Antisense RNA (asRNA) technology and its uses

Abstract

The present invention discloses a novel type of gene modulation technology for modulating target nucleic acid and / or protein levels in cells, tissues, organisms, and animals. This new technology provides compositions for use in gene modulation applications, including the prevention and treatment of human diseases. The compositions comprise short antisense RNA (asRNA) molecules having at least one deoxyribonucleotide monomer interspersed motif. The present invention further provides methods for using the compositions to modulate the expression or function of target genes, or for the treatment or prevention of diseases, as well as for biomedical research, disease diagnosis, and other biological applications.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and the benefit of co-pending U.S. Provisional Patent Application No. 63 / 431,309, filed December 8, 2022, which is incorporated herein by reference in its entirety.

[0002] FIELD OF THE INVENTION The present invention relates to novel designs of short, single-stranded antisense RNA oligonucleotides for use as gene modulation techniques, as well as related compositions and methods that can be used in biological or medical research, in the treatment and prevention of disease, and for the application of gene silencing in other fields of biology. [Background technology]

[0003] Background of the Invention Single-stranded antisense RNA (asRNA) can be found naturally in both prokaryotes and eukaryotes as non-coding RNA transcripts, typically 19–23 nucleotides long, transcribed from the lagging strand of a gene and complementary to and antisense to the mRNA transcript (see Xu, J. et al., 2018). Despite their potential regulatory role in complex biological networks, asRNA remains poorly characterized—the mechanisms underlying their function are still poorly understood, and to date, only a few in vivo functions of asRNA have been demonstrated in known organisms (Xu et al., supra). New breakthroughs are needed to uncover the regulatory potential of antisense activity, first observed in non-coding RNAs more than 30 years ago, both as a research tool and as a therapeutic agent.

[0004] Any reference cited herein is not admitted to be prior art to the claimed invention. Summary of the Invention [Means for solving the problem]

[0005] Summary of the Invention The present invention is based on the surprising discovery that the limited gene silencing efficacy of naturally occurring types of single-stranded antisense RNAs that contain exclusively RNA can be enhanced by introducing deoxyribonucleotide interspersed segments ("ISDs"). Thus, the present invention provides a novel type of gene modulation technology enabled by short, single-stranded antisense RNAs (asRNAs) that contain at least one ISD.

[0006] This novel asRNA with one or more ISDs is a short, single-stranded molecule composed of linked nucleotide monomers, each selected from the group of naturally occurring nucleotides, their analogs, and modified nucleotides (collectively referred to hereinafter as "nucleotide monomers").

[0007] The asRNA molecules of the present invention comprise "ribonucleotide monomers" selected from the group of naturally occurring ribonucleotides, their analogs, and modified ribonucleotides. Furthermore, the gene silencing function of asRNA can be dramatically enabled or enhanced by incorporating one or a few interspersed deoxyribonucleotide monomers. The "deoxyribonucleotide monomers" can be selected from the group of naturally occurring deoxyribonucleotides, their analogs, and modified deoxyribonucleotides.

[0008] The potent gene silencing effect of the novel asRNA-based platform technology contained in the present disclosure is achieved, in one embodiment, by antisense oligoribonucleotides that are substantially complementary to the target RNA sequence. Our data demonstrate that the asRNA molecules of the present invention, due to their unique and novel composition, can induce potent gene silencing, which is more potent than existing gene silencing technologies, thereby enabling reduced dose-dependent toxicity. The asRNA molecules of the present invention are expected to have at least one of the following advantages over existing gene silencing technologies: better penetration; enabling gene silencing not only in the cytoplasm but also in the nucleus(s) and mitochondria(s); reduced off-target effects; better stability; lower synthesis costs; and other improved pharmaceutical properties. Therefore, the asRNA molecules of the present invention have great potential to address various challenges faced by existing gene silencing technologies. The asRNA molecules of the present invention can be used in any area where current gene silencing oligonucleotides are applied or intended for use, including research, diagnosis, disease prevention and treatment, and other applications in the biological field, including agriculture and veterinary medicine.

[0009] In a first aspect, the present invention provides a composition comprising a short antisense RNA (asRNA) molecule having a single strand of linked ribonucleotide monomers, the strand being substantially complementary to a segment targeted by at least one targeting region of an RNA. Furthermore, the asRNA molecule comprises at least one deoxyribonucleotide monomer interspersed segment (ISD) having at least one deoxyribonucleotide monomer. The ribonucleotide monomers in the molecule are selected from the group consisting of naturally occurring ribonucleotides, their analogs, and modified ribonucleotides; the deoxyribonucleotide monomer interspersed segment in the asRNA molecule is selected from the group consisting of naturally occurring deoxyribonucleotides, their analogs, and modified deoxyribonucleotides. In some embodiments, the ISD in the asRNA molecule has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 deoxyribonucleotide monomers. In one aspect, an ISD in an asRNA molecule has at least two, three, four, five, six, seven, or eight consecutive deoxyribonucleotide monomers. In some embodiments, an ISD comprises at least two deoxyribonucleotide monomers. There may be more than one ISD in an asRNA molecule. In one aspect, each ISD, independently of the others, consists of one deoxyribonucleotide monomer or comprises at least two, three, four, five, or more consecutive deoxyribonucleotide monomers. In one aspect, an ISD is located within at least one target region. In some embodiments, the ISD may be located anywhere in the asRNA molecule. In some embodiments, the ISD is located more centrally in the asRNA molecule (at least 1, 2, 3, 4, 5, 6, 7, or 8 nucleotides away from either end, i.e., starting from position 2 or more centrally from either end). In some embodiments, the ISD comprises at least one deoxyribonucleotide monomer located at the 5' and / or 3' end of the asRNA molecule.

[0010] In one aspect, at least one gene modulation or pharmaceutical property is better or more desirable when the single-stranded antisense RNA comprises at least one ISD; the property is selected from the group consisting of efficacy, potency, onset rate, durability, synthesis economy, off-target effects, non-specific immune stimulation, stability, and delivery. More specifically, compared to the corresponding single-stranded antisense RNA without an ISD, improved gene modulation or pharmaceutical properties of the asRNA molecules of the present invention mean, for example, that one or more of the following applies: better efficacy and / or potency, more rapid onset of action, improved pharmacokinetic properties, longer durability, reduced off-target effects, less dosage-dependent typical toxicity, avoidance of non-specific interferon-like responses, as well as lower production costs, better stability, and better delivery.

[0011] The compositions provided by the present invention are used to modulate gene expression or function in eukaryotic cells, and an asRNA having an ISD (asRNA-ISD) is contacted with the cells or administered to a subject.

[0012] In one aspect, the asRNA molecule comprises a plurality of linked nucleotide monomers forming a nucleobase sequence that is at least 70%, 80%, 85%, 90%, 95%, or fully complementary to a targeted segment of a target RNA. In certain embodiments, the target RNA is either an mRNA, pre-mRNA, mt-mRNA, and / or non-coding RNA that encodes a protein involved in a disease, e.g., a mammalian disease, or controls part of a biological pathway involved in a disease, e.g., a mammalian disease. The terms "target" and "targeted" are used interchangeably in this disclosure and share the same meaning.

[0013] In various embodiments, the asRNA molecules have a backbone length of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 and 50 linked nucleotide monomers, or an equivalent thereof, or a range between any two of the foregoing values, inclusive of the endpoints of the range. For example, some of the length ranges of asRNAs are: 8-48 nucleotide monomers; 8-44 nucleotide monomers; 8-42 nucleotide monomers; 8-40 nucleotide monomers; 8-36 nucleotide monomers; 8-33 nucleotide monomers; 10-48 nucleotide monomers; 10-44 nucleotide monomers; 10-42 nucleotide monomers; 10-40 nucleotide monomers; 10-36 nucleotide monomers; 10-30 nucleotide monomers; 10-29 nucleotide monomers; 12-48 nucleotide monomers; 12-44 nucleotide monomers; 12-42 nucleotide monomers; 12-40 nucleotide monomers; 12-36 nucleotide monomers; 12-34 nucleotide monomers; 12-32 nucleotide monomers; 12-30 nucleotide monomers; 12-29 nucleotide monomers; 12-28 nucleotide monomers. -; 12 to 26 nucleotide monomers; 12 to 25 nucleotide monomers; 13 to 48 nucleotide monomers; 13 to 44 nucleotide monomers; 13 to 42 nucleotide monomers; 13 to 40 nucleotide monomers; 13 to 36 nucleotide monomers; 13 to 34 nucleotide monomers; 13 to 32 nucleotide monomers; 13 to 30 nucleotide monomers; 13 to 28 nucleotide monomers; 13 to 26 nucleotide monomers; 13 to 25 nucleotide monomers; 13 to 24 nucleotide monomers; 13 to 23 nucleotide monomers; 14 to 36 nucleotide monomers; 15 to 23 nucleotide monomers; 20 to 36 nucleotide monomers; 21 to 36 nucleotide monomers; 24 to 36 nucleotide monomers; at least 21 nucleotide monomers; at least 24 nucleotide monomers and at least 8 nucleotide monomers.

[0014] In some aspects of the asRNA molecules of the present invention, at least one nucleotide monomer in the strand is a modified nucleotide or nucleotide analog, such as a sugar-modified nucleotide, a backbone-modified nucleotide, and / or a base-modified nucleotide. In some embodiments, such backbone-modified nucleotides have at least one internucleoside linkage modified, for example, to include at least one nitrogen or sulfur heteroatom. In some embodiments, the modified internucleoside linkage is or includes a phosphorothioate (P=S) group, a phosphotriester, a methylphosphonate, or a phosphoramidate.

[0015] In certain embodiments, asRNA molecule comprises at least one modified internucleoside linkage that is phosphorothioate internucleoside linkage.In some embodiments, each internucleoside linkage of asRNA molecule is phosphorothioate internucleoside linkage.In various embodiments, internucleoside linkage is a mixture of phosphorothioate linkage and phosphodiester linkage.

[0016] In certain aspects, the asRNA molecules of the invention have at least one modified nucleotide or nucleotide analog comprising a modified sugar moiety. In certain embodiments, the 2' position of the modified sugar moiety is replaced with a group selected from OR, R, halo, SH, SR, NH, NHR, NR, or CN, where each R is independently C1-C6 alkyl, alkenyl, or alkynyl, and halo is F, Cl, Br, or I. In some embodiments, the 2' position of the modified sugar moiety is selected from allyl, amino, azido, thio, O-allyl, O-C1-C6, 10 Alkyl, OCF3, OCH2F, O(CH2)2SCH3, O(CH2)2-ON(R m )(R n ), O-CH2-C(=O)-N(R m )(R n ), or O-CH2-C(=O)-N(R1)-(CH2)2-N(R m )(R n), wherein each R l , R m and R n are independently H or substituted or unsubstituted C1-C 10 It is alkyl.

[0017] In some embodiments, the modified sugar moiety has a substituent selected from the group of 5'-vinyl, 5'-methyl (R or S), 4'-S, 2'-F, 2'-OCH, 2'-OCHCH, 2'-OCHCHF, 2'-O-aminopropylated (2'-AP), and 2'-O(CH)OCH. In some embodiments, the modified sugar moiety is substituted with a bicyclic sugar selected from the group of 4'-(CH2)-O-2' (LNA); 4'-(CH2)-S-2; 4'-(CH2)2-O-2' (ENA); 4'-CH(CH3)-O-2' (cEt), and 4'-CH(CHOCH3)-O-2', 4'-C(CH3)(CH3)-O-2', 4'-CH2-N(OCH3)-2', 4'-CH2-ON(CH3)-2', 4'-CH2-N(R)-O-2' (where R is H, C1-C12 alkyl, or a protecting group), 4'-CH2-C(H)(CH3)-2', and 4'-CH2-C-(=CH2)-2'. In some embodiments, the modified sugar moiety is selected from the group of 2'-O-methoxyethyl modified sugars (MOE), 4'-(CH2)-O-2' bicyclic sugars (LNA), 2'-deoxy-2'-fluoroarabinose (2'-F-arabino, FANA), and methyl(methyleneoxy) (4'-CH(CH3)-O-2) bicyclic sugars (cEt).

