Multivalent oligonucleotide drugs and methods of use thereof

Abstract

Provided herein are multivalent oligonucleotide agents comprising two or more functional oligonucleotide units independently selected from the group consisting of single-stranded antisense oligonucleotides (ASOs, e.g., gapmers, mixmers, and stereoblock ASOs) and double-stranded (double-stranded) RNA (dsRNA, e.g., siRNA and saRNA), and methods for their preparation. Also provided herein are products comprising the multivalent oligonucleotide agents, and methods of using the multivalent oligonucleotide agents or products to treat diseases, e.g., spinal muscular atrophy (SMA) and cancer.

Description

[Technical field]

[0001] [1] The present disclosure relates to multivalent oligonucleotide agents comprising two or more functional oligonucleotides, which may be selected from single-stranded antisense oligonucleotides (ASOs, such as gapmers and mixmers) and double-stranded (double-stranded) RNA (dsRNA, such as siRNA and saRNA). In some embodiments, the functional oligonucleotides in the multivalent oligonucleotide agent are the same or different, have different targets or the same target, and / or are connected directly or via a linker. In some embodiments, the multivalent oligonucleotide agent is a multitargeted oligonucleotide agent and / or has improved activity. The present disclosure also relates to products, such as compositions and pharmaceuticals, that comprise the multivalent oligonucleotide agent, as well as methods of using the multivalent oligonucleotide agent or product in the treatment of disease. [Background technology]

[0002] [3] Oligonucleotides are a novel class of therapeutic agents currently being developed to treat a variety of diseases with a variety of different mechanisms of action (MOAs). The major categories of oligonucleotide therapeutic agents include single-stranded antisense oligonucleotides (ASOs) and double-stranded (double-stranded) RNA (dsRNA).

[0003] [4] Single-stranded ASOs in the form of "gapmers" can silence gene expression by degrading complementary mRNAs by RNase H hydrolysis. A distinctive "gapmer" ASO has a central DNA region required for RNase H activity and two ribonucleotide wings to enhance target binding affinity. Another category of ASOs, called "mixmers," function as steric blockers, typically composed entirely of ribonucleotide analogs, and bind to pre-mRNA in the nucleus to alter splicing.

[0004] [5] Double-stranded RNAs are divided into two categories: small interfering RNA (siRNA) and small activating RNA (saRNA), both of which require Argonaute (AGO) proteins for function. Small interfering RNAs bind to target mRNAs in the cell's cytoplasm and downregulate gene expression through a post-transcriptional gene silencing mechanism called RNA interference (RNAi). Small activating RNAs have the opposite function, targeting regulatory sequences (i.e. gene promoters) in the cell nucleus and upregulating gene expression through a transcriptional gene activation mechanism called RNAa (RNA activation). [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Coovert, DD, TT Le, et al. (1997). "The survival motor neuron protein in spinal muscular atrophy." Human molecular genetics 6(8): 1205-1214. [Non-Patent Document 2] Crooke, ST (1991). "Antisense technology." Current opinion in biotechnology 2(2): 282-287. [Non-Patent Document 3] Crooke, ST, JL Witztum, et al. (2018). "RNA-Targeted Therapeutics." Cell metabolism 27(4): 714-739. [Non-Patent Document 4] Fire, A., S. Xu, et al. (1998). "Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans." Nature 391(6669): 806-811. [Non-Patent Document 5] Horrobin, DF (2001). "Realism in drug discovery-could Cassandra be right?" Nature biotechnology 19(12): 1099-1100. [Non-Patent Document 6] Hsieh-Li, HM, JG Chang, et al. (2000). "A mouse model for spinal muscular atrophy." Nature genetics 24(1): 66-70. [Non-Patent Document 7] Hua, Y., TA Vickers, et al. (2007). "Enhancement of SMN2 exon 7 inclusion by antisense oligonucleotides targeting the exon." PLoS biology 5(4): e73. [Non-Patent Document 8] Hua, Y., TA Vickers, et al. (2008). "Antisense masking of an hnRNP A1 / A2 intronic splicing silencer corrects SMN2 splicing in transgenic mice." American journal of human genetics 82(4): 834-848. [Non-Patent Document 9] Janowski, B. A., S. T. Younger, et al. (2007). "Activating gene expression in mammalian cells with promoter-targeted duplex RNAs." Nature chemical biology 3(3): 166-173.

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[0006] [6] Many monogenic diseases develop due to insufficient gene expression levels and / or activity of translated proteins, resulting from reduced gene function. “Mixmer” ASOs have been found to be successful in treating several diseases that can be improved by correcting abnormal splicing errors. Several ASO drugs have been approved by the US Food and Drug Administration (FDA), including an exon-embedding ASO called SPINRAZA® (nusinersen) for the treatment of spinal muscular atrophy (SMA) and an exon-exclusion ASO marketed under the name AMONDYS 45 (casimersen) for the treatment of Duchenne muscular dystrophy (DMD). Unfortunately, however, the efficacy of the drug is limited by the physical amount of the target transcript expressed in the patient's cells. Therefore, not all patients respond to the treatment in the same way. Epigenetic modifiers (e.g., HDAC inhibitors) have been tested in SMA models as a means to further increase gene dosage by nonspecifically enhancing global gene transcription to provide Spinraza with a larger pool of target transcripts [Pagliarini,2020#2082]. However, aberrant activation of the transcriptome comes with its own side effects, counteracting the gene-specific precision of oligonucleotide targeted therapy required for monogenic diseases. Our previous work demonstrated an alternative approach by using saRNA to specifically activate transcription of target genes in combination with Spinraza ASO. Treatment with saRNA selectively enhanced the transcriptional output of the target gene SMN2, while Spinraza altered pre-mRNA splicing of SMN2 to produce full-length SMN2 transcripts (SMN2FL) and its cognate protein at levels not achieved by either treatment alone.

[0007] [7] RNA duplexes (e.g., saRNA) are generally larger in mass and structurally more rigid than ASOs, resulting in disparities in their inherent biophysical properties [Crooke, 2017#23; Shen, 2018#40]. Therefore, dsRNA has unique biodistribution and tissue diffusion patterns compared to single-stranded oligonucleotides. Co-therapy with ASOs and saRNA requires that both drug molecules are concentrated in the same intended target tissue / cell in vitro to function cooperatively against a single gene target. Technologies that can guide both drug modalities to the intended target tissue and provide similar distribution properties can enhance the therapeutic efficacy of such combination therapies.

[0008] [8] Therefore, improved oligonucleotide-based therapeutics are needed to address these challenges. [Means for solving the problem]

[0009] [9] Double-stranded RNA (dsRNA) targeting gene regulatory sequences, including promoters, has been shown to upregulate transcription of target genes in a sequence-specific manner through a mechanism known as RNA activation (RNAa) (Li, LC, et al., Small dsRNAs induce transcriptional activation in human cells. PNAS (2006)). Our previous work demonstrated that co-treatment with ASO splicing modulators can further increase gene dosage of specific splicing variants at levels not achieved by either treatment alone. However, double-stranded RNA (e.g., saRNA) and single-stranded ASOs have different biophysical properties and unique patterns of tissue biodistribution, diffusion, and cellular uptake. Typically, ASOs diffuse throughout organ tissues depending on the route of administration. Cellular uptake and ASO activity can be easily measured without the need for adjuvant delivery vehicles, carrier systems, or targeting complexes. On the other hand, double-stranded RNA (e.g., saRNA, siRNA, and miRNA) do not easily penetrate solid tissues or cross cell membranes. In vivo activity usually requires a delivery vehicle, a targeting conjugate, and / or a novel chemical entity. For saRNA and ASO co-treatment to exert a combined therapeutic effect in vivo, both drugs need to have access to the same tissues and / or have similar biodistribution requirements.

[0010]

[10] To overcome the inherent differences in tissue distribution properties, the inventors developed novel chemical constructs that covalently link ASO splicing modulators (e.g., Spinraza) and saRNA in one molecule. The biophysical properties are shared between the different drug modalities, providing uniform biodistribution and broad activity in different tissues. This chemical strategy was further expanded and tested with different oligonucleotide drug combinations, including saRNA-saRNA, saRNA-siRNA, saRNA-saRNA-ASO, ASO-ASO, etc. These novel constructs collectively constitute a group of multifaceted oligonucleotides called multivalent oligonucleotides (MVOs).

[0011]

[11] Embodiments of the present disclosure are based in part on the surprising discovery that two or more functional oligonucleotides, when covalently linked, can upregulate and / or downregulate expression of one or more genes of interest, for example, for treating a disease or condition associated with the one or more genes of interest.

[0012]

[12] In some embodiments, provided herein are multivalent oligonucleotide (MVO) agents comprising two or more covalently linked functional oligonucleotides, wherein the two or more functional oligonucleotides are independently selected from the group consisting of: a) double-stranded RNA (dsRNA); and b) antisense oligonucleotides (ASO).

[0013]

[13] In some embodiments, the MVO agent increases expression of the SMN2 gene or protein. In some MVO agent embodiments, the dsRNA increases expression of the SMN2 gene or protein; and / or the ASO increases production of functional SMN protein by modulating splicing or stability of SMN2 mRNA (SMN2 mRNA modulator).

[0014]

[14] In some embodiments, the MVO agent increases expression of the CDKN1A / p21 gene or protein and / or decreases expression of CD274 / PDL-1. In some embodiments of the MVO agent, the dsRNA is independently selected from the group consisting of: a saRNA that increases expression of the CDKN1A / p21 gene or protein; and a siRNA that decreases expression of CD274 / PDL-1.

[0015]

[15] In some aspects, provided herein is an article of manufacture comprising a multivalent oligonucleotide agent. In some embodiments, the article of manufacture is selected from a pharmaceutical composition or a kit further comprising at least one pharma- ceutically acceptable carrier.

[0016]

[16] In some embodiments, provided herein is a method of disease treatment comprising administering to a subject in need of such treatment a sufficient amount of one or more of the multivalent oligonucleotide agents or products described herein. For example, the method treats or delays the onset or progression of an SMN deficiency-associated condition or a p21 / PDL-1-associated disease in a subject. Also provided herein is the use of one or more of the multivalent oligonucleotide agents or products described herein in the preparation of a product for disease treatment. Also provided herein is one or more of the multivalent oligonucleotide agents or products described herein for disease treatment.

[0017]

[17] In some embodiments, provided herein are methods of preparing a multivalent oligonucleotide agent described herein, comprising providing two or more functional oligonucleotides and covalently linking them; or synthesizing a full-length oligonucleotide agent.

[0018]

[18] Also provided herein is an isolated or synthesized oligonucleotide comprising a nucleotide sequence of a saRNA sense strand having at least 90% identity to the nucleotide sequence of SEQ ID NO:62. In some embodiments, the oligonucleotide further comprises an antisense strand having partial complementarity to the sense saRNA strand. In some embodiments, the oligonucleotide further comprises an antisense strand having at least 90% identity to the nucleotide sequence of SEQ ID NO:63. In some embodiments, the isolated or synthesized oligonucleotide comprises a nucleotide sequence of a saRNA sense strand of SEQ ID NO:62 and a saRNA antisense strand of SEQ ID NO:63. Also provided herein is a pharmaceutical composition or kit comprising an isolated or synthesized oligonucleotide as described herein. Also provided herein is a method of treating a disease comprising administering to a subject in need of such treatment a sufficient amount of one or more of the isolated or synthesized oligonucleotides or pharmaceutical compositions or kits as described herein. Also provided herein is the use of one or more of the isolated or synthesized oligonucleotides or pharmaceutical compositions or kits as described herein in the preparation of a product for treating a disease. Also provided herein is one or more of the isolated or synthesized oligonucleotides or pharmaceutical compositions or kits described herein for treating a disease.

[0019]

[19] Some aspects and embodiments of the present disclosure are as follows: 1. A multivalent oligonucleotide agent comprising two or more covalently linked functional oligonucleotides, wherein said two or more functional oligonucleotides are independently selected from the group consisting of: a) double-stranded RNA (dsRNA); and b) antisense oligonucleotides (ASOs); Multivalent oligonucleotide drugs.

[0020] 2. The multivalent oligonucleotide drug according to item 1, wherein the number of functional oligonucleotides contained in the multivalent oligonucleotide drug is in the range of 2 to X, where X is an integer in the range of 3 to 10.

[0021] 3. The multivalent oligonucleotide agent according to item 2, wherein the number of dsRNAs contained in the agent is 0 to X, and the remaining functional oligonucleotides are ASOs. 4. The multivalent oligonucleotide agent according to item 1, wherein the dsRNA is independently selected from small interfering RNA (siRNA) and small activating RNA (saRNA); and / or the ASO is independently selected from gapmers and mixmers.

[0022] 5. A multivalent oligonucleotide agent according to item 1, wherein the two or more functional oligonucleotides independently regulate the expression of one or more genes, regulate the expression of one or more proteins (e.g., by binding to an mRNA sequence), or regulate a non-coding regulatory nucleic acid sequence.

[0023] 6. The multivalent oligonucleotide agent according to item 5, wherein the non-coding regulatory nucleic acid sequence is a promoter sequence, an enhancer, a silencer, and / or a transcription factor. 7. The multivalent oligonucleotide agent of item 1, wherein each dsRNA comprises a sense strand that is at least 10 contiguous nucleotides and an antisense strand that is at least 10 contiguous nucleotides.

[0024] 8. The multivalent oligonucleotide agent according to item 1, wherein each dsRNA comprises a sense strand having a length of 10 to 60 nucleotides and / or each dsRNA comprises an antisense strand having a length of 10 to 60 nucleotides.

[0025] 9. The multivalent oligonucleotide agent of item 1, wherein each ASO has a nucleotide sequence that is at least 5 contiguous nucleotides in length. 10. The multivalent oligonucleotide agent of item 1, wherein each ASO has a nucleotide sequence between 5 and 30 nucleotides in length.

[0026] 11. The multivalent oligonucleotide agent according to item 1, wherein the two or more functional oligonucleotides have a total length in the range of 12 to 200 nucleotides. 12. The multivalent oligonucleotide agent according to any one of items 1 to 10, wherein any two adjacent functional oligonucleotides of the two or more functional oligonucleotides are covalently linked, either via a linking moiety or without a linking moiety.

[0027] 13. The multivalent oligonucleotide drug according to claim 12, wherein the linking moiety is selected from the following linkers or derivatives thereof:

[0028] [Table 3]

[0029] 14. The multivalent oligonucleotide agent according to item 12, wherein the two adjacent functional oligonucleotides are covalently linked by:

[0030] [ka] , wherein R represents -H, or -OH, or -OMe, or -MOE, or -F, or other 2' chemical modification.

[0031] 15. The multivalent oligonucleotide agent according to item 12, wherein the two adjacent functional oligonucleotides are covalently linked by a phosphodiester or phosphorothioate bond.

[0032] 16. The multivalent oligonucleotide agent according to item 12, wherein the two adjacent functional oligonucleotides are covalently linked by one or more nucleotides. 17. A multivalent oligonucleotide agent according to any one of items 1 to 10, wherein the one or more functional oligonucleotides contain at least one chemically modified nucleotide.

[0033] 18. The multivalent oligonucleotide agent of item 17, wherein the chemical modification of at least one chemically modified nucleotide is one or more 2' sugar modifications selected from the group consisting of a 2'-fluoro-2'-deoxynucleoside (2'-F) modification, a 2'-O-methyl (2'-O-Me) modification, and a 2'-O-(2-methoxyethyl) (2'-O-MOE) modification.

[0034] 19. The multivalent oligonucleotide agent according to item 17, wherein the chemical modification of at least one chemically modified nucleotide is a phosphorothioate (PS) backbone modification. 20. The multivalent oligonucleotide agent according to item 17, wherein the chemical modification of at least one chemically modified nucleotide is the addition of a 5'-phosphate moiety to the 5' end of the nucleotide sequence.

[0035] 21. The multivalent oligonucleotide agent according to item 17, wherein the chemical modification of at least one chemically modified nucleotide is the addition of an (E)-vinylphosphonate moiety to the 5' end of the nucleotide sequence.

[0036] 22. The chemical modification of at least one chemically modified nucleotide is at the 5' end of the nucleotide sequence. 5-Methylcytosine 18. The multivalent oligonucleotide agent of item 17, which is the addition of a moiety.

[0037] 23. A multivalent oligonucleotide agent according to any one of items 1 to 22, wherein each ASO in the agent is covalently linked to an adjacent targeting oligonucleotide in the 3' to 5' or 5' to 3' direction.

[0038] 24. A multivalent oligonucleotide agent according to any one of items 1 to 22, wherein each dsRNA in the agent is covalently linked to an adjacent ASO at the 3' end of the sense or antisense strand, or at the 5' end of the sense or antisense strand.

[0039] 25. A multivalent oligonucleotide agent according to items 1 to 24, wherein the sequences of the two or more functional oligonucleotides are the same or different; and / or the functions of the two or more functional oligonucleotides are the same or different.

[0040] 26. The multivalent oligonucleotide agent comprises: a) a first double-stranded RNA (dsRNA) and a first antisense oligonucleotide (ASO); b) a first double-stranded RNA (dsRNA) and a second dsRNA; c) a first antisense oligonucleotide (ASO) and a second ASO; d) a first double-stranded RNA (dsRNA), a second dsRNA, and a third dsRNA; e) a first double-stranded RNA (dsRNA), a second dsRNA, and a first antisense oligonucleotide (ASO); f) a first double-stranded RNA (dsRNA), a first antisense oligonucleotide (ASO) and a second ASO; or g) a first antisense oligonucleotide (ASO), a second ASO and a third ASO; 26. The multivalent oligonucleotide agent according to items 1 to 25, wherein in any one of a) to g), the functional oligonucleotides are arranged in any order and are covalently linked with or without a linking moiety.

[0041] 27. The multivalent oligonucleotide agent according to item 23, wherein the first dsRNA, the second dsRNA and the third dsRNA, if present, are independently selected from siRNA and saRNA.

[0042] 28. The multivalent oligonucleotide agent of item 23, wherein the first ASO, the second ASO and the third ASO, if present, are independently selected from gapmers and mixmers.

[0043] 29. The multivalent oligonucleotide agent comprises a functional oligonucleotide selected from the group consisting of: a) siRNA-siRNA; b) siRNA-saRNA; c) saRNA-saRNA; d) siRNA-gapmer; e) siRNA-mixmer; f) saRNA-gapmer; g) saRNA-mixmer; h) gapmer-gapmer; i) gapmer-mixmer; j) mixmer-mixmer; k) siRNA-siRNA-siRNA; l) siRNA-siRNA-saRNA; m) siRNA-saRNA-saRNA; n) saRNA-saRNA-saRNA; o) siRNA-siRNA-gapmer; p) siRNA-siRNA-mixmer; q) siRNA-saRNA-gapmer; r) siRNA-saRNA-mixmer;s) saRNA-saRNA-gapmer;t) saRNA-saRNA-mixmer;u) siRNA-gapmer-gapmer;v) saRNA-gapmer-gapmer;w) siRNA-gapmer-mixmer;x) saRNA-gapmer-mixmer;y) siRNA-mixmer-mixmer;z) saRNA-mixmer-mixmer;aa) gapmer-gapmer-gapmer;ab) gapmer-gapmer-mixmer;ac) gapmer-mixmer-mixmer;and ad) mixmer-mixmer-mixmer; However, in any one of a) to ad), the functional oligonucleotides are arranged in any order and are covalently linked with or without a linking moiety; 24. The multivalent oligonucleotide agent of item 23.

[0044] 30. a) the ASO is covalently attached to the 3' end of the sense or antisense strand of the first dsRNA; or b) The multivalent oligonucleotide agent according to any one of items 26 to 29, wherein the ASO is covalently attached to the 5' end of the sense or antisense strand of the first dsRNA.

[0045] 31. A multivalent oligonucleotide agent described in item 27, wherein the 5' end of the ASO is conjugated to a linking moiety; alternatively, the 3' end of the ASO is conjugated to a linking moiety. 32. A multivalent oligonucleotide agent according to any one of items 1 to 31, comprising one or more additional targeting oligonucleotides.

[0046] 33. The multivalent oligonucleotide drug of item 32, wherein the additional targeting oligonucleotides are independently selected from the group consisting of double-stranded RNA (dsRNA) and antisense oligonucleotides (ASO).

[0047] 34. The multivalent oligonucleotide agent according to item 33, wherein the additional double-stranded RNA (dsRNA) is independently selected from small interfering RNA (siRNA) and small activating RNA (saRNA); and / or the targeting oligonucleotide is independently selected from gapmers and mixmers.

[0048] 35. A multivalent oligonucleotide agent according to any one of items 1 to 34, wherein one or more ASOs target the 5'-UTR. 36. A multivalent oligonucleotide agent according to any one of items 1 to 35, wherein the one or more functional oligonucleotides increase expression of the SMN2 gene or protein.

[0049] 37. A multivalent oligonucleotide agent according to item 36, wherein the dsRNA increases expression of the SMN2 gene or protein; and / or the ASO increases production of functional SMN protein by regulating splicing or stability of SMN2 mRNA (SMN2 mRNA modulator).

[0050] 38. The multivalent oligonucleotide agent according to item 36, wherein the dsRNA comprises a saRNA having a nucleotide sequence of a sense strand that has at least 90% identity to a nucleotide sequence selected from the group consisting of: a) DS06-0004 (SEQ ID NO:5); b) DS06-0031 (SEQ ID NO: 7); c) DS06-0067 (SEQ ID NO:9); d) DS06-4A3 (SEQ ID NO: 146); e) R6-04-S1 (SEQ ID NO:59); and f)R6-04(20)-S1V1v(CM-4)(SEQ ID NO:60).

[0051] 39. The multivalent oligonucleotide drug according to item 36, wherein the dsRNA comprises a saRNA having an antisense strand nucleotide sequence that has at least 90% identity to a nucleotide sequence selected from the group consisting of: a) DS06-0004 (SEQ ID NO:6); b) DS06-0031 (SEQ ID NO: 8); c) DS06-0067 (SEQ ID NO: 10); d) DS06-4A3 (SEQ ID NO: 147); e) R6-04-S1 (SEQ ID NO:53); and f)R6-04M1-27A-S1L1V3(CM-26)(SEQ ID NO:17).

[0052] 40. The multivalent oligonucleotide drug according to item 36, wherein the dsRNA comprises a saRNA having a nucleotide sequence pair of a sense strand and an antisense strand that has at least 90% identity to a nucleotide sequence pair selected from the group consisting of: a) DS06-0004: SEQ ID NO:5 and SEQ ID NO:6; b) DS06-0031: SEQ ID NO:7 and SEQ ID NO:8; c) DS06-0067: SEQ ID NO:9 and SEQ ID NO:10; d) DS06-4A3: SEQ ID NO:146 and SEQ ID NO:147; e) R6-04-S1: SEQ ID NO:59 and SEQ ID NO:53; and f) R6-04(20)-S1V1v(CM-4): SEQ ID NO:60 and SEQ ID NO:17.

[0053] 41. The multivalent oligonucleotide agent according to item 36, wherein the dsRNA comprises an siRNA having a nucleotide sequence of a sense strand that has at least 90% identity to the nucleotide sequence of DS06-332i (SEQ ID NO:3) or siSOD1-388-ESC (SEQ ID NO:138).

[0054] 42. The multivalent oligonucleotide agent according to item 36, wherein the dsRNA comprises an siRNA having a nucleotide sequence of the antisense strand that has at least 90% identity to the nucleotide sequence of DS06-332i (SEQ ID NO:4) or siSOD1-388-ESC (SEQ ID NO:139).

[0055] 43. The multivalent oligonucleotide drug according to item 36, wherein the dsRNA comprises an siRNA having a nucleotide sequence pair of a sense strand and an antisense strand that has at least 90% identity to a nucleotide sequence pair selected from the group consisting of: DS06-332i: SEQ ID NO:3 and SEQ ID NO:4; and siSOD1-388-ESC: SEQ ID NO:138 and SEQ ID NO:139.

[0056] 44. The multivalent oligonucleotide agent of item 36, wherein the ASO has a nucleotide sequence that is at least 90% identical to the nucleotide sequence of ASO10-27 (SEQ ID NO:11) or 5'UTR ASO (SEQ ID NO:142).

[0057] 45. The multivalent oligonucleotide drug according to any one of paragraphs 36 to 44, wherein the multivalent oligonucleotide drug has a nucleotide sequence having at least 90% identity to a nucleotide sequence selected from the group consisting of: a) DA06-4A-27A (SEQ ID NO:14) and an antisense saRNA strand having the nucleotide sequence of SEQ ID NO:13 having partial complementarity to the sense saRNA strand of SEQ ID NO:14; b) DA06-4A-27B (SEQ ID NO:15) and an antisense saRNA strand having the nucleotide sequence of SEQ ID NO:13 having partial complementarity to the sense saRNA strand of SEQ ID NO:15; c) R6-04M1-27A-S1L1V3 (SEQ ID NO: 18) and SEQ an antisense saRNA strand having the nucleotide sequence of SEQ ID NO:13, which has partial complementarity to the sense saRNA strand of ID NO:18; d) DA06-31A-27A (SEQ ID NO:19) and an antisense saRNA strand having the nucleotide sequence of SEQ ID NO:8, which has partial complementarity to the sense saRNA strand of SEQ ID NO:19; e) DA06-31B-27A (SEQ ID NO:20) and a sense saRNA strand having the nucleotide sequence of SEQ ID NO:7 having partial complementarity to the antisense saRNA strand of SEQ ID NO:20; f) DA06-67A-27A (SEQ ID NO:21) and an antisense saRNA strand having the nucleotide sequence of SEQ ID NO:10 having partial complementarity to the sense saRNA strand of SEQ ID NO:21; g) DA06-67B-27A (SEQ ID NO:22) and a sense saRNA strand having the nucleotide sequence of SEQ ID NO:9 having partial complementarity to the antisense saRNA strand of SEQ ID NO:22; h)DA6-67A3'L0-27A(SEQ ID NO:23) and SEQ ID an antisense saRNA strand having the nucleotide sequence of SEQ ID NO:10, which has partial complementarity to the sense saRNA strand of NO:23; j)DA6-67A3'L9-27A(SEQ ID NO:24) and SEQ ID an antisense saRNA strand having the nucleotide sequence of SEQ ID NO:10, which has partial complementarity to the sense saRNA strand of NO:24; k)DA6-67A3'L4-27A(SEQ ID NO:25) and SEQ ID an antisense saRNA strand having the nucleotide sequence of SEQ ID NO:10, which has partial complementarity to the sense saRNA strand of NO:25; l) DA6-67B3'L0-27A (SEQ ID NO: 26) and SEQ ID a sense saRNA strand having the nucleotide sequence of SEQ ID NO:9, which has partial complementarity to the antisense saRNA strand of NO:26; m)DA6-67B5'L1-27A(SEQ ID NO:27) and SEQ ID a sense saRNA strand having the nucleotide sequence of SEQ ID NO:9, which has partial complementarity to the antisense saRNA strand of NO:27; o)DA6-67B5'L9-27A(SEQ ID NO:29) and SEQ ID a sense saRNA strand having the nucleotide sequence of SEQ ID NO:9, which has partial complementarity to the antisense saRNA strand of NO:29; p) DA6-67B5'L4-27A (SEQ ID NO: 30) and SEQ ID a sense saRNA strand having the nucleotide sequence of SEQ ID NO:9, which has partial complementarity to the antisense saRNA strand of NO:30; q) DA6-67B3'L9-27A (SEQ ID NO: 31) and SEQ ID a sense saRNA strand having the nucleotide sequence of SEQ ID NO:9, which has partial complementarity to the antisense saRNA strand of NO:31; r) DA6-67B3'L4-27A (SEQ ID NO: 32) and SEQ ID a sense saRNA strand having the nucleotide sequence of SEQ ID NO:9, which has partial complementarity to the antisense saRNA strand of NO:32; s) DA06-67A21L1-27A (SEQ ID NO:33) and an antisense saRNA strand having the nucleotide sequence of SEQ ID NO:34, which has partial complementarity to the sense saRNA strand of SEQ ID NO:33; t) DA06-67B21L1-27A (SEQ ID NO:36) and a sense saRNA strand of SEQ ID NO:35 having partial complementarity to the antisense saRNA strand of SEQ ID NO:36; u) DA6-04A3'L0-27A (SEQ ID NO: 37) and SEQ ID an antisense saRNA strand having the nucleotide sequence of SEQ ID NO:6, which has partial complementarity to the sense saRNA strand of NO:37; v)DA6-04A5'L1-27A(SEQ ID NO:38) and SEQ ID an antisense saRNA strand having the nucleotide sequence of SEQ ID NO:6, which has partial complementarity to the sense saRNA strand of NO:38; w) DA6-04A5'L9-27A (SEQ ID NO: 39) and SEQ ID an antisense saRNA strand having the nucleotide sequence of SEQ ID NO:6, which has partial complementarity to the sense saRNA strand of NO:39; x)DA6-04A5'L4-27A (SEQ ID NO: 40) and SEQ ID an antisense saRNA strand having the nucleotide sequence of SEQ ID NO:6, which has partial complementarity to the sense saRNA strand of NO:40; y) DA6-04A3'L1-27A (SEQ ID NO: 41) and SEQ ID an antisense saRNA strand having the nucleotide sequence of SEQ ID NO:6, which has partial complementarity to the sense saRNA strand of NO:41; z)DA6-04A3'L9-27A(SEQ ID NO:42) and SEQ ID an antisense saRNA strand having the nucleotide sequence of SEQ ID NO:6, which has partial complementarity to the sense saRNA strand of NO:42; aa) DA6-04A3'L4-27A (SEQ ID NO:43) and an antisense saRNA strand having the nucleotide sequence of SEQ ID NO:6 having partial complementarity to the sense saRNA strand of SEQ ID NO:43; bb) DA6-04B3'L0-27A (SEQ ID NO:44) and a sense saRNA strand having the nucleotide sequence of SEQ ID NO:5, which has partial complementarity to the antisense saRNA strand of SEQ ID NO:44; cc) DA6-04B3'L1-27A (SEQ ID NO:45) and a sense saRNA strand having the nucleotide sequence of SEQ ID NO:5 having partial complementarity to the antisense saRNA strand of SEQ ID NO:45; dd) DA6-04B3'L9-27A (SEQ ID NO:46) and a sense saRNA strand having the nucleotide sequence of SEQ ID NO:5 having partial complementarity to the antisense saRNA strand of SEQ ID NO:46; ee) DA6-04B3'L4-27A (SEQ ID NO:47) and a sense saRNA strand having the nucleotide sequence of SEQ ID NO:5 having partial complementarity to the antisense saRNA strand of SEQ ID NO:47; ff) DA06-04A21L1-27A (SEQ ID NO:48) and an antisense saRNA strand having the nucleotide sequence of SEQ ID NO:49 having partial complementarity to the sense saRNA strand of SEQ ID NO:48; gg) DA06-04B21L1-27A (SEQ ID NO:51) and a sense saRNA strand of SEQ ID NO:50 having partial complementarity to the antisense saRNA strand of SEQ ID NO:51; hh) R6-04M1-16nt-S1L1V3v (SEQ ID NO:79) and an antisense saRNA strand of SEQ ID NO:17 having partial complementarity to the sense saRNA strand of SEQ ID NO:79; ii) R6-04M1-15nt-S1L1V3v (SEQ ID NO:80) and an antisense saRNA strand of SEQ ID NO:17 having partial complementarity to the sense saRNA strand of SEQ ID NO:80; jj) R6-04M1-14nt-S1L1V3v (SEQ ID NO:81) and an antisense saRNA strand of SEQ ID NO:17 having partial complementarity to the sense saRNA strand of SEQ ID NO:81; kk) R6-04M1-13nt-S1L1V3v (SEQ ID NO:82) and an antisense saRNA strand of SEQ ID NO:17 having partial complementarity to the sense saRNA strand of SEQ ID NO:82; ll) R6-04M1-(12nt-B)-S1L1V3v (SEQ ID NO:83) and an antisense saRNA strand of SEQ ID NO:17 having partial complementarity to the sense saRNA strand of SEQ ID NO:83; mm) R6-04M1-11nt-S1L1V3v (SEQ ID NO:84) and an antisense saRNA strand of SEQ ID NO:17 having partial complementarity to the antisense saRNA strand of SEQ ID NO:84; nn) R6-04M1-10nt-S1L1V3v (SEQ ID NO:85) and an antisense saRNA strand of SEQ ID NO:17 having partial complementarity to the antisense saRNA strand of SEQ ID NO:85; oo) R6-04M1-9nt-S1L1V3v (SEQ ID NO:86) and an antisense saRNA strand of SEQ ID NO:17 having partial complementarity to the sense saRNA strand of SEQ ID NO:86; pp) R6-04M1-8nt-S1L1V3v (SEQ ID NO:87) and an antisense saRNA strand of SEQ ID NO:17 having partial complementarity with the sense saRNA strand of SEQ ID NO:87; qq) R6-04M1-7nt-S1L1V3v (SEQ ID NO:88) and an antisense saRNA strand of SEQ ID NO:17 having partial complementarity to the sense saRNA strand of SEQ ID NO:88; rr) R6-04M1-6nt-S1L1V3v (SEQ ID NO:89) and an antisense saRNA strand of SEQ ID NO:17 having partial complementarity to the sense saRNA strand of SEQ ID NO:89; ss)DS06-4A-S2L5V(SEQ ID NO:128) and SEQ ID an antisense saRNA strand having the nucleotide sequence of SEQ ID NO:17, which has partial complementarity to the sense saRNA strand of NO:128; ss')DS06-4A-S2L1v(SEQ ID NO:16) and SEQ ID an antisense saRNA strand having the nucleotide sequence of SEQ ID NO:17, which has partial complementarity to the sense saRNA strand of NO:16; tt)DA6-27A-5'UTR(SEQ ID NO:143); uu)DA6-5'UTR-27A(SEQ ID NO:144); vv) R6-67M3-27A-S1L1V3 (SEQ ID NO:129) and an antisense saRNA strand having the nucleotide sequence of SEQ ID NO:130 having partial complementarity to the sense saRNA strand of SEQ ID NO:129; ww) R6-67M3-16nt-S1L1V3 (SEQ ID NO: 129) and an antisense saRNA strand having the nucleotide sequence of SEQ ID NO: 131, which has partial complementarity to the sense saRNA strand of SEQ ID NO: 129; xx) R6-67M3-15nt-S1L1V3 (SEQ ID NO:129) and an antisense saRNA strand having the nucleotide sequence of SEQ ID NO:132 having partial complementarity to the sense saRNA strand of SEQ ID NO:129; yy) R6-67M3-14nt-S1L1V3 (SEQ ID NO:129) and an antisense saRNA strand having the nucleotide sequence of SEQ ID NO:133, which has partial complementarity to the sense saRNA strand of SEQ ID NO:129; zz) R6-67M3-13nt-S1L1V3 (SEQ ID NO:129) and an antisense saRNA strand having the nucleotide sequence of SEQ ID NO:134, which has partial complementarity to the sense saRNA strand of SEQ ID NO:129; aaa) R6-67M3-12nt-S1L1V3 (SEQ ID NO: 129) and SEQ ID NO: 129 having partial complementarity to the sense saRNA strand of SEQ ID NO: 129 NO:Antisense saRNA strand having the nucleotide sequence of 135; bbb) R6-67M3-9nt-S1L1V3 (SEQ ID NO:129) and an antisense saRNA strand having the nucleotide sequence of SEQ ID NO:136, which has partial complementarity to the sense saRNA strand of SEQ ID NO:129; ccc) R6-67M3-8nt-S1L1V3 (SEQ ID NO:129) and an antisense saRNA strand having the nucleotide sequence of SEQ ID NO:137 having partial complementarity to the sense saRNA strand of SEQ ID NO:129; wherein a linker selected from the group consisting of L1, L4 and L9 is present or absent; wherein L1 represents spacer-18; L4 represents spacer-C6; and L9 represents spacer-9.

[0058] 46. ​​The multivalent oligonucleotide drug according to any one of claims 1 to 43, wherein the multivalent oligonucleotide drug has a nucleotide sequence having at least 90% identity to a nucleotide sequence selected from the group consisting of: a)R6-04S1&67S1R-L1V2(SEQ ID NO:52) and SEQ an antisense saRNA strand having a nucleotide sequence of SEQ ID NO:53, which has partial complementarity with the strand of SEQ ID NO:52, and an antisense saRNA strand having a nucleotide sequence of SEQ ID NO:78, which has partial complementarity with the strand of SEQ ID NO:52; b) R6-04S1&67S5-L1V2 (SEQ ID NO:56), an antisense saRNA strand having a nucleotide sequence of SEQ ID NO:53 that has partial complementarity with a strand of SEQ ID NO:56, and an antisense saRNA strand having a nucleotide sequence of SEQ ID NO:78 that has partial complementarity with a strand of SEQ ID NO:56; and c) R6-04M1 & R17-388E-L1V2 (SEQ ID NO:140), an antisense saRNA strand of SEQ ID NO:17 having partial complementarity to a strand of SEQ ID NO:140, and an antisense saRNA strand of SEQ ID NO:141 having partial complementarity to a strand of SEQ ID NO:140.

