Chemically modified sarna compositions and methods of use

Abstract

The disclosure relates to saRNAs useful in upregulating the expression of a target gene and therapeutic compositions comprising the saRNAs, wherein the saRNAs are chemically modified. Methods of using the saRNAs and the therapeutic compositions are also provided.

Description

CHEMICALLY MODIFIED SARNA COMPOSITIONS AND METHODS OF USEREFERENCE TO SEQUENCE LISTING

[0001] The present application is being filed with a Sequence Listing in electronic format. The sequence listing filed, entitled 1600USPR02_SL, was created on November 6, 2024, and is 302,269 bytes in size. The information in electronic format of the Sequence Listing is incorporated herein by reference in its entirety.FIELD OF THE DISCLOSURE

[0002] The disclosure relates to oligonucleotide, specifically saRNA, compositions for modulating gene expression and to the methods of using the compositions in diagnostic and therapeutic applications.BACKGROUND

[0003] It has been found that small duplex RNAs can increase gene expression by targeting ncRNAs that overlap gene promoters. See, e.g., Janowski et al., Nature Chemical Biology, vol.3: 166-173 (2007). Any short RNA that leads to up-regulation of the expression of a target gene by any mechanism is termed a short activating RNA or small activating RNA (saRNA). There remains a need for compositions comprising saRNAs and methods of using saRNAs. There also remains a need to design saRNAs with improved stability, potency, and / or efficacy and less toxicity.BRIEF DESCRIPTION OF THE DRAWINGS

[0004] The foregoing and other objects, features and advantages will be apparent from the following description of particular embodiments of the disclosure, as illustrated in the accompanying drawings in which like reference characters refer to the same parts throughout the different views. The drawings are not necessarily to scale, emphasis instead being placed upon illustrating the principles of various embodiments of the disclosure.

[0005] Fig. 1 shows HNF4a Pl mRNA levels (n=2) in cells after saRNA treatments.

[0006] Fig. 2A-2D shows mouse Hnf4a Pl mRNA levels in cells after treatment with GalNAc- saRNA conjugates.SUMMARY OF THE DISCLOSURE

[0007] The present disclosure provides chemically modified synthetic isolated small activating RNAs (saRNAs) which up-regulate the expression of a target gene. The target gene can be any gene in the human genome, such as any coding gene in Table 1 and any non-coding gene in Table 2 of WO2016 / 170,348. In some embodiments, the saRNA comprises an antisense strand that is at least 80% complementary to a region on a targeted sequence of the target gene, andwherein the antisense strand has 14-30 nucleotides. Pharmaceutical compositions, kits, and devices comprising such saRNAs are also provided.

[0008] The present disclosure provides a double-stranded synthetic isolated small activating RNA (saRNA) which up-regulates the expression of a target gene, comprising an antisense strand and a sense strand, wherein the antisense strand has 19-25 nucleotides (such as 19-22 nucleotides or 21 nucleotides) and the sense strand has 19-25 nucleotides (such as 19-22 nucleotides or 19 nucleotides). In some embodiments, the antisense strand comprises a 2’-0Me modification on the 6thposition of the strand. In some embodiments, the antisense strand has phosphorothioate (-ps-) bonds between the 1stand 2ndpositions, between the 2ndand 3rdpositions, between the last and second to the last positions, and / or between the second to the last and third to the last positions. In some embodiments, the antisense strand comprises 2’-F modifications on the 2nd, 3rd, 4thand / or 14thpositions of the strand. In some embodiments, the antisense strand does not have a 2’-F modification at the 16thposition. In some embodiments, the antisense strand has a 2’-0Me modification at the 16thposition. Optionally, in some embodiments, the antisense strand has 2’-F modifications on the 17thposition and / or the 18thposition. The positions described in the present disclosure are all counted from the 5’ end of the strand and refer to the nucleotide at that position. Further optionally, in some embodiments, the antisense strand may have a E-vinylphosphonate (£-VP) modification at the 5’ end. In some embodiments, the antisense strand of the saRNA has an overhang comprising 2-3 nucleotides at the 3’ end (a 3’ overhang), such as but not limited to -UU, -UUU, -mUmU (m strands for 2’- OMe modification), or -ps-mU-ps-mU. In some embodiments, the sense strand of the saRNA of the present disclosure comprises at least one modification. In some embodiments, the sense strand does not have any 3’ overhang. In other embodiments, the sense strand has an overhang comprising 2-3 nucleotides at the 3’ end (a 3’ overhang), such as but not limited to UU, UUU, or mUmU (m strands for 2’-0Me modification).

[0009] The present disclosure also provides methods of up-regulating the expression of the target gene in a subject. Methods of treating diseases associated with the target gene are also provided. The methods comprise administering saRNAs of the present disclosure to the subject.

[0010] The details of various embodiments of the disclosure are set forth in the description below. Other features, objects, and advantages of the disclosure will be apparent from the description and the drawings, and from the claims.

[0011] Nonlimiting exemplary embodiments of the present disclosure include: Embodiment 1. A double-stranded synthetic isolated small activating RNA (saRNA) which up-regulates the expression of a target gene, comprising an antisense strand and a sensestrand, wherein the sense strand has 19 nucleotides and the antisense strand has 19-22 nucleotides, wherein the antisense strand comprises a 2’-0Me modification on the 6thposition of the strand, and / or wherein the antisense strand has phosphorothioate (-ps-) bonds between the 1stand 2ndpositions and / or between the 2ndand 3rdpositions.Embodiment 2. The saRNA of embodiment 1, wherein the antisense strand of the saRNA comprises at least one additional modification.Embodiment 3. The saRNA of any one of embodiments 1-2, wherein the antisense strand further comprises 2’-F modifications on the 2nd, 3rdand / or 4thpositions of the strand.Embodiment 4. The saRNA of any one of embodiments 1-3, wherein the antisense strand further comprises a 2’-F modification on the 1st, 9thand / or 14thposition of the strand.Embodiment 5. The saRNA of any one of embodiments 1-4, wherein the antisense strand has a 2’-0Me modification at the 10thand / or 13thposition.Embodiment 6. The saRNA of any one of embodiments 1-5, wherein the antisense strand has 2’-F modifications on the 17thposition and / or the 18thposition.Embodiment 7. The saRNA of any one of embodiments 1-6, wherein the antisense strand does not have a 2’-F modification at the 16thposition.Embodiment 8. The saRNA of any one of embodiments 1-7, wherein the antisense strand has a 2’-0Me modification at the 16thposition.Embodiment 9. The saRNA of any one of embodiments 1-8, wherein the antisense strand of the saRNA comprises A- VP at the 5’ end.Embodiment 10. The saRNA of any one of embodiments 1-9, wherein the antisense strand has phosphorothioate (-ps-) bonds between the last and second to the last positions and / or between the second to the last and third to the last positions.Embodiment 11. The saRNA of any one of embodiments 1-10, wherein the antisense strand has an overhang comprising 2-3 nucleotides at the 3’ end.Embodiment 12. The saRNA of embodiment 11, wherein the overhang at the 3’ end of the antisense strand is -UU, -UUU, -mUmU or -ps-mU-ps-mU.Embodiment 13. The saRNA of any one of embodiments 1-12, wherein the antisense strand has 21 nucleotides.Embodiment 14. The saRNA of any one of embodiments 1-13, wherein the antisense strand of the saRNA comprises at least one unmodified nucleotide.Embodiment 15. The saRNA of embodiment 14, wherein the unmodified nucleotide of the antisense strand is A, C or G.Embodiment 16. The saRNA of any one of embodiments 1-15, wherein all the Us of the antisense strand are modified.Embodiment 17. The saRNA of any one of embodiments 1-16, wherein all the Us of the antisense strand are modified either with 2’-0Me or 2’-F.Embodiment 18. The saRNA of any one of embodiments 1-17, wherein the sense strand of the saRNA does not have any 3’ overhang.Embodiment 19. The saRNA of any one of embodiments 1-18, wherein the sense strand of the saRNA comprises at least one modification.Embodiment 20. The saRNA of any one of embodiments 1-19, wherein the sense strand of the saRNA comprises 2’-OMe modifications on the 2ndand 14thpositions of the strand.Embodiment 21. The saRNA of any one of embodiments 1-20, wherein the sense strand of the saRNA comprises a 2’-OMe modification on the 3rdpositions of the strand.Embodiment 22. The saRNA of any one of embodiments 1-21, wherein sense strand has phosphorothioate (-ps-) bonds between the 1stand 2ndpositions, between the 2ndand 3rdpositions, between the 17thand 18thpositions and / or the 18thand 19thpositions.Embodiment 23. The saRNA of any one of embodiments 1-22, wherein the sense strand of the saRNA comprises at least one unmodified nucleotide.Embodiment 24. The saRNA of embodiment 23, wherein the unmodified nucleotide of the sense strand is A, C or G.Embodiment 25. The saRNA of any one of embodiments 1-24, wherein all the Us of the sense strand are modified.Embodiment 26. The saRNA of any one of embodiments 1-25, wherein the Us of the sense strand are modified with either 2’-0Me or 2’-F.Embodiment 27. A conjugate comprising the saRNA of any one of embodiments 1-26 covalently connected to a carbohydrate or lipid moiety.Embodiment 28. The conjugate of embodiment 27, wherein the carbohydrate moiety comprises at least one N-Acetyl-Galactosamine (GalNAc) or derivative thereof.Embodiment 29. The conjugate of embodiment 27, wherein the lipid moiety comprises cholesterol, palmitoyl, or tocopherol group.Embodiment 30. The conjugate of any one of embodiments 27-29, wherein the saRNA is connected to the carbohydrate or lipid moiety with a linker.Embodiment 31. A pharmaceutical composition comprising the saRNA of any one of embodiments 1-26 or the conjugate of any one of embodiments 27-30, and at least one pharmaceutically acceptable excipient.Embodiment 32. A method of up-regulating the expression of a target gene, comprising contacting the target gene with the saRNA of any one of embodiments 1-26, the conjugate of any one of embodiments 27-30, or the pharmaceutical composition of embodiment 31.Embodiment 33. The method of embodiment 32, wherein the expression of the target gene is increased by at least 30%, 40%, or 50%.DETAILED DESCRIPTION

[0012] The present disclosure provides compositions, methods and kits for modulating target gene expression and / or function for therapeutic purposes. These compositions, methods and kits comprise at least one saRNA that upregulates the expression of the target gene.I. Design and Synthesis of saRNA

[0013] One aspect of the present disclosure provides a method to design and synthesize saRNA.

[0014] The terms “small activating RNA”, “short activating RNA”, or “saRNA” in the context of the present disclosure means a single-stranded or double-stranded RNA that upregulates or has a positive effect on the expression of a specific gene. The saRNA may be single-stranded of 14 and up to 50 nucleotides, such as 19, 20, 21, 22, or 23 nucleotides. The saRNA may also be double-stranded, each strand comprising 14 and up to 50 nucleotides, such as 19, 20, 21, 22, or 23 nucleotides. The gene is called the target gene of the saRNA. As used herein, the target gene is a double-stranded DNA comprising a coding strand and a template strand. For example, an saRNA that upregulates the expression of the HNF4a gene is called an “HNF4a-saRNA” and the HNF4a gene is the target gene of the HNF4a-saRNA. A target gene may be any gene of interest.

[0015] saRNAs of the present disclosure may be designed by the method described in WO2016 / 170,348, the contents of which are incorporated herein by reference in their entirety. In some embodiments, the target gene may be any gene in the human genome, such as any coding gene in Table 1 and any non-coding gene in Table 2 of WO2016 / 170,348. In some embodiments, a target gene has a promoter region on the template strand. The method disclosed in US 2013 / 0164846 (saRNA algorithm), may also be used to design saRNA. The design of saRNA is also disclosed in US Pat. No. 8,324,181 and US Pat. No. 7,709,566 to Corey et al., US Pat. Pub. No. 2010 / 0210707 to Li et al., Voutila et al., Mol Ther Nucleic Acids, vol. 1, e35 (2012), and Watts et al., Nucleic Acids Research, 2010, Vol. 38, No. 15, 5242-5259 (2010).

