Synthesis of modified oligonucleotides with improved stability

Abstract

This invention provides a novel type of skeletal modification oligonucleotide in which one or more carbon chains are inserted into the skeletal structure. [Solution] A modified oligonucleotide is provided, comprising a 5' end, a 3' end, and at least one modified subunit linkage of formula I. JPEG2026082842000059.jpg93164 B is the base pair portion; W and Z are O or O(CH2) n n is 1-10; X is H, OH, OR, F, SH, etc.; Y is O - OH, NH - etc.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims the benefits of U.S. Provisional Application No. 63 / 000,328, filed on 26 March 2020, which is incorporated herein by reference.

[0002] Description of research and development funded by the federal government. This invention was made with government support under grant numbers NS104022 and OD020012 granted by the National Institutes of Health. The government has certain rights in this invention.

[0003] Field of Invention This disclosure relates to the synthesis of novel modified oligonucleotides and novel phosphoramidites. [Background technology]

[0004] Currently, the most common metabolically stable skeletal modification used in complex therapeutic RNAs is phosphorothioate (PS) modification. While other available skeletal modification alternatives, such as peptide nucleic acids (PNAs) and phosphorodiamidate morpholino oligonucleotides (PMOs), function well as sterically barrier antisense oligonucleotides, these modifications are unacceptable in many promising RNA-based therapeutic strategies. These strategies include siRNA, miRNA, RNaseH-dependent antisense oligonucleotides, and aptamer-based therapies. This low tolerance stems from the fact that PNAs and PMOs cannot withstand biological mechanisms such as Argonaute proteins (siRNA / miRNAs) and RNaseH, which tightly recognize RNA structures when forming "functional" RNA-protein complexes.

[0005] One of the most common RNA-based therapeutic strategies involves the use of metabolically stable PS-modified RNA or PS / PO-modified chimeric oligonucleotides. However, a significant drawback of this strategy is toxicity due to the nonspecific binding of RNA to various proteins in vivo. Another drawback is that PS-modified, and even PS / PO-modified, RNA is degraded by endogenous nucleases. Therefore, additional skeletal modifications that provide greater metabolic stability without compromising drug efficacy are urgently needed in the field of RNA therapy.

[0006] The availability of synthetic materials is also a crucial factor in the development of therapeutic oligonucleotides. While various other modified skeletons (e.g., boranophosphates, phosphoramidates, etc.) have been reported, many of them require specific synthetic procedures and are not always compatible with conventional phosphoramidite oligonucleotide synthesis cycles. Therefore, it is difficult to freely synthesize / design chimeric skeletons with these modifications, as is difficult to mix PS / PO skeletons with other glycosylation skeletons. This difficulty in synthesis limits the additive diversification of design patterns for functional therapeutic oligonucleotides. Consequently, there is a high demand in this field for new chemical tools that are easy to synthesize and compatible with currently validated chemical modifications.

[0007] A novel type of skeletal modification is provided herein, in which one or more carbon chains are inserted into the skeletal structure. The skeletal modifications provided herein are not expected to have a serious impact on the RNA structure and can therefore offer compatibility with various RNA-binding biological mechanisms. Furthermore, since these modifications are not expected to exhibit toxic nonspecific binding to proteins, they can be incorporated into a wide range of therapeutic RNAs. [Overview of the project]

[0008] In one embodiment, the present disclosure provides a modified oligonucleotide comprising a 5' end, a 3' end, and a linkage between at least one modified subunit of formula I: [Chemical formula] (wherein, B is a base pair moiety; W is O or O(CH2) where n is from 1 to 10; X is selected from the group consisting of H, OH, OR, F, SH, SR, NR 2 2 and C 1-6 -alkoxy; Y is O - , OH, OR, OR 2 , NH - , NH2, NR 2 2, BH3, S - , R 1 , and SH; Z is O or O(CH2) n ; R 1 is alkyl, allyl or aryl; R 2 is alkyl, allyl or aryl).

[0009] In an embodiment of Formula I, Z is O(CH2) n , n is 1, W is O, and Y is O - .

