0.4 nM, 0.5 nM, 0.6 nM, 0.8 nM, 1 nM, 2 nM, 3 nM, 4nM, 5 nM, 10 nM, 25 nM, 50 nM, 75 nM, 100 nM, or 150 nM.

Another aspect of the present application relates to a method for preventing or treating a disorder or condition induced by insufficient expression of plasma complement factor H (CFH) , a CFH gene mutation, and/or low functional CFH levels in plasma in an individual comprising: administering an effective amount of the saRNA, the nucleic acid or isolated polynucleotide encoding the saRNA, or the composition comprising the saRNA disclosed herein to the individual. in certain embodiments, the effective amount of the saRNA disclosed herein can be a concentration ranging from 0.01 nM to 50 nM, e.g., 0.01 nM, 0.02 nM, 0.05 nM, 0.08 nM, 0.1 nM, 0.2 nM, 0.3 nM, 0.4 nM, 0.5 nM, 0.6 nM, 0.8 nM, 1 nM, 5 nM, 10 nM, 25 nM, 50 nM, 75 nM, 100 nM, or 150 nM. In some embodiments, the disorder or condition is CFHD. In some embodiments, the individual is a mammal. In some embodiments, the individual is a human.

In any of the embodiments provided herein, such saRNA, nucleic acids encoding the saRNA of the present application, or compositions comprising such saRNA of the present application may be introduced directly into a cell, or may be produced intracellularly upon introduction of a nucleotide sequence encoding the saRNA into a cell, for example a mammalian cell including, but not limited to, Li-7, Huh-7 and PMH, or a human cell. Such cells may be ex vivo, such as cell lines, and the like, or may be present in mammalian bodies, such as humans. In some embodiments, the human is a patient or individual suffering from a CFH-deficiency-related condition or CFHD. In certain embodiments, a nucleic acid or an isolated polynucleotide encoding an saRNA or a composition comprising the aforementioned saRNA as described herein, in respective amounts sufficient to treat CFHD.

Another aspect of the present application relates administering an effective mount of the saRNA or the composition to an individual using administration pathway as described herein. In some embodiments, the administration pathway is selected from one or more of: parenteral infusions, oral administration, intranasal administration, inhaled administration, vaginal administration, and rectal administration. In some embodiments, the administration pathway is selected from one or more of: intrathecal, intramuscular, intravenous, intra-arterial, intraperitoneal, intravesical, intracerebroventricular, intravitreal and subcutaneous administrations.

Dose regiments and route of administration

Aspects of the present application relate to a pharmaceutical composition comprising the saRNA of the present application. In some embodiments, the pharmaceutical composition comprising the saRNA of the present application and a pharmaceutically acceptable carrier, a therapeutically inert carrier, diluent or pharmaceutically acceptable excipient. The pharmaceutical composition disclosed herein is to be developed into a medicament preventing or treating the CFH-deficiency-related condition or CFHD.

Aspects of the present application also relate to methods of using the saRNAs of the present application to prepare such compositions.

Another aspect of the present application relates to use of the saRNA of the present application in manufacturing the pharmaceutical composition disclosed herein.

Another aspect of the present application relates to use of the saRNA or an isolated polynucleotide, according to any one of the embodiments described herein, or a composition according to any one of the embodiments described herein, in the manufacture of a medicament for the prevention or treatment of gene or protein-related symptom induced by the insufficient expression of CFH protein, a CFH gene mutation, and/or low functional CFH levels in plasma in an individual. The use according to certain embodiments, the condition can include a CFH gene-mutation-related disorder or condition that comprises a CFHD. The use according to certain embodiments, the symptom induced by insufficient expression of CFH protein is aHUS, DDD, C3GN, CFHR5 nephropathy, LN, MPGN1, MPGN2, MPGN3, or AMD. Also related is the use according to certain embodiments wherein the individual is a mammal, for example a human.

The dosage at which the saRNAs or compositions of the present application can be administered can vary within wide limits and will be fitted to the individual requirements in each case. In certain embodiments, a first dose of a pharmaceutical composition according to the present application is administered when the subject is less than one week old, less than one month old, less than 3 months old, less than 6 months old, less than one-year-old, less than 2 years old, less than 15 years old, or older than 15 years old.

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

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

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

In certain embodiments, pharmaceutical compositions comprise a co-solvent system. Certain of such co-solvent systems comprise, for example, benzyl alcohol, a nonpolar surfactant, a water-miscible organic polymer, and an aqueous phase. In certain embodiments, such co-solvent systems are used for hydrophobic compounds. A non-limiting example of such a co-solvent system is the VPD co-solvent system, which is a solution of absolute ethanol comprising 3%w/v benzyl alcohol, 8%w/v of the nonpolar surfactant Polysorbate 80^TM and 65%w/v polyethylene glycol 300. The proportions of such co-solvent systems may be varied considerably without significantly altering their solubility and toxicity characteristics. Furthermore, the identity of co-solvent components may be varied: for example, other surfactants may be used instead of Polysorbate 80^TM; the fraction size of polyethylene glycol may be varied; other biocompatible polymers may replace polyethylene glycol, e.g., polyvinyl pyrrolidone; and other sugars or polysaccharides may substitute for dextrose.

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, drying agents, antimicrobials, needles, syringes, packaging materials, tubes, bottles, flasks, beakers, and the like, for example, for using, modifying, assembling, storing, packaging, preparing, mixing, diluting, and/or preserving the components for a particular use. In embodiments where liquid forms of any of the components are used, the liquid form may be concentrated or ready to use.

In some embodiments, lipid moieties used in nucleic acid therapies can be applied in the present application for delivery of the saRNA molecules disclosed herein. In such methods, the nucleic acid (e.g., one or more saRNAs described herein) is introduced into preformed liposomes or lipoplexes made of mixtures of cationic lipids and neutral lipids. In certain methods, saRNA complexes with mono-or poly-cationic lipids are formed without the presence of a neutral lipid. In certain embodiments, a lipid moiety is selected to increase distribution of a pharmaceutical agent to a particular cell or tissue. In certain embodiments, a lipid moiety is selected to increase distribution of a pharmaceutical agent to fat tissue. In certain embodiments, a lipid moiety is selected to increase distribution of a pharmaceutical agent to muscle tissue.

In certain embodiments, pharmaceutical compositions comprise a delivery system. Examples of delivery systems include, but are not limited to, liposomes and emulsions. Certain delivery systems are useful for preparing certain pharmaceutical compositions including those comprising hydrophobic compounds. In certain embodiments, certain organic solvents such as dimethylsulfoxide are used.

In certain embodiments, pharmaceutical compositions comprise one or more tissue-specific delivery molecules designed to deliver the one or more pharmaceutical agents of the present invention to specific tissues or cell types. For example, in certain embodiments, pharmaceutical compositions include liposomes coated with a tissue-specific antibody.

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

In some embodiments, the vector is a recombinant AAV vector (rAAV) . AAV vectors are DNA viruses of relatively small size that can integrate, in a stable and site-specific manner, into the genome of the cells that they infect. They are able to infect a wide spectrum of cells without inducing any effects on cellular growth, morphology or differentiation, and they do not appear to be involved in human pathologies. The AAV genome has been cloned, sequenced and characterized. It encompasses approximately 4700 bases and contains an inverted terminal repeat (ITR) region of approximately 145 bases at each end, which serves as an origin of replication for the virus. The remainder of the genome is divided into two essential regions that carry the encapsidation functions: the left-hand part of the genome, that contains the rep gene involved in viral replication and expression of the viral genes; and the right-hand part of the genome, that contains the cap gene encoding the capsid proteins of the virus.

AAV vectors may be prepared using standard methods in the art. Adeno-associated viruses of any serotype are suitable (see, e.g., Blacklow, pp. 165-174 of ″Parvoviruses and Human Disease″ J. R. Pattison, ed. (1988) ; Rose, Comprehensive Virology 3: 1, 1974; P. Tattersall ″The Evolution of Parvovirus Taxonomy″ In Parvoviruses (J R Kerr, S F Cotmore. M E Bloom, R M Linden, C R Parrish, Eds. ) p 5-14, Hudder Arnold, London, UK (2006) ; and D E Bowles, J E Rabinowitz, R J Samulski ″The Genus Dependovirus″ (J R Kerr, S F Cotmore. M E Bloom, R M Linden, C R Parrish, Eds. ) p 15-23, Hudder Arnold, London, UK (2006) , the disclosures of which are hereby incorporated by reference herein in their entireties) . Methods for purifying for vectors may be found in, for example, U.S. Pat. Nos. 6,566,118, 6,989,264, and 6,995,006 and WO/1999/011764 titled ″Methods for Generating High Titer Helper-free Preparation of Recombinant AAV Vectors″ , the disclosures of which are herein incorporated by reference in their entirety. Preparation of hybrid vectors is described in, for example, PCT Application No. PCT/US2005/027091, the disclosure of which is herein incorporated by reference in its entirety. The use of vectors derived from the AAVs for transferring genes in vitro and in vivo has been described (See e.g., 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 herein incorporated by reference in their entirety) . These publications describe various AAV-derived constructs in which the rep and/or cap genes are deleted and replaced by a gene of interest, and the use of these constructs for transferring the gene of interest in vitro (into cultured cells) or in vivo (directly into an organism) . The replication defective recombinant AAVs (rAAV) according to the application can be prepared by co-transfecting a plasmid containing the nucleic acid sequence of interest flanked by two AAV inverted terminal repeat (ITR) regions, and a plasmid carrying the AAV encapsulation genes (rep and cap genes) , into a cell line that is infected with a human helper virus (for example an adenovirus) . The AAV recombinants that are produced are then purified by standard techniques.

In some embodiments, the vector (s) for use in the methods of the application are encapsulated into a virus particle (e.g., AAV virus particle including, but not limited to, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, and AAV16) . Accordingly, the application may include a recombinant virus particle (recombinant because it contains a recombinant polynucleotide) comprising any of the vectors described herein. Methods of producing such particles are known in the art and are described in U.S. Pat. No. 6,596,535.

Preparations, pharmaceutical compositions, or medicaments of the present application are formulated, dosed, and administered in a fashion consistent with good medical practice. Factors for consideration in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of administration, the scheduling of administration, and other factors known to medical practitioners.

For the preparations, pharmaceutical compositions, or medicaments of the present application, the delivery can be optionally through parenteral infusions including intrathecal, intramuscular, intravenous, intra-arterial, intraperitoneal, intravesical, intracerebroventricular, intravitreal or subcutaneous administration; or through oral administration, intranasal administration, inhaled administration, vaginal administration, or rectal administration.

A typical formulation of the oligonucleotide modulator in the present application is prepared by mixing a saRNA of the present application and a carrier or excipient. Suitable carriers and excipients are well known to those skilled in the art and are described in detail in, e.g., Ansel H. C. et al., Ansel′s Pharmaceutical Dosage Forms and Drug Delivery Systems (2004) Lippincott, Williams &Wilkins, Philadelphia; Gennaro A. R. et al., Remington: The Science and Practice of Pharmacy (2000) Lippincott, Williams &Wilkins, Philadelphia; and Rowe R. C, Handbook of Pharmaceutical Excipients (2005) Pharmaceutical Press, Chicago. The formulations may also include one or more buffers, stabilizing agents, surfactants, wetting agents, lubricating agents, emulsifiers, suspending agents, preservatives, antioxidants, opaquing agents, glidants, processing aids, colorants, sweeteners, perfuming agents, flavoring agents, diluents and other known additives to provide an elegant presentation of the drug (i.e., a saRNA of the present application or pharmaceutical composition thereof) or aid in the manufacturing of the pharmaceutical product (i.e., medicament) .

Method of diagnosis

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 a cell transfected with the saRNA, the isolated polynucleotide, or the composition comprising the saRNA as disclosed herein. In certain embodiments, the method disclosed herein can be applied in detecting a specific sub-group of patients suffering a disorder or condition induced by insufficient expression of plasma complement factor H (CFH) protein, a CFH gene mutation, and/or low functional CFH levels in plasma. As alternative embodiments of the method disclosed herein, the method can be used in efficacy or safety monitoring of the aforementioned patients treated by the saRNA, nucleic acid or isolated polynucleotide encoding the saRNA, composition, or medicament of the present application.

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

In some embodiments, the saRNA provided herein activates the amount of functional CFH protein in plasma as compared to the baseline measurement aforementioned, 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 some embodiments, the saRNA shows a greater than additive effect or synergy in the treatment, prevention, delaying progression and/or amelioration of diseases caused by the CFH gene mutation. In some embodiments, the saRNA shows a greater than additive effect or synergy in the protection of cells implicated in the pathophysiology of the disease, particularly for the treatment, prevention, delaying progression and/or amelioration CFHD.

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

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

Kit

Another aspect of the present application relates to a kit for performing the method for increasing a level of CFH protein in a cell or a level of functional CFH protein in plasma, comprising the saRNA disclosed herein. In certain embodiments, the kit further comprises means for administering said saRNA to an individual. In certain embodiments, the kit is in a labeled package and the label on said package indicates that the saRNA or the composition can be used in preventing or treating a disease or condition induced by insufficient expression of plasma complement factor H (CFH) , or against CFHD.

A″kit″ as used herein, typically defines a package, assembly, or container (such as an insulated container) including one or more of the components or embodiments of the application, and/or other components associated with the application, for example, as previously described. Any of the agents or components of the kit may be provided in liquid form (e.g., in solution) , or in solid form (e.g., a dried powder, frozen, etc. ) .