[0059] 47. The multivalent oligonucleotide drug according to any one of paragraphs 36 to 44, wherein the multivalent oligonucleotide drug has a nucleotide sequence having at least 90% identity to a nucleotide sequence selected from the group consisting of: a) R6-04S1&27A&67S1R-L1V2 (SEQ ID NO:54), an antisense saRNA strand having a nucleotide sequence of SEQ ID NO:53 that has partial complementarity with a strand of SEQ ID NO:54, and an antisense saRNA strand having a nucleotide sequence of SEQ ID NO:78 that has partial complementarity with a strand of SEQ ID NO:54; b) R6-04S1&67S1R&27A-L1V2 (SEQ ID NO:55), an antisense saRNA strand having a nucleotide sequence of SEQ ID NO:53 that is partially complementary to a strand of SEQ ID NO:55, and an antisense saRNA strand having a nucleotide sequence of SEQ ID NO:78 that is partially complementary to a strand of SEQ ID NO:55; c) R6-04S1&27A&67S5-L1V2 (SEQ ID NO:57), an antisense saRNA strand having a nucleotide sequence of SEQ ID NO:53 that has partial complementarity with a strand of SEQ ID NO:57, and an antisense saRNA strand having a nucleotide sequence of SEQ ID NO:78 that has partial complementarity with a strand of SEQ ID NO:57; and d) R6-04S1&67S5&27A-L1V2 (SEQ ID NO:58), an antisense saRNA strand having a nucleotide sequence of SEQ ID NO:53 that has partial complementarity with a strand of SEQ ID NO:58, and an antisense saRNA strand having a nucleotide sequence of SEQ ID NO:78 that has partial complementarity with a strand of SEQ ID NO:58.

[0060] 48. The multivalent oligonucleotide agent according to any one of paragraphs 36 to 47, wherein the multivalent oligonucleotide agent is selected from those set forth in any one of Tables 7 to 20 or has at least 90% sequence identity thereto, and wherein the linking moieties and / or linking bonds and / or orientations of the multivalent oligonucleotide agent are variable.

[0061] 49. A multivalent oligonucleotide drug according to item 48, wherein the linking moiety and / or linking bond and / or orientation of the multivalent oligonucleotide drug is selected from those defined in any one of items 12 to 25.

[0062] 50. The multivalent oligonucleotide agent according to any one of items 1 to 35, wherein the one or more functional oligonucleotides increase expression of the CDKN1A / p21 gene or protein and / or decrease expression of CD274 / PDL-1.

[0063] 51. The multivalent oligonucleotide agent according to item 50, wherein the dsRNA is independently selected from the group consisting of saRNA that increases the expression of the CDKN1A / p21 gene or protein and siRNA that decreases the expression of CD274 / PDL-1; and / or the ASO is independently selected from the group consisting of ASO that increases the expression of the CDKN1A / p21 gene or protein and / or ASO that decreases the expression of CD274 / PDL-1.

[0064] 52. The multivalent oligonucleotide agent according to item 50, wherein the dsRNA is a saRNA having a nucleotide sequence of the sense strand that has at least 90% identity to the nucleotide sequence of RAG1-40 (SEQ ID NO:62).

[0065] 53. The multivalent oligonucleotide agent according to item 50, wherein the dsRNA is a saRNA having a nucleotide sequence of the antisense strand that has at least 90% identity to the nucleotide sequence of RAG1-40 (SEQ ID NO:63).

[0066] 54. The multivalent oligonucleotide agent according to item 50, wherein the dsRNA is a saRNA having a nucleotide sequence of the sense strand that has at least 90% identity with the nucleotide sequence of RAG1-40 (SEQ ID NO:62) and a nucleotide sequence of the antisense strand that has at least 90% identity with the nucleotide sequence of RAG1-40 (SEQ ID NO:63).

[0067] 55. dsRNA is a) siPDL1-2 (SEQ ID NO:64) and b) siPDL1-3 (SEQ ID NO:66) 51. The multivalent oligonucleotide agent according to item 50, which is an siRNA having a nucleotide sequence of the sense strand that has at least 90% identity to a nucleotide sequence selected from:

[0068] 56. dsRNA is a) siPDL1-2 (SEQ ID NO:65) and b) siPDL1-3 (SEQ ID NO:67) 51. The multivalent oligonucleotide agent according to item 50, which is an siRNA having a nucleotide sequence of the antisense strand that has at least 90% identity to a nucleotide sequence selected from:

[0069] 57. The multivalent oligonucleotide agent according to item 50, wherein the dsRNA is an siRNA selected from the group consisting of: a) an siRNA having a sense strand nucleotide sequence having at least 90% identity to the nucleotide sequence of siPDL1-2 (SEQ ID NO:64) and an antisense strand nucleotide sequence having at least 90% identity to the nucleotide sequence of siPDL1-2 (SEQ ID NO:65); and b) An siRNA having a sense strand nucleotide sequence that has at least 90% identity to the nucleotide sequence of siPDL1-2 (SEQ ID NO:66) and an antisense strand nucleotide sequence that has at least 90% identity to the nucleotide sequence of siPDL1-2 (SEQ ID NO:67).

[0070] 58. ASO is a) aPDL1-1 (SEQ ID NO:68); b) aPDL1-2 (SEQ ID NO:69); and c) aPDL1-3 (SEQ ID NO:70) 51. The multivalent oligonucleotide agent of item 50, having a nucleotide sequence having at least 90% identity to a nucleotide sequence selected from the group consisting of:

[0071] 59. The multivalent oligonucleotide agent according to item 50, wherein the multivalent oligonucleotide agent has a nucleotide sequence having at least 90% identity to a nucleotide sequence selected from the group consisting of: a) saP21-40 / siPDL1-2 (SEQ ID NO:71), an antisense saRNA strand having a nucleotide sequence of SEQ ID NO:63, which has partial complementarity with a strand of SEQ ID NO:71, and an antisense siRNA strand having a nucleotide sequence of SEQ ID NO:65, which has partial complementarity with a strand of SEQ ID NO:71; b) saP21-40 / siPDL1-3 (SEQ ID NO:100) and SEQ an antisense saRNA strand having the nucleotide sequence of SEQ ID NO:63, which has partial complementarity with the strand of SEQ ID NO:100, and an antisense saRNA strand having the nucleotide sequence of SEQ ID NO:65, which has partial complementarity with the strand of SEQ ID NO:100; c) saP21-40 / aPDL1-1 (SEQ ID NO:72) and an antisense saRNA strand having the nucleotide sequence of SEQ ID NO:63, which has partial complementarity to the sense saRNA strand of SEQ ID NO:72; d) saP21-40 / aPDL1-2 (SEQ ID NO:73) and an antisense saRNA strand having the nucleotide sequence of SEQ ID NO:63 having partial complementarity to the sense saRNA strand of SEQ ID NO:73; e) saP21-40 / aPDL1-3 (SEQ ID NO:74) and an antisense saRNA strand having the nucleotide sequence of SEQ ID NO:63 having partial complementarity to the sense saRNA strand of SEQ ID NO:74; f) saP21-40 / aPDL1-1R (SEQ ID NO:75) and an antisense saRNA strand having the nucleotide sequence of SEQ ID NO:63, which has partial complementarity to the sense saRNA strand of SEQ ID NO:75; g) saP21-40 / aPDL1-2R (SEQ ID NO:76) and an antisense saRNA strand having the nucleotide sequence of SEQ ID NO:63 having partial complementarity to the sense saRNA strand of SEQ ID NO:76; and h) saP21-40 / aPDL1-3R (SEQ ID NO:77) and an antisense saRNA strand having the nucleotide sequence of SEQ ID NO:63 which has partial complementarity to the sense saRNA strand of SEQ ID NO:77.

[0072] 60. The multivalent oligonucleotide agent according to item 50, wherein the multivalent oligonucleotide agent is selected from those shown in Table 16 or has at least 90% sequence identity thereto, and the linking moieties and / or linkages and / or orientations of the multivalent oligonucleotide agent are variable.

[0073] 61. A multivalent oligonucleotide drug according to item 50, wherein the linking moiety and / or linking bond and / or orientation of the multivalent oligonucleotide drug is selected from those defined in any one of items 12 to 25.

[0074] 62. A product comprising a multivalent oligonucleotide agent according to any one of items 1 to 61. 63. The product according to item 62, wherein the product is selected from a pharmaceutical composition further comprising at least one pharma- ceutically acceptable carrier, or a kit.

[0075] 64. The product according to item 63, wherein the at least one pharma- ceutically acceptable carrier is selected from the group consisting of aqueous carriers, liposomes, macromolecular polymers, polypeptides, and nanoparticles. 65. The product according to item 63, wherein the kit is used for quality control, screening of drug candidates, drug delivery to tissues or cells, or improving the pharmacological properties of molecules.

[0076] 66. A method for treating a disease, comprising administering to a subject in need of such treatment a sufficient amount of a multivalent oligonucleotide agent according to any one of items 1 to 61 or one or more of the products of items 62 to 65.

[0077] 67. The method according to item 66, further comprising providing to the subject one or more additional pharmaceutical agents or therapies; for example: The additional medication is one or more selected from the group consisting of nusinersen, risdiplam, branapram, zolgensma, fomivirsen, mipomersen, eteplirsen, inotersen, golodirsen, volanesorsen, defibrotide, patisiran, givosiran, lumasiran, inclisiran, or pegaptanib; and / or the additional therapy is one or more selected from the group consisting of physical therapy, dietary management, and surgery.

[0078] 68. The method of item 66, wherein the method treats or delays the onset or progression of an SMN deficiency-associated condition in a subject. 69. The method of claim 66, wherein the subject is suffering from or at risk of having spinal muscular atrophy (SMA); and / or the subject has reduced or abnormal SMN full-length protein expression.

[0079] 70. The method of any one of items 68 to 69, wherein at least one of the two or more targeted oligonucleotides increases expression of the SMN2 gene or protein and / or increases production of functional SMN protein by modulating splicing or stability of SMN2 mRNA (SMN2 mRNA modulator).

[0080] 71. The method of item 68, further comprising providing to the subject one or more additional drugs or therapies for treating or delaying the onset or progression of SMN. 72. The method of item 71, wherein the additional pharmaceutical agent is one or more selected from the group consisting of nusinersen, risdiplam, branapram, and zolgensma.

[0081] 73. The method according to item 71, wherein the additional therapy is one or more selected from the group consisting of physical therapy, dietary management, and surgery. 74. The method of claim 66, wherein the multivalent oligonucleotide agent increases expression of the CDKN1A / p21 gene or protein and decreases expression of CD274 / PDL-1.

[0082] 75. The method according to item 66, wherein the disease is a p21 / PDL-1-associated disease. 76. The method according to item 66, wherein the patient has cancer or is at high risk of having cancer.

[0083] 77. The method according to item 76, wherein the cancer is a solid tumor or a non-solid tumor. 78. Cancers include sarcomas such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, synovium, mesothelioma, Ewing's sarcoma, leiomyosarcoma, rhabdomyosarcoma, colon cancer / colorectal cancer, lymphatic malignancies, pancreatic cancer, breast cancer, lung cancer, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, pheochromocytoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, will 77. The method according to item 76, wherein the tumor is selected from the group consisting of: myeloma, cervical cancer, testicular tumor, seminoma, bladder cancer, melanoma, CNS tumor (e.g., glioma (such as brain stem glioma and mixed glioma), glioblastoma (also called glioblastoma multiforme), astrocytoma, CNS lymphoma, germinoma, medulloblastoma, schwannoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, neuroblastoma, retinoblastoma and metastatic brain tumor.

[0084] 79. The method of any one of items 74 to 78, further comprising administering one or more additional agents or therapies used to treat cancer. 80. The method of item 79, wherein the additional agent or therapy is selected from the group consisting of radiation, chemotherapy, surgery, immunotherapy, and gene therapy.

[0085] 81. The method according to item 80, wherein the chemotherapy is cisplatin, carboplatin, paclitaxel, docetaxel, gemcitabine, vinorelbine, vinblastine, irinotecan, etoposide, pemetrexed, or a combination thereof, or a pharma- ceutically acceptable salt thereof.

[0086] 82. The method of item 79, wherein the additional agent is selected from the group consisting of immunomodulatory antibodies and antibody-drug conjugates. 83. The method of claim 82, wherein the immunomodulatory antibody is selected from the group consisting of an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-CD40 antibody, an anti-CTLA-4 antibody, an anti-OX40 antibody, or a combination thereof; and / or the additional antibody-drug conjugate targets c-Met kinase, LRRC15, EGFR, CS1, or a combination thereof.

[0087] 84. A method for preparing a multivalent oligonucleotide drug according to any one of claims 1 to 61, comprising providing two or more functional oligonucleotides and covalently linking them; or synthesizing a full-length oligonucleotide drug.

[0088] 85. The method according to item 84, wherein the two or more functional oligonucleotides are covalently linked via a linking moiety or without a linking moiety. 86. The method according to item 85, wherein the linking moiety is selected from the following linkers or derivatives thereof:

[0089] [Table 4]

[0090] 87. The method according to item 86, wherein the two adjacent functional oligonucleotides are covalently linked by:

[0091] [ka] , wherein R represents -H, or -OH, or -OMe, or -MOE, or -F, or other 2' chemical modification.

[0092] 88. The method according to item 86, wherein the two adjacent functional oligonucleotides are covalently linked by a phosphodiester bond or a phosphorothioate bond. 89. The method according to item 86, wherein two adjacent functional oligonucleotides are covalently linked by one or more nucleotides.

[0093] 90. The method of item 85, further comprising providing the functional oligonucleotide with one or more chemically modified nucleotides. 91. The method according to item 90, wherein the chemical modification of the at least one chemically modified nucleotide is one or more 2' sugar modifications selected from the group consisting of a 2'-fluoro-2'-deoxynucleoside (2'-F) modification, a 2'-O-methyl (2'-O-Me) modification, and a 2'-O-(2-methoxyethyl) (2'-O-MOE) modification.

[0094] 92. The method according to item 90, wherein the chemical modification of at least one chemically modified nucleotide is a phosphorothioate (PS) backbone modification. 93. The method according to item 90, wherein the chemical modification of the at least one chemically modified nucleotide is the addition of a 5'-phosphate moiety to the 5' end of the nucleotide sequence.

[0095] 94. The method according to item 90, wherein the chemical modification of the at least one chemically modified nucleotide is the addition of an (E)-vinylphosphonate moiety to the 5' end of the nucleotide sequence. 95. The chemical modification of at least one chemically modified nucleotide is at the 5' end of the nucleotide sequence. 5-Methylcytosine 91. The method of claim 90, wherein the moiety is added.

[0096] 96. The method of item 84, wherein each ASO in the agent is covalently linked to an adjacent targeting oligonucleotide in a 3' to 5' or 5' to 3' direction. 97. The method of item 84, wherein each dsRNA in the agent is covalently linked to an adjacent ASO at the 3' end of the sense or antisense strand, or at the 5' end of the sense or antisense strand.

[0097] 98. An isolated or synthesized oligonucleotide comprising a nucleotide sequence of a saRNA sense strand having at least 90% identity to the nucleotide sequence of SEQ ID NO:62.

[0098] 99. The isolated or synthesized oligonucleotide described in item 98, wherein the oligonucleotide further comprises an antisense strand having partial complementarity to the sense saRNA strand described in item 98.

[0099] 100. The isolated or synthesized oligonucleotide according to item 99, wherein the oligonucleotide further comprises an antisense strand having at least 90% identity to the nucleotide sequence of SEQ ID NO:63.

[0100] 101. The isolated or synthesized oligonucleotide according to item 98, wherein the isolated or synthesized oligonucleotide comprises the nucleotide sequence of the saRNA sense strand of SEQ ID NO:62 and the saRNA antisense strand of SEQ ID NO:63.

[0101] 102. A pharmaceutical composition or kit comprising the isolated or synthesized oligonucleotide according to any one of items 98 to 101. 103. A method for treating a disease, comprising administering to a subject in need of such treatment a sufficient amount of one or more of the isolated or synthesized oligonucleotides according to any one of items 98 to 101 or the pharmaceutical composition or kit of item 102. [Brief description of the drawings]

[0102]

[20] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings, in which:

[0103] [Figure 1]

[21] Figure 1 shows the effect of DAO constructs on the expression of full-length (SMN2FL) and exon 7 skipped (SMN2Δ7) SMN2 mRNA in GM03813 cells. GM03813 cells were treated with the indicated concentrations of saRNA, ASO, combo treatment (saRNA+ASO) and DAO (DA06-4A-27A and DA06-4A-27B) for 72 h. As a control treatment, Mock samples were transfected in the absence of oligonucleotides. As an irrelevant oligonucleotide control, dsCon2 was transfected. DS06-332i is an siRNA against SMN2 and was transfected as a control treatment. SMN2FL and SMNΔ7 mRNA levels were determined by RT-qPCR using two sets of primers in separate PCR reactions.

[0104]

[22] Specifically, patient-derived SMA fibroblasts (GM03813 cells) were treated with the indicated oligonucleotides at 20 nM for 72 hours. The exemplary saRNA duplex DS06-0004 is an established activator of the human SMN2 gene. DS06-4A-S2L1A is based on the sequence of DS06-0004 and is a bivalent saRNA structure that covalently links two identically chemically modified duplexes. ASO10-27 is a "mixmer" splicing modulator that increases intracellular levels of SMN2FL transcripts by inclusion of exon 7. A combination of both DS06-4A-S2L1v and ASO10-27 (DS06-4A-S2L1v+ASO10-27) was treated at 20 nM each. DAO constructs were synthesized by covalently linking the ASO10-27 sequence to the saRNA variant of DS06-0004 in conventional (DA-06-4A27A) or reverse (DA-06-4A27B) sequence orientation. Mock treatments were transfected in the absence of oligonucleotide. dsCon2 served as a nonspecific control duplex, while SMN2-specific siRNA (DS06-332i) served as a transfection control by monitoring target gene knockdown.

[0105] In Figure 1A, both full-length (SMN2FL) and Δ7 (SMN2Δ7) splice variants of SMN2 were quantified by RT-qPCR using isoform-specific primer sets. TBP was amplified as an internal reference. Changes in SMN2FL and SMN2Δ7 expression levels relative to mock treatment are shown after normalization to TBP reference levels.

[0106] In Figure 1B, SMN2FL and SMN2Δ7 levels were visualized on agarose gel by semi-quantitative RT-PCR using another primer set spanning exon 7. To distinguish amplicons derived from SMN2 or SMN1 sequences, digestion with DdeI enzyme was performed. The size of the SMN2 product after digestion is shown. Scanning optical densitometry was then performed on the above agarose gel image to quantify the band intensity. The changes in SMN2FL and SMN2Δ7 band intensity levels relative to mock treatment after normalization to TBP reference levels are shown in FIG. 1C.

[0107] [Diagram 2]

[23] Figure 2 shows a dose-dependency study of DAO (DA06-4A-27A) on the expression of SMN2FL and SMN2Δ7 mRNA and SMN protein in GM03813 cells. Mock and dsCon2 were described in Figure 1. SMN2FL and SMNΔ7 mRNA levels were determined by RT-qPCR using two sets of primers in separate PCR reactions. Proteins were harvested from treated cells and immunoblotted in Western blotting assays with an antibody against human SMN protein. As a control for protein loading, an antibody against α / β-tubulin was also blotted.

[0108]

[24] Specifically, we demonstrated the in vitro dose-dependent effect of DAO (DA06-4A-27A) on the transcript levels of SMN2FL and SMN2Δ7. GM03813 cells were treated with the indicated concentrations of DA06-4A-27A for 72 h. Mock treatments were transfected in the absence of oligonucleotide. dsCon2 served as a nonspecific control duplex.

[0109] In Figure 2A, both full-length (SMN2FL) and Δ7 (SMN2Δ7) splice variants of SMN2 were quantified by RT-qPCR using isoform-specific primer sets. TBP was amplified as an internal reference. Changes in SMN2FL and SMN2Δ7 expression levels relative to mock treatment are shown after normalization to TBP reference levels.

[0110] In Figure 2B, SMN2FL and SMN2Δ7 levels were visualized on an agarose gel by semi-quantitative RT-PCR using another primer set spanning exon 7. To distinguish amplicons derived from SMN2 or SMN1 sequences, digestion with DdeI enzyme was performed. The size of the SMN2 product after digestion is shown. Scanning optical densitometry was then performed on the agarose gel images to quantify band intensities. Changes in SMN2FL and SMN2Δ7 band intensity levels after normalization to TBP reference levels relative to mock treatment are shown in Figure 2C.

[0111] [Diagram 3]

[25] Figure 3 shows the dose-dependent effect of DAO (DA06-4A-27A) on the expression of SMN protein in GM03813 cells. Mock and dsCon2 were described in Figure 1. SMN2FL and SMNΔ7 mRNA levels were determined by RT-qPCR using two sets of primers in separate PCR reactions. Proteins were harvested from treated cells and immunoblotted in Western blotting assays with an antibody against human SMN protein. As a control for protein loading, antibodies against α / β-tubulin were also blotted.

[0112]

[26] Specifically, we demonstrated an in vitro dose-dependent effect of DAO (DA06-4A-27A) on total SMN protein levels. GM03813 cells were treated with the indicated concentrations of DA06-4A-27A for 72 h. Mock treatments were transfected in the absence of oligonucleotides. dsCon2 served as a nonspecific control duplex. Whole cell protein extracts were harvested and subjected to immunoblot analysis.

[0113] In Figure 3A, total SMN protein levels were detected using a promiscuous monoclonal antibody that recognizes both SMN1 and SMN2 gene products. Immunodetection of α / β-tubulin served as a protein loading control. Scanning optical densitometry was used to quantitate protein band intensities from the above immunoblots. Changes in total SMN protein levels relative to mock treatment after normalization to α / β-tubulin are shown in Fig. 3B .

[0114] [Figure 4]

[27] Figure 4 shows the efficacy of DAO (DA06-4A-27A) on the expression of SMN2FL and SMN2Δ7 mRNA in SMA-Het mouse brain. RNA was collected from SMA-Het mouse brain by RNAVzol using Qiagen RNeasy columns. PBS represents the untreated control.

[0115]

[28] Specifically, in this figure, SMA heterozygous (Het) mice (Smn1 + / - , SMN2 + / - DAO (DA06-4A-27A) activity in the brain of mice was demonstrated. DA06-4A-27A was administered to mouse pups by ICV injection at the indicated doses on postnatal day 1 (PND1). PBS treatment contained Fast Green as a process control to visually confirm biodistribution in the mouse brain and spinal cord. After 72 hours, mice were sacrificed and whole tissue samples were collected for RNA isolation. n values ​​indicate the number of animals in each treatment group.

[0116] Figure 4A shows SMN2FL and SMN2Δ7 mRNA levels visualized on an agarose gel by semi-quantitative RT-PCR using a primer set spanning exon 7 digested with DdeI enzyme. The amplicon sizes of SMN2FL (392 bp) and SMN2Δ7 (338 bp) after digestion are shown relative to a 100 bp DNA ladder. The Tbp gene was amplified as a loading control. Scanning optical densitometry was then performed on the agarose gel images to quantify band intensities. Mean ± SD SMN2FL and SMN2Δ7 band intensity levels for PBS treatment after normalization to Tbp reference levels are shown in Figure 4B.

[0117] [Diagram 5]

[29] Figure 5 shows the efficacy of DAO (DA06-4A-27A) on the expression of SMN2FL and SMN2Δ7 mRNA in the spinal cord of SMA-Het mice. RNA was collected from the spinal cord of SMA-Het mice by RNAVzol using Qiagen RNeasy columns. PBS represents the untreated control.

[0118]

[30] Specifically, this figure shows the in vitro activity of DAO (DA06-4A-27A) in the spinal cord of SMA-Het mice. Pub mice were treated as in Figure 4 on PND1. After 72 hours, mice were sacrificed and spinal cord samples were collected for RNA isolation. n values ​​indicate the number of animals in each treatment group. SMN2FL and SMN2Δ7 splicing variants were quantified by RT-qPCR using isoform-specific primer sets. Both Gapdh and Tbp were amplified as internal reference controls. Changes in SMN2FL and SMN2Δ7 expression levels relative to mock treatment after normalization to the mean levels of both reference genes are shown.

[0119] [Figure 6]

[31] Figure 6 shows the efficacy of DAO (DA06-4A-27A) on the expression of SMN2FL and SMN2Δ7 mRNA in SMA-Het mouse muscle. RNA was collected from SMA-Het mouse muscle by RNAVzol using Qiagen RNeasy columns. PBS represents the untreated control.

[0120]

[32] Specifically, this figure demonstrates DAO (DA06-4A-27A) activity in muscle of SMA-Het mice. Pub mice were treated as in Figure 4 on PND1. After 72 hours, mice were sacrificed and muscle tissue samples were collected for RNA isolation. n values ​​indicate the number of animals in each treatment group.

[0121] Figure 6A shows SMN2FL and SMN2Δ7 mRNA levels visualized on an agarose gel by semi-quantitative RT-PCR using a primer set spanning exon 7 digested with DdeI enzyme. The amplicon sizes of SMN2FL (392 bp) and SMN2Δ7 (338 bp) after digestion are shown relative to a 100 bp DNA ladder. The Tbp gene was amplified as a loading control. Scanning optical densitometry was then performed on the agarose gel images to quantify band intensities. Mean ± SD SMN2FL and SMN2Δ7 band intensity levels for PBS treatment after normalization to Tbp reference levels are shown in Figure 6B.

[0122] [Figure 7]

[33] Figure 7 shows the effect of different DAO constructs on the expression of SMN2FL and SMN2Δ7 mRNA in GM03813 and GM09677 cells. Mock and dsCon2 were described in Figure 1. SMN2FL and SMNΔ7 mRNA levels were determined by RT-qPCR using two sets of primers in separate PCR reactions. Values ​​(y-axis) are SMN2 band intensities relative to mock treatment after normalization to the band intensity of TBP.

[0123]

[34] Specifically, this figure demonstrates the activity of a DAO (R6-04M1-27A-S1L1V3) with optimized medicinal chemistry in patient-derived SMA fibroblasts. GM03813 and GM09677 cells were treated with 25 nM of the indicated oligonucleotides for 72 h. DS06-4A-S2L5V is a bivalent saRNA variant based on the sequence / scaffold of DS06-4A-S2L1V and with optimized medicinal chemistry. R6-04M1-27A-S1L1V3 is an improved DAO construct based on DA-06-4A27A with similar enhanced chemistry. Mock treatments were transfected in the absence of oligonucleotides. dsCon2 served as a nonspecific control. SMN2FL and SMN2Δ7 splicing variants were quantified by RT-qPCR using isoform-specific primer sets. TBP was amplified as an internal reference. Changes in SMN2FL and SMN2Δ7 expression levels in GM03813 (FIG. 7A) and GM09677 (FIG. 7B) cells, respectively, after normalization to TBP reference levels are shown relative to mock treatment.

[0124] [Figure 8]

[35] Figure 8 shows the effect of DAO structure on SMN2 protein expression in GM03813 and GM09677 cells.

[0125]

[36] Specifically, this figure confirms the activity of DAO (R6-04M1-27A-S1L1V3) on total SMN protein levels in patient-derived SMA fibroblasts. GM03813 and GM09677 cells were treated with 25 nM of the indicated oligonucleotides for 72 h. R6-04(20)-S1V1v(CM-4) is an exemplary saRNA with optimized medicinal chemistry, while R6-04-S1 is its non-chemically modified variant duplex. Mock treatment was transfected in the absence of oligonucleotides. dsCon2 served as a non-specific control duplex. Whole cell protein extracts were harvested and immunoblot analysis was performed. Total SMN protein levels were detected in GM03813 (Figure 8A) and GM09677 (Figure 8C) cells using a promiscuous monoclonal antibody that recognizes both SMN1 and SMN2 gene products. Immunodetection of α / β-tubulin served as a protein loading control. Scanning optical densitometry was used to quantitate protein band intensities from the above immunoblots. The changes in total SMN protein levels in GM03813 and GM09677 cells after normalization to α / β-tubulin are shown in Figure 8B and Figure 8D, respectively, relative to mock treatment.

[0126] [Figure 9]

[37] Figure 9 shows the effect of different DAO structures on the expression of SMN2FL and SMN2Δ7 mRNA in GM03813 cells. Mock and dsCon2 were described in Figure 1. SMN2FL and SMNΔ7 mRNA levels were determined by RT-qPCR using two sets of primers in separate PCR reactions. The novel DAO structures were used for other saRNAs (DS06-0031 and DS06-0067) linked with ASO at different positions.

[0127]

[38] Specifically, this figure shows the effect of DAO constructs on the expression of SMN2FL and SMN2Δ7 mRNA in GM03813 cells. GM03813 cells were treated with 20 nM of the indicated oligonucleotides for 72 h. DS06-0031 and DS06-0067 are saRNA duplexes that preferentially enhance the gene output of the SMN2Δ7 isoform. DAO constructs were synthesized by covalently linking ASO10-27 to the 3' end of the sense strand (DS06-31A-27A and DS06-67A-27A) or antisense strand (DS06-31A-27B and DS06-67B-27B) in both duplexes. Mock treatments were transfected in the absence of oligonucleotides. dsCon2 served as a nonspecific control duplex, while DS06-332i served as a transfection control to monitor SMN2 knockdown. SMN2FL and SMN2Δ7 splice variants were quantified by RT-qPCR using isoform-specific primer sets. TBP was amplified as an internal reference. Changes in SMN2FL and SMN2Δ7 expression levels relative to mock treatment are shown after normalization to TBP reference levels.

[0128] [Figure 10]

[39] Figure 10 shows the effect of DAO constructs with different linkers on the expression of SMN2FL and SMN2Δ7 mRNA in GM03813 cells. GM03813 cells were treated with the indicated concentrations of saRNA, ASO, and DAO with different linkers for 72 h. Mock and dsCon2 were described in Figure 1. SMN2FL and SMNΔ7 mRNA levels were determined by RT-qPCR using two sets of primers in separate PCR reactions.

[0129]

[40] Specifically, this figure shows the effect of DAO structures with different linkers on the expression of SMN2FL and SMN2Δ7 mRNA in GM03813 cells. GM03813 cells were treated with 25 nM of the indicated oligonucleotides (including saRNA, ASO10-27, and DAOs with different linkers) for 72 h. Mock was transfected in the absence of oligonucleotides. As an irrelevant oligonucleotide control, dsCon2 was transfected. SMN2FL and SMNΔ7 mRNA levels were determined by RT-qPCR (Figure 10A) using two sets of primers in separate PCR reactions, as well as by semi-quantitative RT-PCR followed by agarose gel electrophoresis (Figure 10B). As a control for RNA loading, the TBP gene was also amplified.

[0130] FIG. 10A shows SMN2FL and SMNΔ7 mRNA levels determined by RT-qPCR.

[0131] FIG. 10B shows SMN2FL and SMN2Δ7 levels obtained by quantification of PCR product band intensities on agarose gels.

[0132] [Figure 11]

[41] Figure 11 shows the effect of DAO constructs with different linkers on the expression of SMN2FL and SMN2Δ7 mRNA in GM00232 cells. GM00232 cells were treated with the indicated concentrations of saRNA, ASO, and DAO with different linkers for 72 h. Mock and dsCon2 were described in Figure 1. SMN2FL and SMNΔ7 mRNA levels were determined by RT-qPCR using two sets of primers in separate PCR reactions.

[0133]

[42] Specifically, this figure shows the effect of DAO structures with different linkers on the expression of SMN2FL and SMN2Δ7 mRNA in GM00232 cells. GM00232 cells were treated with 25 nM of the indicated oligonucleotides (including saRNA, ASO10-27, and DAOs with different linkers) for 72 h. Mock was transfected in the absence of oligonucleotides. As an irrelevant oligonucleotide control, dsCon2 was transfected. SMN2FL and SMN2Δ7 mRNA levels were determined by RT-qPCR (Figure 11A) using two sets of primers in separate PCR reactions, as well as by semi-quantitative RT-PCR followed by agarose gel electrophoresis (Figure 11B). As a control for RNA loading, the TBP gene was also amplified.

[0134] FIG. 11A shows SMN2FL and SMNΔ7 mRNA levels determined by RT-qPCR.

[0135] FIG. 11B shows SMN2FL and SMN2Δ7 levels obtained by quantification of PCR product band intensities on agarose gels.

[0136] [Figure 12]

[43] Figure 12 shows the effect of different DAO structures on the expression of SMN2FL and SMN2Δ7 mRNA in GM03813 and GM00232 cells. GM03813 and GM00232 cells were treated with the indicated concentrations of saRNA, ASO10-27, and DAO for 72 hours.

[0137]

[44] Specifically, this figure shows the effect of "saRNA-saRNA" DAOs and tripartite DAOs (i.e., bifunctional bivalent saRNAs (trifunctional DAOs) with or without ASO conjugation) on the expression of SMN2FL and SMN2Δ7 mRNA in GM03813 and GM00232 cells. GM03813 and GM00232 cells were transfected with the indicated oligonucleotides at 25 nM for 72 h. Mock was transfected in the absence of oligonucleotide. dsCon2 was transfected as an irrelevant oligonucleotide control. SMN2FL and SMNΔ7 mRNA levels were determined by RT-qPCR using two sets of primers in separate PCR reactions.

[0138] FIG. 12A shows SMN2FL and SMN2Δ7 mRNA levels in GM03813 cells as determined by RT-qPCR.

[0139] FIG. 12B shows SMN2FL and SMN2Δ7 mRNA levels in GM00232 cells as determined by RT-qPCR.

[0140] [Figure 13]

[45] Figure 13 shows the effect of DAO structures on SMN protein expression in GM03813 and GM00232 cells. GM03813 and GM00232 cells were treated with the indicated concentrations of saRNA, ASO, and DAO for 72 hours.

[0141]

[46] Specifically, this figure shows the effect of "saRNA-saRNA" DAO and tripartite DAO (i.e., bifunctional bivalent saRNA (trifunctional DAO) with or without ASO conjugation) on SMN protein expression in GM03813 and GM00232 cells. GM03813 and GM00232 cells were transfected with the indicated oligonucleotides at 25 nM for 72 h. Mock was transfected in the absence of oligonucleotide. dsCon2 was transfected as an irrelevant oligonucleotide control. Whole cell protein extracts were harvested and immunoblot analysis was performed. Total SMN protein levels were detected in GM03813 (Figure 13A) and GM09677 (Figure 13C) cells using a promiscuous monoclonal antibody that recognizes both SMN1 and SMN2 gene products. Immunodetection of α / β-tubulin served as a protein loading control. Scanning optical densitometry was used to quantitate protein band intensities from the above immunoblots. The changes in total SMN protein levels in GM03813 and GM09677 cells after normalization to α / β-tubulin are shown in Figures 13B and 13D, respectively, relative to mock treatment.

[0142] [Figure 14]

[47] Figure 14 shows the effect of DAO constructs with different base numbers of ASO10-27 on the expression of SMN2FL and SMN2Δ7 mRNA in GM03813 and GM09677 cells. GM03813 and GM09677 cells were transfected with DAO at 25 nM for 72 h. Mock was transfected in the absence of oligonucleotide. dsCon2 was transfected as an irrelevant oligonucleotide control. R6-04M1-AC2(8-18 nt)-S1L1V3v was transfected as a control corresponding to R6-04M1-(8-18 nt)-S1L1V3v. SMN2FL and SMNΔ7 mRNA levels were determined by RT-qPCR using two sets of primers in separate PCR reactions. TBP was also amplified as an internal reference.

[0143] FIG. 14A shows SMN2FL and SMN2Δ7 mRNA levels in GM03813 cells as determined by RT-qPCR.

[0144] FIG. 14B shows SMN2FL and SMN2Δ7 mRNA levels in GM09677 cells as determined by RT-qPCR.

[0145]

[48] ​​Specifically, this figure shows the effect of DAOs with different sizes of SMN2 splicing-regulating ASOs on the expression of SMN2FL and SMN2Δ7 mRNA in GM03813 and GM09677 cells. GM03813 and GM09677 cells were transfected with DAOs at 25 nM for 72 h. Mock was transfected in the absence of oligonucleotides. dsCon2 was transfected as an irrelevant oligonucleotide control. ASO10-27 and a series of control DAOs (R6-04M1-AC2(8-18 nt)-S1L1V3v) were transfected as controls. SMN2FL and SMNΔ7 mRNA levels were determined by RT-qPCR using two sets of primers in separate PCR reactions. TBP was also amplified as an internal reference.

[0146] FIG. 14A shows SMN2FL and SMN2Δ7 mRNA levels in GM03813 cells as determined by RT-qPCR.

[0147] FIG. 14B shows SMN2FL and SMN2Δ7 mRNA levels in GM09677 cells as determined by RT-qPCR.

[0148] [Figure 15]

[49] Figure 15 shows the effect of DAOs with different sizes of SMN2 splicing-regulating ASOs on the expression of SMN protein in GM03813 and GM09677 cells. GM03813 and GM09677 cells were treated with 25 nM of the indicated oligonucleotides for 72 hours. Mock was transfected in the absence of oligonucleotides. dsCon2 was transfected as an irrelevant oligonucleotide control. ASO10-27 was transfected as a control.

[0149]

[50] Specifically, this figure shows the effect of DAOs with different sizes of SMN2 splicing-modulating ASOs on SMN protein levels in GM03813 and GM09677 cells. GM03813 and GM09677 cells were treated with 25 nM of the indicated oligonucleotides for 72 hours. Mock was transfected in the absence of oligonucleotides. As an irrelevant oligonucleotide control, dsCon2 was transfected. ASO10-27 was transfected as a positive control. Proteins were harvested from treated cells and immunoblotted in a Western blotting assay with an antibody against human SMN protein. As a control for protein loading, antibodies against α / β-tubulin were also blotted.

[0150] 15A and 15C show a Western blotting membrane with bands for SMN protein and α / β-tubulin protein.

[0151] Figures 15B and 15D show the relative fold change in SMN protein levels obtained by quantifying the band intensities in Figures 15A and 15C. The values ​​(y-axis) in Figures 15B and 15D are the relative band intensities of SMN protein after normalization to α / β-tubulin.

[0152] [Figure 16]

[51] Figure 16 shows the effect of DAO with different sizes of SMN2 splicing regulatory ASO on the expression of SMN2FL and SMN2Δ7 mRNA in GM03813 and GM09677 cells. GM03813 and GM09677 cells were transfected with DAO at 25 nM for 72 hours. Mock was transfected in the absence of oligonucleotide. dsCon2 was transfected as an irrelevant oligonucleotide control. ASO10-27 was transfected as a control. SMN2FL and SMNΔ7 mRNA levels were determined by RT-qPCR using two sets of primers in separate PCR reactions. TBP was also amplified as an internal reference.