[0018] In one aspect of the asRNA molecules of the invention, the sugar moiety of the deoxyribonucleotide monomer is either the sugar moiety of a naturally occurring deoxyribonucleotide (2-H) or 2'-deoxy-2'-fluoroarabinose (FANA).

[0019] In one aspect of the asRNA molecules of the invention, the sugar moiety of the ribonucleotide monomer is selected from the group consisting of naturally occurring ribonucleotides (2-OH), 2'-F modified sugars, 2'-OMe modified sugars, 2'-O-methoxyethyl modified sugars (MOE), 4'-(CH2)-O-2' bicyclic sugars (LNA), and methyl(methyleneoxy) (4'-CH(CH3)-O-2) bicyclic sugars (cEt).

[0020] In another aspect, the asRNA molecules of the invention comprise at least one nucleotide monomer having a modified nucleobase. In some embodiments, the modified nucleobase is 5-methylcytosine (5-Me-C), inosine bases, tritylated bases, 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine; 6-methyl and other alkyl derivatives of adenine and guanine; 2-propyl and other alkyl derivatives of adenine and guanine; 2-thiouracil, 2-thiothymine and 2-thiocytosine, 1-methyl-pseudouracil, 5-halouracil and cytosine; 5-propynyl (-C≡C-CH3) uracil and cytosine and other alkynyl derivatives of pyrimidine bases; 6-azouracil, cytosine and thymine. The modified nucleic acid base is selected from the group consisting of uridine, 5-uracil (pseudouracil), 4-thiouracil, 1-methyl-pseudouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo, particularly 5-bromo, 5-trifluoromethyl, 5-methyluridine and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-aminoadenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine, and 3-deazaguanine and 3-deazaadenine.In certain embodiments, the modified nucleic acid base is 5-methylcytosine.In some embodiments, each cytosine base in the molecule of the present invention is 5-methylcytosine.

[0021] In one aspect of the invention, asRNA molecules are used to modulate gene expression or function in cells, eg, eukaryotic cells, eg, mammalian cells.

[0022] In certain embodiments, the RNA targeted by the asRNA molecules of the present invention is selected from mRNA, pre-mRNA, mt-mRNA, and non-coding RNA. In one aspect, such target RNA encodes a protein involved in a disease or controls a part of a biological pathway involved in a disease. In various embodiments, such target RNA can be selected from, but is not limited to, mRNA, pre-mRNA, mt-mRNA, non-coding RNA, or lncRNA of a gene involved in a human or animal disease or condition; mRNA or pre-mRNA of a gene of a pathogenic microorganism; viral RNA, and RNA involved in a disease selected from the group consisting of autoimmune diseases, inflammatory diseases, degenerative diseases, infectious diseases, proliferative diseases, metabolic diseases, immune-mediated disorders, allergic diseases, dermatological diseases, malignant diseases, gastrointestinal disorders, respiratory disorders, cardiovascular disorders, renal disorders, rheumatoid disorders, neurological disorders, endocrine disorders, and aging-related disorders or diseases.

[0023] In one aspect, the asRNA molecules of the present invention are conjugated to a ligand or moiety. In certain embodiments, the ligand or moiety is selected from the group consisting of peptides / proteins, antibodies, polymers, polysaccharides, lipids, hydrophobic moieties or molecules, cationic moieties or molecules, lipophilic compounds or moieties, oligonucleotides, cholesterol, GalNAc, and aptamers.

[0024] In a second aspect, the present invention provides a pharmaceutical composition, comprising the composition of the first aspect as an active agent and a pharmaceutically acceptable excipient, carrier or diluent.Examples of such carriers include, but are not limited to, pharmaceutical carriers, positively charged carriers, lipid nanoparticles, liposomes, protein carriers, hydrophobic moieties or molecules, cationic moieties or molecules, GalNAc, polysaccharides, polymers, nanoparticles, nanoemulsions, cholesterol, lipids, lipophilic compounds or moieties, and lipids.

[0025] In a third aspect, the present invention provides a method of using the composition of the first aspect or the pharmaceutical composition of the second aspect to treat or prevent a disease or condition by administering a therapeutically effective amount of an asRNA molecule of the present invention or a pharmaceutical composition containing such a molecule to a subject in need thereof, by a route selected from the group consisting of intravenous injection (iv), subcutaneous injection (sc), orally (po), intramuscular (im) injection, oral administration, inhalation, topical, intrathecal, and other localized administration.

[0026] In one aspect, the disease or condition to be treated prophylactically or therapeutically is selected from the group of cancer, autoimmune diseases, inflammatory diseases, degenerative diseases, infectious diseases, proliferative diseases, metabolic diseases, immune-mediated disorders, allergic diseases, dermatological diseases, malignant diseases, gastrointestinal disorders, hepatic disorders, respiratory disorders, cardiovascular disorders, dermatological disorders, nephrological disorders, rheumatoid disorders, neurological disorders, psychiatric disorders, endocrine disorders, and aging-related disorders or diseases.

[0027] In a fourth aspect, the present invention provides a method of using the composition of the first aspect or the pharmaceutical composition of the second aspect to control or modulate gene expression or function in a eukaryotic cell, the method comprising contacting the cell with an effective amount of any asRNA molecule of the present invention or a pharmaceutical composition containing such molecule.

[0028] In one embodiment, the contacting step comprises introducing a composition containing the asRNA molecule into target cells in culture or into target cells in an organism capable of selective gene silencing. In a further embodiment, the introducing step is selected from the group consisting of simple mixing, transfection, lipofection, electroporation, infection, injection, and oral administration, intravenous injection (IV), subcutaneous injection (SC), oral (PO), intramuscular (IM) injection, inhalation, topical, intrathecal, and other localized administration. In another embodiment, the introducing step comprises using a pharmaceutically acceptable excipient, carrier, or diluent selected from the group consisting of pharmaceutical carriers, positively charged carriers, lipid nanoparticles, liposomes, protein carriers, hydrophobic moieties or molecules, cationic moieties or molecules, GalNAc, polysaccharides, polymers, nanoparticles, nanoemulsions, cholesterol, lipids, lipophilic compounds or moieties, and lipids.

[0029] In certain embodiments, the target gene is mRNA. In certain embodiments, the target gene is pre-mRNA. In certain embodiments, the target gene is mt-mRNA. In certain embodiments, the target gene is non-coding RNA, such as microRNA and lncRNA.

[0030] In one embodiment, the target gene is associated with a disease, pathological condition, or undesirable condition in a mammal. In a further embodiment, the target gene is a gene of a pathogenic microorganism. In yet a further embodiment, the target gene is a viral gene. In another embodiment, the target gene is a tumor-associated gene. In yet another embodiment, the target gene is a gene associated with a disease selected from the group listed for the third aspect.

[0031] Other features and advantages of the present invention will be apparent from the additional description provided herein, including the different examples. The provided examples illustrate different components and methodologies useful in practicing the present invention. The examples do not limit the claimed invention. Based on this disclosure, one skilled in the art will be able to identify and utilize other components and methodologies useful in practicing the present invention. While several embodiments have been shown and described, any modifications can be made without departing from the spirit and scope of the present invention. [Brief explanation of the drawings]

[0032] [Figure 1] Figure 1 shows exemplary structures of some embodiments of asRNAs with various motifs of deoxyribonucleotide monomer interspersed segments (ISDs) and corresponding antisense single-stranded RNAs (ASRs) without ISDs, as well as exemplary sequences of asRNAs and ASRs with the exemplary structures shown that target the APOCIII gene.

[0033] [Figure 2] Figure 2 shows the gene silencing efficacy of asRNAs with the structures in Figure 1 targeting the APOCIII gene compared with the corresponding ASRs. After introducing the asRNAs and the corresponding ASRs at 10 nM into HepaRG cells by transfection, the relative mRNA levels of the APOCIII gene were determined.

[0034] [Figure 3] FIG. 3 shows exemplary structures of some embodiments of asRNAs with various ISD positions, as well as exemplary sequences of asRNAs targeting the APOCIII gene.

[0035] [Figure 4] Figure 4 shows the gene silencing efficacy of asRNAs targeting the APOCIII gene, which have the sequences shown in Figure 3. After the asRNAs were introduced into HepaRG cells by transfection at 10 nM, the relative mRNA levels of the APOCIII gene were determined.

[0036] [Figure 5] Figure 5A shows exemplary sequences of some embodiments of asRNAs with various lengths. Figures 5B and 5C show the gene silencing efficacy of the asRNAs shown in Figure 5A targeting the APOCIII gene at different concentrations. After transfection of asRNA at 100 pM and 10 nM into HepaRG cells, the gene silencing efficacy of the relative mRNA level of the APOCIII gene was determined. DETAILED DESCRIPTION OF THE INVENTION

[0037] Detailed Description of the Invention The present invention refers to a new type of short single-stranded antisense RNA that is interspersed with DNA monomers, and uses gene or RNA modulation / silencing technology.This new technology is used to modulate gene expression or function in vitro and in vivo by using the composition of short single-stranded antisense RNA with deoxynucleotide interspersed segments.The present invention also provides the method of using the composition to modulate the expression or function of target gene, or for the treatment or prevention of disease, and for biomedical research and other biological applications.

[0038] 1.Definition As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. For example, the term "a cell" includes a plurality of cells, including mixtures thereof.

[0039] When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below those numerical values. Generally, the term "about" is used herein to modify a numerical value by a variance of 20%, 10%, 5%, or 1% above and below the stated value. In some embodiments, the term "about" is used to modify a numerical value by a variance of 10% above and below the stated value. In some embodiments, the term "about" is used to modify a numerical value by a variance of 5% above and below the stated value. In some embodiments, the term "about" is used to modify a numerical value by a variance of 1% above and below the stated value.

[0040] As used herein, the term " analog " or " analogue " refers to a functional or structural equivalent.For example, nucleoside and nucleotide analogues have been used in the clinical treatment of cancer and viral infection for decades, and new compounds are continuously synthesized and evaluated by researchers and the pharmaceutical industry.See, for example, Jordheim LP et al., Nat Rev Drug Discov 12, 447-464 (2013).

[0041] As used herein, the term "deoxyribonucleoside monomer" refers to a nucleoside monomer, including naturally occurring deoxyribonucleosides, their analogs, and modified deoxyribonucleosides. The term "deoxyribonucleotide monomer" refers to a nucleotide monomer, including naturally occurring deoxyribonucleotides, their analogs, and modified deoxyribonucleotides.

[0042] As used herein, the term "ribonucleoside monomer" refers to a nucleoside monomer, including naturally occurring ribonucleosides, their analogs, and modified ribonucleosides. The term "ribonucleotide monomer" refers to a nucleotide monomer, including naturally occurring ribonucleotides, their analogs, and modified ribonucleotides.