In additional embodiments, a kit can include instructions or instructions to a website or other source in any form that are provided for using the kit in connection with the components and/or methods described herein. For instance, the instructions may include instructions for the use, modification, mixing, diluting, preserving, assembly, storage, packaging, and/or preparation of the components and/or other components associated with the kit. In some cases, the instructions may also include instructions for the delivery of the components, for example, for shipping or storage at room temperature, sub-zero temperatures, cryogenic temperatures, etc. The instructions may be provided in any form that is useful to the user of the kit, such as written or oral (e.g., telephonic) , digital, optical, visual (e.g., videotape, DVD, etc. ) and/or electronic communications (including Internet or web-based communications) , provided in any manner.

Another aspect of the present application relates to a kit for detecting CFH protein or CFH regulated protein in plasma. In certain embodiments, the kit is for detecting CFH protein or CFH regulated protein in a cell transfected with any one or more of the saRNA disclosed herein, or the isolated polynucleotide, or the composition disclosed herein. Also provided herein is a kit for increasing a level of CFH protein in a cell.

saRNA synthesis

The present application provides a method for preparing the oligonucleotide modulator (saRNA) , which comprises sequence design and synthesis.

saRNAs can be chemically synthesized or can be obtained from a biotechnology company specialized in nucleic acid synthesis. Generally speaking, chemical synthesis of nucleic acids comprises the following four steps: a) synthesis of oligomeric ribonucleotides; b) deprotection; c) purification and isolation; d) desalination and annealing. For example, the specific steps for chemically synthesizing saRNAs described are as follows:

Synthesis of oligomeric ribonucleotides:

Synthesis of 1 μM RNA was set in an automatic DNA/RNA synthesizer (e.g.,

Applied Biosystems EXPEDITE8909) , and the coupling time of each cycle was set as 10 to 15 min. With a solid phase-bonded 5′-O-p-dimethoxytriphenylmethyl-thymidine substrate as an initiator, one base was bonded to the solid phase substrate in the first cycle, and then, in the nth (19 ≥ n ≥ 2) cycle, one base was bonded to the base bonded in the n-1th cycle. This process was repeated until the synthesis of the whole nucleic acid sequence was completed.

Deprotection

The solid phase substrate bonded with the saRNA was put into a test tube, and 1 mL of a solution of the mixture of ethanol and ammonium hydroxide (volume ratio: 1: 3) was added to the test tube. The test tube was then sealed and placed in an incubator, and the mixture was incubated at 25-70 ℃ for 2 to 30 h. The solution containing the solid phase substrate bonded with the saRNA was filtered, and the filtrate was collected. The solid phase substrate was rinsed with double distilled water twice (1 mL each time) , and the filtrate was collected. The collected eluents were combined and dried under vacuum for 1 to 12 h. Then the solution was added with 1 mL of a solution of tetrabutylammonium fluoride in tetrahydrofuran (1 M) , let stand at room temperature for 4 to 12 h, followed by addition of 2 mL of n-butanol. Precipitate was collected to give a single stranded crude product of saRNA by high-speed centrifugation.

Purification and isolation

The resulting crude product of saRNA was dissolved in 2 mL of aqueous ammonium acetate solution with a concentration of 1 mol/mL, and the solution was separated by a reversed phase C18 column of high-pressure liquid chromatography to give a purified single-stranded product of saRNA.

Desalination and annealing

Salts were removed by gel filtration (size exclusion chromatography) . A single sense oligomeric ribonucleic acid strand and a single antisense oligomeric ribonucleic acid strand were mixed into 1 to 2 mL of buffer (10 mM Tris, pH = 7.5-8.0, 50 mM NaCl) at a molar ratio of 1: 1. The solution was heated to 95 ℃, and was then slowly cooled to room temperature to give a solution containing saRNA.

Cell culture and treatment

Human hepatocarcinoma Li-7 (TCHu183, Shanghai Institutes for Biological Sciences, China) cells were cultured at 37℃ with 5%CO₂ in modified RPMI1640 medium (Gibco, Thermo Fisher Scientific, Carlsbad, CA) supplemented with 10%bovine calf serum (Sigma-Aldrich) and 1%penicillin/streptomycin (Gibco) . Human hepatocarcinoma Huh-7 (JCRB0403, Cobioer Biosciences CO. LTD, China) cells were cultured at 37℃ with 5%CO₂ in modified DMEM medium (Gibco, Thermo Fisher Scientific, Carlsbad, CA) supplemented with 10%bovine calf serum (Sigma-Aldrich) and 1%penicillin/streptomycin (Gibco) . Mouse primary hepatocytes (PMH) were isolated from the liver of C57BL/6 mice (Beijing Vital River Laboratory Animal Technology Co., Ltd, China) . PMH cells were cultured under the conditions of 5%CO₂ and 37℃ in modified Willian’s Medium E (WME) medium (A12176-01, Gibco, Thermo Fisher Scientific, Carlsbad, CA) supplemented with 1%Insulin (S6955, Selleck, US) and 1%penicillin/streptomycin (Gibco) . The Li-7 and PMH cells were seeded into 96-well plates at 4000 cells/well, respectively. saRNAs were individually transfected into the Li-7 cells in each well at a final concentration of 2.5 nM, 10 nM or 25 nM, or any other concentrations with 0.3 μL of RNAiMAX (Invitrogen, Carlsbad, CA) by following the reverse transfection protocol respectively, and the transfection duration was 72 hours. Mock (blank control) was transfected in the absence of an oligonucleotide. dsCon2 (SEQ ID NO: 617, 926) was served as a non-specific duplex control. DS16A-si5 (SEQ ID NO: 616, 925) was a duplex siRNA for CFH gene and transfected as a negative control. DS16B-si6 (SEQ ID NO: 1310, 1694) was a duplex siRNAs for Cfh gene and transfected as a negative control.

RT-qPCR and ELISA Assays

(1) One-step reverse transcription-quantitative polymerase chain reaction (one-step RT-qPCR)

At the end of transfection, medium was discarded and cells were washed with 150 μL of PBS once per well. After discarding the PBS, 100 μL of cell lysis buffer (Power Green Cellsto-Ct^TM Kit, Life Technologies) was added into each well and incubated at room temperature for 5 min. 0.5 μL of the cell lysis was taken from each well and analyzed by RT-qPCR using One Step TB GreenTM PrimeScripTM RT-PCR kit II (Takara, RR086A) in a Roche Lightcycler 480 real-time PCR machine (Roche, ref: 4729749001, US) . PCR reactions was prepared using Bravo Automated Liquid Handling Platform (Agilent) . Each transfection sample was amplified in 3 repeat wells. PCR reaction conditions are shown in Table 3.

Table 3. PCR reaction preparation

The reaction conditions were as follows: reverse transcription reaction (stage 1) : 42℃ for 5 min, 95℃ for 10 sec; PCR reaction (stage 2) : 95℃ for 5 sec, 59℃ for 20 sec, 72℃ for 10 sec; 40 cycles of amplification; 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 means was used as an internal control for RNA loading. Mouse reference genes (Hmbs and Tbp) were also amplified and their geometric means was used as an internal control for RNA loading. All primer sequences are listed in Table 4.

Table 4. Primer sequences for RT-qPCR assay

(2) Two-step RT-qPCR

For the sake of quantifying mRNA expression in cells, total cellular RNA was isolated from treated cells using a RNeasy Plus Mini kit (Qiagen, Hilden, Germany) according to its manual. The resultant RNA (1 μg) was reverse transcribed into cDNA by using a PrimeScript RT kit containing gDNA Eraser (Takara, Shlga, Japan) . The resultant cDNA was amplified in a Roche LightCycler 480 Multiwell Plate 384 (Roche, ref: 4729749001, US) using SYBR Premix Ex Taq II (Takara, Shlga, Japan) reagents and primers which specifically amplified target genes of interest. Reaction conditions were as follows: reverse transcription reaction (stage 1) : 42℃ for 5 min, 95℃ for 10 sec; PCR reaction (stage 2) : 95℃ for 5 sec, 60℃ for 30 sec, 72℃ for 10 sec; 40 cycles of amplification; Melting curve (stage 3) . PCR reaction conditions are shown in Table 5 and Table 6.

Table 5: RT reaction

Table 6: RT-qPCR reaction

To calculate the expression level (E_rel) of CFH or Cfh (target genes) in an saRNA-transfected sample relative to control treatment (Mock) , the Ct values of the target gene and the two internal reference genes were substituted into formula 1,
E_rel=2 ^(CtTm-CtTs) / ( (2 ^{(CtR1m-CtR1s)} *2 ^{(CtR2m-CtR2s)} ) ^(1/2) )
(Formula 1)

wherein CtT_m was the Ct value of the target gene from the mock-treated sample; CtT_s was the Ct value of the target gene from the saRNA-treated sample; CtR1_m was the Ct value of the internal reference gene 1 from the mock-treated sample; CtR1_s was the Ct value of the internal reference gene 1 from the saRNA-treated sample; CtR2_m was the Ct value of the internal reference gene 2 from the mock-treated sample; and CtR2_s was the Ct value of the internal reference gene 2 from the saRNA treated sample.

(3) ELISA assay

To quantifying human CFH protein expression levels, supernatant of cultivated cells (e.g., Li-7 and Huh-7) were collected and detected by OD value using an ELISA kit (Signalway Antibody, Catalog: EK2066, USA) . The human CFH protein expression levels were conducted according to the following ELISA kit instructions provided by the manufacturer of the kits.

(4) Statistical analysis

Differences between groups of continuous variables were compared using one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparisons. A P value less than 0.05 of were considered significantly different. */#p < 0.05, **/##p < 0.01, ***/###p < 0.001, ****/####p < 0.0001.

Animal procedures

All animal procedures were conducted by certified laboratory personnel using protocols consistent with local and state regulations and approved by the Institutional Animal Care and Use Committee. C57BL/6J mice (B204, Beijing, China) purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd were administered by subcutaneous (SC) injection.

Primary mouse hepatocyte (PMH) isolation

C57BL/6J mice (Beijing Vital River Laboratory Animal Technology Co., Ltd. ) were anesthetized with isoflurane and perfused by initial flushing reagent and digestion reagent successively. The liver was placed into 10 cm dish and teared apart using forceps in culture medium. The cell suspension was collected by filtering through a 70-75-micron membrane in 50 mL conical tube, followed by centrifuging at 4℃ for 2 minutes at 100 × g in a swinging-arm centrifuge. 20 mL cold PBS was pipetted to wash cells after removing the supernatant (Repeat this step twice) . Cells with at least 80%viability which was tested using 0.4%trypan blue were allowed to proceed the assay. Cells were seeded to the cell culture plates which coated collagen I 4 ～ 12 hours in advance, yield a final confluence of 90-95%and started the assay.

It was discovered in this application that after being introduced into a cell, the aforementioned saRNA can effectively increase the expression level of CFH and Cfh mRNA and protein.

The present application will be further illustrated with reference to specific examples and drawings below. It should be understood that these examples are merely intended to illustrate the present application rather than limit the scope of the present application. In the following examples, study methods without specific conditions were generally in accordance with conventional conditions, such as conditions described in Sambrook, et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989) , or conditions recommended by the manufacturer.

EXAMPLES

Example 1: Design and synthesis of saRNAs targeting the human CFH promoter

Coding strand sequence from the promoter of the human CFH gene was retrieved from the UCSC genome database (SEQ ID NO: 1707) . It consisted of a 580 nucleotide sequence ranging from position -1 bp to upstream -580 bp relative to the transcription start site (TSS) (Table 7) .

Table 7. Putative human CFH promoter sequence (5′-3′) (SEQ ID NO: 1707)

A total of 307 small activating RNA (saRNA) duplexes targeting the promoter sequence were synthesized at 19 bp in length according to the following criteria: (i) GC content between 35-65%; (ii) less than 5 consecutive identical nucleotides; (iii) 3 or less total dinucleotide repeats; and (iv) 3 or less total trinucleotide repeats. Dual dTdT overhangs were also added to the 3’ end of each strand for a total length of 21 nts. Strand composition and sequence of each saRNA duplex including cognate target site in the CFH promoter is listed in Table 1.

Example 2: High throughput screening of saRNAs targeting the human CFH promoter

To identify saRNAs capable of upregulating CFH, Li-7 cells were transfected with each of the aforementioned saRNAs at 25 nM concentrations for 72 hours followed by gene expression analysis via one-step RT-qPCR. A non-specific duplex (i.e., dsCon2) served as a non-specific duplex control, while an siRNA (i.e., DS16A-si5) targeting human CFH transcript was used as a negative control for transfection efficiency. Out of the 307 saRNAs, 44 (14.3%) , 53 (17.3%) and 35 (11.4%) saRNAs showed high (≥1.5 fold) , moderate (1.2～1.5 fold) , and

Table 8. Summary of saRNA screen for CFH expression

Whereas 175 (57.0%) saRNAs either reduced or had no obvious impact on CFH levels. Relative changes in CFH expression caused by saRNA treatment are also summarized in Table 1, while expression data organized by gene induction is plotted in FIG. 1. mild activation (1.1～1.2 fold) of CFH expression, respectively (Table 8) . Sorting expression data by target site location within the human CFHpromoter revealed six “hotspots” that were enriched for saRNA activity 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 (FIG. 2) . Nearly 53%of all functional saRNAs targeted sequence within the indicated “hotspots” . Promoter sequence corresponding to each “hotspot” is listed in Table 7.