[0153]

[52] Specifically, this figure shows the effect of DAO with different sizes of SMN2 splicing regulatory ASO on the expression of SMN2FL and SMN2Δ7 mRNA in GM03813 (Figure 16A) and GM09677 (Figure 16B) cells. GM03813 and GM09677 cells were transfected with DAO at 25 nM for 72 hours. Mock was transfected in the absence of oligonucleotide. dsCon2 was transfected as an irrelevant oligonucleotide control. ASO10-27 was transfected as a positive control. SMN2FL and SMNΔ7 mRNA levels were determined by RT-qPCR using two sets of primers in separate PCR reactions. TBP was also amplified as an internal reference.

[0154] [Figure 17]

[53] Figure 17 shows the effect of DAOs with different sizes of SMN2 splicing-regulating ASOs on the expression of SMN protein in GM03813 and GM09677 cells. GM03813 and GM09677 cells were treated with 25 nM of the indicated oligonucleotides for 72 hours. Mock was transfected in the absence of oligonucleotides. dsCon2 was transfected as an irrelevant oligonucleotide control. ASO10-27 was transfected as a control. Proteins were harvested from treated cells and immunoblotted in a Western blotting assay with an antibody against human SMN protein. As a control for protein loading, an antibody against α / β-tubulin was also blotted.

[0155]

[54] Specifically, this figure shows the effect of DAOs with different sizes of SMN2 splicing-modulating ASOs on SMN protein levels in GM03813 and GM09677 cells. GM03813 and GM09677 cells were treated with 25 nM of the indicated oligonucleotides for 72 hours. Mock was transfected in the absence of oligonucleotides. As an irrelevant oligonucleotide control, dsCon2 was transfected. ASO10-27 was transfected as a positive control. Proteins were harvested from treated cells and immunoblotted in a Western blotting assay with an antibody against human SMN protein. As a control for protein loading, antibodies against α / β-tubulin were also blotted.

[0156] 17A and 17C show a Western blotting membrane with bands for SMN protein and α / β-tubulin protein.

[0157] Figures 17B and 17D show the relative fold change in SMN protein levels obtained by quantitating the band intensities in Figures 17A and 17C. Values ​​(y-axis) in Figures 17B and 17D are the relative band intensities of SMN protein after normalization to α / β-tubulin.

[0158] [Figure 18]

[55] Figure 18 shows the effect of DAO with different sizes of SMN2 splicing regulatory ASO on the expression of SMN2FL and SMN2Δ7 mRNA in PMH cells. PMH cells were transfected with DAO at 25 nM for 72 hours. Mock was transfected in the absence of oligonucleotide. dsCon2 was transfected as an irrelevant oligonucleotide control. ASO10-27 was transfected as a positive control. SMN2FL and SMNΔ7 mRNA levels were determined by RT-qPCR using two sets of primers in separate PCR reactions. TBP was also amplified as an internal reference.

[0159] [Figure 19]

[56] Figure 19 shows the effect of "saRNA-siRNA" DAO on the expression of SMN2FL, SMN2Δ7 and SOD1 mRNA in 293A (Figure 19A) and GM03813 (Figure 19B) cells. 293A or GM03813 cells were transfected with the indicated oligonucleotides at 25 nM for 72 hours. Mock was transfected in the absence of oligonucleotide. dsCon2 was transfected as an irrelevant oligonucleotide control. SMN2FL, SMNΔ7 and SOD1 mRNA levels were determined by RT-qPCR. TBP was also amplified as an internal reference.

[0160] [Figure 20]

[57] Figure 20 shows the effect of different DAO structures on the expression of p21 and PDL1 mRNA in PC3 (Figure 20A) and Ku-7 (Figure 20B) cells. PC3 and Ku-7 cells were treated with saRNA, siRNA and DAO at 10 nM for 48 hours, respectively. Mock and dsCon2 were described in Figure 1. p21 and PDL1 mRNA levels were determined by RT-qPCR using two sets of primers in separate PCR reactions.

[0161]

[58] Specifically, this figure shows the effect of DAOs targeting two different genes on the expression of p21 and PD-L1 mRNA. PC3 and KU-7 cells were transfected with the indicated oligonucleotides at 10 nM for 48 h. Mock was transfected in the absence of oligonucleotide. dsCon2 was transfected as an irrelevant oligonucleotide control. p21 and PD-L1 mRNA levels were determined by RT-qPCR using gene-specific primers in separate PCR reactions. GAPDH was also amplified as an internal reference.

[0162] Figure 20A shows p21 and PD-L1 mRNA levels in PC3 cells as determined by RT-qPCR.

[0163] Figure 20B shows p21 and PD-L1 mRNA levels in KU-7 cells as determined by RT-qPCR.

[0164] [Figure 21]

[59] Figure 21 shows the effect of "ASO-ASO" DAO on the expression of SMN2FL and SMN2Δ7 mRNA in GM03813 cells. GM03813 cells were transfected with the indicated oligonucleotides at 25 nM for 72 h. Mock was transfected in the absence of oligonucleotides. dsCon2 was transfected as an irrelevant oligonucleotide control. SMN2FL and SMNΔ7 mRNA levels were determined by RT-qPCR using two sets of primers in separate PCR reactions. TBP was also amplified as an internal reference.

[0165] [Figure 22]

[60] Figure 22 shows the effect of "ASO-ASO" DAO on SMN protein in GM03813 cells. GM03813 cells were transfected with the indicated oligonucleotides at 25 nM for 72 hours. Mock was transfected in the absence of oligonucleotide. dsCon2 was transfected as an irrelevant oligonucleotide control. Proteins were harvested from treated cells and immunoblotted in a Western blotting assay using an antibody against human SMN protein.

[0166] In Figure 22A, total SMN protein levels were detected using a promiscuous monoclonal antibody that recognizes both SMN1 and SMN2 gene products. Immunodetection of α / β-tubulin served as a protein loading control. Scanning optical densitometry was used to quantitate protein band intensities from the above immunoblots. The changes in total SMN protein levels relative to mock treatment after normalization to α / β-tubulin are shown in Figure 22B. Values ​​(y-axis) are the relative band intensities of SMN protein after normalization to α / β-tubulin.

[0167] [Diagram 23]

[61] Figure 23 shows the in vivo knockdown activity of "bivalent" DAO siRNA on Htt mRNA expression in the brain and spinal cord of C57BL / 6 pups (PND4). siRNA was injected by ICV administration at a dose of 40 mg / kg. Saline was injected as a negative control. siHtt-S1V1 lacks the DAO design and serves as a comparison of siHtt-S1L1 activity. Mice were sacrificed on day 3 (Figure 23A) or day 14 (Figure 23B). Brain and spinal cord tissue samples were collected for analysis by RT-qPCR. Htt mRNA levels are the average of 2 animals / group (n=2) relative to saline treatment after normalization to Tbp reference levels.

[0168] [Figure 24]

[62] Figure 24 shows the biodistribution and in vivo knockdown activity of siSOD1M2-S1L1V2v-Qu5 in pup organs by SC injection.

[0169] Figure 24A: Qu5-labeled "bivalent" DAO siRNA (siSOD1M2-S1V1v-Qu5, siSOD1M2-S1L1V2v-Qu5) was administered to C57BL / 6 pups (PND4) by SC injection at a dose of 200 mg / kg. Injection of Qu5-labeled siRNA variant siSOD1M2-S1V1v-Qu5 served as a control. Mice were sacrificed 3 days after treatment and whole organ fluorescence was quantified with an IVIS imaging system using 520 nm excitation and 570 nm emission filters. Examples of IVIS images showing the biodistribution of siSOD1M2-S1L1V1v-Qu5 via Qu5 signal in all major organs compared to siSOD1M2-S1L1V1v-Qu5 after SC injection are shown.

[0170] FIG. 24B: Fluorescence signal intensity of each indicated organ within the same size ROI was plotted.

[0171] Figure 24C: Knockdown of Sod1 mRNA in the indicated organ tissues was quantified by RT-qPCR using gene-specific primer sets. Tbp was amplified as an internal reference. Expression data is shown relative to mRNA levels in organs from untreated animals after normalization to Tbp. Knockdown is relative to saline control.

[0172] [Diagram 25]

[63] Figure 25 shows the biodistribution and in vivo knockdown activity of siSOD1M2-S1L1V2v-Qu5 in mouse pup organs by ICV injection.

[0173] Figure 25A: Qu5-labeled "bivalent" DAO siRNA (siSOD1M2-S1V1v-Qu5, siSOD1M2-S1L1V2v-Qu5) was administered to C57BL / 6 pups (PND4) by ICV injection at a dose of 40 mg / kg. Injection of Qu5-labeled siRNA variant siSOD1M2-S1V1v-Qu5 served as a control. Mice were sacrificed 3 days after treatment and whole organ fluorescence was quantified with an IVIS imaging system using 520 nm excitation and 570 nm emission filters. Examples of IVIS images showing the biodistribution of siSOD1M2-S1L1V1v-Qu5 via Qu5 signal in all major organs compared to siSOD1M2-S1L1V1v-Qu5 after ICV injection are shown.

[0174] FIG. 25B: Fluorescence signal intensity of each indicated organ within the same size ROI was plotted.

[0175] Figure 25C: Knockdown of Sod1 mRNA in the indicated organ tissues was quantified by RT-qPCR using gene-specific primer sets. Tbp was amplified as an internal reference. Expression data is shown relative to mRNA levels in organs from untreated animals after normalization to Tbp. Knockdown is relative to saline control. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0176]

[64] Aspects of the present disclosure include providing improvements in covalently attaching one or more molecules to efficiently target one or more genes associated with a disease or condition, and improvements in chemistry, manufacturing, and quality control (CMC) for gene therapy that can reduce production costs. The inventors have surprisingly found that a novel class of multivalent oligonucleotide (MVO) drugs can be created by covalently attaching oligonucleotides to a single nucleotide molecule that target one or more sequences by the same or different mechanisms of action.

[0177]

[65] In some embodiments, the present invention is based on research into oligonucleotide agents, compositions and methods for activating / upregulating gene expression and / or increasing the amount of full-length genes or proteins expressed to improve the efficacy of treatment for genetic diseases.

[0178]

[66] The present application further demonstrates that combination treatment of SMA patient cells with SMN2 saRNA and an SMN2 mRNA modulator (e.g., an ASO such as nusinersen), or small pyridazine derivatives (including, but not limited to, risdiplam and branapram), significantly enhances full-length SMN2 mRNA and SMN protein levels beyond those achievable by either compound used alone. This combination therapeutic strategy can maximize the treatment outcome for patients, such as SMA patients, by providing enhanced therapeutic benefit compared to monotherapy, for example, by improving clinical symptoms in patients diagnosed with an SMN deficiency-related condition, or by reducing undesirable side effects associated with monotherapy.

[0179]

[67] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention is directed.

[68] In this application, the singular forms "a," "an," "this," etc. include plural referents unless the context clearly dictates otherwise.

[0180]

[69] As used herein, the terms "approximately," "about," "substantially," and similar terms are intended to have a broad meaning consistent with common and accepted usage by those of ordinary skill in the art to which the subject matter of this disclosure pertains.

[0181] 4.1. Definition

[70] As used herein, the terms "multivalent oligonucleotide drug," "MVO drug," "oligonucleotide drug having multiple functional oligonucleotide units," and the like, are used interchangeably and in a broad sense to include any oligonucleotide drug of the invention that includes two or more functional oligonucleotides covalently linked together. The MVO drug may provide improved function or efficacy, or even additive or preferably synergistic effects, compared to any of the functional oligonucleotides used alone. As used herein, the term "functional oligonucleotide," also referred to as "oligonucleotide unit" or "functional oligonucleotide unit," refers to an oligonucleotide unit in a multivalent oligonucleotide drug, which may be selected from the group consisting of single-stranded antisense oligonucleotides (ASOs, e.g., gapmers, mixmers, etc.) and double-stranded (double-stranded) RNA (dsRNA, e.g., siRNAs, saRNAs, etc.), covalently linked to form an assembly molecule. In some embodiments, the functional oligonucleotide units in a multivalent oligonucleotide drug are of the same or different groups, are the same or different, have different or identical targets (e.g., target the same or different genes), and / or are connected directly or via a linker. A multivalent oligonucleotide drug may be a multitargeted oligonucleotide drug having two or more targets of action and having improved activity.

[0182]

[71] As used herein, the term "gapmer" refers to a short DNA antisense oligonucleotide (ASO) structure with modified RNA segments on either side of a central DNA structure. In some embodiments, at least one modified RNA segment comprises one or more modified nucleotides selected from locked nucleic acid (LNA) and 2'-OMe or 2'-F modified nucleotides to increase affinity for the target, increase nuclease resistance, reduce immunogenicity, and / or reduce toxicity. In some embodiments, the gapmer comprises at least one nucleotide modified with a phosphorothioate (PS) group. In some embodiments, the gapmer is designed to hybridize to a target portion of RNA and silence a gene transcript by induction of RNase H cleavage. For example, the ASO drug "Toferson" is a gapmer that knocks down SOD1 mRNA for the treatment of ALS. A possible example of a DAO with a gapmer ASO disclosed in this application is "siSOD1-Toferson".

[0183]

[72] As used herein, the term "mixmer" refers to an antisense oligonucleotide (ASO) characterized by a structural mixture of DNA and chemically modified nucleic acid analogs. Optionally, the mixmer is composed of fully modified nucleotides or nucleic acid analogs. In some embodiments, the mixmer is designed to bind and mask complementary RNA sequences, sterically blocking proteins, factors, or other RNAs from interacting with the target RNA. In some embodiments, the mixmer is designed to alter pre-mRNA splicing by displacing the spliceosome. In some embodiments, the mixmer is designed to bind and sequester microRNAs (miRNAs), which are otherwise referred to as "antagomirs" or "anti-miRs." In some embodiments, examples of DAOs combining saRNAs and mixmers have been described in this application.

[0184]

[73] The terms "gapmer" and "mixmer" may be used to illustrate two different subclasses of single-stranded antisense oligonucleotide (ASO) molecules. In some embodiments, ASOs are either "gapmers" or "mixmers" and serve different molecular functions. In some embodiments, the terms "gapmers" and "mixmers" refer to their chemical design.

[0185]

[74] For a detailed description of gapmers and mixmers, see:Peter H. Hagedorn et al., Locked nucleic acid: modality, diversity, and drug discovery, Drug Discovery Today, Volume 23, Number 1, January 2018;Piotr J. Kamola et al., In silico and in vitro evaluation of exonic and intronic off-target effects form a critical element of therapeutic ASO gapmer optimization, Nucleic Acids Research, Volume 43, Issue 18, 15 October 2015, Pages 8638-8650;Birte Vester et al., LNA (locked nucleic acid): high-affinity targeting of complementary RNA and DNA, Biochemistry 2004, 43, 42, 13233-13241.

[0186]

[75] As used herein, the term "SMN deficiency-associated pathology" refers to a disease caused by a deficiency of full-length SMN protein due to any cause. "SMN deficiency-associated pathology" includes, but is not limited to, spinal muscular atrophy (SMA), arthrogryposis multiplex congenita (AMC), and amyotrophic lateral sclerosis (ALS). For SMN1 (human), the GenBank gene reference is Gene ID: 6606.

[0187]

[76] The term "spinal muscular atrophy" or "SMA" includes, but is not limited to, spinal muscular atrophy (SMA) types 1-4; proximal spinal muscular atrophy; childhood-onset SMA type I (Werdnig-Hoffmann disease); type II (intermediate, chronic), type III (Kugelberg-Welander disease, or juvenile spinal muscular atrophy), and the more recently classified adult-onset type IV. Meeting Report: International SMA Consortium Meeting, Neuromuscul Disord.; 2: 423-428. The term SMA also includes late-onset SMA (also known as SMA types 3 and 4, mild SMA, adult-onset SMA, and Kugelberg-Welander disease). The term SMA also includes other forms of SMA, including X-linked disorders, Spinal Muscular Atrophy with Respiratory Disorders (SMARD), Spino-bulbar Muscular Atrophy (Kennedy's disease, Spino-bulbar Muscular Atrophy), and Distal Spinal Muscular Atrophy. The term SMA includes all forms of SMA described in:Arnold, WD, Kassar, D. & Kissel, JT Spinal muscular atrophy: Diagnosis and management in a new therapeutic era. Muscle and Nerve (2015);Butchbach, MER Copy Number Variations in the Survival Motor Neuron Genes: Implications for Spinal Muscular Atrophy and Other Neurodegenerative Diseases. Front. Mol. Biosci. (2016).

[0188]

[77] If symptoms of SMA are present at birth or by 6 months of age, the disease is diagnosed as SMA. This is called type 1 (also called infantile-onset or Werdnig-Hoffmann). Infants usually have generalized muscle weakness, a weak cry, and labored breathing. They often have difficulty swallowing and sucking, and never reach the developmental milestone of sitting unassisted. These infants are at increased risk for aspiration and growth failure. These infants usually have two or three copies of the SMN2 gene. (Butchbach, MER Copy Number Variations in the Survival Motor Neuron Genes: Implications for Spinal Muscular Atrophy and Other Neurodegenerative Diseases. Front. Mol. Biosci. (2016), incorporated herein in its entirety.)

[0189]

[78] When SMA begins between 3 and 15 months of age and before a child can stand or walk independently, it is called SMA type 2, intermediate SMA, or Dubowitz disease. Children with SMA type 2 typically have three copies of the SMN2 gene (Arnold, WD, Kassar, D. & Kissel, JT Spinal muscular atrophy: Diagnosis and management in a new therapeutic era. Muscle and Nerve (2015), incorporated herein in its entirety). Muscle weakness is predominantly proximal (closer to the center of the body) and involves the lower limbs more than the arms. Facial and eye muscles are usually spared. (Butchbach, MER Copy Number Variations in the Survival Motor Neuron Genes: Implications for Spinal Muscular Atrophy and Other Neurodegenerative Diseases. Front. Mol. Biosci. (2016), incorporated herein in its entirety).

[0190]

[79] Late-onset SMA (also known as SMA types 3 and 4, mild SMA, adult-onset SMA, and Kugelberg-Welander disease) can cause varying degrees of frailty. SMA Patients with type 3 have three to four copies of the SMN2 gene. SMA type 3 (early onset) accounts for 30% of all SMA cases (Arnold, WD, Kassar, D. & Kissel, JT Spinal muscular atrophy: Diagnosis and management in a new therapeutic era. Muscle and Nerve (2015)). Symptoms usually appear between 18 months of age and adulthood. Affected individuals are able to move independently. However, these patients may have proximal muscle weakness that causes falls and difficulty climbing stairs. Over time, many are unable to stand or walk and instead use a wheelchair to get around. Most of these patients develop foot deformities, scoliosis, and respiratory muscle weakness.

[0191]

[80] SMA type 4 is a late-onset disease and accounts for less than 5% of all SMA cases. These patients have 4 to 8 copies of the SMN2 gene (Butchbach, MER Copy Number Variations in the Survival Motor Neuron Genes: Implications for Spinal Muscular Atrophy and Other Neurodegenerative Diseases. Front. Mol. Biosci. (2016)). There is no set age of onset, but it usually occurs after age 30. Since type 4 is a milder form of SMA, lifespan remains normal. Patients are able to achieve motor milestones and maintain motor skills throughout their lives.

[0192]

[81] As used herein, the terms "subject" and "individual" are used interchangeably herein and refer to any living organism that can be treated with the compounds of the present disclosure. The term "patient" refers to a human subject or individual, including an infant, child, or adult, as disclosed herein.

[0193]

[82] A "therapeutically effective amount" of a composition is an amount sufficient to achieve a desired therapeutic effect, not necessarily a cure or complete remission. In an embodiment of the present disclosure, the therapeutic effect is an improvement in any of the disease indicators, and a therapeutically effective amount is sufficient to cause a clinically significant improvement in the condition / symptoms in the treated individual. As used herein, the terms "therapeutically effective amount" and "effective amount" refer to an amount sufficient to reduce by at least about 15%, preferably by at least 50%, more preferably by at least 90%, and most preferably prevent a clinically significant deficit in activity, function, and response in the treated individual.

[0194]

[83] The effective amount may vary depending on factors such as the size and weight of the subject, the type of illness, or the particular compound of the invention. For example, the choice of the compound of the invention may affect what constitutes an "effective amount." One of ordinary skill in the art can study the factors contained herein and make the determination regarding the effective amount of the compound of the invention without undue experimentation.

[0195]

[84] The dosing regimen can affect what constitutes an effective amount. The compounds of the invention can be administered to a subject prior to or after disease diagnosis or symptoms. In addition, they can be administered in divided doses, at staggered times, daily or sequentially, by continuous infusion, or by bolus injection. Furthermore, the dosage of the compounds of the invention can be proportionally increased or decreased as indicated by the exigencies of the therapeutic or prophylactic situation.

[0196]

[85] As used herein, the terms "treat," "treated," "treat," or "treatment" have the meaning commonly understood in the medical arts and, as such, do not require a cure or complete remission but include any beneficial or desired clinical outcome. Non-limiting examples of such beneficial or desired clinical outcomes include extending survival compared to expected survival without treatment, reducing symptoms including one or more of the following: proximal skeletal muscle weakness and atrophy, inability to sit or walk independently, swallowing problems, breathing problems, etc.

[0197]

[86] As used herein, "preventing" or "delaying" a disease refers to inhibiting the full development of the disease.

[0198]

[87] The term "biological sample" refers to tissues, cells, fluids, or other substances derived from an organism (e.g., a human subject). In certain embodiments, the biological sample is serum or blood.

[0199]

[88] As used herein, the term "sequence identity" or "% identity" in the context of oligonucleotide sequences refers to the percentage of residues in the compared sequences that are the same when the sequences are aligned over a specified comparison window. For example, in the context of saRNA, the term "sequence identity" or "sequence homology" means that one oligonucleotide strand (sense or antisense) of saRNA has at least 80% similarity with a region in the coding strand or template strand of the promoter sequence of a target gene. In some embodiments, the percentage identity is measured by using one of the sequence comparison algorithms (e.g., BLASTP and BLASTN, or other algorithms available to those skilled in the art) or by visual inspection.

[0200]

[89] "Promoter sequence of a target gene" refers to a non-coding sequence of a target gene, and in the context of this disclosure, "complementary to the promoter sequence of a target gene" refers to the coding strand, also referred to as the non-template strand, i.e., a nucleic acid sequence that is the same sequence as the coding sequence of the gene.

[0201]

[90] As used herein, the terms "sense strand" and "sense oligonucleotide strand" are interchangeable. The sense oligonucleotide strand of a dsRNA molecule may, for example, comprise the first nucleic acid strand that contains the coding strand of the promoter sequence of a target gene in the saRNA duplex.

[0202]

[91] As used herein, the terms "antisense strand" and "antisense oligonucleotide strand" are interchangeable. The antisense oligonucleotide strand of a dsRNA molecule may comprise the second nucleic acid strand in a saRNA duplex that is complementary to the sense oligonucleotide strand, for example.

[0203]

[92] As used herein, the term "first oligonucleotide strand" may be a sense strand or an antisense strand. The sense strand of a saRNA refers to the oligonucleotide strand that has homology to the coding strand of the promoter DNA sequence of the target gene in the saRNA duplex. The antisense strand refers to the oligonucleotide strand that is complementary to the sense strand in the saRNA duplex.

[0204]

[93] As used herein, the term "second oligonucleotide strand" may also be a sense strand or an antisense strand. If the first oligonucleotide strand is a sense strand, the second oligonucleotide strand is an antisense strand, and if the first oligonucleotide strand is an antisense strand, the second oligonucleotide strand is a sense strand.

[0205]

[94] As used herein, the term "promoter" refers to a nucleic acid sequence that does not code for a protein but serves the role of regulating transcription by spatially associating with a nucleic acid sequence that codes for a protein or RNA. Generally, eukaryotic promoters contain 100-5,000 base pairs, although this length range is not intended to limit the term "promoter" as used herein. Promoter sequences are generally located at the 5' end of the protein or RNA coding sequence, but may also occur in exon and intron sequences.

[0206]

[95] As used herein, the term "coding strand" refers to the DNA strand of a target gene that is not transcribed, the nucleotide sequence of which is identical to the sequence of the RNA produced by transcription (in which the DNA Ts are replaced by Us). The coding strand of a double-stranded DNA sequence of a target gene promoter as described in this disclosure refers to the promoter sequence on the same DNA strand as the DNA coding strand of the target gene.

[0207]

[96] As used herein, the term "template strand" refers to the other strand of double-stranded DNA of a target gene that is complementary to the coding strand and can be transcribed into RNA that is complementary to the bases (AU, GC) of the template transcribed RNA. During transcription, RNA polymerase binds to the template strand, moves along the template strand in a 3'→5' direction, and catalyzes RNA synthesis in a 5'→3' direction. The template strand of the double-stranded DNA sequence of a target gene promoter described in this disclosure refers to the promoter sequence in the same DNA strand as the DNA template strand of the target gene.

[0208]

[97] As used herein, the term "transcription start site" or TSS refers to the nucleotides on the template strand of a gene that mark the start of transcription. A transcription start site may be located on the template strand in the promoter region. A gene may have multiple transcription start sites.

[0209]

[98] As used herein, the term "overhang" refers to an oligonucleotide strand end (5' or 3') that has non-base paired nucleotides due to one strand extending beyond the other strand in a double stranded oligonucleotide. A single stranded region that extends beyond the 3' and / or 5' end of a duplex is called an overhang. In certain embodiments, an overhang is 0-6 nucleotides in length. An overhang of 0 nucleotides is understood to mean there is no overhang.

[0210]

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

[0211]

[0100] As used herein, the terms "small activating RNA", "saRNA" and "small activating ribonucleic acid" are used interchangeably and refer to a ribonucleic acid molecule that can upregulate the expression of a target gene. It may be a double-stranded nucleic acid molecule consisting of a first nucleic acid strand containing a ribonucleotide sequence having sequence homology with a non-coding nucleic acid sequence (such as a promoter or enhancer) of a target gene, and a second nucleic acid strand containing a nucleotide sequence complementary to the first nucleic acid strand. saRNA may also be composed of a synthetic or vector-expressed single-stranded RNA molecule that can form a hairpin structure by two complementary regions within the molecule, where the first region contains a ribonucleotide sequence having sequence homology with a target sequence of the promoter of the gene, and the ribonucleotide sequence contained in the second region is complementary to the first region. The length of the duplex region of the saRNA molecule is typically about 10 to about 60 base pairs, about 10 to about 50 base pairs, about 12 to about 48 base pairs, about 14 to about 46 base pairs, about 16 to about 44 base pairs, about 18 to about 42 base pairs, about 20 to about 40 base pairs, about 22 to about 38 base pairs, about 24 to about 36 base pairs, about 26 to about 34 base pairs, about 28 to about 32 base pairs, typically about 10 base pairs, about 15 base pairs, about 20 base pairs, about 25 base pairs, about 30 base pairs, about 35 base pairs, about 40 base pairs, about 45 base pairs, about 50 base pairs, about 55 base pairs, or about 60 base pairs. Furthermore, the terms "small activating RNA", "saRNA" and "small activating ribonucleic acid" may include nucleic acids other than ribonucleotides, including, but not limited to, modified nucleotides or analogs.

[0212]

[0101] As used herein, the terms "small interfering RNA", "siRNA" and "small interfering ribonucleic acid" are used interchangeably and refer to a ribonucleic acid molecule that can downregulate or even silence the expression of a target gene. It may be a double-stranded nucleic acid molecule consisting of a first nucleic acid strand that contains a ribonucleotide sequence with sequence homology to a non-coding nucleic acid sequence of a target gene and a second nucleic acid strand that contains a nucleotide sequence complementary to the first nucleic acid strand. siRNA may also be composed of a synthetic or vector-expressed single-stranded RNA molecule that can form a hairpin structure by two complementary regions within the molecule, where the first region contains a ribonucleotide sequence with sequence homology to a target sequence of a promoter of the gene, and the ribonucleotide sequence contained in the second region is complementary to the first region. The length of the double-stranded region of the siRNA molecule is typically about 10 to about 60 base pairs, about 10 to about 50 base pairs, about 12 to about 48 base pairs, about 14 to about 46 base pairs, about 16 to about 44 base pairs, about 18 to about 42 base pairs, about 20 to about 40 base pairs, about 20 to about 25 base pairs, typically about 10 base pairs, about 15 base pairs, about 16 base pairs, about 17 base pairs, about 18 base pairs, about 19 base pairs, about 20 base pairs, about 21 base pairs, about 22 base pairs, about 23 base pairs, about 24 base pairs, about 25 base pairs, about 26 base pairs, about 27 base pairs, about 28 base pairs, about 29 base pairs, about 30 base pairs, about 35 base pairs, about 40 base pairs, about 45 base pairs, about 50 base pairs. Furthermore, this term may include nucleic acids other than ribonucleotides, including but not limited to modified nucleotides or analogs.

[0213]

[0102] As used herein, a "covalent linker" refers to a molecule for covalently linking two molecules, such as two dsRNAs. As described in more detail below, this term may include, for example, nucleic acid linkers and peptide linkers, and includes disulfide linkers.

[0214]

[0103] As used herein, the term "synthetic" refers to the means by which an oligonucleotide is synthesized and includes any means by which RNA can be synthesized or chemically modified, such as chemical synthesis, in vitro transcription, vector expression, etc.

[0215] 4.2. Structure and construction of multivalent oligonucleotide (MVO) drugs 4.2.1 Functionalized Oligonucleotides in Multivalent Oligonucleotide Drugs Aspects of the present disclosure include a multivalent oligonucleotide agent comprising two or more functional oligonucleotides covalently linked together.In some embodiments, the two or more functional oligonucleotides are independently selected from double-stranded RNA (dsRNA) and antisense oligonucleotide (ASO).The dsRNA is independently selected from small interfering RNA (siRNA) and small activating RNA (saRNA).The ASO is independently selected from gapmers and mixmers.

[0216] In some embodiments, the multivalent oligonucleotide drug comprises 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, or 7 or more oligonucleotide units. In some embodiments, the multivalent oligonucleotide drug comprises 2 to 10 functional oligonucleotides. In some embodiments, the multivalent oligonucleotide drug is a dual action oligonucleotide (DAO), thus a multiple action oligonucleotide drug.

[0217]

[0106] In some embodiments, the MVO agent may comprise: a) a first double-stranded RNA (dsRNA) and a first antisense oligonucleotide (ASO); b) a first double-stranded RNA (dsRNA) and a second dsRNA; c) a first antisense oligonucleotide (ASO) and a second ASO; d) a first double-stranded RNA (dsRNA), a second dsRNA, and a third dsRNA; e) a first double-stranded RNA (dsRNA), a second dsRNA, and a first antisense oligonucleotide (ASO); f) a first double-stranded RNA (dsRNA), a first antisense oligonucleotide (ASO), and a second ASO; or g) a first antisense oligonucleotide (ASO), a second ASO, and a third ASO; and in any one of a) to g), the functional oligonucleotides are arranged in any order and are covalently linked with or without a linking moiety.

[0218] In some embodiments, dsRNA is small interfering RNA (siRNA). siRNA binds to target mRNA mainly in cytoplasm and downregulates gene expression after transcription by RNA interference (RNAi) mechanism. siRNA can be designed to target the mRNA sequence of gene, such as PDL-1, to silence its expression by RNAi mechanism, for example, to maximize the therapeutic outcome of cancer patients.

[0219]

[0108] siRNAs may be molecules with endogenous RNA bases or chemically modified nucleotides. The modifications do not abolish cellular activity, but rather provide improved stability and / or increased cellular potency. Examples of chemical modifications include phosphorothioate groups, 2'-deoxynucleotides, 2'-OCH 3ribonucleotides, 2'-F-ribonucleotides, 2'-methoxyethyl ribonucleotides, combinations thereof, etc. siRNAs can have a variety of lengths (e.g., 10-200 bps) and structures (e.g., hairpin, single / double stranded, bulge, nick / gap, mismatch) and are processed intracellularly to provide active gene silencing. Double-stranded siRNAs (dsRNAs) can have the same number of nucleotides on each strand (blunt ends) or asymmetric ends (overhangs). For example, 1-2 nucleotide overhangs can be present on the sense and / or antisense strands and can be present on the 5'-end and / or 3'-end of a given strand.

[0220] In some embodiments, the dsRNA is a small activating RNA (saRNA). The saRNA targets regulatory sequences in the nucleus, such as gene promoters, and upregulates gene expression at the transcriptional level through the RNAa (RNA activation) mechanism.

[0221]

[0110] In some embodiments, at least one oligonucleotide is an ASO. The ASO can be designed to target the mRNA of a gene and downregulate its expression through RNase H activity, for example to maximize the efficacy of cancer treatment.

[0222]

[0111] In some embodiments, at least one oligonucleotide is an ASO. The ASO can be designed to target the pre-mRNA of a gene and alter its splicing by steric hindrance, for example, to maximize the expression of the functional protein of the gene.

[0223] In some embodiments, each of the two or more functional oligonucleotides regulates the expression of one or more genes, proteins, or non-coding regulatory nucleic acid sequences by binding to an mRNA sequence. In certain embodiments, the target non-coding regulatory nucleic acid sequence is a promoter sequence.

[0224] In some embodiments, the multivalent oligonucleotide agent comprises a dsRNA comprising a sense strand and an antisense strand.In certain embodiments, the antisense strand of the dsRNA has partial complementarity with the sense dsRNA strand.As used herein, the term "partial complementarity" refers to a partial complementarity of 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more, 17 or more, 18 or more, 19 or more, 20 or more, 21 or more, 22 or more, 23 or more, 24 or more, 25 or more, 26 or more, 27 or more, 28 or more, 29 or more, 30 or more, 31 or more, 32 or more, or a combination of the two or more. The antisense strand of the dsRNA may have complementarity with at least 15 consecutive nucleotides of at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, at least 40, at least 41, at least 42, at least 43, at least 44, at least 45, at least 46, at least 47, at least 48, at least 49, at least 50, at least 51, at least 52, at least 53, at least 54, at least 55, at least 56, at least 57, at least 58, at least 59, or at least 60 consecutive nucleotides. In certain embodiments, the dsRNA comprises an antisense strand having at least 15 consecutive nucleotides that are complementary to at least 15 consecutive nucleotides of the sense strand of the dsRNA. In certain embodiments, the dsRNA comprises an antisense strand having at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, or at least 60 contiguous nucleotides that are complementary to at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, or at least 60 contiguous nucleotides of the sense strand of the dsRNA.

[0225] In some embodiments, the multivalent oligonucleotide agent is a) siRNA-siRNA; b) siRNA-saRNA; c) saRNA-saRNA; d) siRNA-gapmer; e) siRNA-mixmer; f) saRNA-gapmer; g) saRNA-mixmer; h) gapmer-gapmer; i) gapmer-mixmer; j) mixmer-mixmer; k) siRNA-siRNA-siRNA; l) siRNA-siRNA-saRNA; m) siRNA-saRNA-saRNA; n) saRNA-saRNA-saRNA; o) siRNA-siRNA-gapmer; p) siRNA-siRNA-mixmer; q) siRNA-saRNA-gapmer; r) siRNA-saRNA-mixmer; s) saRNA-saRNA-gapmer. and ad) mixmer-mixmer-mixmer; wherein in any one of a) to ad), the functional oligonucleotides are arranged in any order and are covalently linked with or without a linking moiety.

[0226] In some embodiments, the three or more oligonucleotides include three dsRNAs (e.g., a first dsRNA, a second dsRNA, and a third dsRNA). In some embodiments, the three or more oligonucleotides include two dsRNAs and one ASO (e.g., a first dsRNA, a second dsRNA, and an ASO).

[0227]

[0116] In some embodiments, the dsRNA comprises a sense strand that is at least 15 contiguous nucleotides and an antisense strand that is at least 15 contiguous nucleotides.

[0228] In some embodiments, the sense strand has a length in the range of about 10 nucleotides or more, about 15 nucleotides or more, about 20 nucleotides or more, about 25 nucleotides or more, about 30 nucleotides or more, about 35 nucleotides or more, about 40 nucleotides or more, about 45 nucleotides or more, about 50 nucleotides or more, about 55 nucleotides or more, or about 60 nucleotides or more. In some embodiments, the sense strand has a length of 10 to 100 nucleotides (e.g., 10 to 20 nucleotides, 10 to 50 nucleotides, 10 to 90 nucleotides, 20 to 95 nucleotides, 30 to 70 nucleotides, 40 to 80 nucleotides, 50 to 100 nucleotides, 10 to 40 nucleotides, 10 to 30 nucleotides). In some embodiments, the sense strand has a length of 10 to 60 nucleotides (e.g., 10 to 20 nucleotides, 10 to 50 nucleotides, 10 to 40 nucleotides, 10 to 30 nucleotides).

[0229] In some embodiments, the sense strand has a length in the range of about 10 nucleotides or more, about 15 nucleotides or more, about 20 nucleotides or more, about 25 nucleotides or more, about 30 nucleotides or more, about 35 nucleotides or more, about 40 nucleotides or more, about 45 nucleotides or more, about 50 nucleotides or more, about 55 nucleotides or more, about 60 nucleotides or more, about 65 nucleotides or more, about 70 nucleotides or more, about 75 nucleotides or more, about 80 nucleotides or more, about 85 nucleotides or more, about 90 nucleotides or more, about 95 nucleotides or more, or about 100 nucleotides or more. In some embodiments, the sense strand has a length of 10 to 60 nucleotides (e.g., 10 to 20 nucleotides, 10 to 50 nucleotides, 10 to 40 nucleotides, 10 to 30 nucleotides).