[0010] In an embodiment of Formula I, Z is O, W is O(CH2) n , n is 1, and Y is O - .

[0011] In an embodiment of Formula I, Z is O(CH2) n , n is 1, W is O, and Y is O - .

[0012] In an embodiment of Formula I, Z is O(CH2) n , n is 1, W is O(CH2) n , and Y is O - .

[0013] In the embodiment of formula I, Z is O(CH2) n Therefore, n is not 1, and W is O(CH2) n And Y is O - That is the case.

[0014] In the embodiment of formula I, the base pair portion B is selected from the group consisting of adenine, guanine, cytosine, and uracil.

[0015] In another embodiment, the present disclosure provides a modified oligonucleotide comprising a 5' end, a 3' end, and a linkage between at least one modified subunit of formula II: [ka] (In the formula, B is the base pair portion; X is H, OH, OR, F, SH, SR, NR 2 2 and C 1-6 - Selected from the group consisting of alkoxys; Y is O - OH, OR, OR 2 NH - NH2, NR 2 2, BH3, S - , R 1 Selected from the group consisting of , and SH; R 1 These are alkyl, allyl, or aryl compounds; R 2 (These are alkyl, allyl, or aryl compounds.)

[0016] In one embodiment of formula II, Y is O.

[0017] In the embodiment of formula II, the base pair portion B is selected from the group consisting of adenine, guanine, cytosine, and uracil.

[0018] In another embodiment, the present disclosure provides a modified oligonucleotide comprising a 5' end, a 3' end, and a linkage between at least one modified subunit of formula III: [ka] (In the formula, B is the base pair portion; (R is alkyl, allyl, or aryl).

[0019] In one embodiment of Equation III, Y is O.

[0020] In the embodiment of formula III, the base pair portion B is selected from the group consisting of adenine, guanine, cytosine, and uracil.

[0021] In another embodiment, the present disclosure provides a modified oligonucleotide comprising a 5' end, a 3' end, and a linkage between at least one modified subunit of formula IV: [ka] (In the formula, B is the base pair portion; (R is alkyl, allyl, or aryl).

[0022] In the embodiment of formula IV, the base pair portion B is selected from the group consisting of adenine, guanine, cytosine, and uracil.

[0023] In another embodiment, the present disclosure provides a modified oligonucleotide comprising a 5' end, a 3' end, and a linkage between at least one modified subunit of formula V: [ka] (In the formula, B is the base pair portion; (R is alkyl, allyl, or aryl).

[0024] In the embodiment of formula V, the base pair portion B is selected from the group consisting of adenine, guanine, cytosine, and uracil.

[0025] In another aspect, the present disclosure provides a modified oligonucleotide comprising a 5' end, a 3' end, and a linkage between at least one modified subunit of formula VI: [ka] (In the formula, B is the base pair portion; (R is alkyl, allyl, or aryl).

[0026] In the embodiment of formula VI, the base pair portion B is selected from the group consisting of adenine, guanine, cytosine, and uracil.

[0027] In another aspect, the present disclosure provides a phosphoramidite derivative of formula (VII): [ka] (In the formula, B is the base pair portion; X is H, OH, OR, F, SH, SR, NR 2 2. MOE, alkyl, allyl, aryl, and C 1-6 - Selected from the group consisting of alkoxys; Z is either O or OCH2; R is either OMe or OCE (cyanoethyl): R 1 These are alkyl, allyl, or aryl compounds; R 2 (These are alkyl, allyl, or aryl compounds.)

[0028] In the embodiment of formula VII, the base pair portion B is selected from the group consisting of adenine, guanine, cytosine, and uracil.

[0029] In another aspect, the present disclosure provides a phosphoramidite derivative of formula (VIII): [ka] (In the formula, B is the base pair portion; X is H, OH, OR, F, SH, SR, NR 2 2. MOE, alkyl, allyl, aryl, and C 1-6 - Selected from the group consisting of alkoxys; R 1 These are alkyl, allyl, or aryl compounds; R 2 (These are alkyl, allyl, or aryl compounds.)