[0016] The saRNA of the present disclosure may be produced by any suitable method, for example synthetically or by expression in cells using standard molecular biology techniques which are well-known to a person of ordinary skill in the art. For example, the saRNA of the present disclosure may be chemically synthesized or recombinantly produced using methods known in the art. By “upregulation” or “activation” of a gene is meant an increase in the level of expression of a gene, or levels of the polypeptide(s) encoded by a gene or the activity thereof, or levels of the RNA transcript(s) transcribed from the template strand of a gene above that observed in the absence of the saRNA of the present disclosure. The saRNA of the present disclosure may have a direct upregulating effect on the expression of the target gene.

[0017] The saRNAs of the present disclosure may have an indirect upregulating effect on the RNA transcript(s) transcribed from the template strand of the target gene and / or the polypeptide(s) encoded by the target gene or mRNA. The RNA transcript transcribed from the target gene is referred to thereafter as the target transcript. The target transcript may be an mRNA of the target gene. The target transcript may exist in the mitochondria. The saRNAs of the present disclosure may have a downstream effect on a biological process or activity. In such embodiments, a saRNA targeting a first transcript may have an effect (either upregulating or downregulating) on a second, non-target transcript.

[0018] In some embodiments, the saRNA of the present disclosure is a duplex comprising 2 strands. A “strand” in the context of the present disclosure means a contiguous sequence of nucleotides, including non-naturally occurring or modified nucleotides. At least one strand of a saRNA may comprise a region that is complementary to a region on the guide strand of the target gene (targeted sequence) and has sequence identity with a region on the coding strand of the target gene. Such a strand is called an antisense or guide strand of the saRNA duplex. A second strand of a saRNA that comprises a region complementary to the antisense strand of the saRNA is called a sense or passenger strand.

[0019] A saRNA duplex may also be formed from a single molecule that is at least partly self- complementary forming a hairpin structure, including a duplex region. In such case, the term “strand” refers to one of the regions of the saRNA that is complementary to another internal region of the saRNA. The guide strand of the saRNA will have no more than 5, or no more than 4 or 3, or no more than 2, or no more than 1, or no mismatches with the sequence within the region on the template strand of the target gene (targeted sequence).

[0020] In some embodiments, the passenger strand of a saRNA may comprise at least one nucleotide that is not complementary to the corresponding nucleotide on the guide strand, called a mismatch with the guide strand. The mismatch with the guide strand may encouragepreferential loading of the guide strand. See, e.g., Wu et al., PLoS ONE, vol.6 (12):e28580 (2011). In one embodiment, the at least one mismatch with the guide strand may be at 3’ end of the passenger strand. In one embodiment, the 3’ end of the passenger strand may comprise 1-5 mismatches with the guide strand. In one embodiment, the 3’ end of the passenger strand may comprise 2-3 mismatches with the guide strand. In one embodiment, the 3’ end of the passenger strand may comprise 6-10 mismatches with the guide strand.

[0021] In some embodiments, the saRNA may comprise a number of unpaired nucleotides at the 3' end of a strand forming 3' overhangs or tails. The number of unpaired nucleotides forming the 3' overhang of each strand may be in the range of 1 to 5 nucleotides, or 1 to 3 nucleotides, or 2 nucleotides. Non-limiting examples of 3’ overhangs include -UU, -UUU, -mUmU (m strands for 2’-OMe modification) or -ps-mU-ps-mU.Chemical Modi fications o f saRNAs

[0022] Herein, in saRNA, the terms “modification” or, as appropriate, “modified” refer to structural and / or chemical modifications with respect to A, G, U or C ribonucleotides. Nucleotides in the saRNAs of the present disclosure may comprise non-standard nucleotides, such as non-naturally occurring nucleotides or chemically synthesized nucleotides or deoxynucleotides. The saRNA of the present disclosure may include any useful modification, such as to the sugar, the nucleobase, or the intemucleoside linkage (e.g. to a linking phosphate / to a phosphodiester linkage / to the phosphodiester backbone). One or more atoms of a pyrimidine or purine nucleobase may be replaced or substituted with optionally substituted amino, optionally substituted thiol, optionally substituted alkyl (e.g., methyl or ethyl), or halo (e.g., chloro or fluoro). In certain embodiments, modifications (e.g., one or more modifications) are present in each of the sugar and the intemucleoside linkage. Modifications according to the present disclosure may be modifications of ribonucleic acids (RNAs) to deoxyribonucleic acids (DNAs), threose nucleic acids (TNAs), glycol nucleic acids (GNAs), peptide nucleic acids (PNAs), locked nucleic acids (LNAs), unlocked nucleic acid (UNA), or hybrids thereof. In a non-limiting example, the 2’ -OH of U is substituted with 2’-0Me. In some embodiments, the saRNA may comprise nucleobases such as diaminopurine and 2’-thio-uracil / thymine.

[0023] In one embodiment, the saRNAs of the present disclosure may comprise at least one modification described herein.

[0024] In another embodiment, the saRNA is an saRNA duplex and the sense strand and / or antisense sequence may independently comprise at least one modification. As a non-limiting example, the sense sequence may comprise a modification and the antisense strand may be unmodified. As another non-limiting example, the antisense sequence may comprise amodification and the sense strand may be unmodified. As yet another non-limiting example, the sense sequence may comprise more than one modification and the antisense strand may comprise one modification. As a non-limiting example, the antisense sequence may comprise more than one modification and the sense strand may comprise one modification. As yet another non-limiting example, the sense sequence is fully modified, i.e., each nucleotide is chemically modified. As yet another non-limiting example, both the antisense sequence and the sense sequence are fully modified.

[0025] The saRNA of the present disclosure can include a combination of modifications to the sugar, the nucleobase, and / or the intemucleoside linkage. These combinations can include any one or more modifications described herein or in WO2013 / 052523, in particular Formulas (la)- (Ia-5), (Ib)-(If), (Ila)-(IIp), (IIb-1), (IIb-2), (IIc-l)-(IIc-2), (IIn-1), (IIn-2), (IVa)-(IVl), and (IXa)- (IXr)).

[0026] The saRNA of the present disclosure may or may not be uniformly modified along the entire length of the molecule. For example, one or more or all types of nucleotide (e.g., purine or pyrimidine, or any one or more or all of A, G, U, C) may or may not be uniformly modified in the saRNA of the disclosure. In some embodiments, all nucleotides X in an saRNA of the disclosure are modified, wherein X may be any one of or any combinations of nucleotides A, G, U, and C.

[0027] Different sugar modifications, nucleotide modifications, and / or intemucleoside linkages (e.g., backbone structures) may exist at various positions in an saRNA. One of ordinary skill in the art will appreciate that the nucleotide analogs or other modification(s) may be located at any position(s) of an saRNA such that the function of saRNA is not substantially decreased. The saRNA of the present disclosure may contain from about 1% modified nucleotide to about 100% modified nucleotides (either in relation to overall nucleotide content, or in relation to one or more types of nucleotide, i.e. any one or more of A, G, U or C) or any intervening percentage (e.g., from 1% to 20%, from 1% to 25%, from 1% to 50%, from 1% to 60%, from 1% to 70%, from 1% to 80%, from 1% to 90%, from 1% to 95%, from 10% to 20%, from 10% to 25%, from 10% to 50%, from 10% to 60%, from 10% to 70%, from 10% to 80%, from 10% to 90%, from 10% to 95%, from 10% to 100%, from 20% to 25%, from 20% to 50%, from 20% to 60%, from 20% to 70%, from 20% to 80%, from 20% to 90%, from 20% to 95%, from 20% to 100%, from 50% to 60%, from 50% to 70%, from 50% to 80%, from 50% to 90%, from 50% to 95%, from 50% to 100%, from 70% to 80%, from 70% to 90%, from 70% to 95%, from 70% to 100%, from 80% to 90%, from 80% to 95%, from 80% to 100%, from 90% to 95%, from 90% to 100%, and from 95% to 100%).

[0028] In some embodiments, the saRNA comprises at least one sugar modification.Nonlimiting examples of the sugar modification may include the following:cET (methyl LNA)

[0029] In some embodiments, at least one of the 2' positions_of the sugar (OH in RNA or H in DNA) of a nucleotide of the saRNA is substituted with -OMe, referred to as 2’-OMe.

[0030] In some embodiments, at least one of the 2' positions_of the sugar (OH in RNA or H in DNA) of a nucleotide of the saRNA is substituted with -F, referred to as 2’-F.

[0031] In some embodiments, the saRNA comprises at least one phosphorothioate linkage or methylphosphonate linkage between nucleotides.

[0032] In some embodiments, the saRNA comprises 3’ and / or 5’ capping or overhang. In some embodiments, the saRNA of the present disclosure may comprise at least one inverted deoxyribonucleoside or dideoxyribonucleoside overhang (e.g., dT or ddT). The inverted overhang, e.g., dT, may be at the 5’ terminus or 3’ terminus of the passenger (sense) strand. In some embodiments, the saRNA of the present disclosure may comprise inverted abasic (invAb) modifications on the passenger strand. The at least one inverted abasic modification may be on5’ end, or 3’ end, or both ends of the passenger strand. The inverted abasic modification may encourage preferential loading of the guide (antisense) strand. In some embodiments, the overhang may be phosphorothiolated. In some embodiments, the overhang might be 2’-0Me modified nucleosides.

[0033] In some embodiments, the saRNA comprises at least one 5’-(E)-vinylphosphonate (5’- - VP) or 5’-(E)-vinylphosphate modification.E-VP

[0034] In some embodiments, the saRNA comprises at least one glycol nucleic acid (GNA), an acyclic nucleic acid analogue, as a modification.GNA

[0035] In some embodiments, the saRNA comprises at least one locked nucleic acid (LNA).LNA

[0036] In some embodiments, the saRNA comprises at least one unlocked nucleic acid (UNA). At least one nucleic acid of the saRNA may be replaced with UNA. The UNA may locate in the center region of the saRNA, near the 3 ’end of the saRNA, or near the 5’ end of the saRNA. In some embodiments, saRNAs comprising UNAs have improved efficacy and / or improved stability compared to saRNAs without UNAs. wherein the sense strand and / or the antisense strand further comprises at least one additional chemical modification, such as but not limited to 2’-F modification, 2’-OMe modification, alkyl spacer, locked nucleic acid (LNA), a phosphorothioate linkage, 2’F-ANA, 4’S-RNA, 4’S-FANA, 2’-0-M0E, 2’-O-allyl, 2’-O- ethylamine, 2’-O-cyanoetyl, 2’-O-acetalester, 4'-C-aminometyl-2'-O-methyl, 2’ -azido, methylene-cLNA, N-MeO-amino BNA, N-Me-aminooxy BNA, 2’,4’-BNANC[NMe], MC, ONA, DNA, tc-DNA, CeNA, ANA, HNA, or cET.wx CaseUNA

[0037] In some embodiments, the saRNA of the present disclosure comprises an antisense strand and a sense strand, wherein the sense strand has 19 nucleotides and the antisense strand has 19-22 nucleotides, wherein the antisense strand comprises a 2’-OMe modification on the 6thposition of the strand, and / or wherein the antisense strand has phosphor othioate (-ps-) bonds between the 1stand 2ndpositions, between the 2ndand 3rdpositions, between the last and second to the last positions, and / or between the second to the last and third to the last positions. The antisense strand of the saRNA has an overhang comprising 2-3 nucleotides at the 3’ end (a 3’ overhang), such as but not limited to -UU, -UUU, -mUmU, or -ps-mU-ps-mU. Optionally, the antisense strand has at least one additional modification. For example, the antisense strand may have a E-vinylphosphonate (£-VP) modification at the 5 ’ end. The sense strand of the saRNA comprises at least one modification. The sense strand of the saRNA does not have any 3’ overhang. Alternatively, the sense strand has a 3’ overhang comprising 2-3 nucleotides such as but not limited to UU, UUU, or mUmU (m strands for 2’-0Me modification).