By following the design criteria: (i) GC content between 35-65%; (ii) less than 5 consecutive identical nucleotides; (iii) 3 or less total dinucleotide repeats; and (iv) 3 or less total trinucleotide repeats, at least 30%of designed saRNA targeting the provided hotspot sequences can activate the expression of CFH gene by at least 10%.

saRNA duplexes capable of upregulating human CFH expression by 1.1-fold or higher in “hotspot region” H1 (-538 to -500) were 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.

saRNA duplexes capable of upregulating human CFH expression by 1.1-fold or higher in “hotspot region” H2 (-468 to -396) were 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.

saRNA duplexes capable of upregulating human CFH expression by 1.1-fold or higher in “hotspot region” H3 (-329 to -283) were 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.

saRNA duplexes capable of upregulating human CFH expression by 1.1-fold or higher in “hotspot region” H4 (-273 to -192) were 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-141, 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, DS16A-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.

saRNA duplexes capable of upregulating human CFH expression by 1.1-fold or higher in “hotspot region” H5 (-173 to -100) were 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.

saRNA duplexes capable of upregulating human CFH expression by 1.1-fold or higher in “hotspot region” H6 (-64 to -14) were as follows: DS16A-266, DS16A-267, DS16A-268, DS16A-269, DS16A-270, DS16A-271, DS16A-272, DS16A-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-287, 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

Based on the screening results for CFH induction, the top 48 performing saRNAs (see Table 1) were transfected into Li-7 cells at 3 concentrations (i.e., 2.5, 10, and 25 nM) for 72 hours to quantify dose-dependent gene induction via one-step RT-qPCR. Both dsCon2 and DS16A-si5 served as a non-specific duplex control for gene activation and negative control for transfection efficacy, respectively. As shown in FIG. 3, most of the saRNAs demonstrated dose-dependent induction of CFH with the highest levels of activity reaching 9.5-fold by DS16A-274 at the 25 nM treatment concentration. The most potent duplexes included 42 saRNAs (87.5%) that exhibited ≥1.5-fold increases in CFH levels at the lowest concentration (i.e., 2.5 nM) in which 13 (27.1%) activated CFH by over 3-fold.

Example 4: saRNA treatment increases secreted CFH protein levels in Li-7 cells

A subset of the top performers (23 saRNAs in total) were transfected into Li-7 cells at 25 nM concentrations to quantify secretion of CFH protein in media after 72 hours via ELISA. Both dsCon2 and DS16A-si5 served as a non-specific duplex control for gene activation and negative control for transfection efficacy, respectively. As shown in FIG. 4, each saRNA had a measured increase in CFH protein levels after 72 hours treatment compared to baseline (dotted line) in which 9 duplexes recorded statistical significance.

Example 5: Correlation between CFH protein and mRNA induction by saRNA treatment in human hepatocellular carcinoma cell lines

Both CFH mRNA upregulation and protein secretion were evaluated in two hepatocarcinoma cell lines (i.e., Li-7 and Huh-7) using 10 saRNAs (i.e., DS16A-011, -013, -038, 055, -144, -135, -153, -170, -178, and -199) . Each saRNA was transfected at 10 nM concentrations for 4 days in which mRNA expression and CFH protein levels were quantified in the same samples by two-step RT-qPCR and ELISA, respectively. Treatment with dsCon2 served as a non-specific duplex control for gene activation, while DS16A-si5 served as negative control for transfection efficacy and comparison for CFH mRNA and protein knockdown. Both Li-7 (FIG. 5A) and Huh-7 (FIG. 5B) cells shared the same general pattern in which mRNA induction via saRNA treatment correlated with a measurable increase in secreted protein. While relative fold-change in protein levels were generally not as high as mRNA induction, this was likely a result in delayed kinetics in protein translation and secretion compared to transcriptional activation. In support, knockdown of CFH mRNA by DS16A-si5 also correlated with a delay in maximal suppression of protein levels (FIG. 5A-B) . 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 detected.

Example 6: saRNA potency for CFH gene activation in Li-7 cells

Dose response curves were generated via two-step RT-qPCR for 4 of the top saRNAs (i.e., DS16A-013, DS16A-135, DS16A-199 and DS16A-055) 72 hours after treatment using 11 escalating 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 potency (FIG. 6A-D) . Each saRNA demonstrated dose-dependent activation of CFH expression with EC₅₀ values in the low nanomolar range (i.e., ～1.18-2.8 nM) as summarized in Table 11.

Table 11. EC₅₀ values for activating CFH expression in vitro

Example 7. Impact of saRNA structure and sequence on saRNA activity in vitro

To assess impact of duplex structure and sequence specificity on saRNA activity, a series of saRNA variants were synthesized based on 2 of best performers (i.e., DS16A-135 and DS16A-013) for activating human CFH expression. Table 12 lists the sequence composition and design for each saRNA variant. Each duplex was transfected into PLC/PRF/5 or Huh-7 cells for 3 days at 25 nM concentrations and mRNA expression was analyzed via two-step RT-qPCR. Treatment with dsCon2 and DS16A-si5 served as a non- specific duplex control for gene activation and negative control for transfection efficiency, respectively. As shown in FIG. 7A-B, shortening DS16A-135 duplex length to 16 bp (i.e., RD-14694) inactivated saRNA induction of CFH mRNA, while extending duplex length to 20 bp with asymmetric overhangs (i.e., RD-14696) or removal of the overhangs on the 18 bp duplex (i.e., RD-14695) did not significantly impact saRNA activity. However, further extending duplex length to 28 bp with asymmetric overhangs (i.e., RD-14697) notably reduced CFH induction in at least Huh-7 cells (FIG. 7B) . Mutation of the “seed” region on the antisense in DS16A-135 (i.e., RD-14698) only interfered maximal gene induction in PLC/PRF/5 cells, while retained function in Huh-7 cells (FIG. 7A-B) . However, mutation to the “seed” region on the sense strand (i.e., RD-14699) reduced maximal activity in Huh-7 cells implying the sense strand is preferably responsible for gene activation in this cell line (FIG. 7B) . Modifying overhang length to 1, 3, or 5 nts (i.e., RD-14700, RD-14701, and RD-14702, respectively) on the sense strand in asymmetric duplexes performed similarly to the dual nucleotide overhang saRNA (i.e., RD-14696) (FIG. 7B) .

In PLC/PRF/5 cells, DS16A-013 (i.e., RD-11598) did not significantly activate CFH expression; however, extending duplex length to 23 bp with asymmetric overhangs on the sense strand (i.e., RD-14705) did measure a ～1.75-fold increase in mRNA levels (FIG. 7A) . DS16A-013 (i.e., RD-11598) treatment in Huh-7 increased expression levels by nearly 2-fold (FIG. 7B) . Only reducing duplex length to 16 bp (i.e., RD-14703) , extending duplex length to 33 bp (i.e., RD-14708) , or mutating the “seed” region on the antisense strand (i.e., RD-14707) decreased saRNA activity in Huh-7 cells.

Taken together, results indicate that saRNA duplex length can impact gene induction activity in which lengths ≤16 bp or ≥33 bp nucleotide sequences are significantly impaired. Furthermore, mutation to the “seed” region of the sense strand (S) reduced saRNA activity indicating sequence specificity for target gene activation. Lastly, saRNA activity was generally resilient to changes in overhang length although the best performing variants preferably had either dual nucleotide overhangs or blunt structures (no overhangs) .

Table 12. Oligonucleotide sequences and duplex compositions of CFH saRNA variant designs

upper case, RNA. The nucleotides in bold are overhang. The nucleotides in bold and italic are mismatch.

Example 8: Design and synthesis of saRNAs targeting the mouse Cfh promoter

Coding strand sequence from the promoter of the mouse Cfh gene was retrieved from the UCSC genome database (SEQ ID NO: 1719) . It consisted of an 800 nucleotide sequence ranging from position -1 bp to upstream -800 bp relative to the transcription start site (TSS) (Table 13) .

Table 13. Putative mouse Cfh promoter sequence (5′-3′) (SEQ ID NO: 1719)

A total of 383 saRNA duplexes targeting the mouse promoter sequence were synthesized at 19 bp in length according to the following criteria: (i) GC content between 35-65%; (ii) less than 5 consecutive identical nucleotides; (iii) 3 or less total dinucleotide repeats; and (iv) 3 or less total trinucleotide repeats. Dual nucleotide overhangs were also synthesized on the 3’ end of each strand resulting in a total length of 21 nt. Strand composition and sequence of each saRNA duplex targeting the mouse Cfh promoter is listed in Table 2.

Example 9: High throughput screening of saRNAs targeting mouse Cfh promoter

To identify saRNAs capable of activating Cfh expression, primary mouse hepatocytes (PMHs) grown in culture were transfected with each of the aforementioned saRNAs at 25 nM concentrations for 72 hours followed by gene expression analysis via one-step RT-qPCR. An siRNA (i.e., DS16B-si6) targeting the mouse Cfh transcript was also used as a negative control for transfection efficiency. Out of the 383 mouse saRNAs, 89 (23.2 %) , 58 (15.1 %) and 41 (10.7 %) saRNAs showed high (≥1.5 fold) , moderate (1.2～1.5 fold) , and mild activation (1.1～1.2 fold) of Cfh expression, respectively (Table 14) .

Table 14. Summary of activity of all screened Cfh saRNAs

Whereas 195 (51.0 %) saRNAs either reduced or had no obvious effect on the expression of Cfh mRNA. Relative changes in mouse Cfh expression caused by saRNA treatment are also summarized in Table 2, while expression data organized by gene induction is plotted in FIG. 8. Table 15 also compares results between the mouse and human saRNA high throughput screen grouped by fold change of target gene expression.

Table 15. Summary of activity of all screened human CFH and mouse Cfh saRNAs

Sorting expression data by target site location within the mouse Cfh promoter revealed five “hotspots” that were enriched for saRNA activity 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 targeted sequence within the indicated “hotspots” . Promoter sequence corresponding to each “hotspot” is listed in Table 9.

saRNA duplexes capable of upregulating mouse Cfh expression by at least 1.1-fold or higher in “hotpot region” H7 (-754 to -654) were 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, DS16B-082, DS16B-083, DS16B-084, DS16B-085, DS16B-086, DS16B-087, DS16B-088, 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, DS16B-102, DS16B-103, DS16B-104, DS16B-105, DS16B-106, DS16B-107, DS16B-108, DS16B-109, DS16B-110, DS16B-111, DS16B-112, DS16B-113, DS16B-114, DS16B-115, DS16B-116, DS16B-117, DS16B-118, DS16B-119, DS16B-120, DS16B-121.

saRNA duplexes capable of upregulating mouse Cfh expression by at least 1.1-fold or higher in “hotpot region” H8 (-479 to -421) were 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.

saRNA duplexes capable of upregulating mouse Cfh expression by at least 1.1-fold or higher in “hotpot region” H9 (-409 to -370) were 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.

saRNA duplexes capable of upregulating mouse Cfh expression by at least 1.1-fold or higher in “hotpot region” H10 (-208 to -56) were as follows: DS16B-279, DS16B-280, DS16B-281, DS16B-282, DS16B-283, DS16B-284, DS16B-285, DS16B-286, DS16B-287, DS16B-288, DS16B-289, DS16B-290, DS16B-291, DS16B-292, DS16B-293, DS16B-294, DS16B-295, DS16B-296, DS16B-297, DS16B-298, DS16B-299, DS16B-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-321, DS16B-322, DS16B-323, DS16B-324, DS16B-325, DS16B-326, DS16B-327, DS16B-328, DS16B-329, DS16B-330, DS16B-331, DS16B-332, DS16B-333, DS16B-334, DS16B-335.

saRNA duplexes capable of upregulating mouse Cfh expression by at least 1.1-fold or higher in “hotpot region” H11 (-52 to -4) were 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

Based on the screening results for mouse Cfh induction, the top 64 performing saRNAs (see Table 2) were transfected into PMH cells at 3 concentrations (i.e., 2.5, 10, and 25 nM) for 72 hours to quantify dose-dependent gene induction via one-step RT-qPCR. Both dsCon2 and DS16B-si6 served as a non-specific duplex control for gene activation and negative control for transfection efficiency, respectively. As shown in FIG. 10, most of the saRNAs demonstrated dose-dependent induction of Cfh with the highest levels of activity reaching ～4.0-fold by DS16B-070 at the 25 nM treatment concentration. The most potent duplexes included 41 saRNAs (64.1%) that exhibited ≥1.1-fold increases in mouse Cfh levels at the lowest concentration (i.e., 2.5 nM) , however only 4 (6.3%) activated Cfh expression by over 1.5-fold at the same concentration.

Example 11: saRNA treatment increases Cfh mRNA and protein levels over time in PMH cells

A subset of the top performers (19 saRNAs in total) were further assessed for Cfh induction out to five days following transfection in PMH cells at 25 nM concentrations. Both dsCon2 and DS16B-si6 served as a non-specific duplex control for gene activation and negative control for transfection efficiency, respectively. As shown in FIG. 11A, greater fold-changes in mRNA expression were detected at day 5 including 8 saRNAs (42.1%) that induced Cfh levels over 5-fold with DS16B-098 providing the highest measured increase in gene expression at 14.8-fold. Protein levels of Cfh secreted into media were also detected via ELISA at day 5 following saRNA treatment (i.e., DS16B-085, -091, -098, and -237) . Three of the saRNAs (i.e., DS16B-091, DS16B-098, and DS16B-237) measured an increase in Cfh protein level by over 1.5-fold relative to baseline (dotted line) (FIG. 11B) .