[0043] As used herein, the term "nucleoside" refers to a compound comprising a nucleobase moiety and a sugar moiety. Nucleoside monomers include, but are not limited to, naturally occurring nucleosides (e.g., deoxyribonucleosides and ribonucleosides found in DNA and RNA, respectively), their analogs, and modified nucleosides. Nucleoside monomers can be deoxyribonucleoside monomers or ribonucleoside monomers. Nucleoside monomers can be linked to a phosphate moiety, for example, to form nucleotide monomers.

[0044] As used herein, the term "nucleotide" refers to a nucleoside that further comprises a phosphate linking group. Nucleotide monomers include, but are not limited to, naturally occurring nucleotides (e.g., deoxyribonucleotides and ribonucleotides, as found in DNA and RNA, respectively), their analogs, and modified nucleotides. A nucleotide monomer may be a deoxyribonucleotide monomer or a ribonucleotide monomer. A modified nucleotide may be modified in one or more of the following: its nitrogenous nucleobase moiety, its five-carbon sugar moiety, and its phosphate linking group, resulting in a change in the internucleoside linkage.

[0045] As used herein, the term "oligo" or "oligonucleotide" refers to a compound comprising multiple linked nucleoside monomers. In certain embodiments, one or more of the nucleoside monomers or one or more of the internucleoside linkages are modified.

[0046] The terms "deoxynucleoside" and "deoxyribonucleoside" are used interchangeably herein. The terms "deoxynucleotide" and "deoxyribonucleotide" are also used interchangeably herein. As used herein, a "deoxynucleoside" or "deoxynucleotide" is a nucleoside or nucleotide, respectively, that contains a deoxy sugar moiety.

[0047] As used herein, the term "motif" refers to a pattern of chemically distinct regions, for example, within an oligonucleotide strand.

[0048] As used herein, the term "immediately adjacent" means that there are no intervening elements between the two elements, eg, regions, segments, nucleotides and / or nucleosides.

[0049] As used herein, the term "modified nucleotide" means a nucleotide having at least one modified sugar moiety, modified internucleoside linkage, and / or modified nucleobase.

[0050] As used herein, the term "modified nucleoside" means a nucleoside having at least one modified sugar moiety and / or modified nucleobase.

[0051] As used herein, the term "modified oligonucleotide" means an oligonucleotide that contains at least one modified nucleotide.

[0052] As used herein, the term "naturally occurring internucleoside linkage" means a 3' to 5' phosphodiester linkage.

[0053] As used herein, the term "modified internucleoside linkage" refers to a substitution or any change from a naturally occurring internucleoside linkage. For example, a phosphorothioate linkage is a modified internucleoside linkage.

[0054] As used herein, the term "natural sugar moiety" means a sugar that is naturally found in DNA (2-H) or RNA (2-OH).

[0055] As used herein, the term "modified sugar" refers to a substitution or change from a natural sugar. For example, a 2'-O-methoxyethyl modified sugar is a modified sugar.

[0056] As used herein, the term "bicyclic sugar" means a furosyl ring modified by bridging two non-geminal ring atoms. A bicyclic sugar is a modified sugar.

[0057] As used herein, the term "bicyclic nucleic acid," "BNA," "bicyclic nucleoside," or "bicyclic nucleotide" refers to a nucleoside or nucleotide in which the furanose portion of the nucleoside or nucleotide includes a bridge connecting two carbon atoms on the furanose ring, thereby forming a bicyclic ring system.

[0058] As used herein, the term "2'-O-methoxyethyl" (also 2'-MOE, 2'-O(CH2)2-OCH3, and 2'-O-(2-methoxyethyl)) refers to an O-methoxy-ethyl modification at the 2' position of the furosyl ring. A 2'-O-methoxyethyl modified sugar is a modified sugar. As used herein, the term "2'-O-methoxyethyl nucleotide" refers to a modified nucleotide containing a 2'-O-methoxyethyl modified sugar moiety.

[0059] As used herein, the term "modified nucleobase" refers to any nucleobase other than adenine, cytosine, guanine, thymidine or uracil.For example, 5-methylcytosine is a modified nucleobase.On the other hand, as used herein, "unmodified nucleobase" refers to purine bases adenine (A) and guanine (G), and pyrimidine bases thymine (T), cytosine (C) and uracil (U).

[0060] As used herein, the term "5-methylcytosine" means a cytosine modified with a methyl group attached to position 5. For example, 5-methylcytosine is a modified nucleobase.

[0061] As used herein, "RNA-like nucleotides" refers to modified nucleotides that, when incorporated into an oligonucleotide, assume a Northern configuration and function like RNA. RNA-like nucleotides include, but are not limited to, bridged nucleic acids (BNA), LNA, cEt, 2'-O-methylated nucleotides, 2'-O-methoxyethylated (2'-MOE) nucleotides, 2'-fluorinated nucleotides, 2'-O-aminopropylated (2'-AP) nucleotides, tricyclo-DNA (tcDNA), and RNA surrogates.

[0062] As used herein, "DNA-like nucleotides" refers to modified nucleotides that function like DNA when incorporated into an oligonucleotide. DNA-like nucleotides include, but are not limited to, 2'-deoxy-2'-fluoroarabinose (FANA) nucleotides and DNA surrogates.

[0063] As used herein, "non-coding RNA" refers to an RNA molecule that is not translated into protein. Examples of non-coding RNA include transfer RNA (tRNA) and ribosomal RNA (rRNA), as well as small non-coding RNA and long ncRNA (lncRNA). As used herein, examples of "small non-coding RNA" include, but are not limited to, microRNA (miRNA), asRNA, pre-miRNA, pri-miRNA, piRNA, snoRNA, snRNA, exRNA, scaRNA, and mimics of any of the foregoing. As used herein, "lncRNA" and "long non-coding RNA" refer to transcribed RNA molecules containing more than 200 nucleotides that do not encode proteins. lncRNAs can also undergo common post-transcriptional modifications, including 5'-capping, 3'-polyadenylation, and splicing. In general, IncRNAs are a diverse class of molecules that play various roles in modifying gene and genome function. For example, it is known that lncRNAs control gene transcription, translation, and epigenetic regulation. Examples of IncRNAs include, but are not limited to, Kcnqlotl, Xlsirt, Xist, ANRIL, NEAT1, NRON, DANCR, OIP5-AS1, TUG1, CasC7, HOTAIR, and MALAT1. As used herein, "splice" or "splicing" refers to the natural process of removing unnecessary regions of RNA and reforming the RNA. An example of modulation of RNA target function by oligonucleotides is modulation of non-coding RNA function. In some embodiments, asRNAs are designed to target one of the above-mentioned small non-coding RNAs. In some embodiments, asRNAs are designed to target miRNAs. In some embodiments, asRNAs are designed to target pre-miRNAs. In some embodiments, asRNAs are designed to target pri-miRNAs. In some embodiments, asRNAs are designed to target lncRNAs.In some embodiments, the asRNA is designed to target a splice.

[0064] Nuclear targeted RNAs refer to RNA molecules that are synthesized and / or function in the nucleus of a cell. According to preferred embodiments, nuclear targeted RNAs of the present invention include non-coding RNAs, lncRNAs, pre-mRNAs, and pre-miRNAs. As used herein, the term "pre-mRNA" refers to unprocessed or partially processed precursor mRNAs containing introns and exons, which are synthesized by transcription from a cellular DNA template. Pre-mRNAs require intron splicing (removal) to produce mRNA molecules containing only exons. In some embodiments, asRNAs are designed to target pre-mRNAs. The term "mt-mRNA" refers to mRNA molecules transcribed from mitochondrial DNA. In some embodiments, asRNAs are designed to target mt-mRNAs in mitochondria.

[0065] The term "interspersed," as used herein, refers to having portions of different types adjacently spaced apart, e.g., by different types of nucleotides or nucleotide analogs, or by different modifications to the same type of nucleotide or nucleotide analog, e.g., having different modifications to the same type of nucleotide or nucleotide analog next to different types of nucleotides or nucleotide analogs. In various embodiments of the present invention, a "deoxyribonucleotide monomer interspersed segment (ISD)" refers to a section of an oligonucleotide chain in which one or more deoxyribonucleotides are connected to at least one portion that is a different type from the deoxyribonucleotides. For example, if the deoxyribonucleotides are unmodified, the different type portions can be ribonucleotides or analogs thereof, modified ribonucleotides, modified deoxyribonucleotides, or deoxyribonucleotide analogs. If the deoxyribonucleotides are modified, the different type portions can be ribonucleotides or analogs thereof, modified ribonucleotides, unmodified deoxyribonucleotides, differently modified deoxyribonucleotides, or different types of deoxyribonucleotide analogs.

[0066] As used herein, " modulating," "regulating," and its grammatical equivalents refer to either increasing or decreasing (e.g., silencing), in other words, either up-regulating or down-regulating.As used herein, "gene silencing" refers to the reduction of gene expression, and can refer to the reduction of gene expression by about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% of the target gene.

[0067] As used herein, the terms "inhibiting," "to inhibit," and their grammatical equivalents, when used in the context of a biological activity, refer to the downregulation of a biological activity, which may reduce or eliminate a targeted function, such as the production of a protein or the phosphorylation of a molecule. In certain embodiments, inhibition may refer to a reduction of about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% of a targeted activity. When used in the context of a disorder or disease, these terms refer to success in preventing the onset of symptoms, alleviating symptoms, or eliminating a disease, condition, or disorder.

[0068] As used herein, the term "substantially complementary" or "complementary" refers to the complementarity in the base-paired double-stranded region between two strands of linked nucleosides, without any single-stranded regions such as terminal overhangs. Complementarity does not need to be perfect; for example, there may be any number of base pair mismatches between the two strands of linked nucleosides. However, if the number of mismatches is so great that hybridization does not occur even under minimally stringent hybridization conditions, the sequence is not a substantially complementary sequence. When two sequences are referred to herein as "substantially complementary," it means that the sequences are sufficiently complementary to each other to hybridize under selected reaction conditions. The relationship between nucleic acid complementarity and the stringency of hybridization sufficient to achieve specificity is well known in the art. Two substantially complementary strands can be, for example, perfectly complementary, or can contain one to many mismatches, provided that the hybridization conditions are sufficient to allow, for example, discrimination between matched and unmatched sequences. Thus, substantially complementary sequences can refer to sequences having at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% base pair complementarity in the double-stranded region, or any number in between.

[0069] As used herein, "fully complementary" or "100% complementary" means that each nucleobase of a nucleobase sequence of a first strand of linked nucleosides has a complementary nucleobase in a second nucleobase sequence of a second strand of linked nucleosides. In certain embodiments, the first strand of linked nucleosides is a targeting compound and the second strand of linked nucleosides is an antisense compound, or conversely, the first strand of linked nucleosides is an antisense compound and the second strand of linked nucleosides is a targeting compound.

[0070] As used herein, term " targeting region " refers to the region in an oligonucleotide chain, and is substantially or completely complementary to another oligonucleotide chain, and therefore these two chains hybridize or anneal with each other in this targeting region under suitable conditions.For example, antisense chain can comprise targeting region, and can thereby hybridize with target mRNA.

[0071] The terms "administer," "administering," or "administration" are used herein in their broadest sense. These terms refer to any method of introducing a compound or pharmaceutical composition described herein into a subject and may include, for example, introducing a compound into a subject systemically, locally, or in situ. Thus, a compound of the present disclosure produced in a subject from a composition (whether or not the composition contains the compound) is encompassed by these terms. When these terms are used in conjunction with the terms "systemic" or "systemically," they generally refer to in vivo systemic absorption or accumulation of a compound or composition in the bloodstream, followed by distribution throughout the body.