[0038] In some embodiments, the saRNA of the present disclosure comprises an antisense strand and a sense strand, wherein the sense strand has 19 nucleotides and the antisense strand has 19-22 nucleotides, wherein the antisense strand comprises a 2’-0Me modification on the 6thposition of the strand, and / or wherein the antisense strand has phosphor othioate (-ps-) bonds between the 1stand 2ndpositions, between the 2ndand 3rdpositions, between the last and second to the last positions, and / or between the second to the last and third to the last positions, and / or wherein the antisense strand comprises 2’-F modifications on the 2nd, 3rdand / or 4thpositions. The antisense strand of the saRNA has an overhang comprising 2-3 nucleotides at the 3’ end (a 3’ overhang), such as but not limited to -UU, -UUU, -mUmU, or -ps-mU-ps-mU. Optionally, the antisense strand has at least one additional modification. For example, the antisense strand may have a E-vinylphosphonate (£-VP) modification at the 5’ end. The sense strand of the saRNA comprises at least one modification. The sense strand of the saRNA does not have any 3’ overhang. Alternatively, the sense strand has a 3’ overhang comprising 2-3 nucleotides such as but not limited to UU, UUU, or mUmU (m strands for 2’-0Me modification).

[0039] In some embodiments, the saRNA of the present disclosure comprises an antisense strand and a sense strand, wherein the sense strand has 19 nucleotides and the antisense strand has 19-22 nucleotides, wherein the antisense strand comprises a 2’-0Me modification on the 6thposition of the strand, and / or wherein the antisense strand has phosphorothioate (-ps-) bonds between the 1stand 2ndpositions, between the 2ndand 3rdpositions, between the last and second to the last positions, and / or between the second to the last and third to the last positions, and / or wherein the antisense strand comprises 2’-F modifications on the 2nd, 3rdand / or 4thpositions, and / or wherein the antisense strand does not have a 2’-F modification at the 16thposition. In some cases, the antisense strand has a 2’-0Me modification at the 16thposition. The antisense strand of the saRNA has an overhang comprising 2-3 nucleotides at the 3’ end (a 3’ overhang), such as but not limited to -UU, -UUU, -mUmU, or -ps-mU-ps-mU. Optionally, the antisense strand has at least one additional modification. For example, the antisense strand may have a E- vinylphosphonate (E-VP) modification at the 5’ end. The sense strand of the saRNA comprises at least one modification. The sense strand of the saRNA does not have any 3’ overhang. Alternatively, the sense strand has a 3’ overhang comprising 2-3 nucleotides such as but not limited to UU, UUU, or mUmU (m strands for 2’-0Me modification).

[0040] In some embodiments, the saRNA of the present disclosure comprises an antisense strand and a sense strand, wherein the sense strand has 19 nucleotides and the antisense strand has 19-22 nucleotides, wherein the antisense strand comprises a 2’-0Me modification on the 6thand 16thposition of the strand, and / or wherein the antisense strand has phosphorothioate (-ps-) bonds between the 1stand 2ndpositions, between the 2ndand 3rdpositions, between the last and second to the last positions, and / or between the second to the last and third to the last positions, and / or wherein the antisense strand comprises 2’-F modifications on the 2nd, 3rd, 4thand / or 14thpositions. The antisense strand of the saRNA has an overhang comprising 2-3 nucleotides at the 3’ end (a 3’ overhang), such as but not limited to -UU, -UUU, -mUmU, or -ps-mU-ps-mU. Optionally, the antisense strand has at least one additional modification. For example, the antisense strand may have a E-vinylphosphonate (E-VP) modification at the 5’ end. The sense strand of the saRNA comprises at least one modification. The sense strand of the saRNA does not have any 3’ overhang. Alternatively, the sense strand has a 3’ overhang comprising 2-3 nucleotides such as but not limited to UU, UUU, or mUmU (m strands for 2’-0Me modification).

[0041] In some embodiments, the saRNA of the present disclosure comprises an antisense strand and a sense strand, wherein the sense strand has 19 nucleotides and the antisense strand has 19-22 nucleotides, wherein the antisense strand comprises a 2’-0Me modification on the 6thand / or 16thposition of the strand, and / or wherein the antisense strand has phosphorothioate (-ps- ) bonds between the 1stand 2ndpositions, between the 2ndand 3rdpositions, between the last and second to the last positions, and / or between the second to the last and third to the last positions, wherein the antisense strand comprises 2’-F modifications on the 2nd, 3rd, 4thand / or 14thpositions, and / or wherein the antisense strand has 2’-F modifications on the 17thand / or the 18thposition. The antisense strand of the saRNA has an overhang comprising 2-3 nucleotides at the 3’ end (a 3’ overhang), such as but not limited to UU, UUU, mUmU, or -ps-mU-ps-mU. Optionally, the antisense strand has at least one additional modification. The sense strand of the saRNA comprises at least one modification. For example, the antisense strand may have a E- vinylphosphonate (£-VP) modification at the 5’ end. The sense strand of the saRNA does not have any 3’ overhang. Alternatively, the sense strand has a 3’ overhang comprising 2-3 nucleotides such as but not limited to UU, UUU, or mUmU (m strands for 2’-0Me modification).

[0042] In some embodiments, the saRNA of the present disclosure comprises an antisense strand and a sense strand, wherein the sense strand has 19 nucleotides and the antisense strand has 19-22 nucleotides (such as 19, 20, 21 or 22 nucleotides),- wherein the antisense strand comprises a 2’-0Me modification on the 6thposition of the strand,- wherein the antisense strand has phosphorothioate (-ps-) bonds between the 1stand 2ndpositions, between the 2ndand 3rdpositions, between the last and second to the last positions, and / or between the second to the last and third to the last positions,- wherein the antisense strand comprises 2’-F modifications on the 2nd, 3rd, 4thpositions,- wherein the antisense strand of the saRNA comprises at least one unmodified nucleotide, and optionally the unmodified nucleotide is A, C or G,- wherein all the Us of the antisense strand are modified (such as but not limited to with 2’-0Me or 2’-F), and / or- wherein the antisense strand of the saRNA has an overhang comprising 2-3 nucleotides at the 3’ end (a 3’ overhang) (such as but not limited to UU, UUU, mUmU, or -ps-mU-ps-mU). Optionally, the antisense strand has at least one additional modification. For example, the antisense strand might further comprise a 2’-F modification on the 1st, 9thand / or 14thposition of the strand. In another example, the antisense strand has a 2’-0Me modification at the 10thand / or 13thposition. In yet another example, the antisense strand has 2’-F modifications on the 17thposition and / or the 18thposition. In yet another example, the antisense strand does not have a 2’- F modification at the 16thposition. In yet another example, the antisense strand has a 2’-0Me modification at the 16thposition. In yet another example, the antisense strand of the saRNA comprises A- VP at the 5’ end.

[0043] In some embodiments, the saRNA of the present disclosure comprises an antisense strand and a sense strand, wherein the sense strand has 19 nucleotides and the antisense strand has 19-22 nucleotides (such as 19, 20, 21 or 22 nucleotides),- wherein the sense strand of the saRNA comprises 2’-0Me modifications on the 2ndand 14thpositions of the strand,- wherein sense strand has phosphorothioate (-ps-) bonds between the 1stand 2ndpositions, between the 2ndand 3rdpositions, between the 17thand 18thpositions and / or the 18thand 19thpositions,- wherein the sense strand of the saRNA does not have any 3’ overhang,- wherein the sense strand of the saRNA comprises at least one unmodified nucleotide, wherein optionally the unmodified nucleotide is A, C or G,- wherein all the Us of the sense strand are modified (such as but not limited to with 2’-0Me or 2’-F).Optionally, the sense strand has at least one additional modification. For example, the sense strand of the saRNA might comprise a 2’-0Me modification on the 3rdpositions of the strand.

[0044] In some embodiments, In some embodiments, the saRNA of the present disclosure comprises an antisense strand and a sense strand, wherein the sense strand has 19 nucleotides and the antisense strand has 19-22 nucleotides (such as 19, 20, 21 or 22 nucleotides), wherein the antisense strand:- comprises a 2’-0Me modification on the 6thposition of the strand,- has phosphorothioate (-ps-) bonds between the 1stand 2ndpositions, between the 2ndand 3rdpositions, between the last and second to the last positions, and / or between the second to the last and third to the last positions,- comprises 2’-F modifications on the 2nd, 3rd, 4thpositions,- comprises at least one unmodified nucleotide, and optionally the unmodified nucleotide is A, C or G,- all the Us are modified (such as but not limited to with 2’-0Me or 2’-F), and / or- has an overhang comprising 2-3 nucleotides at the 3’ end (a 3’ overhang) (such as but not limited to UU, UUU, mUmU, or -ps-mU-ps-mU), and wherein the sense strand:- comprises 2’-0Me modifications on the 2ndand 14thpositions of the strand,- has phosphorothioate (-ps-) bonds between the 1stand 2ndpositions, between the 2ndand 3rdpositions, between the 17thand 18thpositions and / or the 18thand 19thpositions,- does not have any 3 ’ overhang,- comprises at least one unmodified nucleotide, wherein optionally the unmodified nucleotide is A, C or G, and / or- all the Us are modified (such as with 2’-0Me or 2’-F).

[0045] In some embodiments, the saRNA of the present disclosure comprises an antisense strand and a sense strand, wherein the sense strand has 19 nucleotides and the antisense strand has 19-22 nucleotides (such as 19, 20, 21 or 22 nucleotides),- wherein the antisense strand comprises a 2’-0Me modification on the 6th position of the strand,- wherein the antisense strand has phosphorothioate (-ps-) bonds between the 1st and 2nd positions, between the 2nd and 3rd positions, between the last and second to the last positions, and / or between the second to the last and third to the last positions,- wherein all the Us of the antisense strand are modified (such as but not limited to with 2’-0Me or 2’-F),- wherein the sense strand of the saRNA comprises 2’-0Me modifications on the 2nd and 14th positions of the strand,- wherein all the Us of the sense strand are modified (such as but not limited to with either 2’- OMe modification(s) or 2’-F modification(s)), and- wherein sense strand has phosphorothioate (-ps-) bonds between the 1st and 2nd positions, between the 2nd and 3rd positions, between the 17th and 18th positions and / or the 18th and 19th positions. In some embodiments, the antisense strand further comprises 2’-F modifications on the 2nd, 3rd, 4th positions. In some embodiments, the antisense strand further comprises at least one unmodified nucleotide, and optionally the unmodified nucleotide is A, C or G. In some embodiments, the antisense strand has an overhang comprising 2-3 nucleotides at the 3’ end (a 3’ overhang) (such as but not limited to UU, UUU, mUmU, or -ps-mU-ps-mU). In some embodiments, the antisense strand has at least one additional modification. For example, the antisense strand might further comprise a 2’-F modification on the 1st, 9th and / or 14th position of the strand. In another example, the antisense strand has a 2’-0Me modification at the 10th and / or 13th position. In yet another example, the antisense strand has 2’-F modifications on the 17th position and / or the 18th position. In yet another example, the antisense strand does not have a 2’-F modification at the 16th position. In yet another example, the antisense strand has a 2’- OMe modification at the 16th position. In yet another example, the antisense strand of the saRNA comprises E-VP at the 5’ end. In some embodiments, the sense strand does not have any 3’ overhang. In some embodiments, the sense strand comprises at least one unmodified nucleotide, and optionally the unmodified nucleotide is A, C or G. In some embodiments, the sense strand has at least one additional modification. For example, the sense strand of the saRNA might comprise a 2’-0Me modification on the 3rd positions of the strand.