To quantify Cfh accumulation over time, two saRNAs (i.e., DS16B-089 and DS16B-091) were transfected into PMH cells at 25 nM concentrations in which mRNA and protein levels were measured each day between days 3-6 post-treatment via two-step RT-qPCR and ELISA, respectively. Increases in Cfh mRNA levels ascended and plateaued for both DS16B-089 and DS16B-091 between days 3-6 (FIG. 12A) . While protein levels did not exceed 1.25-fold compared to Mock and dsCon2 control treatments, Cfh protein did correlate with the ramping and plateau of Cfh mRNA following saRNA treatment (FIG. 12B) . Relative fold change in Cfh mRNA and protein levels are also listed in Table 16 and 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: saRNA potency for Cfh gene activation in PMH cells.

Dose response curves were generated via two-step RT-qPCR for 4 of the top saRNAs (i.e., DS16B-070, DS16B -091, DS16B -098, and DS16B -241) 5 days after treatment using 11 escalating 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 potency (FIG. 13A-D) . Each saRNA demonstrated dose-dependent activation of mouse Cfh expression with EC₅₀ values in the low nanomolar range (i.e., ～1.58-15.3 nM) as summarized in Table 18.

Table 18. EC₅₀ values for activating Cfh expression in vitro

Example 13: Development of DEC-conjugated saRNAs for induction of mouse Cfh expression in vivo

Variants of DS16B-098 and DS16B-091 were synthesized with medicinal chemistry (i.e., 2’ F, 2’ Ome, and PS modifications) including conjugation of delivery enhancing compounds (DEC) such as GalNac (i.e., tC2 or tC2x4) on the 3’ end of the antisense strand and/or lipid C5x5 on 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 via Lipofectamine RNAiMax at 25 nM concentrations for 72 hours to confirm retention of saRNA activity following chemical modification. A chemically modified siRNA with tC2x4 conjugation (i.e., RD-13149) targeting Cfh transcript served as a negative control for cell transfection. Compared to the non-conjugated saRNA (i.e., RD-13878) , all DEC- conjugated variants increased mouse Cfh expression as good or better than RD-13878 (FIG. 14) . This data indicates both conjugation and medicinal chemistry are well-tolerated by saRNAs.

A chemically modified variant of DS16B-091 (i.e., RD-14660) with both DEC-conjugates (i.e., tC2x4 and C5x5) was administered at 5 escalating concentrations (i.e., 6.25, 25, 100, 1600, and 6400 nM) to determine if DEC conjugation can enable saRNA delivery to PMH cells in absence of transfection reagent (i.e., free uptake) . A non-specific DEC-saRNA (i.e., RD-14040) served as negative control for saRNA activity. As shown in FIG. 15, RD-14660 demonstrated dose-dependent induction of Cfh up to ～3.0-fold indicating DEC-conjugation can delivery saRNA to target cells in vitro.

To measure activation of Cfh expression in vivo, a tC2x4 DEC-saRNA containing a 5’ VP modification on its antisense strand (i.e., RD-13147) was administered to adult female C57BL/6J mice via SC injection at 50 mg/kg. Saline served as a vehicle control, while treatment with DEC-siRNA (i.e., RD-13149) served as a negative control for liver-targeted delivery in vivo. Mice were sacrificed on day 10 following treatment and Cfh mRNA levels were quantified in liver tissue via two-step RT-qPCR. As summarized in Table 20, RD-13147 increased mean Cfh expression levels in mouse liver by ～1.22-fold compared to saline control animals. In addition, knockdown of Cfh by RD-13149 further supports DEC delivery to liver tissue in vivo. Monitoring animal weight following treatment also revealed no adverse finding supporting DEC-saRNA treatment was generally safe in C57BL/6J mice (FIG. 16) .

Chemical composition and strand sequence of all DEC-conjugated saRNA duplexes are listed in Table 19. The structures of tC2, tC2x4 and C5x5 compound are shown below:

Table 19. Oligonucleotide sequences and duplex compositions

upper case, RNA; *, phosphorothioate (PS) backbone modification; f, 2′-fluoro; m, 2′-O-methyl (2′-OMe) ; VP, 5′- (E) -vinylphosphonate.

Table 20. Relative Cfh mRNA expression in mouse liver tissue (SC, Day 10)

Note: #1, #2 and #3 represent 3 animals in per group.

Example 14: The preparation of compound tC2 of the present disclosure.

1. Segment 1: compound 3 synthesis.

(1) Preparation of compound 2:

The dicarboxylic acid (20 g, 86.8 mmol) was dissolved/susppended in dry CH₂Cl₂ (100 mL) . Then oxalyl chloride (16.2 mL, 190.96 mmol) and DMF (5 drops) were added to the solution. The reaction mixture was stirred at room temperature for 3 h, then concentrated under reduced pressure to provide crude compound 2, which was directly used in the next step without further purification.

(2) Preparation of compound 3:

A solution of benzyl alcohol (9.39 g, 86.8 mmol) and Et₃N (122 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. Then the solution was warmed to room temperature and stirred overnight. A mixture of H₂O (80 mL) , Et₃N (12 mL, 86.8 mmol) and THF (80 mL) was added slowly to the solution over 1 hour, and the stirring was continued for 2 hours. THF was then removed and 20 mL H₂O was added to the residue. The mixture was extracted by ethyl ether (3*100 mL) and the organic phase was combined, dried over Na₂SO₄, and concentrated. Ethyl acetate (20 mL) was added to the residue and the suspension was filtered to remove dodecanedioic acid. Concentration of the filtrate followed by chromatography (Hexane: Ethyl acetate = 4 : 1 to 2: 1) afforded 12.5 g (45%yield) compound 3 as white solid. MW calc. : 320.20; MW Found: 319.02 [M-H] ^-.

2. Segment 2: Compound 6 synthesis.

(1) Preparation of compound 5:

To a solution of Fmoc-L-hydroxyproline compound 4 (13.3 g, 37.6 mmol) in anhydrous THF (250 mL) , was added borane-methyl sulfide complex (6.16 g, 80 mmol) slowly at room temperature. The reaction mixture was stirred for 5 min at room temperature and then heated to reflux for about 1 h. Methanol (15 mL) was carefully added to the reaction mixture, which was refluxed for 15 min. After that, the reaction mixture was concentrated under reduced pressure. Then the crude products were evaporated three times with methanol (100 mL each) to remove boron-related impurity. The obtained crude product compound 5 was directly used in the next step without further purification.

(2) Preparation of compound 6:

To a solution of compound 5 (37.6 mmol) in anhydrous pyridine (200 mL) , was added DMTrCl (14 g, 41.4 mmol) slowly at ice bath. The reaction was stirred at nitrogen atmosphere overnight and then concentrated under reduced pressure. The crude product was dissolved in dry MeCN (300 mL) , after that, the mixture was added Et₃N (112.8 mmol, 15.6 mL) and heated to 60 ℃ for 4 h. After concentrated under reduced pressure, the resulting residue was purified by flash chromatography (silica gel, gradient eluent: 1-8%of MeOH/DCM) to provide compound 6 (7.57 g, 48%yield) as yellow solid. ¹H NMR (400 MHz, CDCl₃) δ 7.41 (d, J = 7.4 Hz, 2H) , 7.30 (d, J = 8.8 Hz, 4H) , 7.28 -7.22 (m, 2H) , 7.18 (t, J = 7.2 Hz, 1H) , 6.80 (d, J = 8.8 Hz, 4H) , 4.34 (s, 1H) , 3.75 (d, J = 11.1 Hz, 6H) , 3.60 (dd, J = 12.7, 6.7 Hz, 1H) , 3.10 -2.92 (m, 5H) , 2.86 (d, J = 11.5 Hz, 1H) , 1.85 (dd, J = 13.5, 7.1 Hz, 1H) , 1.63 (ddd, J = 13.7, 7.9, 5.9 Hz, 1H) .

3. Total synthesis.

(1) Preparation of compound 9:

To a solution of 2-amino-2- (hydroxymethyl) propane-1, 3-diol compound 7 (25 g, 206 mmol) in DMSO (50 mL) , was added sodium hydroxide solution (0.83 g NaOH in 4 mL H₂O) slowly. Then the 3- (tert-butoxy) -3-oxoprop-1-en-1-ylium (90 g, 700 mmol) was added slowly under nitrogen. The reaction was stirred at room temperature for 2 days. Then 100 mL H₂O was added into the reaction, the mixture was extracted by ethyl acetate (3*150 mL) and the organic phase was combined, dried over Na₂SO₄, and concentrated. The resulting residue was purified by flash chromatography (silica gel, gradient eluent: 1-5%of MeOH/DCM) to provide compound 9 (42 g, 40%yield) as colorless oil.

(2) Preparation of compound 10:

To a solution of 12- (benzyloxy) -12-oxododecanoic acid compound 3 (3.3 g, 10.3 mmol) in DCM (50 mL) , was added HBTU (7.81 g, 20.6 mmol) and DIPEA (5.1 mL, 30.9 mmol) under nitrogen atmosphere. 5 minutes later, the reaction was added compound 9 (7.8 g, 15.45 mmol) . After that, the reaction was stirred at room temperature for 4 h. Then 50 mL H₂O was added into the reaction, the mixture was extracted by DCM (3*100 mL) and the organic phase was combined, dried over Na₂SO₄, and concentrated. The resulting residue was purified by flash chromatography (silica gel, gradient eluent: 1-30%of EA/HX) to provide compound 10 (7.16 g, 86%yield) as colorless oil. ¹H NMR (400 MHz, CDCl₃) δ 7.42 -7.27 (m, 5H) , 6.00 (s, 1H) , 5.11 (s, 2H) , 3.70 (s, 6H) , 3.64 (t, J = 6.3 Hz, 6H) , 2.44 (t, J = 6.3 Hz, 6H) , 2.34 (t, J = 7.5 Hz, 2H) , 2.17 -2.09 (m, 2H) , 1.59 (d, J = 11.6 Hz, 4H) , 1.45 (s, 27H) , 1.26 (s, 12H) .

(3) Preparation of compound 12:

To a solution of compound 10 (7.0 g, 8.67 mmol) in DCM (100 mL) , was added CF₃COOH (50 mL) under nitrogen atmosphere. The reaction mixture was stirred overnight at room temperature, then concentrated under reduced pressure to provide crude product as colorless oil. After that, the colorless oil was dissolved in 100 mL DCM, then HBTU (19.73 g, 52.02 mmol) and DIPEA (13 mL, 78.03 mmol) were added into the reaction under nitrogen atmosphere. Five minutes later, tert-butyl (3-aminopropyl) carbamate compound 11 (6.8 g, 39 mmol) was added into the reaction. The reaction mixture was stirred overnight at room temperature. Then 50 mL H₂O was added into the reaction, the mixture was extracted by DCM (3*100 mL) 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%of MeOH/DCM) to provide compound 12 (6.7 g, 70%yield) as colorless oil.

(4) Preparation of compound 14:

To a solution of the compound 12 (1.0 g, 0.9 mmol, 1.0 eq) in DCM (8 mL) , was added HCl/Dioxane (4M, 8 mL) . The reaction mixture was stirred for 3 h at room temperature, then concentrated under reduced pressure to provide crude product as yellow solid. After that, the crude product was dissolved in 15 mL DCM, then compound 13 (1.81 g, 4.05 mmol, 4.5 eq) , HBTU (1.53 g, 4.05 mmol, 4.5 eq) and DIPEA (1.79 mL, 10.8 mmol, 12.0 eq) were added into the reaction under nitrogen atmosphere. The reaction mixture was stirred overnight at room temperature. Then 10 mL H₂O was added into the reaction mixture. The mixture was extracted with DCM (3*20 mL) . The organic phase was combined, dried over Na₂SO₄, and concentrated. The resultant residue was purified with flash chromatography (silica gel, gradient eluent: 1-8%of MeOH/DCM) to provide compound 14 (0.99 g, 52%yield) as white solid. The product was characterized with ¹H NMR. ¹H NMR (400 MHz, CDCl₃) ¹H NMR (400 MHz, CDCl₃) δ 7.37 -7.32 (m, 5H) , 5.34 (d, J = 2.9 Hz, 3H) , 5.29 (s, 2H) , 5.19 (dd, J = 11.2, 3.2 Hz, 3H) , 5.10 (s, 2H) , 4.59 (d, J = 8.4 Hz, 3H) , 4.12 (dd, J = 13.4, 5.8 Hz, 6H) , 3.90 (d, J = 6.2 Hz, 4H) , 3.67 (s, 12H) , 3.47 (s, 6H) , 3.26 (d, J = 5.3 Hz, 10H) , 3.06 (q, J = 7.4 Hz, 4H) , 2.42 (t, J = 5.4 Hz, 6H) , 2.34 (t, J = 7.5 Hz, 3H) , 2.22 (dd, J = 16.2, 7.7 Hz, 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:

To a solution of compound 14 (0.98 g, 0.47 mmol) in MeOH (10 mL) , was added Pd/C (98 mg) under nitrogen atmosphere slowly. Then the reaction atmosphere was replaced with hydrogen gas for three times. After that, the reaction mixture was stirred by purging with a hydrogen balloon at room temperature overnight. Then the reaction mixture was filtered and concentrated under reduced pressure to provide crude product.

