Oligonucleotide conjugate compositions and methods of use

Abstract

The present disclosure relates to a GalNAc moiety comprising at least one GalNAc monomer. The present disclosure also relates to a GalNAc-oligonucleotide conjugate comprising a GalNAc moiety and an oligonucleotide, e.g., saRNA or siRNA, useful for regulating expression of a target gene. Methods of using the GalNAc-oligonucleotide conjugate are also provided.

Description

[Technical Field]

[0001] The present disclosure relates to a GalNAc moiety comprising at least one GalNAc monomer. The present disclosure also relates to a GalNAc-oligonucleotide conjugate comprising a GalNAc moiety and an oligonucleotide, such as a small activating RNA (saRNA) or a small inhibitory RNA (siRNA). [Background technology]

[0002] CCAAT / enhancer-binding protein α (C / EBPα, C / EBP alpha, C / EBPA, or CEBPA) is a leucine zipper protein conserved between humans and rodents. This nuclear transcription factor is abundant in hepatocytes, myelomonocytes, adipocytes, and other types of mammary epithelial cells (Non-Patent Document 1). It consists of two transactivation domains at the N-terminus, a leucine zipper region that mediates dimerization with other C / EBP family members, and a DNA-binding domain at the C-terminus. Binding sites for the C / EBP transcription factor family are present in the promoter regions of numerous genes involved in maintaining normal hepatocyte function and responding to injury. C / EBPα exerts pleiotropic effects on the transcription of several liver-specific genes involved in immune and inflammatory responses, development, cell proliferation, anti-apoptosis, and several metabolic pathways (Non-Patent Document 2). It is essential for maintaining the differentiated state of hepatocytes. It activates the transcription of albumin and regulates the expression of genes encoding several ornithine cycle enzymes involved in ureogenesis, thus playing an important role in normal liver function. [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] Lekstrom-Himes et al., J. Bio. Chem, vol. 273, 28545-28548 (1998) [Non-patent document 2] Darlington et al., Current Opinion of Genetic Development, vol. 5(5), 565-570 (1995) Summary of the Invention [Problem to be solved by the invention]

[0004] There is a need for targeted modulation of CEBPA for therapeutic purposes using saRNA. [Means for solving the problem]

[0005] The present invention provides compositions, methods, and kits for the design, preparation, manufacturing, formulation, and / or use of short (or small) activating RNAs (saRNAs), whether modified or not, to modulate the expression and / or function of target genes for therapeutic purposes, including diagnostics and prognosis. The term "modified" or, where appropriate, "modification" refers to structural and / or chemical modifications of any one or more components of a nucleotide (sugar, base, or backbone). In the case of bases, any of the standard nucleobases: A, G, U, or C ribonucleobases may be modified. Nucleotides in the saRNAs of the present invention may include non-standard nucleotides, such as non-naturally occurring nucleotides or chemically synthesized nucleotides or deoxynucleotides.

[0006] One aspect of the present invention provides an isolated synthetic small activating RNA (saRNA) that upregulates expression of a target gene, wherein the saRNA comprises at least one modification to at least one of the base, sugar, or backbone of the polynucleotide that constitutes the saRNA.

[0007] Another aspect of the present invention provides an N-acetyl-galactosamine (GalNAc) monomer comprising a structure selected from the group consisting of:

[0008] [ka]

[0009] wherein R1, R2, and R3 may be the same or different, and R1, R2, and R3 are independently selected from alkyl, aryl, and alkenyl groups; R4 is a suitable protecting group or a C1-6 linear or branched alkyl group; R5 and R6 are each independently a C1-6 linear or branched alkyl group; and R7 is a suitable protecting group;

[0010] [ka]

[0011] wherein R1, R2, and R3 may be the same or different, and R1, R2, and R3 are independently selected from alkyl, aryl, and alkenyl groups; R4 is a protecting group or a C1-6 linear or branched alkyl group; R5 and R6 are each independently a C1-6 linear or branched alkyl; and R7 is a suitable protecting group;

[0012] [ka]

[0013] wherein R1, R2, and R3 may be the same or different, and R1, R2, and R3 are independently selected from alkyl groups, aryl groups, and alkenyl groups; R7 is a suitable protecting group, and Linker 1 is a cleavable linker; and

[0014] [ka]

[0015] wherein R1, R2, and R3 may be the same or different, and R1, R2, and R3 are independently selected from the group consisting of alkyl groups, aryl groups, and alkenyl groups; R7 is a suitable protecting group, and Linker 1 is a cleavable linker.

[0016] Another aspect of the invention provides a GalNAc moiety comprising at least one GalNAc monomer, wherein said GalNAc monomer is

[0017] [ka]

[0018] where R8 is —H or a C1-6 linear or branched alkyl group;

[0019] [ka]

[0020] wherein R8 is —H or a C1-6 linear or branched alkyl group, and X is O or S;

[0021] [ka]

[0022] where X is O or S;

[0023] [ka]

[0024] is selected from the group consisting of: Another aspect of the present invention provides a conjugate comprising an oligonucleotide connected to a carbohydrate moiety (e.g., an N-acetyl-galactosamine (GalNAc) moiety) via a linker. In the context of this application, the term "moiety" refers to a single unit or component of the entire compound or conjugate. For example, a conjugate may have a GalNAc moiety, a linker moiety, and a saRNA moiety. The GalNAc moiety may comprise one or more GalNAc monomers together. The term "GalNAc cluster" or "GalNAc multimer" refers to two or more GalNAc monomers together. Thus, in some situations (i.e., when two or more molecules are present together), the terms "GalNAc moiety," "GalNAc cluster," and "GalNAc multimer" may be synonymous. The oligonucleotide may be an antisense oligonucleotide (ASO), a small activating RNA (saRNA), a small inhibitory RNA (siRNA), a microRNA (miRNA), a modified mRNA, a self-amplifying RNA, a circular RNA, an aptamer RNA, a ribozyme, a plasmid, or an immunostimulatory nucleic acid. Oligonucleotides may be single-stranded or double-stranded. Oligonucleotides may contain naturally occurring nucleotides, synthetic nucleotides, and / or modified nucleotides. In the context of the present invention, the term "small activator RNA," "short activator RNA," or "saRNA" refers to a single-stranded or double-stranded RNA that upregulates or has a positive effect on the expression of a specific gene. The gene is the target gene of the saRNA. In this context, the term "small interfering RNA," "small inhibitory RNA," or "siRNA" refers to a double-stranded RNA that is involved in the RNA interference (RNAi) pathway and disrupts or inhibits the expression of a specific gene. The gene is the target gene of the siRNA.

[0025] Another aspect of the present invention provides a pharmaceutical composition comprising a modified saRNA or a conjugate comprising a saRNA attached to a carbohydrate moiety (such as a GalNAc moiety) and at least one pharmaceutically acceptable excipient.

[0026] Another aspect of the invention provides a method for delivering saRNA to a cell, comprising administering a conjugate comprising saRNA linked to a carbohydrate moiety (such as a GalNAc moiety).

[0027] Another aspect of the present invention provides a method for upregulating expression of a target gene, comprising administering a conjugate comprising a modified saRNA or saRNA linked to a carbohydrate moiety (such as a GalNAc moiety).

[0028] Another aspect of the present invention provides a method for treating or preventing a disease, comprising administering a conjugate comprising a modified saRNA or a saRNA linked to a carbohydrate moiety (such as a GalNAc moiety), wherein the saRNA upregulates expression of a target gene, and the target gene is associated with the disease.

[0029] Another aspect of the present invention provides a pharmaceutical composition comprising a modified siRNA or a conjugate comprising an siRNA linked to a carbohydrate moiety (e.g., a GalNAc moiety) and at least one pharmaceutically acceptable excipient. The siRNA can downregulate the expression of a target gene, such as, but not limited to, complement C5 (C5) or transthyretin (TTR).

[0030] Another aspect of the invention provides a method of delivering siRNA to a cell, comprising administering a conjugate comprising an siRNA linked to a carbohydrate moiety (such as a GalNAc moiety).

[0031] Another aspect of the invention provides a method for downregulating expression of a target gene comprising administering a modified siRNA or a conjugate comprising an siRNA attached to a carbohydrate moiety (such as a GalNAc moiety).

[0032] Another aspect of the invention provides for the treatment or prevention of a disease comprising administering a conjugate comprising a modified siRNA or an siRNA attached to a carbohydrate moiety (such as a GalNAc moiety), wherein the siRNA downregulates expression of a target gene, and the target gene is associated with the disease.

[0033] Details of various embodiments of the invention are set forth in the description that follows. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

[0034] The foregoing and other objects, features, and advantages will become apparent from the following description of specific embodiments of the present invention 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 present invention. [Brief explanation of the drawings]

[0035] [Figure 1] Figure showing CEBPA mRNA levels after passive delivery of saRNA at 500 nM in primary rat hepatocytes. [Figure 2] Diagram showing albumin mRNA levels after passive delivery of saRNA at 500 nM in primary rat hepatocytes. [Figure 3] Figure showing CEBPA mRNA levels after passive delivery of saRNA at 1 μM in primary rat hepatocytes. [Figure 4] Diagram showing albumin mRNA levels after passive delivery of saRNA at 1 μM in primary rat hepatocytes. [Figure 5] CEBPA mRNA levels in normal mice after 40 mg / kg injections on days 1 and 3 and sacrifice on day 5. CEBPA was normalized to PBS using B2M as housekeeping. RNA was extracted from frozen liver samples and mRNA levels were measured by qPCR. [Figure 6] CEBPA mRNA levels in normal mice after 40 mg / kg injections on days 1 and 3 and sacrifice on day 5. CEBPA was normalized to PBS using B2M as housekeeping. RNA was extracted from frozen liver samples and mRNA levels were measured by qPCR. [Figure 7] Albumin mRNA levels in normal mice after 40 mg / kg injections on days 1 and 3 and sacrifice on day 5. Albumin was normalized to PBS using B2M as housekeeping. RNA was extracted from frozen liver samples and mRNA levels were measured by qPCR. [Figure 8] A diagram showing CEBPA mRNA levels in the liver of normal mice after subcutaneous injection of GalNAc saRNA conjugate at 30 mg / kg on days 1 and 3 and sacrifice on day 5. [Figure 9] Diagram showing the in vitro dose response of CEBPa-saRNA-GalNAc conjugates L80 (XD-14369K1 conjugated to GalNAc cluster G7) and L81 (XD-14369K1 conjugated to GalNAc cluster G8). [Figure 10] A diagram showing C5 mRNA levels after transfection with C5-siRNA-GalNAc conjugates. DETAILED DESCRIPTION OF THE INVENTION

[0036] Detailed Description The present invention provides compositions, methods, and kits for modulating the expression and / or function of a target gene for therapeutic purposes, which comprise at least one saRNA that upregulates the expression of the target gene, wherein the saRNA comprises at least one chemical modification.

[0037] I. Design and synthesis of saRNA In the context of the present invention, the terms "small activator RNA," "short activator RNA," or "saRNA" refer to single- or double-stranded RNA that upregulates or positively affects the expression of a specific gene. saRNA can be single-stranded, consisting of 14 to 30 nucleotides. saRNA can also be double-stranded, with each strand containing 14 to 30 nucleotides. Such genes are referred to as target genes of the saRNA. As used herein, a target gene is a double-stranded DNA comprising a coding strand and a template strand. For example, a saRNA that upregulates the expression of the CEBPA gene is referred to as a "CEBPA-saRNA," and the CEBPA gene is the target gene of the CEBPA-saRNA. A target gene can be any gene of interest. In some embodiments, the target gene has a promoter region on the template strand.

[0038] "Upregulation" or "activation" of a gene or mRNA refers to an increase in the expression level of the gene or mRNA, or the level or activity of the polypeptide encoded by the mRNA. The saRNA of the present invention may have a direct upregulatory effect on the expression of the target gene.

[0039] The saRNA of the present invention may have an indirect upregulatory effect on the RNA transcript transcribed from the template strand of the target gene and / or the polypeptide encoded by the target gene or mRNA. The RNA transcript transcribed from the target gene is then referred to as the target transcript. The target transcript may be the mRNA of the target gene. The target transcript may be present in mitochondria. The saRNA of the present invention may have a downstream effect on a biological process or activity. In such an embodiment, the saRNA targeting a first transcript may have an effect (either upregulation or downregulation) on a second, non-target transcript.

[0040] In one embodiment, the saRNA of the present invention may be effective in proliferating cells. As used herein with respect to cells, "proliferating" refers to cells that are rapidly growing and / or reproducing.

[0041] Targeted antisense RNA transcripts of target genes In one embodiment, the saRNA of the present invention is designed to be complementary to a target antisense RNA transcript of a target gene and can exert an effect on the expression and / or function of the target gene by downregulating the target antisense RNA transcript. The target antisense RNA transcript can be transcribed from the coding strand of the target gene and can be present in the nucleus of the cell.

[0042] In this context, the term "complementary" means capable of hybridizing with a target antisense RNA transcript under stringent conditions. In the context of the present invention, the term "antisense" when used to describe a target antisense RNA transcript means that the sequence is complementary to a sequence on the coding strand of a gene.

[0043] It should be understood that thymidine in DNA is replaced by uridine in RNA, and that this difference does not alter the interpretation of the terms "antisense" or "complementary."

[0044] The target antisense RNA transcript may be transcribed from a locus on the coding strand between 100, 80, 60, 40, 20, or 10 kb upstream of a position corresponding to the transcription start site (TSS) of the target gene and 100, 80, 60, 40, 20, or 10 kb downstream of a position corresponding to the transcription stop site of the target gene.

[0045] In one embodiment, the target antisense RNA transcript is transcribed from a locus on the coding strand located within + / - 1 kb of the transcription start site of the target gene. In another embodiment, the target antisense RNA transcript is transcribed from a locus on the coding strand located within + / -500 nt, + / -250 nt, + / -100 nt, + / -10 nt, ±5 nt, or + / -1 nt of the transcription start site of the target gene.

[0046] In another embodiment, the target antisense RNA transcript is transcribed from a locus on the coding strand located + / - 2000 nucleotides from the transcription start site of the target gene.

[0047] In another embodiment, the locus on the coding strand is no more than 1000 nucleotides upstream or downstream from a position corresponding to the transcription start site of the target gene. In another embodiment, the locus on the coding strand is no more than 500 nucleotides upstream or downstream from a position corresponding to the transcription start site of the target gene.

[0048] As used herein, the term "transcription start site" (TSS) refers to the nucleotides on the template strand of a gene that correspond to or indicate the start position of transcription. The TSS can be located within the promoter region of the template strand of a gene.

[0049] The term "transcription stop site," as used herein, refers to a region, which may be one or more nucleotides on the template strand of a gene, having at least one characteristic feature, such as, but not limited to, a region encoding at least one stop codon of the target transcript, a region encoding the sequence immediately preceding the 3'UTR of the target transcript, a region where RNA polymerase releases the gene, a region encoding a splice site or the portion preceding the splice site, and a region on the template strand where transcription of the target transcript terminates.

[0050] The phrase "transcribed from a particular locus" in reference to a target antisense RNA transcript of the present invention means that transcription of the target antisense RNA transcript is initiated from a particular locus.

[0051] The target antisense RNA transcript is complementary to the coding strand of the genomic sequence of the target gene; any reference herein to the "genomic sequence" is shorthand for "the coding strand of the genomic sequence."

[0052] The "coding strand" of a gene has the same base sequence as the mRNA that is produced, except that Ts are replaced by Us in the mRNA. The "template strand" of a gene is therefore complementary to and antiparallel to the mRNA that is produced.

[0053] Thus, the target antisense RNA transcript may comprise a sequence complementary to a genomic sequence located between 100, 80, 60, 40, 20, or 10 kb upstream of the transcription start site of the target gene and 100, 80, 60, 40, 20, or 10 kb downstream of the transcription stop site of the target gene.

[0054] In one embodiment, the target antisense RNA transcript comprises a sequence complementary to a genomic sequence located between 1 kb upstream of the transcription start site of the target gene and 1 kb downstream of the transcription stop site of the target gene.

[0055] In another embodiment, the target antisense RNA transcript comprises a sequence complementary to a genomic sequence located between 500, 250, 100, 10, 5, or 1 nucleotides upstream of the transcription start site of the target gene and its end 500, 250, 100, 10, 5, or 1 nucleotides downstream of the transcription stop site of the target gene.

[0056] The target antisense RNA transcript may comprise a sequence complementary to the genomic sequence comprising the coding region of the target gene. The target antisense RNA transcript may comprise a sequence complementary to the genomic sequence that aligns with the promoter region of the target gene of the template strand. A gene may have multiple promoter regions, and in this case, the target antisense RNA transcript may align with one, two, or more promoter regions. To identify the promoter region of a gene, an online database of annotated gene loci may be used. When used in relation to a pair of nucleotide sequences, the terms "align" and "alignment" mean that the pair of nucleotide sequences are complementary to each other or have sequence identity with each other.

[0057] The alignment region between the target antisense RNA transcript and the promoter region of the target gene can be partial and can be as short as one nucleotide in length, but can be at least 15 or at least 20 nucleotides in length, or at least 25 nucleotides in length, or at least 30, 35, 40, 45, or 50 nucleotides in length, or at least 55, 60, 65, 70, or 75 nucleotides in length, or at least 100 nucleotides in length. Each of the following specific arrangements is intended to be included within the scope of the term "alignment": a) The target antisense RNA transcript and the promoter region of the target gene are aligned at the same length (i.e., aligned over their entire length).

[0058] b) The target antisense RNA transcript is shorter than the promoter region of the target gene and aligns to the promoter region of the target gene over its entire length (i.e., it aligns to a sequence within the promoter region of the target gene over its entire length).

[0059] c) The target antisense RNA transcript is longer than the promoter region of the target gene, and the promoter region of the target gene is perfectly aligned thereby (i.e., the promoter region of the target gene is aligned to a sequence within the target antisense RNA transcript over its entire length).

[0060] d) The target antisense RNA transcript and the promoter region of the target gene are of the same length or different lengths, and the alignment region is shorter than both the length of the target antisense RNA transcript and the length of the promoter region of the target gene.

[0061] The above definitions of "align" and "alignment" apply mutatis mutandis to descriptions of other overlapping sequences, e.g., aligned sequences, throughout this specification. It is clear that when a target antisense RNA transcript is described as aligning to a region of a target gene other than the promoter region, the sequence of the target antisense RNA transcript aligns to sequences within the described region, but not within the promoter region of the target gene.

[0062] In one embodiment, the target antisense RNA transcript is at least 1 kb or at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 kb, for example, 20, 25, 30, 35, or 40 kb in length.

[0063] In one embodiment, the target antisense RNA transcript comprises a sequence that is at least 75%, or at least 85%, or at least 90%, or at least 95% complementary along its entire length to the sequence of the coding strand of the target gene.

[0064] The present invention provides saRNA that targets target antisense RNA transcripts, and can effectively and specifically downregulate such target antisense RNA transcripts.This can be achieved by saRNA that has a high degree of complementarity with the region in the target antisense RNA transcript.The saRNA may have 5 or less mismatches with the region in the targeted antisense RNA transcript, or 4 or 3 or less mismatches, or 2 or less mismatches, or no mismatches at all.

[0065] Because the target antisense RNA transcript has sequence identity with a region of the template strand of the target gene, the target antisense RNA transcript will be partially identical to a region within the template strand of the target gene, and may be referred to either as the template strand of the gene or as the target antisense RNA transcript. The position at which the saRNA hybridizes or binds to the target antisense RNA transcript (i.e., the same position on the template strand) is referred to as the "targeted sequence" or "target site."

[0066] The guide strand or antisense strand of the saRNA (whether single-stranded or double-stranded) can be at least 80%, 90%, 95%, 98%, 99% or 100% identical to the reverse complement of the targeted sequence on the template strand of the target gene. In other words, the guide strand or antisense strand of the saRNA can be at least 80%, 90%, 95%, 98%, 99% or 100% complementary to the targeted sequence. Thus, the reverse complement of the guide strand or antisense strand of the saRNA has a high degree of sequence identity with the targeted sequence. The targeted sequence can have the same length, i.e., the same number of nucleotides, as the saRNA and / or the reverse complement of the saRNA.

[0067] In some embodiments, the targeted sequence comprises at least 14 and fewer than 30 nucleotides. In some embodiments, the targeted sequence has 17, 18, 19, 20, 21, 22, or 23 nucleotides.

[0068] In some embodiments, the location of the targeted sequence is within the promoter region of the template strand. In some embodiments, the targeted sequence is located within the TSS (transcription start site) core of template strand.As used herein, "TSS core" or "TSS core sequence" refers to the region between 2000 nucleotides upstream and 2000 nucleotides downstream of TSS (transcription start site).Therefore, the TSS core comprises 4001 nucleotides, and the TSS is located at position 2001 from the 5' end of the TSS core sequence.

[0069] In some embodiments, the targeted sequence is located between 1000 nucleotides upstream and 1000 nucleotides downstream of the TSS. In some embodiments, the targeted sequence is located between 500 nucleotides upstream and 500 nucleotides downstream of the TSS.

[0070] In some embodiments, the targeted sequence is located between 250 nucleotides upstream and 250 nucleotides downstream of the TSS. In some embodiments, the targeted sequence is located between 100 nucleotides upstream and 100 nucleotides downstream of the TSS.

[0071] In some embodiments, the targeted sequence is located between 10 nucleotides upstream and 10 nucleotides downstream of the TSS. In some embodiments, the targeted sequence is located between 5 nucleotides upstream and 5 nucleotides downstream of the TSS.

[0072] In some embodiments, the targeted sequence is located between one nucleotide upstream and one nucleotide downstream of the TSS. In some embodiments, the targeted sequence is located upstream of the TSS in the TSS core. The targeted sequence can be less than 2000, 1000, 500, 250, 100, 100, or 5 nucleotides upstream of the TSS.

[0073] In some embodiments, the targeted sequence is located downstream of the TSS of the TSS core. The targeted sequence can be less than 2000 nucleotides, less than 1000 nucleotides, less than 500 nucleotides, less than 250 nucleotides, less than 100 nucleotides, less than 10 nucleotides, or less than 5 nucleotides downstream of the TSS.

[0074] In some embodiments, the targeted sequence is located within + / -50 nucleotides surrounding the TSS of the TSS core. In some embodiments, the targeted sequence substantially overlaps with the TSS of the TSS core. In some embodiments, the overlap of the targeted sequence begins or ends with the TSS of the TSS core. In some embodiments, the targeted sequence overlaps with the TSS of the TSS core by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 nucleotides in either the upstream or downstream direction.

[0075] The position of the targeted sequence on the template strand is defined by the position of the 5' end of the targeted sequence. The 5' end of the targeted sequence may be at any position of the TSS core, and the targeted sequence may start at any position selected from positions 1 to 4001 of the TSS core. For reference, if the 5'-most end of the targeted sequence is between positions 1 and 2000 of the TSS core, the targeted sequence is considered to be upstream of the TSS, and if the 5'-most end of the targeted sequence is between positions 2002 and 4001, the targeted sequence is considered to be downstream of the TSS. If the 5'-most end of the targeted sequence is at nucleotide 2001, the targeted sequence is considered to be a sequence in the middle of the TSS, and is neither upstream nor downstream of the TSS.

[0076] For further reference, for example, if the 5' end of the targeted sequence is at position 1600 of the TSS core, i.e., it is the 1600th nucleotide of the TSS core, then the targeted sequence starts at position 1600 of the TSS core and is considered to be upstream of the TSS.