[0046] In some embodiments, the target gene of the saRNAs is HNF4a. Non-limiting examples of chemically modified HNF4a-saRNAs are shown in Table 4.1. All the saRNAs have the same base sequence (HNF4A-PR3) but have different combinations of chemical modifications. The first strand of a double-stranded saRNA may have at least 60%, 70%, 80% or 90% identity with a sequence selected from the sequences of the guide strands in Table 4.1. In one embodiment, the first strand of the double-stranded saRNA comprises a sequence selected from the sequences of the guide strands in Table 4.1. The second strand of a double-stranded saRNA may have at least 60%, 70%, 80% or 90% identity with a sequence selected from the sequences of the passenger strands in Table 4.1. In one embodiment, the second strand of the double-stranded saRNA comprises a sequence selected from the sequences of the passenger strands in Table 4.1. In some embodiments, the antisense strand of the HNF4a-saRNA is selected from HNF4A-PR3- AS06, AS07 and AS08. In some embodiments, the antisense strand of the HNF4a-saRNA is HNF4A-PR3-AS06 or AS08.Table 4.1. Sequences of non-limiting HNF4a-saRNA examples (with chemical modification(s))m: 2'-OMe modification f: 2'-F modification-ps-: phosphorothioate bond

[0047] In some embodiments, the target gene of the saRNAs is HBG1 and / or HBG2 (also be referred to as H G) gene. Non-limiting examples of chemically modified HBG-saRNAs are shown in Table 4.2. The first strand of a double-stranded saRNA may have at least 60%, 70%, 80% or 90% identity with a sequence selected from the sequences of the guide strands in Table 4.2. In one embodiment, the first strand of the double-stranded saRNA comprises a sequence selected from the sequences of the guide strands in Table 4.2. The second strand of a doublestranded saRNA may have at least 60%, 70%, 80% or 90% identity with a sequence selected from the sequences of the passenger strands in Table 4.2. In one embodiment, the second strandof the double-stranded saRNA comprises a sequence selected from the sequences of the passenger strands in Table 4.2.Table 4.2. Sequences of non-limiting HBG-saRNA examples (with chemical modification(s))m: 2'-0Me modification f: 2'-F modification-ps-: phosphorothioate bond saRNA Conjugates and Combinations

[0048] Conjugation may result in increased stability and / or half-life and may be particularly useful in targeting the saRNA of the present disclosure to specific sites in the cell, tissue or organism. The saRNA of the present disclosure can be designed to be conjugated to other polynucleotides, dyes, intercalating agents (e.g. acridines), cross-linkers (e.g. psoralene, mitomycin C), porphyrins (TPPC4, texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g. EDTA), alkylating agents, phosphate, amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]?, polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes, haptens (e.g. biotin), transport / absorption facilitators (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases, proteins, e.g., glycoproteins, or peptides, e.g., molecules having a specific affinity for a co-ligand, or antibodies e.g., an antibody, that binds to a specified cell type such as a cancer cell, endothelial cell, or bone cell, hormones and hormone receptors, non-peptidic species, such as lipids, lectins, carbohydrates, vitamins, cofactors, or a drug. Suitable conjugates for nucleic acid molecules are disclosed in International Publication WO 2013 / 090648.

[0049] According to the present disclosure, saRNA of the present disclosure may be administered with, or further include one or more of RNAi agents, small interfering RNAs (siRNAs), small hairpin RNAs (shRNAs), long non-coding RNAs (IncRNAs), enhancer RNAs, enhancer-derived RNAs or enhancer-driven RNAs (eRNAs), microRNAs (miRNAs), miRNA binding sites, antisense RNAs, ribozymes, catalytic DNA, tRNA, RNAs that induce triple helix formation, aptamers or vectors, and the like to achieve different functions. The one or more RNAi agents, small interfering RNAs (siRNAs), small hairpin RNAs (shRNAs), long noncoding RNAs (IncRNA), microRNAs (miRNAs), miRNA binding sites, antisense RNAs,ribozymes, catalytic DNA, tRNA, RNAs that induce triple helix formation, aptamers or vectors may comprise at least one modification or substitution.

[0050] In some embodiments, the modification is selected from a chemical substitution of the nucleic acid at a sugar position, a chemical substitution at a phosphate position and a chemical substitution at a base position. In other embodiments, the chemical modification is selected from incorporation of a modified nucleotide; 3' capping; conjugation to a high molecular weight, non- immunogenic compound; conjugation to a lipophilic compound; and incorporation of phosphorothioate into the phosphate backbone. In one embodiment, the high molecular weight, non-immunogenic compound is polyalkylene glycol, or polyethylene glycol (PEG).

[0051] The saRNA-aptamer conjugate may be formed using any known method for linking two moieties, such as direct chemical bond formation, or via a linker such as streptavidin and so on.

[0052] The saRNA of the present disclosure may be provided in combination with other active ingredients known to have an effect in the particular method being considered. The other active ingredients may be administered simultaneously, separately, or sequentially with the saRNA of the present disclosure. In one embodiment, saRNA of the present disclosure is administered with saRNA modulating a different target gene.

[0053] In one embodiment, the saRNA is conjugated with a carbohydrate ligand, such as any carbohydrate ligand disclosed in US Pat Nos. 8106022 and 8828956 to Manoharan et al.. For example, the carbohydrate ligand may be monosaccharide, disaccharide, tri saccharide, tetrasaccharide, oligosaccharide, or polysaccharide. These carbohydrate-conjugated RNA agents may target the parenchymal cells of the liver. In one embodiment, the saRNA is conjugated with more than one carbohydrate ligand, preferably two or three. In one embodiment, the saRNA is conjugated with one or more galactose moiety. In another embodiment, the saRNA is conjugated at least one (e.g., two or three or more) lactose molecules (lactose is a glucose coupled to a galactose). In another embodiment, the saRNA is conjugated with at least one (e.g., two or three or more) N-Acetyl-Galactosamine (GalNAc), N-Ac-Glucosamine (GluNAc), or mannose (e.g., mannose-6-phosphate). In one embodiment, the saRNA is conjugated with at least one mannose ligand, and the conjugated saRNA targets macrophages.GalNAc-nucleotide (GalNAc-saRNA) Conjugates

[0054] In some embodiments, the saRNA is covalently connected to a carbohydrate moiety, wherein the moiety comprises at least one (e.g., two or three or more) N-Acetyl-Galactosamine (GalNAc) or derivative thereof, to form a GalNAc-saRNA conjugate. GalNAc is an amino sugarderivative of galactose comprising a structureeffective moiety to carry nucleic acids construct into hepatocytes. It has been shown that discrete structure of ternary GalNAc may be used for efficacious delivery of single stranded and double stranded oligonucleotides for gene silencing. The GalNAc-nucleotide conjugate may be delivered to cells expressing asialoglycoprotein receptor without any transfection agent. The nucleotide may be part of a saRNA, and the GalNAc-nucleotide conjugate is referred to as a GalNAc-saRNA conjugate.

[0055] In some embodiments, the present disclosure provides a GalNAc-saRNA conjugate comprising a small activating RNA (saRNA) connected to a GalNAc moiety, wherein the saRNA comprises at least one modification, such modification which may optionally be independent of the connected GalNAc. The saRNA may comprise at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 modifications for each strand.

[0056] In some embodiments, the saRNA of the conjugate is at least about 50% modified, i.e., at least about 50% of the nucleotides are modified. In some embodiments, the saRNA is at least about 75% modified, i.e., at least about 75% of the nucleotides are modified. In some embodiments, both strands of the saRNA may be modified across the whole length (100% modified). It is to be understood that since a nucleotide (sugar, base and phosphate moiety, e.g., linker) may each be modified, any modification to any portion of a nucleotide, or nucleoside, will constitute a modification. In some embodiments, each nucleotide of the saRNA sense strand and / or antisense strand is modified.

[0057] In some embodiments, the saRNA is at least about 10% modified in only one component of the nucleotide, with such component being selected from the nucleobase, sugar or linkage between nucleosides. For example, modifications of an saRNA may be made to at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90% or about 100% of the nucleobases, sugars or linkages of said saRNA.

[0058] In some embodiments, the saRNA of the conjugate comprises at least one sugar modification. In some embodiments, at least one of the 2' positions of the sugar (OH in RNA or H in DNA) of a nucleotide of the saRNA is substituted with -OMe, referred to as 2’-0Me. In some embodiments, at least one of the 2' positions of the sugar (OH in RNA or H in DNA) of a nucleotide of the saRNA is substituted with -F, referred to as 2’-F.

[0059] In some embodiments, the saRNA of the conjugate comprises 3’ and / or 5’ capping or overhang. In some embodiments, the saRNA of the present invention may comprise at least one inverted deoxyribonucleoside overhang. The inverted overhang, e.g., dT, may be at 5’ terminus or 3’ terminus of the passenger (sense) strand. In some embodiments, the saRNA of the present invention may comprise inverted abasic modifications on the passenger strand. The at least one inverted abasic modification may be on 5’ end, or 3’ end, or both ends of the passenger strand. The inverted abasic modification may encourage preferential loading of the guide strand.

[0060] In some embodiments, the saRNA comprises at least one phosphorothioate (ps) linkage or methylphosphonate (mps) linkage between nucleotides.

[0061] In some embodiments, the GalNAc moiety is attached to the 2'- or 3'- position of the ribosugar, or to a nucleobase of a nucleotide of a saRNA. A phosphodiester or phosphorothioate linkage may be between the GalNAc moiety and the nucleotide.

[0062] In some embodiments, the GalNAc moiety is attached to a nucleotide of a saRNA via a linker. The linker may be attached to any appropriate position of a nucleotide of the saRNA. The linker may bind to the GalNAc moiety covalently or non-covalently.

[0063] In some cases, the linker is connected to the terminal of a strand of the saRNA. In some cases, the linker is connected to the 5’ end of the sense strand or antisense strand. In some cases, the linker is connected to the 3’ end of the sense strand (shown below) or antisense strand.3'or 5' end of SSLinker — GalNAc

[0064] In some cases, the linker is connected to an internal nucleotide of a strand of the saRNA. In some cases, the linker is connected to an internal nucleotide of the sense strand of the saRNA (shown below). In some cases, the linker is connected to an internal nucleotide of the anti-sense strand of the saRNA.Linker — GalNAc

[0065] Any attachment method disclosed in Manoharan et al., Chemical Biology, vol.10(5): 1181, (2015) or Manoharan et al., ChemBioChem, vol,16(6):903, (2015), the contents of each of which are incorporated herein by reference in their entirety, may be used to attach the GalNAc moiety to the saRNA.