The crude product was dissolved in DCM (20 mL) , then HBTU (267 mg, 0.705 mmol, 1.5 eq) and DIPEA (265 μL, 1.598 mmol, 3.4 eq) were added into the reaction under nitrogen atmosphere. After five minutes, compound 6 (236 mg, 0.564 mmol, 1.2 eq) was added into the reaction mixture. The reaction mixture was stirred overnight at room temperature. Then H₂O (10 mL) was added into the reaction mixture. The mixture was extracted with DCM (3*20 mL) . The organic phase was combined, dried over Na₂SO₄, and concentrated. The resultant residue was purified with flash chromatography (silica gel, gradient eluent: 1-10%of MeOH/DCM) to provide compound 15 (0.79 g, 69%yield) as yellow solid. The product was characterized with ¹H NMR. ¹H NMR (400 MHz, CDCl₃) δ 7.34 (d, J = 8.5 Hz, 2H) , 7.25 -7.20 (m, 5H) , 6.97 -6.89 (m, 2H) , 6.80 (t, J = 8.6 Hz, 4H) , 5.33 (s, 3H) , 5.17 (ddd, J = 11.0, 7.7, 3.2 Hz, 3H) , 4.60 (t, J = 9.1 Hz, 3H) , 4.19 -4.06 (m, 9H) , 3.95 -3.85 (m, 6H) , 3.78 (d, J = 3.5 Hz, 6H) , 3.67 (s, 12H) , 3.48 (t, J = 10.8 Hz, 4H) , 3.26 (s, 12H) , 2.42 (t, J = 5.2 Hz, 6H) , 2.24 (dd, J = 26.4, 11.2 Hz, 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 = 8.6 Hz, 12H) .

(6) Preparation of compound 16:

To a solution of compound 15 (780 mg, 0.324 mmol, 1.0 eq) in anhydrous in DCM (6 mL) were added DMAP (138 mg, 1.13 mmol, 3.5 eq) , succinic anhydride (97 mg, 0.97 mmol, 3.0 eq) under nitrogen atmosphere. The reaction mixture was stirred at room temperature overnight, and then H₂O (10 mL) was added into the reaction. The mixture was extracted with DCM (3*20 mL) . The organic phase was combined, dried over Na₂SO₄, and concentrated. The resultant residue was purified with flash chromatography (silica gel, gradient eluent: 1-15%of MeOH/DCM) to provide compound 16 (690 mg, 85%yield) as white solid. The product was characterized with ¹H NMR. ¹H NMR (400 MHz, CDCl₃) δ 7.38 -7.31 (m, 4H) , 7.21 -7.12 (m, 5H) , 6.80 (dd, J = 13.7, 5.2 Hz, 4H) , 5.33 (d, J = 2.7 Hz, 3H) , 5.19 (d, J = 11.2 Hz, 3H) , 4.63 (dd, J = 8.2, 4.9 Hz, 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 = 7.7 Hz, 4H) , 2.43 (t, J = 5.2 Hz, 6H) , 2.25 (td, J = 15.1, 7.7 Hz, 11H) , 2.13 (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 = 12.0 Hz, 12H) .

(7) Preparation of compound tC2:

To a solution of compound 16 (690 mg, 0.275 mmol, 1.0 eq) , Controlled Pore Glass (CPG) (Code: C3006-1000, Hebei DNA chem Biotechnology Co., Ltd, China) (4.8 g) , N, N-Diisopropylethylamine (DIPEA) (137 μL, 0.825 mmol, 3.0 eq) in acetonitrile (40 mL) was added HBTU (208 mg, 0.55 mmol, 2.0 eq) under nitrogen atmosphere. The reaction mixture was shaked at 25 ℃ overnight and then washed with DCM and ethyl ether to produce crude support material.

To a solution of acetic anhydride (15 mL) , pyridine (34 mL) , NEt₃ (500 μL) in acetonitrile (22 mL) was added the crude support material under nitrogen atmosphere. The reaction mixture was shaked at 25 ℃ 1h and then washed with DCM and ethyl ether to produce Compound tC2 of the present disclosure (4.7 g) .

Example 15: The preparation of oligonucleotide linked with the conjugation group derived from the compound tC2 of the present disclosure.

General synthesis method of oligonucleotide

(1) Single strand synthesis method

The single strand oligonucleotide was synthesized on a K&A DNA synthesizer (K&A Laborgeraete GbR, chaafheim, Germany) by a support material synthesis technique. The starting material was universal support material or special support material commercially available or synthesis as disclosure in previous context. In general, phosphoramidite monomers including various linkers and conjugates (0.1M in acetonitrile or dichloromethane) , were added sequentially onto a support material in the DNA synthesizer to generate the desired full-length oligonucleotides. Each cycle of amidite addition consisted of four chemical reactions including detritylation, coupling, oxidation/thiolation and capping. In first step, the detritylation was performed by using 3%dichloroacetic acid (TCA) in DCM for 45 seconds. In the second step, Phosphoramidite coupling was conducted for 6 minutes for all amidites by 12 eq. ; In the third step, oxidation was performed by using 0.02 M iodine in THF: pyridine: water (70: 20: 10, v/v/v) for 1 minute; if phosphorothioate modification needed then replace oxidation by thiolation which was carried out with 0.1 M solution of xanthane hydride in pyridine: ACN (50: 50, v/v) for 3 minutes; In the fourth step, the capping was performed by using a THF: acetic anhydride: Pyridine (80: 10: 10, v/v/v) (CAP A) and N-methylimidazole: THF (10: 90, v/v) , (CAP B) for 20 seconds. The Cycles of four chemical reaction will be depended by the length of single of oligonucleotide.

Deprotection I (Nucleobase Deprotection) : After completion of the synthesis, the support material was transferred to a screw-cap microcentrifuge tube. For a 1 μmol synthesis scale, 1 ml of a mixture of methylamine and ammonium hydroxide was added. The tube containing the support material was then heated in an oven at 60℃ to 65℃ for 15 min and then allowed to cool to room temperature. The cleavage solution was collected and evaporated to dryness in a speedvac to provide crude single strand of oligonucleotide.

Deprotection II (Removal of 2’ -TBDMS Group) : If The crude RNA oligonucleotide, still carrying the 2’ -TBDMS groups, then dissolved in 0.1 ml of DMSO. After adding 1 ml of Triethylamine trihydrofluoride, the tube was capped, and the mixture was shaken vigorously to ensure complete dissolutionand then heated in an oven at 65℃ for 15 minutes. The tube was removed from the oven and cooled down to room temperature. The solution containing the completely desilylated oligonucleotide was cooled on dry ice. 2 ml of ice-cold n-butanol (-20℃) were carefully added in 0.5 ml portions to precipitate the oligonucleotides. The precipitate was filtered, washed with 1 ml ice-cold n-butanol, and subsequently dissolved in 0.01 M Tris (hydroxymethyl) aminomethanol hydrochloride buffer.

(2) single strand purification

The purification of oligonucleotides was performed on an AKTA explorer 10 equipped with a Source 15Q 4.6/100 PE column using the following conditions: buffer A: (10 mM Tris-HCl, 1 mM EDTA, pH 7.5) , B: (10 mM Tris-HCl, 1 mM EDTA, 2M NaCl, pH 7.5) , gradient: 10%B to 60%B in 25 min, flow rate: 1 ml/min. The pure oligonucleotides were collected and desalting by a HiPrep 26/10 Desalting column.

(3) Annealing to form duplex

For duplex, after the generation of desalted purified single strand solutions, sense strand and antisense strand were mixed by equal volumes at equimolar concentration in the tube. Place the tube in a heatblock at 95℃ for 5 min and then cool to room temperature then were subsequently lyophilized to powder.

The O1 was generated by using a conjugation group derived from the compound tC2 as starting support material according to the above methods of general synthesis method of oligonucleotide. Exemplary structure of the DEC-conjugated oligonucleotide is O1 as illustrated below:

It can be seen that in the structures of O1, the conjugation group derived from the delivery enhancing compound tC2 is linked with double-stranded RNA (dsRNA) duplexes (including but not limited to saRNA or siRNA) at the 3’-end of the sense strand (S) via 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 16: The preparation of compound tC2x4 of the present disclosure.

(1) The compound 18 was prepared by using the start material of (2S,3R,4R,5R,6R) -3-acetamido-6- (acetoxymethyl)tetrahydro-2H-pyran-2,4,5-triyl triacetate compound 17.

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) , was added TMSOTf (15.3 mL, 84.7 mmol, 1.1 eq) under nitrogen atmosphere. The reaction mixture was stirred at room temperature for 6 h. Then NaHCO₃ solution (21 g NaHCO₃ in 200 mL water) was added under ice bath and the reaction was transferred to room temperature. After 30 minutes of stirring, the mixture was extracted 3 times with DCM, then the organic phase was combined and washed with brine, dried over Na₂SO₄, and concentrated. The resultant residue was purified with flash chromatography (silica gel, gradient eluent: 1-50% of EA/Hexane) to provide compound 18(19.8 g, 78% yield) . The product was characterized with mass spectrometry and ¹H NMR. MW calc.: 329.11; MW Found: 330.33 [M+H]⁺. ¹H NMR (400 MHz, CDCl3) δ 5.97 (d, J = 9.5 Hz, 1H) , 5.39 (s, 1H) , 5.26 (dd, J = 11.4, 3.2 Hz, 1H) , 4.53 (td, J = 11.4, 3.4 Hz, 1H) , 4.43 (t, J = 6.5 Hz, 1H) , 4.27 (s, 1H) , 4.11 - 4.08 (m, 1H) , 2.17 (s, 3H) , 2.05 (s, 3H) , 2.00 (d, J = 7.3 Hz, 6H) .

(2) The preparation of compound 20

To a solution of compound 18 (19.7 g, 59.8 mmol, 1.0 eq) in DCE (260 mL) , was added 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) under nitrogen atmosphere. The reaction mixture was stirred at room temperature for 6 h. Then saturated NaHCO₃ solution (100 mL) was added, the mixture was extracted 3 times with DCM. The organic phase was combined and washed with brine, dried over Na₂SO₄, and concentrated. The resultant residue was purified with flash chromatography (silica gel, gradient eluent: 1-5%of MeOH/DCM) to provide compound 20 (18.7 g, 73%yield) .

(3) The preparation of compound 13

To a solution of compound 20 (18.7g, 43.6 mmol, 1.0 eq) in ACN/DCM/H₂O (140mL/140mL/210mL) under ice bath, was added NaIO₄ (37.3 g, 174.4 mol, 4.0 eq) and RuCl₃·3H₂O (1.8 g, 8.72 mol, 0.2 eq) . After 10 minutes, the reaction mixture was transferred to room temperature and stirred overnight. Then the reaction mixture was filtered and concentrated under reduced pressure. After that, water (100 mL) was added into the mixture, then the mixture was extracted 3 times with DCM, the organic phase was washed 1 time with brine. After being dried with anhydrous Na₂SO₄ and concentrated under reduced pressure, the resultant residue was purified with flash chromatography (silica gel, gradient eluent: 1-8%of MeOH/DCM) to provide compound 13 (19 g, 97%yield) . The product was characterized with mass spectrometry and ¹H NMR. MW calc. : 447.17; MW Found: 448.36 [M+H] ⁺. ¹H NMR (400 MHz, CDCl3) δ 6.15 (d, J = 8.7 Hz, 1H) , 5.33 (d, J = 3.0 Hz, 1H) , 5.29 -5.26 (m, 1H) , 4.66 (d, J = 8.4 Hz, 1H) , 4.13 (dd, J = 9.3, 6.8 Hz, 2H) , 3.92 (dd, J = 8.4, 5.4 Hz, 2H) , 3.50 (dd, J = 5.7, 4.0 Hz, 1H) , 2.35 (dd, J = 14.3, 7.0 Hz, 2H) , 2.13 (s, 3H) , 2.03 (s, 3H) , 1.98 (s, 3H) , 1.95 (s, 3H) , 1.69 -1.61 (m, 4H) .

(4) This step comprises the preparation of compound 23 from methyl 4-fluoro-3-nitrobenzoate.

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) , under nitrogen atmosphere, was added benzyl (3-aminopropyl) carbamate compound 22 (8.3 g, 40.2 mmol, 1.0 eq) . The reaction mixture was stirred at 25 ℃ for 6 h, then cold water (100 mL) was added. The mixture was extracted 3 times by ethyl acetate, then the organic phase was washed 3 times by saturated LiCl solution and 1 time by brine. Then dried by anhydrous Na₂SO₄ and concentrated under reduced pressure to form yellow solid compound 23 which was directly used in the next step without further purification.

(5) The preparation of compound 24

To a solution of compound 23 (10 g, 25.84 mmol, 1.0 eq) in THF/H₂O (9: 1, 100 mL) , under ice bath, was added HCOONH₄ (9.78 g, 155.04 mmol, 6.0 eq) and Zn powder (10.14 g, 155.04 mmol, 6.0 eq) . After 10 minutes, the reaction mixture was moved to room temperature and stirred overnight. Then the reaction mixture was filtered and concentrated under reduced pressure. After that, water (100 mL) was added into the mixture, then extracted 3 times by ethyl acetate, the organic phase was washed 1 time by brine. After dried by anhydrous Na₂SO₄ and concentrated under reduced pressure, the light red solid compound 24 formed then was directly used in the next step without further purification.