[0072] The term "effective amount" or "therapeutically effective amount" refers to an amount of a compound or pharmaceutical compound described herein that is sufficient to affect an intended outcome, including, but not limited to, disease treatment, as described below. In some embodiments, a "therapeutically effective amount" is an amount that is effective in detectably killing or inhibiting the growth or spread of cancer cells, tumor size or number, and / or other measures of the level, stage, progression, and / or severity of cancer. In some embodiments, a "therapeutically effective amount" refers to an amount administered systemically, locally, or in situ (e.g., the amount of compound produced in situ in a subject). A therapeutically effective amount may vary depending on the intended application (in vitro or in vivo), or the subject and condition to be treated, such as the subject's weight and age, the severity of the condition, or the mode of administration, and can be readily determined by one of ordinary skill in the art. The term also applies to a dose that will induce a specific response in target cells, such as reduced cell migration. The specific dose may vary depending, for example, on the particular pharmaceutical composition, the subject and their age and pre-existing health conditions or risk of health conditions, the dosing regimen to be followed, the severity of the disease, whether it is administered in combination with other agents, the timing of administration, the tissue to which it is administered, and the physical delivery system in which it is carried.

[0073] The term "cancer" in a subject refers to the presence of cells that have properties typical of cancer-causing cells, such as uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rate, and certain morphological characteristics. Often, cancer cells will exist in the form of a tumor or mass, but such cells may exist alone within a subject or circulate in the bloodstream as independent cells, such as leukemia or lymphoma cells. Examples of cancer as used herein include, but are not limited to, lung cancer, pancreatic cancer, bone cancer, skin cancer, head or neck cancer, cutaneous or intraocular melanoma, breast cancer, uterine cancer, ovarian cancer, peritoneal cancer, colon cancer, rectal cancer, colorectal adenocarcinoma, anal cancer, stomach cancer, gastric cancer, gastrointestinal cancer, gastric adenocarcinoma, adrenocorticoid cancer, and the like. carcinoma), uterine cancer, fallopian tube cancer, endometrial cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, esophageal cancer, gastroesophageal junction cancer, gastroesophageal adenocarcinoma, chondrosarcoma, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal gland cancer, soft tissue sarcoma, Ewing's sarcoma, urethral cancer, penile cancer, prostate cancer, bladder cancer, testicular cancer, ureteral cancer, renal pelvis cancer, mesothelioma, hepatocellular carcinoma, biliary tract cancer, kidney cancer, renal cell carcinoma, chronic or acute leukemia, lymphocytic lymphoma, neoplasms of the central nervous system (CNS), spinal axis tumor, brain stem glioma, glioblastoma multiforme, astrocytoma, schwannoma, ependymoma, medulloblastoma, meningioma, squamous cell carcinoma, pituitary adenoma, including refractory versions of any of the foregoing cancers or combinations of one or more of the foregoing cancers. Some of the exemplified cancers are encompassed by the general term and are included in this term. For example, the general term urinary cancer includes bladder cancer, prostate cancer, kidney cancer, testicular cancer, etc., and another general term, hepatobiliary cancer, includes liver cancer (which itself is a general term including hepatocellular carcinoma or biliary tract cancer), gallbladder cancer, biliary tract cancer, or pancreatic cancer. Both urinary cancer and hepatobiliary cancer are contemplated by the present disclosure and are included in the term "cancer."

[0074] The term "pharmaceutical composition" refers to a formulation containing an active ingredient, such as a molecule or composition disclosed herein, in a form suitable for administration to a subject, often in admixture with other substances, e.g., a pharmaceutical carrier such as a sterile aqueous solution. In one embodiment, the pharmaceutical composition is in bulk or in unit dosage form. The unit dosage form may be in any of a variety of forms, including, for example, a capsule, an IV bag, a tablet, a single pump on an aerosol inhaler, or a vial. The amount of active ingredient in a unit dose of the composition is an effective amount and will vary according to the particular treatment involved. Those skilled in the art will understand that routine variations in dosage may sometimes be necessary depending on the age and condition of the patient. Dosage will also depend on the route of administration. Various routes are contemplated, including oral, pulmonary, rectal, parenteral, transdermal, subcutaneous, intravenous, intramuscular, intraperitoneal, and intranasal routes. Dosage forms for topical or transdermal administration of the asRNA of the present invention include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches, and inhalants.

[0075] The term "pharmaceutical agent" means a substance that provides a therapeutic benefit when administered to an individual.

[0076] The term " pharmaceutically acceptable carrier " refers to a medium or diluent that does not interfere with the structure of the compound. Some of these carriers allow pharmaceutical compositions to be formulated as, for example, tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and lozenges for oral ingestion by subjects. Some of these carriers allow pharmaceutical compositions to be formulated for injection, infusion or local administration. For example, a pharmaceutically acceptable carrier is a sterile aqueous solution.

[0077] The term "pharmaceutically acceptable derivatives" includes derivatives of the compounds described herein, such as solvates, hydrates, esters, prodrugs, polymorphs, isomers, isotopically labeled variants, pharmaceutically acceptable salts, and other derivatives known in the art.

[0078] The term "pharmaceutically acceptable salt" refers to a physiologically and pharmaceutically acceptable salt of a compound, i.e., a salt that retains the desired biological activity of the parent compound and does not impart undesired toxicological effects thereto. The term "pharmaceutically acceptable salt" or "salt" includes salts prepared by reacting the parent compound with a pharmaceutically acceptable non-toxic acid or base, including inorganic or organic acids and bases. Pharmaceutically acceptable salts of the compounds described herein can be prepared by methods well known in the art. For a review of pharmaceutically acceptable salts, see Stahl and Wermuth, Handbook of Pharmaceutical Salts: Properties, Selection and Use (Wiley-VCH, Weinheim, Germany, 2002). Pharmaceutically acceptable salts can include, but are not limited to, acid addition salts, including hydrochloride, hydrobromide, phosphate, sulfate, hydrogen sulfate, alkylsulfonate, arylsulfonate, acetate, benzoate, citrate, maleate, fumarate, succinate, lactate and tartrate; alkali metal cations, for example, Na, K, Li, alkaline earth metal salts, for example, Mg or Ca, or organic amine salts.In particular, the sodium salt of oligonucleotide has been proven to be useful and is well tolerated for therapeutic administration to humans.Therefore, in one embodiment, the compound described herein is in the form of sodium salt.

[0079] As used herein, the term "subject" refers to any animal (e.g., mammal), including but not limited to humans, non-human primates, rodents, etc., who will be the recipient of a particular treatment. Typically, the terms "subject" and "patient" are used interchangeably herein in reference to a human subject.

[0080] The terms "treating" or "treatment" or "to treat" or "alleviating" or "to alleviate," as used herein, refer to both (1) therapeutic measures that cure, slow, reduce the symptoms of, and / or halt the progression of a diagnosed pathological condition or disorder, and (2) prophylactic or preventative measures that prevent or slow the onset of the targeted pathological condition or disorder. Thus, those in need of treatment include those who already have the disorder; those who are susceptible to the disorder; and those in whom the disease is to be prevented. A subject is successfully "treated" according to the methods of the present invention if the patient exhibits one or more of the following: a reduction in the number of cancer cells or a complete absence of cancer cells; a reduction in tumor size; inhibition or absence of cancer cell invasion into peripheral organs, including the spread of cancer to soft tissue and bone; inhibition or absence of tumor metastasis; inhibition or absence of tumor growth; a reduction in one or more symptoms associated with the specific cancer; a reduction in morbidity and mortality; and an improvement in quality of life.

[0081] The term "carrier," as used herein, means a pharmaceutically acceptable material, composition, or vehicle, such as, for example, a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, which is involved in or capable of carrying or transporting a subject pharmaceutical compound from one organ or part of the body to another. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Non-limiting examples of pharmaceutically acceptable carriers, carriers, and / or diluents include sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethylcellulose, ethyl cellulose, and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols such as propylene glycol; polyols such as glycerin, sorbitol, mannitol, and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffer solutions; and other non-toxic compatible substances utilized in pharmaceutical formulations. Wetting agents, emulsifiers, and lubricants, such as sodium lauryl sulfate, magnesium stearate, and polyethylene oxide-polypropylene oxide copolymers, as well as coloring agents, release agents, coating agents, sweetening, flavoring, and perfuming agents, preservatives, and antioxidants can also be present in the composition. 2. Certain Embodiments

[0082] Certain embodiments of the present invention provide asRNA compositions comprised of linked ribonucleoside monomers with at least one deoxyribonucleoside monomer interspersed segment, referred to as an ISD. Some or all of the nucleoside monomers and / or internucleoside linkages contained therein may be modified from those found in natural RNA or DNA. One or more ISDs may be found in the asRNA of the present invention. In some embodiments, each ISD independently consists of one deoxyribonucleotide monomer or at least two, three, four, five, six, seven, eight, nine, or ten consecutive deoxyribonucleotide monomers. In some embodiments, an ISD has at least two consecutive linked deoxyribonucleotide monomers.

[0083] Exemplary structures and sequences of asRNA molecules of the present invention are shown in Figures 1, 3 and 5A.

[0084] The compositions of the present invention can be used to modulate gene expression or function in eukaryotic cells in at least three ways: (i) contacting a cell with one type of asRNA molecule or administering it to a subject; (ii) contacting a cell with different types of asRNA molecules ...

[0085] In certain embodiments, the asRNA comprises a nucleobase sequence region, referred to as the "targeting region," that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% complementary to the target segment of the target gene to which it is targeted, including mRNA and non-coding RNA. In certain embodiments, the asRNA molecule has a nucleobase sequence that contains the complete complement of the target segment of the target gene to which it is targeted. In certain embodiments, the asRNA molecule has a nucleobase sequence that contains at most one, two, or three mismatches when hybridized to the target segment of the target gene to which it is targeted. In certain embodiments, the target gene is selected from mRNA or non-coding RNA involved in mammalian disease. In some embodiments, at least one ISD is located within the targeting region of the asRNA. In certain embodiments, the ISD is located at or near the 5' end of the asRNA or at or near the 3' end of the strand. In other embodiments, the ISD is located more centrally in the asRNA (i.e., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleobases away from either end, i.e., starting from position 2 or more centrally from the end). In some embodiments, at least one ISD can be located anywhere in the asRNA.

[0086] In various embodiments, the asRNA has a backbone length of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 and 50 linked nucleotide monomers, or an equivalent thereof, or a range between any two of the foregoing values, inclusive of the endpoints of the range. For example, some of the length ranges for asRNA strands are: 8 to 48 nucleotide monomers; 8 to 44 nucleotide monomers; 8 to 42 nucleotide monomers; 8 to 40 nucleotide monomers; 8 to 36 nucleotide monomers; 8 to 33 nucleotide monomers; 10 to 48 nucleotide monomers; 10 to 44 nucleotide monomers; 10 to 42 nucleotide monomers; 10 to 40 nucleotide monomers; 10 to 36 nucleotide monomers; 10 to 30 nucleotide monomers; 10 to 29 nucleotide monomers; 12 to 48 nucleotide monomers; 12 to 44 nucleotide monomers; 12 to 42 nucleotide monomers; 12 to 40 nucleotide monomers; 12 to 36 nucleotide monomers; 12 to 34 nucleotide monomers; 12 to 32 nucleotide monomers; 12 to 30 nucleotide monomers; 12 to 29 nucleotide monomers; 12 to 28 nucleotide monomers. mer; 12 to 26 nucleotide monomers; 12 to 25 nucleotide monomers; 13 to 48 nucleotide monomers; 13 to 44 nucleotide monomers; 13 to 42 nucleotide monomers; 13 to 40 nucleotide monomers; 13 to 36 nucleotide monomers; 13 to 34 nucleotide monomers; 13 to 32 nucleotide monomers; 13 to 30 nucleotide monomers; 13 to 28 nucleotide monomers; 13 to 26 nucleotide monomers; 13 to 25 nucleotide monomers; 13 to 24 nucleotide monomers; 13 to 23 nucleotide monomers; 14 to 36 nucleotide monomers; 15 to 23 nucleotide monomers; 20 to 36 nucleotide monomers; 21 to 36 nucleotide monomers; 24 to 36 nucleotide monomers; at least 21 nucleotide monomers; at least 24 nucleotide monomers and at least 8 nucleotide monomers.