[0066] In some cases, the linker of the GalNAc-saRNA conjugate is a direct bond or an atom such as oxygen or sulfur, a unit such as -NH-, -C(O)-, -C(O)NH-, -S(O)-, -SO2-, -SO2NH-or a chain of atoms, such as, but not limited to, alkyl, alkenyl, alkynyl, arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl, heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, alkylarylalkyl, alkylarylalkenyl, alkylarylalkynyl, alkenylarylalkyl, alkenylarylalkenyl, alkenylarylalkynyl, alkynylarylalkyl, alkynylarylalkenyl, alkynylarylalkynyl, alkylheteroarylalkyl, alkylheteroarylalkenyl, alkylheteroarylalkynyl, alkenylheteroarylalkyl, alkenylheteroarylalkenyl, alkenylheteroarylalkynyl, alkynylheteroarylalkyl, alkynylheteroarylalkenyl, alkynylheteroarylalkynyl, alkylheterocyclylalkyl, alkylheterocyclylalkenyl, alkylhererocyclylalkynyl, alkenylheterocyclylalkyl, alkenylheterocyclylalkenyl, alkenylheterocyclylalkynyl, alkynylheterocyclylalkyl, alkynylheterocyclylalkenyl, alkynylheterocyclylalkynyl, alkylaryl, alkenylaryl, alkynylaryl, alkylheteroaryl, alkenylheteroaryl, alkynylhereroaryl, wherein each group may be substituted or unsubstituted.

[0067] In some cases, the linker is a cleavable linker. The cleavable linker may be cleaved at a certain pH, by a certain enzyme, or at a certain redox environment. The cleavable linker may comprise an ester bond, an acid-labile bond, a disulfide bond, or a phosphate bond by way of example.

[0068] In some cases, the linker is a non-cleavable linker.

[0069] In some cases, the linker comprises an amine group, such as -NH-(CH2)e- or NH2-(CH2)e- (referred to as NH2C6, C6NH2, or C6). For GalNAc clusters that comprise a carboxylic acid at the terminus, the carboxylic acid reacts with the amine on the linker and the GalNAc cluster is directly attached to the saRNA-C6NH-.

[0070] In some cases, the linker comprises a carboxylic acid group, such as -O-CO-(CH2)n- CO-NH-(CH2)e-, n=2, 3, 4, 5 or 6. For GalNAc clusters that comprise an amine at the terminus, the amine reacts with the carboxylic acid on the linker and the GalNAc cluster is directed attached to saRNA-(CH2)6-NH-CO-(CH2)n-CO-.

[0071] In some cases, a phosphorothioate linkage is between the linker and the sense strand.

[0072] In some cases, the GalNAc moiety may be a triantennary GalNAc-cluster. AnyGalNAc cluster disclosed in Prakash et al., Journal of Medicinal Chemistry, vol.59:2718-2733 (2016), the contents of which are incorporated herein by reference in their entirety, such as Tris based GalNAc clusters, Triacid based GalNAc clusters, Lys-Lys based GalNAc clusters, Lys- Gly based GalNAc clusters, Trebler based clusters, hydroxyprolinol based clusters in Fig. 2 of Prakash et al., may be used according to the current disclosure. The GalNAc cluster may have astructure of:

[0073] When a linker is used to connect the 3’ or 5’ end of the sense strand to the GalNAc moiety, the GalNAc-saRNA conjugate comprises a structure of:

[0074] For example, the GalNAc-saRNA conjugate may have a structure of:whereinX is O or S.

[0075] Non-limiting examples of GalNAc-saRNA conjugates are shown in Table 5. All the saRNAs have the same base sequence (Hnf4a-PR2) but have different combinations of chemical modifications.Table 5. Sequences of non-limiting GalNAc-NHF4a-saRNA conjugate examplesm: 2'-0Me modification f: 2'-F modification ps: phosphorothioate bond C6: (CH2)e linkerGalNAc3: tri-GalNAc moietyVP: E-vinylphosphonateII. Composition of the disclosure

[0076] One aspect of the present disclosure provides pharmaceutical compositions comprising a small activating RNA (saRNA) that upregulates a target gene, and at least one pharmaceutically acceptable carrier.Formulation, Delivery, Administration, and Dosins

[0077] Pharmaceutical formulations may additionally comprise a pharmaceutically acceptable excipient, which, as used herein, includes, but is not limited to, any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, and the like, as suited to the particular dosage form desired. Various excipients for formulating pharmaceutical compositions and techniques for preparing the composition are known in the art (see Remington: The Science and Practice of Pharmacy, 21stEdition, A. R. Gennaro, Lippincott, Williams & Wilkins, Baltimore, MD, 2006). The use of a conventional excipient medium may be contemplated within the scope of the present disclosure, except insofar as any conventional excipient medium may be incompatible with a substance or its derivatives, such as by producing any undesirable biological effect or otherwise interacting in a deleterious manner with any other component s) of the pharmaceutical composition.

[0078] In some embodiments, compositions are administered to humans, human patients or subjects. For the purposes of the present disclosure, the phrase “active ingredient” generally refers to saRNA to be delivered as described herein.

[0079] Although the descriptions of pharmaceutical compositions provided herein are principally directed to pharmaceutical compositions which are suitable for administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to any other animal, e.g., to non-human animals, e.g. non-human mammals. Modification of pharmaceutical compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and / or perform such modification with merely ordinary, if any, experimentation. Subjects to which administration of the pharmaceutical compositions is contemplated include, but are not limited to, humans and / or other primates; mammals, including commercially relevant mammals such as cattle, pigs, horses, sheep, cats, dogs, mice, and / or rats; and / or birds, including commercially relevant birds such as poultry, chickens, ducks, geese, and / or turkeys.

[0080] Formulations of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, suchpreparatory methods include the step of bringing the active ingredient into association with an excipient and / or one or more other accessory ingredients, and then, if necessary and / or desirable, dividing, shaping and / or packaging the product into a desired single- or multi-dose unit.

[0081] A pharmaceutical composition in accordance with the disclosure may be prepared, packaged, and / or sold in bulk, as a single unit dose, and / or as a plurality of single unit doses. As used herein, a “unit dose” is discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient. The amount of the active ingredient is generally equal to the dosage of the active ingredient which would be administered to a subject and / or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage.

[0082] Relative amounts of the active ingredient, the pharmaceutically acceptable excipient, and / or any additional ingredients in a pharmaceutical composition in accordance with the disclosure will vary, depending upon the identity, size, and / or condition of the subject treated and further depending upon the route by which the composition is to be administered. By way of example, the composition may comprise between 0.1% and 100%, e.g., between .5 and 50%, between 1-30%, between 5-80%, at least 80% (w / w) active ingredient.Dosage Forms

[0083] A pharmaceutical composition described herein can be formulated into a dosage form described herein, such as a topical, intranasal, intratracheal, or injectable (e.g., intravenous, intraocular, intravitreal, intramuscular, intracardiac, intraperitoneal, subcutaneous). Liquid dosage forms, injectable preparations, pulmonary forms, and solid dosage forms described in WO 2013 / 090648 may be used as dosage forms for the saRNA of the present disclosure.III. Methods of Use

[0084] One aspect of the present disclosure provides methods of using saRNA of the present disclosure and pharmaceutical compositions comprising the saRNA and at least one pharmaceutically acceptable carrier. The saRNA of the present disclosure modulates the expression of its target gene. In one embodiment is provided a method of regulating the expression of a target gene in vitro and / or in vivo comprising administering the saRNA of the present disclosure. In one embodiment, the expression of the target gene is increased by at least 5, 10, 20, 30, 40%, or at least 45, 50, 55, 60, 65, 70, 75%, or at least 80% in the presence of the saRNA of the present disclosure compared to the expression of the target gene in the absence of the saRNA of the present disclosure. In a further embodiment, the expression of the target gene is increased by a factor of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or by a factor of at least 15, 20, 25, 30, 35, 40, 45, 50, or by a factor of at least 60, 70, 80, 90, 100, in the presence of the saRNA of thepresent disclosure compared to the expression of the target gene in the absence of the saRNA of the present disclosure.IV. Kits and DevicesKits

[0085] The disclosure provides a variety of kits for conveniently and / or effectively carrying out methods of the present disclosure. Typically, kits will comprise sufficient amounts and / or numbers of components to allow a user to perform multiple treatments of a subject(s) and / or to perform multiple experiments.

[0086] In one embodiment, the present disclosure provides kits for regulate the expression of genes in vitro or in vivo, comprising saRNA of the present disclosure or a combination of saRNA of the present disclosure, saRNA modulating other genes, siRNAs, miRNAs or other oligonucleotide molecules.

[0087] The kit may further comprise packaging and instructions and / or a delivery agent to form a formulation composition. The delivery agent may comprise a saline, a buffered solution, a lipidoid, a dendrimer or any delivery agent disclosed herein.

[0088] In one non-limiting example, the buffer solution may include sodium chloride, calcium chloride, phosphate and / or EDTA. In another non-limiting example, the buffer solution may include, but is not limited to, saline, saline with 2mM calcium, 5% sucrose, 5% sucrose with 2mM calcium, 5% Mannitol, 5% Mannitol with 2mM calcium, Ringer’s lactate, sodium chloride, sodium chloride with 2mM calcium and mannose (See U.S. Pub. No. 20120258046). In yet another non-limiting example, the buffer solutions may be precipitated or it may be lyophilized. The amount of each component may be varied to enable consistent, reproducible higher concentration saline or simple buffer formulations. The components may also be varied in order to increase the stability of saRNA in the buffer solution over a period of time and / or under a variety of conditions.Devices

[0089] The present disclosure provides for devices which may incorporate saRNA of the present disclosure. These devices contain in a stable formulation available to be immediately delivered to a subject in need thereof, such as a human patient.

[0090] Non-limiting examples of the devices include a pump, a catheter, a needle, a transdermal patch, a pressurized olfactory delivery device, iontophoresis devices, multi-layered microfluidic devices. The devices may be employed to deliver saRNA of the present disclosure according to single, multi- or split-dosing regiments. The devices may be employed to deliver saRNA of the present disclosure across biological tissue, intradermal, subcutaneously, orintramuscularly. More examples of devices suitable for delivering oligonucleotides are disclosed in WO 2013 / 090648.Definitions

[0091] For convenience, the meaning of certain terms and phrases used in the specification, examples, and appended claims, are provided below. If there is an apparent discrepancy between the usage of a term in other parts of this specification and its definition provided in this section, the definition in this section shall prevail.

[0092] About: As used herein, the term “about” means + / - 10% of the recited value.

[0093] Administered in combination: As used herein, the term “administered in combination” or “combined administration” means that two or more agents are administered to a subject at the same time or within an interval such that there may be an overlap of an effect of each agent on the patient. In some embodiments, they are administered within about 60, 30, 15, 10, 5, or 1 minute of one another. In some embodiments, the administrations of the agents are spaced sufficiently close together such that a combinatorial (e.g., a synergistic) effect is achieved.

[0094] Amino acid: As used herein, the terms "amino acid" and "amino acids" refer to all naturally occurring L-alpha-amino acids. The amino acids are identified by either the one-letter or three-letter designations as follows: aspartic acid (Asp:D), isoleucine (Ile:I), threonine (Thr:T), leucine (Leu:L), serine (Ser:S), tyrosine (Tyr:Y), glutamic acid (Glu:E), phenylalanine (Phe:F), proline (Pro:P), histidine (His:H), glycine (Gly:G), lysine (Lys:K), alanine (Ala:A), arginine (Arg:R), cysteine (Cys:C), tryptophan (Trp:W), valine (Val:V), glutamine (Gln:Q) methionine (Met:M), asparagines (Asn:N), where the amino acid is listed first followed parenthetically by the three and one letter codes, respectively.

[0095] Animal: As used herein, the term “animal” refers to any member of the animal kingdom. In some embodiments, “animal” refers to humans at any stage of development. In some embodiments, “animal” refers to non-human animals at any stage of development. In certain embodiments, the non-human animal is a mammal (e.g., a rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, a sheep, cattle, a primate, or a pig). In some embodiments, animals include, but are not limited to, mammals, birds, reptiles, amphibians, fish, and worms. In some embodiments, the animal is a transgenic animal, genetically-engineered animal, or a clone.

[0096] Approximately: As used herein, the term “approximately” or “about,” as applied to one or more values of interest, refers to a value that is similar to a stated reference value. In certain embodiments, the term “approximately” or “about” refers to a range of values that fallwithin 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).