[0077] In some embodiments, the TSS core is a sequence of a target gene as set forth in Tables 1 and 2 of WO2016170348, the contents of which are incorporated herein by reference in their entirety.

[0078] In one embodiment, the TSS core is a sequence such as, but not limited to, SEQ ID NOs: 1-4047, 315236-318726, 584785-589061, 913310-917531, 1241080-1245401, 1559932-1564372, and 1879189-1889207 of WO2016170348, the contents of which are incorporated herein by reference in their entirety.

[0079] In one non-limiting example, the target gene is CCAAT / enhancer-binding protein alpha (C / EBPα, C / EBP alpha, C / EBPA, or CEBPA). CEBPA-saRNA is provided in the present application to upregulate the expression of CEBPA. CEBPA is an intronless gene with a single TSS and a length of 2591 nucleotides. The TSS core sequence of CEBPA is shown in Table 1.

[0080] [Table 1]

[0081] In one embodiment, the saRNA of the present invention can have two strands that form a duplex, and one strand can be a guide strand.The saRNA duplex is also called double-stranded saRNA.As used herein, double-stranded saRNA or saRNA duplex refers to saRNA that contains more than one strand, preferably two strands, where hybridization between strands can form a duplex structure region.The two strands of double-stranded saRNA are called antisense strand or guide strand and sense strand or passenger strand.

[0082] The antisense strand of saRNA duplex, which is referred to interchangeably as the guide strand of saRNA, antisense strand saRNA, or antisense saRNA, has high complementarity to the region in the target antisense RNA transcript.The antisense strand may have no more than 5, no more than 4, no more than 3, no more than 2, no more than 1, or no more than none with the region in the target antisense RNA transcript or the sequence to be targeted.Therefore, the antisense strand is highly complementary to the sequence to be targeted on the template strand.The sense strand of saRNA duplex, which is referred to interchangeably as the sense strand saRNA or sense saRNA, has high sequence identity to the sequence to be targeted on the template strand.In some embodiments, the sequence to be targeted is located in the promoter region of the template strand.In some embodiments, the sequence to be targeted is located in the TSS core of the template strand.

[0083] The position of the antisense and / or sense strands of the saRNA duplex relative to the target sequence is defined by referring to the TSS core sequence. For example, if the target sequence is downstream of the TSS, the antisense saRNA and sense saRNA will start downstream of the TSS. In another example, if the target sequence starts at position 200 of the TSS core, the antisense saRNA and sense saRNA will start upstream of the TSS.

[0084] In the context of the present invention, a "strand" refers to a continuous sequence of nucleotides, including non-naturally occurring or modified nucleotides. Two or more strands may be separate molecules, each forming a part of another, or they may be covalently linked by a spacer, for example, a polyethylene glycol linker. At least one strand of the saRNA may contain a region complementary to the target antisense RNA. Such a strand is called the antisense strand or guide strand of the saRNA duplex. The second strand of the saRNA, which contains a region complementary to the antisense strand of the saRNA, is called the sense strand or passenger strand.

[0085] The saRNA duplex can also be formed from a single molecule that is at least partially self-complementary, forming a hairpin structure containing the double-stranded region. In such cases, 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 may have no more than 5, 4, 3, 2, 1, or no mismatches with sequences in the target antisense RNA transcript.

[0086] In some embodiments, the passenger strand of the saRNA may contain at least one nucleotide that is not complementary to the corresponding nucleotide on the guide strand, referred to as a mismatch with the guide strand. Mismatches with the guide strand may promote preferential loading of the guide strand (Wu et al., PLoS ONE, vol. 6(12):e28580 (2011), the entire contents of which are incorporated herein by reference). In one embodiment, at least one mismatch with the guide strand may be at the 3' end of the passenger strand. In one embodiment, the 3' end of the passenger strand may contain 1 to 5 mismatches with the guide strand. In one embodiment, the 3' end of the passenger strand may contain 2 to 3 mismatches with the guide strand. In one embodiment, the 3' end of the passenger strand may contain 6 to 10 mismatches with the guide strand.

[0087] In one embodiment, the saRNA duplex may exhibit efficacy in proliferating cells. The saRNA duplex may have siRNA-like complementarity to a region of the target antisense RNA transcript, i.e., 100% complementarity between nucleotides 2-6 from the 5' end of the guide strand of the saRNA duplex and the region of the target antisense RNA transcript. Additionally, other nucleotides of the saRNA may have at least 80%, 90%, 95%, 98%, 99%, or 100% complementarity to the region of the target antisense RNA transcript. For example, nucleotides 7 (counting from the 5' end) through the 3' end of the saRNA may have at least 80%, 90%, 95%, 98%, 99%, or 100% complementarity to the region of the target antisense RNA transcript.

[0088] In this context, the term "small interfering RNA" or "siRNA" refers to a double-stranded RNA, typically 20-25 nucleotides in length, that participates in the RNA interference (RNAi) pathway and disrupts or inhibits the expression of a specific gene. The gene is the target gene of the siRNA. For example, an siRNA that disrupts the expression of the A3GALT2 gene is called an "A3GALT2-siRNA," and the A3GALT2 gene is the target gene. The siRNA is typically about 21 nucleotides in length, with 3' overhangs (e.g., 2 nucleotides) on both ends of the two strands.

[0089] siRNA binds to one or more RNA transcripts of target gene at specific sequence and promotes its cleavage, thereby inhibiting the expression of target gene.Typically, in RNAi, the RNA transcript is mRNA, so the cleavage of mRNA leads to the downregulation of gene expression.In the present invention, without wishing to be bound by any theory, one possible mechanism is that the saRNA of the present invention can regulate target gene expression by binding to target antisense RNA transcript.Target antisense RNA transcript can be cleaved or not cleaved.

[0090] A double-stranded saRNA may contain one or more single-stranded nucleotide overhangs. The term "overhang" or "tail" in the context of double-stranded saRNA and siRNA refers to at least one unpaired nucleotide protruding from the double-stranded structure of the saRNA or siRNA. For example, a nucleotide overhang exists when the 3' end of one strand of the saRNA extends beyond the 5' end of the other strand, or vice versa. The saRNA may contain at least one nucleotide overhang; alternatively, the overhang may contain at least two nucleotides, at least three nucleotides, at least four nucleotides, at least five nucleotides, or more nucleotides. The nucleotide overhang may comprise or consist of nucleotide / nucleoside analogs, including deoxynucleotides / nucleosides. The overhang may be present in the sense strand, the antisense strand, or any combination thereof. Furthermore, the overhanging nucleotides may be present at the 5' end, the 3' end, or both ends of either the antisense or sense strand of the saRNA. When two oligonucleotides are designed to form one or more single-stranded overhangs during hybridization, such overhangs shall not be considered as mismatches in determining complementarity.For example, saRNA comprising one oligonucleotide of 19 nucleotides in length and another oligonucleotide of 21 nucleotides in length, if the longer oligonucleotide comprises a 19-nucleotide sequence that is completely complementary to the shorter oligonucleotide, it can still be considered as "completely complementary" for the purposes described herein.Overhang nucleotides can be natural or non-natural nucleotides.Overhangs can be modified nucleotides as defined herein.

[0091] In one embodiment, the antisense strand of the double-stranded saRNA has a 1-10 nucleotide overhang at its 3'-end and / or 5'-end. In one embodiment, the antisense strand of the double-stranded saRNA has a 1-4 nucleotide overhang at its 3'-end or a 1-2 nucleotide overhang at its 3'-end. In one embodiment, the sense strand of the double-stranded saRNA has a 1-10 nucleotide overhang at its 3'-end and / or 5'-end. In one embodiment, the sense strand of the double-stranded saRNA has a 1-4 nucleotide overhang at its 3'-end or a 1-2 nucleotide overhang at its 3'-end. In one embodiment, both the sense and antisense strands of the double-stranded saRNA have a 3' overhang. The 3' overhang may contain one or more uracils, for example, the sequence UU or UUU. In one embodiment, one or more nucleotides in the overhang are replaced with a nucleoside thiophosphate, where the internucleoside linkage is a thiophosphate. In one embodiment, the overhang comprises one or more deoxyribonucleosides, for example, the sequence dTdT or dTdTdT. In one embodiment, the overhang comprises the sequence dT*dT, where "*" is a phosphorothioate internucleoside linkage (sometimes referred to as "s"). In one embodiment, the overhang comprises at least one 2'-OMe modified U (referred to as u). In one embodiment, the overhang comprises u*u (also referred to as usu). In one embodiment, the overhang comprises uu. In one embodiment, the overhang comprises an inverted nucleotide or nucleoside connected to the strand by an inverted linkage (3'-3' or 5'-5' linkage). For example, the overhang can comprise an inverted dT or an inverted abasic nucleoside. An inverted abasic nucleoside has no base moiety.

[0092] Those skilled in the art will understand that it is convenient to define the saRNA of the present invention by referring to the target antisense RNA transcript or the sequence being targeted, regardless of the mechanism by which the saRNA regulates target gene expression. However, the saRNA of the present invention may alternatively be defined by referring to the target gene. Because the target antisense RNA transcript is complementary to a genomic region of the coding strand of the target gene, and the saRNA of the present invention is then complementary to a region of the target antisense RNA transcript, the saRNA of the present invention may be defined as having sequence identity with a region of the coding strand of the target gene. All of the characteristics discussed herein regarding the definition of the saRNA of the present invention by referring to the target antisense RNA transcript also apply, mutatis mutandis, to the definition of the saRNA of the present invention by referring to the target gene, and therefore any discussion of complementarity with the target antisense RNA transcript should be understood to include identity to the genomic sequence of the target gene. Thus, the saRNA of the present invention may have a high percentage identity with the genomic sequence of the target gene, for example, at least 80%, 90%, 95%, 98%, or 99% or 100% identity. The genomic sequence can be up to 2000, 1000, 500, 250, or 100 nucleotides upstream or downstream of the transcription start site of the target gene. It can be aligned to the promoter region of the target gene. Thus, the saRNA can have sequence identity with the sequence aligned to the promoter region of the target gene.

[0093] In one embodiment, it is not necessary to determine the presence of a target antisense RNA transcript to design a saRNA of the present invention. In other words, designing a saRNA does not require identifying a target antisense RNA transcript. For example, the nucleotide sequence of the TSS core, i.e., the sequence of the region 2000 nucleotides upstream of the transcription start site of the target gene to 2000 nucleotides downstream of the transcription start site of the target gene, can be obtained by sequencing or database search using the genomic sequence of the coding strand of the target gene. A targeted sequence within the TSS core starting at any position between 1 and 4001 of the TSS core of the template strand can be selected and then used to design a saRNA sequence. As discussed above, saRNAs have a high degree of sequence identity with the reverse complement of the targeted sequence.

[0094] The number of off-target hits, zero-mismatch (0 mm) hits, and one-mismatch (1 mm) hits for the saRNA sequences across the entire genome are then determined. The term "off-target hits" refers to the number of other sites across the entire genome that are identical to the sequence targeted by the saRNA on the template strand of the target gene. The term "0 mm hits" refers to the number of known protein-coding transcripts other than the saRNA target transcript to which the saRNA can hybridize or bind with zero mismatches. In other words, the "0 mm hits" counts the number of known protein-coding transcripts other than the saRNA target transcript that contain a region completely identical to the saRNA sequence. The term "1 mm hits" refers to the number of known protein-coding transcripts other than the saRNA target transcript that can hybridize or bind with one mismatch to their complement. In other words, the "1 mm hits" counts the number of known protein-coding transcripts other than the saRNA target transcript that contain a region identical to the saRNA sequence with only one mismatch. In one embodiment, only saRNA sequences with no off-target hits, no 0 mm hits, and no 1 mm hits are selected. The saRNA sequences disclosed in this application have no off-target hits, no 0 mm hits, and no 1 mm hits, respectively.

[0095] The method disclosed in U.S. Patent Application Publication No. 2013 / 0164846 (saRNA Algorithm), filed June 23, 2011, the entire contents of which are incorporated herein by reference, can also be used to design saRNAs. saRNA design is also disclosed in U.S. Patent Nos. 8,324,181 and 7,709,566 to Corey et al., U.S. Patent Application Publication No. 2010 / 0210707 to Li et al., and Voutila et al., Mol Ther Nucleic Acids, vol. 1, e35 (2012), the entire contents of each of which are incorporated herein by reference.

[0096] "Determining the presence" means either searching a database of ESTs and / or antisense RNA transcripts surrounding the locus of the target gene to identify a suitable target antisense RNA transcript, or confirming the physical presence of the target antisense RNA transcript in the cell using RT-PCR or any other known technique.

[0097] In some embodiments, the saRNA of the present invention can be single-stranded or double-stranded. A double-stranded molecule comprises a first strand and a second strand. If double-stranded, each strand of the duplex can be at least 14 or at least 18 nucleotides in length, for example, 19, 20, 21, or 22 nucleotides in length. The duplex can hybridize over the entire length of at least 12, at least 15, at least 17, or at least 19 nucleotides. Each strand can be exactly 19 nucleotides in length. Because oligonucleotide duplexes longer than this length may increase the risk of inducing an interferon response, the saRNA is preferably less than 30 nucleotides in length. In one embodiment, the saRNA is 19-25 nucleotides in length. The strands forming the saRNA duplex can be of equal or unequal length.

[0098] In one embodiment, the saRNA of the present invention comprises a sequence of at least 14 nucleotides and less than 30 nucleotides that has at least 80%, 90%, 95%, 98%, 99%, or 100% complementarity to the targeted sequence. In one embodiment, the sequence that has at least 80%, 90%, 95%, 98%, 99%, or 100% complementarity to the targeted sequence is at least 15, 16, 17, 18, or 19 nucleotides in length, or 18-22, or 19-21, or exactly 19 nucleotides in length.

[0099] The saRNA of the present invention may contain a short 3' or 5' sequence that is not complementary to the target antisense RNA transcript. In one embodiment, such a sequence is at the 3' end of the strand. The sequence may be 1 to 5 nucleotides long, or 2 or 3 nucleotides long. The sequence may include uracil, thus providing a 3' stretch of 2 or 3 uracils. The sequence may contain one or more deoxyribonucleosides, such as dT. In one embodiment, one or more nucleotides in the sequence are replaced with a nucleoside thiophosphate, where the internucleoside linkage is a thiophosphate. As a non-limiting example, the sequence may include the sequence dT*dT, where * is a thiophosphate internucleoside linkage. This non-complementary sequence may be referred to as a "tail." If a 3' tail is present, the strand may be longer, for example, a 3' tail that can be 19 nucleotides plus UU or UUU. Such a 3' tail shall not be considered a mismatch with respect to determining complementarity between the saRNA and the target antisense RNA transcript.

[0100] Thus, the saRNA of the present invention may comprise (i) a sequence having at least 80% complementarity to a region of the target antisense RNA transcript and (ii) a 1-5 nucleotide 3' tail that may contain or consist of uracil residues. Thus, the saRNA will typically have complementarity to a region of the target antisense RNA transcript over its entire length, excluding the 3' tail, if present. Any of the saRNA sequences disclosed in the present application may optionally include such a 3' tail. Thus, any of the saRNA sequences disclosed in the saRNA tables and sequence listings may optionally include such a 3' tail. The saRNA of the present invention may further comprise a substrate sequence for Dicer or Drosha.

[0101] The saRNA of the present invention may contain a flanking sequence. The flanking sequence may be inserted at the 3' or 5' end of the saRNA of the present invention. In one embodiment, the flanking sequence is a miRNA sequence that gives the saRNA miRNA structure and can be processed by Drosha and Dicer. In a non-limiting example, the saRNA of the present invention has two strands and is cloned into a microRNA precursor, for example, the miR-30 backbone flanking sequence.

[0102] The saRNA of the present invention may contain a substrate or recognition sequence for a restriction enzyme. The restriction enzyme recognition sequence may be at the 3' or 5' end of the saRNA of the present invention. Non-limiting examples of restriction enzymes include NotI and AscI.

[0103] In one embodiment, the saRNA of the present invention consists of two stably base-paired, integrated strands. In some embodiments, the passenger strand may contain at least one nucleotide that is not complementary to the corresponding nucleotide on the guide strand, referred to as a mismatch with the guide strand. In one embodiment, at least one mismatch with the guide strand may be at the 3' end of the passenger strand. In one embodiment, the 3' end of the passenger strand may contain one to five mismatches with the guide strand. In one embodiment, the 3' end of the passenger strand may contain two to three mismatches with the guide strand. In one embodiment, the 3' end of the passenger strand may contain six to ten mismatches with the guide strand.

[0104] In some embodiments, the double-stranded saRNA may contain several unpaired nucleotides at the 3' end of each strand forming a 3' overhang. The number of unpaired nucleotides forming the 3' overhang of each strand may be in the range of 1 to 5 nucleotides, 1 to 3 nucleotides, or 2 nucleotides. The 3' overhang may be formed on the 3' tail described above, and thus the 3' tail may be the 3' overhang of the double-stranded saRNA.

[0105] Thus, the saRNA of the present invention can be single-stranded and comprise (i) a sequence having at least 80% complementarity to a region of the target antisense RNA transcript and (ii) a 1-5 nucleotide 3' tail, which may contain uracil residues. The saRNA of the present invention can be complementary to a region of the target antisense RNA transcript over its entire length, excluding the 3' tail, if present. As noted above, instead of being "complementary to the target antisense RNA transcript," the saRNA of the present invention can also be defined as having "identity" with the coding strand of the target gene. The saRNA of the present invention can be double-stranded and comprise (i) a first strand comprising a first sequence having at least 80% complementarity to a region of the target antisense RNA transcript and (ii) a 1-5 nucleotide 3' overhang, and (i) a second sequence that forms a duplex with the first sequence and (ii) a 1-5 nucleotide 3' overhang.

[0106] As described herein, the genomic sequence of target gene can be used to design saRNA of target gene.The sequence of target antisense RNA transcript can be determined from the sequence of target gene to design saRNA of target gene.However, it is not necessary to determine the existence of such target antisense RNA transcript.

[0107] One aspect of the present invention provides saRNAs that regulate the expression of target genes. Also provided are saRNAs that regulate the level of target transcripts. In some embodiments, the target transcript is a coding transcript, such as an mRNA. Another aspect of the present invention provides saRNAs that regulate the level of a protein encoded by a target coding transcript. In one embodiment, the expression of a target gene is increased by at least 20, 30, 40%, or at least 45, 50, 55, 60, 65, 70, 75%, or at least 80% in the presence of a saRNA of the present invention compared to the expression of the target gene in the absence of a saRNA of the present invention. In a further embodiment, the expression of a target gene is increased by at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 times, or at least 15, 20, 25, 30, 35, 40, 45, 50 times, or at least 60, 70, 80, 90, or 100 times in the presence of a saRNA of the present invention compared to the expression of the target gene in the absence of a saRNA of the present invention. Modulation of expression of a target gene can be reflected or determined by changes in the level of mRNA encoding the target gene.

[0108] The saRNA of the invention can be produced by any suitable method, for example, synthetically or by expression in a cell using standard molecular biology techniques known to those skilled in the art. For example, the saRNA of the invention can be chemically synthesized or recombinantly produced using methods known in the art.

[0109] The saRNA of the present invention may be single-stranded and contain 14 to 30 nucleotides. The sequence of the single-stranded saRNA may have at least 60%, 70%, 80%, or 90% identity to sequences such as, but not limited to, SEQ ID NOs: 4048-315235, 318727-584784, 589062-913309, 917532-1241079, 1245402-1559931, 1564373-1879188, and 1889208-2585259 of WO 2016170348, the entire contents of which are incorporated herein by reference.

[0110] In one embodiment, the single-stranded saRNA includes sequences such as, but not limited to, SEQ ID NOs: 4048-315235, 318727-584784, 589062-913309, 917532-1241079, 1245402-1559931, 1564373-1879188, and 1889208-2585259 of International Publication No. WO 2016170348, the contents of which are incorporated herein by reference in their entirety.

[0111] In one embodiment, the saRNA is a single-stranded saRNA comprising an antisense sequence, such as, but not limited to, any of the antisense sequences set forth in the sequence listing referenced at the beginning of this application.

[0112] In one embodiment, the saRNA is a single-stranded saRNA comprising an antisense sequence, such as, but not limited to, any of the sense sequences set forth in the sequence listing referenced at the beginning of this application.

[0113] The single-stranded saRNA of the present invention may be modified or unmodified. In one embodiment, the single-stranded saRNA may have a 3' tail. In one embodiment, the saRNA can be double-stranded. The two strands form a duplex, also known as a saRNA duplex, with each strand containing 14 to 30 nucleotides. The first strand of the double-stranded saRNA can have at least 60%, 70%, 80%, or 90% identity to a sequence such as, but not limited to, SEQ ID NOs: 4048-315235, 318727-584784, 589062-913309, 917532-1241079, 1245402-1559931, 1564373-1879188, and 1889208-2585259 in WO 2016170348, the contents of which are incorporated herein by reference in their entirety. In one embodiment, the first strand of the double-stranded saRNA includes sequences such as, but not limited to, SEQ ID NOs: 4048-315235, 318727-584784, 589062-913309, 917532-1241079, 1245402-1559931, 1564373-1879188, and 1889208-2585259. The second strand of the double-stranded saRNA may have at least 60%, 70%, 80%, or 90% identity to sequences such as, but not limited to, SEQ ID NOs: 4048-315235, 318727-584784, 589062-913309, 917532-1241079, 1245402-1559931, 1564373-1879188, and 1889208-2585259 of WO 2016170348, the contents of which are incorporated herein by reference in their entirety. In one embodiment, the second strand of the double-stranded saRNA comprises a sequence such as, but not limited to, SEQ ID NOs: 4048-315235, 318727-584784, 589062-913309, 917532-1241079, 1245402-1559931, 1564373-1879188, and 1889208-2585259 of International Publication No. WO 2016170348, the contents of which are incorporated herein by reference in their entirety. In one embodiment, the double-stranded saRNA may have a 3' overhang on each strand.

[0114] In one embodiment, the saRNA of the present invention is a saRNA duplex.The saRNA duplex can be a pair of sense and antisense sequences, such as, but not limited to, any of the sense sequences and corresponding antisense sequences listed in the sequence listing at the beginning of this application.The saRNA of the present invention can be a pair of sense and antisense sequences listed in the sequence listing at the beginning of this application.

[0115] The double-stranded saRNA of the present invention may be modified or unmodified. Bifunctional oligonucleotides Bifunctional or dual-functional oligonucleotides, such as saRNA, may be designed to upregulate the expression of a first gene and downregulate the expression of at least one second gene. One strand of the dual-functional oligonucleotide activates the expression of the first gene, and the other strand inhibits the expression of the second gene. Each strand may further comprise a Dicer substrate sequence.

[0116] Chemical modification of saRNA As used herein, the term "modified" or, where appropriate, "modified" in saRNA refers to structural and / or chemical modifications to A, G, U, or C ribonucleotides. Nucleotides in the saRNA of the present invention may include non-standard nucleotides, such as non-naturally occurring nucleotides, chemically synthesized nucleotides, or deoxynucleotides. The saRNA of the present invention may include any useful modification, for example, modifications to the sugar, nucleobase, or internucleoside linkage (e.g., phosphate linkage / phosphodiester linkage / phosphodiester backbone). One or more atoms of a pyrimidine nucleobase may be replaced or substituted with an optionally substituted amino, an optionally substituted thiol, an optionally substituted alkyl (e.g., methyl or ethyl), or a halo (e.g., chloro or fluoro). In certain embodiments, a modification (e.g., one or more modifications) is present in each of the sugar and the internucleoside linkage. The modification according to the present invention can be a modification of ribonucleic acid (RNA) resulting in deoxyribonucleic acid (DNA), threose nucleic acid (TNA), glycol nucleic acid (GNA), peptide nucleic acid (PNA), locked nucleic acid (LNA), or a hybrid thereof. In a non-limiting example, the 2'-OH of U is replaced with 2'-OMe.