[0030] In the embodiment of formula (VIII), the base pair portion B is selected from the group consisting of adenine, guanine, cytosine, and uracil.

[0031] In another aspect, the present disclosure provides a phosphoramidite derivative of formula (IX): [ka] (In the formula, B is the base pair portion; X is H, OH, OR, F, SH, SR, NR 2 2. MOE, alkyl, allyl, aryl, and C 1-6 - Selected from the group consisting of alkoxys; R 1 These are alkyl, allyl, or aryl compounds; R 2 (These are alkyl, allyl, or aryl compounds.)

[0032] In the embodiment of formula (IX), the base pair portion B is selected from the group consisting of adenine, guanine, cytosine, and uracil.

[0033] In another aspect, the present disclosure provides a method for synthesizing a modified oligonucleotide comprising a 5' end, a 3' end, and at least one modified subunit linkage, the method is (a) To provide a nucleoside having a 5'-protecting group bonded to a solid support; (b) removing the protecting group; (c) Combining a deprotected nucleoside with a phosphoramidite derivative of formula (VII) to form a triester phosphite; [ka] (d) capping with phosphite triester; (e) Oxidizing phosphite triesters; (f) Repeat steps (b) through (e) using additional phosphoramidite; (g) including cutting from a solid support.

[0034] In another aspect, the present disclosure provides a method for coupling a phosphoramidite derivative of formula (VII) to the 5' end of a nucleoside or oligonucleotide, [ka] This method involves adding a phosphoramidite derivative of formula (VII) to a nucleoside or oligonucleotide in an organic solvent containing an aromatic heterocyclic acid.

[0035] In another aspect, the present disclosure provides: a method for synthesizing an exNA phosphoramidite, the method is: (a) To provide a nucleoside having a 3'-protecting group; (b) Oxidizing the 5'-hydroxyl group of a nucleoside to a 5'-aldehyde group; (c) Wittig olefinization converts the 5'-aldehyde group of the nucleoside to a 5'-vinyl group; (d) Hydroboration / oxidation of the 5'-vinyl group to produce a 6'-hydroxyl group; (e) Protecting the 6'-hydroxyl group with a DMTr group; (f) Removal of the 3'-protecting group of the nucleoside; (g) The process includes phosphytylating the 3'-hydroxyl group to produce a 3'-phosphoamidite.

[0036] The above and other features and advantages of this disclosure will be better understood from the detailed description of the exemplary embodiments in conjunction with the accompanying drawings. This patent or application document includes at least one drawing drawn in color. Copies of this patent or patent application publication, including the color drawing, are available from the Patent Office upon request and payment of the necessary fees. [Brief explanation of the drawing]