[0097] Associated with: As used herein, the terms “associated with,” “conjugated,” “linked,” “attached,” and “tethered,” when used with respect to two or more moieties, means that the moieties are physically associated or connected with one another, either directly or via one or more additional moieties that serves as a linking agent, to form a structure that is sufficiently stable so that the moieties remain physically associated under the conditions in which the structure is used, e.g., physiological conditions. An “association” need not be strictly through direct covalent chemical bonding. It may also suggest ionic or hydrogen bonding or a hybridization based connectivity sufficiently stable such that the “associated” entities remain physically associated.

[0098] Bijunction or Bifunctional: As used herein, the terms “bifunction” and “bifunctional” refers to any substance, molecule or moiety which is capable of or maintains at least two functions. The functions may affect the same outcome or a different outcome. The structure that produces the function may be the same or different. For example, bifunctional saRNA of the present disclosure may comprise a cytotoxic peptide (a first function) while those nucleosides which comprise the saRNA are, in and of themselves, cytotoxic (second function).

[0099] Biocompatible'. As used herein, the term “biocompatible” means compatible with living cells, tissues, organs or systems posing little to no risk of injury, toxicity or rejection by the immune system.

[0100] Biologically active'. As used herein, the phrase “biologically active” refers to a characteristic of any substance that has activity in a biological system and / or organism. For instance, a substance that, when administered to an organism, has a biological effect on that organism, is considered to be biologically active. In particular embodiments, the saRNA of the present disclosure may be considered biologically active if even a portion of the saRNA is biologically active or mimics an activity considered biologically relevant.

[0101] Cancer: As used herein, the term "cancer" in an individual refers to the presence of cells possessing characteristics typical of cancer-causing cells, such as uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rate, and certain characteristic morphological features. Often, cancer cells will be in the form of a tumor, but such cells may exist alone within an individual, or may circulate in the blood stream as independent cells, such as leukemic cells.

[0102] Cell type: As used herein, the term "cell type" refers to a cell from a given source (e.g., a tissue, organ) or a cell in a given state of differentiation, or a cell associated with a given pathology or genetic makeup.

[0103] Chromosome: As used herein, the term “chromosome” refers to an organized structure of DNA and protein found in cells.

[0104] Complementary: As used herein, the term “complementary” as it relates to nucleic acids refers to hybridization or base pairing between nucleotides or nucleic acids, such as, for example, between the two strands of a double-stranded DNA molecule or between an oligonucleotide probe and a target are complementary.

[0100] Condition: As used herein, the term “condition” refers to the status of any cell, organ, organ system or organism. Conditions may reflect a disease state or simply the physiologic presentation or situation of an entity. Conditions may be characterized as phenotypic conditions such as the macroscopic presentation of a disease or genotypic conditions such as the underlying gene or protein expression profiles associated with the condition. Conditions may be benign or malignant.

[0101] Delivery: As used herein, “delivery” refers to the act or manner of delivering a compound, substance, entity, moiety, cargo or payload.

[0102] Delivery Agent. As used herein, “delivery agent” refers to any substance which facilitates, at least in part, the in vivo delivery of a saRNA of the present disclosure to targeted cells.

[0103] Encapsulate: As used herein, the term “encapsulate” means to enclose, surround or encase.

[0104] Expression'. As used herein, “expression” of a nucleic acid sequence refers to one or more of the following events: (1) production of an RNA template from a DNA sequence (e.g., by transcription); (2) processing of an RNA transcript (e.g., by splicing, editing, 5’ cap formation, and / or 3’ end processing); (3) translation of an RNA into a polypeptide or protein; and (4) post-translational modification of a polypeptide or protein.

[0105] Feature: As used herein, a “feature” refers to a characteristic, a property, or a distinctive element.

[0106] Formulation'. As used herein, a “formulation” includes at least one saRNA of the present disclosure and a delivery agent.

[0107] Fragment: A “fragment,” as used herein, refers to a portion. For example, fragments of proteins may comprise polypeptides obtained by digesting full-length protein isolated fromcultured cells. Fragments of oligonucleotides may comprise nucleotides, or regions of nucleotides.

[0108] Functional'. As used herein, a “functional” biological molecule is a biological molecule in a form in which it exhibits a property and / or activity by which it is characterized.

[0109] Gene: As used herein, the term "gene" refers to a nucleic acid sequence that comprises control and most often coding sequences necessary for producing a polypeptide or precursor. Genes, however, may not be translated and instead code for regulatory or structural RNA molecules.

[0110] A gene may be derived in whole or in part from any source known to the art, including a plant, a fungus, an animal, a bacterial genome or episome, eukaryotic, nuclear or plasmid DNA, cDNA, viral DNA, or chemically synthesized DNA. A gene may contain one or more modifications in either the coding or the untranslated regions that could affect the biological activity or the chemical structure of the expression product, the rate of expression, or the manner of expression control. Such modifications include, but are not limited to, mutations, insertions, deletions, and substitutions of one or more nucleotides. The gene may constitute an uninterrupted coding sequence or it may include one or more introns, bound by the appropriate splice junctions.[OHl] Gene expression: As used herein, the term "gene expression" refers to the process by which a nucleic acid sequence undergoes successful transcription and in most instances translation to produce a protein or peptide. For clarity, when reference is made to measurement of “gene expression”, this should be understood to mean that measurements may be of the nucleic acid product of transcription, e.g., RNA or mRNA or of the amino acid product of translation, e.g., polypeptides or peptides or proteins. Methods of measuring the amount or levels of RNA, mRNA, polypeptides and peptides are well known in the art.

[0112] Genome: The term "genome" is intended to include the entire DNA complement of an organism, including the nuclear DNA component, chromosomal or extrachromosomal DNA, as well as the cytoplasmic domain (e.g., mitochondrial DNA).

[0113] Homology: As used herein, the term “homology” refers to the overall relatedness between polymeric molecules, e.g. between nucleic acid molecules (e.g. DNA molecules and / or RNA molecules) and / or between polypeptide molecules. In some embodiments, polymeric molecules are considered to be “homologous” to one another if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical or similar. The term “homologous” necessarily refers to a comparison between at least two sequences (polynucleotide or polypeptide sequences). In accordance with the disclosure,two polynucleotide sequences are considered to be homologous if the polypeptides they encode are at least about 50%, 60%, 70%, 80%, 90%, 95%, or even 99% for at least one stretch of at least about 20 amino acids. In some embodiments, homologous polynucleotide sequences are characterized by the ability to encode a stretch of at least 4-5 uniquely specified amino acids. For polynucleotide sequences less than 60 nucleotides in length, homology is determined by the ability to encode a stretch of at least 4-5 uniquely specified amino acids. In accordance with the disclosure, two protein sequences are considered to be homologous if the proteins are at least about 50%, 60%, 70%, 80%, or 90% identical for at least one stretch of at least about 20 amino acids.

[0114] Identity. As used herein, the term “identity” refers to the overall relatedness between polymeric molecules, e.g., between oligonucleotide molecules (e.g. DNA molecules and / or RNA molecules) and / or between polypeptide molecules. Calculation of the percent identity of two polynucleotide sequences, for example, can be performed by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second nucleic acid sequences for optimal alignment and non-identical sequences can be disregarded for comparison purposes). In certain embodiments, the length of a sequence aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% of the length of the reference sequence. The nucleotides at corresponding nucleotide positions are then compared. When a position in the first sequence is occupied by the same nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which needs to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. For example, the percent identity between two nucleotide sequences can be determined using methods such as those described in Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991. For example, the percent identity between two nucleotide sequences can be determined using the algorithm of Meyers and Miller (CABIOS, 1989, 4: 11-17), which has been incorporated into the ALIGN program (version 2.0)using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. The percent identity between two nucleotide sequences can, alternatively, be determined using the GAP program in the GCG software package using an NWSgapdna.CMP matrix. Methods commonly employed to determine percent identity between sequences include, but are not limited to those disclosed in Carillo, H., and Lipman, D., SIAM J Applied Math., 48: 1073 (1988). Techniques for determining identity are codified in publicly available computer programs. Exemplary computer software to determine homology between two sequences include, but are not limited to, GCG program package, Devereux, J., et cd., Nucleic Acids Research, 12(1), 387 (1984)), BLASTP, BLASTN, and FASTA Altschul, S. F. el al., J. Molec. Biol., 215, 403 (1990)).

[0115] Inhibit expression of a gene: As used herein, the phrase “inhibit expression of a gene” means to cause a reduction in the amount of an expression product of the gene. The expression product can be an RNA transcribed from the gene (e.g., an mRNA) or a polypeptide translated from an mRNA transcribed from the gene. Typically a reduction in the level of an mRNA results in a reduction in the level of a polypeptide translated therefrom. The level of expression may be determined using standard techniques for measuring mRNA or protein.

[0116] In vitro'. As used herein, the term “in vitro" refers to events that occur in an artificial environment, e.g., in a test tube or reaction vessel, in cell culture, in a Petri dish, etc., rather than within an organism (e.g., animal, plant, or microbe).

[0117] In vivo'. As used herein, the term “in vivo" refers to events that occur within an organism (e.g., animal, plant, or microbe or cell or tissue thereof).

[0118] Isolated'. As used herein, the term “isolated” refers to a substance or entity that has been separated from at least some of the components with which it was associated (whether in nature or in an experimental setting). Isolated substances may have varying levels of purity in reference to the substances from which they have been associated. Isolated substances and / or entities may be separated from at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or more of the other components with which they were initially associated. In some embodiments, isolated agents are more than about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% pure. As used herein, a substance is “pure” if it is substantially free of other components. Substantially isolated'. By “substantially isolated” is meant that the compound is substantially separated from the environment in which it was formed or detected. Partial separation can include, for example, a composition enriched in the compound of the present disclosure. Substantial separation caninclude compositions containing at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 97%, or at least about 99% by weight of the compound of the present disclosure, or salt thereof. Methods for isolating compounds and their salts are routine in the art.

[0119] Label: The term “label” refers to a substance or a compound which is incorporated into an object so that the substance, compound or object may be detectable.

[0120] Linker: As used herein, a linker refers to a group of atoms, e.g., 10-1,000 atoms, and can be comprised of the atoms or groups such as, but not limited to, carbon, amino, alkylamino, oxygen, sulfur, sulfoxide, sulfonyl, carbonyl, and imine. The linker can be attached to a modified nucleoside or nucleotide on the nucleobase or sugar moiety at a first end, and to a payload, e.g., a detectable or therapeutic agent, at a second end. The linker may be of sufficient length as to not interfere with incorporation into a nucleic acid sequence. The linker can be used for any useful purpose, such as to form saRNA conjugates, as well as to administer a payload, as described herein.

[0121] Examples of chemical groups that can be incorporated into the linker include, but are not limited to, alkyl, alkenyl, alkynyl, amido, amino, ether, thioether, ester, alkylene, heteroalkylene, aryl, or heterocyclyl, each of which can be optionally substituted, as described herein. Examples of linkers include, but are not limited to, unsaturated alkanes, polyethylene glycols (e.g., ethylene or propylene glycol monomeric units, e.g., diethylene glycol, dipropylene glycol, triethylene glycol, tripropylene glycol, tetraethylene glycol, or tetraethylene glycol), and dextran polymers and derivatives thereof. Other examples include, but are not limited to, cleavable moieties within the linker, such as, for example, a disulfide bond (-S-S-) or an azo bond (-N=N-), which can be cleaved using a reducing agent or photolysis. Non-limiting examples of a selectively cleavable bond include an amido bond can be cleaved for example by the use of tris(2-carboxyethyl)phosphine (TCEP), or other reducing agents, and / or photolysis, as well as an ester bond can be cleaved for example by acidic or basic hydrolysis. In some embodiments, the linker may comprise a native phosphate that can be cleaved by nucleases.