[0117] In one embodiment, the saRNA of the present invention may contain at least one modification described herein. In another embodiment, the saRNA is a saRNA duplex, and the sense strand and the antisense sequence can independently contain at least one modification.As a non-limiting example, the sense sequence can contain a modification, and the antisense strand can be unmodified.As another non-limiting example, the antisense sequence can contain a modification, and the sense strand can be unmodified.As yet another non-limiting example, the sense sequence can contain two or more modifications, and the antisense strand can contain one modification.As a non-limiting example, the antisense sequence can contain two or more modifications, and the sense strand can contain one modification.

[0118] The saRNAs of the invention may contain combinations of modifications to the sugar, nucleobase, and / or internucleoside linkage, including any one or more of the modifications described herein or in WO 2013 / 052523, filed October 3, 2012, particularly those represented by formulae (Ia)-(Ia-5), (Ib)-(If), (IIa)-(IIp), (IIb-1), (IIb-2), (IIc-1)-(IIc-2), (IIn-1), (IIn-2), (IVa)-(IVl), and (IXa)-(IXr), the contents of which are incorporated herein by reference in their entirety.

[0119] The saRNA of the present invention may or may not be uniformly modified along the entire length of the molecule. For example, the saRNA of the present invention may or may not have uniformly modified one or more or all types of nucleotides (e.g., purines or pyrimidines, or one or more or all of A, G, U, and C). In some embodiments, every nucleotide X of the saRNA of the present invention is modified, where X can be any one of the nucleotides A, G, U, or C, or any one of the combinations A+G, A+U, A+C, G+U, G+C, U+C, A+G+U, A+G+C, G+U+C, or A+G+C.

[0120] Various sugar modifications, nucleotide modifications, and / or internucleoside linkages (e.g., backbone structures) can be present at various positions in the saRNA. One of skill in the art will recognize that nucleotide analogs or other modifications can be placed at any position in the saRNA such that the function of the saRNA is not substantially reduced. The saRNA of the present invention may contain modified nucleotides in an amount ranging from about 1% to about 100% (based on the total nucleotide content or based on one or more types of nucleotides, i.e., A, G, U, or C) or any intervening percentage (e.g., 1% to 20%, 1% to 25%, 1% to 50%, 1% to 60%, 1% to 70%, 1% to 80%, 1% to 90%, 1% to 95%, 10% to 20%, 10% to 25%, 10% to 50%, 10% to 60%, 10% to 70%, 10% to 80%, 10% to 90% , 10% to 95%, 10% to 100%, 20% to 25%, 20% to 50%, 20% to 60%, 20% to 70%, 20% to 80%, 20% to 90%, 20% to 95%, 20% to 100%, 50% to 60%, 50% to 70%, 50% to 80%, 50% to 90%, 50% to 95%, 50% to 100%, 70% to 80%, 70% to 90%, 70% to 95%, 70% to 100%, 80% to 90%, 80% to 95%, 80% to 100%, 90% to 95%, 90% to 100%, and 95% to 100%.

[0121] In some embodiments, the saRNA of the present invention can be modified to become a circular nucleic acid. The ends of the saRNA of the present invention can be linked using chemical reagents or enzymes to generate a circular saRNA with no free ends. Circular saRNAs are expected to be more stable than their linear counterparts and more resistant to digestion by RNase R exonuclease. Circular saRNAs can also contain other structural and / or chemical modifications of A, G, U, or C ribonucleotides.

[0122] The saRNA of the present invention may be modified with any of the oligonucleotide or polynucleotide modifications disclosed on pages 136-247 of PCT Publication No. WO 2013 / 151666, published October 10, 2013, the entire contents of which are incorporated herein by reference.

[0123] The saRNA of the invention may contain a combination of modifications. The saRNA may contain 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 on each strand.

[0124] In some embodiments, the saRNA is at least 50% modified, e.g., at least 50% of the nucleotides are modified. In some embodiments, the saRNA is at least 75% modified, e.g., at least 75% of the nucleotides are modified. In some embodiments, both strands of the saRNA may be modified along their entire length (100% modified). It should be understood that any modification to any part of a nucleotide or nucleoside constitutes a modification, as each nucleotide (sugar, base, and phosphate moiety, e.g., linker) can be modified.

[0125] In some embodiments, the saRNA is modified in at least 10% of only one component of the nucleotides, and such component is selected from the nucleobase, sugar, or internucleoside linkage. For example, the saRNA can be modified in at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the nucleobases, sugars, or linkages of the saRNA.

[0126] In some embodiments, the saRNA comprises at least one sugar modification. Non-limiting examples of sugar modifications can include:

[0127] [ka]

[0128] [ka]

[0129] In some embodiments, at least one of the 2' positions (OH in RNA, H in DNA) of the sugars of the nucleotides of the saRNA is substituted with -OMe, referred to as 2'-OMe.

[0130] [ka]

[0131] In some embodiments, at least one of the 2' positions (OH in RNA, H in DNA) of the sugar of the nucleotides of the saRNA is substituted with -F, referred to as 2'-F.

[0132] [ka]

[0133] In some embodiments, the saRNA contains at least one phosphorothioate or methylphosphonate bond between nucleotides. In some embodiments, the saRNA comprises a 3' and / or 5' capping or overhang. In some embodiments, the saRNA of the present invention may comprise at least one inverted deoxyribonucleoside overhang (e.g., dT). The inverted overhang, e.g., dT, may be at the 5' or 3' end of the passenger (sense) strand. In some embodiments, the saRNA of the present invention may comprise an inverted abasic modification on the passenger strand. The at least one inverted abasic modification may be at the 5' end, the 3' end, or both ends of the passenger strand. The inverted abasic modification may promote preferential loading of the guide (antisense) strand.

[0134] In some embodiments, the saRNA comprises at least one 5'-(E)-vinylphosphonate (5'-E-VP) modification.

[0135] [ka]

[0136] In some embodiments, the saRNA includes as a modification at least one glycol nucleic acid (GNA), which is an acyclic nucleic acid analog.

[0137] [ka]

[0138] In some embodiments, the saRNA comprises at least one motif of at least two consecutive nucleotides with the same sugar modification. In one example, such a motif may comprise two or three consecutive nucleotides. In some embodiments, the consecutive nucleotides of the motif comprise a 2'-F modification. In some embodiments, the consecutive nucleotides of the motif comprise a 2'-OMe modification.

[0139] In some embodiments, when the saRNA is double-stranded, the passenger strand and guide strand of the saRNA each contain at least one motif of consecutive nucleotides having the same sugar modification.

[0140] In some embodiments, the passenger strand and guide strand of the saRNA each contain at least two motifs of consecutive nucleotides with the same sugar modification. In some embodiments, at least two motifs on a given strand independently have different sugar modifications. For example, the passenger strand or guide strand may have at least one motif with a 2'-OMe modification and at least one motif with a 2'-F modification. In some embodiments, at least two motifs on a given strand are separated by at least one nucleotide (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20). In some embodiments, at least two motifs on a given strand are connected. In some embodiments, at least one motif on the passenger strand and its complementary motif on the guide strand have different sugar modifications. For example, the nucleotides of the motif on the passenger strand have a 2'-F modification and the nucleotides of the motif on the guide strand have a 2'-OMe modification, where the two motifs are complementary to each other. In another example, the nucleotides of the motif on the passenger strand have a 2'-OMe modification and the nucleotides of the motif on the guide strand have a 2'-F modification, where the two motifs are complementary to each other.

[0141] In some embodiments, the modification of a motif differs from the modification of the immediately adjacent nucleotides on either side of the motif. In some embodiments, the saRNA comprises at least one motif with alternating sugar modifications. In one example, the alternating sugar modification motif comprises 2-30 nucleotides. In some embodiments, the motif comprises alternating 2'-F and 2'-OMe modifications.

[0142] In some embodiments, when the saRNA is double-stranded, the passenger strand and the guide strand each contain at least one motif with alternating sugar modifications.In some embodiments, at least one nucleotide on the passenger strand and its complementary nucleotide on the guide strand have different sugar modifications.For example, one nucleotide of a base pair on the passenger strand has a 2'-F modification, and the other nucleotide of a base pair on the guide strand has a 2'-OMe modification.In another example, one nucleotide of a base pair on the passenger strand has a 2'-OMe modification, and the other nucleotide of a base pair on the guide strand has a 2'-F modification.

[0143] In some embodiments, the saRNA is double-stranded and has the following general formula: Passenger (sense or SS): 5' overhang 1-NT1-(XXX-NT2)n-overhang 2 3', Guide (antisense or AS): 3' overhang 3-NT1'-(YYY-NT2')n-overhang 4 5', (I) where: Each strand is 14 to 30 nucleotides in length. overhang 1, overhang 2, overhang 3, and overhang 4 each independently represent an oligonucleotide sequence containing 0 to 5 nucleotides; NT1 and NT1' represent oligonucleotide sequences containing 0 to 20 nucleotides, NT1 being complementary to NT1'; XXX-NT2 and YYY-NT2' each independently represent a motif of consecutive nucleotides, wherein the first three consecutive nucleotides have the same chemical modification, followed by an oligonucleotide sequence comprising 0 to 20 nucleotides, wherein XXX is complementary to YYY and NT2 is complementary to NT2'; each of NT1, NT2, NT1', and NT2' comprises at least one chemical modification; n is a number from 1 to 5.

[0144] The guide strand of the saRNA having formula (I) comprises a sequence that is at least 80% identical to the reverse complement of the targeted sequence located in the TSS core on the template strand of the target gene. In other words, the guide strand of the saRNA having formula (I) comprises a sequence that is at least 80% complementary to the targeted sequence located in the TSS core on the template strand of the target gene. "Targeted sequence" and "TSS core" are defined above.

[0145] The three consecutive nucleotides in XXX and YYY do not have to be the same; they just have to have the same chemical modification. In some cases, each strand comprises 14-17 nucleotides, 17-25 nucleotides, 17-23 nucleotides, 23-27 nucleotides, 19-21 nucleotides, 21-23 nucleotides, or 27-30 nucleotides.

[0146] In some cases, each chain includes at least one sugar modification. In some cases, at least one nucleotide on the passenger strand and its complementary nucleotide on the guide strand have different sugar modifications.

[0147] In some cases, NT1, NT2, NT1', and NT2' have alternating modifications, such as alternating 2'-OMe and 2'-F modifications. In some cases, three consecutive nucleotides of XXX have a 2'-OMe modification and three consecutive nucleotides of YYY have a 2'-F modification.

[0148] In some cases, three consecutive nucleotides of XXX have a 2'-F modification and three consecutive nucleotides of YYY have a 2'-OMe modification. In some cases, the modification of XXX or YYY is different from the modification of the immediately adjacent nucleotides on either side of the XXX or YYY.

[0149] In some cases, the YYY motif may begin at the 8th, 9th, 10th, 11th, 12th, or 13th position from the 5' end of the antisense strand. In some cases, the XXX motif may start at the 8th, 9th, 10th, 11th, 12th, or 13th position from the 3' end of the sense strand.

[0150] In some cases, overhang 1, overhang 2, overhang 3 and / or overhang 4 comprise uu. In some cases, overhang 1, overhang 2, and / or overhang 3 comprise an inverted dT.

[0151] In some cases, overhang 1, overhang 2, and / or overhang 3 comprise an inverted abasic nucleoside. In some cases, the saRNA contains at least one phosphorothioate bond between nucleotides (

[0152] [ka]

[0153] , referred to in the sequence as s) or a methylphosphonate bond. The phosphorothioate or methylphosphonate bond can be at the 3' end of one strand, for example, the sense strand or the antisense strand. For example, the overhang at the 3' end of the antisense strand can be usu.

[0154] In some cases, the passenger strand of the saRNA contains a linker at its 3' or 5' end, which allows a moiety to be attached to the 3' or 5' end of the passenger strand. Overhang 1 or Overhang 2 may contain a linker. The linker may be any suitable linker, such as NH2-(CH2)6-- (referred to in the sequence as NH2C6). A phosphorothioate bond may be present between the linker and the passenger strand.

[0155] In some embodiments, the modified saRNA has improved stability compared to the unmodified version.The serum half-life of the modified saRNA can be at least about 6 hours, about 12 hours, about 18 hours, about 24 hours, about 30 hours, about 36 hours, about 42 hours, about 48 hours, about 54 hours, 60 hours, 66 hours, 72 hours, 78 hours, 84 hours, 90 hours, or 96 hours longer than the unmodified version.In some embodiments, the modified saRNA has a half-life of at least 48 hours, 60 hours, 72 hours, 84 hours, or 96 hours.

[0156] In some embodiments, the saRNA upregulates CEBPA. Non-limiting examples of CEBPA-saRNA sequences with at least one modification include the saRNAs in Table 2. The parent sequence does not have the modification.

[0157] [Table 2]

[0158] The (NH2C6) linker on any modified saRNA can be replaced with another suitable linker. In one embodiment, the CEBPA-saRNA comprises formula (I). The antisense strand of the CEBPA-saRNA is at least 80% identical to the reverse complement of a region on the CEBPA TSS core. Non-limiting examples of CEBPA-saRNAs having general formula (I) include S6 (XD-06414, SEQ ID NOs: 14 and 15).

[0159] saRNA conjugates and combinations Conjugation can result in increased stability and / or half-life, and can be particularly useful for targeting the saRNA of the present invention to specific sites in cells, tissues, or organisms. The saRNA of the present invention can be conjugated to other polynucleotides, dyes, intercalating agents (e.g., acridine), crosslinking agents (e.g., psoralen, 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]2, polyamino, alkyl, substituted alkyl, radiolabels, etc. They can be designed to be conjugated to markers, enzymes, haptens (e.g., biotin), transport / absorption enhancers (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases, proteins, e.g., glycoproteins, or peptides, e.g., molecules with specific affinity for co-ligands, or antibodies, e.g., antibodies that bind to specific cell types such as cancer cells, endothelial cells, bone cells, etc., hormones and hormone receptors, non-peptide species, e.g., lipids, lectins, carbohydrates, vitamins, cofactors, or drugs. Suitable conjugates for nucleic acid molecules are disclosed in WO 2013 / 090648, filed December 14, 2012, the entire contents of which are incorporated herein by reference.

[0160] According to the present invention, the saRNA of the present invention may be administered together with or further comprise one or more of an RNAi agent, small interfering RNA (siRNA), short hairpin RNA (shRNA), long non-coding RNA (lncRNA), enhancer RNA, enhancer-derived RNA or enhancer-driven RNA (eRNA), microRNA (miRNA), miRNA-binding site, antisense RNA, ribozyme, catalytic DNA, tRNA, RNA that induces triple helix formation, aptamer, vector, etc. To achieve various functions, one or more of the RNAi agent, small interfering RNA (siRNA), short hairpin RNA (shRNA), long non-coding RNA (lncRNA), microRNA (miRNA), miRNA-binding site, antisense RNA, ribozyme, catalytic DNA, tRNA, RNA that induces triple helix formation, aptamer, or vector may comprise at least one modification or substitution.

[0161] In some embodiments, the modification is selected from chemical substitution of the nucleic acid at the sugar position, chemical substitution at the phosphate position, and chemical substitution at the base position. In other embodiments, the chemical modification is selected from incorporation of modified nucleotides, 3'-capping, conjugation to a high molecular weight non-immunogenic compound, conjugation to a lipophilic compound, and incorporation of phosphorothioates into the phosphate backbone. In one embodiment, the high molecular weight non-immunogenic compound is polyalkylene glycol or polyethylene glycol (PEG).

[0162] In one embodiment, saRNA containing at least one modification may exhibit efficacy in proliferating cells. In one embodiment, the saRNA of the present invention can be linked to a transgene so that it can be co-expressed from an RNA polymerase II promoter. In a non-limiting example, the saRNA of the present invention is linked to the green fluorescent protein gene (GFP).

[0163] In one embodiment, the saRNA of the present invention can bind to a DNA or RNA aptamer to generate a saRNA-aptamer conjugate. Aptamers are oligonucleotides or peptides with high selectivity, affinity, and stability. They provide highly specific and tight binding to target molecules by adopting a specific and stable three-dimensional shape. Aptamers can be nucleic acid species engineered through repeated rounds of in vitro selection or equivalently, SELEX (Systematic Evolution of Ligands by Exponential Enrichment) to bind to various molecular targets, including small molecules, proteins, and nucleic acids, as well as cells, tissues, and organisms. Nucleic acid aptamers possess specific binding affinity to molecules through interactions other than typical Watson-Crick base pairing. Nucleic acid aptamers, like peptides generated by phage display or monoclonal antibodies (mAbs), can specifically bind to a selected target and, through binding, block the target's ability to function. In some cases, aptamers can also be peptide aptamers. For any specific molecular target, nucleic acid aptamers can be identified from a combinatorial library of nucleic acids, for example, by SELEX. Peptide aptamers can be identified using the yeast two-hybrid system. Therefore, those skilled in the art can design suitable aptamers for delivering the saRNA of the present invention or cells to target cells, such as hepatocytes. DNA aptamers, RNA aptamers, and peptide aptamers are contemplated. It is preferable to administer the saRNA of the present invention to the liver using a liver-specific aptamer.

[0164] As used herein, a typical nucleic acid aptamer is approximately 10-15 kDa in size (20-45 nucleotides), binds to its target with at least nanomolar affinity, and discriminates against highly related targets. Nucleic acid aptamers can be ribonucleic acid, deoxyribonucleic acid, or a mixture of ribonucleic acid and deoxyribonucleic acid. Aptamers can be single-stranded ribonucleic acid, deoxyribonucleic acid, or a mixture of ribonucleic acid and deoxyribonucleic acid. Aptamers can include at least one chemical modification.

[0165] Suitable nucleotide lengths for aptamers range from about 15 to about 100 nucleotides (nt), and in various other embodiments, are 15-30 nt, 20-25 nt, 30-100 nt, 30-60 nt, 25-70 nt, 25-60 nt, 40-60 nt, 25-40 nt, 30-40 nt, any of the following values: 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nt, or 40-70 nt. However, sequences can be designed with sufficient flexibility to accommodate interactions between the aptamer and two targets at the distances described herein. Aptamers can be further modified to provide protection from nuclease and other enzymatic activity. Aptamer sequences can be modified by any suitable method known in the art.

[0166] The saRNA-aptamer conjugate can be formed using any known method for linking two moieties, such as direct chemical bond formation, linkage via a linker such as streptavidin, etc.

[0167] In one embodiment, the saRNA of the present invention may be conjugated to an antibody. Methods for generating antibodies against target cell surface receptors are well known. The saRNA molecules of the present invention can be conjugated to such antibodies using known methods, for example, using RNA carrier proteins. The resulting complex can then be administered to a subject and taken up by the target cells via receptor-mediated endocytosis.

[0168] In one embodiment, the saRNA of the invention comprises a lipid moiety, such as a cholesterol moiety (Letsinger et al., Proc. Natl. Acids Sci. USA, 1989, 86:6553-6556), cholic acid (Manoharan et al., Bior. Med. Chem. Let., 1994, 4:1053-1060), a thioether, such as beryl-5-tritylthiol (Manoharan et al., Ann. NY Acad. Sci., 1992, 660:306-309; Manoharan et al., Bior. Med. Chem. Let., 1993, 3:2765-2770), or a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20:533-538), aliphatic chains, such as dodecanediol or undecyl residues (Saison-Behmoaras et al., EMBO J, 1991, 10:1111-1118; Kabanov et al., FEBS Lett., 1990, 259:327-330; Svinarchuk et al., Biochimie, 1993, 75:49-54), phospholipids, such as di-hexadecyl-rac-glycerol or triethyl-ammonium 1,2-di-O-hexadecyl-rac-glycero-3-H phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654; Shea et al., Nucl. Acids Res., 1990, 18:3777-3783), polyamine or polyethylene glycol chains (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654), palmityl moieties (Mishra et al., Biochim. Biophys.Acta, 1995, 1264:229-237), or octadecylamine or hexylaminocarbonyloxycholesterol moieties (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923-937), the contents of each of which are incorporated herein by reference in their entirety.

[0169] In one embodiment, the saRNA of the present invention is conjugated to a ligand. In one non-limiting example, the ligand may be any of the ligands disclosed in U.S. Patent Application Publication No. 20130184328 to Manoharan et al., the entire contents of which are incorporated herein by reference. The conjugate may be a ligand-[linker] 任意選択 -[Tether] 任意選択 -oligonucleotide agent having the formula: The oligonucleotide agent can include a subunit having formula (I) as disclosed in U.S. Patent Application Publication No. 2013 / 0184328 to Manoharan et al., the entire contents of which are incorporated herein by reference. In another non-limiting example, the ligand can be any of the ligands disclosed in U.S. Patent Application Publication No. 20130317081 to Akinc et al., the entire contents of which are incorporated herein by reference, such as lipid, protein, hormone, or carbohydrate ligands of Formulas II-XXVI. The ligand can also be attached to the saRNA using a bivalent or trivalent branched linker of Formulas XXXI-XXXV as disclosed in Akinc.

[0170] Representative United States patents that teach the preparation of such nucleic acid / lipid conjugates include U.S. Pat. Nos. 4,828,979, 4,948,882, 5,218,105, 5,525,465, 5,541,313, 5,545,730, 5,552,538, 5,578,717, 5,580,731, 5,591,584, 5,109,124, 5,118,802, 5,138,000, and the like. Specification No. 45, Specification No. 5,414,077, Specification No. 5,486,603, Specification No. 5,512,439, Specification No. 5,578,718, Specification No. 5,608,046, Specification No. 4,587,044, Specification No. 4,605,735, Specification No. Specification No. 4,667,025, Specification No. 4,762,779, Specification No. 4,789,737, Specification No. 4,824,941, Specification No. 4,835,263, Specification No. 4,876,335, Specification No. 4,904,582, Specification No. 4,958,01 Specification No. 3, Specification No. 5,082,830, Specification No. 5,112,963, Specification No. 5,214,136, Specification No. 5,082,830, Specification No. 5,112,963, Specification No. 5,214,136, Specification No. 5,245,022, Specification No. Specification No. 5,254,469, Specification No. 5,258,506, Specification No. 5,262,536, Specification No. 5,272,250, Specification No. 5,292,873, Specification No. 5,317,098, Specification No. 5,371,241, Specification No. 5,391,72 3, 5,416,203, 5,451,463, 5,510,475, 5,512,667, 5,514,785, 5,565,552, 5,567,810, 5,574,142, 5,585,481, 5,587,371, 5,595,726, 5,597,696, 5,599,923, 5,599,928, and 5,688,Examples of suitable ion exchangers include, but are not limited to, US Pat. No. 5,941,241 (the entire contents of each of which are incorporated herein by reference).