[0087] In certain embodiments, the asRNA is 8 to 36 nucleotide monomers in length (inclusive). In other words, the asRNA is 8 to 36 linked nucleobase monomers (inclusive). In certain embodiments, the asRNA is composed of 20 to 36 linked nucleoside monomers (inclusive). In other embodiments, the asRNA comprises oligonucleotides composed of 8 to 100, 10 to 80, 12 to 50, 14 to 30, 15 to 23, 16 to 22, 16 to 21, or 20 linked nucleobases (inclusive).

[0088] In the asRNA molecule of the present invention, at least one nucleotide monomer can be a modified nucleotide or nucleotide analog, such as a sugar-modified nucleotide, a backbone-modified nucleotide, and / or a base-modified nucleotide.In some embodiments, such backbone-modified nucleotide has at least one internucleoside linkage modified, for example, to include at least one nitrogen or sulfur heteroatom.In some embodiments, the modified internucleoside linkage is or includes a phosphorothioate (P=S) group, a phosphotriester, a methylphosphonate, or a phosphoramidate.

[0089] In certain embodiments, the asRNA contains at least one modified internucleoside linkage. Such modified internucleoside linkage can be between two deoxyribonucleoside monomers, between two ribonucleoside monomers, or between one deoxyribonucleoside monomer and one ribonucleoside monomer. Alternatively, the phosphate group on at least one of the terminal nucleoside monomers can be modified. In certain embodiments, the internucleoside linkage is a phosphorothioate internucleoside linkage. In certain embodiments, the internucleoside linkage is a thiophosphoramidate internucleoside linkage. In certain embodiments, each internucleoside linkage in the oligonucleotide chain is a phosphorothioate internucleoside linkage. In certain embodiments, all of the internucleoside linkages in the asRNA are phosphorothioate internucleoside linkages, or a mixture of phosphorothioate and phosphodiester linkages.

[0090] In certain embodiments, the asRNA comprises at least one nucleoside monomer having a modified sugar moiety. Such a nucleoside monomer may be a deoxyribonucleoside monomer or a ribonucleoside monomer.

[0091] In certain embodiments, the 2'-position of the modified sugar moiety is replaced with a group selected from OR, R, halo, SH, SR, NH, NHR, NR, or CN, where each R is independently C-C alkyl, alkenyl, or alkynyl, and halo is F, Cl, Br, or I. In some embodiments, the 2'-position of the modified sugar moiety is replaced with a group selected from allyl, amino, azido, thio, O-allyl, O-C-C 10 Alkyl, OCF3, OCH2F, O(CH2)2SCH3, O(CH2)2-ON(R m )(R n ), O-CH2-C(=O)-N(R m )(R n ), or O-CH2-C(=O)-N(R1)-(CH2)2-N(R m )(R n), wherein each R l , R m and R n are independently H or substituted or unsubstituted C1-C 10 In some embodiments, the modified sugar moiety is selected from the group of 5'-vinyl, 5'-methyl (R or S), 4'-S, 2'-F, 2'-OCH, 2'-OCHCH, 2'-OCHCHF, and 2'-O(CH)OCH substituents. In some embodiments, the modified sugar moiety is substituted with a bicyclic sugar selected from the group of 4'-(CH2)-O-2' (LNA); 4'-(CH2)-S-2; 4'-(CH2)2-O-2' (ENA); 4'-CH(CH3)-O-2' (cEt), and 4'-CH(CHOCH3)-O-2', 4'-C(CH3)(CH3)-O-2', 4'-CH2-N(OCH3)-2', 4'-CH2-ON(CH3)-2', 4'-CH2-N(R)-O-2' (where R is H, C1-C12 alkyl, or a protecting group), 4'-CH2-C(H)(CH3)-2', and 4'-CH2-C-(=CH2)-2'.

[0092] In some embodiments, the modified sugar moiety is selected from the group of 2'-O-methoxyethyl modified sugars (MOE), 4'-(CH2)-O-2' bicyclic sugars (LNA), 2'-deoxy-2'-fluoroarabinose (FANA), and methyl(methyleneoxy) (4'-CH(CH3)-O-2) bicyclic sugars (cEt).

[0093] In some embodiments, the asRNA of the present invention comprises at least one nucleoside monomer having a modified nucleobase. Such a nucleoside monomer may be a deoxyribonucleoside monomer or a ribonucleoside monomer.

[0094] In some embodiments, modified nucleobases are 5-methylcytosine (5-Me-C), inosine bases, tritylated bases, 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine; 6-methyl and other alkyl derivatives of adenine and guanine; 2-propyl and other alkyl derivatives of adenine and guanine; 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine; 5-propynyl (—C≡C—CH3) uracil and cytosine and other alkynyl derivatives of pyrimidine bases; 6-azouracil, cytosine and thymine, Selected from the group of 5-uracil (pseudouracil), 4-thiouracil, 1-methyl-pseudo-uracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo, particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-aminoadenine, 8-azaguanine and 8-azaadenine, and 7-deazaguanine and 7-deazaadenine, and 3-deazaguanine and 3-deazaadenine.

[0095] In certain embodiments, the modified nucleobase in the molecule of the present invention is 5-methylcytosine.In some embodiments, each cytosine base in the molecule of the present invention is 5-methylcytosine.In certain embodiments, the modified nucleobase is 5-methyluracil.In certain embodiments, each uracil is 5-methyluracil.

[0096] In one aspect, in the molecules of the present invention, the asRNA comprises linked ribonucleoside monomers and, in addition to these linked ribonucleoside monomers, further comprises an ISD consisting of one or more linked deoxyribonucleoside monomers. Furthermore, there may be even more ISD segments. The ISD may be located anywhere in the asRNA. In some embodiments, one or more ISDs comprise a terminal nucleoside monomer or a penultimate terminal nucleoside monomer. In some embodiments, one or more ISDs are inserted into a segment of ribonucleoside monomers, thereby separating them into multiple segments. In certain embodiments, each ISD independently consists of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 linked ribonucleoside monomers.

[0097] In certain embodiments, at least one or each of the linked deoxyribonucleoside monomers within an ISD is a modified deoxyribonucleotide or deoxyribonucleotide analog. The deoxyribonucleotides may be modified in the same or similar manner as follows: to have a modified internucleoside linkage, a modified sugar moiety, and / or a modified nucleobase.

[0098] In some embodiments, the asRNA molecules of the present invention may contain at least one CpG motif that can be recognized by a pattern recognition receptor (PRR), such as a Toll-like receptor.

[0099] In some embodiments, the sugar moieties of the deoxyribonucleotide monomers in the asRNA molecules are either sugar moieties of naturally occurring deoxyribonucleotides (2-H) or 2'-deoxy-2'-fluoroarabinose (FANA).

[0100] In some embodiments, the sugar moiety of the ribonucleotide monomer in the asRNA molecule is selected from the group of naturally occurring ribonucleotides (2-OH), 2'-F modified sugars, 2'-OMe modified sugars, 2'-O-methoxyethyl modified sugars (MOE), 4'-(CH2)-O-2' bicyclic sugars (LNA), and methyl(methyleneoxy) (4'-CH(CH3)-O-2) bicyclic sugars (cEt).

[0101] In certain embodiments, each ribonucleoside monomer of an asRNA molecule has a 2'-O-methoxyethyl modified sugar, where each cytosine is 5-methylcytosine, each uracil is 5-methyluracil or methyl-pseudouracil, and each internucleoside linkage is a phosphorothioate linkage. In certain embodiments, each deoxyribonucleoside monomer within an ISD has a 2'-deoxy-2'-fluoroarabinose (FANA) modified sugar moiety, where each cytosine is 5-methylcytosine, and each internucleoside linkage is a phosphorothioate linkage.

[0102] In certain embodiments, the molecules of the present invention can be stabilized against degradation by at least one chemical modification or secondary structure.Not only can any or all of the nucleotide monomers in asRNA be chemically modified, but they can also be conjugated to one or more moieties or ligands, such as peptides, antibodies, antibody fragments, polymers, polysaccharides, lipids, hydrophobic moieties or molecules, cationic moieties or molecules, lipophilic compounds or moieties, oligonucleotides, cholesterol, GalNAc, and aptamers, to enhance their functionality.

[0103] In certain embodiments, the targeting region of the molecule of the present invention does not contain any mismatch or bulge, and is completely complementary to the target oligonucleotide in the targeting region.In another embodiment, the targeting region of asRNA contains mismatch and / or bulge when hybridized with target RNA.In some embodiments, the entire asRNA is completely complementary to target RNA.

[0104] As known to those skilled in the art, it is possible to introduce mismatched bases without losing activity.Similarly, when asRNA of the present invention is base-paired with target RNA, it can contain unmatched or mismatched region.The mismatch in asRNA may be desirable to reduce off-target effects, or may enable other features for asRNA.

[0105] In certain embodiments, the target is mRNA or non-coding RNA involved in mammalian disease.In certain embodiments, the target is mRNA.In certain embodiments, the target is non-coding RNA, such as microRNA and lncRNA.As long as asRNA and target sequence are substantially complementary to each other, asRNA can occupy the target and inactivate the target gene by hybridizing.

[0106] 3. Modifications Nucleoside monomers are base-sugar compositions. The nucleobase (also known as base) portion of a nucleoside monomer is usually a heterocyclic base moiety. Nucleotide monomers are nucleoside monomers that further contain a phosphate group covalently linked to the sugar portion of the nucleoside. For nucleoside monomers containing a pentofuranosyl sugar, the phosphate group may be linked to the 2', 3', or 5' hydroxyl moiety of the sugar. Oligonucleotides are formed by covalently linking adjacent nucleoside monomers to each other to form a linear polymeric oligonucleotide. Within the oligonucleotide structure, the phosphate groups are commonly referred to as forming the internucleoside linkages of the oligonucleotide.

[0107] Modifications to the asRNA molecules of the present invention include substitutions or changes to internucleoside linkages, sugar moieties, or nucleobases. Modified asRNAs are sometimes preferred over native forms due to desirable properties, such as increased inhibitory activity, enhanced cellular uptake, enhanced strand affinity, solubility, reduced nonspecific interactions, and resistance to RNase degradation or other enhanced stability. Therefore, equivalent results can often be obtained with short asRNAs having such chemically modified nucleoside monomers. One or more of the natural nucleotides in the asRNA of the present invention can be replaced with modified nucleotides or nucleotide analogs. The substitution can occur anywhere in the asRNA.

[0108] Modifications of oligonucleotide molecules have been investigated to improve the stability of various oligonucleotide molecules, including antisense oligonucleotides, ribozymes, aptamers, and RNAi (Chiu and Rana, 2003; Czauderna et al., 2003; de Fougerolles et al., 2007; Kim and Rossi, 2007; Mack, 2007; Zhang et al., 2006; Schrnidt, 2007; Setten RL et al., 2020; Crooke ST et al., 2018; and Roberts TC et al., 2020).