[0037] [Figure 1] This figure summarizes the modified subunit linkers provided herein. [Figure 2] This figure illustrates the synthesis of 2'-OMe-exNA phosphoramidite 9a. [Figure 3] This figure illustrates the synthesis of 2'-F-exNA phosphoramidite 9a. [Figure 4] This figure illustrates the synthesis of exNA-C phosphoramidite. [Figure 5] This figure illustrates the synthesis of exNA-G and exNA-A phosphoramidites. [Figure 6] This figure illustrates the synthesis of 5'-3'-bis-methylene-exNA phosphoramidite. [Figure 7] This figure illustrates the synthesis of exNA-ribo-uridine phosphoramidite. [Figure 8] This figure illustrates the synthesis of exNA-ribocytosine phosphoramidite. [Figure 9] This figure illustrates the synthesis of exNA-riboguanosine or exNA-riboadenine phosphoramidite. [Figure 10] This figure illustrates the synthesis of phosphoramidite monomers. [Figure 11] This figure provides a universal scheme for exNA conversion of sugar-modified nucleotides. [Figure 12] This figure illustrates the synthesis of oligonucleotides incorporating the exNA skeleton. [Figure 13]This figure provides a chart of synthesized exNA-modified RNA nucleotides. [Figure 14] This figure shows the results of in vitro silencing of target mRNA using double-stranded siRNA containing exNA with subunit links at various positions. [Figure 15] This figure provides a model demonstrating the increased 3' exonuclease stability of oligonucleotides with increasing numbers of exNA and phosphorothioate subunit linkages. [Figure 16] This figure shows the results of the 3'-exonuclease stability test. Each oligonucleotide (17.5 mM) was incubated at 37°C in a buffer containing 10 mM Tris-HCl (pH 8.0), 2 mM MgCl2, and snake venom phosphodiesterase I (20 mU / mL). [Figure 17] This figure shows the results of 3'-exonuclease stability tests of ex-NA subunit linkages in association with polyuridyl sequences containing phosphodiester (PO) and phosphorothioate (PS) oligonucleotides. Oligonucleotides were tested with 1, 2, 3, 4, or 5 ex-NA subunit linkages. [Figure 18] This figure shows the results of the 5'-phosphate-dependent 5'-exonuclease stability test. Each oligonucleotide was incubated with RNase-free water or 3.3 units of Terminator® (EpiCentre) exonuclease in Buffer A (provided by EpiCentre with Terminator® enzyme) at 37°C. [Figure 19] This figure shows the results from a 5'-phosphate-independent 5'-exonuclease stability test. Each oligonucleotide (10 μM) was incubated at 37°C in RNase-free water or 30 mM NaOAc (pH 6.0) buffer containing 0.25 U / mL bovine spleen phosphodiesterase II (BSP). [Figure 20A]This figure shows the in vitro silencing activity of several siRNA double-stranded structures containing inter-linking of 3'-terminal exNA subunits of one or more antisense strands. Antisense strands containing one, two, three, or four inter-linking of 3'-terminal exNA subunits were used in the dose-response curves. [Figure 20B] This figure shows the in vitro silencing activity of several siRNA double-stranded siRNAs containing inter-exNA subunit ligations at the 3' end of one or more antisense strands. The percentage change in potency compared to a control siRNA double-stranded siRNA without inter-exNA subunit ligations was also determined. [Figure 21A] This figure shows the in vivo silencing activity of several siRNA duplexes containing inter-subunit linkages between one or more antisense strands at the 3' end of the exNA subunit. The siRNA duplexes were in a Di-siRNA format targeting ApoE mRNA. Each siRNA duplex was administered to mice at 5 nmol by ICV injection, and ApoE mRNA levels were measured after 1 month. ApoE mRNA levels were measured in the following brain region: medial cortex. [Figure 21B] This figure shows the in vivo silencing activity of several siRNA duplexes containing inter-subunit linkages between one or more antisense strands at the 3' end of the exNA subunit. The siRNA duplexes were in a Di-siRNA format targeting ApoE mRNA. Each siRNA duplex was administered to mice at 5 nmol by ICV injection, and ApoE mRNA levels were measured after 1 month. ApoE mRNA levels were measured in the following brain region: striatum. [Figure 21C] This figure shows the in vivo silencing activity of several siRNA duplexes containing inter-subunit linkages between one or more antisense strands at the 3' end of exNA subunits. The siRNA duplexes were in a Di-siRNA format targeting ApoE mRNA. Each siRNA duplex was administered to mice at 5 nmol by ICV injection, and ApoE mRNA levels were measured after 1 month. ApoE mRNA levels were measured in the following brain region: hippocampus. [Figure 21D]This figure shows the in vivo silencing activity of several siRNA duplexes containing inter-subunit linkages between one or more antisense strands at the 3' end of exNA subunits. The siRNA duplexes were in a Di-siRNA format targeting ApoE mRNA. Each siRNA duplex was administered to mice at 5 nmol by ICV injection, and ApoE mRNA levels were measured after 1 month. ApoE mRNA levels were measured in the following brain region: thalamus. [Figure 21E] This figure shows the in vivo silencing activity of several siRNA duplexes containing inter-subunit linkages between one or more antisense strands at the 3' end of exNA subunits. The siRNA duplexes were in a Di-siRNA format targeting ApoE mRNA. Each siRNA duplex was administered to mice at 5 nmol by ICV injection, and ApoE mRNA levels were measured after 1 month. ApoE mRNA levels were measured in the following brain region: cerebellum. [Figure 22] This figure shows the in vivo silencing activity of several siRNA duplexes containing inter-subunit linkages between one or more antisense strands at the 3' end of exNA subunits. The siRNA duplexes targeted Htt mRNA. Each siRNA duplex was administered to mice at approximately 60 μg by ICV injection, and Htt mRNA levels were measured after 2 months. Htt mRNA levels were measured in the following brain regions: medial cortex (A), striatum (B), hippocampus (C), prefrontal cortex (D), and thalamus (E). The numbers 1-5 along the X-axis correspond to the chemical modification patterns of the siRNAs shown in Example 14. [Figure 23]This figure shows the in vivo silencing activity of several siRNA duplexes containing inter-subunit linkages between one or more antisense strands at the 3' end of exNA subunits. The siRNA duplexes targeted Htt mRNA. Each siRNA duplex was administered to mice at approximately 60 μg by ICV injection, and Htt protein levels were measured after 2 months. Htt protein levels were measured in the following brain regions: medial cortex (A), striatum (B), hippocampus (C), prefrontal cortex (D), and thalamus (E). The numbers 1–5 along the X axis correspond to the chemical modification patterns of the siRNAs shown in Example 14. [Modes for carrying out the invention]