[0171] The saRNA of the present invention may be provided in combination with other active ingredients known to be effective in the particular method under consideration. The other active ingredients may be administered simultaneously, separately, or sequentially with the saRNA of the present invention. In one embodiment, the saRNA of the present invention is administered with saRNAs that regulate different target genes. Non-limiting examples include saRNAs that regulate albumin, insulin, or HNF4A genes. Regulation of any gene can be achieved using a single saRNA or a combination of two or more different saRNAs. Non-limiting examples of saRNAs that can be administered with the saRNA of the present invention include saRNAs that regulate albumin or HNF4A disclosed in International Publication No. 2012 / 175958, filed June 20, 2012; saRNAs that regulate insulin disclosed in both International Publication Nos. 2012 / 046084 and 2012 / 046085, filed October 10, 2011; U.S. Patent No. 7,709,709, filed November 13, 2006;saRNA regulating the human progesterone receptor, human major vault protein (hMVP), E-cadherin gene, p53 gene, or PTEN gene disclosed in U.S. Patent Application Publication No. 456 and U.S. Patent Application Publication No. 2010 / 0273863 filed on April 23, 2010; p21 gene disclosed in International Publication No. WO 2006 / 113246 filed on April 11, 2006; and saRNA regulating the human progesterone receptor, human major vault protein (hMVP), E-cadherin gene, p53 gene, or PTEN gene disclosed in International Publication No. WO 2012 / 065143 filed on November 12, 2011. any nucleic acid that upregulates the expression of a gene in Table 8 of WO 2013 / 173635 filed on May 16, 2013, or increases the expression of a tumor suppressor; any oligonucleotide that activates a target gene in Table 4 of WO 2013 / 173637 filed on May 16, 2013; any oligonucleotide that activates a target gene in Table 4 of WO 2013 / 173637 filed on May 16, 2013; any sequence selected from the sequences of SEQ ID NOs: 1 to 1212 of WO 2013 / 173652 filed on May 16, 2013; any oligonucleotide that regulates the expression of APOA1 and ABCA1 genes disclosed in International Publication No. 20133173647 filed on May 16, 2013; any oligonucleotide that regulates the expression of SMN family genes disclosed in International Publication No. 20133173638 filed on May 16, 2013; any oligonucleotide that regulates the expression of PTEN gene disclosed in International Publication No. 2013173605 filed on May 16, 2013 any oligonucleotide that regulates the expression of the MECP2 gene disclosed in International Publication No. 2013173608 filed on May 16, 2013; any oligonucleotide that regulates the expression of the ATP2A2 gene disclosed in International Publication No. 2013173598 filed on May 16, 2013; any oligonucleotide that regulates the expression of the UTRN gene disclosed in International Publication No. 2013173645 filed on May 16, 2013; U.S. Patent No. 8,288, filed on December 28, 2006;any nucleic acid molecule that modulates the expression of CD97, TS-α, C / EBP delta, CDC23, PINK1, HIF1α, Gnbp3g, adrenomedullin AM1 receptor, 3-oxoacid CoA transferase, cathepsin W, or BACE1 disclosed in U.S. Patent Application Publication No. 2013 / 0245099 filed November 17, 2011; an antagomir NAT having formula (I) disclosed in U.S. Patent Application Publication No. 2013 / 0245099 filed April 30, 2010; Examples of suitable saRNAs include any antagomir NAT that upregulates expression of hemoglobin (HBF / HBG) polynucleotides as disclosed in U.S. Patent No. 18,690, and any antisense oligonucleotide targeting an antisense oligonucleotide that increases expression of apolipoprotein (ApoA1) polynucleotides as disclosed in U.S. Patent No. 8,153,696 (CURNA), filed October 2, 2009, the contents of each of which are incorporated herein by reference in their entirety.

[0172] In one embodiment, the saRNA is conjugated to a carbohydrate ligand, such as any of the carbohydrate ligands disclosed in U.S. Pat. Nos. 8,106,022 and 8,828,956 to Manoharan et al. (Alnylam Pharmaceuticals), the entire contents of which are incorporated herein by reference. For example, the carbohydrate ligand can be a monosaccharide, disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, or polysaccharide. These carbohydrate-conjugated RNA agents can target liver parenchymal cells. In one embodiment, the saRNA is conjugated to two or more, preferably two or three, carbohydrate ligands. In one embodiment, the saRNA is conjugated to one or more galactose moieties. In another embodiment, the saRNA is conjugated to at least one (e.g., two or more) lactose molecule (lactose is glucose linked to galactose). In other embodiments, the saRNA is conjugated to at least one (e.g., two or more) N-acetyl-galactosamine (GalNAc), N-Ac-glucosamine (GluNAc), or mannose (e.g., mannose-6-phosphate). In one embodiment, the saRNA is conjugated to at least one mannose ligand, and the conjugated saRNA targets macrophages.

[0173] GalNAc-nucleotide (GalNAc-saRNA / GalNAc-siRNA) conjugates In some embodiments, the saRNA is covalently linked to a carbohydrate moiety that contains at least one (e.g., two or more) N-acetyl-galactosamine (GalNAc) or a derivative thereof to form a GalNAc-saRNA conjugate.

[0174] [ka]

[0175] GalNAc is an amino sugar derivative of galactose containing the following structure. GalNAc is an effective moiety for delivering nucleic acid constructs to hepatocytes. The discrete triantennary GalNAc structure has been shown to be optimal for the effective delivery of single- and double-stranded oligonucleotides for gene silencing. GalNAc-nucleotide conjugates can be delivered to cells expressing asialoglycoprotein receptors without the use of transfection agents. The nucleotides can also be part of saRNA, and GalNAc-nucleotide conjugates are referred to as GalNAc-saRNA conjugates. The nucleotides can also be part of small inhibitory RNAs (also known as small interfering RNAs or siRNAs) that inhibit gene expression, and GalNAc-nucleotide conjugates are referred to as GalNAc-siRNA conjugates.

[0176] In some embodiments, the present disclosure provides GalNAc-saRNA conjugates comprising a small activating RNA (saRNA) attached to a GalNAc moiety, wherein the saRNA comprises at least one modification, which may optionally be independent of the attached 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 on each strand.

[0177] In some embodiments, the saRNA of the conjugate is at least 50% modified, i.e., at least 50% of the nucleotides are modified. In some embodiments, the saRNA is at least 75% modified, i.e., at least 75% of the nucleotides are modified. In some embodiments, both strands of the saRNA may be modified along their entire length (100% modified). It should be understood that any modification to any part of the nucleotide or nucleoside constitutes a modification, as each nucleotide (sugar, base, and phosphate moiety, e.g., linker) may be modified.

[0178] In some embodiments, the saRNA is modified in at least 10% of only one component of the nucleotides, and such component is selected from the nucleobase, sugar, or internucleoside linkage. For example, the saRNA can be modified in at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the nucleobases, sugars, or linkages of the saRNA.

[0179] 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 sugars of the nucleotides of the saRNA (OH in RNA, H in DNA) is substituted with -OMe, referred to as 2'-OMe. In some embodiments, at least one of the 2'-positions of the sugars of the nucleotides of the saRNA (OH in RNA, H in DNA) is substituted with -F, referred to as 2'-F.

[0180] In some embodiments, the saRNA of the conjugate comprises a 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 the 5' or 3' end of the passenger (sense) strand. In some embodiments, the saRNA of the present invention may comprise an inverted abasic modification on the passenger strand. At least one inverted abasic modification may be at the 5' end, 3' end, or both ends of the passenger strand. The inverted abasic modification may promote preferential loading of the guide strand.

[0181] In some embodiments, the saRNA comprises at least one motif of at least two consecutive nucleotides with the same sugar modification. In one example, such a motif may comprise two or three consecutive nucleotides. In some embodiments, the consecutive nucleotides of the motif comprise a 2'-F modification. In some embodiments, the consecutive nucleotides of the motif comprise a 2'-OMe modification.

[0182] In some embodiments, when the saRNA is double-stranded, the passenger strand and guide strand of the saRNA each contain at least one motif of consecutive nucleotides having the same sugar modification.

[0183] In some embodiments, the passenger strand and guide strand of the saRNA each contain at least two motifs of consecutive nucleotides with the same sugar modification. In some embodiments, at least two motifs on a given strand independently have different sugar modifications. For example, the passenger strand or guide strand may have at least one motif with a 2'-OMe modification and at least one motif with a 2'-F modification. In some embodiments, at least two motifs on a given strand are separated by at least one nucleotide (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20). In some embodiments, at least two motifs on a given strand are connected. In some embodiments, at least one motif on the passenger strand and its complementary motif on the guide strand have different sugar modifications. For example, the nucleotides of the motif on the passenger strand have a 2'-F modification and the nucleotides of the motif on the guide strand have a 2'-OMe modification, where the two motifs are complementary to each other. In another example, the nucleotides of the motif on the passenger strand have a 2'-OMe modification and the nucleotides of the motif on the guide strand have a 2'-F modification, where the two motifs are complementary to each other.

[0184] In some embodiments, the modification of a motif differs from the modification of the immediately adjacent nucleotides on either side of the motif. In some embodiments, the saRNA comprises at least one motif with alternating sugar modifications. For example, the alternating sugar modification motif comprises 2-30 nucleotides. In some embodiments, the motif comprises alternating 2'-F and 2'-OMe modifications.

[0185] In some embodiments, when the saRNA is double-stranded, the passenger strand and the guide strand each contain at least one motif with alternating sugar modifications.In some embodiments, at least one nucleotide on the passenger strand and its complementary nucleotide on the guide strand have different sugar modifications.For example, one nucleotide of a base pair on the passenger strand has a 2'-F modification, and the other nucleotide of a base pair on the guide strand has a 2'-OMe modification.In another example, one nucleotide of a base pair on the passenger strand has a 2'-OMe modification, and the other nucleotide of a base pair on the guide strand has a 2'-F modification.

[0186] In some embodiments, the saRNA contains at least one phosphorothioate or methylphosphonate bond between nucleotides. In some embodiments, the saRNA of the conjugate comprises the general formula of formula (I) described herein.

[0187] In some embodiments, the present disclosure provides GalNAc-siRNA conjugates comprising a small inhibitory RNA (siRNA) attached to a GalNAc moiety. In some embodiments, the GalNAc moiety is linked to the 2' or 3' position of the ribosugar or to the nucleobase of a nucleotide of the saRNA or siRNA. A phosphodiester or phosphorothioate bond can be between the GalNAc moiety and the nucleotide.

[0188] In some embodiments, the GalNAc moiety is linked to the nucleotide of the saRNA or siRNA via a linker. The linker can be linked to any suitable position of the nucleotide of the saRNA or siRNA. The linker can be linked to the GalNAc moiety via a covalent or non-covalent bond.

[0189] In some cases, the linker is attached to the end of the saRNA or siRNA strand. In some cases, the linker is attached to the 5' end of the sense strand. In some cases, the linker is attached to the 3' end of the sense strand.

[0190] [ka]

[0191] In some cases, the linker is attached to an internal nucleotide of the saRNA or siRNA strand. In some cases, the linker is attached to an internal nucleotide of the sense strand of the saRNA or siRNA. In some cases, the linker is attached to an internal nucleotide of the antisense strand of the saRNA or siRNA.

[0192] [ka]

[0193] Any of the attachment methods 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, can be used to attach the GalNAc moiety to the saRNA.

[0194] In some cases, the linker of a GalNAc-saRNA conjugate or a GalNAc-siRNA conjugate may be a direct bond or an atom such as oxygen or sulfur, a unit such as -NH-, -C(O)-, -C(O)NH-, -S(O)-, -SO2-, -SONH-, or a unit such as 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, alkylheteroalkyl, alkynylarylalkenyl, alkynylarylalkynyl, alkylheteroalkyl, alkynylarylalkenyl, alkynylarylalkynyl, alkylheteroalkyl, alkynylarylalkynyl, alkylhetero ... and a chain of atoms such as, but not limited to, alkylaryl, alkylheteroarylalkenyl, alkylheteroarylalkynyl, alkenylheteroarylalkyl, alkenylheteroarylalkenyl, alkenylheteroarylalkynyl, alkynylheteroarylalkyl, alkynylheteroarylalkenyl, alkynylheteroarylalkynyl, alkylheterocyclylalkyl, alkylheterocyclylalkenyl, alkylheterocyclylalkynyl, alkenylheterocyclylalkyl, alkenylheterocyclylalkenyl, alkenylheterocyclylalkynyl, alkynylheterocyclylalkyl, alkynylheterocyclylalkenyl, alkynylheterocyclylalkynyl, alkylaryl, alkenylaryl, alkynylaryl, alkylheteroaryl, alkenylheteroaryl, alkynylheteroaryl, where each group can be substituted or unsubstituted.

[0195] In some cases, the linker is a cleavable linker. A cleavable linker can be cleaved at a specific pH, by a specific enzyme, or in a specific redox environment. Cleavable linkers can include, for example, an ester bond, an acid-labile bond, a disulfide bond, or a phosphate bond.

[0196] In some cases, the linker is a non-cleavable linker. In some cases, the linker contains an amine group (referred to as NH2C6, C6NH2, or C6), such as -NH-(CH2)6- or NH2-(CH2)6-. In the case of a GalNAc cluster containing a terminal carboxylic acid, the carboxylic acid reacts with an amine on the linker, directly attaching the GalNAc cluster to the saRNA-C6NH- or siRNA-C6NH.

[0197] In some cases, the linker is -O-CO-(CH2) n -CO-NH-(CH2)6-, where n = 2, 3, 4, 5, or 6. In the case of a GalNAc cluster containing an amine at its terminal, the amine reacts with the carboxylic acid on the linker, forming the GalNAc cluster into saRNA-(CH2)6-NH-CO-(CH2). n -CO- or siRNA-(CH2)6-NH-CO-(CH2) n It is directly bonded to -CO-.

[0198] In some cases, there is a phosphorothioate bond between the linker and the sense strand. In some cases, the GalNAc moiety can be a triantennary GalNAc cluster. Any GalNAc cluster disclosed in Prakash et al., Journal of Medicinal Chemistry, vol. 59:2718-2733 (2016), Figure 2, the contents of which are incorporated herein by reference in their entirety, can be used in accordance with the present disclosure, such as the tris-based GalNAc cluster, the tri-acid-based GalNAc cluster, the Lys-Lys-based GalNAc cluster, the Lys-Gly-based GalNAc cluster, the treble-based cluster, and the hydroxyprolinol-based cluster. The GalNAc cluster can have the following structure:

[0199] [ka]

[0200] , n=1, 2, 3, 4, 5, or 6. When a linker is used to connect the 3' or 5' end of the sense strand to the GalNAc site, the GalNAc-saRNA or GalNAc-SiRNA conjugate may be:

[0201] [ka]

[0202] The structure of, e.g.

[0203] [ka]

[0204] Includes the structure of For example, a GalNAc-saRNA conjugate or a GalNAc-siRNA conjugate may be:

[0205] [ka]

[0206] The structure of, for example,

[0207] [ka]

[0208] where X is O or S. It may have the structure: In some embodiments, the GalNAc-saRNA conjugate or GalNAc-siRNA conjugate comprises at least one GalNAc monomer selected from M1, M1', M2, M2', M3, M3', M4, M4', M5, M5', M6, M6', or a derivative thereof. The GalNAc-saRNA conjugate may comprise 1, 2, 3, 4, 5, 6, 7, 8, or 9 GalNAc monomers selected from M1, M1', M2, M2', M3, M3', M4, M4', M5, M5', M6, M6', or a derivative thereof.

[0209] In one embodiment, the GalNAc-saRNA or GalNAc-siRNA conjugate comprises at least one GalNAc monomer selected from M1, M1', or derivatives thereof.

[0210] [ka]

[0211] wherein R1, R2, and R3 may be the same or different, and R1, R2, and R3 are independently selected from alkyl, aryl, and alkenyl groups. In some embodiments, at least one of R1, R2, and R3 is -CH3. In some embodiments, R1, R2, and R3 are all -CH3.

[0212] where R4 is a suitable protecting group or a C1-6 straight or branched alkyl, including, but not limited to, methyl, ethyl, n-propyl, 2-propyl, n-butyl, isobutyl, and other alkyl groups. In some embodiments, R4 is -CH3 or CH2CH3. In some embodiments, R4 is -CH2CH2CN.

[0213] wherein R5 and R6 are each independently C1-6 straight or branched alkyl, including, but not limited to, methyl, ethyl, n-propyl, 2-propyl, n-butyl, isobutyl, and similar alkyl groups. In some embodiments, R5 and R6 are both 2-propyl.

[0214] and, wherein R7 is a suitable protecting group. In some embodiments, the protecting group is 4,4'-dimethoxytrityl.

[0215] [ka]

[0216] where R8 is -H or C1-6 straight or branched alkyl, including, but not limited to, methyl, ethyl, n-propyl, 2-propyl, n-butyl, isobutyl, and similar alkyl groups; and where X is O or S. In some embodiments, R8 is -CH3 or -CH2CH3.

[0217] In another embodiment, the GalNAc-saRNA or GalNAc-siRNA conjugate comprises at least one GalNAc monomer selected from M2, M2', or derivatives thereof.

[0218] [ka]

[0219] wherein R1, R2, and R3 may be the same or different, and R1, R2, and R3 are independently selected from alkyl, aryl, and alkenyl groups. In some embodiments, at least one of R1, R2, and R3 is -CH3. In some embodiments, R1, R2, and R3 are all -CH3.

[0220] wherein R4 is a protecting group or a C1-6 straight or branched alkyl, including, but not limited to, methyl, ethyl, n-propyl, 2-propyl, n-butyl, isobutyl, and similar alkyl groups. In some embodiments, R4 is -CH3 or CH2CH3. In some embodiments, R4 is -CH2CH2CN.

[0221] wherein R5 and R6 are each independently C1-6 straight or branched alkyl, including, but not limited to, methyl, ethyl, n-propyl, 2-propyl, n-butyl, isobutyl, and other alkyl groups. In some embodiments, R5 and R6 are both 2-propyl.

[0222] and, wherein R7 is a suitable protecting group. In some embodiments, the protecting group is 4,4'-dimethoxytrityl.

[0223] [ka]

[0224] where R8 is -H or C1-6 straight or branched alkyl, including, but not limited to, methyl, ethyl, n-propyl, 2-propyl, n-butyl, isobutyl, and similar alkyl groups; and where X is O or S. In some embodiments, R8 is -CH3 or -CH2CH3.

[0225] In one embodiment, the GalNAc-saRNA conjugate or GalNAc-siRNA conjugate comprises at least one GalNAc monomer selected from M3, M3', or derivatives thereof.

[0226] [ka]

[0227] where X is O or S. In one embodiment, the GalNAc-saRNA or GalNAc-siRNA conjugate comprises at least one GalNAc monomer selected from M4, M4', or derivatives thereof.

[0228] [ka]

[0229] wherein R1, R2, and R3 may be the same or different, and R1, R2, and R3 are independently selected from alkyl, aryl, and alkenyl groups. In some embodiments, at least one of R1, R2, and R3 is -CH3. In some embodiments, R1, R2, and R3 are all -CH3.

[0230] wherein R7 is a protecting group. In some embodiments, the protecting group is 4,4'-dimethoxytrityl. and, wherein Linker 1 is a cleavable linker. In some embodiments, Linker 1 is succinyl.

[0231] [ka]

[0232] In one embodiment, the GalNAc-saRNA or GalNAc-siRNA conjugate comprises at least one GalNAc monomer selected from M5, M5', or derivatives thereof.

[0233] [ka]

[0234] wherein R1, R2, and R3 may be the same or different, and R1, R2, and R3 are independently selected from alkyl, aryl, and alkenyl groups. In some embodiments, at least one of R1, R2, and R3 is -CH3. In some embodiments, R1, R2, and R3 are all -CH3.

[0235] wherein R7 is a suitable protecting group. In some embodiments, the protecting group is 4,4'-dimethoxytrityl. and, wherein Linker 1 is a cleavable linker. In some embodiments, Linker 1 is succinyl.

[0236] [ka]

[0237] In one embodiment, the GalNAc-saRNA or GalNAc-siRNA conjugate comprises at least one GalNAc monomer selected from M6, M6', or derivatives thereof.

[0238] [ka]

[0239] [ka]

[0240] GalNAc monomers are used to construct GalNAc moieties that are conjugated to saRNAs to deliver the saRNAs to targeted organs, such as the liver. The GalNAc moiety contains at least one GalNAc monomer. In some embodiments, the GalNAc moiety can be a GalNAc cluster (or multimer) containing at least two GalNAc monomers. In some embodiments, the GalNAc cluster can be a GalNAc dimer cluster containing two GalNAc monomers. In some embodiments, the GalNAc cluster can be a triantennary GalNAc cluster containing three GalNAc monomers. GalNAc monomer building blocks are compatible with standard oligonucleotide synthesis using the phosphoramidite method. The monomers can be used to provide functionalized supports or can be added in-line during oligonucleotide synthesis. The GalNAc monomer can be attached in-line to the 5' end of a ligated oligonucleotide to form a GalNAc conjugate. As used herein, "in-line" refers to the automated process of oligonucleotide elongation during synthesis. GalNAc monomers can be added alone or in combination with other GalNAc monomers at any position in the oligonucleotide. They can be added sequentially without a linker or separated by nucleotides or other linkers.

[0241] In some embodiments, the GalNAc moiety comprises at least one GalNAc monomer and at least one spacer (which may also be referred to in some contexts as a linker), where the GalNAc monomer is linked to the spacer via a bond (such as a phosphate or phosphorothioate bond). In some embodiments, the GalNAc moiety comprises at least two GalNAc monomers (such as two, three, four, five, or six monomers) and, optionally, at least one spacer, where the monomers are linked to each other or to the spacer via a bond (such as a phosphate or phosphorothioate bond). The spacer can be a non-cleavable linker, such as, but not limited to, hexaethylene glycol (HEG), C12, abasic furan, triethylene glycol (TEG), C3, or a derivative thereof (e.g., with an appropriate protecting group): 1) HEG spacer (HEG) shown in fully deprotected oligonucleotide

[0242] [ka]

[0243] , where X is O or S. In this disclosure, when "HEG" is used, X is O. When "S-HEG" is used, X is S. 2). C12 spacer (C12) shown in fully deprotected oligonucleotide:

[0244] [ka]

[0245] , where X is O or S. In this disclosure, when "C12" is used, X is O. When "S-C12" is used, X is S. 3). Abasic spacer (ab) shown in fully deprotected oligonucleotide:

[0246] [ka]

[0247] , where X is O or S. In this disclosure, when "ab" is used, X is O. When "S-ab" is used, X is S. 4). TEG spacer (TEG) shown in fully deprotected oligonucleotide:

[0248] [ka]

[0249] , where X is O or S. In this disclosure, when "TEG" is used, X is O. When "S-TEG" is used, X is S. 5). C3 spacer (C3) shown in fully deprotected oligonucleotide:

[0250] [ka]

[0251] , where X is O or S. In this disclosure, when "C3" is used, X is O. When "S-C3" is used, X is S. The GalNAc moiety can be prepared by a process comprising the following steps: 1). providing at least one GalNAc monomer selected from the group consisting of M1', M2', M3', M4', M5' and M6'; and 2). Synthesizing a GalNAc moiety from the GalNAc monomer(s) in step 1), optionally adding at least one spacer, and optionally removing the protecting group.