[0109] Any stabilizing modification known to those skilled in the art can be used to improve the stability of oligonucleotide molecules.In oligonucleotide molecules, chemical modifications can be introduced into the phosphate backbone (e.g., phosphorothioate linkage), sugar (e.g., locked nucleic acid, glycerol nucleic acid, cEt, 2'-MOE, 2'-fluorouridine, 2'-O-methyl), and / or base (e.g., 2'-fluoropyrimidine).

[0110] Some examples of such chemical modifications are summarized in the following sections.

[0111] In various embodiments, the modified nucleotide or nucleotide analog is a sugar-modified nucleotide, a backbone-modified nucleotide, and / or a base-modified nucleotide.

[0112] 3.1 Modified Internucleoside Linkages or Backbone-Modified Nucleotides Naturally occurring internucleoside linkages in RNA and DNA are 3' to 5' phosphodiester linkages. AsRNA molecules of the invention having one or more modified, i.e., non-naturally occurring, internucleoside linkages in one or both strands may be chosen over corresponding molecules having only naturally occurring internucleoside linkages because of desirable properties, such as, for example, enhanced cellular uptake, increased affinity for target nucleic acids, and increased stability in the presence of nucleases.

[0113] AsRNA with modified internucleoside linkages includes not only internucleoside linkages that retain phosphorus atoms, but also internucleoside linkages that do not have phosphorus atoms. In some embodiments, phosphodiester internucleoside linkages are modified to contain at least nitrogen and / or sulfur heteroatoms. Representative phosphorus-containing internucleoside linkages include, but are not limited to, phosphodiester, phosphotriester, methylphosphonate, phosphoramidate, thiophosphoramidate, and phosphorothioate. Methods for preparing phosphorus-containing and non-phosphorus-containing linkages are well known.

[0114] In one embodiment, the modified nucleotide or nucleotide analog is a backbone-modified nucleotide. The backbone-modified nucleotide may have a modified phosphodiester internucleoside linkage. In a further embodiment, the backbone-modified nucleotide is a phosphorothioate internucleoside linkage. In certain embodiments, each internucleoside linkage is a phosphorothioate internucleoside linkage.

[0115] 3.2 Modified sugar moieties The asRNA of the present invention may optionally contain one or more nucleoside monomers in which the sugar group has been modified. Such sugar-modified nucleoside monomers may confer enhanced nuclease stability, increased binding affinity, or some other beneficial biological property to the chain. In certain embodiments, the nucleoside monomer comprises a chemically modified ribofuranose ring moiety. Examples of chemically modified ribofuranose rings include, but are not limited to, the addition of substituents (including 5' and 2' substituents), bridging of non-geminal ring atoms to form bicyclic nucleic acids (BNAs), and the modification of ribosyl ring oxygen atoms to S, N(R), or C(R1)(R2) (where R, R1, and R2 are each independently H, C1-C1). 12 Examples of chemically modified sugars include 2'-F-5'-methyl substituted nucleosides (see PCT International Application WO2008 / 101157, published August 21, 2008, for other disclosed 5',2'-disubstituted nucleosides), or replacement of the ribosyl ring oxygen atom with S with further substitution at the 2' position (see U.S. Patent Application Publication No. 2005-0130923, published June 16, 2005), or alternatively, 5' substitution of BNAs (see PCT International Application WO2007 / 134181, published November 22, 2007, in which LNAs are substituted, for example, with a 5'-methyl or 5'-vinyl group).

[0116] Examples of nucleoside monomers having modified sugar moieties include, but are not limited to, nucleosides containing 5'-vinyl, 5'-methyl (R or S), 4'-S, 2'-F, 2'-OCH, 2'-OCHCH, 2'-OCHCHF, and 2'-O(CH)OCH substituents. The substituent at the 2' position can be any of allyl, amino, azido, thio, O-allyl, O-C1-C2 10and O—CH—C(═O)—N(R)(R), and O—CH—C(═O)—N(R)—(CH)—N(R)(R), where each R, R, and R is independently H or a substituted or unsubstituted C to C 10 It is alkyl.

[0117] Bicyclic nucleosides are modified nucleosides having a bicyclic sugar moiety. Examples of bicyclic nucleic acids (BNAs) include, but are not limited to, nucleosides comprising a bridge between the 4'-ribosyl ring atom and the 2'-ribosyl ring atom. In certain embodiments, the asRNA provided herein comprises one or more BNA nucleosides, wherein the bridge comprises one of the following formulas: 4'-(CH2)-O-2' (LNA); 4'-(CH2)-S-2; 4'-(CH2)2-O-2' (ENA); 4'-CH(CH3)-O-2' and 4'-CH(CHOCH3)-O-2' (and analogs thereof, see U.S. Patent No. 7,399,845, issued July 15, 2008); 4'-C(CH3)(CH3)-O-2' (and analogs thereof, see U.S. Patent No. 7,399,845, issued July 15, 2008). See PCT / US2008 / 068922, published as WO / 2009 / 006478, published on December 8, 2008; 4'-CH2-N(OCH3)-2' (and its analogs, see PCT / US2008 / 064591, published as WO / 2008 / 150729, published on December 11, 2008); 4'-CH2-ON(CH3)-2' (see U.S. Patent Application Publication No. 2004-0171570, published on September 2, 2004); 4'-CH2-N(R)-O-2' (wherein R is H, C1-C 12alkyl, or a protecting group) (see U.S. Pat. No. 7,427,672, issued Sep. 23, 2008); 4'-CH2-C(H)(CH3)-2' (see Chattopadhyaya et al., J. Org. Chem., 2009, 74, 118-134); and 4'-CH2-C-(=CH2)-2' (and analogs thereof, see PCT / US2008 / 066154, published Dec. 8, 2008, published as WO2008 / 154401).

[0118] In certain embodiments, the bicyclic nucleoside is: (A) α-L-methyleneoxy(4′-CH2-O-2)BNA (B) β-D-methyleneoxy(4′-CH2-O-2)BNA These include, but are not limited to, (C) ethyleneoxy (4'-(CH)-O-2') BNAs, (D) aminooxy (4'-CH-ON(R)-2') BNAs, (E) oxyamino (4'-CH-N(R)-O-2) BNAs, (F) methyl (methyleneoxy) (4'-CH(CH)-O-2) BNAs (referred to as constrained ethyl or cEt), (G) methylene-thio (4'-CH-S-2') BNAs, (H) methylene-amino (4'-CH-N(R)-2') BNAs, (I) methyl carbocyclic (4'-CH-CH(CH)-2) BNAs, (J) propylene carbocyclic (4'-(CH)-2') BNAs, and (K) vinyl BNAs.

[0119] In certain embodiments, the modified nucleotide or nucleotide analog is a sugar-modified ribonucleotide in which the 2'-OH group is replaced by a group selected from H, OR, R, halo, SH, SR, NH2, NHR, NR2, and CN, where each R is independently selected from the group consisting of C1-C6 alkyl, alkenyl, and alkynyl, and halo is selected from the group of F, Cl, Br, and I. In certain embodiments, the sugar-modified ribonucleotide is selected from the group of 2'-OMe-modified nucleotides, 2'-F-modified nucleotides, 2'-O-methoxyethyl (2'MOE)-modified nucleotides, LNA (locked nucleic acid)-modified nucleotides, GNA (glycerol nucleic acid)-modified nucleotides, and cEt (constrained ethyl)-modified nucleotides.

[0120] The molecules of the present invention can be stabilized by adopting chemical modifications at the 2' position of ribose, such as 2'-O-methylpurine and 2'-fluoropyrimidine, which increase the resistance to endonuclease activity in serum.The position for introducing modifications should be carefully selected so as to avoid the significant reduction of the silencing / regulating efficacy of molecules.In certain embodiments, the first nucleotide monomer adjacent to the 5'-end nucleotide monomer of chain is 2'-fluoro-ribonucleotide.

[0121] 3.3 Modified Nucleobases AsRNA can also have nucleobase (or base) modifications or substitutions. The nucleobase (or base) modifications or substitutions are structurally distinct from naturally occurring or synthetic unmodified nucleobases, but functionally interchangeable. Both natural and modified nucleobases can participate in hydrogen bonds. Such nucleobase modifications can confer nuclease stability, binding affinity, or some other beneficial biological properties to asRNA molecules. Modified nucleobases include, for example, synthetic and natural nucleobases, such as 5-methylcytosine (5-Me-C). Certain nucleobase substitutions, including 5-methylcytosine substitutions, are particularly useful for increasing the binding affinity of antisense strands. For example, 5-methylcytosine substitution has been shown to increase nucleic acid duplex stability by 0.6-1.2°C (Sanghvi, YS, Crooke, ST and Lebleu, B., eds., Antisense Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278).

[0122] Additional modified nucleobases include 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine; 6-methyl and other alkyl derivatives of adenine and guanine; 2-propyl and other alkyl derivatives of adenine and guanine; 2-thiouracil, 1-methylpseudouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine; 5-propynyl (-C≡C-CH3) uracil and cytosine and other alkynyl derivatives of pyrimidine bases; 6-azouracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo, particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-aminoadenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine, and 3-deazaguanine and 3-deazaadenine.

[0123] Heterocyclic base moiety can comprise purine or pyrimidine base replaced with other heterocycles, such as 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone.The nucleic acid base that is particularly useful for increasing the binding affinity of antisense strand includes 5-substituted pyrimidine, 6-azapyrimidine and N-2, N-6 and O-6 substituted purine, and said substituted purine includes 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine.

[0124] In certain embodiments, the modified nucleotide or nucleotide analog is a base-modified nucleotide. In certain embodiments, the modified nucleotide or nucleotide analog has an unusual or modified base. In certain embodiments, the modified base is 5-methylcytosine (5'-Me-C). In certain embodiments, each cytosine is a 5-methylcytosine. In certain embodiments, the modified base is 5-methyluracil (5'-Me-U). In certain embodiments, each uracil is a 5-methyluracil.

[0125] Any modified nucleotide or analog that may aid in stability or affinity can be made without departing from the spirit and scope of the present invention. Some examples of such chemical modifications are the same as those summarized above.

[0126] 4. Pharmaceutical Compositions In some embodiments, the present invention also provides pharmaceutical formulations comprising the asRNA of the present invention or a pharmaceutically acceptable derivative thereof and at least one pharmaceutically acceptable excipient or carrier. As used herein, "pharmaceutically acceptable excipient" or "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Suitable carriers are described in "Remington: The Science and Practice of Pharmacy, Twentieth Edition," Lippincott Williams & Wilkins, Philadelphia, PA, incorporated herein by reference. Examples of such carriers or diluents include, but are not limited to, water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Liposomes and non-aqueous vehicles, such as fixed oils, may also be used. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the asRNA molecule, use thereof in the compositions is contemplated.

[0127] Examples of pharmaceutically acceptable carriers that can be used with the molecules of the invention include, but are not limited to, pharmaceutical carriers, positively charged carriers, liposomes, lipid nanoparticles, protein carriers, hydrophobic moieties or molecules, cationic moieties or molecules, GalNAc, polysaccharides, polymers, nanoparticles, nanoemulsions, cholesterol, lipids, lipophilic compounds or moieties, and lipids.