[0230] In some embodiments, the antisense strand has a length in the range of about 10 nucleotides or more, about 15 nucleotides or more, about 20 nucleotides or more, about 25 nucleotides or more, about 30 nucleotides or more, about 35 nucleotides or more, about 40 nucleotides or more, about 45 nucleotides or more, about 50 nucleotides or more, about 55 nucleotides or more, or about 60 nucleotides or more. In some embodiments, the antisense strand has a length of 10 to 100 nucleotides (e.g., 10 to 20 nucleotides, 10 to 50 nucleotides, 10 to 90 nucleotides, 20 to 95 nucleotides, 30 to 70 nucleotides, 40 to 80 nucleotides, 50 to 100 nucleotides, 10 to 40 nucleotides, 10 to 30 nucleotides). In some embodiments, the antisense strand has a length of 10 to 60 nucleotides (e.g., 10 to 20 nucleotides, 10 to 50 nucleotides, 10 to 40 nucleotides, 10 to 30 nucleotides).

[0231] In some embodiments, the antisense strand has a length in the range of about 10 nucleotides or more, about 15 nucleotides or more, about 20 nucleotides or more, about 25 nucleotides or more, about 30 nucleotides or more, about 35 nucleotides or more, about 40 nucleotides or more, about 45 nucleotides or more, about 50 nucleotides or more, about 55 nucleotides or more, about 60 nucleotides or more, about 65 nucleotides or more, about 70 nucleotides or more, about 75 nucleotides or more, about 80 nucleotides or more, about 85 nucleotides or more, about 90 nucleotides or more, about 95 nucleotides or more, or about 100 nucleotides or more.In some embodiments, the antisense strand has a length of 10 to 60 nucleotides (e.g., 10 to 20 nucleotides, 10 to 50 nucleotides, 10 to 40 nucleotides, 10 to 30 nucleotides).

[0232]

[0121] In some embodiments, the two or more covalently linked functional oligonucleotides have a total nucleotide length in the range of 10 nucleotides to 500 nucleotides (e.g., 10 nucleotides to 100 nucleotides, 50 nucleotides to 100 nucleotides, 50 nucleotides to 200 nucleotides, 20 nucleotides to 100 nucleotides, 20 nucleotides to 200 nucleotides, 20 nucleotides to 300 nucleotides, 50 nucleotides to 300 nucleotides, 20 nucleotides to 80 nucleotides, 100 nucleotides to 300 nucleotides, 300 nucleotides to 500 nucleotides).

[0233] 4.2.2 Nucleotide Modifications

[0122] All nucleotides of the oligonucleotides described herein may be natural, i.e., non-chemically modified nucleotides, but at least one nucleotide may be chemically modified.Non-limiting examples of chemical modifications may include one or a combination of the following: (1) modification of the phosphodiester bond of the nucleotide in the nucleotide sequence of the functional oligonucleotide (e.g., SMN2 saRNA); (2) modification of the 2'-OH of the ribose in the nucleotide sequence of the functional oligonucleotide; (3) modification of the base of the nucleotide sequence of the functional oligonucleotide.

[0234]

[0123] The chemical modification of nucleotide or oligonucleotide in the present disclosure is well known to those skilled in the art, and the modification of phosphodiester bond refers to the modification of oxygen in phosphodiester bond, including phosphorothioate modification and boronized phosphate modification.Both modifications stabilize the structure of oligonucleotide and maintain high specificity and high affinity for base pairing.

[0235]

[0124] Ribose modification is a modification of the 2'-OH of a nucleotide pentose, i.e., the introduction of a specific substituent at the hydroxyl position of ribose, and examples include 2'-fluoro modification, 2'-oxomethyl modification, 2'-oxyethylenemethoxy modification, 2,4'-dinitrophenol modification, locked nucleic acid (LNA), 2'-amino modification, and 2'-deoxy modification.

[0236]

[0125] Base modification refers to modification of the base of a nucleotide, and examples include 5'-bromouracil modification, 5'-iodouracil modification, N-methyluracil modification, and 2,6-diaminopurine modification.

[0237]

[0126] These modifications may increase the bioavailability of the oligonucleotide, increase the affinity for the target sequence, and improve resistance to intracellular nuclease hydrolysis.

[0238]

[0127] Furthermore, based on the above modifications, in order to facilitate entry of the oligonucleotide into cells, a lipophilic group such as cholesterol can be introduced to the end of the sense strand or antisense strand of the oligonucleotide, making it easier for the oligonucleotide to penetrate and act through the cell membrane and nuclear membrane, which are made of lipid bilayers, and the gene promoter region in the nucleus.

[0239]

[0128] The oligonucleotide agents disclosed herein, upon contact with a cell, are effective in activating or upregulating expression of one or more genes in the cell, preferably by at least 10% (e.g., at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%).

[0240] One aspect of the present disclosure provides a cell comprising two or more functional oligonucleotides of the present disclosure, or a nucleic acid encoding at least two oligonucleotides of the present disclosure.In one embodiment, the cell is a mammalian cell, preferably a human cell.Such cells may be ex vivo, such as a cell line, or may be present in a mammalian body, such as a human, including an infant, a child, or an adult.

[0241] In some embodiments, at least one oligonucleotide in at least two oligonucleotides of a multivalent oligonucleotide agent may comprise at least one modified nucleotide, such as 2'-O-methyl modified nucleotide, nucleotide comprising 5'-phosphorothioate group, terminal nucleotide linked to cholesteryl derivative or dodecanoic acid bisdecylamide group, 2'-deoxy-2'-fluoro modified nucleotide, 2'-deoxy modified nucleotide, locked nucleotide, abasic nucleotide, 2'-amino modified nucleotide, 2'-alkyl modified nucleotide, morpholino nucleotide, phosphoramidate, and nucleotide comprising unnatural base. In some embodiments, the first and second dsRNA comprise "end-right" modification with 2'-O-methyl modified nucleotide and nucleotide comprising 5'-phosphorothioate group.

[0242] In some embodiments, the chemical modification of at least one chemically modified nucleotide is the addition of an (E)-vinyl phosphonate moiety to the 5' end of the nucleotide sequence. In some embodiments, the chemical modification of at least one chemically modified nucleotide is the addition of an (E)-vinyl phosphonate moiety to the 5' end of the nucleotide sequence. 5-Methylcytosine It is the addition of a part.

[0243] In some embodiments, at least one oligonucleotide of the multivalent agent is chemically modified to enhance stability or other beneficial properties. The nucleic acids featured in this disclosure can be synthesized and / or modified by conventional methods, such as those described in "Current protocols in nucleic acid chemistry" Beaucage, SL et al. (Edrs.), John Wiley & Sons, Inc., New York, NY, USA, which is incorporated herein by reference. Modifications include, for example, (a) end modifications, such as 5' end modifications (phosphorylation, conjugation, reverse linkage, etc.) and 3' end modifications (conjugation, DNA nucleotides, reverse linkage, etc.), (b) base modifications, such as stabilizing bases, destabilizing bases, or bases that base pair with an expanded repertoire of partners, removal of bases (abasic nucleotides), or complexed bases, (c) sugar modifications or replacement of sugars (e.g., at the 2' or 4' positions), and (d) backbone modifications, including modification or replacement of phosphodiester bonds. Specific examples of RNA compounds that can be used in the present disclosure include, but are not limited to, RNAs that contain modified backbones or RNAs that do not contain natural internucleoside linkages. In some embodiments, RNAs with modified backbones include those that do not have a phosphorus atom in the backbone. In some embodiments, modified RNAs that do not have a phosphorus atom in the internucleoside backbone can also be considered oligonucleosides. In some embodiments, modified oligonucleotides have a phosphorus atom in their internucleoside backbone.

[0244] Modified oligonucleotide backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl phosphotriesters, methyl and other alkyl phosphonates including 3'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3'-amino phosphoramidates and aminoalkyl phosphoramidates, thionophosphoramidates, thioalkyl phosphonates, thioalkyl phosphotriesters, and boranophosphates with normal 3'-5' linkages, their 2'-5' linkage analogs, and those with reverse polarity where adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'. Also includes various salts, mixed salts, and free acid forms.

[0245]

[0134] Non-limiting examples of the preparation of phosphorus-containing linkages are described in U.S. Patent Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,195; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5 ,399,676;5,405,939;5,453,496;5,455,233;5,466,677;5,476,925;5,519,126;5,536,821;5,541,316;5,550,111;5,563,253;5,571,799;5,587,361;5,625, 050; 6,028,188; 6,124,445; 6,160,109; 6,169,170; 6,172,209; 6,239,265; 6,277,603; 6,326,199; 6,346,614; 6,444,423; 6,531,590, 6,534,639, 6,608,035, 6,683,167, 6,858,715, 6,867,294, 6,878,805, 7,015,315, 7,041,816, 7,273,933, 7,321,029, and U.S. Patent RE39464, which are incorporated by reference in their entireties.

[0246] 4.2.3 Covalent bonds

[0135] Aspects of the present disclosure include multivalent oligonucleotide agents that include two or more functional oligonucleotides covalently linked by a linking moiety, a phosphodiester bond, or one or more nucleotides.

[0247] In some embodiments, the functional oligonucleotide units in the MVO agent are linked by a covalent linker.In some embodiments, the linker is a disulfide linker.Various combinations of chains can be linked, for example, the first and second dsRNA sense strands are covalently linked, or for example, the first and second dsRNA antisense strands are covalently linked.In some embodiments, any of the multivalent oligonucleotide agents of the present disclosure comprises a ligand.

[0248]

[0137] In some embodiments, the linking components can include, but are not limited to, the following: --spacer phosphoramidite 18 (phosphoramidic acid, N,N-bis(1-methylethyl)-,19,19-bis(4-methoxyphenyl)-19-phenyl-3,6,9,12,15,18-hexaoxanonadecyl-1-yl 2-cyanoethyl ester); -- Spacer-9 (3- [2- [2- [2- [bis(4-methoxyphenyl)-phenylmethoxy]ethoxy]ethoxy]ethoxy- [di(propan-2-yl)amino]phosphanyl]oxypropanenitrile); Spacer phosphoramidite C3 (6-(4,4'-dimethoxytrityl)hexyl-1- [(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite); --Spacer C6 phosphoramidite (6-(4,4'-dimethoxytrityl)hexyl-1-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite).

[0249] In some embodiments, the linking moiety comprises a compound structure shown in Table 1.

[0250] [Table 5]

[0251] In some embodiments, two or more functional oligonucleotides are covalently linked by a phosphodiester bond. In some embodiments, two or more functional oligonucleotides are covalently linked by a phosphorothioate bond.

[0252]

[0140] In some embodiments, two or more functional oligonucleotides are covalently linked by one or more nucleotides.

[0253]

[0141] Non-limiting examples of covalent linkers can be found in US Patent Publication No. 20200332292, which is incorporated herein by reference in its entirety. Covalent linkers can link two or more functional oligonucleotides. In some embodiments, covalent linkers can link two sense strands, two antisense strands, one sense strand and one antisense strand, two sense strands and one antisense strand, two antisense strands and one sense strand, two sense strands and two antisense strands, antisense strand and ASO, sense strand and ASO, etc.

[0254] In certain embodiments, the covalent linker may comprise RNA and / or DNA and / or peptide. The linker may be single-stranded, double-stranded, partially single-stranded, or partially double-stranded. In some embodiments, the linker comprises a disulfide bond. The linker may be cleavable or non-cleavable.

[0255] In certain embodiments, the covalent linker may be, for example, dTsdTuu = (5'-2' deoxythymidyl-3'-thiophosphate-5'-2' deoxythymidyl-3'-phosphate-5'-uridyl-3'-phosphate); rUsrU (thiophosphate linker: 5'-uridyl-3'-thiophosphate-5'-uridyl-3'-phosphate); rUrU linker; dTsdTaa (aadTsdT, 5'-2' deoxythymidyl-3'-thiophosphate-5'-2' deoxythymidyl-3'-phosphate dTsdT (5'-2'deoxythymidyl-3'-thiophosphate-5'-2'deoxythymidyl-3'-phosphate); dTsdTuu = uudTsdT = 5'-2'deoxythymidyl-3'-thiophosphate-5'-2'deoxythymidyl-3'-phosphate-5'-uridyl-3'-phosphate-5'-uridyl-3'-phosphate.

[0256] In some embodiments, the covalent linker may comprise a polyRNA, such as poly(5'-adenyi-3'-phosphate-AAAAAAA) or poly(5'-cytidyl-3'-phosphate-5'-uridyl-3'-phosphate-CUCUCU)), e.g., a Xn single-stranded polyRNA linker, where n is an integer between 2 and 50 (inclusive), preferably between 4 and 15 (inclusive), and most preferably between 7 and 8 (inclusive). Modified nucleotides or mixtures of nucleotides may also be present in the polyRNA linker. The covalent linker may be a polyDNA, such as poly(5'-2'deoxythymidyl-3'-phosphate-TTTTTTTT), where n is an integer between 2 and 50 (inclusive), preferably between 4 and 15 (inclusive), and most preferably between 7 and 8 (inclusive). Modified nucleotides or mixtures of nucleotides may also be present in the polyDNA linker. In the single stranded poly DNA linker, n is an integer between 2 and 50 (inclusive), preferably between 4 and 15 (inclusive), most preferably between 7 and 8 (inclusive). Modified nucleotides or mixtures of nucleotides may also be present in said poly DNA linker.

[0257]

[0145] In some embodiments, the covalent linker may include a disulfide bond, and is optionally a bis-hexyl-disulfide linker. In one embodiment, the disulfide linker is as shown below:

[0258] [ka]

[0259] C 12 H 26 O 4 P.S. 2 (Accurate mass: 329.1010; Molecular weight: 329.4362)

[0260]

[0146] In some embodiments, the covalent linker may comprise a peptide bond, for example an amino acid. In one embodiment, the covalent linker is a long-chain linker of 1-10 amino acids, preferably 4-5 amino acids, optionally X-Gly-Phe-Gly-Y, where X and Y represent any amino acid.

[0261]

[0147] In some embodiments, the covalent linker may include a hexaethylene glycol linker, HEG.

[0262] 4.2.4 Covalent bond orientation Aspects of the present disclosure include covalently linking two or more functional oligonucleotides to form a multivalent oligonucleotide drug.The inventors have surprisingly found that the position and linkage orientation of two or more functional oligonucleotides affect the activity of the multivalent oligonucleotide drug in inducing or silencing or regulating target gene expression.In some embodiments, the orientation of two or more functional oligonucleotides and linkers can enhance stability, oligonucleotide activity, or other beneficial properties, such as maximizing target gene output or increasing or decreasing the activity or expression (e.g., mRNA expression, protein expression, etc.) of one or more target genes.

[0263] In some embodiments, two adjacent functional oligonucleotides are covalently linked at the 5' end of the first functional oligonucleotide and the 3' end of the second functional oligonucleotide, with or without a linker between them.In some embodiments, two adjacent functional oligonucleotides are covalently linked at the 3' end of the first functional oligonucleotide and the 5' end of the second functional oligonucleotide, with or without a linker between them.

[0264] For example, in some embodiments, the first ASO is covalently linked to the 3' end of the sense or antisense strand of the first dsRNA; or b) the first ASO is covalently linked to the 5' end of the sense or antisense strand of the first dsRNA. As another example, in certain embodiments, the 5' end of the first or second ASO is conjugated to a linking moiety. As another example, in certain embodiments, the 3' end of the first or second ASO is conjugated to a linking moiety.

[0265] 4.2.5 ASO Size in Multivalent Oligonucleotide Drugs

[0151] ASO usually has a length of 15-25 nucleotides. The present inventors have surprisingly found that the size of single-stranded ASO in a multivalent oligonucleotide drug affects the activity of the multivalent oligonucleotide drug in inducing or silencing target gene expression. For example, the size of ASO can be reduced from about 20nt to 12nt, or even 6nt, and the activity of the whole drug can be increased.

[0266]

[0152] In some embodiments, multivalent oligonucleotide drugs containing ASOs of a defined size can enhance stability, oligonucleotide activity, or other beneficial properties, such as maximizing target gene output or increasing or decreasing the activity or expression (e.g., mRNA expression, protein expression, etc.) of one or more target genes.

[0267]

[0153] In some embodiments, the ASO in the MVO drug has a nucleotide sequence that is at least 5 consecutive nucleotides in length, for example, 5 to 30 nucleotides in length, for example, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 nucleotides in length.

[0268]

[0154] The size of the ASO can be selected by testing and comparing the effect or activity of an MVO drug that includes said ASO.

[0269] 4.2.5 Subtypes of Multivalent Oligonucleotide Drugs

[0155] Disclosed herein are multivalent oligonucleotide agents comprising two or more covalently linked functional oligonucleotides, wherein the two or more functional oligonucleotides are independently selected from the group consisting of: a) double-stranded RNA (dsRNA); and b) antisense oligonucleotides (ASO).

[0270] The following is a description of bivalent and trivalent oligonucleotide drugs, including various functional oligonucleotides.However, such embodiments do not necessarily represent the full scope of the present invention, so refer to claims and this specification to interpret the scope of the present invention.

[0271] 4.2.5.1 Bivalent Oligonucleotide Drugs, Including dsRNA and ASOs In some embodiments, the present specification discloses a bivalent oligonucleotide drug that comprises two functional oligonucleotides, namely, a first double-stranded RNA (dsRNA) and a first antisense oligonucleotide (ASO).In such a bivalent oligonucleotide drug, dsRNA and ASO can be arranged in any order, for example, dsRNA-ASO or ASO-dsRNA.

[0272] In some embodiments, the dsRNA is selected from small interfering RNA (siRNA) or small activating RNA (saRNA), and the ASO is a gapmer or mixmer. In some embodiments, the two functional oligonucleotides independently regulate the expression of one or more genes, regulate the expression of one or more proteins (e.g., by binding to an mRNA sequence), or regulate a non-coding regulatory nucleic acid sequence (e.g., a promoter sequence, an enhancer, a silencer, and / or a transcription factor).

[0273] In some embodiments, the dsRNA comprises a sense strand that is at least 10 contiguous nucleotides and an antisense strand that is at least 10 contiguous nucleotides. In some embodiments, the dsRNA comprises a sense strand that is 10-60 nucleotides in length and / or an antisense strand that is 10-60 nucleotides in length. In some embodiments, the ASO has a nucleotide sequence that is at least 5 contiguous nucleotides in length. In some embodiments, the ASO has a nucleotide sequence that is 5-30 nucleotides in length. In some embodiments, the bivalent oligonucleotide agent has a total length in the range of 15-100 nucleotides.

[0274] In some embodiments, two adjacent functional oligonucleotides are covalently linked via a linking moiety or without a linking moiety. The linking moiety may be selected from the group consisting of spacer-9, spacer-18, spacer-C3 and spacer-C6, or derivatives thereof, or any suitable linking moiety disclosed herein or known in the art. In some embodiments, two adjacent functional oligonucleotides are covalently linked by:

[0275] [ka] , wherein R represents -H, or -OH, or -OMe, or -MOE, or -F, or other 2' chemical modification. In some embodiments, two adjacent functional oligonucleotides are covalently linked by a phosphodiester bond, or a phosphorothioate bond, or one or more nucleotides.

[0276]

[0161] In some embodiments, the functional oligonucleotide comprises at least one chemically modified nucleotide. The modification to the chemically modified nucleotide is one or more 2' sugar modifications selected from the group consisting of 2'-fluoro-2'-deoxynucleoside (2'-F) modification, 2'-O-methyl (2'-O-Me) modification, and 2'-O-(2-methoxyethyl) (2'-O-MOE) modification. In some embodiments, the chemical modification of the at least one chemically modified nucleotide is a phosphorothioate (PS) backbone modification. In some embodiments, the chemical modification of the at least one chemically modified nucleotide is a 5'-phosphate moiety, an (E)-vinylphosphonate moiety, or a 5'-phosphate moiety to the 5' end of the nucleotide sequence. 5-Methylcytosine In some embodiments, the functional oligonucleotide comprises one or more of the above modifications at one or more nucleotides (e.g., modification of one up to all nucleotides).

[0277] In some embodiments, the ASO in the agent is covalently linked to the adjacent dsRNA in a 3' to 5' or 5' to 3' direction. In some embodiments, the dsRNA in the agent is covalently linked to the ASO at the 3' end of the sense or antisense strand, or at the 5' end of the sense or antisense strand.

[0278] In some embodiments, the dsRNA is siRNA or saRNA, and the ASO is gapmer and mixmer.In some embodiments, the multivalent oligonucleotide agent comprises a functional oligonucleotide selected from the group consisting of: (a) siRNA-gapmer; (b) siRNA-mixmer; (c) saRNA-gapmer; and (d) saRNA-mixmer; where in any one of (a) to (d), the functional oligonucleotide is arranged in any order and is covalently linked via or without a linking moiety.In some embodiments, the ASO targets 5'-UTR.

[0279] In some aspects, the dsRNA and / or ASO increases expression of the SMN2 gene or protein. In some embodiments, the dsRNA increases expression of the SMN2 gene or protein; and / or the ASO increases production of functional SMN protein by modulating splicing or stability of SMN2 mRNA (SMN2 mRNA modulators).

[0280]

[0165] In some embodiments, the dsRNA comprises a saRNA having a nucleotide sequence of the sense strand that has at least 90% identity to a nucleotide sequence selected from the group consisting of: a) DS06-0004 (SEQ ID NO:5); b) DS06-0031 (SEQ ID NO:7); c) DS06-0067 (SEQ ID NO:9); d) DS06-4A3 (SEQ ID NO:146); e) R6-04-S1 (SEQ ID NO:59); and f) R6-04(20)-S1V1v(CM-4) (SEQ ID NO:60).

[0281]

[0166] In some embodiments, the dsRNA comprises a saRNA having an antisense strand nucleotide sequence that has at least 90% identity to a nucleotide sequence selected from the group consisting of: a) DS06-0004 (SEQ ID NO:6); b) DS06-0031 (SEQ ID NO:8); c) DS06-0067 (SEQ ID NO:10); d) DS06-4A3 (SEQ ID NO:147); e) R6-04-S1 (SEQ ID NO:53); and f) R6-04M1-27A-S1L1V3 (CM-26) (SEQ ID NO:17).

[0282]

[0167] In some embodiments, the dsRNA comprises a saRNA having a sense and antisense nucleotide sequence pair that has at least 90% identity to a nucleotide sequence pair selected from the group consisting of: a) DS06-0004: SEQ ID NO:5 and SEQ ID NO:6; b) DS06-0031: SEQ ID NO:7 and SEQ ID NO:8; c) DS06-0067: SEQ ID NO:9 and SEQ ID NO:10; d) DS06-4A3: SEQ ID NO:146 and SEQ ID NO:147; e) R6-04-S1: SEQ ID NO:59 and SEQ ID NO:53; and f) R6-04(20)-S1V1v(CM-4): SEQ ID NO:60 and SEQ ID NO:17.

[0283]

[0168] In some embodiments, the dsRNA comprises an siRNA having a nucleotide sequence of a sense strand that has at least 90% identity with the nucleotide sequence of DS06-332i (SEQ ID NO:3) or siSOD1-388-ESC (SEQ ID NO:138). In some embodiments, the dsRNA comprises an siRNA having a nucleotide sequence of an antisense strand that has at least 90% identity with the nucleotide sequence of DS06-332i (SEQ ID NO:4) or siSOD1-388-ESC (SEQ ID NO:139). In some embodiments, the dsRNA comprises an siRNA having a nucleotide sequence pair of a sense strand and an antisense strand that has at least 90% identity with a nucleotide sequence pair selected from the group consisting of: DS06-332i: SEQ ID NO:3 and SEQ ID NO:4; and siSOD1-388-ESC: SEQ ID NO:138 and SEQ ID NO:139.

[0284] In some embodiments, a multivalent oligonucleotide agent has a nucleotide sequence having at least 90% identity to a nucleotide sequence selected from the group consisting of: a) DA06-4A-27A (SEQ ID NO:14) and an antisense saRNA strand having the nucleotide sequence of SEQ ID NO:13 having partial complementarity to the sense saRNA strand of SEQ ID NO:14; b) DA06-4A-27B (SEQ ID NO:15) and an antisense saRNA strand having the nucleotide sequence of SEQ ID NO:13 having partial complementarity to the sense saRNA strand of SEQ ID NO:15; c) R6-04M1-27A-S1L1V3 (SEQ ID NO: 18) and SEQ an antisense saRNA strand having the nucleotide sequence of SEQ ID NO:13, which has partial complementarity to the sense saRNA strand of ID NO:18; d) DA06-31A-27A (SEQ ID NO:19) and an antisense saRNA strand having the nucleotide sequence of SEQ ID NO:8, which has partial complementarity to the sense saRNA strand of SEQ ID NO:19; e) DA06-31B-27A (SEQ ID NO:20) and a sense saRNA strand having the nucleotide sequence of SEQ ID NO:7 having partial complementarity to the antisense saRNA strand of SEQ ID NO:20; f) DA06-67A-27A (SEQ ID NO:21) and an antisense saRNA strand having the nucleotide sequence of SEQ ID NO:10 having partial complementarity to the sense saRNA strand of SEQ ID NO:21; g) DA06-67B-27A (SEQ ID NO:22) and a sense saRNA strand having the nucleotide sequence of SEQ ID NO:9 having partial complementarity to the antisense saRNA strand of SEQ ID NO:22; h)DA6-67A3'L0-27A(SEQ ID NO:23) and SEQ ID an antisense saRNA strand having the nucleotide sequence of SEQ ID NO:10, which has partial complementarity to the sense saRNA strand of NO:23; j)DA6-67A3'L9-27A(SEQ ID NO:24) and SEQ ID an antisense saRNA strand having the nucleotide sequence of SEQ ID NO:10, which has partial complementarity to the sense saRNA strand of NO:24; k)DA6-67A3'L4-27A(SEQ ID NO:25) and SEQ ID an antisense saRNA strand having the nucleotide sequence of SEQ ID NO:10, which has partial complementarity to the sense saRNA strand of NO:25; l) DA6-67B3'L0-27A (SEQ ID NO: 26) and SEQ ID a sense saRNA strand having the nucleotide sequence of SEQ ID NO:9, which has partial complementarity to the antisense saRNA strand of NO:26; m)DA6-67B5'L1-27A(SEQ ID NO:27) and SEQ ID a sense saRNA strand having the nucleotide sequence of SEQ ID NO:9, which has partial complementarity to the antisense saRNA strand of NO:27; o)DA6-67B5'L9-27A(SEQ ID NO:29) and SEQ ID a sense saRNA strand having the nucleotide sequence of SEQ ID NO:9, which has partial complementarity to the antisense saRNA strand of NO:29; p) DA6-67B5'L4-27A (SEQ ID NO: 30) and SEQ ID a sense saRNA strand having the nucleotide sequence of SEQ ID NO:9, which has partial complementarity to the antisense saRNA strand of NO:30; q) DA6-67B3'L9-27A (SEQ ID NO: 31) and SEQ ID a sense saRNA strand having the nucleotide sequence of SEQ ID NO:9, which has partial complementarity to the antisense saRNA strand of NO:31; r) DA6-67B3'L4-27A (SEQ ID NO: 32) and SEQ ID a sense saRNA strand having the nucleotide sequence of SEQ ID NO:9, which has partial complementarity to the antisense saRNA strand of NO:32; s) DA06-67A21L1-27A (SEQ ID NO:33) and an antisense saRNA strand having the nucleotide sequence of SEQ ID NO:34, which has partial complementarity to the sense saRNA strand of SEQ ID NO:33; t) DA06-67B21L1-27A (SEQ ID NO:36) and a sense saRNA strand of SEQ ID NO:35 having partial complementarity to the antisense saRNA strand of SEQ ID NO:36; u) DA6-04A3'L0-27A (SEQ ID NO: 37) and SEQ ID an antisense saRNA strand having the nucleotide sequence of SEQ ID NO:6, which has partial complementarity to the sense saRNA strand of NO:37; v)DA6-04A5'L1-27A(SEQ ID NO:38) and SEQ ID an antisense saRNA strand having the nucleotide sequence of SEQ ID NO:6, which has partial complementarity to the sense saRNA strand of NO:38; w) DA6-04A5'L9-27A (SEQ ID NO: 39) and SEQ ID an antisense saRNA strand having the nucleotide sequence of SEQ ID NO:6, which has partial complementarity to the sense saRNA strand of NO:39; x)DA6-04A5'L4-27A (SEQ ID NO: 40) and SEQ ID an antisense saRNA strand having the nucleotide sequence of SEQ ID NO:6, which has partial complementarity to the sense saRNA strand of NO:40; y) DA6-04A3'L1-27A (SEQ ID NO: 41) and SEQ ID an antisense saRNA strand having the nucleotide sequence of SEQ ID NO:6, which has partial complementarity to the sense saRNA strand of NO:41; z)DA6-04A3'L9-27A(SEQ ID NO:42) and SEQ ID an antisense saRNA strand having the nucleotide sequence of SEQ ID NO:6, which has partial complementarity to the sense saRNA strand of NO:42; aa) DA6-04A3'L4-27A (SEQ ID NO:43) and an antisense saRNA strand having the nucleotide sequence of SEQ ID NO:6 having partial complementarity to the sense saRNA strand of SEQ ID NO:43; bb) DA6-04B3'L0-27A (SEQ ID NO:44) and a sense saRNA strand having the nucleotide sequence of SEQ ID NO:5, which has partial complementarity to the antisense saRNA strand of SEQ ID NO:44; cc) DA6-04B3'L1-27A (SEQ ID NO:45) and a sense saRNA strand having the nucleotide sequence of SEQ ID NO:5 having partial complementarity to the antisense saRNA strand of SEQ ID NO:45; dd) DA6-04B3'L9-27A (SEQ ID NO:46) and a sense saRNA strand having the nucleotide sequence of SEQ ID NO:5 having partial complementarity to the antisense saRNA strand of SEQ ID NO:46; ee) DA6-04B3'L4-27A (SEQ ID NO:47) and a sense saRNA strand having the nucleotide sequence of SEQ ID NO:5 having partial complementarity to the antisense saRNA strand of SEQ ID NO:47; ff) DA06-04A21L1-27A (SEQ ID NO:48) and an antisense saRNA strand having the nucleotide sequence of SEQ ID NO:49 having partial complementarity to the sense saRNA strand of SEQ ID NO:48; gg) DA06-04B21L1-27A (SEQ ID NO:51) and a sense saRNA strand of SEQ ID NO:50 having partial complementarity to the antisense saRNA strand of SEQ ID NO:51; hh) R6-04M1-16nt-S1L1V3v (SEQ ID NO:79) and an antisense saRNA strand of SEQ ID NO:17 having partial complementarity to the sense saRNA strand of SEQ ID NO:79; ii) R6-04M1-15nt-S1L1V3v (SEQ ID NO:80) and an antisense saRNA strand of SEQ ID NO:17 having partial complementarity to the sense saRNA strand of SEQ ID NO:80; jj) R6-04M1-14nt-S1L1V3v (SEQ ID NO:81) and an antisense saRNA strand of SEQ ID NO:17 having partial complementarity to the sense saRNA strand of SEQ ID NO:81; kk) R6-04M1-13nt-S1L1V3v (SEQ ID NO:82) and an antisense saRNA strand of SEQ ID NO:17 having partial complementarity to the sense saRNA strand of SEQ ID NO:82; ll) R6-04M1-(12nt-B)-S1L1V3v (SEQ ID NO:83) and an antisense saRNA strand of SEQ ID NO:17 having partial complementarity to the sense saRNA strand of SEQ ID NO:83; mm) R6-04M1-11nt-S1L1V3v (SEQ ID NO:84) and an antisense saRNA strand of SEQ ID NO:17 having partial complementarity to the antisense saRNA strand of SEQ ID NO:84; nn) R6-04M1-10nt-S1L1V3v (SEQ ID NO:85) and an antisense saRNA strand of SEQ ID NO:17 having partial complementarity to the antisense saRNA strand of SEQ ID NO:85; oo) R6-04M1-9nt-S1L1V3v (SEQ ID NO:86) and an antisense saRNA strand of SEQ ID NO:17 having partial complementarity to the sense saRNA strand of SEQ ID NO:86; pp) R6-04M1-8nt-S1L1V3v (SEQ ID NO:87) and an antisense saRNA strand of SEQ ID NO:17 having partial complementarity with the sense saRNA strand of SEQ ID NO:87; qq) R6-04M1-7nt-S1L1V3v (SEQ ID NO:88) and an antisense saRNA strand of SEQ ID NO:17 having partial complementarity to the sense saRNA strand of SEQ ID NO:88; rr) R6-04M1-6nt-S1L1V3v (SEQ ID NO:89) and an antisense saRNA strand of SEQ ID NO:17 having partial complementarity to the sense saRNA strand of SEQ ID NO:89; ss)DS06-4A-S2L5V(SEQ ID NO:128) and SEQ ID an antisense saRNA strand having the nucleotide sequence of SEQ ID NO:17, which has partial complementarity to the sense saRNA strand of NO:128; ss')DS06-4A-S2L1v(SEQ ID NO:16) and SEQ ID an antisense saRNA strand having the nucleotide sequence of SEQ ID NO:17, which has partial complementarity to the sense saRNA strand of NO:16; tt)DA6-27A-5'UTR(SE tt)DA6-27A-5'UTR(SEQ ID NO:143); uu)DA6-5'UTR-27A(SEQ ID NO:144); vv) R6-67M3-27A-S1L1V3 (SEQ ID NO:129) and an antisense saRNA strand having the nucleotide sequence of SEQ ID NO:130 having partial complementarity to the sense saRNA strand of SEQ ID NO:129; ww) R6-67M3-16nt-S1L1V3 (SEQ ID NO: 129) and an antisense saRNA strand having the nucleotide sequence of SEQ ID NO: 131, which has partial complementarity to the sense saRNA strand of SEQ ID NO: 129; xx) R6-67M3-15nt-S1L1V3 (SEQ ID NO:129) and an antisense saRNA strand having the nucleotide sequence of SEQ ID NO:132 having partial complementarity to the sense saRNA strand of SEQ ID NO:129; yy) R6-67M3-14nt-S1L1V3 (SEQ ID NO:129) and an antisense saRNA strand having the nucleotide sequence of SEQ ID NO:133, which has partial complementarity to the sense saRNA strand of SEQ ID NO:129; zz) R6-67M3-13nt-S1L1V3 (SEQ ID NO:129) and an antisense saRNA strand having the nucleotide sequence of SEQ ID NO:134, which has partial complementarity to the sense saRNA strand of SEQ ID NO:129; aaa) R6-67M3-12nt-S1L1V3 (SEQ ID NO: 129) and SEQ ID NO: 129 having partial complementarity to the sense saRNA strand of SEQ ID NO: 129 NO:Antisense saRNA strand having the nucleotide sequence of 135; bbb) R6-67M3-9nt-S1L1V3 (SEQ ID NO:129) and an antisense saRNA strand having the nucleotide sequence of SEQ ID NO:136, which has partial complementarity to the sense saRNA strand of SEQ ID NO:129; ccc) R6-67M3-8nt-S1L1V3 (SEQ ID NO:129) and an antisense saRNA strand having the nucleotide sequence of SEQ ID NO:137 having partial complementarity to the sense saRNA strand of SEQ ID NO:129; wherein a linker selected from the group consisting of L1, L4 and L9 is present or absent; wherein L1 represents spacer-18; L4 represents spacer-C6; and L9 represents spacer-9.

[0285] In some embodiments, the multivalent oligonucleotide agent is as listed in Tables 7-11 and Tables 13-14. In some embodiments, the linking moieties and / or linking bonds and / or orientations of the multivalent oligonucleotide agents described above can be varied.

[0286] In some embodiments, the one or more functional oligonucleotides increase expression of the CDKN1A / p21 gene or protein and / or decrease expression of CD274 / PDL-1. In some embodiments, the dsRNA is a saRNA that increases expression of the CDKN1A / p21 gene or protein; and / or a siRNA that decreases expression of CD274 / PDL-1. In some embodiments, the ASO is an ASO that increases expression of the CDKN1A / p21 gene or protein and / or decreases expression of CD274 / PDL-1.

[0287] In some embodiments, the dsRNA is RAG1-40 (SEQ ID NO: In some embodiments, the dsRNA is a saRNA having a nucleotide sequence of the sense strand that has at least 90% identity to the nucleotide sequence of RAG1-40 (SEQ ID NO:62). In some embodiments, the dsRNA is a saRNA having a nucleotide sequence of the antisense strand that has at least 90% identity to the nucleotide sequence of RAG1-40 (SEQ ID NO:63). In some embodiments, the dsRNA is a saRNA having a nucleotide sequence of the sense strand that has at least 90% identity to the nucleotide sequence of RAG1-40 (SEQ ID NO:62) and a nucleotide sequence of the antisense strand that has at least 90% identity to the nucleotide sequence of RAG1-40 (SEQ ID NO:63).

[0288] In some embodiments, the dsRNA is a) siPDL1-2(SEQ ID NO:1) In some embodiments, the dsRNA is an siRNA having a nucleotide sequence of the sense strand that has at least 90% identity to a nucleotide sequence selected from a) siPDL1-2 (SEQ ID NO:65) and b) siPDL1-3 (SEQ ID NO:67). In some embodiments, the dsRNA is an siRNA selected from the group consisting of: a) an siRNA having a sense strand nucleotide sequence that has at least 90% identity to the nucleotide sequence of siPDL1-2 (SEQ ID NO:64) and an antisense strand nucleotide sequence that has at least 90% identity to the nucleotide sequence of siPDL1-2 (SEQ ID NO:65); and b) an siRNA having a sense strand nucleotide sequence that has at least 90% identity to the nucleotide sequence of siPDL1-2 (SEQ ID NO:66) and an antisense strand nucleotide sequence that has at least 90% identity to the nucleotide sequence of siPDL1-2 (SEQ ID NO:67).