[0038] Novel modified oligonucleotides and their synthesis are provided. Novel phosphoramidites and their synthesis are also provided.

[0039] Unless otherwise specified, the nomenclature used herein in relation to cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization is well known and commonly used in the art. Unless otherwise specified, the methods and techniques provided herein are carried out in accordance with conventional methods well known in the art, and unless otherwise specified, as described in the various general and more specific references cited and discussed throughout this specification. Enzyme reactions and purification techniques are carried out as described herein, in accordance with the manufacturer's specifications or as commonly achieved in the art. The terminology used herein in relation to analytical chemistry, synthetic organic chemistry, and pharmaceutical / medical chemistry, as well as laboratory procedures and techniques, are well known and commonly used in the art. Standard techniques are used in chemical synthesis, chemical analysis, preparation, formulation, and delivery of pharmaceuticals, and in the treatment of patients.

[0040] Unless otherwise defined herein, scientific and technical terms used herein have the meanings generally understood by those skilled in the art. In the event of any potential ambiguity, the definitions provided herein shall prevail over any dictionary or external definitions. Unless otherwise required by context, singular terms shall include plural forms, and plural terms shall include singular forms. The use of "or" shall mean "and / or" unless otherwise stated. The use of the term "including," as well as other forms such as "include" and "included," is not limited to these.

[0041] To make this disclosure easier to understand, certain terms are defined first.

[0042] The term "nucleoside" refers to a molecule having a purine or pyrimidine base covalently linked to a ribose or deoxyribose sugar. Exemplary nucleosides include adenosine, guanosine, cytidine, uridine, and thymidine. Further exemplary nucleosides include inosine, 1-methylinosine, pseudouridine, 5,6-dihydrouridine, ribothymidine, 2N-methylguanosine, and 2,2N,N-dimethylguanosine (also known as "rare" nucleosides). The term "nucleotide" refers to a nucleoside having one or more phosphate groups attached to the sugar moiety within an ester linkage. Exemplary nucleotides include nucleoside monophosphates, diphosphates, and triphosphates. The terms "polynucleotide" and "nucleic acid molecule" are used interchangeably herein and refer to polymers of nucleotides linked together by phosphodiester or phosphorothioate linkages between the 5' and 3' carbon atoms.

[0043] The terms “RNA,” “RNA molecule,” or “ribonucleic acid molecule” refer to polymers of ribonucleotides (e.g., 2, 3, 4, 5, 10, 15, 20, 25, 30, or more ribonucleotides). The terms “DNA,” “DNA molecule,” or “deoxyribonucleic acid molecule” refer to polymers of deoxyribonucleotides. DNA and RNA can be synthesized spontaneously (e.g., by DNA replication or DNA transcription, respectively). RNA can be modified after transcription. DNA and RNA can also be synthesized chemically. DNA and RNA can be single-stranded (i.e., ssRNA and ssDNA, respectively) or multi-stranded (e.g., double-stranded, i.e., dsRNA and dsDNA, respectively). “mRNA” or “messenger RNA” is single-stranded RNA that identifies the amino acid sequence of one or more polypeptide chains. This information is converted during protein synthesis when ribosomes bind to mRNA.