(6) The preparation of compound 26

To a solution of compound 24 (8.87 g, 24.84 mmol, 1.0 eq) in EtOH (220 mL) under nitrogen atmosphere, was added 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) . The reaction mixture was stirred at 80℃ overnight, and then concentrated under reduced pressure. Then saturated NaHCO₃ solution (100 mL) was added therein, the mixture was extracted 3 times with ethyl acetate. The organic phase was combined and washed by brine, dried over Na₂SO₄, and concentrated. The resultant residue was purified with flash chromatography (silica gel, gradient eluent: 1-5%of MeOH/DCM) to provide the compound 26 (6.3 g, 46%yield) as red solid. The product was characterized with mass spectrometry, ¹H NMR and ¹³C NMR. MW calc. : 553.30; MW Found: 554.29 [M+H] ⁺. ¹H NMR (400 MHz, CDCl3) δ 8.42 (d, J = 0.8 Hz, 1H) , 7.95 (dd, J = 8.5, 1.1 Hz, 1H) , 7.44 -7.28 (m, 5H) , 7.26 (d, J = 6.8 Hz, 1H) , 5.12 (s, 2H) , 4.16 (dd, J = 12.6, 5.3 Hz, 2H) , 3.93 (s, 3H) , 3.67 (t, J = 6.3 Hz, 2H) , 3.28 (d, J = 6.2 Hz, 2H) , 2.87 (t, J = 7.6 Hz, 2H) , 2.04 -1.88 (m, 4H) , 1.67 (dd, J = 14.7, 6.6 Hz, 2H) , 0.88 (s, 9H) , 0.04 (s, 6H) . ¹³C NMR (100 MHz, CDCl₃) δ 167.72, 156.63, 142.35, 138.19, 136.30, 128.60, 128.24, 124.10, 123.87, 121.63, 108.66, 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) The preparation of compound 27

To a solution of compound 26 (6.3 g, 11.4 mmol, 1.0 eq) in anhydrous THF (50 mL) under nitrogen atmosphere, was added 1 M TBAF THF solution (17.1 mL, 17.1 mmol, 1.5 eq) . The reaction mixture was stirred at room temperature for 1 h, and then concentrated under reduced pressure. Then water (100 mL) was added, the mixture was extracted 3 times with DCM, then the organic phase was combined and washed with brine, dried over Na₂SO₄, and concentrated. The resultant residue was dissolved in 50 mL pyridine, and DMTrCl (4.6 g, 13.68 mmol, 1.2 eq) was added therein. The reaction mixture was stirred at room temperature for 6 h, after which it was concentrated under reduced pressure. The resultant residue was purified with flash chromatography (silica gel, gradient eluent: 1-5%of MeOH/DCM) to provide compound 27 (6.23 g, 74%yield) as yellow solid. The product was characterized with mass spectrometry, ¹H NMR and ¹³C NMR. MW calc. : 741.34; MW. Found: 303.11 [DMT] -, 440.14 [DMT off + H] ⁺. ¹H NMR (400 MHz, CDCl₃) δ 8.63 (dd, J = 5.7, 1.5 Hz, 1H) , 8.43 (d, J = 1.0 Hz, 1H) , 7.97 (dd, J = 8.5, 1.1 Hz, 1H) , 7.45 (d, J = 7.4 Hz, 2H) , 7.37 -7.28 (m, 11H) , 7.21 (dd, J = 8.1, 6.1 Hz, 1H) , 6.83 (t, J = 5.9 Hz, 4H) , 5.13 (s, 2H) , 4.12 (t, J = 7.2 Hz, 2H) , 3.95 (s, 3H) , 3.79 (s, 6H) , 3.24 (d, J = 6.2 Hz, 2H) , 3.14 (t, J = 6.2 Hz, 2H) , 2.83 (t, J = 7.4 Hz, 2H) , 2.01 (dd, J = 16.0, 8.5 Hz, 4H) , 1.79 (dd, J = 14.1, 6.6 Hz, 2H) . ¹³C NMR (100 MHz, CDCl3) δ 167.81, 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 was reduced to methylene-oxy group so as to produce compound 28.

To a solution of compound 27 (5.0 g, 6.74 mmol, 1.0 eq) in anhydrous THF (60 mL) under nitrogen atmosphere and ice bath, was added LiAlH₄ (384 mg, 10.11 mmol, 1.5 eq) . The mixture was then transferred to room temperature after 10 minutes and stirred for about 1 h. Then the reaction was transferred to ice bath, and saturated potassium sodium tartrate solution (30 mL) was added slowly into the mixture. After reacting for 30 minutes, the reaction was extracted 3 times with Et₂O, then the organic phase was combined and washed with brine, dried over Na₂SO₄, and concentrated. The resultant residue was dissolved in 20 mL DMF, then imidazole (688 mg, 10.11 mmol, 1.5 eq) and TBSCl (1.524 g, 10.11 mmol, 1.5 eq) were added. The reaction mixture was stirred at room temperature for 1 h. After being concentrated under reduced pressure, the resultant residue was purified with flash chromatography (silica gel, gradient eluent: 1-5%of MeOH/DCM) to provide compound 28 (4.78 g, 86%yield) as yellow solid. The product was characterized with mass spectrometry, ¹H NMR and ¹³C NMR. MW calc. : 827.43; MW. Found: 303.16 [DMT] -, 526.50 [DMT off + H] ⁺. ¹H NMR (400 MHz, CDCl₃) δ 7.69 (s, 1H) , 7.47 -7.42 (m, 2H) , 7.40 -7.28 (m, 11H) , 7.22 (t, J = 3.5 Hz, 3H) , 6.86 -6.80 (m, 4H) , 5.13 (s, 2H) , 4.86 (s, 2H) , 4.15 (dd, J = 14.3, 7.2 Hz, 2H) , 3.79 (s, 6H) , 3.13 (t, J = 6.3 Hz, 2H) , 2.82 (t, J = 7.5 Hz, 2H) , 2.05 -1.94 (m, 4H) , 1.81 -1.74 (m, 2H) , 1.29 (t, J = 7.1 Hz, 2H) , 0.97 (s, 9H) , 0.13 (s, 6H) . ¹³C NMR (100 MHz, CDCl₃) δ 171.27, 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) Preparation of compound 30 from the starting 4-fluoro-3-nitrobenzoic acid compound 29.

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) under nitrogen atmosphere, was added benzyl bromide (35.6 g, 207.98 mmol, 1.1 eq) slowly. The reaction mixture was stirred at 45℃ for 3 h, then cold water (200 mL) was added therein. The mixture was extracted 3 times with ethyl acetate, then the organic phase was washed 3 times with saturated LiCl solution and 1 time with brine. Then the organic phase was dried with anhydrous Na₂SO₄ and concentrated under reduced pressure to form a yellow oil compound 30 which was directly used in the next step without further purification.

(10) The preparation of compound 31

To a solution of compound 30 (52 g, 189.1 mmol, 1.0 eq) and K₂CO₃ (26.13 g, 189.1 mmol, 1.0 eq) in anhydrous DMF (500 mL) under nitrogen atmosphere, was added tert-butyl (3-aminopropyl) carbamate compound 11 (32.9 g, 189.1 mmol, 1.0 eq) . The reaction mixture was stirred at room temperature for 3 h, then cold water (200 mL) was added. The mixture was extracted 3 times by ethyl acetate, then the organic phase was washed 3 times by saturated LiCl solution and 1 time by brine. After dried by anhydrous Na₂SO₄ and concentrated under reduced pressure, the yellow solid compound 31 was formed and then directly used in the next step without further purification.

(11) Compound 32 was generated by reduction of compound 31.

To a solution of compound 31 (81.16 g, 189.1 mmol, 1.0 eq) in THF/H₂O (9: 1, 666 mL) under ice bath, was added HCOONH₄ (71.58 g, 1.134 mol, 6.0 eq) and Zn powder (74.19 g, 1.134 mol, 6.0 eq) . After 10 minutes, the reaction mixture was moved to room temperature and stirred overnight. Then the reaction mixture was filtered and concentrated under reduced pressure. After that, water (200 mL) was added into the mixture, then the mixture was extracted 3 times with ethyl acetate, the organic phase was washed 1 time with brine. The organic brine was dried with anhydrous NaSO₄ and concentrated under reduced pressure, a light red solid compound 32 was formed and was directly used 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.

To a solution of compound 32 (16.8 g, 42 mmol, 1.0 eq) in DCM (300 mL) , was added 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) under nitrogen atmosphere. The reaction mixture was stirred overnight at room temperature. Then 200 mL H₂O was added into the reaction mixture, and the mixture was extracted with DCM (3*100 mL) . The organic phase was combined, dried over Na₂SO₄, and concentrated. The resultant residue was purified by using flash chromatography (silica gel, gradient eluent: 1-50%of EA/Hexane) to provide compound 34 (27.5 g, 90%yield) as yellow solid. The product was characterized with mass spectrometry and ¹H NMR. MW calc. : 727.42; MW Found: 728.47 [M+H] ⁺. ¹H NMR (400 MHz, CDCl3) δ 7.88 (dd, J = 8.6, 1.9 Hz, 1H) , 7.79 (s, 1H) , 7.35 (ddt, J = 9.8, 7.1, 5.5 Hz, 5H) , 6.62 (d, J = 8.6 Hz, 1H) , 5.29 (s, 2H) , 4.16 (s, 1H) , 3.37 -2.97 (m, 6H) , 1.78 (d, J = 25.4 Hz, 4H) , 1.51 (s, 2H) , 1.48 -1.35 (m, 27H) , 1.36 -1.13 (m, 2H) .

(13) The compound 35, which comprises a benzimidazole structure was prepared from compound 34.

The compound 34 (20 g, 27.5 mmol, 1.0 eq) was added into AcOH (100 mL) under nitrogen atmosphere. The reaction mixture was stirred at 95℃ for 4 h. Then saturated NaHCO₃ solution (100 mL) was added therein, the mixture was extracted 3 times with ethyl acetate, then the organic phase was combined and washed with saturated NaHCO₃ solution (3*100 mL) , dried over Na₂SO₄, and concentrated. The resultant residue was purified by flash chromatography (silica gel, gradient eluent: 1-50%of EA/Hexane) to provide compound 35 (8.78 g, 45%yield) as white solid. The product was characterized with mass spectrometry and ¹H NMR. MW calc. : 709.41; MW Found: 710.57 [M+H] ⁺. ¹H NMR (400 MHz, CDCl3) δ 8.50 (s, 1H) , 8.04 (dd, J = 8.5, 1.4 Hz, 1H) , 7.46 (d, J = 7.0 Hz, 2H) , 7.41 (s, 1H) , 7.38 -7.34 (m, 3H) , 5.39 (s, 2H) , 5.02 (dd, J = 15.8, 7.7 Hz, 1H) , 3.20 -3.10 (m, 6H) , 2.08 -1.96 (m, 4H) , 1.57 -1.48 (m, 4H) , 1.41 (d, J = 8.6 Hz, 27H) .

(14) The preparation of compound 36

To a solution of compound 35 (1.8 g, 2.54 mmol, 1.0 eq) in DCM (10 mL) , was added HCl/Dioxane (4M, 20 mL) . The reaction mixture was stirred for 3 h at room temperature, then concentrated under reduced pressure to provide crude product as yellow solid. After that, the crude product was dissolved in 25 mL DCM, then compound 13 (3.4 g, 7.62 mmol, 3.0 eq) , HBTU (3.37 g, 8.89 mmol, 3.5 eq) and DIPEA (5.05 mL, 30.48 mmol, 12.0 eq) were added into the reaction under nitrogen atmosphere. The reaction mixture was stirred overnight at room temperature. Then 20 mL H₂O was added into the reaction mixture. The mixture was extracted with DCM (3*30 mL) . The organic phase was combined, dried over Na₂SO₄, and concentrated. The resultant residue was purified with flash chromatography (silica gel, gradient eluent: 1-8%of MeOH/DCM) to provide compound 36 (3.67 g, 85%yield) as yellow solid. The product was characterized with mass spectrometry and ¹H NMR. MW calc. : 1697.80; MW Found: 1698.53 [M+H] ⁺. ¹H NMR (400 MHz, CDCl3) δ 8.41 (d, J = 22.5 Hz, 1H) , 8.04 (d, J = 8.5 Hz, 1H) , 7.44 (d, J = 7.0 Hz, 2H) , 7.40 -7.31 (m, 4H) , 5.42 -5.26 (m, 7H) , 5.19 (td, J = 11.0, 3.3 Hz, 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 = 6.9 Hz, 9H) , 2.03 (s, 9H) , 1.99 (s, 9H) , 1.92 (dd, J = 11.6, 5.2 Hz, 9H) , 1.78 (s, 2H) , 1.71 -1.61 (m, 6H) , 1.51 (dd, J = 12.6, 6.5 Hz, 8H) , 1.42 (d, J = 6.6 Hz, 4H) .

(15) The compound 36 was linked with compound 37, which was derived from compound 28

To a solution of compound 36 (1.85 g, 1.09 mmol, 1.0 eq) in MeOH (20 mL) , was added Pd/C (185 mg) under nitrogen atmosphere slowly. Then the reaction atmosphere was replaced with hydrogen gas for three times. After that, the reaction mixture was stirred by purging with a hydrogen balloon at room temperature overnight. Then the reaction mixture was filtered and concentrated under reduced pressure to provide crude product.