[0128] In certain embodiments, the present invention provides a method of treatment comprising administering a therapeutically effective amount of a pharmaceutical composition to a subject in need thereof. In certain embodiments, the pharmaceutical composition is administered by a route selected from the group consisting of intravenous injection (iv), subcutaneous injection (sc), orally (po), intramuscular (im) injection, oral administration, inhalation, topical, intrathecal, and other localized administration. In other embodiments, the therapeutically effective amount is 1 ng to 1 g per day, 100 ng to 1 g per day, or 1 μg to 1000 mg per day.

[0129] Methods for formulation are described in PCT International Application PCT / US02 / 24262 (WO03 / 01 1224), U.S. Patent Application Publication Nos. 2003 / 0091639 and 2004 / 0071775, each of which is incorporated herein by reference.

[0130] The asRNA molecules of the present invention are administered in a suitable dosage form prepared by combining a therapeutically effective amount (e.g., an effective level sufficient to achieve the desired therapeutic effect by inhibiting tumor growth, killing tumor cells, treating or preventing a cell proliferative disorder, etc.) of the asRNA molecules of the present invention (as the active ingredient) with standard pharmaceutical carriers or diluents according to conventional procedures (i.e., by producing a pharmaceutical composition of the present invention).

[0131] These procedures may include mixing, granulating, and compressing or dissolving the ingredients, as appropriate, to achieve the desired preparation. In another embodiment, a therapeutically effective amount of an asRNA molecule is administered in a suitable dosage form without standard pharmaceutical carriers or diluents. In some embodiments, a therapeutically effective amount of a molecule of the present invention is administered in a suitable dosage form. Pharmaceutically acceptable carriers include solid carriers such as lactose, terra alba, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate, stearic acid, and the like. Exemplary liquid carriers include syrup, peanut oil, olive oil, water, and the like. Similarly, carriers or diluents include time-delay materials known in the art, such as glyceryl monostearate or glyceryl distearate, alone or with wax, ethylcellulose, hydroxypropylmethylcellulose, methyl methacrylate, and the like. Other fillers, excipients, flavorants, and other additives, such as those known in the art, may also be included in pharmaceutical compositions according to the present invention.

[0132] The pharmaceutical composition of the present invention can be prepared in a generally known manner, for example, by conventional mixing, dissolving, granulating, sugar-coating, elutriating, emulsifying, encapsulating, entrapping or lyophilization process.Pharmaceutical composition can be formulated in a conventional manner using one or more physiologically acceptable carriers, including excipients and / or auxiliary agents that facilitate the processing of antisense oligonucleotide into pharmaceutical preparations that can be used.Of course, suitable formulation depends on the selected route of administration.

[0133] The compositions, compounds, combinations, or pharmaceutical compositions of the present invention can be administered to subjects using many of the well-known methods currently used in chemotherapy treatment.For example, for the treatment of cancer, the asRNA molecules of the present invention can be injected directly into tumors, injected into the bloodstream or body cavities, or taken orally, or applied through the skin using a patch.For the treatment of psoriasis, systemic administration (e.g., oral administration) or topical administration to the affected area of ​​the skin is the preferred administration route.The selected dose should be sufficient to constitute effective treatment, but not so high as to cause unacceptable side effects.The patient's condition (e.g., cancer, psoriasis, etc.) and health status should be closely monitored during and for a reasonable period after treatment.

[0134] 5.Usefulness 5.1 How to use The present invention also provides a method for modulating gene expression or function in a cell or organism. The cell can be a eukaryotic cell, such as a mammalian cell. The method includes contacting the cell or organism with an asRNA molecule disclosed herein under conditions that allow selective gene silencing, and mediating selective gene silencing exerted by the asRNA molecule on a target nucleic acid having a sequence portion substantially complementary to the antisense strand. The target nucleic acid can be RNA, such as mRNA or non-coding RNA, which encodes a protein involved in a disease or controls part of a biological pathway involved in a disease.

[0135] In some embodiments, the contacting step includes introducing the asRNA molecule into target cells in culture or into target cells in an organism capable of selective gene silencing. In further embodiments, the introducing step includes mixing, transfection, lipofection, infection, electroporation, or other delivery techniques. In other embodiments, the introducing step includes administering the asRNA molecule by intravenous, subcutaneous, intrathecal, oral, inhalation, topical, or other clinically acceptable administration method using a pharmaceutically acceptable excipient, carrier, or diluent selected from the group consisting of pharmaceutical carriers, positively charged carriers, liposomes, lipid nanoparticles, protein carriers, polymers, nanoparticles, nanoemulsions, lipids, N-acetyl-galactosamine (GalNAc), lipophilic compounds or moieties, and lipoids.

[0136] In certain embodiments, the silencing method is used to determine the function or utility of a gene in a cell or organism.

[0137] In some embodiments, the gene or RNA targeted by the compositions of the present invention is associated with or involved in a disease, such as a human disease or animal disease, a pathological condition, or an undesirable condition. In a further embodiment, the target gene or RNA is of a pathogenic microorganism. In yet a further embodiment, the target gene or RNA is of viral origin. In another embodiment, the target gene or RNA is tumor-associated.

[0138] In alternative embodiments, the gene or RNA targeted by the compositions of the invention is a gene or RNA associated with, or more specifically involved in, a cancer, an autoimmune disease, an inflammatory disease, a degenerative disease, an infectious disease, a proliferative disease, a metabolic disease, an immune-mediated disorder, an allergic disease, a dermatological disease, a malignant disease, a gastrointestinal disorder, a hepatic disorder, a respiratory disorder, a cardiovascular disorder, a dermatological disorder, a nephrological disorder, a rheumatoid disorder, a neurological disorder, a psychiatric disorder, an endocrine disorder, or an aging-related disorder or disease.

[0139] 5.2 Treatment method The present invention also provides methods for treating or preventing various diseases or conditions, including those summarized for ASOs and siRNAs (Czech, 2006; de Fougerolles et al., 2007; Dykxhoorn et al., 2003; Kim and Rossi, 2007; Mack, 2007; Crooke ST et al., 2018; Setten RL et al., 2019; Roberts TC et al., 2020). The method includes administering an effective amount of an asRNA molecule to a subject in need thereof under conditions that allow for the desired gene inhibition described in the section immediately above.

[0140] In an exemplary embodiment, a pharmaceutical composition having an asRNA molecule and a pharmaceutically acceptable excipient, carrier, or diluent is administered in a therapeutically effective amount to a patient in need thereof to treat or prevent a disease or undesirable condition.

[0141] In some embodiments, the present invention can be used as cancer treatment or to prevent cancer.AsRNA composition can be used to silence or knock down the gene involved in cell proliferation or other cancer phenotypes.Examples of these genes are k-Ras, β-catenin, Stat3.These oncogenes are involved in many types of cancer.

[0142] The novel compositions of the present invention can also be used to treat or prevent ophthalmic diseases (e.g., age-related macular degeneration (AMD) and diabetic retinopathy (DR)); infectious diseases (e.g., HIV / AIDS, hepatitis B virus (HBV), hepatitis C virus (HCV), human papillomavirus (HPV), herpes simplex virus (HSV), RCV, cytomegalovirus (CMV), dengue fever, West Nile virus); respiratory diseases (e.g., respiratory syncytial virus (RSC), asthma, cystic fibrosis); neurological diseases (e.g., Huntington's disease (HD), amyotrophic lateral sclerosis (ALS), spinal cord injury, Parkinson's disease, Alzheimer's disease, pain); cardiovascular diseases; metabolic disorders (e.g., hyperlipidemia, hypercholesterolemia, and diabetes); genetic disorders; and inflammatory conditions (e.g., inflammatory bowel disease (IBD), arthritis, rheumatoid diseases, autoimmune disorders), dermatological diseases.

[0143] In alternative embodiments, the method of administration is a route selected from the group of intravenous injection (iv), subcutaneous injection (sc), orally (po), intrathecal, inhalation, topical, and other localized administration. [Example]

[0144] Examples are provided below to further illustrate different features of the present invention. The examples also illustrate useful methodologies for practicing the invention. These examples do not limit the claimed invention. Methods and Materials cell culture

[0145] HepaRG cells were grown in Williams' medium supplemented with 10% FBS, 10 mg / ml hydrocortisone, and 4 mg / ml human recombinant insulin. Other suitable commercially available cell lines can be purchased and used, as known to those skilled in the art.

[0146] Transfection of asRNA into cells 24 hours before transfection, HepaRG cells or other commonly used cell lines were seeded in 6-well plates (1 x 105 cells / 2mL / well). AsRNA was transfected at different final concentrations, for example, 100pM or 10nM, using Lipofectamine® RNAiMAX (Thermo Fisher, USA) as described in the manufacturer's instructions. Briefly, asRNA and RNAiMAX were incubated in serum-free OPTI-MEM (Thermo Fisher) for 20 minutes, and then added to the cells together with the culture medium.

[0147] quantitative PCR Cells transfected with the indicated asRNAs were harvested 48 hours posttransfection. RNA was isolated with TRIZOL and subjected to qRT-PCR using TaqMan one-step RT-PCR reagents and the APOCIII assay for APOCIII mRNA detection. GAPDH mRNA levels were used as an internal control.

[0148] Target sequence

[0149] In order to investigate the gene silencing effect of asRNA disclosed in the present invention, asRNA was designed and produced to target different genes.The target genes, target sequences and exemplary asRNA sequences designed and used are listed in Table 1 below. Table 1. Target sequences and exemplary asRNA sequences used in the examples below [Table 1]

[0150] Example 1 Structure-activity relationship (SAR) for asRNAs with various ISDs Figure 1 shows various structural designs of a series of asRNA embodiments (labeled asRNA_1-13) with various ISDs. asRNA_1-13 were designed to target the APOCIII gene. Corresponding antisense single-stranded RNAs (ASRs) without ISDs were also designed for comparison (their structures and sequences are shown in Figure 1). The gene silencing activity of these asRNAs and the corresponding ASRs was tested at 10 nM in HepaRG cells (Figure 2).

[0151] In Figure 1, all letters "D" in the structures shown represent DNA residues or deoxyribonucleotide monomers; all letters "R" in the structures shown represent RNA residues or ribonucleotide monomers. All letters "dA, dT, dG, dC" in the sequences represent DNA residues; all letters "mA, mU, mG, mC" in the sequences represent 2'-MOE modified RNA residues; and all "*" in the structures and sequences shown represent PS (phosphorothioate internucleoside linkages).

[0152] The results suggest that the gene silencing activities of all designed asRNAs with at least one ISD are more potent and effective than the corresponding ASRs without an ISD.

[0153] Example 2 SAR of asRNAs with ISDs at various positions Figure 3 shows the different structural designs of another series of asRNA embodiments (labeled D10-SH1 to D10-SH11) by varying the position of the ISD while keeping the length of the asRNA and the number of deoxyribonucleotide monomers in the ISD constant. Exemplary sequences of asRNAs targeting the APOCIII gene are also shown in Figure 3. The gene silencing activity of these asRNAs D10-SH1 to D10-SH11 targeting the APOCIII gene was tested at 10 nM in HepaRG cells. The results are shown in Figure 4.

[0154] In FIG. 3, all letters "D," "R," "dA, dT, dG, dC," "mA, mU, mG, mC," and all "*" in the structures and sequences shown represent the same as in FIG. 1.

[0155] The results suggest that all the designed asRNAs with ISDs at various positions have potent gene silencing activity.

[0156] Example 3 SAR of asRNAs with various lengths Figure 5A shows the different structural designs of another series of asRNA embodiments. These asRNA molecules of various lengths were designed to target the APOCIII gene (labeled AS-8nt to AS-36nt, and their structures and sequences are shown in Figure 5A). The gene silencing activity of asRNAs ranging from 8 to 36 nt in length was tested to target the APOCIII gene. The gene silencing activity of these asRNAs was tested in HepaRG cells at 100 pM and 10 nM concentrations (Figures 5B and 5C).