[0044] As used herein, the term “small interfering RNA” (“siRNA”) (also known in the art as “short interfering RNA”) refers to RNA (or RNA analogues) containing about 10 to 50 nucleotides (or nucleotide analogues) that can direct or mediate RNA interference. Preferably, siRNA contains about 15 to 30 nucleotides or nucleotide analogues, more preferably about 16 to 25 nucleotides (or nucleotide analogues), even more preferably about 18 to 23 nucleotides (or nucleotide analogues), and even more preferably about 19 to 22 nucleotides (or nucleotide analogues) (e.g., 19, 20, 21, or 22 nucleotides or nucleotide analogues). The term “short” siRNA refers to siRNA containing about 21 nucleotides (or nucleotide analogues), e.g., 19, 20, 21, or 22 nucleotides. The term “long” siRNA refers to siRNA containing about 24 to 25 nucleotides, e.g., 23, 24, 25, or 26 nucleotides. Short siRNAs may, in some cases, contain fewer than 19 nucleotides, e.g., 16, 17, or 18 nucleotides, provided that the short siRNA retains its ability to mediate RNAi. Similarly, long siRNAs may, in some cases, contain more than 26 nucleotides, provided that the longer siRNA retains its ability to mediate RNAi without further processing of the short siRNA, e.g., enzymatic processing.

[0045] The terms “nucleotide analog,” “modified nucleotide,” or “modified nucleotide” refer to non-standard nucleotides, such as ribonucleotides or deoxyribonucleotides, which do not exist in nature. Exemplary nucleotide analogs are modified at any position to alter the specific chemical properties of a nucleotide, while retaining the ability of the nucleotide analog to perform its intended function. Examples of nucleotide positions that can be derivatized include the 5-position, e.g., 5-(2-amino)propyluridine, 5-bromouridine, 5-propyneuridine, 5-propenyluridine; the 6-position, e.g., 6-(2-amino)propyluridine; and the 8-position of adenosine and / or guanosine, e.g., 8-bromoguanosine, 8-chloroguanosine, 8-fluoroguanosine. Furthermore, examples of nucleotide analogs include deazanucleotides, such as 7-deaza-adenosine; O- and N-modified (e.g., alkylated, e.g., N6-methyladenosine, or those known in the art) nucleotides; and other heterocyclic modified nucleotide analogs, such as those described in Herdewijn, Antisense Nucleic Acid Drug Dev., 2000 Aug.10(4):297-310.

[0046] Nucleotide analogs may also involve modifications to the sugar moiety of the nucleotide. For example, the 2'OH group may be substituted with a group selected from H, OR, R, F, Cl, Br, I, SH, SR, NH2, NHR, NR2, or COOR, where R is a substituted or unsubstituted C1-C6 alkyl, alkenyl, alkynyl, aryl, etc. Other possible modifications are described in U.S. Patents No. 5,858,988 and No. 6,291,438.

[0047] The phosphate group of a nucleotide can also be modified, for example, by substituting one or more oxygen atoms of the phosphate group with sulfur (e.g., a phosphorothioate), or by making other substitutions so that the nucleotide can perform its intended function. For example, see Eckstein, Antisense Nucleic Acid Drug Dev. 2000 Apr. 10(2):117-21, Rusckowski et al. Antisense Nucleic Acid Drug Dev. 2000 Oct. 10(5):333-45, Stein, Antisense Nucleic Acid Drug Dev. 2001 Oct. 11(5):317-25, Vorobjev et al. Antisense Nucleic Acid Drug Dev. 2001 Apr. 11(2):77-85, and U.S. Patent No. 5,684,143. The specific modifications referenced above (e.g., phosphate group modifications) preferably reduce the hydrolysis rate of polynucleotides, including analogs, in vivo or in vitro.