The crude product was dissolved in DCM (20 mL) , then HBTU (826 mg, 2.18 mmol, 2.0 eq) and DIPEA (614 μL, 3.7 mmol, 3.4 eq) were added into the reaction under nitrogen atmosphere. Five minutes later, compound 37 (849 mg, 1.2 mmol, 1.1 eq) , which was produced by reducing compound 28, was added into the reaction mixture. The reaction mixture was stirred overnight at room temperature. Then H₂O (20 mL) was added into the reaction, the mixture was extracted with DCM (3*50 mL) . The organic phase was combined, dried over Na₂SO₄, and concentrated. The resultant residue was purified with flash chromatography (silica gel, gradient eluent: 1-8%of MeOH/DCM) to provide compound 38 (1.8 g, 72%yield) as white solid. The product was characterized with mass spectrometry and ¹H NMR. MW calc. : 2282.08; MW Found: 1142.33 [M+2H] +/2. ¹H NMR (400 MHz, CDCl3) δ 8.08 (s, 1H) , 7.64 (s, 1H) , 7.38 (dd, J = 12.7, 8.1 Hz, 4H) , 7.28 (d, J = 8.8 Hz, 5H) , 7.23 (d, J = 7.8 Hz, 2H) , 7.20 -7.15 (m, 2H) , 6.78 (d, J = 8.8 Hz, 4H) , 5.33 (s, 3H) , 5.23 -5.11 (m, 2H) , 4.81 (s, 2H) , 4.58 (dd, J = 8.4, 2.1 Hz, 2H) , 4.18 (d, J = 10.6 Hz, 3H) , 4.17 -4.04 (m, 8H) , 3.96 -3.80 (m, 6H) , 3.75 (s, 6H) , 3.63 (dd, J = 13.3, 6.6 Hz, 1H) , 3.57 -3.40 (m, 5H) , 3.22 (s, 3H) , 3.07 (dd, J = 10.9, 4.7 Hz, 2H) , 2.85 (t, J = 7.5 Hz, 2H) , 2.42 (s, 6H) , 2.20 (dd, J = 34.7, 6.1 Hz, 8H) , 2.10 (d, J = 7.6 Hz, 9H) , 2.06 (d, J = 9.1 Hz, 2H) , 2.01 (d, J = 3.3 Hz, 9H) , 1.97 (dd, J = 7.5, 2.4 Hz, 9H) , 1.89 (dd, J = 28.9, 13.9 Hz, 9H) , 1.80 -1.64 (m, 6H) , 1.62 -1.46 (m, 12H) , 1.40 (d, J = 6.0 Hz, 3H) , 0.92 (s, 9H) , 0.08 (s, 6H) .

(16) Compound 39 was generated by deprotecting TBS group of compound 38 then acylated with succinic anhydride.

To a solution of compound 38 (1.57 g, 0.687 mmol, 1.0 eq) in anhydrous THF (10 mL) under nitrogen atmosphere, was added 1 M TBAF THF solution (2.06 mL, 2.06 mmol, 3.0 eq) . The reaction mixture was stirred at room temperature for 1 h, and then concentrated under reduced pressure to produce the crude product.

The crude product was dissolved in DCM (15 mL) then DMAP (587 mg, 4.8 mmol, 3.5 eq) , succinic anhydride (137 mg, 1.374 mmol, 2.0 eq) were added under nitrogen atmosphere. The reaction mixture was stirred at room temperature overnight, then H₂O (20 mL) was added into the reaction mixture. The mixture was extracted with DCM (3*50 mL) . The organic phase was combined, dried over Na₂SO₄, and concentrated. The resultant residue was purified with flash chromatography. The resultant residue was purified with flash chromatography (silica gel, gradient eluent: 1-10%of MeOH/DCM) to provide compound 39 (1.0 g, 64%yield) as colorless oil. The product was characterized with mass spectrometry and ¹H NMR. MW calc. : 2268.01; MW Found: 1135.22 [M+2H] +/2. ¹H NMR (400 MHz, CDCl₃) δ 8.17 (d, J = 6.6 Hz, 2H) , 7.40 (d, J = 7.6 Hz, 2H) , 7.30 -7.26 (m, 5H) , 7.24 (s, 2H) , 7.18 (d, J = 6.8 Hz, 2H) , 6.79 (d, J = 8.8 Hz, 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, 8.3 Hz, 2H) , 4.17 (d, J = 10.0 Hz, 2H) , 4.14 -3.96 (m, 8H) , 3.87 (dd, J = 15.1, 6.5 Hz, 5H) , 3.76 (s, 6H) , 3.45 (s, 6H) , 3.19 (d, J = 8.7 Hz, 2H) , 3.08 (t, J = 6.0 Hz, 2H) , 3.01 (s, 10H) , 2.90 -2.77 (m, 2H) , 2.63 (d, J = 17.4 Hz, 4H) , 2.19 (dd, J = 24.8, 17.2 Hz, 9H) , 2.12 -2.08 (m, 9H) , 2.01 (d, J = 3.7 Hz, 9H) , 1.98 -1.94 (m, 9H) , 1.87 (dd, J = 24.8, 13.3 Hz, 9H) , 1.76 -1.62 (m, 6H) , 1.55 (d, J = 15.4 Hz, 12H) .

(17) The compound 39 was linked with a support material, Controlled Pore Glass (CPG) , to produce Compound tC2x4 of the present disclosure.

To a solution of compound 39 (233 mg, 0.103 mmol, 1.0 eq) , Controlled Pore Glass (CPG) (1.8 g) , N, N-diisopropylethylamine (DIPEA) (51 μL, 0.309 mmol, 3.0 eq) in acetonitrile (14 mL) was added HBTU (78 mg, 0.206 mmol, 2.0 eq) under nitrogen atmosphere. The reaction mixture was shaked at 25 ℃ overnight and then washed with DCM and ethyl ether to generate crude support material.

To a solution of acetic anhydride (6.2 mL) , pyridine (12 mL) , NEt₃ (186 uL) in acetonitrile (7.9 mL) was added the crude support material under nitrogen atmosphere. The reaction mixture was shaked at 25 ℃ 1h and then washed with DCM and ethyl ether to produce Compound tC2x4 of the present disclosure (1.84 g) .

Example 17:澔The preparation of conjugated oligonucleotide linked with the conjugation group derived from compound tC2x4 of the present disclosure.

The O2 was generated by using a conjugation group derived from the compound tC2x4 as starting support material according to the above methods of general synthesis method of oligonucleotide which presented in Example 15. Exemplary structure of the DEC-conjugated oligonucleotide is O2 as illustrated below:

It can be seen that in the structures of O2, the conjugation group derived from the delivery enhancing compound is linked with double-stranded RNA (dsRNA) duplexes (including but not limited to saRNA or siRNA) at the 3’-end of the sense strand (S) via 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:澔The preparation of compound C5x5 of the present disclosure.

Compound C5x5 was prepared in this Example by using the following procedures.

(1) The preparation of compound 42

To a solution of methyl methyl 2- (4-fluoro-3-nitrophenyl) acetate compound 41 (17.3 g, 81 mmol, 1.0 eq) and K₂CO₃ (11.2 g, 81 mmol) in anhydrous DMF (200 mL) , under nitrogen atmosphere, was added compound 40 (19.56 g, 81 mmol, 1.0 eq) . The reaction mixture was stirred at 55 ℃ for 6 h, then cold water (100 mL) was added. The mixture was extracted three times by ethyl acetate, then the organic phase was washed three times by saturated LiCl solution and one time by brine. Then dried by anhydrous Na₂SO₄ and concentrated under reduced pressure to form yellow oil compound 42 which was directly used in the next step without further purification.

(2) The preparation of compound 43

To a solution of compound 42 (35.18 g, 81 mmol, 1.0 eq) in THF/H₂O (9: 1, 280 mL) , under ice bath, was added HCOONH₄ (30.67 g, 486 mmol, 6.0 eq) and Zn powder (31.78 g, 486 mmol, 6.0 eq) . After 10 minutes, the reaction mixture was moved to room temperature and stirred overnight. Then the reaction mixture was filtered and concentrated under reduced pressure. After that, water (200 mL) was added into the mixture, then extracted three times by ethyl acetate, the organic phase was washed one time by brine. After dried by anhydrous Na₂SO₄ and concentrated under reduced pressure, the compound 43 formed then was directly used in the next step without further purification. The compound 43 was characterized with mass spectrometry. MW calc. : 404.34; MW. Found: 405.3 [M+H] ⁺.

(3) The preparation of compound 45

To a solution of compound 43 (19.36 g, 48 mmol, 1.0 eq) in EtOH (200 mL) under nitrogen atmosphere, was added 3- ( (tert-butyldimethylsilyl) oxy) propanal compound 44 (9.0 g, 48 mmol, 1.0 eq) and AcOH (11 mL, 192 mmol, 4.0 eq) . The reaction mixture was stirred at 80℃ overnight, and then concentrated under reduced pressure. Then a saturated NaHCO₃ solution (100 mL) was added, the mixture was extracted three times by ethyl acetate, then the organic phase was combined and washed by brine, dried over Na₂SO₄, and concentrated. The resultant residue compound 45 was directly used in the next step without further purification.

(4) The preparation of compound 46

To a solution of compound 45 (10 g, 17.5 mmol, 1.0 eq) in anhydrous THF (50 mL) under nitrogen atmosphere, was added 1 M TBAF THF solution (26.3 mL, 26.3 mmol, 1.5 eq) . The reaction mixture was stirred at room temperature for 1 h, and then concentrated under reduced pressure. Then water (100 mL) was added, the mixture was extracted three times with DCM, then the organic phase was combined and washed with brine, dried over Na₂SO₄, and concentrated. The resultant residue was dissolved in 50 mL pyridine, and DMTrCl (7.12 g, 21 mmol, 1.2 eq) was added therein. The reaction mixture was stirred at room temperature for 6 h, after which it was concentrated under reduced pressure. The resultant residue was purified with flash chromatography (silica gel, gradient eluent: 1-3%of MeOH/DCM) to provide compound 46 (8.1 g, 61%yield) as yellow solid. The product was characterized with mass spectrometry and ¹H NMR. MW calc. : 760.48; MW.Found: 761.8 [M + H] ⁺. ¹H NMR (400 MHz, CDCl₃) δ 7.59 -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, 5.6 Hz, 4H) , 4.17 -4.02 (m, 2H) , 3.76 (s, 6H) , 3.73 (s, 2H) , 3.67 (s, 3H) , 3.59 (t, J = 7.0 Hz, 2H) , 3.19 -3.05 (m, 2H) , 1.29 -1.25 (m, 28H) , 0.88 (t, J = 6.5 Hz, 3H) .

(5) The preparation of compound C5x5

To a solution of compound 46 (2.7 g, 3.55 mmol, 1.0 eq) in anhydrous THF (20 mL) under nitrogen atmosphere and ice bath, was added LiAlH₄ (202 mg, 5.33 mmol, 1.5 eq) . The mixture was moved to room temperature after 10 minutes and stirred for 1 h. Then the reaction was moved to ice bath, saturated potassium sodium tartrate solution (20 mL) was added slowly into the mixture. After 30 minutes, the reaction was extracted three times with Et₂O, then the organic phase was combined and washed by brine, dried over Na₂SO₄, and concentrated. The crude product (300 mg, 0.41 mmol, 1.0 eq) was dissolved in anhydrous DCM (5 mL) then DIPEA (204 μL, 1.23 mmol, 3.0 eq) , 3- ( (chloro(diisopropylamino)phosphanyl)oxy)propanenitrile compound 47 (274 μL, 1.23 mmol, 3.0 eq.) were added under nitrogen atmosphere at 25℃. The reaction mixture was stirred for 1 h. The mixture was extracted two times with DCM, then washed with brine and dried with anhydrous Na₂SO₄. The organic layer was concentrated under reduced pressure and the resultant residue was purified with flash chromatography (silica gel, gradient eluent: 1-5% of MeOH/DCM, 1% Et₃N) to provide compound C5x5 (299 mg, 78% yield) as colorless oil. The product was characterized with mass spectrometry and ¹H NMR. MW calc.: 946.61; MW Found: 303.2 [DMT]^-.¹H NMR (400 MHz, CDCl₃) δ 7.62 (s, 1H) , 7.32 (dd, J = 7.6, 4.1 Hz, 2H) , 7.27 - 7.11 (m, 9H) , 6.73 (dd, J = 7.9, 5.6 Hz, 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, 5.1 Hz, 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, 3.2 Hz, 12H) , 0.88 (t, J = 6.5 Hz, 3H) .

Example 19:澔The preparation of conjugated oligonucleotide linked with the conjugation group derived from compound C5x5 of the present disclosure.

The O3 was generated by using a conjugation group derived from the compound C5x5 as terminus amidite according to the above methods of general synthesis method of oligonucleotide which presented in Example 15. Exemplary structure of the DEC-conjugated oligonucleotide is O3 as illustrated below:

It can be seen that in the structures of O3, the conjugation derived from the delivery enhancing compound C5x5 is linked with double-stranded RNA (dsRNA) duplexes (including but not limited to saRNA or siRNA) at the 5’-end of the sense strand (S) via a linking moiety, such as -OP (O) 2O-or -P (O) -O-, wherein (S) is the sense strand and (AS) is the antisense strand.

In summary, the high throughput screening data revealed a plurality of “hotspots” for saRNA activity in the promoter of both human CFe and mouse Cfh gene. Exemplary saRNAs increased expression of both mRNA and protein levels while demonstrating dose-dependent gene induction. Moreover, saRNA activity readily tolerated medicinal chemistry and DEC conjugation enabling delivery in vivo. These results provide evidence that targeted activation of CFH expression in the liver via saRNA administration is a promising strategy to treat CFHD.