[0289] In some embodiments, the ASO is a) aPDL1-1 (SEQ ID NO: a) aPDL1-2 (SEQ ID NO:69); and c) aPDL1-3 (SEQ ID NO:70).

[0290] In some embodiments, a multivalent oligonucleotide agent has a nucleotide sequence having at least 90% identity to a nucleotide sequence selected from the group consisting of: a) saP21-40 / aPDL1-1 (SEQ ID NO:72) and an antisense saRNA strand having the nucleotide sequence of SEQ ID NO:63, which has partial complementarity to the sense saRNA strand of SEQ ID NO:72; b) saP21-40 / aPDL1-2 (SEQ ID NO:73) and an antisense saRNA strand having the nucleotide sequence of SEQ ID NO:63, which has partial complementarity to the sense saRNA strand of SEQ ID NO:73; c) saP21-40 / aPDL1-3 (SEQ ID NO:74) and an antisense saRNA strand having the nucleotide sequence of SEQ ID NO:63, which has partial complementarity to the sense saRNA strand of SEQ ID NO:74; d) saP21-40 / aPDL1-1R (SEQ ID NO:75) and an antisense saRNA strand having the nucleotide sequence of SEQ ID NO:63, which has partial complementarity to the sense saRNA strand of SEQ ID NO:75; e) saP21-40 / aPDL1-2R (SEQ ID NO:76) and an antisense saRNA strand having the nucleotide sequence of SEQ ID NO:63 having partial complementarity to the sense saRNA strand of SEQ ID NO:76; and f) saP21-40 / aPDL1-3R (SEQ ID NO:77) and an antisense saRNA strand having the nucleotide sequence of SEQ ID NO:63 which has partial complementarity to the sense saRNA strand of SEQ ID NO:77.

[0291] In some embodiments, the multivalent oligonucleotide agents are selected from, or have at least 90% sequence identity to, those depicted in Table 16. In some embodiments, the linking moieties and / or linking bonds and / or orientations of those multivalent oligonucleotide agents are variable.

[0292] 4.2.5.2 dsRNA and dsRNA-Containing Bivalent Oligonucleotide Drugs In some embodiments, the present specification discloses a bivalent oligonucleotide drug that comprises two functional oligonucleotides, namely a first double-stranded RNA (dsRNA) and a second double-stranded RNA (dsRNA).In such a bivalent oligonucleotide drug, dsRNA can be arranged in any order, for example, dsRNA1-dsRNA2 or dsRNA2-dsRNA1; and two functional oligonucleotides can be covalently linked via linker or bond.

[0293]

[0178] In some embodiments, the dsRNA is selected from a small interfering RNA (siRNA) or a small activating RNA (saRNA), and the two functional oligonucleotides independently regulate the expression of one or more genes, regulate the expression of one or more proteins (e.g., by binding to an mRNA sequence), or regulate a non-coding regulatory nucleic acid sequence (e.g., a promoter sequence, enhancer, silencer, and / or transcription factor).

[0294] In some embodiments, the dsRNA comprises a sense strand that is at least 10 consecutive nucleotides and an antisense strand that is at least 10 consecutive nucleotides. In some embodiments, the dsRNA comprises a sense strand that is 10-60 nucleotides in length and / or an antisense strand that is 10-60 nucleotides in length. In some embodiments, the bivalent oligonucleotide agent has a total length in the range of 20-200 nucleotides.

[0295] In some embodiments, two adjacent functional oligonucleotides are covalently linked via a linking moiety or without a linking moiety. The linking moiety may be selected from the group consisting of spacer-9, spacer-18, spacer-C3 and spacer-C6, or derivatives thereof, or any suitable linking moiety disclosed herein or known in the art. In some embodiments, two adjacent functional oligonucleotides are covalently linked by:

[0296] [ka] , wherein R represents -H, or -OH, or -OMe, or -MOE, or -F, or other 2' chemical modification. In some embodiments, two adjacent functional oligonucleotides are covalently linked by a phosphodiester bond, or a phosphorothioate bond, or one or more nucleotides.

[0297]

[0181] In some embodiments, the functional oligonucleotide comprises at least one chemically modified nucleotide. The modification to the chemically modified nucleotide is one or more 2' sugar modifications selected from the group consisting of 2'-fluoro-2'-deoxynucleoside (2'-F) modification, 2'-O-methyl (2'-O-Me) modification, and 2'-O-(2-methoxyethyl) (2'-O-MOE) modification. In some embodiments, the chemical modification of the at least one chemically modified nucleotide is a phosphorothioate (PS) backbone modification. In some embodiments, the chemical modification of the at least one chemically modified nucleotide is a 5'-phosphate moiety, an (E)-vinylphosphonate moiety, or a 5'-phosphate moiety to the 5' end of the nucleotide sequence. 5-Methylcytosine In some embodiments, the functional oligonucleotide comprises one or more of the above modifications at one or more nucleotides (e.g., modification of one up to all nucleotides).

[0298] In some embodiments, the dsRNA in the agent is covalently linked in the 3' to 5' direction or the 5' to 3' direction.In some embodiments, the first dsRNA in the agent is covalently linked to the second dsRNA at the 3' end of the sense strand or the antisense strand, or at the 5' end of the sense strand or the antisense strand.

[0299] In some embodiments, dsRNA is siRNA or saRNA.In some embodiments, the multivalent oligonucleotide drug comprises a functional oligonucleotide selected from the group consisting of: (a) siRNA-siRNA; (b) siRNA-saRNA; (c) saRNA-saRNA; provided that in any one of (a) to (c), the functional oligonucleotide is arranged in any order and is covalently linked via or without a linking moiety.In some embodiments, the functional oligonucleotides in the same drug can be the same or different.

[0300]

[0184] In some embodiments, the dsRNA increases / suppresses expression of the SMN2 gene or protein.

[0301]

[0185] In some embodiments, the dsRNA comprises a saRNA having a nucleotide sequence of the sense strand that has at least 90% identity to a nucleotide sequence selected from the group consisting of: a) DS06-0004 (SEQ ID NO:5); b) DS06-0031 (SEQ ID NO:7); c) DS06-0067 (SEQ ID NO:9); d) DS06-4A3 (SEQ ID NO:146); e) R6-04-S1 (SEQ ID NO:59); and f) R6-04(20)-S1V1v(CM-4) (SEQ ID NO:60).

[0302]

[0186] In some embodiments, the dsRNA comprises a saRNA having a nucleotide sequence of the antisense strand that has at least 90% identity to a nucleotide sequence selected from the group consisting of: a) DS06-0004 (SEQ ID NO:6); b) DS06-0031 (SEQ ID NO:8); c) DS06-0067 (SEQ ID NO:10); d) DS06-4A3 (SEQ ID NO:147); e) R6-04-S1 (SEQ ID NO:53); and f) R6-04M1-27A-S1L1V3 (CM-26) (SEQ ID NO:17).

[0303]

[0187] In some embodiments, the dsRNA comprises a saRNA having a sense and antisense nucleotide sequence pair that has at least 90% identity to a nucleotide sequence pair selected from the group consisting of: a) DS06-0004: SEQ ID NO:5 and SEQ ID NO:6; b) DS06-0031: SEQ ID NO:7 and SEQ ID NO:8; c) DS06-0067: SEQ ID NO:9 and SEQ ID NO:10; d) DS06-4A3: SEQ ID NO:146 and SEQ ID NO:147; e) R6-04-S1: SEQ ID NO:59 and SEQ ID NO:53; and f) R6-04(20)-S1V1v(CM-4): SEQ ID NO:60 and SEQ ID NO:17.

[0304]

[0188] In some embodiments, the dsRNA comprises an siRNA having a nucleotide sequence of a sense strand that has at least 90% identity with the nucleotide sequence of DS06-332i (SEQ ID NO:3) or siSOD1-388-ESC (SEQ ID NO:138). In some embodiments, the dsRNA comprises an siRNA having a nucleotide sequence of an antisense strand that has at least 90% identity with the nucleotide sequence of DS06-332i (SEQ ID NO:4) or siSOD1-388-ESC (SEQ ID NO:139). In some embodiments, the dsRNA comprises an siRNA having a nucleotide sequence pair of a sense strand and an antisense strand that has at least 90% identity with a nucleotide sequence pair selected from the group consisting of: DS06-332i: SEQ ID NO:3 and SEQ ID NO:4; and siSOD1-388-ESC: SEQ ID NO:138 and SEQ ID NO:139.

[0305] In some embodiments, a multivalent oligonucleotide agent has a nucleotide sequence having at least 90% identity to a nucleotide sequence selected from the group consisting of: a)R6-04S1&67S1R-L1V2(SEQ ID NO:52) and SEQ an antisense saRNA strand having a nucleotide sequence of SEQ ID NO:53, which has partial complementarity with the strand of SEQ ID NO:52, and an antisense saRNA strand having a nucleotide sequence of SEQ ID NO:78, which has partial complementarity with the strand of SEQ ID NO:52; b) R6-04S1&67S5-L1V2 (SEQ ID NO:56), an antisense saRNA strand having a nucleotide sequence of SEQ ID NO:53 that has partial complementarity with the strand of SEQ ID NO:56, and an antisense saRNA strand having a nucleotide sequence of SEQ ID NO:78 that has partial complementarity with the strand of SEQ ID NO:56; c) R6-04M1&R17-388E-L1V2 (SEQ ID NO:140), an antisense saRNA strand of SEQ ID NO:17 having partial complementarity with a strand of SEQ ID NO:99, and an antisense saRNA strand of SEQ ID NO:141 having partial complementarity with a strand of SEQ ID NO:99; d) DS06-4A-S2L1v (SEQ ID NO:16) and an antisense saRNA strand of SEQ ID NO:17 having partial complementarity to the strand of SEQ ID NO:16; and e) DS06-4A-S2L5V (SEQ ID NO:128) and an antisense saRNA strand having the nucleotide sequence of SEQ ID NO:17 which has partial complementarity to the sense saRNA strand of SEQ ID NO:128.

[0306] In some embodiments, the multivalent oligonucleotide agent is as listed in Tables 12 and 15. In some embodiments, the linking moieties and / or linking bonds and / or orientations of the multivalent oligonucleotide agents described above are variable.

[0307] In some embodiments, the one or more functional oligonucleotides increase expression of the CDKN1A / p21 gene or protein and / or decrease expression of CD274 / PDL-1. In some embodiments, the dsRNA is a saRNA that increases expression of the CDKN1A / p21 gene or protein; and / or a siRNA that decreases expression of CD274 / PDL-1.

[0308] In some embodiments, the dsRNA is RAG1-40 (SEQ ID NO: In some embodiments, the dsRNA is a saRNA having a nucleotide sequence of the sense strand that has at least 90% identity to the nucleotide sequence of RAG1-40 (SEQ ID NO:62). In some embodiments, the dsRNA is a saRNA having a nucleotide sequence of the antisense strand that has at least 90% identity to the nucleotide sequence of RAG1-40 (SEQ ID NO:63). In some embodiments, the dsRNA is a saRNA having a nucleotide sequence of the sense strand that has at least 90% identity to the nucleotide sequence of RAG1-40 (SEQ ID NO:62) and a nucleotide sequence of the antisense strand that has at least 90% identity to the nucleotide sequence of RAG1-40 (SEQ ID NO:63).

[0309] In some embodiments, the dsRNA is a) siPDL1-2(SEQ ID NO:1) In some embodiments, the dsRNA is an siRNA having a nucleotide sequence of the sense strand that has at least 90% identity to a nucleotide sequence selected from a) siPDL1-2 (SEQ ID NO:65) and b) siPDL1-3 (SEQ ID NO:67). In some embodiments, the dsRNA is an siRNA selected from the group consisting of: a) an siRNA having a sense strand nucleotide sequence that has at least 90% identity to the nucleotide sequence of siPDL1-2 (SEQ ID NO:64) and an antisense strand nucleotide sequence that has at least 90% identity to the nucleotide sequence of siPDL1-2 (SEQ ID NO:65); and b) an siRNA having a sense strand nucleotide sequence that has at least 90% identity to the nucleotide sequence of siPDL1-2 (SEQ ID NO:66) and an antisense strand nucleotide sequence that has at least 90% identity to the nucleotide sequence of siPDL1-2 (SEQ ID NO:67).

[0310] In some embodiments, a multivalent oligonucleotide agent has a nucleotide sequence having at least 90% identity to a nucleotide sequence selected from the group consisting of: a) saP21-40 / siPDL1-2 (SEQ ID NO:71), an antisense saRNA strand having a nucleotide sequence of SEQ ID NO:63 that has partial complementarity with a strand of SEQ ID NO:71, and an antisense siRNA strand having a nucleotide sequence of SEQ ID NO:65 that has partial complementarity with a strand of SEQ ID NO:71; and b) saP21-40 / siPDL1-3 (SEQ ID NO:100) and SEQ an antisense saRNA strand having the nucleotide sequence of SEQ ID NO:63, which has partial complementarity with the strand of SEQ ID NO:100, and an antisense saRNA strand having the nucleotide sequence of SEQ ID NO:65, which has partial complementarity with the strand of SEQ ID NO:100.

[0311] In some embodiments, the multivalent oligonucleotide agents are selected from, or have at least 90% sequence identity to, those shown in Table 16. In some embodiments, the linking moieties and / or linking bonds and / or orientations of those multivalent oligonucleotide agents are variable.

[0312] 4.2.5.3 ASOs and bivalent oligonucleotide drugs that contain ASOs In some embodiments, the present specification discloses a bivalent oligonucleotide agent that comprises two functional oligonucleotides, namely, a first antisense oligonucleotide (ASO1) and a second antisense oligonucleotide (ASO2).In such a bivalent oligonucleotide agent, dsRNA and ASO can be arranged in any order, for example, ASO1-ASO2 or ASO2-ASO1.Both ASOs can be the same or different.

[0313] In some embodiments, the ASO is independently selected from a gapmer or a mixmer. In some embodiments, the two functional oligonucleotides independently regulate the expression of one or more genes, regulate the expression of one or more proteins (e.g., by binding to an mRNA sequence), or regulate a non-coding regulatory nucleic acid sequence (e.g., a promoter sequence, an enhancer, a silencer, and / or a transcription factor).

[0314] In some embodiments, the ASO has a nucleotide sequence that is at least 5 contiguous nucleotides in length. In some embodiments, the ASO has a nucleotide sequence that is 5-30 nucleotides in length. In some embodiments, the bivalent oligonucleotide agent has a total length in the range of 10-100 nucleotides.

[0315] In some embodiments, two adjacent functional oligonucleotides are covalently linked via a linking moiety or without a linking moiety. The linking moiety may be selected from the group consisting of spacer-9, spacer-18, spacer-C3 and spacer-C6, or derivatives thereof, or any suitable linking moiety disclosed herein or known in the art. In some embodiments, two adjacent functional oligonucleotides are covalently linked by:

[0316] [ka] , wherein R represents -H, or -OH, or -OMe, or -MOE, or -F, or other 2' chemical modification. In some embodiments, two adjacent functional oligonucleotides are covalently linked by a phosphodiester bond, or a phosphorothioate bond, or one or more nucleotides.

[0317]

[0200] In some embodiments, the functional oligonucleotide comprises at least one chemically modified nucleotide. The modification to the chemically modified nucleotide is one or more 2' sugar modifications selected from the group consisting of 2'-fluoro-2'-deoxynucleoside (2'-F) modification, 2'-O-methyl (2'-O-Me) modification, and 2'-O-(2-methoxyethyl) (2'-O-MOE) modification. In some embodiments, the chemical modification of the at least one chemically modified nucleotide is a phosphorothioate (PS) backbone modification. In some embodiments, the chemical modification of the at least one chemically modified nucleotide is a 5'-phosphate moiety, an (E)-vinylphosphonate moiety, or a 5'-phosphate moiety to the 5' end of the nucleotide sequence. 5-Methylcytosine In some embodiments, the functional oligonucleotide comprises one or more of the above modifications at one or more nucleotides (e.g., modification of one up to all nucleotides).

[0318] In some embodiments, the first ASO in the agent is covalently linked to the second ASO in the 3' to 5' or 5' to 3' direction. In some embodiments, the first ASO in the agent is covalently linked to the second ASO at its 3' end or its 5' end.

[0319] In some embodiments, ASO is selected from gapmer and mixmer.In some embodiments, multivalent oligonucleotide drug comprises functional oligonucleotide selected from the group consisting of: (a) mixmer-mixmer; (b) gapmer-mixmer; (c) gapmer-gapmer; provided that in any one of (a) to (c), functional oligonucleotide is arranged in any order and is covalently linked via linking moiety or not via linking moiety.In some embodiments, ASO targets 5'-UTR.

[0320] In some aspects, the ASO increases expression of the SMN2 gene or protein. In some embodiments, the ASO increases production of functional SMN protein by modulating splicing or stability of SMN2 mRNA (SMN2 mRNA modulator).

[0321]

[0204] In some embodiments, the ASO has a nucleotide sequence that is at least 90% identical to the nucleotide sequence of ASO10-27 (SEQ ID NO:11) or 5'UTR ASO (SEQ ID NO:142).

[0322] In some embodiments, a multivalent oligonucleotide agent has a nucleotide sequence having at least 90% identity to a nucleotide sequence selected from the group consisting of: a) DA6-27A-5'UTR (SEQ ID NO:143); and b)DA6-5'UTR-27A(SEQ ID NO:144).

[0323] In some embodiments, the multivalent oligonucleotide agents are selected from, or have at least 90% sequence identity to, those shown in Table 17. In some embodiments, the linking moieties and / or linking bonds and / or orientations of those multivalent oligonucleotide agents are variable.

[0324] 4.2.5.4 Trivalent oligonucleotide drugs containing two dsRNAs and one ASO In some embodiments, the present specification discloses a trivalent oligonucleotide drug comprising three functional oligonucleotides, namely a first double-stranded RNA (dsRNA), a second dsRNA and a first antisense oligonucleotide (ASO).In such a trivalent oligonucleotide drug, dsRNA and ASO can be arranged in any order, for example, dsRNA1-dsRNA2-ASO, dsRNA2-dsRNA1-ASO, dsRNA1-ASO-dsRNA2, dsRNA2-ASO-dsRNA1, ASO-dsRNA1-dsRNA2, ASO-dsRNA2-dsRNA1.

[0325] In some embodiments, the dsRNA is selected from small interfering RNA (siRNA) or small activating RNA (saRNA), and the ASO is a gapmer or mixmer. In some embodiments, the two functional oligonucleotides independently regulate the expression of one or more genes, regulate the expression of one or more proteins (e.g., by binding to an mRNA sequence), or regulate a non-coding regulatory nucleic acid sequence (e.g., a promoter sequence, an enhancer, a silencer, and / or a transcription factor).

[0326] In some embodiments, the dsRNA comprises a sense strand that is at least 10 contiguous nucleotides and an antisense strand that is at least 10 contiguous nucleotides. In some embodiments, the dsRNA comprises a sense strand that is 10-60 nucleotides in length and / or an antisense strand that is 10-60 nucleotides in length. In some embodiments, the ASO has a nucleotide sequence that is at least 5 contiguous nucleotides in length. In some embodiments, the ASO has a nucleotide sequence that is 5-30 nucleotides in length. In some embodiments, the trivalent oligonucleotide agent has a total length in the range of 15-100 nucleotides.

[0327] In some embodiments, two adjacent functional oligonucleotides are covalently linked via a linking moiety or without a linking moiety. The linking moiety may be selected from the group consisting of spacer-9, spacer-18, spacer-C3 and spacer-C6, or derivatives thereof, or any suitable linking moiety disclosed herein or known in the art. In some embodiments, two adjacent functional oligonucleotides are covalently linked by:

[0328] [ka] , wherein R represents -H, or -OH, or -OMe, or -MOE, or -F, or other 2' chemical modification. In some embodiments, two adjacent functional oligonucleotides are covalently linked by a phosphodiester bond, or a phosphorothioate bond, or one or more nucleotides.

[0329]

[0211] In some embodiments, the functional oligonucleotide comprises at least one chemically modified nucleotide. The modification to the chemically modified nucleotide is one or more 2' sugar modifications selected from the group consisting of 2'-fluoro-2'-deoxynucleoside (2'-F) modification, 2'-O-methyl (2'-O-Me) modification, and 2'-O-(2-methoxyethyl) (2'-O-MOE) modification. In some embodiments, the chemical modification of the at least one chemically modified nucleotide is a phosphorothioate (PS) backbone modification. In some embodiments, the chemical modification of the at least one chemically modified nucleotide is a 5'-phosphate moiety, an (E)-vinylphosphonate moiety, or a 5'-phosphate moiety to the 5' end of the nucleotide sequence. 5-Methylcytosine In some embodiments, the functional oligonucleotide comprises one or more of the above modifications at one or more nucleotides (e.g., modification of one up to all nucleotides).

[0330] In some embodiments, the ASO in the agent is covalently linked to the adjacent dsRNA in the 3' to 5' or 5' to 3' direction. In some embodiments, the dsRNA in the agent is covalently linked to the ASO at the 3' end of the sense or antisense strand, or at the 5' end of the sense or antisense strand.

[0331] In some embodiments, the dsRNA is siRNA or saRNA, and the ASO is gapmer and mixmer.In some embodiments, the multivalent oligonucleotide agent comprises a functional oligonucleotide selected from the group consisting of: (a) siRNA-siRNA-gapmer; (b) siRNA-siRNA-mixmer; (c) siRNA-saRNA-gapmer; (d) siRNA-saRNA-mixmer; (e) saRNA-saRNA-gapmer; (f) saRNA-saRNA-mixmer; provided that in any one of (a) to (f), the functional oligonucleotide is arranged in any order and is covalently linked via or without a linking moiety.In some embodiments, the ASO targets 5'-UTR.

[0332] In some aspects, the dsRNA and / or ASO increases expression of the SMN2 gene or protein. In some embodiments, the dsRNA increases expression of the SMN2 gene or protein; and / or the ASO increases production of functional SMN protein by modulating splicing or stability of SMN2 mRNA (SMN2 mRNA modulators).

[0333]

[0215] In some embodiments, the dsRNA comprises a saRNA having a nucleotide sequence of the sense strand that has at least 90% identity to a nucleotide sequence selected from the group consisting of: a) DS06-0004 (SEQ ID NO:5); b) DS06-0031 (SEQ ID NO:7); c) DS06-0067 (SEQ ID NO:9); d) DS06-4A3 (SEQ ID NO:146); e) R6-04-S1 (SEQ ID NO:59); and f) R6-04(20)-S1V1v(CM-4) (SEQ ID NO:60).

[0334]

[0216] In some embodiments, the dsRNA comprises a saRNA having an antisense strand nucleotide sequence that has at least 90% identity to a nucleotide sequence selected from the group consisting of: a) DS06-0004 (SEQ ID NO:6); b) DS06-0031 (SEQ ID NO:8); c) DS06-0067 (SEQ ID NO:10); d) DS06-4A3 (SEQ ID NO:147); e) R6-04-S1 (SEQ ID NO:53); and f) R6-04M1-27A-S1L1V3 (CM-26) (SEQ ID NO:17).

[0335]

[0217] In some embodiments, the dsRNA comprises a saRNA having a sense and antisense nucleotide sequence pair that has at least 90% identity to a nucleotide sequence pair selected from the group consisting of: a) DS06-0004: SEQ ID NO:5 and SEQ ID NO:6; b) DS06-0031: SEQ ID NO:7 and SEQ ID NO:8; c) DS06-0067: SEQ ID NO:9 and SEQ ID NO:10; d) DS06-4A3: SEQ ID NO:146 and SEQ ID NO:147; e) R6-04-S1: SEQ ID NO:59 and SEQ ID NO:53; and f) R6-04(20)-S1V1v(CM-4): SEQ ID NO:60 and SEQ ID NO:17.

[0336]

[0218] In some embodiments, the dsRNA comprises an siRNA having a nucleotide sequence of a sense strand that has at least 90% identity with the nucleotide sequence of DS06-332i (SEQ ID NO:3) or siSOD1-388-ESC (SEQ ID NO:138). In some embodiments, the dsRNA comprises an siRNA having a nucleotide sequence of an antisense strand that has at least 90% identity with the nucleotide sequence of DS06-332i (SEQ ID NO:4) or siSOD1-388-ESC (SEQ ID NO:139). In some embodiments, the dsRNA comprises an siRNA having a nucleotide sequence pair of a sense strand and an antisense strand that has at least 90% identity with a nucleotide sequence pair selected from the group consisting of: DS06-332i: SEQ ID NO:3 and SEQ ID NO:4; and siSOD1-388-ESC: SEQ ID NO:138 and SEQ ID NO:139.

[0337] In some embodiments, the multivalent oligonucleotide agent has a nucleotide sequence having at least 90% identity to a nucleotide sequence selected from the group consisting of: a) R6-04S1&27A&67S1R-L1V2 (SEQ ID NO:54), an antisense saRNA strand having a nucleotide sequence of SEQ ID NO:53 which has partial complementarity with a strand of SEQ ID NO:54, and an antisense saRNA strand having a nucleotide sequence of SEQ ID NO:78 which has partial complementarity with a strand of SEQ ID NO:54; b) R6-04S1&67S1R&27A-L1V2 (SEQ ID NO:55), an antisense saRNA strand having a nucleotide sequence of SEQ ID NO:53 which has partial complementarity with a strand of SEQ ID NO:55, and an antisense saRNA strand having a nucleotide sequence of SEQ ID NO:78 which has partial complementarity with a strand of SEQ ID NO:55. c) R6-04S1&27A&67S5-L1V2 (SEQ ID NO:57), an antisense saRNA strand having a nucleotide sequence of SEQ ID NO:53 which has partial complementarity with the strand of SEQ ID NO:57, and an antisense saRNA strand having a nucleotide sequence of SEQ ID NO:78 which has partial complementarity with the strand of SEQ ID NO:57; and d) R6-04S1&67S5&27A-L1V2 (SEQ ID NO:58), an antisense saRNA strand having a nucleotide sequence of SEQ ID NO:53 which has partial complementarity with the strand of SEQ ID NO:58, and an antisense saRNA strand having a nucleotide sequence of SEQ ID NO:78 which has partial complementarity with the strand of SEQ ID NO:58; wherein L1 represents spacer-18; L4 represents spacer-C6; and L9 represents spacer-9.

[0338]

[0220] In some embodiments, the multivalent oligonucleotide agent is as listed in Table 12. In some embodiments, the linking moieties and / or linking bonds and / or orientations of the multivalent oligonucleotide agents described above are variable.

[0339] In some embodiments, the one or more functional oligonucleotides increase expression of the CDKN1A / p21 gene or protein and / or decrease expression of CD274 / PDL-1. In some embodiments, the dsRNA is a saRNA that increases expression of the CDKN1A / p21 gene or protein; and / or a siRNA that decreases expression of CD274 / PDL-1. In some embodiments, the ASO is an ASO that increases expression of the CDKN1A / p21 gene or protein and / or decreases expression of CD274 / PDL-1.

[0340] In some embodiments, the dsRNA is RAG1-40 (SEQ ID NO: In some embodiments, the dsRNA is a saRNA having a nucleotide sequence of the sense strand that has at least 90% identity to the nucleotide sequence of RAG1-40 (SEQ ID NO:62). In some embodiments, the dsRNA is a saRNA having a nucleotide sequence of the antisense strand that has at least 90% identity to the nucleotide sequence of RAG1-40 (SEQ ID NO:63). In some embodiments, the dsRNA is a saRNA having a nucleotide sequence of the sense strand that has at least 90% identity to the nucleotide sequence of RAG1-40 (SEQ ID NO:62) and a nucleotide sequence of the antisense strand that has at least 90% identity to the nucleotide sequence of RAG1-40 (SEQ ID NO:63).

[0341] In some embodiments, the dsRNA is a) siPDL1-2(SEQ ID NO:1) In some embodiments, the dsRNA is an siRNA having a nucleotide sequence of the sense strand that has at least 90% identity to a nucleotide sequence selected from a) siPDL1-2 (SEQ ID NO:65) and b) siPDL1-3 (SEQ ID NO:67). In some embodiments, the dsRNA is an siRNA selected from the group consisting of: a) an siRNA having a sense strand nucleotide sequence that has at least 90% identity to the nucleotide sequence of siPDL1-2 (SEQ ID NO:64) and an antisense strand nucleotide sequence that has at least 90% identity to the nucleotide sequence of siPDL1-2 (SEQ ID NO:65); and b) an siRNA having a sense strand nucleotide sequence that has at least 90% identity to the nucleotide sequence of siPDL1-2 (SEQ ID NO:66) and an antisense strand nucleotide sequence that has at least 90% identity to the nucleotide sequence of siPDL1-2 (SEQ ID NO:67).

[0342] In some embodiments, the ASO is a) aPDL1-1 (SEQ ID NO: a) aPDL1-2 (SEQ ID NO:69); and c) aPDL1-3 (SEQ ID NO:70).

[0343]

[0225] In some embodiments, the linking moieties and / or linking bonds and / or orientations of these multivalent oligonucleotide agents are variable.

[0226] In some embodiments, a trivalent oligonucleotide drug may be generated by attaching a single dsRNA or ASO to a bivalent oligonucleotide drug (e.g., a bivalent oligonucleotide drug described in 4.2.5.1 or 4.2.5.2).

[0344] 4.2.5.5 Trivalent oligonucleotide drugs containing one dsRNA and two ASOs In some embodiments, the present specification discloses a trivalent oligonucleotide drug comprising three functional oligonucleotides, namely a first double-stranded RNA (dsRNA), a first antisense oligonucleotide (ASO) and a second ASO.In such a trivalent oligonucleotide drug, dsRNA and ASO can be arranged in any order, for example, dsRNA-ASO1-ASO2, dsRNA-ASO2-ASO1, ASO1-dsRNA-ASO2, ASO1-ASO2-dsRNA, ASO2-dsRNA-ASO1, ASO2-ASO1-dsRNA.

[0345] In some embodiments, the dsRNA is selected from small interfering RNA (siRNA) or small activating RNA (saRNA), and the ASO is a gapmer or mixmer. In some embodiments, the two functional oligonucleotides independently regulate the expression of one or more genes, regulate the expression of one or more proteins (e.g., by binding to an mRNA sequence), or regulate a non-coding regulatory nucleic acid sequence (e.g., a promoter sequence, an enhancer, a silencer, and / or a transcription factor).

[0346] In some embodiments, the dsRNA comprises a sense strand that is at least 10 contiguous nucleotides and an antisense strand that is at least 10 contiguous nucleotides. In some embodiments, the dsRNA comprises a sense strand that is 10-60 nucleotides in length and / or an antisense strand that is 10-60 nucleotides in length. In some embodiments, the ASO has a nucleotide sequence that is at least 5 contiguous nucleotides in length. In some embodiments, the ASO has a nucleotide sequence that is 5-30 nucleotides in length. In some embodiments, the trivalent oligonucleotide agent has a total length in the range of 15-100 nucleotides.

[0347] In some embodiments, two adjacent functional oligonucleotides are covalently linked via a linking moiety or without a linking moiety. The linking moiety may be selected from the group consisting of spacer-9, spacer-18, spacer-C3 and spacer-C6, or derivatives thereof, or any suitable linking moiety disclosed herein or known in the art. In some embodiments, two adjacent functional oligonucleotides are covalently linked by:

[0348] [ka] , wherein R represents -H, or -OH, or -OMe, or -MOE, or -F, or other 2' chemical modification. In some embodiments, two adjacent functional oligonucleotides are covalently linked by a phosphodiester bond, or a phosphorothioate bond, or one or more nucleotides.

[0349]

[0231] In some embodiments, the functional oligonucleotide comprises at least one chemically modified nucleotide. The modification to the chemically modified nucleotide is one or more 2' sugar modifications selected from the group consisting of 2'-fluoro-2'-deoxynucleoside (2'-F) modification, 2'-O-methyl (2'-O-Me) modification, and 2'-O-(2-methoxyethyl) (2'-O-MOE) modification. In some embodiments, the chemical modification of the at least one chemically modified nucleotide is a phosphorothioate (PS) backbone modification. In some embodiments, the chemical modification of the at least one chemically modified nucleotide is a 5'-phosphate moiety, an (E)-vinylphosphonate moiety, or a 5'-phosphate moiety to the 5' end of the nucleotide sequence. 5-Methylcytosine In some embodiments, the functional oligonucleotide comprises one or more of the above modifications at one or more nucleotides (e.g., modification of one up to all nucleotides).

[0350] In some embodiments, the ASO in the agent is covalently linked to the adjacent dsRNA or ASO in the 3' to 5' or 5' to 3' direction. In some embodiments, the dsRNA in the agent is covalently linked to the ASO or dsRNA at the 3' end of the sense or antisense strand, or at the 5' end of the sense or antisense strand.

[0351] In some embodiments, the dsRNA is siRNA or saRNA, and the ASO is gapmer and mixmer.In some embodiments, the multivalent oligonucleotide agent comprises a functional oligonucleotide selected from the group consisting of: (a) siRNA-gapmer-gapmer; (b) saRNA-gapmer-gapmer; (c) siRNA-gapmer-mixmer; (d) saRNA-gapmer-mixmer; (e) siRNA-mixmer-mixmer; (f) saRNA-mixmer-mixmer; provided that in any one of (a) to (f), the functional oligonucleotide is arranged in any order and is covalently linked via or without a linking moiety.In some embodiments, the ASO targets 5'-UTR.

[0352] In some aspects, the dsRNA and / or ASO increases expression of the SMN2 gene or protein. In some embodiments, the dsRNA increases expression of the SMN2 gene or protein; and / or the ASO increases production of functional SMN protein by modulating splicing or stability of SMN2 mRNA (SMN2 mRNA modulators).

[0353]

[0235] In some embodiments, the dsRNA comprises a saRNA having a nucleotide sequence of the sense strand that has at least 90% identity to a nucleotide sequence selected from the group consisting of: a) DS06-0004 (SEQ ID NO:5); b) DS06-0031 (SEQ ID NO:7); c) DS06-0067 (SEQ ID NO:9); d) DS06-4A3 (SEQ ID NO:146); e) R6-04-S1 (SEQ ID NO:59); and f) R6-04(20)-S1V1v(CM-4) (SEQ ID NO:60).

[0354]

[0236] In some embodiments, the dsRNA comprises a saRNA having an antisense strand nucleotide sequence that has at least 90% identity to a nucleotide sequence selected from the group consisting of: a) DS06-0004 (SEQ ID NO:6); b) DS06-0031 (SEQ ID NO:8); c) DS06-0067 (SEQ ID NO:10); d) DS06-4A3 (SEQ ID NO:147); e) R6-04-S1 (SEQ ID NO:53); and f) R6-04M1-27A-S1L1V3 (CM-26) (SEQ ID NO:17).

[0355]

[0237] In some embodiments, the dsRNA comprises a saRNA having a sense and antisense nucleotide sequence pair that has at least 90% identity to a nucleotide sequence pair selected from the group consisting of: a) DS06-0004: SEQ ID NO:5 and SEQ ID NO:6; b) DS06-0031: SEQ ID NO:7 and SEQ ID NO:8; c) DS06-0067: SEQ ID NO:9 and SEQ ID NO:10; d) DS06-4A3: SEQ ID NO:146 and SEQ ID NO:147; e) R6-04-S1: SEQ ID NO:59 and SEQ ID NO:53; and f) R6-04(20)-S1V1v(CM-4): SEQ ID NO:60 and SEQ ID NO:17.

[0356]

[0238] In some embodiments, the dsRNA comprises an siRNA having a nucleotide sequence of a sense strand that has at least 90% identity with the nucleotide sequence of DS06-332i (SEQ ID NO:3) or siSOD1-388-ESC (SEQ ID NO:138). In some embodiments, the dsRNA comprises an siRNA having a nucleotide sequence of an antisense strand that has at least 90% identity with the nucleotide sequence of DS06-332i (SEQ ID NO:4) or siSOD1-388-ESC (SEQ ID NO:139). In some embodiments, the dsRNA comprises an siRNA having a nucleotide sequence pair of a sense strand and an antisense strand that has at least 90% identity with a nucleotide sequence pair selected from the group consisting of: DS06-332i: SEQ ID NO:3 and SEQ ID NO:4; and siSOD1-388-ESC: SEQ ID NO:138 and SEQ ID NO:139.

[0357] In some embodiments, the linking moieties and / or linking bonds and / or orientations of the multivalent oligonucleotide agents described above are variable.

[0358] In some embodiments, the one or more functional oligonucleotides increase expression of the CDKN1A / p21 gene or protein and / or decrease expression of CD274 / PDL-1. In some embodiments, the dsRNA is a saRNA that increases expression of the CDKN1A / p21 gene or protein; and / or a siRNA that decreases expression of CD274 / PDL-1. In some embodiments, the ASO is an ASO that increases expression of the CDKN1A / p21 gene or protein and / or decreases expression of CD274 / PDL-1.

[0359] In some embodiments, the dsRNA is RAG1-40 (SEQ ID NO: In some embodiments, the dsRNA is a saRNA having a nucleotide sequence of the sense strand that has at least 90% identity to the nucleotide sequence of RAG1-40 (SEQ ID NO:62). In some embodiments, the dsRNA is a saRNA having a nucleotide sequence of the antisense strand that has at least 90% identity to the nucleotide sequence of RAG1-40 (SEQ ID NO:63). In some embodiments, the dsRNA is a saRNA having a nucleotide sequence of the sense strand that has at least 90% identity to the nucleotide sequence of RAG1-40 (SEQ ID NO:62) and a nucleotide sequence of the antisense strand that has at least 90% identity to the nucleotide sequence of RAG1-40 (SEQ ID NO:63).