[0048] The term “oligonucleotide” refers to a short polymer of nucleotides and / or nucleotide analogs. The term “RNA analog” refers to a polynucleotide (e.g., a chemically synthesized polynucleotide) that has at least one modified or altered nucleotide compared to the corresponding unmodified or unaltered RNA, but retains the same or similar properties or functions as the corresponding unmodified or unaltered RNA. As described above, oligonucleotides may be linked by linkages that, compared to RNA molecules with phosphodiester linkages, result in a reduced hydrolysis rate of the RNA analog. For example, the nucleotides of the analog may include methylenediol, ethylenediol, oxymethylthio, oxyethylthio, oxycarbonyloxy, phosphorodiamidate, phosphoramidate, and / or phosphorothioate linkages. Preferred RNA analogs include sugar and / or skeletal-modified ribonucleotides and / or deoxyribonucleotides. Such alterations or modifications may further include the addition of non-nucleotide substances to the terminal(s) or internal(s) (one or more nucleotides) of the RNA. The RNA analog only needs to be sufficiently similar to native RNA that has the ability to mediate RNA interference.

[0049] As used herein, the term “RNA interference” (“RNAi”) refers to the selective intracellular degradation of RNA. RNAi occurs naturally within cells and removes foreign RNA (e.g., viral RNA). Natural RNAi proceeds via fragments cleaved from free dsRNA, directing the degradation mechanism to other similar RNA sequences. Alternatively, RNAi can be initiated by humans, for example, to silencing the expression of a target gene.

[0050] RNAi agents, such as RNA silencing agents, have a chain that is "sufficiently complementary to the target mRNA sequence in order to direct target-specific RNA interference (RNAi)." This means that the chain has a sequence sufficient to cause the destruction of the target mRNA by the RNAi mechanism or process.

[0051] As used herein, “isolated RNA” (e.g., “isolated siRNA” or “isolated siRNA precursor”) refers to an RNA molecule that, if produced by recombinant technology, is substantially free of other cell material or culture medium, and, if chemically synthesized, is substantially free of chemical precursors or other chemicals.

[0052] As used herein, the term “RNA silencing” refers to a group of sequence-specific regulatory mechanisms mediated by RNA molecules (e.g., RNA interference (RNAi), transcriptional gene silencing (TGS), post-transcriptional gene silencing (PTGS), querring, co-repression, and translational repression) that result in inhibition or “silencing” of the expression of the corresponding protein-coding gene. RNA silencing has been observed in many types of organisms, including plants, animals, and fungi.

[0053] The term “discriminative RNA silencing” refers to the ability of an RNA molecule to substantially inhibit the expression of a “first” or “target” polynucleotide sequence while not substantially inhibiting the expression of a “second” or “non-target” polynucleotide sequence, for example, when both polynucleotide sequences are present in the same cell. In certain embodiments, the target polynucleotide sequence corresponds to a target gene, and the non-target polynucleotide sequence corresponds to a non-target gene. In other embodiments, the target polynucleotide sequence corresponds to a target allele, and the non-target polynucleotide sequence corresponds to a non-target allele. In certain embodiments, the target polynucleotide sequence is a DNA sequence encoding a regulatory region (e.g., a promoter or enhancer element) of the target gene. In other embodiments, the target polynucleotide sequence is the target mRNA encoded by the target gene.

[0054] The term "in vitro" has the meaning recognized in the art, including, for example, purified reagents or extracts, such as cell extracts. The term "in vivo" also has the meaning recognized by those skilled in the art, including, for example, living cells, such as immortalized cells, primary cells, cell lines, and / or cells within an organism.