[0252] In some embodiments, the GalNAc moiety comprises at least one M1 monomer (such as exactly one, exactly two, or exactly three M1 monomers) and at least one spacer. In some embodiments, the GalNAc moiety comprises at least one M1 monomer; at least one M2 or M3 monomer (such as three M3 monomers), or one M4, M5, or M6 monomer. In some embodiments, the GalNAc moiety comprises at least one M1 monomer; at least one spacer; and at least one M2 or M3 monomer (such as three M3 monomers), or one M4, M5, or M6 monomer. In some embodiments, the GalNAc moiety comprises at least one M1 monomer (such as exactly one, exactly two, or exactly three M1 monomers), but no spacer.

[0253] In some embodiments, the GalNAc moiety comprises at least one M2 monomer (such as exactly one, exactly two, or exactly three M2 monomers) and at least one spacer. In some embodiments, the GalNAc moiety comprises at least one M2 monomer; at least one M1 or M3 monomer (such as three M3 monomers), or one M4, M5, or M6 monomer. In some embodiments, the GalNAc moiety comprises at least one M2 monomer; at least one spacer; at least one M1 or M3 monomer (such as three M3 monomers), or one M4, M5, or M6 monomer. In some embodiments, the GalNAc moiety comprises at least one M2 monomer (such as exactly one, exactly two, or exactly three M2 monomers), but no spacer.

[0254] In some embodiments, the GalNAc moiety comprises at least one M3 monomer and at least one spacer. In some embodiments, the GalNAc moiety comprises at least one M3 monomer; and at least one M1 or M2 monomer, or one M4, M5, or M6 monomer. In some embodiments, the GalNAc moiety comprises at least one M3 monomer; at least one spacer; and at least one M1 or M2 monomer, or one M4, M5, or M6 monomer. In some embodiments, the GalNAc moiety comprises three M3 monomers with at least one spacer. In some embodiments, the GalNAc moiety comprises three M3 monomers but no spacer. In some embodiments, the GalNAc moiety excludes GalNAc moieties consisting of only one M3 monomer.

[0255] In some embodiments, the GalNAc moiety does not comprise two or more M4 monomers. In some embodiments, the GalNAc moiety comprises one M4 monomer. In some embodiments, the GalNAc moiety does not comprise any M4 monomers. In some embodiments, the GalNAc moiety comprises one M4 monomer; and at least one M1, M2, or M3 monomer, or one M5 or M6 monomer.

[0256] In some embodiments, the GalNAc moiety does not contain two or more M5 monomers. In some embodiments, the GalNAc moiety contains one M5 monomer. In some embodiments, the GalNAc moiety does not contain any M5 monomers. In some embodiments, the GalNAc moiety contains one M5 monomer; and at least one M1, M2, or M3 monomer, or one M4 or M6 monomer.

[0257] In some embodiments, the GalNAc moiety does not contain two or more M6 monomers. In some embodiments, the GalNAc moiety contains one M6 monomer. In some embodiments, the GalNAc moiety does not contain any M6 monomers. In some embodiments, the GalNAc moiety contains one M6 monomer; at least one M1, M2, or M3 monomer (such as three M3 monomers), or one M4 or M5 monomer. In some embodiments, the GalNAc moiety excludes GalNAc moieties containing one M6 monomer and two M3 monomers.

[0258] In some embodiments, the GalNAc moiety is a triantennary GalNAc cluster having the following structure:

[0259] [ka]

[0260] or any of the structures in Table 3. Here, the GalNAc moiety is also referred to as a GalNAc cluster.

[0261] [Table 3]

[0262] [ka]

[0263] [ka]

[0264] [ka]

[0265] [ka]

[0266]

change

[0267]

change

[0268]

change

[0269]

change

[0270]

change

[0271]

change

[0272]

change

[0273]

change

[0274]

change

[0275]

change

[0276] [ka]

[0277] [ka]

[0278] [ka]

[0279] [ka]

[0280] [ka]

[0281] [ka]

[0282] The GalNAc moiety can be attached to an oligonucleotide sequence (e.g., the sense strand of a double-stranded saRNA) by a bond (such as a phosphodiester or phosphorothioate bond) with or without a cleavable linker to form a conjugate. The GalNAc moiety can be attached to the 5'- or 3'-O of the oligonucleotide sequence. In some embodiments, the cleavable linker is a C6ssC6 linker having the following structure:

[0283] [ka]

[0284] (C6ssC6 in the fully deprotected oligonucleotide), where X is O or S. In some embodiments, the cleavable linker is a dT linker having the following structure:

[0285] [ka]

[0286] in a fully deprotected oligonucleotide. GalNAc-saRNA conjugates can be prepared by a process comprising the following steps: 1). Providing at least one GalNAc monomer selected from the group consisting of M1', M2', M3', M4', M5' and M6'; optionally adding at least one spacer; 2). Providing at least one saRNA (such as any saRNA in Table 2); optionally adding at least one linker; and 3) Synthesizing a GalNAc-saRNA conjugate from the GalNAc monomer(s) of step 1) and the saRNA of step 2), optionally removing the protecting group.

[0287] In some embodiments, the GalNAc moiety is attached to the 5' end of the sense strand of a double-stranded saRNA (saRNA duplex) to form a conjugate, wherein the saRNA is CEBPA-saRNA. In some embodiments, the GalNAc moiety is attached to the 3' end of the sense strand of a double-stranded saRNA to form a conjugate, wherein the saRNA is CEBPA-saRNA. The saRNA can be any saRNA in Table 2. In one embodiment, the saRNA has the following sequence: XD-14369K1 duplex: Antisense: 5'-GfsAfscCfaGfuGfaCfaauGfaCfcGfcsUfsu-3' (SEQ ID NO: 25) Sense: 5'-GfscsGfgUfcAfUfUfgUfcAfcUfgGfuCf-3' (SEQ ID NO: 24) or XD-06414 duplex: Antisense: 5'-gAfcCfaGfuGfaCfaauGfaCfcGfcsusu-3' (SEQ ID NO: 15) Sense: 5'-sGfcGfgUfcAfUfUfgUfcAfcUfgGfuCfuu(invdT)-3' (SEQ ID NO: 14) (Nf = nucleotide N (N can be A, U, C, or G) has a 2'-fluoro (2'-F) modification; Lowercase = nucleotide has 2'-O-methyl (2'-OMe) modification; s: phosphorothioate bond; and invdT: inverted deoxyT (dT). Non-limiting examples of GalNAc-saRNA or GalNAc-siRNA conjugates include the conjugates of the genus and species in Table 4. It is understood that the sense strand of the saRNA or siRNA forms a duplex with the antisense strand of the saRNA or siRNA.

[0288] [Table 4]

[0289] In some embodiments, a GalNAc moiety is attached to the 5' end of the sense strand of the XD-06414 duplex to form the conjugate. Non-limiting examples of conjugates include any of the conjugates in Table 5. Conjugates L1 through L19 each contain a cleavable linker. Conjugates L40 through L58 do not contain a cleavable linker.

[0290] [Table 5]

[0291] [Table 6-1]

[0292] [Table 6-2]

[0293] In some embodiments, the GalNAc-nucleotide conjugate (such as a GalNAc-saRNA conjugate or a GalNAc-siRNA conjugate) has one of the following structures: (L=optional linker such as C6ssC6 for conjugates L1 to L19, dT for L66 to L71; L is absent for conjugates L40 to L58, L60 to 65, and L72 to 75).

[0294] Po = phosphodiester bond; Ps = phosphorothioate bond; and Nuc = nucleotide or oligonucleotide such as double-stranded saRNA (e.g., XD-06414) or the sense strand of a double-stranded siRNA) 1). C6-GalNAc (including the GalNAc-saRNA conjugate disclosed in Example 2 of PCT / EP2018 / 074211 filed September 7, 2018):

[0295] [ka]

[0296] 2). GalNAc-Clv (including the GalNAc-saRNA conjugate disclosed in PCT / EP2018 / 074211 filed September 7, 2018):

[0297] [ka]

[0298] 3). CJ1 (including L1 and L40 in Table 5):

[0299] [ka]

[0300] 4). CJ2 (including L2 and L41 in Table 5):

[0301] [ka]

[0302] 5). CJ3 (including L3 and L42 in Table 5):

[0303] [ka]

[0304] 6). CJ4 (including L4 and L43 in Table 5):

[0305] [ka]

[0306] 7). CJ5 (including L5 and L44 in Table 5):

[0307] [ka]

[0308] 8). CJ6 (including L6 and L45 in Table 5):

[0309] [ka]

[0310] 9).CJ7 (including L14 and L53 in Table 5 and L80 in Table 6):

[0311] [ka]

[0312] 10). CJ8 (including L15 and L54 in Table 5):

[0313] [ka]

[0314] 11).CJ9 (including L16 and L55 in Table 5 and L81 in Table 6):

[0315] [ka]

[0316] 12). CJ10 (including L17 and L56 in Table 5):

[0317] [ka]

[0318] 13). CJ11 (including L18 and L57 in Table 5):

[0319] [ka]

[0320] 14). CJ12 (including L19 and L58 in Table 5):

[0321] [ka]

[0322] 15). CJ13 (including L60 and L66 in Table 6):

[0323] [ka]

[0324] 16). CJ14 (including L61 and L67 in Table 6):

[0325] [ka]

[0326] 17). CJ15 (including L62 and L68 in Table 6):

[0327] [ka]

[0328] 18). CJ16 (including L63 and L69 in Table 6):

[0329] [ka]

[0330] 19). CJ17 (including L64 and L70 in Table 6):

[0331] [ka]

[0332] 20). CJ18 (including L65 and L71 in Table 6):

[0333] [ka]

[0334] 21). CJ19 (including L72 and L73 in Table 6):

[0335] [ka]

[0336] 22). CJ20 (including L73 in Table 6):

[0337] [ka]

[0338] 23). CJ21 (including L74 in Table 6):

[0339] [ka]

[0340] 24). CJ22 (including L75 in Table 6):

[0341] [ka]

[0342] 25). CJ23 (including L76 and L77 in Table 6):

[0343] [ka]

[0344] 26). CJ24 (including L78 in Table 6):

[0345] [ka]

[0346] 27). CJ25 (including L79 in Table 6):

[0347] [ka]

[0348] It has. In some cases, the GalNAc-saRNA conjugate upregulates the expression of CEBPA, where the saRNA is a CEBPA-saRNA. For example, the CEBPA-saRNA can be any saRNA in Table 2, such as XD-06414 (SEQ ID NOs: 14 and 15).

[0349] In some embodiments, the GalNAc-saRNA conjugate or GalNAc-siRNA conjugate is delivered to liver cells of a subject. The liver cells can be hepatoma cells.

[0350] GalNAc-saRNA conjugates can be synthesized by any suitable method known in the art. For example, GalNAc-saRNA conjugates can be synthesized according to the method described in the experimental section of Prakash et al., Journal of Medicinal Chemistry, vol. 59: 2718-2733 (2016), the entire contents of which are incorporated herein by reference.

[0351] In some embodiments, the GalNAc moiety is conjugated to an siRNA to form a GalNAc-siRNA conjugate. In some cases, the GalNAc-siRNA conjugate downregulates expression of the targeted gene.

[0352] In some embodiments, the GalNAc-siRNA conjugate is delivered to a subject's hepatocytes, which can be hepatoma cells. GalNAc-siRNA conjugates can be synthesized by any suitable method known in the art. For example, GalNAc-siRNA conjugates can be synthesized according to the method described in the experimental section of Prakash et al., Journal of Medicinal Chemistry, vol. 59: 2718-2733 (2016), the entire contents of which are incorporated herein by reference.

[0353] II. Pharmaceutical Compositions One aspect of the present invention provides a pharmaceutical composition comprising a small activating RNA (saRNA) that upregulates a target gene and at least one pharmaceutically acceptable carrier.

[0354] Formulation, delivery, administration, and dosage Pharmaceutical formulations may additionally contain pharmaceutically acceptable excipients, which, as used herein, include, but are not limited to, any solvent, dispersion medium, diluent, or other liquid medium, dispersing or suspending aid, surfactant, isotonicity agent, thickener, or emulsifier, preservative, and the like, suitable for the particular dosage form desired. Various excipients for formulating pharmaceutical compositions and techniques for preparing compositions are known in the art (see Remington: The Science and Practice of Pharmacy, 21st Edition, A.R. Gennaro, Lippincott, Williams & Wilkins, Baltimore, MD, 2006, incorporated herein by reference in its entirety). Except insofar as any conventional excipient medium may be incompatible with a substance or its derivatives, such as by producing some undesirable biological effect or otherwise adversely interacting with any other component of the pharmaceutical composition, the use of any conventional excipient medium is contemplated within the scope of the present disclosure.

[0355] In some embodiments, the composition is administered to a human, human patient, or subject. For purposes of this disclosure, the phrase "active ingredient" generally refers to the saRNA delivered as described herein.

[0356] While the description of pharmaceutical compositions provided herein is primarily directed to pharmaceutical compositions suitable for administration to humans, those skilled in the art will understand that such compositions are generally suitable for administration to any other animal, e.g., non-human animals, e.g., non-human mammals. Modifications of pharmaceutical compositions suitable for administration to humans to make them suitable for administration to a variety of animals are well understood, and a veterinary pharmacologist of ordinary skill can design and / or implement such modifications with no more than routine experimentation, if any. Subjects to which pharmaceutical compositions are contemplated include, but are not limited to, humans and / or other primates, mammals (including commercially relevant mammals such as cows, 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).

[0357] In one embodiment, the efficacy of the formulated saRNA described herein can be determined in proliferating cells. Formulations of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such methods include the step of bringing into association the active ingredient with an excipient and / or one or more other accessory ingredients, and then, if necessary and / or desirable, dividing, shaping, and / or packaging the resulting product into a desired single- or multi-dose unit.

[0358] Pharmaceutical compositions of the invention 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 a 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 dose of the active ingredient which would be administered to a subject and / or a convenient fraction of such a dose, e.g., one-half or one-third of such a dose.

[0359] The relative amounts of active ingredient, pharmaceutically acceptable excipient, and / or any additional ingredients in a pharmaceutical composition of the invention will vary depending on the identity, size, and / or condition of the subject being treated, as well as the route of administration of the composition. By way of example, the composition may contain from 0.1% to 100%, e.g., 0.5 to 50%, 1 to 30%, 5 to 80%, at least 80% (w / w) active ingredient.

[0360] In some embodiments, the formulations described herein may contain at least one saRNA. Non-limiting examples include formulations containing 1, 2, 3, 4, or 5 saRNAs with different sequences. In one embodiment, the formulation contains at least 3 saRNAs with different sequences. In one embodiment, the formulation contains at least 5 saRNAs with different sequences.

[0361] The saRNA of the present invention can be formulated with one or more excipients to (1) improve stability, (2) improve cell transfection, (3) allow for sustained or delayed release (e.g., from a depot formulation of the saRNA), (4) alter biodistribution (e.g., to target the saRNA to specific tissues or cell types), (5) increase translation of the encoded protein in vivo, and / or (6) alter the release profile of the encoded protein in vivo.

[0362] In addition to conventional excipients, such as any solvent, dispersion medium, diluent or other liquid vehicle, dispersing or suspending aid, surfactant, tonicity agent, thickener or emulsifier, and preservative, excipients of the present invention may include, but are not limited to, lipidoids, liposomes, lipid nanoparticles, polymers, lipoplexes, core-shell nanoparticles, peptides, proteins, saRNA-transfected cells (e.g., for transplantation into a subject), hyaluronidase, nanoparticle mimics, and combinations thereof. Thus, formulations of the present invention may contain one or more excipients in amounts that, combined together, improve the stability of the saRNA and / or improve cell transfection by the saRNA. Furthermore, the saRNA of the present invention may be formulated using self-assembling nucleic acid nanoparticles. Pharmaceutically acceptable carriers, excipients, and delivery agents for nucleic acids that can be used in formulations containing the saRNA of the present invention are disclosed in International Publication No. WO 2013 / 090648, filed December 14, 2012, the entire contents of which are incorporated herein by reference.

[0363] delivery The present disclosure contemplates future advances in the science of drug delivery and encompasses delivery of saRNA for either therapeutic, prophylactic, pharmaceutical, diagnostic, or imaging purposes by any suitable route, whether naked or formulated.

[0364] The saRNA of the present invention can be delivered to cells naked. As used herein, "naked" refers to delivery of the saRNA without the use of agents that promote transfection. For example, the saRNA delivered to cells can be unmodified. Naked saRNA can be delivered to cells using routes of administration known in the art and described herein.

[0365] The saRNA of the present invention can be formulated using the methods described herein. The formulation can contain modified and / or unmodified saRNA. The formulation can further include, but is not limited to, a cell-penetrating agent, a pharmaceutically acceptable carrier, a delivery agent, a bioerodible or biocompatible polymer, a solvent, and a sustained-release delivery depot. The formulated saRNA can be delivered to cells using administration routes known in the art and described herein.

[0366] In some embodiments, the saRNA of the present invention is delivered by non-encapsulation techniques, such as agents containing an N-acetylgalactosamine (GalNAc) group or a derivative thereof, or clusters containing two or more GalNAc groups or derivatives thereof linked via a bivalent or trivalent branched linker.

[0367] The compositions can also be formulated for direct delivery to organs or tissues using any of several methods in the art, including, but not limited to, direct soaking or bathing, via a catheter, by gels, powders, ointments, creams, gels, lotions, and / or drops, by using substrates such as fabrics or biodegradable materials coated or impregnated with the composition, etc. The saRNAs of the present invention can also be cloned into retroviral replicating vectors (RRVs) and transduced into cells.

[0368] Administration The saRNA of the present invention can be administered by any route that results in a therapeutically effective outcome. These include, but are not limited to, enteral, gastrointestinal, epidural, oral, transdermal, epidural (outside the dura), intracerebral (in the brain), intraventricular (in the ventricles of the brain), epicutaneous (applied to the skin), intradermal (in the skin itself), subcutaneous (under the skin), nasal administration (through the nose), intravenous (into a vein), intraarterial (into an artery), intramuscular (into a muscle), intracardiac (into the heart), intraosseous injection (into the bone marrow), intrathecal (into the spinal canal), intraperitoneal (infusion or injection into the peritoneum), intravesical instillation, intravitreal (through the eye), intracavernosal injection, (into the base of the penis), intravaginal administration, intrauterine, extra-amniotic administration, transdermal (diffusion through intact skin for systemic distribution), transmucosal (diffusion through mucous membranes), insufflation (inhaled through the nose), sublingual, sublabial, enema, ophthalmic (on the conjunctiva), or ear drops. In certain embodiments, compositions can be administered in a manner that allows them to cross the blood-brain barrier, vascular barrier, or other epithelial barrier. The routes of administration disclosed in International Publication No. WO 2013 / 090648, filed December 14, 2012, the entire contents of which are incorporated herein by reference, can be used to administer the saRNAs of the invention.

[0369] Dosage form The pharmaceutical compositions described herein can be formulated into dosage forms described herein, such as topical, intranasal, intratracheal, or injectable (e.g., intravenous, intraocular, intravitreal, intramuscular, intracardiac, intraperitoneal, subcutaneous) formulations. The liquid, injectable, pulmonary, and solid formulations described in International Publication No. WO 2013 / 090648, filed December 14, 2012, the entire contents of which are incorporated herein by reference, can be used as dosage forms for the saRNA of the present invention.

[0370] III.How to use One aspect of the present invention provides a method for delivering saRNA to cells using GalNAc-saRNA conjugates without transfection agents.The cells express asialoglycoprotein receptors.In some embodiments, targeted delivery of saRNA to cells is achieved using the GalNAc-saRNA conjugates of the present invention.In some cases, the cells are hepatocytes.In some cases, the cells are liver cancer cells.

[0371] Another aspect of the present invention provides methods for using the saRNA or GalNAc-saRNA conjugates of the present invention, and pharmaceutical compositions comprising the saRNA or GalNAc-saRNA conjugate and at least one pharmaceutically acceptable carrier. The saRNA or GalNAc-saRNA conjugates of the present invention regulate the expression of their target genes. In one embodiment, a method for regulating the expression of a target gene in vitro and / or in vivo is provided, comprising administering the saRNA of the present invention. 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 invention, compared to the expression of the target gene in the absence of the saRNA of the present invention. In further embodiments, in the presence of the saRNA of the present invention, expression of the target gene is increased by at least 2, 3, 4, 5, 6, 7, 8, 9, 10-fold, or at least 15, 20, 25, 30, 35, 40, 45, 50-fold, or at least 60, 70, 80, 90, 100-fold compared to expression of the target gene in the absence of the saRNA of the present invention.

[0372] In one embodiment, increased gene expression of the saRNAs described herein is exhibited in proliferating cells. In one embodiment, the saRNAs described herein can be used as spacers in CRISPR (clustered regularly interspaced short palindromic repeats) systems, such as the CRISPR / Cas9 system. The CRISPR system comprising the saRNAs described herein can be used to cut and edit target genes.

[0373] In one embodiment, increased gene expression upon treatment with the saRNA or GalNAc-saRNA conjugates described herein is demonstrated in proliferating cells. hyperproliferative disorders In one embodiment of the present invention, the saRNA or GalNAc-saRNA conjugates of the present invention are used to reduce cell proliferation in hyperproliferative cells. Examples of hyperproliferative cells include cancerous cells such as carcinomas, sarcomas, lymphomas, and blastomas. Such cancerous cells can be benign or malignant. Hyperproliferative cells may result from autoimmune conditions such as rheumatoid arthritis, inflammatory bowel disease, or psoriasis. Hyperproliferative cells can also occur in patients with an overactive immune system who have come into contact with an allergen. Such conditions involving an overactive immune system include, but are not limited to, allergic reactions such as asthma, allergic rhinitis, eczema, and allergic anaphylaxis. In one embodiment, the development and / or proliferation of tumor cells is inhibited. In a preferred embodiment, the proliferation of solid tumor cells is inhibited. In another preferred embodiment, the metastasis of tumor cells is prevented. In another preferred example, the proliferation of undifferentiated tumor cells is inhibited.

[0374] Inhibiting or reducing cell proliferation means that proliferation is reduced or completely stopped. Therefore, "reducing proliferation" is one embodiment of "inhibiting proliferation." Compared to the proliferation of the cells before treatment with the saRNA or GalNAc-saRNA conjugate of the present invention, or compared to the proliferation of equivalent untreated cells, in the presence of the saRNA or GalNAc-saRNA conjugate of the present invention, cell proliferation is reduced by at least 20%, 30%, 40%, preferably at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, and more preferably at least 80%, 90%, or 95%. In embodiments in which cell proliferation is inhibited in hyperproliferative cells, the "equivalent" cells are also hyperproliferative cells. In a preferred embodiment, proliferation is reduced to a rate comparable to the proliferation rate of equivalent healthy (non-hyperproliferative) cells. In another aspect, a preferred embodiment of "inhibiting cell proliferation" is the inhibition of hyperproliferation or the regulation of cell proliferation to reach a normal, healthy level of proliferation.

[0375] As a non-limiting example, the saRNA or GalNAc-saRNA conjugate of the present invention is used to reduce the proliferation of leukemia and lymphoma cells.Preferably, the cells include Jurkat cells (acute T-cell lymphoma cell line), K562 cells (erythroid leukemia cell line), U373 cells (glioblastoma cell line), and 32Dp210 cells (myeloid leukemia cell line).

[0376] In another non-limiting example, the saRNA or GalNAc-saRNA conjugate of the present invention is used to reduce the proliferation of ovarian cancer cells, liver cancer cells, pancreatic cancer cells, breast cancer cells, prostate cancer cells, rat liver cancer cells, and insulinoma cells.Preferably, the cells include PEO1 and PEO4 (ovarian cancer cell lines), HepG2 (hepatocellular carcinoma cell line), Panc1 (human pancreatic cancer cell line), MCF7 (human breast cancer cell line), DU145 (human metastatic prostate cancer cell line), rat liver cancer cells, and MIN6 (rat insulinoma cell line).