[0122] Modified: As used herein “modified” refers to a changed state or structure of a molecule of the disclosure. Molecules may be modified in many ways including chemically, structurally, and functionally. In one embodiment, the saRNAs of the present disclosure are modified by the introduction of non-natural nucleosides and / or nucleotides.

[0123] Naturally occurring: As used herein, “naturally occurring” means existing in nature without artificial aid.

[0124] Nucleic acid: The term "nucleic acid" as used herein, refers to a molecule comprised of one or more nucleotides, i.e., ribonucleotides, deoxyribonucleotides, or both. The term includes monomers and polymers of ribonucleotides and deoxyribonucleotides, with the ribonucleotides and / or deoxyribonucleotides being bound together, in the case of the polymers, via 5' to 3' linkages. The ribonucleotide and deoxyribonucleotide polymers may be single or double-stranded. However, linkages may include any of the linkages known in the art including, for example, nucleic acids comprising 5' to 3' linkages. The nucleotides may be naturally occurring or may be synthetically produced analogs that are capable of forming base-pair relationships with naturally occurring base pairs. Examples of non-naturally occurring bases that are capable of forming base-pairing relationships include, but are not limited to, aza and deaza pyrimidine analogs, aza and deaza purine analogs, and other heterocyclic base analogs, wherein one or more of the carbon and nitrogen atoms of the pyrimidine rings have been substituted by heteroatoms, e.g., oxygen, sulfur, selenium, phosphorus, and the like.

[0125] Patient: As used herein, “patient” refers to a subject who may seek or be in need of treatment, requires treatment, is receiving treatment, will receive treatment, or a subject who is under care by a trained professional for a particular disease or condition.

[0126] Peptide: As used herein, “peptide” is less than or equal to 50 amino acids long, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long.

[0127] Pharmaceutically acceptable'. The phrase “pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0128] Pharmaceutically acceptable excipients: The phrase “pharmaceutically acceptable excipient,” as used herein, refers any ingredient other than the compounds described herein (for example, a vehicle capable of suspending or dissolving the active compound) and having the properties of being substantially nontoxic and non-inflammatory in a patient. Excipients may include, for example: anti adherents, antioxidants, binders, coatings, compression aids, disintegrants, dyes (colors), emollients, emulsifiers, fillers (diluents), film formers or coatings, flavors, fragrances, glidants (flow enhancers), lubricants, preservatives, printing inks, sorbents, suspensing or dispersing agents, sweeteners, and waters of hydration. Exemplary excipients include, but are not limited to: butylated hydroxytoluene (BHT), calcium carbonate, calcium phosphate (dibasic), calcium stearate, croscarmellose, crosslinked polyvinyl pyrrolidone, citric acid, crospovidone, cysteine, ethylcellulose, gelatin, hydroxypropyl cellulose, hydroxypropylmethylcellulose, lactose, magnesium stearate, maltitol, mannitol, methionine, methylcellulose, methyl paraben, microcrystalline cellulose, polyethylene glycol, polyvinyl pyrrolidone, povidone, pregelatinized starch, propyl paraben, retinyl palmitate, shellac, silicon dioxide, sodium carboxymethyl cellulose, sodium citrate, sodium starch glycolate, sorbitol, starch (corn), stearic acid, sucrose, talc, titanium dioxide, vitamin A, vitamin E, vitamin C, and xylitol.

[0129] Pharmaceutically acceptable salts'. The present disclosure also includes pharmaceutically acceptable salts of the compounds described herein. As used herein, “pharmaceutically acceptable salts” refers to derivatives of the disclosed compounds wherein the parent compound is modified by converting an existing acid or base moiety to its salt form (e.g., by reacting the free base group with a suitable organic acid). Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids; and the like. Representative acid addition salts include acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptonate, hexanoate, hydrobromide, hydrochloride, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3 -phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, toluenesulfonate, undecanoate, valerate salts, and the like. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like, as well as nontoxic ammonium, quaternary ammonium, and amine cations, including, but not limited to ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, and the like. The pharmaceutically acceptable salts of the present disclosure include the conventional non-toxic salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. The pharmaceutically acceptable salts of the present disclosure can be synthesized from the parent compound which contains a basic or acidic moiety by conventional chemical methods. Generally, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two; generally, nonaqueous media like ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred. Lists of suitable salts are found in Remington ’s Pharmaceutical Sciences, 17thed., Mack Publishing Company, Easton, Pa., 1985, p. 1418, Pharmaceutical Salts: Properties, Selection, and Use, P.H. Stahl andC.G. Wermuth (eds.), Wiley-VCH, 2008, and Berge et al., Journal of Pharmaceutical Science, 66, 1-19 (1977).

[0130] Pharmaceutically acceptable solvate'. The term “pharmaceutically acceptable solvate,” as used herein, means a compound of the disclosure wherein molecules of a suitable solvent are incorporated in the crystal lattice. A suitable solvent is physiologically tolerable at the dosage administered. For example, solvates may be prepared by crystallization, recrystallization, or precipitation from a solution that includes organic solvents, water, or a mixture thereof. Examples of suitable solvents are ethanol, water (for example, mono-, di-, and tri-hydrates), A-methylpyrrolidinone (NMP), dimethyl sulfoxide (DMSO), N,N’- dimethylformamide (DMF), N,N ’-dimethylacetamide (DMAC), l,3-dimethyl-2-imidazolidinone (DMEU), l,3-dimethyl-3,4,5,6-tetrahydro-2-(lH)-pyrimidinone (DMPU), acetonitrile (ACN), propylene glycol, ethyl acetate, benzyl alcohol, 2-pyrrolidone, benzyl benzoate, and the like. When water is the solvent, the solvate is referred to as a “hydrate.”

[0131] Protein: A "protein" means a polymer of amino acid residues linked together by peptide bonds. The term, as used herein, refers to proteins, polypeptides, and peptides of any size, structure, or function. Typically, however, a protein will be at least 50 amino acids long. In some instances the protein encoded is smaller than about 50 amino acids. In this case, the polypeptide is termed a peptide. If the protein is a short peptide, it will be at least about 10 amino acid residues long. A protein may be naturally occurring, recombinant, or synthetic, or any combination of these. A protein may also comprise a fragment of a naturally occurring protein or peptide. A protein may be a single molecule or may be a multi-molecular complex. The term protein may also apply to amino acid polymers in which one or more amino acid residues are an artificial chemical analogue of a corresponding naturally occurring amino acid.

[0132] Protein expression: The term "protein expression" refers to the process by which a nucleic acid sequence undergoes translation such that detectable levels of the amino acid sequence or protein are expressed.

[0133] Purified: As used herein, “purify,” “purified,” “purification” means to make substantially pure or clear from unwanted components, material defilement, admixture or imperfection.

[0134] Sample: As used herein, the term “sample” or “biological sample” refers to a subset of its tissues, cells or component parts (e.g. body fluids, including but not limited to blood, mucus, lymphatic fluid, synovial fluid, cerebrospinal fluid, saliva, amniotic fluid, amniotic cord blood, urine, vaginal fluid and semen). A sample further may include a homogenate, lysate or extract prepared from a whole organism or a subset of its tissues, cells or component parts, or afraction or portion thereof, including but not limited to, for example, plasma, serum, spinal fluid, lymph fluid, the external sections of the skin, respiratory, intestinal, and genitourinary tracts, tears, saliva, milk, blood cells, tumors, organs. A sample further refers to a medium, such as a nutrient broth or gel, which may contain cellular components, such as proteins or nucleic acid molecule.

[0135] Signal Sequences: As used herein, the phrase “signal sequences” refers to a sequence which can direct the transport or localization of a protein.

[0136] Single unit dose. As used herein, a “single unit dose” is a dose of any therapeutic administered in one dose / at one time / single route / single point of contact, i.e., single administration event.

[0137] Similarity: As used herein, the term “similarity” refers to the overall relatedness between polymeric molecules, e.g. between polynucleotide molecules (e.g. DNA molecules and / or RNA molecules) and / or between polypeptide molecules. Calculation of percent similarity of polymeric molecules to one another can be performed in the same manner as a calculation of percent identity, except that calculation of percent similarity takes into account conservative substitutions as is understood in the art.

[0138] Split dose: As used herein, a “split dose” is the division of single unit dose or total daily dose into two or more doses.

[0139] Stable: As used herein “stable” refers to a compound that is sufficiently robust to survive isolation to a useful degree of purity from a reaction mixture, and in one embodiment, capable of formulation into an efficacious therapeutic agent.

[0140] Stabilized: As used herein, the term “stabilize”, “stabilized,” “stabilized region” means to make or become stable.

[0141] Subject: As used herein, the term “subject” or “patient” refers to any organism to which a composition in accordance with the disclosure may be administered, e.g, for experimental, diagnostic, prophylactic, and / or therapeutic purposes. Typical subjects include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and humans) and / or plants.

[0142] Substantially: As used herein, the term “substantially” refers to the qualitative condition of exhibiting total or near-total extent or degree of a characteristic or property of interest. One of ordinary skill in the biological arts will understand that biological and chemical phenomena rarely, if ever, go to completion and / or proceed to completeness or achieve or avoid an absolute result. The term “substantially” is therefore used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena.

[0143] Substantially equal'. As used herein as it relates to time differences between doses, the term means plus / minus 2%.

[0144] Substantially simultaneously. As used herein and as it relates to plurality of doses, the term means within 2 seconds.

[0145] Suffering from'. An individual who is “suffering from” a disease, disorder, and / or condition has been diagnosed with or displays one or more symptoms of a disease, disorder, and / or condition.

[0146] Synthetic. The term “synthetic” means produced, prepared, and / or manufactured by the hand of man. Synthesis of polynucleotides or polypeptides or other molecules of the present disclosure may be chemical or enzymatic.

[0147] Targeted Cells: As used herein, “targeted cells” refers to any one or more cells of interest. The cells may be found in vitro, in vivo, in situ or in the tissue or organ of an organism. The organism may be an animal, in one embodiment, a mammal, or a human and in one embodiment, a patient.

[0148] Therapeutic Agent: The term “therapeutic agent” refers to any agent that, when administered to a subject, has a therapeutic, diagnostic, and / or prophylactic effect and / or elicits a desired biological and / or pharmacological effect.

[0149] Therapeutically effective amount: As used herein, the term “therapeutically effective amount” means an amount of an agent to be delivered (e.g., nucleic acid, drug, therapeutic agent, diagnostic agent, prophylactic agent, etcf that is sufficient, when administered to a subject suffering from or susceptible to an infection, disease, disorder, and / or condition, to treat, improve symptoms of, diagnose, prevent, and / or delay the onset of the infection, disease, disorder, and / or condition.

[0150] Therapeutically effective outcome'. As used herein, the term “therapeutically effective outcome” means an outcome that is sufficient in a subject suffering from or susceptible to an infection, disease, disorder, and / or condition, to treat, improve symptoms of, diagnose, prevent, and / or delay the onset of the infection, disease, disorder, and / or condition.

[0151] Total daily dose: As used herein, a “total daily dose” is an amount given or prescribed in 24 hour period. It may be administered as a single unit dose.

[0152] Transcription factor: As used herein, the term “transcription factor” refers to a DNA- binding protein that regulates transcription of DNA into RNA, for example, by activation or repression of transcription. Some transcription factors effect regulation of transcription alone, while others act in concert with other proteins. Some transcription factor can both activate and repress transcription under certain conditions. In general, transcription factors bind a specifictarget sequence or sequences highly similar to a specific consensus sequence in a regulatory region of a target gene. Transcription factors may regulate transcription of a target gene alone or in a complex with itself (as a homodimer) other with other molecules (as a heterodimer). Each of these complex formation is able to induce multiple regulatory function from a single transcription factor.