[0360] In some embodiments, the dsRNA is a) siPDL1-2(SEQ ID NO:1) In some embodiments, the dsRNA is an siRNA having a nucleotide sequence of the sense strand that has at least 90% identity to a nucleotide sequence selected from a) siPDL1-2 (SEQ ID NO:65) and b) siPDL1-3 (SEQ ID NO:67). In some embodiments, the dsRNA is an siRNA selected from the group consisting of: a) an siRNA having a sense strand nucleotide sequence that has at least 90% identity to the nucleotide sequence of siPDL1-2 (SEQ ID NO:64) and an antisense strand nucleotide sequence that has at least 90% identity to the nucleotide sequence of siPDL1-2 (SEQ ID NO:65); and b) an siRNA having a sense strand nucleotide sequence that has at least 90% identity to the nucleotide sequence of siPDL1-2 (SEQ ID NO:66) and an antisense strand nucleotide sequence that has at least 90% identity to the nucleotide sequence of siPDL1-2 (SEQ ID NO:67).

[0361] In some embodiments, the ASO is a) aPDL1-1 (SEQ ID NO: a) aPDL1-2 (SEQ ID NO:69); and c) aPDL1-3 (SEQ ID NO:70).

[0362]

[0244] In some embodiments, the linking moieties and / or linking bonds and / or orientations of these multivalent oligonucleotide agents are variable.

[0245] In some embodiments, a trivalent oligonucleotide drug may be generated by attaching a single dsRNA or ASO to a bivalent oligonucleotide drug (e.g., a bivalent oligonucleotide drug described in 4.2.5.1 or 4.2.5.3).

[0363] 4.2.5.6 Trivalent Oligonucleotide Agents Containing Three dsRNAs In some embodiments, the present disclosure provides a trivalent oligonucleotide drug comprising three functional oligonucleotides, namely a first double-stranded RNA (dsRNA), a second dsRNA, and a third dsRNA.In such a trivalent oligonucleotide drug, the dsRNAs can be arranged in any order, for example, dsRNA1-dsRNA2-dsRNA3, dsRNA1-dsRNA3-dsRNA2, dsRNA2-dsRNA1-dsRNA3, dsRNA2-dsRNA3-dsRNA1, dsRNA3-dsRNA1-dsRNA2, dsRNA3-dsRNA2-dsRNA1.In some embodiments, the three functional oligonucleotides can be covalently linked via a linker or bond.

[0364]

[0247] In some embodiments, the dsRNA is selected from a small interfering RNA (siRNA) or a small activating RNA (saRNA), and the three functional oligonucleotides independently regulate the expression of one or more genes, regulate the expression of one or more proteins (e.g., by binding to an mRNA sequence), or regulate a non-coding regulatory nucleic acid sequence (e.g., a promoter sequence, enhancer, silencer, and / or transcription factor).

[0365] In some embodiments, each dsRNA comprises a sense strand of at least 10 consecutive nucleotides and an antisense strand of at least 10 consecutive nucleotides. In some embodiments, each dsRNA comprises a sense strand of 10-60 nucleotides in length and / or an antisense strand of 10-60 nucleotides in length. In some embodiments, the trivalent oligonucleotide agent has a total length in the range of 30-250 nucleotides.

[0366] In some embodiments, two adjacent functional oligonucleotides are covalently linked via a linking moiety or without a linking moiety. The linking moiety may be selected from the group consisting of spacer-9, spacer-18, spacer-C3 and spacer-C6, or derivatives thereof, or any suitable linking moiety disclosed herein or known in the art. In some embodiments, two adjacent functional oligonucleotides are covalently linked by:

[0367] [ka] , wherein R represents -H, or -OH, or -OMe, or -MOE, or -F, or other 2' chemical modification. In some embodiments, two adjacent functional oligonucleotides are covalently linked by a phosphodiester bond, or a phosphorothioate bond, or one or more nucleotides.

[0368]

[0250] In some embodiments, the functional oligonucleotide comprises at least one chemically modified nucleotide. The modification to the chemically modified nucleotide is one or more 2' sugar modifications selected from the group consisting of 2'-fluoro-2'-deoxynucleoside (2'-F) modification, 2'-O-methyl (2'-O-Me) modification, and 2'-O-(2-methoxyethyl) (2'-O-MOE) modification. In some embodiments, the chemical modification of the at least one chemically modified nucleotide is a phosphorothioate (PS) backbone modification. In some embodiments, the chemical modification of the at least one chemically modified nucleotide is a 5'-phosphate moiety, an (E)-vinylphosphonate moiety, or a 5'-phosphate moiety to the 5' end of the nucleotide sequence. 5-Methylcytosine In some embodiments, the functional oligonucleotide comprises one or more of the above modifications at one or more nucleotides (e.g., modification of one up to all nucleotides).

[0369] In some embodiments, the dsRNA in the agent is covalently linked in the 3' to 5' or 5' to 3' direction. In some embodiments, one dsRNA in the agent is covalently linked to another dsRNA at the 3' end of the sense or antisense strand, or at the 5' end of the sense or antisense strand.

[0370] In some embodiments, dsRNA is siRNA or saRNA.In some embodiments, the multivalent oligonucleotide drug comprises a functional oligonucleotide selected from the group consisting of: (a) siRNA-siRNA-siRNA; (b) siRNA-siRNA-saRNA; (c) siRNA-saRNA-saRNA; (d) saRNA-saRNA-saRNA; provided that in any one of (a) to (d), the functional oligonucleotides are arranged in any order and are covalently linked via or without a linking moiety.In some embodiments, the two or three functional oligonucleotides in the same drug can be the same or different.

[0371]

[0253] In some embodiments, the dsRNA increases / suppresses expression of the SMN2 gene or protein.

[0254] In some embodiments, the dsRNA comprises a saRNA having a nucleotide sequence of the sense strand that has at least 90% identity to a nucleotide sequence selected from the group consisting of: a) DS06-0004 (SEQ ID NO:5); b) DS06-0031 (SEQ ID NO:7); c) DS06-0067 (SEQ ID NO:9); d) DS06-4A3 (SEQ ID NO:146); e) R6-04-S1 (SEQ ID NO:59); and f) R6-04(20)-S1V1v(CM-4) (SEQ ID NO:60).

[0372]

[0255] In some embodiments, the dsRNA comprises a saRNA having an antisense strand nucleotide sequence that has at least 90% identity to a nucleotide sequence selected from the group consisting of: a) DS06-0004 (SEQ ID NO:6); b) DS06-0031 (SEQ ID NO:8); c) DS06-0067 (SEQ ID NO:10); d) DS06-4A3 (SEQ ID NO:147); e) R6-04-S1 (SEQ ID NO:53); and f) R6-04M1-27A-S1L1V3 (CM-26) (SEQ ID NO:17).

[0373]

[0256] In some embodiments, the dsRNA comprises a saRNA having a sense and antisense nucleotide sequence pair that has at least 90% identity to a nucleotide sequence pair selected from the group consisting of: a) DS06-0004: SEQ ID NO:5 and SEQ ID NO:6; b) DS06-0031: SEQ ID NO:7 and SEQ ID NO:8; c) DS06-0067: SEQ ID NO:9 and SEQ ID NO:10; d) DS06-4A3: SEQ ID NO:146 and SEQ ID NO:147; e) R6-04-S1: SEQ ID NO:59 and SEQ ID NO:53; and f) R6-04(20)-S1V1v(CM-4): SEQ ID NO:60 and SEQ ID NO:17.

[0374]

[0257] In some embodiments, the dsRNA comprises an siRNA having a nucleotide sequence of a sense strand that has at least 90% identity with the nucleotide sequence of DS06-332i (SEQ ID NO:3) or siSOD1-388-ESC (SEQ ID NO:138). In some embodiments, the dsRNA comprises an siRNA having a nucleotide sequence of an antisense strand that has at least 90% identity with the nucleotide sequence of DS06-332i (SEQ ID NO:4) or siSOD1-388-ESC (SEQ ID NO:139). In some embodiments, the dsRNA comprises an siRNA having a nucleotide sequence pair of a sense strand and an antisense strand that has at least 90% identity with a nucleotide sequence pair selected from the group consisting of: DS06-332i: SEQ ID NO:3 and SEQ ID NO:4; and siSOD1-388-ESC: SEQ ID NO:138 and SEQ ID NO:139.

[0375] In some embodiments, the one or more functional oligonucleotides increase expression of the CDKN1A / p21 gene or protein and / or decrease expression of CD274 / PDL-1. In some embodiments, the dsRNA is a saRNA that increases expression of the CDKN1A / p21 gene or protein; and / or a siRNA that decreases expression of CD274 / PDL-1.

[0376] In some embodiments, the dsRNA is RAG1-40 (SEQ ID NO: In some embodiments, the dsRNA is a saRNA having a nucleotide sequence of the sense strand that has at least 90% identity to the nucleotide sequence of RAG1-40 (SEQ ID NO:62). In some embodiments, the dsRNA is a saRNA having a nucleotide sequence of the antisense strand that has at least 90% identity to the nucleotide sequence of RAG1-40 (SEQ ID NO:63). In some embodiments, the dsRNA is a saRNA having a nucleotide sequence of the sense strand that has at least 90% identity to the nucleotide sequence of RAG1-40 (SEQ ID NO:62) and a nucleotide sequence of the antisense strand that has at least 90% identity to the nucleotide sequence of RAG1-40 (SEQ ID NO:63).

[0377] In some embodiments, the dsRNA is a) siPDL1-2(SEQ ID NO:1) In some embodiments, the dsRNA is an siRNA having a nucleotide sequence of the sense strand that has at least 90% identity to a nucleotide sequence selected from a) siPDL1-2 (SEQ ID NO:65) and b) siPDL1-3 (SEQ ID NO:67). In some embodiments, the dsRNA is an siRNA selected from the group consisting of: a) an siRNA having a sense strand nucleotide sequence that has at least 90% identity to the nucleotide sequence of siPDL1-2 (SEQ ID NO:64) and an antisense strand nucleotide sequence that has at least 90% identity to the nucleotide sequence of siPDL1-2 (SEQ ID NO:65); and b) an siRNA having a sense strand nucleotide sequence that has at least 90% identity to the nucleotide sequence of siPDL1-2 (SEQ ID NO:66) and an antisense strand nucleotide sequence that has at least 90% identity to the nucleotide sequence of siPDL1-2 (SEQ ID NO:67).

[0378] In some embodiments, the linking moieties and / or linkage bonds and / or orientations of these multivalent oligonucleotide drugs can be varied. In some embodiments, a trivalent oligonucleotide drug can be produced by adding a single dsRNA to a bivalent oligonucleotide drug (e.g., a bivalent oligonucleotide drug described in 4.2.5.2).

[0379] 4.2.5.7 Trivalent oligonucleotide drugs containing three ASOs In some embodiments, the present specification discloses a trivalent oligonucleotide drug that comprises three functional oligonucleotides, namely a first antisense oligonucleotide (ASO), a second ASO, and a third ASO.In such a trivalent oligonucleotide drug, the ASOs can be arranged in any order, for example, ASO1-ASO2-ASO3, ASO1-ASO3-ASO2, ASO2-ASO1-ASO3, ASO2-ASO3-ASO1, ASO3-ASO1-ASO2, ASO3-ASO2-ASO1.Two or three of the ASOs can be the same or different.

[0380] In some embodiments, the ASO is independently selected from a gapmer or a mixmer. In some embodiments, the three functional oligonucleotides independently regulate the expression of one or more genes, regulate the expression of one or more proteins (e.g., by binding to an mRNA sequence), or regulate a non-coding regulatory nucleic acid sequence (e.g., a promoter sequence, an enhancer, a silencer, and / or a transcription factor).

[0381] In some embodiments, each ASO has a nucleotide sequence that is at least 5 contiguous nucleotides in length. In some embodiments, the ASO has a nucleotide sequence that is 5-30 nucleotides in length. In some embodiments, the trivalent oligonucleotide agent has a total length in the range of 15-150 nucleotides.

[0382] In some embodiments, two adjacent functional oligonucleotides are covalently linked via a linking moiety or without a linking moiety. The linking moiety may be selected from the group consisting of spacer-9, spacer-18, spacer-C3 and spacer-C6, or derivatives thereof, or any suitable linking moiety disclosed herein or known in the art. In some embodiments, two adjacent functional oligonucleotides are covalently linked by:

[0383] [ka] , wherein R represents -H, or -OH, or -OMe, or -MOE, or -F, or other 2' chemical modification. In some embodiments, two adjacent functional oligonucleotides are covalently linked by a phosphodiester bond, or a phosphorothioate bond, or one or more nucleotides.

[0384]

[0266] In some embodiments, the functional oligonucleotide comprises at least one chemically modified nucleotide. The modification to the chemically modified nucleotide is one or more 2' sugar modifications selected from the group consisting of 2'-fluoro-2'-deoxynucleoside (2'-F) modification, 2'-O-methyl (2'-O-Me) modification, and 2'-O-(2-methoxyethyl) (2'-O-MOE) modification. In some embodiments, the chemical modification of the at least one chemically modified nucleotide is a phosphorothioate (PS) backbone modification. In some embodiments, the chemical modification of the at least one chemically modified nucleotide is a 5'-phosphate moiety, an (E)-vinylphosphonate moiety, or a 5'-phosphate moiety to the 5' end of the nucleotide sequence. 5-Methylcytosine In some embodiments, the functional oligonucleotide comprises one or more of the above modifications at one or more nucleotides (e.g., modification of one up to all nucleotides).

[0385] In some embodiments, one ASO in the agent is covalently linked to another ASO in the 3' to 5' or 5' to 3' direction, hi some embodiments, one ASO in the agent is covalently linked to another ASO at its 3' end or at its 5' end.

[0386] In some embodiments, the ASO is selected from gapmers and mixmers.In some embodiments, the multivalent oligonucleotide agent comprises a functional oligonucleotide selected from the group consisting of: (a) gapmer-gapmer-gapmer; (b) gapmer-gapmer-mixmer; (c) gapmer-mixmer-mixmer; (d) mixmer-mixmer-mixmer;; in any one of (a) to (d), the functional oligonucleotides are arranged in any order and are covalently linked via or without a linking moiety.In some embodiments, the ASO targets the 5'-UTR.

[0387] In some embodiments, the ASO increases expression of the SMN2 gene or protein. In some embodiments, the ASO increases production of functional SMN protein by modulating splicing or stability of SMN2 mRNA (SMN2 mRNA modulators). In some embodiments, the ASO has a nucleotide sequence that has at least 90% identity to the nucleotide sequence of ASO10-27 (SEQ ID NO:11) or 5'UTR ASO (SEQ ID NO:142).

[0388] In some embodiments, the linking moieties and / or linkage bonds and / or orientation of these multivalent oligonucleotide drugs can be varied. In some embodiments, a trivalent oligonucleotide drug can be produced by adding a single dsRNA to a bivalent oligonucleotide drug (e.g., a bivalent oligonucleotide drug described in 4.2.5.3).

[0389] 4.2.5.8 Multivalent Oligonucleotide Agents Containing More Than Three Functional Oligonucleotides In some embodiments, disclosed herein are multivalent oligonucleotide agents comprising more than three covalently linked functional oligonucleotides, where the four or more functional oligonucleotides are independently selected from the group consisting of a) double-stranded RNA (dsRNA) and b) antisense oligonucleotides (ASO). In such multivalent oligonucleotide agents, the dsRNA and ASO may be arranged in any order.

[0390] In some embodiments, the number of functional oligonucleotides contained in a multivalent oligonucleotide agent ranges from 4 to X, where X is an integer ranging from 5 to 10. In some embodiments, the number of dsRNAs contained in the agent ranges from 0 to X, and the remaining functional oligonucleotides are ASOs.

[0391] In some embodiments, the dsRNA is selected from small interfering RNA (siRNA) or small activating RNA (saRNA), and the ASO is a gapmer or mixmer. In some embodiments, the two functional oligonucleotides independently regulate the expression of one or more genes, regulate the expression of one or more proteins (e.g., by binding to an mRNA sequence), or regulate a non-coding regulatory nucleic acid sequence (e.g., a promoter sequence, an enhancer, a silencer, and / or a transcription factor).

[0392] In some embodiments, each dsRNA comprises a sense strand that is at least 10 contiguous nucleotides and an antisense strand that is at least 10 contiguous nucleotides. In some embodiments, each dsRNA comprises a sense strand that is 10-60 nucleotides in length and / or an antisense strand that is 10-60 nucleotides in length. In some embodiments, each ASO has a nucleotide sequence that is at least 5 contiguous nucleotides in length. In some embodiments, each ASO has a nucleotide sequence that is 5-30 nucleotides in length. In some embodiments, the multivalent oligonucleotide agent has a total length in the range of 20-200 nucleotides.

[0393] In some embodiments, two adjacent functional oligonucleotides are covalently linked with or without a linking moiety. The linking moiety may be selected from the group consisting of spacer-9, spacer-18, spacer-C3 and spacer-C6, or derivatives thereof, or any suitable linking moiety disclosed herein or known in the art. In some embodiments, two adjacent functional oligonucleotides are covalently linked by: wherein R represents -H, or -OH, or -OMe, or -MOE, or -F, or other 2' chemical modification. In some embodiments, two adjacent functional oligonucleotides are covalently linked by a phosphodiester bond, or a phosphorothioate bond, or one or more nucleotides.

[0394]

[0276] In some embodiments, the functional oligonucleotide comprises at least one chemically modified nucleotide. The modification to the chemically modified nucleotide is one or more 2' sugar modifications selected from the group consisting of 2'-fluoro-2'-deoxynucleoside (2'-F) modification, 2'-O-methyl (2'-O-Me) modification, and 2'-O-(2-methoxyethyl) (2'-O-MOE) modification. In some embodiments, the chemical modification of the at least one chemically modified nucleotide is a phosphorothioate (PS) backbone modification. In some embodiments, the chemical modification of the at least one chemically modified nucleotide is a 5'-phosphate moiety, an (E)-vinylphosphonate moiety, or a 5'-phosphate moiety to the 5' end of the nucleotide sequence. 5-Methylcytosine In some embodiments, the functional oligonucleotide comprises one or more of the above modifications at one or more nucleotides (e.g., modification of one up to all nucleotides).

[0395]

[0277] In some embodiments, the sequences of two or more functional oligonucleotides in a multivalent oligonucleotide drug are the same or different; and / or the functions of two or more functional oligonucleotides are the same or different.

[0396] In some embodiments, the linking moieties and / or linking bonds and / or orientation of these multivalent oligonucleotide drugs can be varied. In some embodiments, a multivalent oligonucleotide drug can be produced by attaching one or more dsRNAs and / or ASOs to a bivalent oligonucleotide drug (e.g., a bivalent oligonucleotide drug described in 4.2.5.1-4.2.5.3) or to a trivalent oligonucleotide drug (e.g., a trivalent oligonucleotide drug described in 4.2.5.4-4.2.5.7).

[0397] 4.3 Exemplary Functions of Multivalent Oligonucleotide Agents

[0279] In addition, the present specification discloses multivalent oligonucleotide drugs with various functions. In some embodiments, depending on the functional oligonucleotide in the multivalent oligonucleotide drug, the drug can be used to target a desired gene related to a specific disease to produce a therapeutic effect. Therefore, a method for treating a disease using a multivalent oligonucleotide drug and a product containing a multivalent oligonucleotide drug for treating a disease are also provided.

[0398]

[0280] Multivalent oligonucleotide drugs can be used to modulate and / or regulate the expression and / or activity of a target of interest (potentially associated with a particular disease). Based on the teachings and spirit of the present disclosure, suitable multivalent oligonucleotide drugs that specifically bind, modulate and / or regulate the expression and / or activity of a target of interest can be designed, and the effect of the multivalent oligonucleotide drug can be evaluated by routine experimentation.

[0399]

[0281] Some exemplary aspects and embodiments of the present invention are as follows: However, such aspects and embodiments do not necessarily represent the full scope of the present invention, and therefore reference is made to the claims and this specification to interpret the scope of the present invention.

[0400] 4.3.1 Multivalent Oligonucleotide Agents That Increase SMN2 Gene or Protein Expression In some embodiments, the two or more functional oligonucleotides increase expression of the SMN2 gene or protein. Administering the multivalent oligonucleotide agent to a patient treats or delays the onset of an SMN deficiency-associated condition, such as spinal muscular atrophy. In certain embodiments, the described multivalent oligonucleotide agents increase the amount of full-length SMN protein, for example by activating / upregulating SMN2 transcription in combination with modulating splicing of exon 7 inclusion to increase the amount of full-length SMN2 mRNA. In certain embodiments, full-length SMN protein is increased in an amount sufficient to alleviate symptoms associated with an SMN deficiency-associated condition. In certain embodiments, full-length SMN protein is increased by at least 10% (e.g., at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%).

[0401] In certain embodiments, at least one of the two or more functional oligonucleotides of a multivalent oligonucleotide agent that increases expression of the SMN2 gene or protein is a saRNA. The SMN2 saRNA activates or upregulates expression of the SMN2 gene in cells in which it is normally expressed.

[0402]

[0284] In a typical embodiment, the first strand of the SMN2 saRNA contains a segment having at least 75% sequence identity or sequence complementarity to a 16-35 nucleotide fragment of the promoter region of the SMN2 gene, thereby resulting in activation or upregulation of expression of the gene.

[0403]

[0285] In particular, the first strand of the SMN2 saRNA has homology or complementarity to a region of the SMN2 gene promoter selected from the region from -1639 to -1481 of the SMN2 gene promoter (SEQ ID NO:124), the region from -1090 to -1008 of the SMN2 gene promoter (SEQ ID NO:125), the region from -994 to -180 of the SMN2 gene promoter (SEQ ID NO:126), or the region from -144 to -37 of the SMN2 gene promoter (SEQ ID NO:127), and has at least 75%, for example at least about 79%, about 80%, about 85%, about 90%, about 95%, or about 99% homology or complementarity. More specifically, one strand of the SMN2 saRNA has at least 75%, for example at least about 79%, or about 99% homology or complementarity to any nucleotide sequence selected from the group consisting of SEQ ID NOs:315-471.

[0404]

[0286] In the present disclosure, the SMN2 saRNA comprises a sense nucleic acid fragment and an antisense nucleic acid fragment. The sense nucleic acid fragment and the antisense nucleic acid fragment comprise a complementary region capable of forming a double-stranded nucleic acid structure that promotes the expression of the SMN2 gene in cells by an RNA activation mechanism. The sense nucleic acid fragment and the antisense nucleic acid fragment of the saRNA may be present in two different nucleic acid strands, or may be present in the same nucleic acid strand. When the sense and antisense nucleic acid fragments are present in two strands, at least one strand of the saRNA has a 3' overhang of 0 to 6 nucleotides in length, and preferably both strands have a 3' overhang of 2 or 3 nucleotides in length, and preferably the nucleotide of the overhang is deoxythymine (dT). When the sense nucleic acid fragment and the antisense nucleic acid fragment of the saRNA are present in the same nucleic acid strand, the saRNA is preferably a single-stranded hairpin structure nucleic acid molecule, provided that the complementary regions of the sense nucleic acid fragment and the antisense nucleic acid fragment form a double-stranded nucleic acid structure. In such saRNAs, the sense and antisense nucleic acid fragments are 10 to 60 nucleotides in length and may be 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 nucleotides in length.

[0405]

[0287] In certain embodiments of the present disclosure, the SMN2 saRNA comprises a sense nucleic acid strand and an antisense nucleic acid strand, wherein the sense nucleic acid strand comprises at least one region complementary to at least one region in the antisense nucleic acid strand, forming a double-stranded nucleic acid structure capable of activating expression of the SMN2 gene in a cell.

[0406] In certain embodiments of the present disclosure, the sense nucleic acid strand and the antisense nucleic acid strand are located on two different nucleic acid strands.

[0289] In certain embodiments of the present disclosure, the sense nucleic acid fragment and the antisense nucleic acid fragment are located on the same nucleic acid strand and form a hairpin single-stranded nucleic acid molecule, provided that the complementary regions of the sense nucleic acid fragment and the antisense nucleic acid fragment form a double-stranded nucleic acid structure.

[0407] In certain embodiments of the present disclosure, at least one nucleic acid strand has a 3' overhang with a length of 0-6 nucleotides. In certain embodiments of the present disclosure, both nucleic acid strands have a 3' overhang with a length of 2-3 nucleotides. In certain embodiments of the present disclosure, the sense and antisense nucleic acid strands are each 16-35 nucleotides in length.

[0408]

[0291] In some embodiments, the first, second and / or third dsRNA increases expression of the SMN2 gene or protein, and the ASO increases production of functional SMN protein by regulating splicing or stability of SMN2 mRNA (SMN2 mRNA modulator).

[0409] As used herein, the term "SMN2 mRNA modulator" refers to a modulator of splicing or stability of SMN2 mRNA that increases the production of functional SMN2 mRNA and functional SMN protein. The term "SMN2 mRNA modulator" includes agents that alter the splicing mode of the SMN2 pre-mRNA so that it contains all the information necessary to make a functional full-length SMN protein, for example, by inhibiting the effect of the intronic inhibitory splicing region in intron 7 of the SMN2 gene. SMN2 mRNA modulators include agents that increase the desired splicing and subsequent protein production by stabilizing the interaction between the spliceosome and SMN2 pre-mRNA (J Med Chem, 2018 Dec 27; 61(24): 11021-11036), as well as agents that enhance stabilization of the transient double-stranded RNA structure formed in the complex of SMN2 pre-mRNA and U1 small nuclear ribonucleoprotein (snRNP) (Nat Chem Biol, 2015 Jul;11(7):511-7). In certain instances, SMN2 mRNA modulators regulate splicing of SMN2 pre-mRNA to include exon 7 in the processed transcript. Alternatively, SMN2 mRNA modulators of the present disclosure include agents capable of increasing functional SMN protein levels by inhibiting exon 7 from being spliced ​​out of the mature SMN mRNA during splicing. SMN2 mRNA modulators according to the present disclosure also include those described in US Pat. Nos. 10,436,802 and 10,420,753, which are hereby incorporated by reference in their entireties.

[0410]

[0293] Examples of SMN2 mRNA modulators according to the present disclosure include pyridazine derivatives, such as those described in WO2014028459A1, the entire contents of which are incorporated herein by reference. Specific examples of SMN2 mRNA modulators include branapram (also known as LMI070) and risdiplam (also known as RG7916 or RO7034067).

[0411] [ka]

[0412] Further examples of SMN2 mRNA modulators according to the present disclosure include antisense oligonucleotides, such as those that can antisense target, replace and / or disrupt intronic sequences of the SMN2 gene to enhance production of the SMN2 full-length (SMN2FL) transcript (a transcript that includes exon 7) upon splicing. In certain embodiments, nusinersen, commercially available as SPINRAZA®, is suitable for use according to the disclosed combinations.

[0413] In some embodiments, the dsRNA is a small activating RNA (saRNA). The saRNA targets regulatory sequences in the nucleus, such as gene promoters, and upregulates gene expression at the transcriptional level by the RNAa (RNA activation) mechanism. For example, a small activating ribonucleic acid (saRNA) that activates or upregulates the expression of the SMN2 gene in cells (also referred to herein as "SMN2 saRNA") can be covalently linked to one or more modulators of splicing or stability of SMN2 mRNA (also referred to herein as "SMN2 mRNA modulators") that increase the production of functional SMN2 mRNA, thereby achieving a significant increase in the level of full-length SMN2 mRNA and full-length SMN protein. Covalent linking of the multivalent oligonucleotides provided herein can enhance the therapeutic effect compared to monotherapy, for example, maximizing the treatment outcome of SMA patients.

[0414]

[0296] In some aspects, the present disclosure provides an isolated SMN2 gene saRNA targeting site having any 16 to 35 contiguous nucleotide sequence in the promoter region of the SMN2 gene (NCBI GeneID: 6607) (UCSC Genome Browser coordinates: chr5:70,044,612-70,049,522).

[0415]

[0297] In some embodiments, the saRNA may comprise a nucleotide sequence of the sense strand having at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100%) identity to a nucleotide sequence selected from the group consisting of: DS06-0004 (SEQ ID NO:5); b) DS06-0031 (SEQ ID NO:7); c) DS06-0067 (SEQ ID NO:9); d) DS06-4A3 (SEQ ID NO:146); e) R6-04-S1 (SEQ ID NO:59); and f) R6-04(20)-S1V1v(CM-4) (SEQ ID NO:60). In some embodiments, the saRNA may comprise a nucleotide sequence of the sense strand that has at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100%) identity to the nucleotide sequence of DS06-0004 (SEQ ID NO:5). In some embodiments, the saRNA may comprise a nucleotide sequence of the sense strand that has at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100%) identity to the nucleotide sequence of DS06-0031 (SEQ ID NO:7). In some embodiments, the saRNA may comprise a nucleotide sequence of a sense strand that has at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100%) identity to the nucleotide sequence of DS06-0067 (SEQ ID NO:9).In some embodiments, the saRNA may comprise a nucleotide sequence of the sense strand that has at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100%) identity to the nucleotide sequence of DS06-4A3 (SEQ ID NO:12). In some embodiments, the saRNA may comprise a nucleotide sequence of the sense strand that has at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100%) identity to the nucleotide sequence of R6-04-S1 (SEQ ID NO:59). In some embodiments, the saRNA may comprise a nucleotide sequence of a sense strand that has at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100%) identity to the nucleotide sequence of R6-04(20)-S1V1v(CM-4) (SEQ ID NO:60).

[0416] In some embodiments, the saRNA may comprise a nucleotide sequence of the antisense strand that has at least 17% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100%) identity to a nucleotide sequence selected from the group consisting of: a) DS06-0004 (SEQ ID NO: 1) NO:6); b) DS06-0031 (SEQ ID NO:8); c) DS06-0067 (SEQ ID NO:10); d) DS06-4A3 (SEQ ID NO:147); e) R6-04-S1 (SEQ ID NO:53); and f) R6-04M1-27A-S1L1V3 (CM-26) (SEQ ID NO:17). In certain embodiments, the saRNA may comprise a nucleotide sequence of the antisense strand having at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100%) identity to the nucleotide sequence of DS06-0004 (SEQ ID NO:6). In certain embodiments, the saRNA may comprise a nucleotide sequence of the antisense strand that has at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100%) identity to the nucleotide sequence of DS06-0031 (SEQ ID NO:8). In certain embodiments, the saRNA may comprise a nucleotide sequence of the antisense strand that has at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100%) identity to the nucleotide sequence of DS06-0067 (SEQ ID NO:10). In certain embodiments, the saRNA may comprise a nucleotide sequence of the antisense strand that has at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100%) identity to the nucleotide sequence of DS06-4A3 (SEQ ID NO:147).In certain embodiments, the saRNA may comprise a nucleotide sequence of the antisense strand that has at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100%) identity to the nucleotide sequence of R6-04-S1 (SEQ ID NO:53). In certain embodiments, the saRNA may comprise a nucleotide sequence of the antisense strand that has at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100%) identity to the nucleotide sequence of R6-04M1-27A-S1L1V3 (CM-26) (SEQ ID NO:17).

[0417]

[0301] In some embodiments, the dsRNA is an siRNA, and the siRNA may comprise a nucleotide sequence of a sense strand that has at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100%) identity to a nucleotide sequence selected from DS06-332i (SEQ ID NO:3).

[0418] In certain embodiments, the siRNA is selected from the group consisting of DS06-332i (SEQ ID NO: 1) and DS06-332i (SEQ ID NO: 2). No. 4), the nucleotide sequence of the antisense strand may have at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100%) identity to a nucleotide sequence selected from the group consisting of

[0419]

[0301] In some embodiments, at least one oligonucleotide is an ASO. The ASO can be designed to target the mRNA of a gene and downregulate its expression through RNase H activity, for example to maximize the efficacy of cancer treatment.

[0420]

[0302] In some embodiments, the ASO may comprise a nucleotide sequence having at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100%) identity to the nucleotide sequence of ASO10-27 (SEQ ID NO:11).

[0421] In some embodiments, the two or more functional oligonucleotides include a first dsRNA and a second dsRNA. In certain embodiments, the first and second dsRNA are each siRNA.

[0422] In certain embodiments, the first and second dsRNAs are each saRNA. The saRNA can be designed to target the promoter sequence of a gene and induce its transcription through the RNAa mechanism, for example, to treat various cancers.

[0423] In some embodiments, the first dsRNA is a saRNA and the second dsRNA is a siRNA. In some embodiments, the second dsRNA is a siRNA and the second dsRNA is a saRNA.

[0424]

[0306] In some embodiments, the two or more functional oligonucleotides comprise a first ASO and a second ASO.

[0307] In some embodiments, the first dsRNA is a siRNA and the second oligonucleotide is an ASO.

[0425] In some embodiments, the first dsRNA is a saRNA and the second oligonucleotide is an ASO. In certain embodiments, the multivalent oligonucleotide agent comprises a nucleotide sequence having at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100%) identity to the nucleotide sequence of DA06-4A-27A (SEQ ID NO:14) and a nucleotide sequence having at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100%) identity to the nucleotide sequence of SEQ ID NO:14. The antisense saRNA strand has a nucleotide sequence of SEQ ID NO: 13 that is partially complementary to the sense saRNA strand of ID NO: 14. In certain embodiments, the multivalent oligonucleotide agent has a nucleotide sequence that is at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100%) identical to the nucleotide sequence of DA06-4A-27B (SEQ ID NO: 15), and an antisense saRNA strand has a nucleotide sequence of SEQ ID NO: 13 that is partially complementary to the sense saRNA strand of SEQ ID NO: 15. In certain embodiments, a multivalent oligonucleotide agent has a nucleotide sequence having at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100%) identity to the nucleotide sequence of R6-04M1-27A-S1L1V3 (SEQ ID NO:18) and a nucleotide sequence having at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100%) identity to the nucleotide sequence of SEQ ID and an antisense saRNA strand having a nucleotide sequence of SEQ ID NO:13 that is partially complementary to the sense saRNA strand of SEQ ID NO:18. In certain embodiments, the multivalent oligonucleotide agent has a nucleotide sequence that is at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100%) identical to the nucleotide sequence of DA06-31A-27A (SEQ ID NO:19), and an antisense saRNA strand having a nucleotide sequence of SEQ ID NO:8 that is partially complementary to the sense saRNA strand of SEQ ID NO:19. In certain embodiments, the multivalent oligonucleotide agent has a nucleotide sequence having at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100%) identity to the nucleotide sequence of DA06-31B-27A (SEQ ID NO:20) and a sense saRNA strand having the nucleotide sequence of SEQ ID NO:7 which has partial complementarity to the antisense saRNA strand of SEQ ID NO:20.

[0426]

[0309] In certain embodiments, the multivalent oligonucleotide drug has a nucleotide sequence having at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100%) identity to the nucleotide sequence of DA06-67A-27A (SEQ ID NO:21) and an antisense saRNA strand having the nucleotide sequence of SEQ ID NO:10 which has partial complementarity to the sense saRNA strand of SEQ ID NO:21.

[0427]

[0310] In certain embodiments, the multivalent oligonucleotide drug has a nucleotide sequence having at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100%) identity to the nucleotide sequence of DA06-67B-27A (SEQ ID NO:22) and a sense saRNA strand having the nucleotide sequence of SEQ ID NO:9 which has partial complementarity to the antisense saRNA strand of SEQ ID NO:22.

[0428]

[0311] In certain embodiments, the multivalent oligonucleotide drug has a nucleotide sequence having at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100%) identity to the nucleotide sequence of DA6-67A3'L0-27A (SEQ ID NO:23) and an antisense saRNA strand having the nucleotide sequence of SEQ ID NO:10 which has partial complementarity to the sense saRNA strand of SEQ ID NO:23.

[0429]

[0312] In certain embodiments, the multivalent oligonucleotide agent has a nucleotide sequence having at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100%) identity to the nucleotide sequence of DA6-67A3'L9-27A (SEQ ID NO:24) and an antisense saRNA strand having the nucleotide sequence of SEQ ID NO:10 which has partial complementarity to the sense saRNA strand of SEQ ID NO:24.

[0430]

[0313] In certain embodiments, the multivalent oligonucleotide agent has a nucleotide sequence having at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100%) identity to the nucleotide sequence of DA6-67A3'L4-27A (SEQ ID NO:25) and an antisense saRNA strand having the nucleotide sequence of SEQ ID NO:10 which has partial complementarity to the sense saRNA strand of SEQ ID NO:25.

[0431]

[0314] In certain embodiments, the multivalent oligonucleotide agent has a nucleotide sequence having at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100%) identity to the nucleotide sequence of DA6-67B3'L0-27A (SEQ ID NO:26) and a sense saRNA strand having the nucleotide sequence of SEQ ID NO:9 which has partial complementarity to the antisense saRNA strand of SEQ ID NO:26.

[0432]

[0315] In certain embodiments, the multivalent oligonucleotide agent has a nucleotide sequence having at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100%) identity to the nucleotide sequence of DA6-67B5'L1-27A (SEQ ID NO:27) and a sense saRNA strand having the nucleotide sequence of SEQ ID NO:9 which has partial complementarity to the antisense saRNA strand of SEQ ID NO:27.

[0433]

[0316] In certain embodiments, the multivalent oligonucleotide agent has a nucleotide sequence having at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100%) identity to the nucleotide sequence of DA6-67B3'L1-27A (SEQ ID NO:28) and a sense saRNA strand having the nucleotide sequence of SEQ ID NO:9 which has partial complementarity to the antisense saRNA strand of SEQ ID NO:28.

[0434]

[0317] In certain embodiments, the multivalent oligonucleotide agent has a nucleotide sequence having at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100%) identity to the nucleotide sequence of DA6-67B5'L9-27A (SEQ ID NO:29) and a sense saRNA strand having the nucleotide sequence of SEQ ID NO:9 which has partial complementarity to the antisense saRNA strand of SEQ ID NO:29.