[0377] In one embodiment, the saRNA or GalNAc-saRNA conjugates of the present invention are used to treat hyperproliferative disorders. Tumors and cancers represent hyperproliferative disorders of particular interest, including all types of tumors and cancers, such as solid tumors and hematological cancers. Examples of cancer include, but are not limited to, cervical cancer, endometrial cancer, ovarian cancer, kidney cancer, gallbladder cancer, liver cancer, head and neck cancer, squamous cell carcinoma, gastrointestinal cancer, breast cancer, prostate cancer, testicular cancer, lung cancer, non-small cell lung cancer, non-Hodgkin's lymphoma, multiple myeloma, leukemia (such as acute lymphocytic leukemia, chronic lymphocytic leukemia, acute myeloid leukemia, and chronic myeloid leukemia), brain tumors (e.g., astrocytoma, neurocytoma, medulloblastoma), neuroblastoma, sarcoma, colon cancer, rectal cancer, gastric cancer, anal cancer, bladder cancer, endometrial cancer, plasmacytoma, lymphoma, retinoblastoma, Wilm's tumor, Ewing's sarcoma, melanoma, and other skin cancers. Liver cancers include, but are not limited to, cholangiocarcinoma, hepatoblastoma, angiosarcoma, and hepatocellular carcinoma (HCC). HCC is of particular interest.

[0378] Primary liver cancer is the fifth most common cancer worldwide and the third leading cause of cancer-related deaths. HCC accounts for the majority of primary liver cancers (El-Serag et al., Gastroenterology, vol. 132(7), 2557-2576 (2007), the entire contents of which are incorporated herein by reference). HCC is influenced by the interplay of several factors, including cancer cell biology, the immune system, and various etiologies (viral, toxic, and general). The majority of HCC patients develop malignant tumors on the backdrop of liver cirrhosis. Currently, most patients are diagnosed at an advanced stage, so the 5-year survival rate for the majority of HCC patients remains dismal. Currently, surgical resection, locoregional resection, and liver transplantation are the only potentially curative treatment options for HCC. However, based on individual assessments of liver function and tumor burden, only approximately 5–15% of patients are candidates for surgical intervention. The present invention utilizes saRNA or GalNAc-saRNA conjugates to regulate the expression of target genes and treat cirrhosis and HCC.

[0379] The methods of the present invention may reduce tumor volume by at least 10, 20, 30, 40, 50, 60, 70, 80, or 90%. Preferably, the development of one or more new tumors is inhibited; for example, a subject treated according to the present invention develops fewer and / or smaller tumors. Fewer tumors means that the subject develops fewer tumors than a comparable subject over a period of time. For example, the subject develops at least 1, 2, 3, 4, or 5 fewer tumors than a comparable control (untreated) subject. Smaller tumors means that the tumors are at least 10, 20, 30, 40, 50, 60, 70, 80, or 90% smaller in weight and / or volume than the tumors of a comparable subject. The methods of the present invention reduce tumor burden by at least 10, 20, 30, 40, 50, 60, 70, 80, or 90%.

[0380] The period of time can be any suitable period of time, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 months or years. In one non-limiting example, a method for treating undifferentiated tumors is provided, comprising contacting cells, tissues, organs, or subjects with the saRNA or GalNAc-saRNA conjugate of the present invention.Undifferentiated tumors generally have a poorer prognosis than differentiated tumors.Since the degree of differentiation in tumors is related to prognosis, it is hypothesized that the use of biologics that promote differentiation can be a useful antiproliferative drug.Undifferentiated tumors that can be treated with saRNA or GalNAc-saRNA conjugates include undifferentiated small cell lung cancer, undifferentiated pancreatic adenocarcinoma, undifferentiated human pancreatic cancer, undifferentiated human metastatic prostate cancer, and undifferentiated human breast cancer.

[0381] In one embodiment, the saRNA or GalNAc-saRNA conjugate of the present invention is used to regulate oncogenes and tumor suppressor genes. Preferably, the expression of oncogenes can be downregulated. Compared to the expression in the absence of the saRNA or GalNAc-saRNA conjugate of the present invention, the expression of oncogenes is reduced by at least 20, 30, 40%, more preferably at least 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95% in the presence of the saRNA or GalNAc-saRNA conjugate of the present invention. In a more preferred embodiment, in the presence of a saRNA or GalNAc-saRNA conjugate of the present invention, the expression of an oncogene is reduced by at least 2, 3, 4, 5, 6, 7, 8, 9, or 10-fold, more preferably at least 15, 20, 25, 30, 35, 40, 45, or 50-fold, and even more preferably at least 60, 70, 80, 90, or 100-fold, compared to the expression in the absence of the saRNA or GalNAc-saRNA conjugate of the present invention. Preferably, the expression of a tumor suppressor gene can be inhibited. In the presence of a saRNA or GalNAc-saRNA conjugate of the present invention, the expression of a tumor suppressor gene is increased by at least 20, 30, or 40%, more preferably at least 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95%, and even more preferably at least 100%, compared to the expression in the absence of the saRNA or GalNAc-saRNA conjugate of the present invention. In a further preferred embodiment, expression of a tumor suppressor gene is increased by at least 2, 3, 4, 5, 6, 7, 8, 9, 10-fold, more preferably at least 15, 20, 25, 30, 35, 40, 45, 50-fold, and even more preferably at least 60, 70, 80, 90, 100-fold in the presence of a saRNA or GalNAc-saRNA conjugate of the present invention compared to expression in the absence of the saRNA or GalNAc-saRNA conjugate of the present invention.

[0382] In one embodiment, the saRNA or GalNAc-saRNA conjugate of the present invention is used to regulate microRNA (miRNA or miR) in the treatment of hepatocellular carcinoma. MicroRNAs are small non-coding RNAs that regulate gene expression. They are involved in important physiological functions and may be involved in all stages of carcinogenesis. Typically, they contain 21 nucleotides and regulate gene expression at the post-transcriptional level by binding to the 3' untranslated region (3'-UTR) of the mRNA, thereby blocking mRNA translation or inducing mRNA degradation.

[0383] In tumors, the regulation of miRNA expression influences tumor development. In HCC, as in other cancers, miRNAs function as either oncogenes or tumor suppressor genes, affecting cell growth and proliferation, cell metabolism and differentiation, apoptosis, angiogenesis, metastasis, and ultimately prognosis. (Lin et al., Biochemical and Biophysical Research Communications, vol. 375, 315-320 (2008); Kutay et al., J. Cell. Biochem., vol. 99, 671-678 (2006); Meng et al., Gastroenterology, vol. 133(2), 647-658 (2007), the entire contents of each of which are incorporated herein by reference.) The saRNA or GalNAc-saRNA conjugate of the present invention regulates the expression and / or function of target genes and also regulates miRNA levels in HCC cells. Non-limiting examples of miRNAs that can be regulated by the saRNA or GalNAc-saRNA conjugates of the present invention include hsa-let-7a-5p, hsa-miR-133b, hsa-miR-122-5p, hsa-miR-335-5p, hsa-miR-196a-5p, hsa-miR-142-5p, hsa-miR-96-5p, hsa-miR-184, hsa-miR-214-3p, hsa-miR-15a-5p, hsa-let-7b-5p, hsa-miR-205-5p, hsa-miR-181a-5p, hsa-miR-140-5p, hsa-miR-146b-5p, hsa -miR-34c-5p, hsa-miR-134, hsa-let-7g-5p, hsa-let-7c, hsa-miR-218-5p, hsa-m iR-206, hsa-miR-124-3p, hsa-miR-100-5p, hsa-miR-10b-5p, hsa-miR-155-5p, hsa -miR-1, hsa-miR-150-5p, hsa-let-7i-5p, hsa-miR-27b-3p, hsa-miR-127-5p, hsa -miR-191-5p, hsa-let-7f-5p, hsa-miR-10a-5p, hsa-miR-15b-5p, hsa-miR-16-5p,It includes hsa-miR-34a-5p, hsa-miR-144-3p, hsa-miR-128, hsa-miR-215, hsa-miR-193a-5p, hsa-miR-23b-3p, hsa-miR-203a, hsa-miR-30c-5p, hsa-let-7e-5p, hsa-miR-146a-5p, hsa-let-7d-5p, hsa-miR-9-5p, hsa-miR-181b-5p, hsa-miR-181c-5p, hsa-miR-20b-5p, hsa-miR-125a-5p, hsa-miR-148b-3p, hsa-miR-92a-3p, hsa-miR-378a-3p, hsa-miR-130a-3p, hsa-miR-20a-5p, hsa-miR-132-3p, hsa-miR-193b-3p, hsa-miR-183-5p, hsa-miR-148a-3p, hsa-miR-138-5p, hsa-miR-373-3p, hsa-miR-29b-3p, hsa-miR-135b-5p, hsa-miR-21-5p, hsa-miR-181d, hsa-miR-301a-3p, hsa-miR-200c-3p, hsa-miR-7-5p, hsa-miR-29a-3p, hsa-miR-210, hsa-miR-17-5p, hsa-miR-98-5p, hsa-miR-25-3p, hsa-miR-143-3p, hsa-miR-19a-3p, hsa-miR-18a-5p, hsa-miR-125b-5p, hsa-miR-126-3p, hsa-miR-27a-3p, hsa-miR-372, hsa-miR-149-5p, and hsa-miR-32-5p.,

[0384] In one non-limiting example, the miRNA is an oncogenic miRNA and is downregulated by at least 0.01, 0.02, 0.05, 0.1, 0.2, 0.3, 0.5, 1, 1.5, 2, 2.5, and 3-fold in the presence of a saRNA or GalNAc-saRNA conjugate of the present invention compared to the absence of the saRNA or GalNAc-saRNA conjugate. In another non-limiting example, the miRNA is a tumor-suppressing miRNA and is upregulated in the presence of a saRNA or GalNAc-saRNA conjugate of the present invention by at least 0.01, 0.02, 0.05, 0.1, 0.2, 0.3, 0.5, or 1-fold, more preferably at least 2, 3, 4, 5, 6, 7, 8, 9, or 10-fold, more preferably at least 15, 20, 25, 30, 35, 40, 45, or 50-fold, and even more preferably at least 60, 70, 80, 90, or 100-fold compared to the absence of the saRNA or GalNAc-saRNA conjugate.

[0385] IV. Kits and Devices kit The present invention provides various kits for conveniently and / or effectively practicing the methods of the present invention. Typically, the kits will contain components in quantities and / or numbers sufficient to allow the user to perform multiple treatments of subjects and / or perform multiple experiments.

[0386] In one embodiment, the present invention provides a kit for regulating gene expression in vitro or in vivo, comprising a saRNA or GalNAc-saRNA conjugate of the present invention, or a combination of a saRNA or GalNAc-saRNA conjugate of the present invention and another gene-regulating saRNA, siRNA, miRNA or other oligonucleotide molecule.

[0387] The kit may further include packaging and instructions for forming the pharmaceutical composition and / or a delivery agent, which may include saline, a buffer, a lipidoid, a dendrimer, or any delivery agent disclosed herein.

[0388] Non-limiting examples of genes are listed in Table 1 herein. In one embodiment, kits containing the saRNA or GalNAc-saRNA conjugates described herein can be used with proliferating cells to demonstrate efficacy.

[0389] In one non-limiting example, the buffer solution may contain sodium chloride, calcium chloride, phosphate, and / or EDTA. In another non-limiting example, the buffer solution may contain, but is not limited to, saline, saline with 2 mM calcium, 5% sucrose, 5% sucrose with 2 mM calcium, 5% mannitol, 5% mannitol with 2 mM calcium, Ringer's lactate, sodium chloride, sodium chloride with 2 mM calcium, and mannose (see U.S. Patent Application Publication No. 20120258046, incorporated herein by reference in its entirety). In yet another non-limiting example, the buffer solution may be precipitated or lyophilized. The amount of each component may be varied to allow for consistent and reproducible formulation of highly concentrated saline or simple buffers. Components may also be varied to enhance the stability of the saRNA or GalNAc-saRNA conjugate in the buffer solution over a period of time and / or under various conditions.

[0390] device The present invention provides devices that can incorporate the saRNA or GalNAc-saRNA conjugates of the present invention. These devices contain stable formulations that are readily available for delivery to subjects in need thereof, such as human patients.

[0391] Non-limiting examples of devices include pumps, catheters, needles, transdermal patches, pressurized olfactory delivery devices, iontophoresis devices, and multi-layer microfluidic devices. The devices can be used to deliver the saRNA or GalNAc-saRNA conjugates of the present invention in a single, multiple, or divided dose regimen. The devices can be used to deliver the saRNA or GalNAc-saRNA conjugates of the present invention to the entire biological tissue, intradermally, subcutaneously, or intramuscularly. Many examples of devices suitable for delivering oligonucleotides are disclosed in International Publication No. WO 2013 / 090648, filed December 14, 2012, the entire contents of which are incorporated herein by reference.

[0392] definition For convenience, the meanings of certain terms and phrases used in the specification, examples, and appended claims are provided below. If there is an apparent discrepancy between the use of a term in other parts of this specification and its definition provided in this section, the definition in this section shall control.

[0393] About: As used herein, the term "about" means + / - 10% of the stated value. 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 simultaneously or at intervals such that the effects of each agent on the patient can overlap. In some embodiments, they are administered within about 60, 30, 15, 10, 5, or 1 minute of each other. In some embodiments, the administration of the agents is spaced sufficiently close together so that a combined (e.g., synergistic) effect is achieved.

[0394] Amino acid: As used herein, the terms "amino acid" and "amino acids" refer to all naturally occurring L-alpha-amino acids. Amino acids are identified by one-letter or three-letter codes, where the amino acid is listed first, followed by the three-letter and one-letter codes, respectively: 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), and asparagine (Asn:N).

[0395] Animal: As used herein, the term "animal" refers to any member of the animal kingdom. In some embodiments, "animal" refers to a human at any stage of development. In some embodiments, "animal" refers to a non-human animal at any stage of development. In certain embodiments, the non-human animal is a mammal (e.g., a rodent, mouse, rat, rabbit, monkey, dog, cat, sheep, cow, primate, or pig). In some embodiments, animals include, but are not limited to, mammals, birds, reptiles, amphibians, fish, and nematodes. In some embodiments, the animal is a transgenic animal, a genetically engineered animal, or a clone.

[0396] Approximately: As used herein, the term "approximately" or "about," when applied to one or more values ​​of interest, refers to a value similar to a stated reference value. In certain embodiments, unless otherwise stated or clear from the context, the term "approximately" or "about" refers to a range of values ​​that falls within 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 or lesser than) the stated reference value (except where such number would exceed 100% of a possible value).

[0397] Associated: As used herein, the terms "associated," "conjugated," "bonded," "attached," and "tethered," when used in reference to two or more moieties, mean physically associated or connected to one another, either directly or through one or more additional moieties that function as linking agents, 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 a direct covalent chemical bond; it can also imply ionic, hydrogen, or hybridization-based bonding that is sufficiently stable so that the "associated" elements remain physically associated.

[0398] Bifunctional or bifunctional: As used herein, the terms "bifunctional" and "bifunctional" refer to any substance, molecule, or moiety capable of or maintaining at least two functions. The functions may affect the same outcome or different outcomes. The structures that exert the functions may be the same or different. For example, a bifunctional saRNA of the present invention may contain a cytotoxic peptide (first function), while the nucleosides that make up the saRNA are themselves cytotoxic (second function).

[0399] Biocompatible: As used herein, the term "biocompatible" means compatible with living cells, tissues, organs or systems presenting little or no risk of injury, toxicity, or rejection by the immune system.

[0400] Biodegradable: As used herein, the term "biodegradable" means capable of being broken down into harmless products by the action of living organisms. Biological activity: As used herein, the term "biological activity" refers to any property of a substance that has activity in a biological system and / or organism. For example, a substance that, when administered to an organism, has a biological effect on the organism is considered to be biologically active. In certain embodiments, a saRNA of the present invention may be considered biologically active if even a portion of the saRNA is biologically active or mimics an activity that is considered biologically relevant.

[0401] Cancer: As used herein, the term "cancer" in an individual refers to the presence of cells that have characteristics typical of cancer-causing cells, such as uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rate, and characteristic morphological characteristics. Often, cancer cells will be in the form of a tumor, but such cells may exist alone within an individual or circulate in the bloodstream as independent cells, such as leukemia cells.

[0402] Cell growth: As used herein, the term "cell growth" refers primarily to an increase in cell number resulting from cell replication (i.e., proliferation), the rate of which is greater than the rate of cell death (e.g., by apoptosis or necrosis), resulting in an increase in the size of the cell population, although under certain circumstances a small portion of the growth may also result from an increase in cell size or cytoplasmic volume of individual cells. Thus, agents that inhibit cell growth may do so in a way that alters the balance between these two opposing processes by either inhibiting proliferation or promoting cell death, or both.

[0403] Cell type: As used herein, the term "cell type" refers to cells of a given origin (e.g., tissue, organ), or of a given differentiation state, or associated with a given pathology or genetic makeup.

[0404] Chromosome: As used herein, the term "chromosome" refers to an organized structure of DNA and proteins found in cells. Complementary: As used herein, the term "complementary" with respect to nucleic acids refers to hybridization or base pairing between nucleotides or nucleic acids, such as between the two strands of a double-stranded DNA molecule or between an oligonucleotide probe and a target.

[0405] Pathology: As used herein, the term "pathology" refers to the state of any cell, organ, organ system, or organism. A pathology may reflect a disease state or simply the physiological presentation or status of an entity. A pathology may be characterized as a phenotypic pathology, such as the macroscopic presentation of a disease, or a genotypic pathology, such as the underlying gene or protein expression profile associated with the pathology. A pathology may be benign or malignant.

[0406] Controlled Release: As used herein, the term "controlled release" refers to a release profile of a pharmaceutical composition or compound that conforms to a particular release pattern that results in a therapeutic outcome.

[0407] Cytostatic: As used herein, "cytostatic" means inhibiting, reducing, or suppressing the growth, division, or proliferation of a cell (e.g., a mammalian cell (e.g., a human cell)), bacterium, virus, fungus, protozoan, parasite, prion, or combination thereof.

[0408] Cytotoxic: As used herein, "cytotoxic" means killing or causing a deleterious, toxic, or lethal effect on a cell (e.g., a mammalian cell (e.g., a human cell)), bacterium, virus, fungus, protozoan, parasite, prion, or combination thereof.

[0409] Delivery: As used herein, "delivery" means the act or manner of delivering a compound, substance, element, moiety, cargo, or payload. Delivery agent: As used herein, "delivery agent" means any substance that at least partially facilitates the in vivo delivery of the saRNA of the present invention to targeted cells.

[0410] Destabilization: As used herein, the terms "unstable," "destabilize," or "destabilizing region" refer to a region or molecule that is less stable than an initial, wild-type, or native form of the same region or molecule.

[0411] Detectable Label: As used herein, "detectable label" refers to one or more markers, signals, or moieties attached to, incorporated into, or associated with another entity that are readily detected by methods known in the art, including radiography, fluorescence, chemiluminescence, enzymatic activity, absorbance, and the like. Detectable labels include radioisotopes, fluorophores, chromophores, enzymes, dyes, metal ions, ligands (e.g., biotin, avidin, streptavidin, and haptens), quantum dots, and the like. Detectable labels can be placed at any position in the oligonucleotides disclosed herein. They can be within the nucleotide or located at the 5' or 3' terminus.

[0412] Encapsulation: As used herein, the term "encapsulation" means to seal, surround, or contain. Engineered: As used herein, an embodiment of the invention is "engineered" if it is designed to have characteristics or properties, whether structural or chemical, that differ from the starting, wild-type, or naturally occurring molecule.

[0413] Equivalent subject: As used herein, an "equivalent subject" can be, for example, a subject of similar age, sex, and health, e.g., liver health or cancer stage, or the same subject prior to treatment with the present invention. An equivalent subject is "untreated" in that it has not received treatment with the saRNA of the present invention. However, subjects treated with the saRNA of the present invention may also receive conventional anti-cancer treatment, provided that they receive the same or equivalent conventional anti-cancer treatment.

[0414] Exosome: As used herein, an "exosome" is a vesicle secreted by mammalian cells. 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 the RNA transcript (e.g., by splicing, editing, 5' capping, and / or 3' end processing), (3) translation of the RNA into a polypeptide or protein, and (4) post-translational modification of the polypeptide or protein.

[0415] Characteristic: As used herein, "characteristic" means a property, quality, or distinguishing element. Formulation: As used herein, a "formulation" comprises at least one saRNA of the present invention and a delivery agent.

[0416] Fragment: As used herein, "fragment" means a portion. For example, a fragment of a protein may include a polypeptide obtained by digesting a full-length protein isolated from a cultured cell. A fragment of an oligonucleotide may include a nucleotide or a region of a nucleotide.

[0417] 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. Gene: As used herein, the term "gene" refers to a nucleic acid sequence that comprises control sequences and, in most cases, coding sequences necessary for the production of a polypeptide or precursor. However, a gene need not be translated, but instead may encode a regulatory or structural RNA molecule.

[0418] A gene may be derived in whole or in part from any source known in the art, including plant, fungal, animal, or bacterial genomes or episomes, 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 untranslated regions that may affect the biological activity or 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. A gene may constitute an uninterrupted coding sequence or may contain one or more introns bounded by appropriate splice junctions.

[0419] Gene Expression: As used herein, the term "gene expression" refers to the process by which a nucleic acid sequence is successfully transcribed and, in most cases, translated to produce a protein or peptide. For clarity, when reference is made to measuring "gene expression," it should be understood to mean that the measurement can be of the nucleic acid product of transcription, e.g., RNA or mRNA, or the amino acid product of translation, e.g., a polypeptide or peptide. Methods for measuring the amount or levels of RNA, mRNA, polypeptides, and peptides are well known in the art.

[0420] Genome: The term "genome" is intended to include the entire DNA complement of an organism, including nuclear DNA components, chromosomal and extrachromosomal DNA, as well as cytoplasmic domains (e.g., mitochondrial DNA).

[0421] Homology: As used herein, the term "homology" refers to the overall relatedness between polymers, e.g., between nucleic acid molecules (e.g., DNA molecules and / or RNA molecules) and / or between polypeptide molecules. In some embodiments, polymers are considered to be "homologous" to one another if the 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 sequences or polypeptide sequences). According to the present invention, 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% identical over at least one stretch of at least about 20 amino acids. In some embodiments, homologous polynucleotide sequences are characterized by their ability to encode a uniquely specified stretch of at least 4-5 amino acids. For polynucleotide sequences less than 60 nucleotides in length, homology is determined by their ability to encode a uniquely specified stretch of at least 4-5 amino acids. According to the present invention, two protein sequences are considered to be homologous if the proteins are at least about 50%, 60%, 70%, 80%, or 90% identical over at least one stretch of at least about 20 amino acids.

[0422] The term "hyperproliferative cell" can refer to any cell that proliferates at an abnormally high rate compared to the proliferation rate of an equivalent healthy cell (which may be referred to as a "control"). An "equivalent healthy" cell is a normal, healthy counterpart of a cell. It is therefore a cell of the same type, e.g., a cell of the same organ that performs the same function as a comparator cell. For example, the proliferation of hyperproliferative liver cells should be assessed relative to healthy liver cells, while the proliferation of hyperproliferative prostate cells should be assessed relative to healthy prostate cells.