[0157] In Figure 5A, all lowercase letters "a, c, g, t" represent DNA residues, and all uppercase letters "A, C, G, U" represent 2'-MOE modified RNA residues, where all "U"s represent 5-methyluridine 2'-MOE RNA residues, all "C"s and "c"s represent 5-Me-C; and all "*"s represent PS (phosphorothioate internucleoside linkages).

[0158] The results suggest that all designed asRNAs with a length of at least 10 linked nucleotide monomers possess potent gene silencing activity. It is a surprising finding that longer asRNAs are more potent in terms of gene silencing activity, and that asRNAs 20 nt or longer are particularly potent. In contrast, the typical length of an ASO is 16-20 nt. AsRNAs longer than 20 nt are far more potent than typical ASOs optimized with the most advanced state-of-the-art know-how.

[0159] The results in Examples 1 to 3 strongly suggest that asRNAs with ISDs designed according to the principles of the present invention can achieve high gene silencing efficacy.

[0160] equivalent The representative examples are intended to serve to illustrate the invention and are not intended to, and should not be construed as, limiting the scope of the invention. Indeed, various modifications of the invention and many further embodiments thereof, in addition to those shown and described herein, will become apparent to those skilled in the art from the complete contents of this document, including the examples and references to scientific and patent literature contained herein. The examples contain important additional information, exemplification and guidance that may be adapted to the practice of this invention in its various embodiments and equivalents thereof.

[0161] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of this disclosure, the preferred methods and materials are now described. The methods described herein can be carried out in any order that is logically possible, in addition to the specific order disclosed.

[0162] Incorporation by Reference References to and citations of other documents, such as patents, patent applications, patent publications, periodicals, books, academic papers, and web content, have been made in this disclosure. All such documents are hereby incorporated by reference in their entirety for all purposes. Any material or portion thereof that is stated to be incorporated by reference herein but that contradicts existing definitions, descriptions, or other disclosed material expressly set forth herein, is incorporated only to the extent that no contradiction arises between the incorporated material and the disclosed material. In the event of a conflict, the conflict should be resolved in favor of the present disclosure as the preferred disclosure. [ka]

Claims

1. 1. A short antisense RNA (asRNA) molecule comprising a single strand of linked nucleotide monomers, the strand is substantially complementary to a segment targeted by at least one targeting region of an RNA; the asRNA molecule comprises at least one deoxyribonucleotide monomer interspersed segment (ISD) comprising at least one deoxyribonucleotide monomer; asRNA molecule.

2. The asRNA molecule of claim 1, wherein the asRNA has at least one improved gene modulation or pharmaceutical property compared to a corresponding single-stranded antisense RNA without an ISD.

3. The asRNA molecule of claim 1, wherein at least one of the ISDs comprises at least one deoxyribonucleotide monomer or at least two, three, four, five, six, seven, or eight consecutive deoxyribonucleotide monomers.

4. The asRNA molecule of claim 1, wherein the ISD comprises at least two consecutive deoxyribonucleotide monomers.

5. 5. The asRNA molecule of claim 1, wherein at least one ISD is located within at least one targeted region.

6. 6. The asRNA molecule of claim 1, wherein the strand is at least 70%, 80%, 85%, 90%, 95% complementary or fully complementary to the targeted segment of the targeted RNA.

7. 7. The asRNA molecule of claim 1, wherein the asRNA has a length selected from the group consisting of 6, 7, 8, 9, 10, 12, 13, 14, 15, 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 and 50 nucleotide monomers.

8. The asRNA is a) 8 to 50 nucleotide monomers; b) 10 to 48 nucleotide monomers; c) 10 to 36 nucleotide monomers; d) 12 to 36 nucleotide monomers; e) 12 to 25 nucleotide monomers, and f) 21-36 nucleotide monomers The asRNA molecule of claim 7, having a length selected from the group consisting of:

9. The asRNA molecule according to any one of claims 1 to 8, wherein at least one nucleotide monomer is a modified nucleotide or a nucleotide analog.

10. The asRNA molecule according to claim 9, wherein the modified nucleotide or nucleotide analog is a sugar-modified nucleotide, a backbone-modified nucleotide, and / or a base-modified nucleotide.

11. The backbone-modified nucleotide has a modification of the internucleoside linkage, (a) the internucleoside linkage is modified to include at least one of a nitrogen or sulfur heteroatom; (b) the modified internucleoside linkage is selected from the group consisting of phosphorothioate (P=S) groups, phosphotriesters, methylphosphonates, and phosphoramidates; and / or (c) the asRNA includes at least one modified internucleoside linkage, and the modified internucleoside linkage is a phosphorothioate internucleoside linkage, The asRNA molecule according to claim 10.

12. The asRNA molecule according to claim 11(c), wherein each internucleoside linkage is a phosphorothioate internucleoside linkage.

13. The asRNA molecule according to claim 9, wherein the modified nucleotide or nucleotide analog includes a modified sugar moiety, (a) the 2' position of the modified sugar moiety is selected from the group consisting of OR, R, halo, SH, SR, NH ２ , N.H.R., N.R. ２ and CN, wherein each R is independently selected from the group consisting of C １ ~C ６ alkyl, alkenyl, or alkynyl, and halo is F, Cl, Br, or I; (b) the 2'-position of the modified sugar moiety is selected from the group consisting of allyl, amino, azido, thio, O-allyl, O-C １ ~C １０ Alkyl, OCF ３ , OCH ２ F, O(CH2) ２ SCH ３ , O(CH ２ ) ２ -O-N(R ｍ ) (R ｎ ), O-CH ２ -C(=O)-N(R ｍ ) (R ｎ ), and O—CH ２ -C(=O)-N(R ｌ )-(CH ２ ) ２ -N(R ｍ ) (R ｎ ) wherein R ｌ , R ｍ and R ｎ Each of is independently H or substituted or unsubstituted C １ ~C １０ is alkyl; (c) the modified sugar moiety is 5'-vinyl, 5'-methyl (R or S), 4'-S, 2'-F, 2'-OCH ３ , 2'-OCH ２ CH ３ , 2'-OCH ２ CH ２ F and 2'-O(CH2) ２ OCH ３ The substituents are selected from the group consisting of: (d) the modified sugar moiety is 4'-(CH ２ )-O-2'(LNA);4'-(CH ２ )-S-2;4'-(CH ２ )2-O-2'(ENA);4'-CH(CH ３ )-O-2'(cEt) and 4'-CH(CH ２ OCH ３ )-O-2', 4'-C(CH ３ ) (CH ３ )-O-2', 4'-CH ２ -N(OCH ３ )-2',4'-CH ２ -O-N(CH ３ )-2',4'-CH ２ -N(R)-O-2' (wherein R is H, C1-C12 alkyl, or a protecting group), 4'-CH ２ -C(H)(CH ３ )-2', and 4'-CH ２ -C-(=CH ２ )-2'; and / or (e) the modified sugar moiety is a 2'-O-methyl modified sugar, a 2'-O-methoxyethyl modified sugar (MOE), a 4'-(CH ２ )-O-2' bicyclic sugar (LNA), 2'-deoxy-2'-fluoroarabinose (FANA), and methyl(methyleneoxy) (4'-CH(CH ３ )-O-2) bicyclic sugars (cEt), The asRNA molecule according to claim 9.

14. The asRNA molecule according to claim 9, wherein the modified nucleotide or nucleotide analog includes a modified nucleic acid base, (a) the modified nucleobase is 5-methylcytosine (5-Me-C), inosine bases, tritylated bases, 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 1-methyl-pseudo-uracil, 5-halouracil and cytosine, 5-propynyl (-C≡C-CH ３ ) uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azouracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo, in particular 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-aminoadenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine, and 3-deazaguanine and 3-deazaadenine; and / or (b) the modified nucleic acid base is 5-methylcytosine, The asRNA molecule according to claim 9.

15. The asRNA molecule according to any one of claims 1 to 14, wherein the asRNA is used to modulate gene expression or function in a cell, the cell is a eukaryotic cell, and the eukaryotic cell is a mammalian cell.

16. The asRNA molecule according to claim 1, wherein the target RNA is any one of mRNA, pre-mRNA, mt-mRNA or non-coding RNA, and such RNA encodes a protein involved in a disease or controls a part of a biological pathway involved in a disease.

17. The target RNA is a) mRNA, pre-mRNA or mt-RNA of a gene involved in a human or animal disease or condition, b) mRNA or pre-mRNA of a gene of a pathogenic microorganism, c) viral RNA, d) lncRNA, e) miRNA, and f) RNA involved in a disease or disorder selected from the group consisting of an autoimmune disease, an inflammatory disease, a degenerative disease, an infectious disease, a proliferative disease, a metabolic disease, an immune-mediated disorder, an allergic disease, a dermatological disease, a malignant disease, a gastrointestinal disorder, a respiratory disorder, a cardiovascular disorder, a nephrological disorder, a rheumatoid disorder, a neurological disorder, an endocrine disorder, and an aging-related disorder. The asRNA molecule of claim 1, selected from the group consisting of:

18. 18. The asRNA molecule of claim 1, wherein the asRNA is conjugated to a ligand or moiety.

19. 19. The asRNA molecule of claim 18, wherein the ligand or moiety is selected from the group consisting of a peptide, an antibody, a polymer, a polysaccharide, a lipid, a hydrophobic moiety or molecule, a cationic moiety or molecule, a lipophilic compound or moiety, an oligonucleotide, cholesterol, GalNAc, and an aptamer.

20. A pharmaceutical composition comprising an asRNA molecule according to any one of claims 1 to 19 as an active agent and a pharmaceutically acceptable excipient, carrier or diluent.

21. 21. The pharmaceutical composition of claim 20, wherein the carrier is selected from the group consisting of a pharmaceutical carrier, a positively charged carrier, a lipid nanoparticle, a liposome, a protein carrier, a hydrophobic moiety or molecule, a cationic moiety or molecule, GalNAc, a polysaccharide polymer, a nanoparticle, a nanoemulsion, cholesterol, a lipid, a lipophilic compound or moiety, and a lipid.

22. A method for treating or preventing a disease or condition, comprising administering a therapeutically effective amount of an asRNA molecule described in any one of claims 1 to 19 or a pharmaceutical composition described in any one of claims 20 or 21 to a subject in need thereof.

23. 23. The method of claim 22, wherein the disease or condition is selected from the group consisting of cancer, an autoimmune disease, an inflammatory disease, a degenerative disease, an infectious disease, a proliferative disease, a metabolic disease, an immune-mediated disorder, an allergic disease, a dermatological disease, a malignant disease, a gastrointestinal disorder, a hepatic disorder, a respiratory disorder, a cardiovascular disorder, a dermatological disorder, a nephrological disorder, a rheumatoid disorder, a neurological disorder, a psychiatric disorder, an endocrine disorder, and an aging-related disorder.

24. 24. The method of claim 23, wherein the asRNA molecule or pharmaceutical composition is administered by a route selected from the group consisting of intravenous injection (iv), subcutaneous injection (sc), orally (po), intramuscular (im) injection, oral administration, inhalation, topical, intrathecal, and other localized administration.

25. 22. A method for modulating gene expression or gene function in a eukaryotic cell, comprising contacting the cell with an effective amount of an asRNA molecule described in any one of claims 1 to 19 or a pharmaceutical composition described in any one of claims 20 or 21.

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