[0153] Treating'. As used herein, the term “treating” refers to partially or completely alleviating, ameliorating, improving, relieving, delaying onset of, inhibiting progression of, reducing severity of, and / or reducing incidence of one or more symptoms or features of a particular infection, disease, disorder, and / or condition. For example, “treating” cancer may refer to inhibiting survival, growth, and / or spread of a tumor. Treatment may be administered to a subject who does not exhibit signs of a disease, disorder, and / or condition and / or to a subject who exhibits only early signs of a disease, disorder, and / or condition for the purpose of decreasing the risk of developing pathology associated with the disease, disorder, and / or condition.

[0154] The phrase "a method of treating" or its equivalent, when applied to, for example, cancer refers to a procedure or course of action that is designed to reduce, eliminate or prevent the number of cancer cells in an individual, or to alleviate the symptoms of a cancer. "A method of treating" cancer or another proliferative disorder does not necessarily mean that the cancer cells or other disorder will, in fact, be completely eliminated, that the number of cells or disorder will, in fact, be reduced, or that the symptoms of a cancer or other disorder will, in fact, be alleviated. Often, a method of treating cancer will be performed even with a low likelihood of success, but which, given the medical history and estimated survival expectancy of an individual, is nevertheless deemed an overall beneficial course of action.

[0155] Unmodified. As used herein, “unmodified” refers to any substance, compound or molecule prior to being changed in any way. Unmodified may, but does not always, refer to the wild type or native form of a biomolecule. Molecules may undergo a series of modifications whereby each modified molecule may serve as the “unmodified” starting molecule for a subsequent modification.Equivalents and Scope

[0156] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments in accordance with the disclosure described herein. The scope of the present disclosure is not intended to be limited to the above Description, but rather is as set forth in the appended claims.

[0157] In the claims, articles such as “a,” “an,” and “the” may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include “or” between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The disclosure includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The disclosure includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process.

[0158] It is also noted that the term “comprising” is intended to be open and permits the inclusion of additional elements or steps.

[0159] Where ranges are given, endpoints are included. Furthermore, it is to be understood that unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or subrange within the stated ranges in different embodiments of the disclosure, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.

[0160] In addition, it is to be understood that any particular embodiment of the present disclosure that falls within the prior art may be explicitly excluded from any one or more of the claims. Since such embodiments are deemed to be known to one of ordinary skill in the art, they may be excluded even if the exclusion is not set forth explicitly herein. Any particular embodiment of the compositions of the disclosure (e.g., any nucleic acid or protein encoded thereby; any method of production; any method of use; etc.) can be excluded from any one or more claims, for any reason, whether or not related to the existence of prior art.

[0161] In case of conflicting statements of a cited source and the instant application, the statement in the instant application shall control.

[0162] The disclosure is further illustrated by the following non-limiting examples.EXAMPLESExample 1. HNF4a Upregulation by chemically modified saRNAsMaterials and Procedures:Transfection of saRNA

[0163] Sense and antisense strands of saRNAs (such as HNF4A-PR3-ESC, MC01, MC02, MC03, MC04, MC05, MC06, MC07, and MC08) in Table 4 were synthesized. They were first annealed in a 1 Ox PBS buffer following a denaturing step at 95°C, followed by a gradual anneal step to room temperature.

[0164] HepG2 cells were seeded at 0.16xl05per well in a 96-well plate and transfected using Lipofectamine RNAiMAX (Life Technologies). Transfection was performed immediately after seeding and again 24 hours later with the indicated oligonucleotide concentration using 0.2uL of Lipofectamine RNAiMAX. Two biological replicates were measured for each saRNA.RT-qPCR (qPCR).

[0165] Total RNA was harvested at 72 hours post seeding as indicated by each experiment. RNA was recovered using the RNeasy 96 Kit (QIAGEN) following the manufacturer's recommendation. Equal volumes of RNAs were added and relative expression levels were determined by 1-step real-time PCR using TaqPath 1-Step Multiplex Master Mix (Thermo Fisher) with multiplexed B2M-VIC and HNF4a Pl-FAM Taqman assays. B2M was used as a reference gene.

[0166] Fig. 1 shows HNF4a Pl mRNA levels (n=2) in cells after saRNA treatments. As shown in Fig. 1, saRNAs having antisense strands with a 2’-OMe modification on the 6thposition of the strands (such as MC01, MC02, MC04, MC05, MC06, MC07 and MC08) showed better activity than saRNAs having antisense strands without a 2’-OMe modification on the 6thposition of the strands (such as MC03 and ESC).Example 2. Activity of GalNAc-saRNA Conjugates by ASGPR Receptor-media Uptake in Primary Rat Hepatocytes

[0167] GalNAc-saRNA conjugates described in Table 5 were synthesized and their activity in primary rat hepatocytes was measured. Cryopreserved plateable primary rat hepatocytes (Thermo Fisher) were seeded at 0.4xl05per well in a 96-well plate and GalNAc-saRNA conjugates were added directly to the cell culture medium at the indicated concentration. One biological replicate was measured for AS05-containing conjugates, three biological replicates were measured for AS07- or AS08-containing conjugates, or four biological replicates were measured for AS06-containing conjugates.RT-qPCR (qPCR).

[0168] Total RNA was harvested at 72 hours post seeding as indicated by each experiment. RNA was recovered using the RNeasy 96 Kit (QIAGEN) following the manufacturer's recommendation. Equal volumes of RNAs were added and relative expression levels were determined by 1-step real-time PCR using TaqPath 1-Step Multiplex Master Mix (Thermo Fisher) with RplpO and Hnf4a Pl Taqman assays. RplpO was used as a reference gene.

[0169] Fig. 2A-2D shows rat Hnf4a Pl mRNA levels in cells after treatment with GalNAc- saRNA conjugates. MC62, MC63, MC68 and MC69 showed better activity and potency than DV22.Example 3. Upregulation of HB HBG2 gene expression with saRNAs in vitro

[0001] In this study, upregulation of HBG1 / 2 (i.e., HBG1 and HBG2 may also be referred to as H G) gene expression by saRNA treatment was tested in primary bone marrow-derived CD34+ cells (a marker of human hematopoietic stem cells and all colony -forming activity of human bone marrow (BM) cells). In brief, the total HBGl-mRNA and HBG2-mRNA level (i.e., HBG1 / 2 mRNA or HBG mRNA) was measured by RT-qPCR relative to a housekeeper gene (POLR2A). In brief, a qPCR assay that detects both HBGl-mRNA and HBG2-mRNA was used to measure total HBG1 / 2 mRNA. All samples were normalized to mock Nucleofected cells that contained Nucleofection buffer but no oligo during Nucleofection. The relative expressions of HBG1 / 2 genes (mean of each biological replicate and n number shown) in expanded CD34+ cells are shown in Table 8 below.Table 8. Relative expression of HBG1 / 2 genes (relative to P0LR2A) in CD34+ cells - 48hrOTHER EMBODIMENTS

[0170] It is to be understood that while the present disclosure has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the present disclosure, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

Claims1. A double-stranded synthetic isolated small activating RNA (saRNA) which up-regulates the expression of a target gene, comprising an antisense strand and a sense strand, wherein the sense strand has 19 nucleotides and the antisense strand has 19-22 nucleotides, wherein the antisense strand comprises a 2’-0Me modification on the 6thposition of the strand, and / or wherein the antisense strand has phosphorothioate (-ps-) bonds between the 1stand 2ndpositions and / or between the 2ndand 3rdpositions.

2. The saRNA of claim 1, wherein the antisense strand of the saRNA comprises at least one additional modification.

3. The saRNA of any one of claims 1-2, wherein the antisense strand further comprises 2’-F modifications on the 2nd, 3rdand / or 4thpositions of the strand.

4. The saRNA of any one of claims 1-3, wherein the antisense strand further comprises a 2’-F modification on the 1st, 9thand / or 14thposition of the strand.

5. The saRNA of any one of claims 1-4, wherein the antisense strand has a 2’-0Me modification at the 10thand / or 13thposition.

6. The saRNA of any one of claims 1-5, wherein the antisense strand has 2’-F modifications on the 17thposition and / or the 18thposition.

7. The saRNA of any one of claims 1-6, wherein the antisense strand does not have a 2’-F modification at the 16thposition.

8. The saRNA of any one of claims 1-7, wherein the antisense strand has a 2’-0Me modification at the 16thposition.

9. The saRNA of any one of claims 1-8, wherein the antisense strand of the saRNA comprises A- VP at the 5’ end.

10. The saRNA of any one of claims 1-9, wherein the antisense strand has phosphorothioate (-ps-) bonds between the last and second to the last positions and / or between the second to the last and third to the last positions.

11. The saRNA of any one of claims 1-10, wherein the antisense strand has an overhang comprising 2-3 nucleotides at the 3’ end.

12. The saRNA of claim 11, wherein the overhang at the 3’ end of the antisense strand is - UU, -UUU, -mUmU or -ps-mU-ps-mU.

13. The saRNA of any one of claims 1-12, wherein the antisense strand has 21 nucleotides.

14. The saRNA of any one of claims 1-13, wherein the antisense strand of the saRNA comprises at least one unmodified nucleotide.

15. The saRNA of claim 14, wherein the unmodified nucleotide of the antisense strand is A, C or G.

16. The saRNA of any one of claims 1-15, wherein all the Us of the antisense strand are modified.

17. The saRNA of any one of claims 1-16, wherein all the Us of the antisense strand are modified either with 2’-0Me or 2’-F.

18. The saRNA of any one of claims 1-17, wherein the sense strand of the saRNA does not have any 3’ overhang.

19. The saRNA of any one of claims 1-18, wherein the sense strand of the saRNA comprises at least one modification.

20. The saRNA of any one of claims 1-19, wherein the sense strand of the saRNA comprises 2’-0Me modifications on the 2ndand 14thpositions of the strand.

21. The saRNA of any one of claims 1-20, wherein the sense strand of the saRNA comprises a 2’-0Me modification on the 3rdpositions of the strand.

22. The saRNA of any one of claims 1-21, wherein sense strand has phosphorothioate (-ps-) bonds between the 1stand 2ndpositions, between the 2ndand 3rdpositions, between the 17thand 18thpositions and / or the 18thand 19thpositions.

23. The saRNA of any one of claims 1-22, wherein the sense strand of the saRNA comprises at least one unmodified nucleotide.

24. The saRNA of claim 23, wherein the unmodified nucleotide of the sense strand is A, C or G.

25. The saRNA of any one of claims 1-24, wherein all the Us of the sense strand are modified.

26. The saRNA of any one of claims 1-25, wherein the Us of the sense strand are modified with either 2’-0Me or 2’-F.

27. A conjugate comprising the saRNA of any one of claims 1-26 covalently connected to a carbohydrate or lipid moiety.

28. The conjugate of claim 27, wherein the carbohydrate moiety comprises at least one N- Acetyl-Galactosamine (GalNAc) or derivative thereof.

29. The conjugate of claim 27, wherein the lipid moiety comprises cholesterol, palmitoyl, or tocopherol group.

30. The conjugate of any one of claims 27-29, wherein the saRNA is connected to the carbohydrate or lipid moiety with a linker.

31. A pharmaceutical composition comprising the saRNA of any one of claims 1-26 or the conjugate of any one of claims 27-30, and at least one pharmaceutically acceptable excipient.

32. A method of up-regulating the expression of a target gene, comprising contacting the target gene with the saRNA of any one of claims 1-26, the conjugate of any one of claims 27-30, or the pharmaceutical composition of claim 31.

33. The method of claim 32, wherein the expression of the target gene is increased by at least 30%, 40%, or 50%.

Images