[0423] An "abnormally high" rate of proliferation means that the proliferation rate of the hyperproliferative cells is increased by at least 20, 30, 40%, or at least 45, 50, 55, 60, 65, 70, 75%, or at least 80% compared to the proliferation rate of an equivalent healthy (non-hyperproliferative) cell. An "abnormally high" rate of proliferation can also mean a rate that is increased by at least 2, 3, 4, 5, 6, 7, 8, 9, 10-fold, or at least 15, 20, 25, 30, 35, 40, 45, 50-fold, or at least 60, 70, 80, 90, 100-fold compared to the proliferation rate of an equivalent healthy cell.

[0424] Hyperproliferative disorder: As used herein, a "hyperproliferative disorder" can be any disorder involving hyperproliferative cells as defined above. Examples of hyperproliferative disorders include neoplastic disorders such as cancer, psoriatic arthritis, rheumatoid arthritis, gastric hyperproliferative disorders such as inflammatory bowel disease, skin disorders such as psoriasis, Reiter's syndrome, pityriasis rubra pilaris, and hyperproliferative variants of keratinization disorders.

[0425] Those skilled in the art are familiar with how to identify hyperproliferative cells.The existence of hyperproliferative cells in animals can be identified by scanning such as X-ray, MRI, CT scan, etc. Hyperproliferative cells can also be identified by culturing samples in vitro or assaying cell proliferation using cell proliferation assays such as MTT, XTT, MTS or WST-1 assay.In vitro cell proliferation can also be determined by flow cytometry.

[0426] Identity: As used herein, the term "identity" refers to the overall relatedness between polymers, e.g., between oligonucleotide molecules (e.g., DNA molecules and / or RNA molecules) and / or between polypeptide molecules. For example, the percent identity of two polynucleotide sequences can be calculated by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced in one or both of the first and second nucleic acid sequences to ensure optimal alignment, and different sequences can be ignored for comparison purposes). In certain embodiments, the length of the sequences 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. 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, the molecules are identical at that position. The percent identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps and the length of each gap that must be introduced to optimally align 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 calculated using the methods described in Computational Molecular Biology, Lesk, AM (ed.), Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, DW (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, AM and Griffin, HG (eds.), Humana Press, New Jersey, 1994; and Sequence Analysis: An Introduction to Sequence Analysis. Primer), Gribskov, M. and Devereux, J. (eds.), M Stockton Press, New York, 1991, each of which is incorporated herein by reference. For example, the percent identity between two nucleotide sequences can be determined by the Meyers and Miller algorithm (CABIOS, 1989, 4:11-17) as 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 by the GAP program in the GCG software package using the NWSgapdna.CMP matrix.Commonly utilized methods for determining 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), which is incorporated herein by reference. Techniques for determining identity have been codified in the form of publicly available computer programs. Exemplary computer software for determining homology between two sequences include, but are not limited to, the GCG program package; Devereux, J. et al., Nucleic Acids Research, 12(1), 387 (1984); BLASTP, BLASTN, and FASTA; Altschul, S. F. et al., J. Molec. Biol., 215, 403 (1990).

[0427] Inhibition of gene expression: As used herein, the phrase "inhibition of gene expression" means causing a reduction in the amount of an expression product of a gene. The expression product can be an RNA (e.g., mRNA) transcribed from the gene or a polypeptide translated from an mRNA transcribed from the gene. Typically, a reduction in the level of mRNA results in a reduction in the level of the polypeptide translated from it. The level of expression can be determined using standard techniques for measuring mRNA or protein.

[0428] In vitro: As used herein, the term "in vitro" refers to events that take place not within a living organism (e.g., an animal, plant, or microorganism) but in an artificial environment, such as a test tube or reaction vessel, cell culture, petri dish, etc.

[0429] In vivo: As used herein, the term "in vivo" refers to events that take place within an organism (e.g., an animal, plant, or microorganism, or cells or tissues thereof).

[0430] 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 its natural environment or in an experimental setting). Isolated substances can have various levels of purity relative to the substance with which it was associated. Isolated substances and / or entities can 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 it was initially associated. In some embodiments, an isolated agent is greater 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 greater than about 99% pure. As used herein, a substance is "pure" if it is substantially free of other components. Substantially isolated: "Substantially isolated" means that the compound is substantially separated from the environment in which it was produced or detected. Partial isolation can include, for example, compositions enriched in a compound according to the present disclosure. Substantial isolation can include 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 a compound according to the present disclosure or a salt thereof. Methods for isolating compounds and salts thereof are routine in the art.

[0431] Label: The term "label" refers to a substance, compound, or substance or compound that is incorporated into an object so that the object can be detected. Linker: As used herein, linker refers to a group of atoms (e.g., 10-1,000 atoms) that can be composed of atoms or groups, including, but not limited to, carbon, amino, alkylamino, oxygen, sulfur, sulfoxide, sulfonyl, carbonyl, and imine. The linker can attach at one end to the nucleobase or sugar moiety of a modified nucleoside or nucleotide and at the second end to a payload, such as a detectable or therapeutic agent. The linker can be of sufficient length so as not to interfere with incorporation into a nucleic acid sequence. The linker can be used for any useful purpose, such as forming a saRNA conjugate or further administering a payload, as described herein.

[0432] Examples of chemical groups that can be incorporated into linkers and / or spacers include, but are not limited to, alkyl, alkenyl, alkynyl, amide, amino, ether, thioether, ester, alkylene, heteroalkylene, aryl, or heterocyclyl, each of which can be optionally substituted as described herein. Examples of spacers include, but are not limited to, unsaturated alkanes, polyethylene glycols (e.g., ethylene or propylene glycol monomer units, e.g., diethylene glycol, dipropylene glycol, triethylene glycol, tripropylene glycol, tetraethylene glycol, or tetraethylene glycol), and dextran polymers, and derivatives thereof. Examples of linkers include, but are not limited to, those with moieties within the linker that can be cleaved by reducing agents or photolysis, such as disulfide bonds (-SS-) and azo bonds (-N=N-). Non-limiting examples of selectively cleavable bonds include disulfide bonds that can be cleaved by the use of, for example, tris(2-carboxyethyl)phosphine (TCEP) or other reducing agents.

[0433] Metastasis: As used herein, the term "metastasis" refers to the process by which cancer spreads to distant locations in the body from where it first began as a primary tumor. Metastasis also refers to cancer resulting from the spread of a primary tumor. For example, a patient with breast cancer may develop metastases in their lymphatic system, liver, bones, or lungs.

[0434] Modified: As used herein, "modified" refers to an altered state or structure of a molecule of the invention. Molecules can be modified in many ways, including chemically, structurally, and functionally. In one embodiment, the saRNA of the invention is modified by the introduction of non-natural nucleosides and / or nucleotides.

[0435] Naturally occurring: As used herein, "naturally occurring" means existing in nature without artificial assistance. Nucleic Acid: As used herein, the term "nucleic acid" refers to a molecule composed of one or more nucleotides, i.e., ribonucleotides, deoxyribonucleotides, or both. This term includes ribonucleotide and deoxyribonucleotide monomers and polymers, where the ribonucleotides and / or deoxyribonucleotides are linked together via 5' to 3' linkages. Ribonucleotide and deoxyribonucleotide polymers can be single-stranded or double-stranded. However, the linkages can include any linkage known in the art; for example, nucleic acids include 5' to 3' linkages. Nucleotides can be naturally occurring or synthetically produced analogs capable of forming base-pairing relationships with naturally occurring base pairs. Examples of non-naturally occurring bases capable of forming base-pairing relationships include, but are not limited to, aza and deazapyrimidine analogs, aza and deazapurine analogs, and other heterocyclic base analogs in which one or more of the carbon and nitrogen atoms of the pyrimidine ring are replaced by a heteroatom, such as oxygen, sulfur, selenium, phosphorus, etc.

[0436] Patient: As used herein, "patient" means a subject who is seeking or believed to be in need of treatment, who needs treatment, who is undergoing treatment, or who will receive treatment, or who is receiving care from a trained professional for a particular disease or condition.

[0437] Peptide: As used herein, a "peptide" is 50 amino acids or less in length, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids in length.

[0438] Pharmaceutically acceptable: The phrase "pharmaceutically acceptable," as used herein, means those compounds, materials, compositions, and / or dosage forms that 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.

[0439] Pharmaceutically acceptable excipient: As used herein, the term "pharmaceutically acceptable excipient" refers to any ingredient, other than the compounds described herein, that is substantially non-toxic and non-inflammatory in a patient (e.g., a vehicle capable of suspending or dissolving an active compound). Excipients can include, for example, antiadhesives, antioxidants, binders, coating agents, compression aids, disintegrants, dyes (colorants), emollients, emulsifiers, fillers (diluents), film-forming or coating agents, flavors, fragrances, glidants (glidants), lubricants, preservatives, printing inks, sorbents, suspending or dispersing agents, sweeteners, and water for hydration. Exemplary excipients include, but are not limited to, butylated hydroxytoluene (BHT), calcium carbonate, calcium phosphate (dibasic), calcium stearate, croscarmellose, cross-linked polyvinylpyrrolidone, citric acid, crospovidone, cysteine, ethylcellulose, gelatin, hydroxypropyl cellulose, hydroxypropylmethylcellulose, lactose, magnesium stearate, maltitol, mannitol, methionine, methylcellulose, methylparaben, microcrystalline cellulose, polyethylene glycol, polyvinylpyrrolidone, povidone, pregelatinized starch, propylparaben, retinyl palmitate, shellac, silicon dioxide, sodium carboxymethylcellulose, sodium citrate, sodium starch glycolate, sorbitol, starch (corn), stearic acid, sucrose, talc, titanium dioxide, vitamin A, vitamin E, vitamin C, and xylitol.

[0440] Pharmaceutically acceptable salts: The present disclosure also encompasses pharmaceutically acceptable salts of the compounds described herein. As used herein, "pharmaceutically acceptable salts" refers to derivatives of the disclosed compounds in which the parent compound is modified by converting an existing acid or base moiety into 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 acid 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, and 2-hydroxyethanesulfonate. salts include sodium, 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, and the like. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like, as well as non-toxic ammonium, quaternary ammonium, and amine cations, such as, 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 form of these compounds with a stoichiometric amount of the appropriate base or acid in water or an organic solvent, or a mixture of the two; generally, non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred. Lists of suitable salts can be found in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa., 1985, p. 1418; Pharmaceutical Salts: Properties, Selection, and Use, edited by P.H. Stahl and C.G. Wermuth, Wiley-VCH, 2008; and Berge et al., Journal of Pharmaceutical Science, 66, 1-19 (1977), each of which is incorporated herein by reference in its entirety.

[0441] Pharmaceutically acceptable solvate: As used herein, the term "pharmaceutically acceptable solvate" refers to a compound of the present invention wherein molecules of a suitable solvent are incorporated into the crystal lattice. A suitable solvent is physiologically tolerable at the administered dosage. For example, solvates may be prepared by crystallization, recrystallization, or precipitation from a solution containing an organic solvent, water, or a mixture thereof. Examples of suitable solvents are ethanol, water (e.g., mono-, di-, and trihydrates), N-methylpyrrolidinone (NMP), dimethyl sulfoxide (DMSO), N,N'-dimethylformamide (DMF), N,N'-dimethylacetamide (DMAC), 1,3-dimethyl-2-imidazolidinone (DMEU), 1,3-dimethyl-3,4,5,6-tetrahydro-2-(1H)-pyrimidinone (DMPU), acetonitrile (ACN), propylene glycol, ethyl acetate, benzyl alcohol, 2-pyrrolidone, benzyl benzoate, etc. When water is the solvent, the solvate is referred to as a "hydrate."

[0442] Pharmacological effect: As used herein, a "pharmacological effect" refers to a measurable biological phenomenon in an organism or system that occurs after the organism or system has contacted or been exposed to an exogenous agent. A pharmacological effect can result in a therapeutically effective outcome, such as treatment or amelioration of one or more symptoms, diagnosis, prevention, and delay of the onset of a disease, disorder, condition, or infection. Measurements of such biological phenomena can be quantitative, qualitative, or relative to other biological phenomena. Quantitative measurements can be statistically significant. Qualitative measurements can be of degree or kind and can differ by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more. They can be observable as present or absent, better or worse, more or less. An exogenous agent, when referring to a pharmacological effect, is an agent that is foreign in whole or in part to the organism or system. For example, modifications to a wild-type biomolecule, whether structural or chemical, would result in an exogenous agent. Similarly, the incorporation of or combination of a wild-type molecule with a compound, molecule, or substance not naturally found in the organism or system would also result in an exogenous agent.

[0443] The saRNA of the present invention includes exogenous agents. Examples of pharmacological effects include, but are not limited to, changes in cell numbers, such as an increase or decrease in neutrophils, reticulocytes, granulocytes, erythrocytes (red blood cells), megakaryocytes, platelets, monocytes, connective tissue macrophages, epidermal Langerhans cells, osteoclasts, dendritic cells, microglial cells, neutrophils, eosinophils, basophils, mast cells, helper T cells, suppressor T cells, cytotoxic T cells, natural killer T cells, B cells, natural killer cells, or reticulocytes. Pharmacological effects also include changes in blood chemistry, pH, hemoglobin, hematocrit, changes in enzymes, such as, but not limited to, the levels of the liver enzymes AST and ALT, lipid profiles, electrolytes, metabolic markers, hormones, or other markers or profiles known to those skilled in the art.

[0444] Physicochemical: As used herein, "physicochemical" means of or relating to physical and / or chemical properties. Prevention: As used herein, the term "prevention" means partially or completely delaying the onset of an infection, disease, disorder, and / or condition; partially or completely delaying the onset of one or more symptoms, characteristics, or clinical lesions of a particular infection, disease, disorder, and / or condition; partially or completely delaying the onset of one or more symptoms, characteristics, or lesions of a particular infection, disease, disorder, and / or condition; partially or completely delaying the progression from an infection, a particular disease, disorder, and / or condition, and / or reducing the risk of developing pathology associated with an infection, disease, disorder, and / or condition.

[0445] Prodrugs: The present disclosure also encompasses prodrugs of the compounds described herein. As used herein, "prodrug" refers to any substance, molecule, or entity that undergoes a chemical or physical change to assume a form that is considered to act as a therapeutic agent. Prodrugs may be covalently attached or entrapped in some manner and release or are converted to an active drug moiety before, during, or after administration to a mammalian subject. Prodrugs can be prepared by modifying functional groups present in the compound in such a way that the modifications are cleaved, either by routine manipulation or in vivo, to yield the parent compound. Prodrugs include compounds in which a hydroxyl, amino, sulfhydryl, or carboxyl group is bonded to any group that is cleaved to generate a free hydroxyl, amino, sulfhydryl, or carboxyl group, respectively, upon administration to a mammalian subject. The preparation and use of prodrugs are discussed in T. Higuchi and V. Stella, "Pro-drugs as Novel Delivery Systems," Vol. 14 of the ACS Symposium Series, and in Edward B. Roche (ed.), "Bioreversible Carriers in Drug Design," American Pharmaceutical Association and Pergamon Press, 1987, both of which are incorporated herein by reference in their entireties.

[0446] Prognosis: As used herein, the term "prognosis" means the statement or assertion that a particular biological event will occur in the future or is highly likely to occur.

[0447] Progression: As used herein, the term "progression" or "cancer progression" refers to the advancement or worsening of or toward a disease or condition. Proliferative: As used herein, the term "proliferative" means to grow, expand, or increase, or to cause rapid growth, expansion, or increase. "Proliferative" means having the ability to proliferate. "Antiproliferative" means having properties that are opposed to or incompatible with proliferative properties.

[0448] Protein: "Protein" refers to a polymer of amino acid residues joined together by peptide bonds. As used herein, the term refers to proteins, polypeptides, and peptides of any size, structure, or function. Typically, however, a protein will be at least 50 amino acids in length. In some cases, the encoded protein is less than about 50 amino acids. In this case, the polypeptide is referred to as a peptide. If the protein is a short peptide, it will be at least about 10 amino acid residues in length. Proteins can be naturally occurring, recombinant, or synthetic, or any combination thereof. Proteins can also include fragments of naturally occurring proteins or peptides. Proteins can be single molecules or multimolecular complexes. The term protein can also apply to amino acid polymers in which one or more amino acid residues are artificial chemical analogs of the corresponding naturally occurring amino acids.

[0449] Protein expression: The term "protein expression" refers to the process by which a nucleic acid sequence undergoes translation such that detectable levels of an amino acid sequence or protein are expressed. Purified: As used herein, "purify," "purified," and "purification" mean to make substantially pure or clear from undesired components, contaminating, adulterated, or imperfect materials.

[0450] Regression: As used herein, the term "regression" or "degree of regression" refers to the reversal of cancer progression, either phenotypically or genotypically. A slowing or halting of cancer progression may be considered regression.

[0451] Sample: As used herein, the term "sample" or "biological sample" refers to a subset of its tissues, cells, or components (e.g., bodily fluids, including but not limited to, blood, mucus, lymph, synovial fluid, cerebrospinal fluid, saliva, amniotic fluid, amniotic cord blood, urine, vaginal fluid, and semen). Samples can further include homogenates, lysates, or extracts prepared from a whole organism, or a subset of its tissues, cells, or components, or a fraction or portion thereof, including, for example, but not limited to, plasma, serum, spinal fluid, lymph, outer skin sections, respiratory, intestinal, and genitourinary tracts, tears, saliva, milk, blood cells, tumors, organs. Samples also refer to media, e.g., nutrient broths or gels, that may contain cellular components, such as protein or nucleic acid molecules.

[0452] Signal sequence: As used herein, the phrase "signal sequence" refers to a sequence capable of directing the transport or localization of a protein. Single unit dose: As used herein, a "single unit dose" refers to a dose of any therapeutic agent administered at one time / at one time / by one route / at one point of contact, i.e., in a single administration event.

[0453] Similarity: As used herein, the term "similarity" refers to the overall relatedness between polymers, e.g., between polynucleotide molecules (e.g., DNA molecules and / or RNA molecules) and / or between polypeptide molecules. Calculation of percent similarity of polymers to each other can be performed in the same manner as calculating percent identity, except that percent similarity is calculated taking into account conservative substitutions, as understood in the art.

[0454] Split dose: As used herein, a "split dose" refers to a single unit dose or total daily dose divided into two or more doses. 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, amenable to formulation into an effective therapeutic agent.

[0455] Stabilized: As used herein, the terms "stabilize," "stabilized," and "stabilization region" mean to make stable or to become stable. Subject: As used herein, the term "subject" or "patient" refers to any living organism to which a composition of the invention can 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.

[0456] Substantially: As used herein, the term "substantially" refers to the qualitative condition of exhibiting the whole or nearly whole extent or degree of a characteristic or property of interest. Those skilled in the biological arts will understand that biological and chemical phenomena rarely, if ever, reach an end state and / or proceed perfectly or achieve or avoid an absolute result. Thus, the term "substantially" is used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena.

[0457] Substantially equal: As used herein in reference to the time difference between doses, this term means ±2%. Substantially simultaneously: When used herein in connection with multiple doses, this term means within 2 seconds.

[0458] Suffering from: An individual "suffering from" a disease, disorder, and / or condition has been diagnosed with or exhibits one or more symptoms of the disease, disorder, and / or condition. Susceptible to: An individual who is "susceptible to" a disease, disorder, and / or condition may not have been diagnosed with and / or exhibit symptoms of the disease, disorder, and / or condition, but has a tendency to develop the disease or its symptoms. In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition (e.g., cancer) can be characterized by one or more of the following: (1) a genetic mutation associated with the development of the disease, disorder, and / or condition; (2) a genetic polymorphism associated with the development of the disease, disorder, and / or condition; (3) an increase and / or decrease in the expression and / or activity of a protein and / or nucleic acid associated with the disease, disorder, and / or condition; (4) a behavior and / or lifestyle associated with the development of the disease, disorder, and / or condition; (5) a family history of the disease, disorder, and / or condition; and (6) exposure to and / or infection with a microorganism associated with the development of the disease, disorder, and / or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition will develop the disease, disorder, and / or condition, hi some embodiments, an individual who is susceptible to a disease, disorder, and / or condition will not develop the disease, disorder, and / or condition.

[0459] Sustained release: As used herein, the term "sustained release" refers to a release profile of a pharmaceutical composition or compound that matches the release rate over a specific period of time. Synthetic: The term "synthetic" means produced, prepared, and / or manufactured by the hand of man. Synthesis of polynucleotides or polypeptides or other molecules according to the invention can be chemical or enzymatic.

[0460] Targeted Cell: As used herein, "targeted cell" 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 most embodiments, a patient.

[0461] Therapeutic Agent: The term "therapeutic agent" means any agent that has a therapeutic, diagnostic, and / or prophylactic effect and / or induces a desired biological and / or pharmacological effect when administered to a subject.

[0462] Therapeutically effective amount: As used herein, the term "therapeutically effective amount" means an amount of a delivery agent (e.g., nucleic acid, drug, therapeutic agent, diagnostic agent, prophylactic agent, etc.) sufficient to treat, ameliorate the symptoms of, diagnose, prevent, and / or delay the onset of, the infection, disease, disorder, and / or condition when administered to a subject suffering from or susceptible to the infection, disease, disorder, and / or condition.

[0463] Therapeutically Effective Outcome: As used herein, the term "therapeutically effective outcome" means an outcome sufficient to treat, ameliorate the symptoms of, diagnose, prevent, and / or delay the onset of an infection, disease, disorder, and / or condition in a subject suffering from or susceptible to the infection, disease, disorder, and / or condition.

[0464] Total daily dose: As used herein, a "total daily dose" refers to the amount administered or prescribed in a 24-hour period. It may be administered as a single unit dose. Transcription factor: As used herein, the term "transcription factor" refers to a DNA-binding protein that regulates transcription of DNA to RNA, for example, by activating or repressing transcription. Some transcription factors regulate transcription only, while others act in concert with other proteins. Some transcription factors are capable of both activating and repressing transcription under certain conditions. Generally, transcription factors bind to one or more specific target sequences that closely resemble particular consensus sequences in the regulatory regions of target genes. Transcription factors can regulate the transcription of target genes alone or in complexes with other molecules.

[0465] Treatment: As used herein, the term "treatment" means to partially or completely alleviate, ameliorate, improve, or relieve a particular infection, disease, disorder, and / or condition, delay its onset, inhibit its progression, reduce its severity, and / or reduce the appearance of one or more symptoms or characteristics thereof. For example, "treating" cancer can mean inhibiting the survival, growth, and / or spread of a tumor. Treatment can be administered to subjects who do not exhibit signs of the disease, disorder, and / or condition, and / or to subjects who exhibit early signs of the disease, disorder, and / or condition, with the intent of reducing the risk of developing pathologies associated with the disease, disorder, and / or condition.

[0466] The phrase "treatment method" or its equivalents, when applied to, for example, cancer, refers to a procedure or course of action designed to reduce, eliminate, or prevent the number of cancer cells or alleviate the symptoms of cancer in an individual. A "treatment method" of cancer or other proliferative disorder does not necessarily mean that the cancer cells or other disorder are effectively completely eliminated, that the number of cells or disorder is effectively reduced, or that the symptoms of the cancer or other disorder are effectively alleviated. In many cases, a treatment method for cancer will be undertaken even if the likelihood of success is low, but is nevertheless considered an overall beneficial course of action taking into account the individual's medical history and estimated life expectancy.