Table 1. Human CFH gene saRNA sequences

n.b. : Target sequence is identical to the identified sense sequence but the nucleotide ″U ″ is converted to ″T″ and excluding the overhang 2 nucleotides ″TT″ . DS16A-si5 is a negative control.

Table 2. Mouse Cfh gene saRNA sequences

n.b. : Sense and antisense sequences are including the overhang 2-nt natural overhang selected from or complementary to the corresponding nucleotides on the DNA target. DS16B-si6 is a negative control.

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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, and T. H. Goodship. 2013. ′Genotype/phenotype correlations in complement factor H deficiency arising from uniparental isodisomy′, Am J Kidney Dis, 62: 978-83.

Claims

An oligonucleotide modulator (saRNA) comprising an oligonucleotide sequence having a length ranging from 16 to 35 consecutive nucleotides, wherein the continuous 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 of SEQ ID NO: 1707, wherein the saRNA activates the expression of CFH gene by at least 10%as compared to baseline expression of the CFH gene.
The saRNA of claim 1, wherein the equal length portion of SEQ ID NO: 1707 is located in the region -538 to -500 (SEQ ID NO: 1708) , region -468 to -396 (SEQ ID NO: 1709) , region -329 to -283 (SEQ ID NO: 1710) , region -273 to -192 (SEQ ID NO: 1711) , region -173 to -100 (SEQ ID NO: 1712) , or region -64 to -14 (SEQ ID NO: 1713) upstream of the transcription start site of CFH gene.
The saRNA of any one of claims 1-2, wherein the saRNA (1) has a GC content between 35%and 65%; (2) with less than 5 consecutive identical nucleotides; (3) with 3 or less dinucleotide repeats; and (4) with 3 or less trinucleotide repeats.
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 complementary regions, wherein the complementary regions of the sense strand and the antisense strand form a double-stranded nucleic acid structure.
The saRNA of any one of claims 1-3, wherein the sense strand and the antisense strand have a complementarity of at least 90%.
The saRNA of claim 4, wherein the sense strand and the antisense strand are located on two different nucleic acid strands.
The saRNA of claim 4, wherein the sense strand and the antisense strand are located on a contiguous nucleic acid strand, optionally 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.
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.
The saRNA of claim 8, wherein the sense strand and the antisense strand comprise a 3′ overhang of ranging from 2 to 3 nucleotides in length.
The saRNA of claim 8, wherein at least one of the nucleotides of the overhang is thymine deoxyribonucleotide.
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 consecutive nucleotides.
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.
The saRNA of claim 12, wherein the sense strand comprises a nucleotide sequence selected from SEQ ID NOs: 309-615, and the antisense strand comprises a nucleotide sequence selected from SEQ ID NOs: 618-924.
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.
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.
The saRNA of claim 1, wherein the antisense strand of the oligonucleotide sequence has at least 75%sequence complementarity to a nucleotide sequence selected from SEQ ID NOs: 1-307.
The saRNA of any one of claims 1-16, wherein at least one nucleotide of the saRNA is a chemically modified nucleotide.
The saRNA of claim 17, wherein at least one nucleotide of the antisense and/or sense strand of the saRNA is chemically modified.
The saRNA of claim 17, wherein the chemically modified nucleotide is a nucleotide with at least one the following modifications:

a) modification of a phosphodiester bond connecting nucleotides in the nucleotide sequence of the saRNA;

b) modification of 2′-OH of a ribose in the nucleotide sequence of the saRNA; and

c) modification of a base in the nucleotide sequence of the saRNA.
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 non-natural base comprising nucleotide.
The saRNA of claim 17, wherein the chemical modification of the at least one chemically modified nucleotide is an addition of a (E) -vinylphosphonate moiety at the 5’ end of the sense strand or the antisense strand.
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 conjugation groups selected from a lipid, a fatty acid, a fluorophore, a ligand, a saccharide, a peptide, and an antibody.
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 a cell-penetrating peptide, polyethylene glycol, an alkaloid, a tryptamine, a benzimidazole, a quinolone, an amino acid, a cholesterol, glucose, and N-acetylgalactosamine.
The saRNA of claim 23, wherein said conjugation group is derived from a compound selected from tC2, tC2x4, and C5x5,

wherein represents a support material.
The saRNA of claim 22, wherein said conjugation group is a lipid selected from fatty acid comprising a carbon chain length of from 4 to 30 carbon atoms.
The saRNA of claim 25, wherein said conjugation group is fatty acid comprising a carbon chain length of 16 carbon atoms.
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 conjugation groups, wherein the two conjugation groups are a lipid and a N-acetylgalactosamine.
The saRNA of claim 27, wherein said two conjugation groups are C5x5 and tC2x4.
An isolated polynucleotide of the saRNA of claim 1, wherein the isolated polynucleotide is a nucleotide sequence having a length ranging from 16 to 35 consecutive nucleotides of SEQ ID NO: 1707.
The isolated polynucleotide of claim 29, wherein the isolated polynucleotide is a nucleic acid sequence selected from SEQ ID NO: 1-307.
An isolated oligonucleotide complex comprising the antisense strand of the saRNA of any of claim 1-28 and the sense strand of the isolated polynucleotide of any of claim 29-30.
The isolated oligonucleotide complex of claim 31, wherein the isolated oligonucleotide complex activates the expression of CFH gene by at least 10%as compared to baseline expression of the CFH gene.
An isolated nucleic acid sequence upstream of the transcription start site of CFH gene, wherein the isolated nucleic acid sequence is selected from SEQ ID NO: 1708-1713.
An isolated nucleic acid sequence selected from the group consisting of region -538 to -500, region -468 to -396, region -329 to -283, region -273 to -192, region -173 to -100 or region -64 to -14 upstream of the transcription start site of the CFH gene.
The isolated nucleic acid sequence of claim 33 or 34, wherein the isolated nucleic acid sequence comprises the isolated polynucleotide of any one of claims 29-30.
The isolated nucleic acid sequence of claim 33 or 34, wherein at least 30%of designed saRNA targeting the isolated nucleic acid sequence can activate the expression of CFH gene by at least 10%, wherein the designed saRNA (1) having a GC content between 35%and 65%; (2) with less than 5 consecutive identical nucleotides; (3) with 3 or less dinucleotide repeats; and (4) with 3 or less trinucleotide repeats.
An isolated nucleic acid complex comprising the antisense strand of the saRNA of any of claim 1-28 and the sense strand of the isolated nucleic acid sequence of any of claim 33-36.
The isolated nucleic acid complex of claim 37, wherein the isolated nucleic acid complex activates the expression of CFH gene by at least 10%as compared to baseline expression of the CFH gene.
An isolated polynucleotide encoding the saRNA of any one of claims 1-28.
The isolated polynucleotide of claim 39, wherein the isolated polynucleotide is a DNA.
A vector comprising the isolated polynucleotide of any one of claims 39-40.
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.
A composition comprising the saRNA of any one of claims 1-28, or the isolated polynucleotide of claim 39 or 40 and optionally, a pharmaceutically acceptable carrier.
The composition of claim 43, wherein the pharmaceutically acceptable carrier is selected from the group consisting of an aqueous carrier, a liposome, a high-molecular polymer, a polypeptide and an antibody.
The composition of claim 43 or 44, wherein the composition comprises 0.001-150 nM of the saRNA.
The composition of claim 45, wherein the composition comprises 1-150 nM of the saRNA.
An saRNA comprising an oligonucleotide sequence with a length ranging from 16 to 35 continuous 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 complementary to an equal length portion of SEQ ID NO: 1707, wherein the saRNA activates the expression of CFH gene by at least 10%as compared to baseline expression of the CFH gene.
The saRNA of claim 47, wherein the equal length region of SEQ ID NO: 1707 is located in the region -538 to -500 (SEQ ID NO: 1708) , region -468 to -396 (SEQ ID NO: 1709) , region -329 to -283 (SEQ ID NO: 1710) , region -273 to -192 (SEQ ID NO: 1711) , region -173 to -100 (SEQ ID NO: 1712) , or region -64 to -14 (SEQ ID NO: 1713) upstream of the transcription start site of CFH gene.
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 SEQ ID NOs: 309-615, and the antisense strand comprises or is a nucleotide sequence selected from SEQ ID NOs: 618-924.
A method for preventing or treating a disease or condition induced by insufficient expression of plasma complement factor H (CFH) protein, a CFH gene mutation, and/or low functional CFH levels in plasma in an individual comprising: administering 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 to the individual.
The method of claim 50, wherein the disease or condition is a complement factor H deficiency (CFHD) .
The method of claim 50, wherein the individual is a mammal.
The method of claim 52, wherein the individual is a human.
The method of claim 50, wherein the individual suffers from a symptom induced by insufficient expression of CFH protein, a CFH gene mutation, and/or low functional CFH levels in plasma.
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 administrated to an individual by an administration pathway selected from one or more of: parenteral infusions, oral administration, intranasal administration, inhaled administration, vaginal administration, and rectal administration.
The method of claim 55, wherein the administration pathway is selected from one or more of intrathecal, intramuscular, intravenous, intraarterial, intraperitoneal, intravesical, intracerebroventricular, intravitreal and subcutaneous administrations.
The method of claim 50, wherein the method activates/up-regulates expression of the CFH gene or Cfh gene mRNA in the individual by at least 10%as compared to baseline expression of the CFH gene or cfh gene.
The method of claim 50, wherein the method increases a level of CFH protein in the individual by at least 10%as compared to baseline expression of the CFH gene.
A method for detecting CFH protein or CFH regulated protein in the cell of claim 42.
A kit for performing the method of claim 59, comprising the saRNA of any one of claims 1-28.
The kit of claim 60, wherein the instruction for use comprising means for administering the saRNA of any one of claims 1-28 to an individual.
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 and the label on package indicates that the saRNA, the isolated polynucleotide, the vector or the composition can be used in preventing or treating a disease or condition induced by insufficient expression of plasma complement factor H (CFH) , or against CFHD.
A kit for detecting CFH protein or CFH regulated protein in the cell of claim 42.
The 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 preparing a medicament for preventing or treating a disease or condition induced by insufficient expression of CFH protein, a CFH gene mutation, and/or low functional CFH levels in plasma in an individual.
The use of claim 64, wherein the disease or condition is a CFHD.
The use of claim 64, wherein the individual is a mammal, optionally wherein the mammal is a human.
The 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 preparing a preparation for activating/up-regulating expression of CFH gene in a cell.
The use of claim 67, wherein the saRNA of any one of claims 1-28, or 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.
The use of claim 68, wherein the saRNA is produced in the cell after a nucleotide sequence encoding the saRNA is introduced into the cell.
The use of any of claims 67-69, wherein the cell is a mammalian cell, optionally wherein the mammalian cell is a human cell.
The use of claim 70, wherein the cell is in a human body.
The use of claim 71, wherein the human body is a patient suffering from a symptom induced by the insufficient expression of CFH protein, a CFH gene mutation, and/or low functional CFH levels in plasma, wherein the saRNA, the isolated polynucleotide or the composition is administered in a sufficient amount to treat the symptom.
The use of claim 72, wherein the symptom induced by insufficient expression of CFH protein is a CFHD.
A method for activating/up-regulating expression of CFH gene in a cell comprising: administering 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 to the cell.
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 introduced directly into the cell.
The method of claim 75, wherein the method for introducing directly into the cell comprises:

1) composing 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 saRNA in the composition of 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 polymer, a polypeptide and an antibody, and

2) conjugating 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 saRNA in the composition of any one of claims 43-46 to one or more conjugation groups selected from a cell-penetrating peptide, polyethylene glycol, an alkaloid, a tryptamine, a benzimidazole, a quinolone, an amino acid, a cholesterol, glucose, and N-acetylgalactosamine.
The method of claim 76, wherein said conjugation group is derived from a compound selected from tC2, tC2x4, and C5x5,

wherein represents a support material.
The method of claim 76, wherein said conjugation group is a lipid selected from fatty acid comprising a carbon chain length of from 4 to 30 carbon atoms.
The method of claim 78, wherein said conjugation group is fatty acid comprising a carbon chain length of 16 carbon atoms.
The method of claim 76, wherein the sense strand or the antisense strand of the saRNA is conjugated to two conjugation groups, wherein the two conjugation groups are a lipid and a N-acetylgalactosamine.
The method of claim 80, wherein said two conjugation groups are derived from C5x5 and tC2x4.
The method of any of claims 74-81, wherein the cell is a mammalian cell, preferably a cell from a human body.
The method of claim 82, wherein the human body is a patient suffering from a symptom induced by insufficient expression of the CFH protein, a CFH gene mutation, and/or low functional CFH levels in plasma, wherein the saRNA, the isolated polynucleotide or the composition is administered in a sufficient amount to treat the symptom.
The method of claim 83, wherein the symptom caused by insufficient expression of CFH protein is atypical hemolytic uremic syndrome (aHUS) , dense deposit disease (DDD) , C3 glomerulonephritis (C3GN) , CFHR5 nephropathy, lupus nephritis (LN) , type I MPGN with pure complement C3 deposition (MPGN1) , membranoproliferative glomerulonephritis type II (MPGN2) , familial type III MPGN (MPGN3) , and age-related macular degeneration (AMD) .
A method for increasing a level of CFH protein in a cell, comprising introducing 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 saRNA in the composition of any one of claims 43-46 into the cell, wherein the saRNA, the isolated polynucleotide or the composition activate expression of CFH gene by at least 10%as compared to baseline expression of the CFH gene.
A compound selected from tC2, tC2x4, and C5x5,

wherein represents a support material.