[0435]

[0318] In certain embodiments, the multivalent oligonucleotide agent has a nucleotide sequence having at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100%) identity to the nucleotide sequence of DA6-67B5'L4-27A (SEQ ID NO:30) and a sense saRNA strand having the nucleotide sequence of SEQ ID NO:9 which has partial complementarity to the antisense saRNA strand of SEQ ID NO:30.

[0436]

[0319] In certain embodiments, the multivalent oligonucleotide agent has a nucleotide sequence having at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100%) identity to the nucleotide sequence of DA6-67B3'L9-27A (SEQ ID NO:31) and a sense saRNA strand having the nucleotide sequence of SEQ ID NO:9 which has partial complementarity to the antisense saRNA strand of SEQ ID NO:31.

[0437]

[0320] In certain embodiments, the multivalent oligonucleotide agent has a nucleotide sequence having at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100%) identity to the nucleotide sequence of DA6-67B3'L4-27A (SEQ ID NO:32) and a sense saRNA strand having the nucleotide sequence of SEQ ID NO:9 which has partial complementarity to the antisense saRNA strand of SEQ ID NO:32.

[0438]

[0321] In certain embodiments, the multivalent oligonucleotide drug has a nucleotide sequence having at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100%) identity to the nucleotide sequence of DA06-67A21L1-27A (SEQ ID NO:33) and an antisense saRNA strand having the nucleotide sequence of SEQ ID NO:34 which has partial complementarity to the sense saRNA strand of SEQ ID NO:33.

[0439]

[0322] In certain embodiments, the multivalent oligonucleotide agent has a nucleotide sequence having at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100%) identity to the nucleotide sequence of DA06-67B21L1-27A (SEQ ID NO:36) and a sense saRNA strand of SEQ ID NO:35 that has partial complementarity to the antisense saRNA strand of SEQ ID NO:36.

[0440]

[0323] In certain embodiments, the multivalent oligonucleotide agent has a nucleotide sequence having at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100%) identity to the nucleotide sequence of DA6-04A3'L0-27A (SEQ ID NO:37) and an antisense saRNA strand having the nucleotide sequence of SEQ ID NO:6 which has partial complementarity to the sense saRNA strand of SEQ ID NO:37.

[0441]

[0324] In certain embodiments, the multivalent oligonucleotide agent has a nucleotide sequence having at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100%) identity to the nucleotide sequence of DA6-04A5'L1-27A (SEQ ID NO:38) and an antisense saRNA strand having the nucleotide sequence of SEQ ID NO:6 which has partial complementarity to the sense saRNA strand of SEQ ID NO:38.

[0442]

[0325] In certain embodiments, the multivalent oligonucleotide agent has a nucleotide sequence having at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100%) identity to the nucleotide sequence of DA6-04A5'L9-27A (SEQ ID NO:39) and an antisense saRNA strand having the nucleotide sequence of SEQ ID NO:6 which has partial complementarity to the sense saRNA strand of SEQ ID NO:39.

[0443]

[0326] In certain embodiments, the multivalent oligonucleotide agent has a nucleotide sequence having at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100%) identity to the nucleotide sequence of DA6-04A5'L4-27A (SEQ ID NO:40) and an antisense saRNA strand having the nucleotide sequence of SEQ ID NO:6 which has partial complementarity to the sense saRNA strand of SEQ ID NO:40.

[0444]

[0327] In certain embodiments, the multivalent oligonucleotide agent has a nucleotide sequence having at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100%) identity to the nucleotide sequence of DA6-04A3'L1-27A (SEQ ID NO:41) and an antisense saRNA strand having the nucleotide sequence of SEQ ID NO:6 which has partial complementarity to the sense saRNA strand of SEQ ID NO:41.

[0445]

[0328] In certain embodiments, the multivalent oligonucleotide agent has a nucleotide sequence having at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100%) identity to the nucleotide sequence of DA6-04A3'L9-27A (SEQ ID NO:42) and an antisense saRNA strand having the nucleotide sequence of SEQ ID NO:6 which has partial complementarity to the sense saRNA strand of SEQ ID NO:42.

[0446]

[0329] In certain embodiments, the multivalent oligonucleotide agent has a nucleotide sequence having at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100%) identity to the nucleotide sequence of DA6-04A3'L4-27A (SEQ ID NO:43) and an antisense saRNA strand having the nucleotide sequence of SEQ ID NO:6 which has partial complementarity to the sense saRNA strand of SEQ ID NO:43.

[0447]

[0330] In certain embodiments, the multivalent oligonucleotide agent has a nucleotide sequence having at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100%) identity to the nucleotide sequence of DA6-04B3'L0-27A (SEQ ID NO:44) and a sense saRNA strand having the nucleotide sequence of SEQ ID NO:5 which has partial complementarity to the antisense saRNA strand of SEQ ID NO:44.

[0448]

[0331] In certain embodiments, the multivalent oligonucleotide agent has a nucleotide sequence having at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100%) identity to the nucleotide sequence of DA6-04B3'L1-27A (SEQ ID NO:45) and a sense saRNA strand having the nucleotide sequence of SEQ ID NO:5 which has partial complementarity to the antisense saRNA strand of SEQ ID NO:45.

[0449]

[0332] In certain embodiments, the multivalent oligonucleotide agent has a nucleotide sequence having at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100%) identity to the nucleotide sequence of DA6-04B3'L9-27A (SEQ ID NO:46) and a sense saRNA strand having the nucleotide sequence of SEQ ID NO:5 which has partial complementarity to the antisense saRNA strand of SEQ ID NO:46.

[0450]

[0333] In certain embodiments, the multivalent oligonucleotide agent has a nucleotide sequence having at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100%) identity to the nucleotide sequence of DA6-04B3'L4-27A (SEQ ID NO:47) and a sense saRNA strand having the nucleotide sequence of SEQ ID NO:5 which has partial complementarity to the antisense saRNA strand of SEQ ID NO:47.

[0451]

[0334] In certain embodiments, the multivalent oligonucleotide agent has a nucleotide sequence having at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100%) identity to the nucleotide sequence of DA06-04A21L1-27A (SEQ ID NO:48) and an antisense saRNA strand having the nucleotide sequence of SEQ ID NO:49 which has partial complementarity to the sense saRNA strand of SEQ ID NO:48.

[0452]

[0335] In certain embodiments, the multivalent oligonucleotide agent has a nucleotide sequence having at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100%) identity to the nucleotide sequence of DA06-04B21L1-27A (SEQ ID NO:51) and a sense saRNA strand of SEQ ID NO:50 that has partial complementarity to the antisense saRNA strand of SEQ ID NO:51.

[0453]

[0336] In certain embodiments, the multivalent oligonucleotide agent has a nucleotide sequence having at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100%) identity to the nucleotide sequence of R6-04M1-27A-S1L1V3(CM-26)(SEQ ID NO:61) and an antisense saRNA strand of SEQ ID NO:17 having partial complementarity to the sense saRNA strand of SEQ ID NO:61.

[0454]

[0337] In certain embodiments, the multivalent oligonucleotide agent has a nucleotide sequence having at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100%) identity to the nucleotide sequence of R6-04M1-16nt-S1L1V3v(CM-27) (SEQ ID NO:79) and an antisense saRNA strand of SEQ ID NO:17 having partial complementarity to the sense saRNA strand of SEQ ID NO:79.

[0455] In certain embodiments, a multivalent oligonucleotide agent comprises a nucleotide sequence having at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100%) identity to the nucleotide sequence of R6-04M1-15nt-S1L1V3v (SEQ ID NO:80) and ... It has a sense saRNA strand of SEQ ID NO:80 and an antisense saRNA strand of SEQ ID NO:17 which has partial complementarity.

[0456] In certain embodiments, a multivalent oligonucleotide agent comprises a nucleotide sequence having at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100%) identity to the nucleotide sequence of R6-04M1-14nt-S1L1V3v (SEQ ID NO:81) and ... It has a sense saRNA strand of SEQ ID NO:81 and an antisense saRNA strand of SEQ ID NO:17 which has partial complementarity.

[0457] In certain embodiments, a multivalent oligonucleotide agent comprises a nucleotide sequence having at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100%) identity to the nucleotide sequence of R6-04M1-13nt-S1L1V3v (SEQ ID NO:82) and ... It has a sense saRNA strand of SEQ ID NO:82 and an antisense saRNA strand of SEQ ID NO:17 which has partial complementarity.

[0458]

[0341] In certain embodiments, the multivalent oligonucleotide agent has a nucleotide sequence having at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100%) identity to the nucleotide sequence of R6-04M1-(12nt-B)-S1L1V3v (SEQ ID NO:83) and an antisense saRNA strand of SEQ ID NO:17 having partial complementarity to the sense saRNA strand of SEQ ID NO:83.

[0459] In certain embodiments, a multivalent oligonucleotide agent comprises a nucleotide sequence having at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100%) identity to the nucleotide sequence of R6-04M1-11nt-S1L1V3v (SEQ ID NO:84) and ... It has a sense saRNA strand of SEQ ID NO:84 and an antisense saRNA strand of SEQ ID NO:17 which has partial complementarity.

[0460] In certain embodiments, a multivalent oligonucleotide agent comprises a nucleotide sequence having at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100%) identity to the nucleotide sequence of R6-04M1-10nt-S1L1V3v (SEQ ID NO:85) and ... It has a sense saRNA strand of SEQ ID NO:85 and an antisense saRNA strand of SEQ ID NO:17 which has partial complementarity.

[0461]

[0344] In certain embodiments, the multivalent oligonucleotide agent has a nucleotide sequence having at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100%) identity to the nucleotide sequence of R6-04M1-9nt-S1L1V3v (SEQ ID NO:86) and a sense saRNA strand of SEQ ID NO:17 that has partial complementarity to the sense saRNA strand of SEQ ID NO:86.

[0462]

[0345] In certain embodiments, the multivalent oligonucleotide agent has a nucleotide sequence having at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100%) identity to the nucleotide sequence of R6-04M1-8nt-S1L1V3v (SEQ ID NO:87) and a sense saRNA strand of SEQ ID NO:17 that has partial complementarity to the sense saRNA strand of SEQ ID NO:87.

[0463]

[0346] In certain embodiments, the multivalent oligonucleotide agent has a nucleotide sequence having at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100%) identity to the nucleotide sequence of R6-04M1-7nt-S1L1V3v (SEQ ID NO:88) and an antisense saRNA strand of SEQ ID NO:17 having partial complementarity to the sense saRNA strand of SEQ ID NO:88.

[0464]

[0347] In certain embodiments, the multivalent oligonucleotide agent has a nucleotide sequence having at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100%) identity to the nucleotide sequence of R6-04M1-6nt-S1L1V3v (SEQ ID NO:89) and an antisense saRNA strand of SEQ ID NO:17 having partial complementarity to the sense saRNA strand of SEQ ID NO:89.

[0465] In certain embodiments, a multivalent oligonucleotide agent comprises a nucleotide sequence having at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100%) identity to the nucleotide sequence of R6-04M1-AC2(18)-S1L1V3v (SEQ ID NO:90), and a nucleotide sequence having at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100%) identity to the nucleotide sequence of SEQ It has a sense saRNA strand of ID NO:90 and an antisense saRNA strand of SEQ ID NO:17 which has partial complementarity.

[0466] In certain embodiments, the multivalent oligonucleotide agent comprises a nucleotide sequence having at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100%) identity to the nucleotide sequence of R6-04M1-AC2(16)-S1L1V3v (SEQ ID NO:91), and a nucleotide sequence having at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100%) identity to the nucleotide sequence of SEQ It has a sense saRNA strand of ID NO:91 and an antisense saRNA strand of SEQ ID NO:17 which has partial complementarity.

[0467] In certain embodiments, a multivalent oligonucleotide agent comprises a nucleotide sequence having at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100%) identity to the nucleotide sequence of R6-04M1-AC2(15)-S1L1V3v (SEQ ID NO:92), and a nucleotide sequence having at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100%) identity to the nucleotide sequence of SEQ It has a sense saRNA strand of ID NO:92 and an antisense saRNA strand of SEQ ID NO:17 which has partial complementarity.

[0468]

[0351] In certain embodiments, the multivalent oligonucleotide agent has a nucleotide sequence having at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100%) identity to the nucleotide sequence of R6-04M1-AC2(14)-S1L1V3v (SEQ ID NO:93) and an antisense saRNA strand of SEQ ID NO:17 having partial complementarity to the sense saRNA strand of SEQ ID NO:93;

[0469] In certain embodiments, a multivalent oligonucleotide agent comprises a nucleotide sequence having at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100%) identity to the nucleotide sequence of R6-04M1-AC2(13)-S1L1V3v (SEQ ID NO:94), and a nucleotide sequence having at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100%) identity to the nucleotide sequence of SEQ having a sense saRNA strand of SEQ ID NO:94 and an antisense saRNA strand of SEQ ID NO:17 having partial complementarity;

[0470] In certain embodiments, a multivalent oligonucleotide agent comprises a nucleotide sequence having at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100%) identity to the nucleotide sequence of R6-04M1-AC2(12)-S1L1V3v (SEQ ID NO:95), and a nucleotide sequence having at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100%) identity to the nucleotide sequence of SEQ having a sense saRNA strand of SEQ ID NO:95 and an antisense saRNA strand of SEQ ID NO:17 having partial complementarity;

[0471] In certain embodiments, a multivalent oligonucleotide agent comprises a nucleotide sequence having at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100%) identity to the nucleotide sequence of R6-04M1-AC2(11)-S1L1V3v (SEQ ID NO:96), and a nucleotide sequence having at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100%) identity to the nucleotide sequence of SEQ having a sense saRNA strand of SEQ ID NO:96 and an antisense saRNA strand of SEQ ID NO:17 having partial complementarity;

[0472] In certain embodiments, a multivalent oligonucleotide agent comprises a nucleotide sequence having at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100%) identity to the nucleotide sequence of R6-04M1-AC2(10)-S1L1V3v (SEQ ID NO:97), and a nucleotide sequence having at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100%) identity to the nucleotide sequence of SEQ having a sense saRNA strand of SEQ ID NO:97 and an antisense saRNA strand of SEQ ID NO:17 having partial complementarity;

[0473] In certain embodiments, the multivalent oligonucleotide agent has a nucleotide sequence having at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100%) identity to the nucleotide sequence of R6-04M1-AC2(9)-S1L1V3v (SEQ ID NO:98) and an antisense saRNA strand of SEQ ID NO:17 having partial complementarity to the sense saRNA strand of SEQ ID NO:98; and

[0474]

[0357] In certain embodiments, the multivalent oligonucleotide agent has a nucleotide sequence having at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100%) identity to the nucleotide sequence of R6-04M1-AC2(8)-S1L1V3v (SEQ ID NO:99) and an antisense saRNA strand of SEQ ID NO:17 having partial complementarity to the sense saRNA strand of SEQ ID NO:99.

[0475] In some embodiments, the multivalent oligonucleotide agent comprises a third oligonucleotide. In certain embodiments, the third oligonucleotide is selected from the group consisting of siRNA, saRNA and ASO. In certain embodiments, the multivalent oligonucleotide agent has a nucleotide sequence having at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100%) identity with the nucleotide sequence of R6-04S1&27A&67S1R-L1V2 (SEQ ID NO:54) and an antisense saRNA strand having the nucleotide sequence of SEQ ID NO:53 having partial complementarity with the DS06-0004 sense saRNA strand of SEQ ID NO:54.

[0476] In certain embodiments, a multivalent oligonucleotide agent comprises a nucleotide sequence having at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100%) identity to the nucleotide sequence of R6-04S1&67S1R&27A-L1V2 (SEQ ID NO:55) and a SEQ ID NO:55 having partial complementarity to the DS06-0004 sense saRNA strand of SEQ ID NO:55. and an antisense saRNA strand having the nucleotide sequence of ID NO:53.

[0477]

[0360] In certain embodiments, the multivalent oligonucleotide agent has a nucleotide sequence having at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100%) identity to the nucleotide sequence of R6-04S1&27A&67S5-L1V2 (SEQ ID NO:57) and an antisense saRNA strand having the nucleotide sequence of SEQ ID NO:53 which has partial complementarity to the DS06-0004 sense saRNA strand of SEQ ID NO:57.

[0478]

[0361] In certain embodiments, the multivalent oligonucleotide agent has a nucleotide sequence having at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100%) identity to the nucleotide sequence of R6-04S1&67S5&27A-L1V2 (SEQ ID NO:58) and an antisense saRNA strand having the nucleotide sequence of SEQ ID NO:53 which has partial complementarity to the DS06-0004 sense saRNA strand of SEQ ID NO:58.

[0479] 4.3.2 Agents that increase the expression of the p21 gene or protein and decrease the expression of the PDL-1 gene or protein

[0362] Aspects of the present disclosure include multivalent oligonucleotide agents that increase expression of the p21 gene or protein and decrease expression of the PDL-1 gene or protein.

[0480] CDKN1A (p21) and CD274 (PD-L1, programmed death-ligand 1) are two important genes involved in tumorigenesis. p21 is both a negative cell cycle regulator and a putative tumor suppressor, and its activation by saRNA leads to tumor suppression. PD-L1 is an important target in cancer therapy, and inhibition of PD-L1 can promote T cell-mediated immune surveillance against cancer, showing great clinical benefit in cancer patients.

[0481]

[0364] In one embodiment, the multivalent oligonucleotide agents of the present disclosure contain two or more functional oligonucleotides covalently linked to combine the tumor-suppressing effects of p21 activation and PD-L1 blockade in a single agent.

[0482] In some embodiments, the two or more functional oligonucleotides comprise a first dsRNA and a second dsRNA. In certain embodiments, the first dsRNA is a saRNA that increases the expression of CDKN1A / p21 gene or protein.

[0483] In certain embodiments, the second dsRNA comprises an siRNA that reduces expression of CD274 / PDL-1. In other embodiments, the second dsRNA comprises an siRNA that reduces expression of CD274 / PDL-1.

[0484] In some embodiments, the two or more functional oligonucleotides comprise a first dsRNA and a first ASO. In certain embodiments, the second dsRNA comprises a first ASO that reduces the expression of CD274 / PDL-1.

[0485]

[0368] In some embodiments, the dsRNA is an siRNA. In certain embodiments, the siRNA has a nucleotide sequence of a sense strand that has at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100%) identity to a nucleotide sequence selected from siPDL1-2 (SEQ ID NO:64) and siPDL1-3 (SEQ ID NO:66). In some embodiments, the dsRNA is an siRNA. In certain embodiments, the siRNA has a nucleotide sequence of a sense strand that has at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100%) identity to a nucleotide sequence of siPDL1-2 (SEQ ID NO:64). In some embodiments, the dsRNA is an siRNA. In certain embodiments, the siRNA has a nucleotide sequence of the sense strand that has at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100%) identity to the nucleotide sequence of siPDL1-3 (SEQ ID NO:66).

[0486]

[0369] In some embodiments, the dsRNA is an siRNA. In certain embodiments, the siRNA has a nucleotide sequence of the antisense strand that has at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100%) identity with a nucleotide sequence selected from siPDL1-2 (SEQ ID NO:65) and siPDL1-3 (SEQ ID NO:67). In some embodiments, the dsRNA is an siRNA. In certain embodiments, the siRNA has a nucleotide sequence of the antisense strand that has at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100%) identity with a nucleotide sequence of siPDL1-2 (SEQ ID NO:65). In some embodiments, the dsRNA is an siRNA. In certain embodiments, the siRNA has a nucleotide sequence of the antisense strand that has at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100%) identity to the nucleotide sequence of siPDL1-3 (SEQ ID NO:67).

[0487] In some embodiments, the second oligonucleotide of the at least two oligonucleotides comprises an ASO. In certain embodiments, the siRNA has a nucleotide sequence that has at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100%) identity to a nucleotide sequence selected from a) aPDL1-1 (SEQ ID NO:72); b) aPDL1-2 (SEQ ID NO:73); and c) aPDL1-3 (SEQ ID NO:74). In certain embodiments, the ASO has a nucleotide sequence that has at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100%) identity to the nucleotide sequence of aPDL1-1 (SEQ ID NO:72). In certain embodiments, the ASO has a nucleotide sequence that is at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100%) identical to the nucleotide sequence of aPDL1-2 (SEQ ID NO:73). In certain embodiments, the ASO has a nucleotide sequence that is at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100%) identical to the nucleotide sequence of aPDL1-3 (SEQ ID NO:74).

[0488] In some embodiments, the multivalent oligonucleotide agent comprises saRNA and siRNA. In certain embodiments, the multivalent oligonucleotide agent has a nucleotide sequence that has at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100%) identity with a nucleotide sequence selected from the group consisting of: a) saP21-40 / siPDL1-2 (SEQ ID NO:71) and an antisense saRNA strand having a nucleotide sequence of SEQ ID NO:63 that has partial complementarity with the sense saRNA strand of SEQ ID NO:71; and b) saP21-40 / siPDL1-3 (SEQ ID NO:100) and an antisense saRNA strand having a nucleotide sequence of SEQ ID NO:63 that has partial complementarity with the sense saRNA strand of SEQ ID NO:100.

[0489]

[0372] In some embodiments, the multivalent oligonucleotide agent includes saRNA and ASO. In certain embodiments, the multivalent oligonucleotide agent has a nucleotide sequence having at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100%) identity to a nucleotide sequence selected from the group consisting of: a) saP21-40 / aPDL1-1 (SEQ ID NO:72) and an antisense saRNA strand having a nucleotide sequence of SEQ ID NO:63 which has partial complementarity to the sense saRNA strand of SEQ ID NO:72; b) saP21-40 / aPDL1-2 (SEQ ID NO:73) and an antisense saRNA strand having a nucleotide sequence of SEQ ID NO:63 which has partial complementarity to the sense saRNA strand of SEQ ID NO:73; c) saP21-40 / aPDL1-3 (SEQ ID NO:74) and an antisense saRNA strand having a nucleotide sequence of SEQ ID NO:63 which has partial complementarity to the sense saRNA strand of SEQ ID NO:73; d) an antisense saRNA strand having the nucleotide sequence of SEQ ID NO:63, which has partial complementarity with the sense saRNA strand of SEQ ID NO:74; d) saP21-40 / aPDL1-1R (SEQ ID NO: NO:75) and an antisense saRNA strand having the nucleotide sequence of SEQ ID NO:63 having partial complementarity to the sense saRNA strand of SEQ ID NO:75; e) saP21-40 / aPDL1-2R (SEQ ID NO:76) and an antisense saRNA strand having the nucleotide sequence of SEQ ID NO:63 having partial complementarity to the sense saRNA strand of SEQ ID NO:75; and f) saP21-40 / aPDL1-3R (SEQ ID NO:77) and an antisense saRNA strand having the nucleotide sequence of SEQ ID NO:63 that is partially complementary to the sense saRNA strand of SEQ ID NO:77.

[0490]

[0373] In certain embodiments, the multivalent oligonucleotide agent has a nucleotide sequence that is at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100%) identical to the nucleotide sequence of saP21-40 / aPDL1-1 (SEQ ID NO:72) and an antisense saRNA strand having the nucleotide sequence of SEQ ID NO:63, which has partial complementarity to the sense saRNA strand of SEQ ID NO:72.

[0491]

[0374] In certain embodiments, the multivalent oligonucleotide agent has a nucleotide sequence that is at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100%) identical to the nucleotide sequence of saP21-40 / aPDL1-2 (SEQ ID NO:73) and an antisense saRNA strand having the nucleotide sequence of SEQ ID NO:63 which has partial complementarity to the sense saRNA strand of SEQ ID NO:73.

[0492]

[0375] In certain embodiments, the multivalent oligonucleotide agent has a nucleotide sequence that is at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100%) identical to the nucleotide sequence of saP21-40 / aPDL1-3 (SEQ ID NO:74) and an antisense saRNA strand having the nucleotide sequence of SEQ ID NO:63 which has partial complementarity to the sense saRNA strand of SEQ ID NO:74.

[0493]

[0376] In certain embodiments, the multivalent oligonucleotide agent has a nucleotide sequence that is at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100%) identical to the nucleotide sequence of saP21-40 / aPDL1-1R (SEQ ID NO:75) and an antisense saRNA strand having the nucleotide sequence of SEQ ID NO:63 which has partial complementarity to the sense saRNA strand of SEQ ID NO:75.

[0494]

[0377] In certain embodiments, the multivalent oligonucleotide agent has a nucleotide sequence that is at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100%) identical to the nucleotide sequence of saP21-40 / aPDL1-2R (SEQ ID NO:76) and an antisense saRNA strand having the nucleotide sequence of SEQ ID NO:63 which has partial complementarity to the sense saRNA strand of SEQ ID NO:76.

[0495]

[0378] In certain embodiments, the multivalent oligonucleotide agent has a nucleotide sequence that is at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100%) identical to the nucleotide sequence of saP21-40 / aPDL1-3R (SEQ ID NO:77) and an antisense saRNA strand having the nucleotide sequence of SEQ ID NO:63 which has partial complementarity to the sense saRNA strand of SEQ ID NO:77.

[0496] 4.4 Compositions of Multivalent Oligonucleotide Agents

[0379] Another aspect of the present disclosure provides pharmaceutical compositions comprising one or more multivalent oligonucleotide agents that include two or more functional oligonucleotides described in this disclosure.

[0497]

[0380] In some embodiments, the pharmaceutical composition further comprises at least one pharma- ceutically acceptable carrier.

[0498]

[0381] In one embodiment, the pharma- ceutically acceptable carrier comprises one or more of an aqueous carrier, a liposome, a high molecular weight polymer, and a polypeptide. In one embodiment, the pharma- ceutically acceptable carrier comprises one or more of an aqueous carrier, a liposome, a high molecular weight polymer, or a polypeptide. In one embodiment, the aqueous carrier may be, for example, RNase-free water or an RNase-free buffer. The composition may comprise 1-150 nM, for example, 1-100 nM, for example, 1-50 nM, for example, 1-20 nM, for example, 10-100 nM, 10-50 nM, 20-50 nM, 20-100 nM, for example, 50 nM of the oligonucleotide or a nucleic acid encoding the oligonucleotide according to the present disclosure.

[0499]

[0382] In some embodiments, the composition comprises 1-150 nM of a first dsRNA comprising a first siRNA, and 1-150 nM of a second dsRNA comprising a second siRNA. In some embodiments, the composition comprises 1-150 nM of a first dsRNA comprising a first saRNA, and 1-150 nM of a second dsRNA comprising a second saRNA. In some embodiments, the composition comprises 1-150 nM of a first dsRNA comprising an siRNA, and 1-150 nM of a second dsRNA comprising an saRNA. In some embodiments, the composition comprises 1-150 nM of a first dsRNA comprising an saRNA, and 1-150 nM of a first ASO. In some embodiments, the composition comprises 1-150 nM of a first dsRNA comprising an siRNA, and 1-150 nM of a first ASO. In some embodiments, the composition comprises 1-150 nM of a first ASO and 1-150 nM of a second ASO. In some embodiments, the composition comprises 1-150 nM of a first dsRNA comprising a first saRNA, 1-150 nM of a second dsRNA comprising a second saRNA, and 1-150 nM of a third ASO.

[0500]

[0383] Another aspect of the present disclosure relates to the use of two or more functional oligonucleotides described herein, nucleic acids encoding two or more functional oligonucleotides described herein, or compositions comprising two or more functional oligonucleotides or nucleic acids encoding two or more functional oligonucleotides described herein, where the two or more functional oligonucleotides are covalently linked to prepare one or more compositions for modulating expression of one or more genes or proteins expressed by a cell.

[0501] Another embodiment provides a pharmaceutical composition or medicament comprising a compound of the present disclosure and a therapeutically inert carrier, diluent or pharmaceutically acceptable excipient, and a method of using the compound of the present disclosure to prepare such a composition and medicament.In certain embodiments, two or more functional oligonucleotides of the present disclosure are covalently linked, so that the two or more functional oligonucleotides are in the same pharmaceutical composition.

[0502]

[0385] The compositions of the present disclosure are formulated, dosed, and administered in a manner consistent with good medical practice. Factors to be considered in this context include the particular disorder being treated, the particular mammal being treated, the individual clinical condition of the patient, the cause of the disorder, the site of administration of the agent, the method of administration, the administration schedule, and other factors known to medical practitioners.

[0503] Compositions comprising any of the small molecule compounds described herein, such as risdiplam and branapram, may be administered by any suitable means, including oral, topical (including buccal and sublingual), rectal, vaginal, transdermal, parenteral, subcutaneous, intraperitoneal, intrapulmonary, intradermal, intrathecal, epidural and intranasal, as well as local administration when localized treatment is desired. In the case of multivalent oligonucleotide compositions, delivery may be by parenteral injection, including intrathecal, intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration.

[0504]

[0387] The small molecule compounds described herein, such as risdiplam and branapram, can be administered in any convenient dosage form, such as tablets, powders, capsules, solutions, dispersions, suspensions, syrups, powders, suppositories, gels, emulsions, patches, etc. Such compositions may contain ingredients conventionally used in pharmaceutical formulations, such as diluents, carriers, pH adjusters, preservatives, solubilizers, stabilizers, wetting agents, emulsifiers, sweeteners, colorants, flavorings, salts for varying osmotic pressure, buffers, masking agents, antioxidants, and additional active agents. Such compositions may further contain other therapeutically valuable substances.

[0505] Typical preparation is prepared by mixing the compound of the present invention and carrier or excipient.Suitable carrier and excipient are well known to those skilled in the art, and their details are described in, for example, Ansel HC et al., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems (2004) Lippincott, Williams & Wilkins, Philadelphia;Gennaro AR et al., Remington: The Science and Practice of Pharmacy (2000) Lippincott, Williams & Wilkins, Philadelphia;and Rowe R. C, Handbook of Pharmaceutical Excipients (2005) Pharmaceutical Press, Chicago. The formulation may also include one or more buffers, stabilizers, surfactants, wetting agents, lubricants, emulsifiers, suspending agents, preservatives, antioxidants, opacifying agents, glidants, processing aids, colorants, sweeteners, flavors, flavorings, diluents, and other known additives to provide an elegant presentation of the drug (i.e., a compound of the invention or a pharmaceutical composition thereof) or to aid in the manufacture of a pharmaceutical product (i.e., a drug product).

[0506] In another aspect, the present invention provides the use of a combination of two or more functional oligonucleotides of a covalently linked multivalent oligonucleotide agent according to any one of the embodiments described herein, or a composition according to any one of the embodiments described herein, in the manufacture of a medicament for treating a gene or protein-related condition in an individual. In certain embodiments, the condition may include an SMN deficiency-related condition, including a genetic neuromuscular disease, preferably spinal muscular atrophy. In other embodiments, the condition may include an immune-related condition, such as cancer. Also provided is the use according to certain embodiments, in which the individual is a mammal, preferably a human.

[0507] 4.5 Multivalent Oligonucleotide Drug Kits In another aspect, any of the multivalent oligonucleotide agents or compositions described herein may be provided in one or more kits, optionally including instructions for use of the agent or composition. That is, the kit may include a description of the use of the multivalent oligonucleotide agent or composition in any of the methods described herein. As used herein, a "kit" typically defines a package, assembly, or container (e.g., an insulated container) that contains one or more of the components or embodiments of the present invention and / or other components related to the present invention, for example, as described above. Any precursor or component of the kit may be provided in liquid form (e.g., in solution) or in solid form (e.g., dry powder, frozen, etc.).

[0508] In some cases, the kit includes one or more components, which may be present in the same container or in two or more containers, and / or any combination thereof. The containers can contain liquids, and non-limiting examples include bottles, vials, jars, tubes, flasks, beakers, etc. In some cases, the containers are spill-proof (when closed, liquids cannot exit the container, regardless of the orientation of the container).

[0509]

[0392] Examples of other compositions or components related to the agents, compounds and methods described herein include, but are not limited to, diluents, salts, buffers, chelating agents, preservatives, desiccants, antimicrobial agents, needles, syringes, packaging materials, tubes, bottles, flasks, beakers, etc., for example, using, modifying, assembling, storing, packaging, preparing, mixing, diluting, and / or preserving the components for a particular use. In embodiments in which a liquid form of any component is used, the liquid form may be concentrated or ready to use.

[0510] In additional embodiments, kits are provided for using the kit in conjunction with the components and / or methods described herein and may include any form of instruction manual or instructions to a website or other source. For example, the instruction manual may include instructions for use, modification, mixing, dilution, preservation, assembly, storage, packaging, and / or preparation of the components and / or other components associated with the kit. In some cases, the instruction manual may include instructions for delivery, such as shipping or storage of the components at room temperature, below freezing, cryogenic temperatures, etc. The instructions may be provided in any form useful to a user of the kit, such as written or verbal (e.g., telephone), digital, optical, visual (e.g., videotape, DVD, etc.), and / or electronic communication (including internet or web-based communication).

[0511] 4.5 Uses / Methods of Use of Multivalent Oligonucleotide Agents

[0394] The multivalent oligonucleotide agents of the present disclosure are useful in therapeutic approaches to treat diseases associated with the agent's target.

[0512]

[0395] Provided herein is a method for treating a disease comprising administering to a subject in need of treatment one or more multivalent oligonucleotide agents, compositions or kits of the present disclosure, wherein the target of the multivalent oligonucleotide agent is associated with the disease.

[0513]

[0396] Some exemplary aspects and embodiments of the present invention are as follows: However, such aspects and embodiments do not necessarily represent the full scope of the present invention, and therefore reference is made to the claims and this specification to interpret the scope of the present invention.

[0514] 4.5.1 Methods for regulating splicing and gene expression

[0397] The multivalent oligonucleotides of the present disclosure are useful in approaches to treat diseases such as spinal muscular atrophy (SMA), an autosomal recessive disorder that affects approximately 1 in 6,000-8,000 newborns and is the leading genetic cause of infant mortality. SMA is caused by reduced levels of the survival of motor neuron (SMN) protein as a result of homozygous deletion or mutation of the telomeric copy of the survival of motor neuron gene (SMN1) located on chromosome 5q13.4.

[0515]

[0398] The SMN protein is encoded by two SMN genes (SMN1 and SMN2), which differ in the coding sequence by a single nucleotide in exon 7 in that the SMN2 gene has a cytosine (C) changed to thymine (T) (Coovert, DD et al., The survival motor neuron protein in spinal muscular atrophy. Human Mol Genet (1997)). This critical difference creates a cryptographic splice site such that exon 7 is skipped in approximately 90% of mature SMN mRNAs transcribed from the SMN2 gene. SMN2 mRNA lacking exon 7 (SMN2Δ7) produces a truncated SMN protein that is unstable and rapidly degraded. In SMA patients, the SMN1 gene no longer produces the SMN protein, and the amount of full-length SMN protein produced by SMN2 is insufficient to compensate for the loss of SMN1, leading to apoptotic death of motor neurons in the anterior horn of the spinal cord, atrophy of skeletal muscles, and loss of muscle strength (Monani, UR et al., The human centromeric survival motor neuron gene (SMN2) rescues embryonic lethality in Smn(- / -) mice and results in a mouse with spinal muscular atrophy. Human Mol Genet (2000)). The severity of symptoms in SMA patients is determined by the number of copies of the SMN2 gene in the patient's cells, and the higher the copy number, the milder the symptoms (Harada, Y. et al., Correlation between SMN2 copy number and clinical phenotype of spinal muscular atrophy: three SMN2 copies fail to rescue some patients from the disease severity. J Neurol (2002)).

[0516] One strategy to develop a treatment for SMA is to stimulate the inclusion of exon 7 in the splicing process using splicing modulators (SMs). In this regard, the antisense oligonucleotide (ASO) drug Spinraza® has already been approved for sale by the US Food and Drug Administration (FDA) (Hua, Y. and AR Krainer. Antisense-mediated exon inclusion. Methods Mol Biol (2012), and Stein, CA and D. Castanotto. FDA-Approved Oligonucleotide Therapies in 2017. Mol Thera (2017)). Another drug, risdiplam (RG7916), is an investigational, orally administered, small molecule drug that is currently pending a New Drug Application (NDA) with the FDA (amdas, S. and L. Servais. New treatments in spinal muscular atrophy: an overview of currently available data. Expert Opin Pharmaco (2020)). These two drugs have transformed the treatment of SMA by significantly extending patient survival and improving motor milestones. Despite these improvements, treated patients, especially children, are far from leading normal lives. There are several plausible reasons to interpret the poor efficacy of SM. One is the ceiling effect, which limits the maximal achievable level of full-length SMN protein restored by treatment. SM does not affect SMN2 transcription and therefore does not increase the amount of available SMN2 pre-mRNA. To restore SMN protein to normal physiological levels, SM would optimally need to achieve 100% efficiency in vivo in converting SMN2Δ7 mRNA to full-length mRNA, an ideal effect that is unlikely to occur in reality. Thus, the maximum efficacy that SM can provide to patients is limited by the availability of SMN2 pre-mRNA.

[0517] Another therapeutic approach to treat SMA is to stimulate transcription of SMN2 to increase the levels of full-length SMN protein. So far, various epigenetic modifiers, such as histone deacetylase (HDAC) inhibitors (e.g., sodium butyrate, valproic acid) and non-HDAC inhibitors (e.g., hydroxyurea, celecoxib, albuterol), have been tested in vitro and in mouse SMA models, but they have failed to show significant clinical efficacy (Lunke, S. and A. El-Osta. The emerging role of epigenetic modifications and chromatin remodeling in spinal muscular atrophy. J Neurochem (2009)). An explanation for this failure is the lack of target specificity of epigenetic modifiers. There is a need for improved methods and compositions for treating SMN deficiency-related pathologies, such as spinal muscular atrophy.