[0467] Tumor growth: As used herein, the term "tumor growth" or "tumor metastasis growth" is used as commonly used in oncology unless otherwise specified, except that the term primarily relates to the increased mass or volume of a tumor or tumor metastasis as a result of tumor cell growth.

[0468] Tumor burden: As used herein, the term "tumor burden" means the total tumor volume of all tumor nodules greater than 3 mm in diameter that a subject has. Tumor volume: As used herein, the term "tumor volume" refers to the size of a tumor. 3The tumor volume in units is calculated using the formula: volume = (width) 2 It is calculated by × length / 2.

[0469] Unmodified: As used herein, "unmodified" refers to any substance, compound, or molecule before it has been altered in any way. Unmodified refers to the wild-type or unmodified form of a biomolecule, but this is not always the case. A molecule can undergo a series of modifications, whereby each modified molecule can serve as the "unmodified" starting molecule for subsequent modifications.

[0470] Equivalents and Scope Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. The scope of the invention is not limited to the foregoing description, but is instead set forth in the appended claims.

[0471] In the claims, articles such as "a," "an," and "the" may mean one or more unless indicated to the contrary or otherwise apparent from the context. A claim or description including "or" between one or more members of a group is deemed satisfied if one, more than one, or all of the group members are present in, utilized in, or otherwise adapted to a given product or process, unless indicated to the contrary or otherwise apparent from the context. The invention includes embodiments in which exactly one member of a group is present in, utilized in, or otherwise adapted to a given product or process. The invention includes embodiments in which more than one or all of the group members are present in, utilized in, or otherwise adapted to a given product or process.

[0472] It should also be noted that the term "comprising" is intended to be open, allowing for the incorporation of additional elements or steps. When ranges are given, the endpoints are included. Furthermore, unless otherwise specified, or otherwise apparent from the context and the understanding of one of ordinary skill in the art, values ​​expressed as ranges should be understood to be able to assume any particular value or subrange within the stated range in various embodiments of the invention, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.

[0473] In addition, it should be understood that any specific embodiment of the present invention that is encompassed by the prior art may be explicitly excluded from any one or more of the claims. Such embodiments may be excluded even if the exclusion is not expressly stated herein because they are deemed to be known to those of skill in the art. Any specific embodiment of the compositions of the present invention (e.g., any nucleic acid or protein encoded thereby, any method of production, any method of use, etc.) may be excluded from any one or more of the claims for any reason, regardless of the existence of prior art.

[0474] All cited sources, such as references, publications, databases, database entries, and techniques cited herein, are incorporated by reference, even if not expressly stated in the citation. In the event of a conflict between the contents of a cited source and the contents of this application, the contents of this application shall control.

[0475] The present invention is further illustrated by the following non-limiting examples. [Example]

[0476] Example 1: Synthesis of GalNAc Monomer (3aS,5R,6R,7R,7aR)-5-(acetoxymethyl)-2-methyl-3a,6,7,7a-tetrahydro-5H-pyrano[3,2-d]oxazole-6,7-diyl diacetate 2 To a stirred suspension of GalNAc (50 g, 129 mmol) in dichloromethane (580 mL) at room temperature, trimethylsilyl trifluoromethanesulfonate (47 mL, 316 mmol, 2.46 equiv.) is added and the reaction mixture is heated to reflux. The reaction is stirred for 24 h and then cooled to 0 °C. The reaction is quenched with triethylamine, washed with saturated aqueous NaHCO3, dried over Na2SO4, filtered, and concentrated in vacuo to give 2 as a crude brown gum that is used directly in the next step.

[0477] (2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-(allyloxy)tetrahydro-2H-pyran-3,4-diyl diacetate 3 A solution of 2 (42 g, 128 mmol) in dichloromethane (1000 mL) was stirred over activated 4A molecular sieves (160 g) at room temperature, and allyl alcohol (9.6 mL, 141 mmol, 1.1 equiv.) was added. The reaction mixture was stirred for 30 min, followed by the addition of trimethylsilyl trifluoromethanesulfonate (20.5 mL, 138 mmol, 1.0 equiv.). The reaction mixture was stirred for an additional 3 h 15 min, then filtered through Celite and washed with saturated aqueous NaHCO3. The mixture was dried over Na2SO4, filtered, and concentrated in vacuo. The crude product was recrystallized from ethyl acetate / diethyl ether, then ethyl acetate, washed with ethyl acetate (×4), diethyl ether (×2), and dried under high vacuum to give 3 as a brown solid in 35% yield from GalNAc.

[0478] 2-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)acetic acid 4 To a stirred solution of 3 (19.2 g, 49.6 mmol) in 1:1 dichloromethane / acetonitrile (192 mL) at room temperature was added sodium periodate (40.3 g, 189 mmol, 3.8 equiv.) and water (45 mL). The mixture was cooled to 5 °C, and ruthenium chloride (1.03 g, 4.96 mmol, 0.1 equiv.) was added in one portion. The reaction was allowed to warm to room temperature and stirred for 16 h. The organic solvent was removed in vacuo, and the aqueous phase was extracted with dichloromethane (×9). The organic phases were combined, dried over Na2SO4, filtered, and concentrated in vacuo. The crude product was recrystallized from ethyl acetate, washed with ethyl acetate (×2), diethyl ether (×2), and dried under high vacuum to give 4 as an off-white solid in 70% yield.

[0479] (2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-(2-((6-(((2R,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-hydroxytetrahydrofuran-2-yl)oxy)hexyl)amino)-2-oxoethoxy)tetrahydro-2H-pyran-3,4-diyl diacetate 6 To a stirred suspension of 4 (4.65 g, 11.5 mmol) and 5 (6.15 g, 11.5 mmol) in tetrahydrofuran (100 mL) at room temperature, hydroxybenzotriazole (1.86 g, 13.8 mmol, 1.2 equiv.) was added, followed by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (2.65 g, 13.8 mmol, 1.2 equiv.), and the reaction mixture was stirred for 16 h. The mixture was concentrated in vacuo, dissolved in ethyl acetate, washed with 10:3 water / brine, back-extracted with ethyl acetate, dried over Na2SO4, filtered, and concentrated in vacuo. The crude oil was purified by flash column chromatography (silica, dichloromethane / acetone gradient) to afford 6 as a yellow solid in 68% yield.

[0480] (2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-(2-((6-(((2R,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-(((2-cyanoethoxy)(diisopropylamino)phosphaneyl)oxy)tetrahydrofuran-2-yl)oxy)hexyl)amino)-2-oxoethoxy)tetrahydro-2H-pyran-3,4-diyl diacetate M1′ (where R1 = R2 = R3 = Ac and R4 = OCH2CH2CN, R5 = R6 = 2-propyl).

[0481] 6 (6.88 g, 7.38 mmol) was azeotroped with dichloromethane (×3) and then dissolved in dichloromethane (70 mL) and stirred at room temperature. To this mixture was added a solution of 2-cyanoethoxy-bis(N,N-diisopropylamino)phosphine (2.45 g, 8.12 mmol, 1.1 equiv.) in dichloromethane, followed by diisopropylammonium tetrazolide (0.63 g, 3.69 mmol, 0.5 equiv.), and the mixture was stirred at room temperature for 16 h. The reaction mixture was washed with water, then brine, dried over Na2SO4, filtered, and concentrated in vacuo. The crude oil was precipitated with pentane (×5) and then purified by flash column chromatography (silica, ethyl acetate) to give a yellow gum, which was dissolved in acetonitrile, filtered, and concentrated in vacuo to give 7 as a yellow solid in 63% yield.

[0482] Triethylammonium 4-(((2R,3S,5R)-5-((6-(2-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)acetamido)hexyl)oxy)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)tetrahydrofuran-3-yl)oxy)-4-oxobutanoate 8 To a stirred suspension of 6 (2 g, 2.17 mmol) in dichloromethane (6 mL) at room temperature, succinic anhydride (0.54 g, 5.42 mmol, 2.5 equiv.) and triethylamine (0.76 mL, 5.42 mmol, 2.5 equiv.) were added, and the mixture was stirred for 16 h at room temperature. The mixture was diluted with dichloromethane and washed with saturated aqueous NaHCO3, then with brine. The aqueous phases were combined, back-extracted with dichloromethane, dried over NaSO4, filtered, and concentrated in vacuo. The crude product was purified by flash column chromatography (silica, dichloromethane / methanol gradient) to give 8 in 29% yield.

[0483] β-dR-GalNAc-succinyl-LCAA-CPG(1000Å)M4′ (where R1 = R2 = R3 = Ac, L1 = succinyl, and the support is 1000Å LCAA-CPG) To a stirred suspension of 8 (2.38 g, 2.11 mmol) in 2% triethylamine / dichloromethane (8 mL) was added 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethylaminium tetrafluoroborate (1.02 g, 3.18 mmol, 1.5 equiv.), and the mixture was stirred at room temperature for 15 min. The reaction mixture was added to pre-washed Amino SynBase™ LCAA CPG 1000 / 100 (49 g) and mixed by bubbling with a stream of nitrogen for 2 h. The CPG was filtered, washed with dichloromethane (×3), and then suspended in a solution of dimethylaminopyridine (0.25 g) and acetic anhydride (3.8 mL) in pyridine (150 mL). The mixture is allowed to stand for 30 minutes with occasional gentle stirring, then filtered, washed with methanol (x3), dichloromethane (x3) and diethyl ether (x3) and air dried to give a free-flowing white solid.

[0484] Example 2: Synthesis of saRNA-GalNAc conjugate The monomeric GalNAc building block is compatible with standard oligonucleotide synthesis via the phosphoramidite method. Solid supports functionalized with phosphoramidites are used during the synthesis process. GalNAc phosphoramidites can be added to any position of the oligonucleotide, either alone or in combination with other GalNAc monomers. They can be added sequentially without a spacer or linker, or separated by nucleotides, spacers, or linkers. GalNAc solid supports are used to incorporate GalNAc modifications at the 3' end of the oligonucleotide.

[0485] The saRNA-GalNAc conjugate was prepared using typical oligonucleotide synthesis, deprotection, purification, and annealing protocols for this type of modified oligonucleotide.

[0486] Example 3: In vitro and in vivo studies using CEBPA-saRNA-GalNAc conjugates The 24 GalNAc-CEBPA-saRNA conjugates in Table 5 were synthesized and tested for activity in vitro in primary hepatocytes by passive transfection against a previously described fully modified GalNAc-C6-CEBPA saRNA conjugate (Example 2 of PCT / EP2018 / 074211, filed September 7, 2018), which contains the C6-GalNAc structure described herein. All novel designs resulted in equivalent or greater upregulation of CEBPA and albumin mRNA than GalNAc-C6-CEBPA by passive transfection in primary rat hepatocytes at 500 nM (Figures 1 and 2) and 1 μM (Figures 3 and 4).

[0487] L1:

[0488] [ka]

[0489] L2:

[0490]

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[0491] L3:

[0492]

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[0493] L4:

[0494]

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[0495] L5:

[0496]

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[0497] L6:

[0498]

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[0499] L14:

[0500]

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[0501] L15:

[0502]

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[0503] L16:

[0504]

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[0505] L17:

[0506]

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[0507] L18:

[0508]

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[0509] L19:

[0510]

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[0511] L40:

[0512]

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[0513] L41:

[0514]

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[0515] L42:

[0516]

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[0517] L43:

[0518]

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[0519] L44:

[0520]

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[0521] L45:

[0522]

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[0523] L53:

[0524]

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[0525] L54:

[0526]

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[0527] L55:

[0528]

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[0529] L56:

[0530]

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[0531] L57:

[0532]

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[0533] L58:

[0534] [ka]

[0535] Following in vitro experiments, the most promising compounds were administered in vivo to normal mice. Conjugates L1, L2, L3, L4, L5, L16, L40, L41, L42, L43, and L55 were injected intravenously (IV) at 30 mg / kg on days 1 and 3, and livers were harvested on day 5 to examine upregulation of CEBPA mRNA (Figure 5). Only L55 showed upregulation of CEBPA in the liver, whereas the GalNAc-C6-CEBPA conjugate showed no effect with this administration method. Unexpectedly, L1 showed downregulation of CEBPA mRNA.

[0536] L14, L53, and L54 were then intravenously injected according to the same protocol as in the previous experiment (Figures 6 and 7). The original GalNAc-C6-CEBPA conjugate showed significant upregulation of CEBPA mRNA, while L53 showed significant upregulation of both CEBPA and albumin mRNA. In this experiment, L53 was more effective than GalNAc-C6-CEBPA.

[0537] Finally, L6, L18, L19, L56, L57, and L58 were injected subcutaneously (SC) at 30 mg / kg into normal mice on days 1 and 3, and livers were harvested on day 5. None of the conjugates tested showed upregulation in these conditions.

[0538] In further studies, L1, L2, L3, L16, L40, L41, L42, and L55 were synthesized and injected subcutaneously (SC). Normal mice were injected SC with 30 mg / kg of GalNAc saRNA conjugates on days 1 and 3, and livers were harvested on day 5. As shown in Figure 8, L55 showed significantly greater upregulation of CEBPA mRNA than the original GalNAc-C6-CEBPA conjugate, even with SC administration.

[0539] In a further study, CEBPa-saRNA-GalNAc conjugates L80 (XD-14369K1 conjugated to GalNAc cluster G7) and L81 (XD-14369K1 conjugated to GalNAc cluster G8) were administered to cells at various doses up to 1000 nM. CEBPA mRNA levels were measured. Figure 9 shows the in vitro dose response of L80 and L81.

[0540] Example 4: In vitro testing of C5-siRNA-GalNAc conjugates In this in vitro study, siRNA targeting the complement C5 gene (C5-siRNA) was conjugated to a GalNAc cluster. C5-siRNA was delivered to cells by passive transfection, and C5 mRNA levels were subsequently measured. The sequence of the siRNA was as follows:

[0541] [Table 7]

[0542] The C5-siRNA-GalNAc conjugates tested in this study were: 1. C5-siRNA-C6-GalNAc having the following structure (GalNAc-C6-siC5 in Figure 10)

[0543] [ka]

[0544] 2. C5-siRNA-G7 (GalNAc-53-siC5 in Figure 10) having the following structure:

[0545] [ka]

[0546] and 3. C5-siRNA-G9 having the following structure (GalNAc-55-siC5 in Figure 10)

[0547] [ka]

[0548] Includes: GalNAc-C6-siC5, GalNAc-53-siC5, GalNAc-55-siC5, and controls (GalNAc-C6-FLUC, GalNAc-53-FLUC, GalNAc-55-FLUC) were administered to primary rat hepatocytes at doses ranging from 0.3125 nM to 20 nM. Intracellular C5 mRNA levels were then measured by qPCR. As shown in Figure 10, C5-siRNA conjugated to GalNAc cluster G7 (GalNAc-53-siC5), C5-siRNA conjugated to GalNAc cluster G9 (GalNAc-55-siC5), and GalNAc-C6-siC5 all reduced C5 mRNA levels.

[0549] Other embodiments While the present disclosure has been described in conjunction with its detailed description, it is to be understood that the foregoing description is intended to be illustrative and not limiting of the scope of the disclosure, which is defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims. (Addendum) The technical ideas that can be understood from the above-described embodiment and modified examples will be described. [Item 1] N-acetyl-galactosamine (GalNAc) monomers,

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Claims

1. N-acetyl-galactosamine (GalNAc) monomers, such as: 【Chemistry 1】 wherein R1, R2, and R3 may be the same or different, and R1, R2, and R3 are independently selected from alkyl, aryl, and alkenyl groups; R4 is a suitable protecting group or a C1-6 linear or branched alkyl group; R5 and R6 are each independently a C1-6 linear or branched alkyl group; and R7 is a suitable protecting group; 【Chemistry 2】 wherein R1, R2, and R3 may be the same or different, and R1, R2, and R3 are independently selected from alkyl, aryl, and alkenyl groups; R4 is a protecting group or a C1-6 linear or branched alkyl group; R5 and R6 are each independently a C1-6 straight or branched alkyl; and R7 is a suitable protecting group; 【Transformation 3】 wherein R1, R2, and R3 may be the same or different, and R1, R2, and R3 are independently selected from alkyl, aryl, and alkenyl groups; R7 is a suitable protecting group, and Linker 1 is a cleavable linker; and 【Chemistry 4】 wherein R1, R2, and R3 may be the same or different, and R1, R2, and R3 are independently selected from the group consisting of alkyl groups, aryl groups, and alkenyl groups; R7 is a suitable protecting group, and Linker 1 is a cleavable linker; An N-acetyl-galactosamine (GalNAc) monomer comprising a structure selected from the group consisting of:

2. 1. A compound or conjugate comprising a GalNAc moiety comprising at least one GalNAc monomer, wherein said GalNAc monomer is: 【Transformation 5】 Here, R ８ is —H or a C1-6 straight or branched alkyl group; 【Transformation 6】 Here, R ８ is —H or a C1-6 straight or branched alkyl group, and X is O or S; 【Transformation 7】 and 【Transformation 8】 A compound or conjugate selected from the group consisting of:

3. the GalNAc moiety comprises a spacer; The spacer is HEG; 【Chemistry 8-1】 where X is O or S; 【Chemistry 8-2】 where X is O or S; TEG; or 【Chemistry 8-3】 where X is O or S; 3. The compound or conjugate of claim 2, comprising the structure:

4. The compound or conjugate of claim 2, wherein the GalNAc moiety comprises two or three GalNAc monomers.

5. The compound or conjugate of claim 2, wherein the GalNAc moiety comprises three M1 or M2 monomers.

6. The compound or conjugate of claim 5, wherein the GalNAc moiety comprises three M1 monomers.

7. The GalNAc cluster is: 【Chemistry 9-1】 【Chemistry 9-2】 【Chemistry 9-3】 【Chemistry 9-4】 【Chemistry 9-5】 【Chemistry 9-6】 【Chemistry 9-7】 【Chemistry 9-8】 【Chemistry 9-9】 [Chemistry 9-10] 【Chemistry 9-11】 【Chemistry 9-12】 【Chemistry 9-13】 [Chemistry 9-14] 【Chemistry 9-15】 【Chemistry 9-16】 3. The compound or conjugate of claim 2, comprising the structure:

8. A method for preparing a compound or conjugate comprising a GalNAc moiety, comprising: The following steps: 1) Providing at least one GalNAc monomer according to claim 1; and 2). Synthesizing the GalNAc moiety from the GalNAc monomer in step 1); optionally adding at least one spacer, and optionally removing a protecting group. A preparation method comprising:

9. The conjugate further comprising an oligonucleotide, The conjugate of any one of claims 2 to 7, wherein the oligonucleotide regulates expression of a target gene, and the oligonucleotide and the GalNAc moiety are connected by a bond or a cleavable linker.

10. The conjugate of claim 9 , wherein the oligonucleotide and the GalNAc moiety are connected by a cleavable linker.

11. The conjugate of claim 10, wherein the linker is C6ssC6 or dT.

12. 10. The conjugate of claim 9, wherein the oligonucleotide and the GalNAc moiety are connected by a bond, the bond being a phosphodiester bond or a phosphorothioate bond. 【Request Item 13】 【Chemistry 10-1】 【Chemistry 10-2】 【Chemistry 10-3】 [Chemistry 10-4] 【Transformation 10-5】 【Chemistry 10-6】 【Chemistry 10-7】 [Transformation 10-8] 【Chemistry 10-9】 【Chemistry 10-10】 【Chemistry 10-11】 [Chemistry 10-12] [Chemistry 10-13] [Chemistry 10-14] [Chemistry 10-15] [Chemistry 10-16] or 【Chemistry 10-17】 The structure of L is an optional linker; 10. The conjugate of claim 9, wherein Nuc is a nucleotide or an oligonucleotide.

14. The conjugate of any one of claims 9 to 13, wherein the oligonucleotide is an isolated synthetic small activating RNA (saRNA).

15. The conjugate of claim 14, wherein the target gene is CEBPA.

16. The conjugate of claim 14, wherein the saRNA is a double-stranded saRNA.

17. 17. The conjugate of claim 16, wherein the GalNAc cluster is attached to the 5' or 3' end of the sense strand.

18. 17. The conjugate of claim 16, wherein the double-stranded saRNA is selected from the group consisting of XD-03302 (SEQ ID NOs: 2 and 3), S1 (XD-06409) (SEQ ID NOs: 4 and 5), S2 (XD-06410) (SEQ ID NOs: 6 and 7), S3 (XD-06411) (SEQ ID NOs: 8 and 9), S4 (XD-06412) (SEQ ID NOs: 10 and 11), S5 (XD-06413) (SEQ ID NOs: 12 and 13), S6 (XD-06414) (SEQ ID NOs: 14 and 15), S7 (XD-06415) (SEQ ID NOs: 16 and 17), S8 (SEQ ID NOs: 18 and 19), XD-07139 (SEQ ID NOs: 20 and 21), XD-03934 (SEQ ID NOs: 22 and 23), and XD-14369K1 (SEQ ID NOs: 24 and 25).

19. 17. The conjugate of claim 16, wherein the saRNA is XD-06414 having an antisense strand comprising SEQ ID NO: 15 and a sense strand comprising SEQ ID NO:

14.

20. The conjugate may comprise: L14: 【Chemistry 11】 L15: 【Chemistry 12】 L16: 【Chemistry 13】 L17: 【Chemistry 14】 L18: 【Chemistry 15】 L19: 【Chemistry 16】 L53: 【Chemistry 17】 L54: [Chemistry 18] L55: 【Chemistry 19】 L56: 【Chemistry 20】 L57: 【Chemistry 21】 and L58: 【Chemistry 22】 20. The conjugate of claim 19, selected from the group consisting of:

21. 20. The conjugate of claim 19, which is L53.

22. 20. The conjugate of claim 19, which is L55.

23. The conjugate of any one of claims 9 to 13, wherein the oligonucleotide is an isolated synthetic small inhibitory RNA (siRNA).

24. 24. The conjugate of claim 23, wherein the siRNA is a double-stranded siRNA.

25. 25. The conjugate of claim 24, wherein the GalNAc cluster is attached to the 5' or 3' end of the sense strand.

26. A pharmaceutical composition comprising the conjugate of any one of claims 9 to 25 and at least one pharmaceutically acceptable excipient.

27. 27. The pharmaceutical composition of claim 26, wherein the conjugate comprises saRNA.

28. 27. The pharmaceutical composition of claim 26, wherein the conjugate comprises an siRNA.

29. A composition comprising the conjugate of any one of claims 9 to 25 for delivering an oligonucleotide to a cell without the use of a transfection agent.

30. 30. The composition of claim 29, wherein the oligonucleotide comprises saRNA.

31. 30. The composition of claim 29, wherein the oligonucleotide comprises an siRNA.

32. A composition comprising a conjugate described in any one of claims 9 to 25 for regulating expression of a target gene in a patient in need of such regulation.

33. 33. The composition of claim 32, wherein the conjugate comprises saRNA.

34. 33. The composition of claim 32, wherein expression of the target gene is increased.

35. 34. The composition of claim 33, wherein the target gene is CEBPA.

36. The composition of claim 35, wherein expression of albumin is increased in the patient.

37. 33. The composition of claim 32, wherein the conjugate comprises an siRNA.

38. 33. The composition of claim 32, wherein expression of the target gene is reduced.

39. 39. The composition of claim 38, wherein the target gene is C5.

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