[0518] Another aspect of the disclosure relates to a method of treating or delaying the onset of an SMN deficiency-associated condition in an individual, comprising administering to the subject a therapeutically effective amount of a multivalent oligonucleotide agent described herein, a nucleic acid encoding a multivalent oligonucleotide agent described herein, or a composition comprising a multivalent oligonucleotide agent of the invention or a nucleic acid encoding a multivalent oligonucleotide agent described herein. The subject may be a mammal, such as a human. The subject may be an infant, a child, or an adult. In one embodiment, the disease caused by under-expression of the full-length SMN protein or a mutation in the SMN1 gene may include, for example, SMA. In one embodiment, the disease caused by under-expression of the full-length SMN protein, a mutation or deletion in the SMN1 gene, and / or under-expression of the full-length SMN protein is SMA. In one embodiment, the SMA of the present invention includes SMA type I, SMA type II, SMA type, and SMA type IV.

[0519]

[0402] In some embodiments, the methods disclosed herein include a method of increasing expression of the SMN2 gene or protein while also increasing functional SMN protein levels in a subject in need thereof, comprising administering to the subject a pharmaceutical composition comprising a multivalent agent of the disclosure and a pharma- ceutically acceptable carrier.

[0520]

[0403] In some embodiments, the production of functional SMN protein is increased by modulating the splicing or stability of SMN2 mRNA (SMN2 mRNA modulators).

[0521]

[0404] In some embodiments, the methods disclosed herein further include administering at least one or more SMN2 mRNA modulators selected from the group consisting of nusinersen, risdiplam, and branapram.

[0522] Another aspect of the present invention relates to the use of a multivalent oligonucleotide drug of the present disclosure, a nucleic acid encoding two or more functional oligonucleotides of a multivalent oligonucleotide drug of the present disclosure, or a composition comprising a multivalent oligonucleotide drug of the present disclosure or a nucleic acid encoding two or more functional oligonucleotides of a multivalent oligonucleotide drug of the present disclosure, in the preparation of a medicament for treating or delaying the onset of an SMN deficiency-associated condition. The subject may be a mammal, such as a human. The subject may be an infant, a child, or an adult. In one embodiment, the SMN deficiency-associated condition includes, for example, SMA. In one embodiment, the SMA of the present invention includes SMA type I, SMA type II, SMA type, and SMA type IV.

[0523] Also provided is the use of any one of the combinations of SMN2 dsRNA and SMN2 mRNA modulators described herein, or any one of the compositions of the combinations of SMN2 dsRNA and SMN2 mRNA modulators described herein, in the manufacture of a formulation for increasing the amount of full-length SMN protein in a cell. In certain embodiments, the cell is a mammalian cell, preferably a human cell. In certain embodiments, the cell is in a human. In certain embodiments, the human is a patient suffering from a condition caused by an SMN deficiency-related condition. In certain embodiments, the combination or composition thereof is administered in an amount effective to treat the SMN deficiency-related condition. In certain embodiments, the condition caused by an SMN deficiency-related condition is a condition associated with an inherited neuromuscular disease, preferably spinal muscular atrophy.

[0524]

[0407] In certain embodiments, two or more covalently linked functional oligonucleotides achieve an increase in full-length SMN protein that is greater than that achieved by administration of the same amount of either substance used individually, while reducing toxicity or undesirable side effects. In certain embodiments, two or more functional oligonucleotides achieve an increase in full-length SMN protein that is greater than the additive effect of treatment with the same amount of either oligonucleotide used individually. In certain embodiments, when used in the covalent combination embodiments described herein, the amount of each of the oligonucleotides administered is less than that used in conventional treatment. In certain embodiments, the multivalent oligonucleotide agent optionally includes an SMN2 mRNA modulator.

[0525] In certain embodiments, the effect of the combination of two or more functional oligonucleotides achieves a greater clinical improvement than the effect of the same amount of either substance used individually. In certain embodiments, the effect of two or more covalently linked functional oligonucleotides achieves a greater than additive clinical improvement than the effect of the same amount of either substance used individually.

[0526]

[0409] The present disclosure also relates to a method for increasing the amount of full-length SMN protein in a cell, comprising administering to the cell a multivalent oligonucleotide comprising two or more functional oligonucleotides described herein, a nucleic acid encoding two or more functional oligonucleotides described herein, or a composition comprising two or more functional oligonucleotides or a nucleic acid encoding two or more functional oligonucleotides described herein.

[0527] In any of the embodiments provided herein, such multivalent agents, nucleic acids encoding the disclosed multivalent agents, or compositions comprising such multivalent agents or nucleic acids encoding the disclosed multivalent agents may be directly introduced into a cell, or may be produced intracellularly by introducing a nucleotide sequence encoding the multivalent agent into a cell, preferably a mammalian cell, more preferably a human cell. Such cells may be ex vivo, such as a cell line, or may be present in a mammal, such as a human, including an infant, child, or adult. In some embodiments, the human is a patient or individual suffering from an SMN deficiency-associated condition. In certain embodiments, the nucleic acid encoding the disclosed multivalent agent, or the composition comprising the aforementioned multivalent agent or nucleic acid encoding the multivalent agent, is in an amount sufficient to effectively treat an SMN deficiency-associated condition. In one embodiment, the SMN deficiency-associated condition is SMA. In one embodiment, the SMA of the present disclosure includes SMA type I, SMA type II, SMA type, and SMA type IV.

[0528]

[0411] In certain embodiments, the combination of SMN2 dsRNA and SMN2 mRNA modulator, when covalently linked, achieves an increase in full-length SMN protein that is greater than that achieved by administration of the same amount of either agent used individually. In certain embodiments, the combination of two or more functional oligonucleotides in a multivalent drug results in reduced toxicity and / or reduced undesirable side effects compared to monotherapy treatment. In certain embodiments, the two or more functional oligonucleotides in a multivalent drug achieve an increase in full-length SMN protein that is greater than the additive effect of treatment with the same amount of either agent used individually. In certain embodiments, each oligonucleotide, when covalently linked, is administered in an amount less than that used in conventional monotherapy treatment.

[0529] In certain embodiments, two or more functional oligonucleotides of a multivalent agent achieve a greater clinical improvement than the effect of the same amount of either agent used individually. In certain embodiments, two or more functional oligonucleotides of a multivalent agent achieve a greater than additive clinical improvement than the effect of the same amount of either agent used individually.

[0530] In certain embodiments, the baseline measurement is obtained from a biological sample as defined herein and is obtained from an individual before administering the therapy described herein.In certain embodiments, the biological sample is peripheral blood mononuclear cells, plasma, serum, skin tissue, cerebrospinal fluid (CSF).In certain embodiments, the increase in SMN protein level in peripheral blood mononuclear cells and skin correlates with the increase in neurons in the central nervous system (CNS), indicating that the change in these levels in blood or skin can be used as a non-invasive surrogate to determine the change in SMN protein level in the CNS.In further embodiments, the combinations provided herein increase or decrease the amount of full length SMN protein by at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 110%, at least 115%, at least 120%, at least 125%, at least 130%, at least 135%, at least 140%, at least 145%, at least 150%, at least 155%, at least 160%, at least 165%, at least 170%, at least 175%, at least 180%, at least 185%, at least 190%, at least 200%, at least 210%, at least 220%, at least 230%, at least 240%, at least 250%, at least 260%, at least 270%, at least 280%, at least 290%, at least 300%, at least 310%, at least 320%, at least 330%, at least 340%, at least 350%, at least 355%, at least 360%, at least 370%, at least 380%, at least 390%, at least 400%, at least 410%, at least 420%, at least 430%, at least 440%, at least 450%, at least 460%, at least 470%, at least 480%, at least 490%, at least 500%, at least 510%, at least 520%, at least 530%, at least 540%, at least 550%, at least increase by 5%, at least 200%, at least 210%, at least 215%, at least 220%, at least 225%, at least 230%, at least 235%, at least 240%, at least 245%, at least 250%, at least 255%, at least 260%, at least 265%, at least 270%, at least 275%, at least 280%, at least 285%, at least 290%, at least 295%, at least 300%, at least 310%, at least 315%, at least 320%, at least 325%, at least 330%, at least 335%, at least 340%, at least 345%, at least 350%, at least 355%, at least 360%, at least 365%, at least 370%, at least 375%, at least 380%, at least 385%, at least 390%, at least 395%, at least 400%.

[0531]

[0414] The dosage at which the compositions of the present disclosure can be administered can vary within wide limits and will, of course, be adapted to the individual requirements in each case.

[0532]

[0415] In certain embodiments, the multivalent agent exhibits greater than additive or synergistic effects in the treatment, prevention, delay of progression and / or amelioration of diseases caused by inactivating mutations or deletions in the SMN1 gene and / or associated with loss or deficiency of SMN1 gene function, and for the protection of cells involved in the pathophysiology of the disease, in particular for the treatment, prevention, delay of progression and / or amelioration of spinal muscular atrophy (SMA).

[0533]

[0416] In certain embodiments, the first dose of a pharmaceutical composition of the present disclosure is administered when the subject is less than 1 week, less than 1 month, less than 3 months, less than 6 months, less than 1 year, less than 2 years, less than 15 years, or more than 15 years.

[0534] In certain embodiments...

Claims

1. 1. A multivalent oligonucleotide drug comprising two or more covalently linked functional oligonucleotides, wherein said two or more functional oligonucleotides are independently selected from the group consisting of: a) double-stranded RNA (dsRNA); and b) Antisense oligonucleotides (ASOs).

2. the number of functional oligonucleotides contained in the multivalent oligonucleotide agent is in the range of 2 to X, where X is an integer in the range of 3 to 10; and / or 2. The multivalent oligonucleotide agent of claim 1, wherein the number of dsRNAs contained in the agent ranges from 0 to X, and the remaining functional oligonucleotides are ASOs.

3. The dsRNA is independently selected from small interfering RNA (siRNA) and small activating RNA (saRNA); and / or The ASOs are independently selected from gapmers and mixmers. The multivalent oligonucleotide drug of claim 1 .

4. Each dsRNA is characterized in that it comprises a sense strand of at least 10 consecutive nucleotides and an antisense strand of at least 10 consecutive nucleotides. and / or Each ASO is characterized in that it has a nucleotide sequence that is at least 5 contiguous nucleotides in length; and / or The multivalent oligonucleotide agent of claim 1, wherein the two or more functional oligonucleotides have a total length in the range of 12 to 200 nucleotides.

5. The multivalent oligonucleotide drug of claim 1, wherein any two adjacent functional oligonucleotides among the two or more functional oligonucleotides are covalently linked with or without a linking moiety.

6. 6. The multivalent oligonucleotide drug of claim 5, wherein the linking moiety is selected from the following linkers or derivatives thereof: 【Table 1】

7. The multivalent oligonucleotide drug of claim 5, characterized in that two adjacent functional oligonucleotides are covalently linked by: 【Chemistry 1】 wherein R represents -H, or -OH, or -OMe, or -MOE, or -F, or other 2' chemical modification; and / or phosphodiester or phosphorothioate bonds; and / or One or more nucleotides.

8. The multivalent oligonucleotide drug of claim 1, wherein one or more functional oligonucleotides contain at least one chemically modified nucleotide.

9. Each ASO in the agent is covalently linked to an adjacent targeting oligonucleotide in a 3' to 5' or 5' to 3' direction; and / or The multivalent oligonucleotide agent of claim 1, wherein each dsRNA in the agent is covalently linked to an adjacent ASO at the 3' end of the sense or antisense strand or at the 5' end of the sense or antisense strand.

10. The multivalent oligonucleotide drug of claim 1, characterized in that the multivalent oligonucleotide drug comprises: a) a first double-stranded RNA (dsRNA) and a first antisense oligonucleotide (ASO); b) a first double-stranded RNA (dsRNA) and a second dsRNA; c) a first antisense oligonucleotide (ASO) and a second ASO; d) a first double-stranded RNA (dsRNA), a second dsRNA, and a third dsRNA; e) a first double-stranded RNA (dsRNA), a second dsRNA, and a first antisense oligonucleotide (ASO); f) a first double-stranded RNA (dsRNA), a first antisense oligonucleotide (ASO) and a second ASO; or g) a first antisense oligonucleotide (ASO), a second ASO and a third ASO; With the proviso that in any one of a) to g), the functional oligonucleotides are arranged in any order and are covalently linked with or without a linking moiety.

11. the first dsRNA, the second dsRNA and the third dsRNA, if present, are independently selected from siRNA and saRNA; and / or The first ASO, the second ASO and the third ASO, if present, are independently selected from gapmers and mixmers. A multivalent oligonucleotide drug according to claim 10.

12. 12. The multivalent oligonucleotide drug of claim 11, wherein the multivalent oligonucleotide drug comprises a functional oligonucleotide selected from the group consisting of: a) siRNA-siRNA; b) siRNA-saRNA; c) saRNA-saRNA; d) siRNA-gapmer; e) siRNA-mixmer; f) saRNA-gapmer; g) saRNA-mixmer; h) gapmer-gapmer; i) gapmer-mixmer; j) mixmer-mixmer; k) siRNA-siRNA-siRNA; l) siRNA-siRNA-saRNA; m) siRNA-saRNA-saRNA; n) saRNA-saRNA-saRNA; o) siRNA-siRNA-gapmer; p) siRNA-siRNA-mixmer; q) siRNA-saRNA-gapmer; r) siRNA-saRNA-mixmer; s) saRNA-saRNA-gapmer; t) saRNA-saRNA-mixmer; u) siRNA-gapmer-gapmer; v) saRNA-gapmer-gapmer; w) siRNA-gapmer-mixmer; x) saRNA-gapmer-mixmer; y) siRNA-mixmer-mixmer; z) saRNA-mixmer-mixmer; aa) gapmer-gapmer-gapmer; ab) gapmer-gapmer-mixmer; ac) gapmer-mixmer-mixmer; and ad) mixmer-mixmer-mixmer; However, in any one of a) to ad), the functional oligonucleotides are arranged in any order and are covalently linked with or without a linking moiety.

13. one or more additional targeting oligonucleotides, The multivalent oligonucleotide drug of claim 1, wherein the additional targeting oligonucleotides are independently selected from the group consisting of double-stranded RNA (dsRNA) and antisense oligonucleotides (ASO).

14. one or more ASOs are characterized in that they target the 5'-UTR; and / or The multivalent oligonucleotide drug of claim 1, wherein the one or more functional oligonucleotides increase expression of the SMN2 gene or protein.

15. The multivalent oligonucleotide drug of claim 14, wherein the dsRNA increases expression of the SMN2 gene or protein; and / or the ASO increases production of functional SMN protein by modulating splicing or stability of SMN2 mRNA (SMN2 mRNA modulator).

16. The dsRNA includes a saRNA having a nucleotide sequence of the sense strand that has at least 90% identity to a nucleotide sequence selected from the group consisting of: a) DS06-0004 (SEQ ID NO: 5); b) DS06-0031 (SEQ ID NO: 7); c) DS06-0067 (SEQ ID NO:9); d) DS06-4A3 (SEQ ID NO: 146); e) R6-04-S1 (SEQ ID NO:59); and f) R6-04(20)-S1V1v (CM-4) (SEQ ID NO: 60); and / or The dsRNA includes a saRNA having an antisense strand nucleotide sequence that has at least 90% identity to a nucleotide sequence selected from the group consisting of: a') DS06-0004 (SEQ ID NO: 6); b') DS06-0031 (SEQ ID NO: 8); c') DS06-0067 (SEQ ID NO: 10); d') DS06-4A3 (SEQ ID NO: 147); e') R6-04-S1 (SEQ ID NO:53); and f') R6-04M1-27A-S1L1V3 (CM-26) (SEQ ID NO: 17) A multivalent oligonucleotide drug according to claim 14.

17. The dsRNA comprises an siRNA having a sense strand nucleotide sequence that has at least 90% identity to the nucleotide sequence of DS06-332i (SEQ ID NO: 3) or siSOD1-388-ESC (SEQ ID NO: 138); and / or The dsRNA includes an siRNA having an antisense nucleotide sequence that has at least 90% identity with the nucleotide sequence of DS06-332i (SEQ ID NO: 4) or siSOD1-388-ESC (SEQ ID NO: 139). A multivalent oligonucleotide drug according to claim 14.

18. The multivalent oligonucleotide agent of claim 14, wherein the ASO has a nucleotide sequence that has at least 90% identity to the nucleotide sequence of ASO10-27 (SEQ ID NO: 11) or 5'UTR ASO (SEQ ID NO: 142).

19. The multivalent oligonucleotide drug of claim 14, characterized in that the multivalent oligonucleotide drug has a nucleotide sequence having at least 90% identity to a nucleotide sequence selected from the group consisting of: a) DA06-4A-27A (SEQ ID NO:14) and an antisense saRNA strand having the nucleotide sequence of SEQ ID NO:13 which has partial complementarity to the sense saRNA strand of SEQ ID NO:14; b) DA06-4A-27B (SEQ ID NO:15) and an antisense saRNA strand having the nucleotide sequence of SEQ ID NO:13 having partial complementarity to the sense saRNA strand of SEQ ID NO:15; c) R6-04M1-27A-S1L1V3 (SEQ ID NO: 18) and SEQ an antisense saRNA strand having the nucleotide sequence of SEQ ID NO: 13, which has partial complementarity to the sense saRNA strand of ID NO: 18; d) DA06-31A-27A (SEQ ID NO:19) and an antisense saRNA strand having the nucleotide sequence of SEQ ID NO:8, which has partial complementarity to the sense saRNA strand of SEQ ID NO:19; e) DA06-31B-27A (SEQ ID NO:20) and a sense saRNA strand having the nucleotide sequence of SEQ ID NO:7, which has partial complementarity to the antisense saRNA strand of SEQ ID NO:20; f) DA06-67A-27A (SEQ ID NO:21) and an antisense saRNA strand having the nucleotide sequence of SEQ ID NO:10, which has partial complementarity to the sense saRNA strand of SEQ ID NO:21; g) DA06-67B-27A (SEQ ID NO:22) and a sense saRNA strand having the nucleotide sequence of SEQ ID NO:9 which has partial complementarity to the antisense saRNA strand of SEQ ID NO:22; h) DA6-67A3'L0-27A (SEQ ID NO: 23) and SEQ ID an antisense saRNA strand having the nucleotide sequence of SEQ ID NO: 10, which has partial complementarity to the sense saRNA strand of SEQ ID NO: 23; j) DA6-67A3'L9-27A (SEQ ID NO: 24) and SEQ ID an antisense saRNA strand having the nucleotide sequence of SEQ ID NO: 10, which has partial complementarity to the sense saRNA strand of SEQ ID NO: 24; k) DA6-67A3'L4-27A (SEQ ID NO: 25) and SEQ ID an antisense saRNA strand having the nucleotide sequence of SEQ ID NO: 10, which has partial complementarity to the sense saRNA strand of SEQ ID NO: 25; l) DA6-67B3'L0-27A (SEQ ID NO: 26) and SEQ ID a sense saRNA strand having the nucleotide sequence of SEQ ID NO:9, which has partial complementarity to the antisense saRNA strand of SEQ ID NO:26; m) DA6-67B5'L1-27A (SEQ ID NO: 27) and SEQ ID a sense saRNA strand having the nucleotide sequence of SEQ ID NO:9, which has partial complementarity to the antisense saRNA strand of SEQ ID NO:27; o) DA6-67B5'L9-27A (SEQ ID NO: 29) and SEQ ID a sense saRNA strand having the nucleotide sequence of SEQ ID NO: 9, which has partial complementarity to the antisense saRNA strand of SEQ ID NO: 29; p) DA6-67B5'L4-27A (SEQ ID NO: 30) and a sense saRNA strand having the nucleotide sequence of SEQ ID NO:9, which has partial complementarity to the antisense saRNA strand of SEQ ID NO:30; q) DA6-67B3'L9-27A (SEQ ID NO: 31) and a sense saRNA strand having the nucleotide sequence of SEQ ID NO: 9, which has partial complementarity to the antisense saRNA strand of SEQ ID NO: 31; r) DA6-67B3'L4-27A (SEQ ID NO: 32) and a sense saRNA strand having the nucleotide sequence of SEQ ID NO: 9, which has partial complementarity to the antisense saRNA strand of SEQ ID NO: 32; s) DA06-67A21L1-27A (SEQ ID NO:33) and an antisense saRNA strand having the nucleotide sequence of SEQ ID NO:34, which has partial complementarity to the sense saRNA strand of SEQ ID NO:33; t) DA06-67B21L1-27A (SEQ ID NO:36) and a sense saRNA strand of SEQ ID NO:35 having partial complementarity to the antisense saRNA strand of SEQ ID NO:36; u) DA6-04A3'L0-27A (SEQ ID NO: 37) and SEQ ID an antisense saRNA strand having the nucleotide sequence of SEQ ID NO: 6, which has partial complementarity to the sense saRNA strand of SEQ ID NO: 37; v) DA6-04A5'L1-27A (SEQ ID NO: 38) and SEQ ID an antisense saRNA strand having the nucleotide sequence of SEQ ID NO: 6, which has partial complementarity to the sense saRNA strand of SEQ ID NO: 38; w) DA6-04A5'L9-27A (SEQ ID NO: 39) and SEQ ID an antisense saRNA strand having the nucleotide sequence of SEQ ID NO: 6, which has partial complementarity to the sense saRNA strand of SEQ ID NO: 39; x) DA6-04A5'L4-27A (SEQ ID NO: 40) and SEQ ID an antisense saRNA strand having the nucleotide sequence of SEQ ID NO:6, which has partial complementarity to the sense saRNA strand of SEQ ID NO:40; y) DA6-04A3'L1-27A (SEQ ID NO: 41) and SEQ ID an antisense saRNA strand having the nucleotide sequence of SEQ ID NO: 6, which has partial complementarity to the sense saRNA strand of SEQ ID NO: 41; z) DA6-04A3'L9-27A (SEQ ID NO: 42) and SEQ ID an antisense saRNA strand having the nucleotide sequence of SEQ ID NO: 6, which has partial complementarity to the sense saRNA strand of SEQ ID NO: 42; aa) DA6-04A3'L4-27A (SEQ ID NO:43) and an antisense saRNA strand having the nucleotide sequence of SEQ ID NO:6 having partial complementarity to the sense saRNA strand of SEQ ID NO:43; bb) DA6-04B3'L0-27A (SEQ ID NO:44) and a sense saRNA strand having the nucleotide sequence of SEQ ID NO:5 which has partial complementarity to the antisense saRNA strand of SEQ ID NO:44; cc) DA6-04B3'L1-27A (SEQ ID NO:45) and a sense saRNA strand having the nucleotide sequence of SEQ ID NO:5 which has partial complementarity to the antisense saRNA strand of SEQ ID NO:45; dd) DA6-04B3'L9-27A (SEQ ID NO:46) and a sense saRNA strand having the nucleotide sequence of SEQ ID NO:5 which has partial complementarity to the antisense saRNA strand of SEQ ID NO:46; ee) DA6-04B3'L4-27A (SEQ ID NO:47) and a sense saRNA strand having the nucleotide sequence of SEQ ID NO:5 which has partial complementarity to the antisense saRNA strand of SEQ ID NO:47; ff) DA06-04A21L1-27A (SEQ ID NO:48) and an antisense saRNA strand having the nucleotide sequence of SEQ ID NO:49 which has partial complementarity to the sense saRNA strand of SEQ ID NO:48; gg) DA06-04B21L1-27A (SEQ ID NO:51) and a sense saRNA strand of SEQ ID NO:50 having partial complementarity to the antisense saRNA strand of SEQ ID NO:51; hh) R6-04M1-16nt-S1L1V3v (SEQ ID NO:79) and an antisense saRNA strand of SEQ ID NO:17 having partial complementarity to the sense saRNA strand of SEQ ID NO:79; ii) R6-04M1-15nt-S1L1V3v (SEQ ID NO:80) and an antisense saRNA strand of SEQ ID NO:17 having partial complementarity to the sense saRNA strand of SEQ ID NO:80; jj) R6-04M1-14nt-S1L1V3v (SEQ ID NO:81) and an antisense saRNA strand of SEQ ID NO:17 having partial complementarity to the sense saRNA strand of SEQ ID NO:81; kk) R6-04M1-13nt-S1L1V3v (SEQ ID NO:82) and an antisense saRNA strand of SEQ ID NO:17 having partial complementarity to the sense saRNA strand of SEQ ID NO:82; ll) R6-04M1-(12nt-B)-S1L1V3v (SEQ ID NO:83) and an antisense saRNA strand of SEQ ID NO:17 having partial complementarity to the sense saRNA strand of SEQ ID NO:83; mm) R6-04M1-11nt-S1L1V3v (SEQ ID NO:84) and an antisense saRNA strand of SEQ ID NO:17 having partial complementarity to the antisense saRNA strand of SEQ ID NO:84; nn) R6-04M1-10nt-S1L1V3v (SEQ ID NO: 85) and a SEQ ID NO: 85 having partial complementarity to the antisense saRNA strand. Antisense saRNA strand of ID NO: 17; oo) R6-04M1-9nt-S1L1V3v (SEQ ID NO:86) and an antisense saRNA strand of SEQ ID NO:17 having partial complementarity to the sense saRNA strand of SEQ ID NO:86; pp) R6-04M1-8nt-S1L1V3v (SEQ ID NO: 87) and an antisense saRNA strand of SEQ ID NO: 17 having partial complementarity to the sense saRNA strand of SEQ ID NO: 87; qq) R6-04M1-7nt-S1L1V3v (SEQ ID NO:88) and an antisense saRNA strand of SEQ ID NO:17 having partial complementarity to the sense saRNA strand of SEQ ID NO:88; rr) R6-04M1-6nt-S1L1V3v (SEQ ID NO:89) and an antisense saRNA strand of SEQ ID NO:17 having partial complementarity to the sense saRNA strand of SEQ ID NO:89; ss) DS06-4A-S2L5V (SEQ ID NO: 128) and SEQ ID an antisense saRNA strand having the nucleotide sequence of SEQ ID NO: 17, which has partial complementarity to the sense saRNA strand of SEQ ID NO: 128; ss') DS06-4A-S2L1v (SEQ ID NO: 16) and SEQ ID an antisense saRNA strand having the nucleotide sequence of SEQ ID NO: 17, which has partial complementarity to the sense saRNA strand of SEQ ID NO: 16; tt) DA6-27A-5'UTR (SEQ ID NO: 143); uu) DA6-5'UTR-27A (SEQ ID NO: 144); vv) R6-67M3-27A-S1L1V3 (SEQ ID NO: 129) and an antisense saRNA strand having the nucleotide sequence of SEQ ID NO: 130, which has partial complementarity to the sense saRNA strand of SEQ ID NO: 129; ww) R6-67M3-16nt-S1L1V3 (SEQ ID NO: 129) and an antisense saRNA strand having the nucleotide sequence of SEQ ID NO: 131, which has partial complementarity to the sense saRNA strand of SEQ ID NO: 129; xx) R6-67M3-15nt-S1L1V3 (SEQ ID NO: 129) and an antisense saRNA strand having the nucleotide sequence of SEQ ID NO: 132, which has partial complementarity to the sense saRNA strand of SEQ ID NO: 129; yy) R6-67M3-14nt-S1L1V3 (SEQ ID NO: 129) and an antisense saRNA strand having the nucleotide sequence of SEQ ID NO: 133, which has partial complementarity to the sense saRNA strand of SEQ ID NO: 129; zz) R6-67M3-13nt-S1L1V3 (SEQ ID NO: 129) and an antisense saRNA strand having the nucleotide sequence of SEQ ID NO: 134, which has partial complementarity to the sense saRNA strand of SEQ ID NO: 129; aaa) R6-67M3-12nt-S1L1V3 (SEQ ID NO: 129) and a SEQ ID NO having partial complementarity to the sense saRNA strand of SEQ ID NO: 129 NO: an antisense saRNA strand having a nucleotide sequence of 135; bbb) R6-67M3-9nt-S1L1V3 (SEQ ID NO:129) and an antisense saRNA strand having the nucleotide sequence of SEQ ID NO:136, which has partial complementarity to the sense saRNA strand of SEQ ID NO:129; ccc) R6-67M3-8nt-S1L1V3 (SEQ ID NO:129) and an antisense saRNA strand having the nucleotide sequence of SEQ ID NO:137, which has partial complementarity to the sense saRNA strand of SEQ ID NO:129; with the proviso that a linker selected from the group consisting of L1, L4 and L9 is present or absent; with the proviso that L1 represents spacer-18; L4 represents spacer-C6; and L9 represents spacer-9.

20. The multivalent oligonucleotide agent has a nucleotide sequence having at least 90% identity to a nucleotide sequence selected from the group consisting of: a) R6-04S1&67S1R-L1V2 (SEQ ID NO:52) and SEQ an antisense saRNA strand having the nucleotide sequence of SEQ ID NO: 53, which has partial complementarity with the strand of SEQ ID NO: 52, and an antisense saRNA strand having the nucleotide sequence of SEQ ID NO: 78, which has partial complementarity with the strand of SEQ ID NO: 52; b) R6-04S1&67S5-L1V2 (SEQ ID NO:56), an antisense saRNA strand having the nucleotide sequence of SEQ ID NO:53 which has partial complementarity with the strand of SEQ ID NO:56, and an antisense saRNA strand having the nucleotide sequence of SEQ ID NO:78 which has partial complementarity with the strand of SEQ ID NO:56; and c) R6-04M1 & R17-388E-L1V2 (SEQ ID NO: 140), an antisense saRNA strand of SEQ ID NO: 17 having partial complementarity with a strand of SEQ ID NO: 140, and an antisense saRNA strand of SEQ ID NO: 141 having partial complementarity with a strand of SEQ ID NO: 140; and / or The multivalent oligonucleotide agent has a nucleotide sequence having at least 90% identity to a nucleotide sequence selected from the group consisting of: a') R6-04S1&27A&67S1R-L1V2 (SEQ ID NO:54), an antisense saRNA strand having the nucleotide sequence of SEQ ID NO:53 which has partial complementarity with the strand of SEQ ID NO:54, and an antisense saRNA strand having the nucleotide sequence of SEQ ID NO:78 which has partial complementarity with the strand of SEQ ID NO:54; b') R6-04S1&67S1R&27A-L1V2 (SEQ ID NO:55), an antisense saRNA strand having the nucleotide sequence of SEQ ID NO:53 which has partial complementarity with the strand of SEQ ID NO:55, and an antisense saRNA strand having the nucleotide sequence of SEQ ID NO:78 which has partial complementarity with the strand of SEQ ID NO:55; c') R6-04S1&27A&67S5-L1V2 (SEQ ID NO:57), an antisense saRNA strand having the nucleotide sequence of SEQ ID NO:53 which has partial complementarity with the strand of SEQ ID NO:57, and an antisense saRNA strand having the nucleotide sequence of SEQ ID NO:78 which has partial complementarity with the strand of SEQ ID NO:57; and d') R6-04S1&67S5&27A-L1V2 (SEQ ID NO:58), an antisense saRNA strand having the nucleotide sequence of SEQ ID NO:53 which has partial complementarity with the strand of SEQ ID NO:58, and an antisense saRNA strand having the nucleotide sequence of SEQ ID NO:78 which has partial complementarity with the strand of SEQ ID NO:

58. A multivalent oligonucleotide drug according to claim 14.

21. The multivalent oligonucleotide drug of claim 14, wherein the multivalent oligonucleotide drug is selected from those set forth in any one of Tables 7-20 or has at least 90% sequence identity thereto, and the linking moieties and / or linking bonds and / or orientation of the multivalent oligonucleotide drug are variable.

22. The multivalent oligonucleotide drug of claim 1, wherein the one or more functional oligonucleotides increase the expression of the CDKN1A / p21 gene or protein and / or decrease the expression of CD274 / PDL-1.

23. 23. A multivalent oligonucleotide agent according to claim 22, characterized in that the dsRNA is independently selected from the group consisting of: saRNA that increases the expression of the CDKN1A / p21 gene or protein; and siRNA that decreases the expression of CD274 / PDL-1; and / or The ASOs are independently selected from the group consisting of ASOs that increase expression of the CDKN1A / p21 gene or protein and / or decrease expression of CD274 / PDL-1.

24. The dsRNA is a saRNA having a nucleotide sequence of the sense strand that has at least 90% identity to the nucleotide sequence of RAG1-40 (SEQ ID NO:62); and / or The dsRNA is a saRNA having an antisense strand nucleotide sequence that has at least 90% identity to the nucleotide sequence of RAG1-40 (SEQ ID NO: 63).

23. The multivalent oligonucleotide drug of claim 22.

25. The dsRNA is a) siPDL1-2 (SEQ ID NO: 64) and b) siPDL1-3 (SEQ ID NO: 66) and / or a siRNA having a sense strand nucleotide sequence having at least 90% identity to a nucleotide sequence selected from The dsRNA is a) siPDL1-2 (SEQ ID NO: 65) and b) siPDL1-3 (SEQ ID NO: 67) and an siRNA having an antisense strand nucleotide sequence having at least 90% identity with a nucleotide sequence selected from the group consisting of 23. The multivalent oligonucleotide drug of claim 22.

26. The dsRNA is an siRNA selected from the group consisting of: a) an siRNA having a sense strand nucleotide sequence having at least 90% identity with the nucleotide sequence of siPDL1-2 (SEQ ID NO: 64) and an antisense strand nucleotide sequence having at least 90% identity with the nucleotide sequence of siPDL1-2 (SEQ ID NO: 65); and b) an siRNA having a sense strand nucleotide sequence having at least 90% identity with the nucleotide sequence of siPDL1-2 (SEQ ID NO: 66) and an antisense strand nucleotide sequence having at least 90% identity with the nucleotide sequence of siPDL1-2 (SEQ ID NO: 67); and / or ASO is a") aPDL1-1 (SEQ ID NO: 68); b") aPDL1-2 (SEQ ID NO:69); and c") has a nucleotide sequence having at least 90% identity with a nucleotide sequence selected from the group consisting of aPDL1-3 (SEQ ID NO: 70); and / or The multivalent oligonucleotide agent has a nucleotide sequence having at least 90% identity to a nucleotide sequence selected from the group consisting of: a') saP21-40 / siPDL1-2 (SEQ ID NO: 71) and SEQ an antisense saRNA strand having the nucleotide sequence of SEQ ID NO: 63, which has partial complementarity with the strand of SEQ ID NO: 71, and an antisense siRNA strand having the nucleotide sequence of SEQ ID NO: 65, which has partial complementarity with the strand of SEQ ID NO: 71; b') saP21-40 / siPDL1-3 (SEQ ID NO:100), an antisense saRNA strand having the nucleotide sequence of SEQ ID NO:63, which has partial complementarity with the strand of SEQ ID NO:100, and an antisense saRNA strand having the nucleotide sequence of SEQ ID NO:65, which has partial complementarity with the strand of SEQ ID NO:100; c') saP21-40 / aPDL1-1 (SEQ ID NO:72) and an antisense saRNA strand having the nucleotide sequence of SEQ ID NO:63, which has partial complementarity to the sense saRNA strand of SEQ ID NO:72; d') saP21-40 / aPDL1-2 (SEQ ID NO:73) and an antisense saRNA strand having the nucleotide sequence of SEQ ID NO:63 having partial complementarity to the sense saRNA strand of SEQ ID NO:73; e') saP21-40 / aPDL1-3 (SEQ ID NO:74) and an antisense saRNA strand having the nucleotide sequence of SEQ ID NO:63 having partial complementarity to the sense saRNA strand of SEQ ID NO:74; f') saP21-40 / aPDL1-1R (SEQ ID NO: 75) and SEQ an antisense saRNA strand having the nucleotide sequence of SEQ ID NO: 63, which has partial complementarity to the sense saRNA strand of ID NO: 75; g') saP21-40 / aPDL1-2R (SEQ ID NO: 76) and SEQ an antisense saRNA strand having the nucleotide sequence of SEQ ID NO: 63, which has partial complementarity to the sense saRNA strand of ID NO: 76; and h') saP21-40 / aPDL1-3R (SEQ ID NO: 77) and SEQ The multivalent oligonucleotide drug of claim 22, characterized in that it has an antisense saRNA strand having the nucleotide sequence of SEQ ID NO: 63, which has partial complementarity with the sense saRNA strand of ID NO:

77.

27. 13. An article of manufacture comprising the multivalent oligonucleotide agent of claim 1.

28. Use of a multivalent oligonucleotide drug as described in claim 1, or use of the product of claim 27 for the preparation of a pharmaceutical agent, characterized in that it treats or delays the onset or progression of an SMN deficiency associated pathology in a subject.

29. The pharmaceutical agent is for treating a p21 / PDL-1 associated disease; or for the treatment of cancer or at high risk of developing cancer; and / or The cancer is a solid or non-solid tumor.

29. The use according to claim 28.

30. The cancer is selected from the group consisting of sarcomas such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, and osteosarcoma, synovium, mesothelioma, Ewing's sarcoma, leiomyosarcoma, rhabdomyosarcoma, colon cancer / colorectal cancer, lymphatic malignant tumors, pancreatic cancer, breast cancer, lung cancer, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, pheochromocytoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, medullary carcinoma, bronchial carcinoma, renal cell carcinoma, liver cancer, bile duct carcinoma, choriocarcinoma, and Wilms' tumor.

30. The use according to claim 29, characterized in that the tumor is selected from the group consisting of cervical cancer, testicular tumor, seminoma, bladder cancer, melanoma, CNS tumors (e.g. gliomas (such as brain stem gliomas and mixed gliomas), glioblastomas (also called glioblastoma multiforme), astrocytomas, CNS lymphomas, germinomas, medulloblastomas, schwannomas, craniopharyngiomas, ependymoma, pinealomas, hemangioblastomas, acoustic neuroma, oligodendroglioma, meningiomas, neuroblastomas, retinoblastomas and metastatic brain tumors.

31. A method for preparing a multivalent oligonucleotide drug according to claim 1, comprising providing two or more functional oligonucleotides and covalently linking them together; or synthesizing a full-length oligonucleotide drug.

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