[0055] As used herein, the term “transgene” refers to any nucleic acid molecule that is artificially inserted into a cell and becomes part of the genome of an organism that develops from the cell. Such a transgene may include a gene that is partially or completely heterologous (i.e., exotic) to the transgenic organism, or it may represent a gene that is homologous to the organism’s endogenous genes. “Transgene” also means a nucleic acid molecule containing one or more selected nucleic acid sequences, e.g., DNA, that encodes one or more manipulated RNA precursors expressed in a transgenic organism, e.g., an animal, which is either partially or completely heterologous (i.e., exogenous) to the transgenic animal, or homologous to the transgenic animal’s endogenous genes but designed to be inserted into the animal’s genome at a location different from that of the native genes. The transgene may include one or more promoters and any other DNA, e.g., introns, all of which are operably ligated to the selected nucleic acid sequence and may include enhancer sequences.

[0056] Genes “involved” in a disease or disorder include genes whose normal or abnormal expression or function affects or causes a disease or disorder, or at least one symptom of a disease or disorder.

[0057] As used herein, the term “gain-of-function mutation” refers to any mutation in a gene that causes or contributes to a disease or disorder by causing a protein encoded by that gene (i.e., a mutant protein) to acquire a function not normally associated with that protein (i.e., a wild-type protein). A gain-of-function mutation may be a deletion, addition, or substitution of nucleotides in a gene that causes a change in the function of the encoded protein. In one embodiment, a gain-of-function mutation alters the function of the mutant protein or causes an interaction with another protein. In another embodiment, a gain-of-function mutation causes, for example, a reduction or removal of the normal wild-type protein through interaction between the modified mutant protein and the normal wild-type protein.

[0058] As used herein, the term “target gene” refers to a gene whose expression is substantially inhibited or “silenced.” This silencing can be achieved, for example, by cleaving the mRNA of the target gene or by silencing the RNA by translational repression of the target gene. The term “non-target gene” refers to a gene whose expression is not substantially silenced. In one embodiment, the polynucleotide sequences of the target gene and the non-target gene (e.g., the mRNA encoded by the target gene and the non-target gene) may differ by one or more nucleotides. In another embodiment, the target and non-target genes may differ by one or more polymorphisms (e.g., single nucleotide polymorphisms or SNPs). In yet another embodiment, the target and non-target genes may share less than 100% sequence identity. In yet another embodiment, the non-target gene may be a homolog (e.g., an ortholog or paralog) of the target gene.

[0059] A “target allele” is an allele (e.g., an SNP allele) whose expression is selectively inhibited or “silenced.” This silencing can be achieved by RNA silencing, for example, by cleaving the target gene or the mRNA of the target allele with siRNA. A “non-target allele” is an allele whose expression is substantially not silenced. In certain embodiments, the target allele and the non-target allele may correspond to the same target gene. In other embodiments, the target allele may correspond to or be associated with the target gene, and the non-target allele may correspond to or be associated with the non-target gene. In one embodiment, the polynucleotide sequences of the target allele and the non-target allele may differ by one or more nucleotides. In another embodiment, the target allele and the non-target allele may differ by one or more allele polymorphisms (e.g., one or more SNPs). In yet another embodiment, the target allele and the non-target allele may share less than 100% sequence identity.

[0060] As used herein, the term “polymorphism” refers to a variation in a gene sequence (e.g., one or more deletions, insertions, or substitutions) that is identified or detected when comparing the same gene sequence from different sources or subjects (but from the same organism). For example, polymorphisms can be identified when comparing the same gene sequence from different subjects. Identification of such polymorphisms is commonplace in the art, and the methodology is similar to that used, for example, to detect point mutations in breast cancer. Identification can be performed, for example, from DNA extracted from lymphocytes of the subject, and the polymorphic region can then be amplified using a specific primer for the polymorphic region. Alternatively, polymorphisms can be identified when comparing two alleles of the same gene. In certain embodiments, the polymorphism is a single nucleotide polymorphism (SNP).

[0061] A variation in the sequence between two alleles of the same gene within an organism is referred to herein as “allelic polymorphism.” In certain embodiments, allelic polymorphisms correspond to SNP alleles. For example, an allelic polymorphism may include a single-nucleotide variation between two alleles of an SNP. Polymorphisms may be located in nucleotides within a coding region, but due to the degeneracy of the genetic code, changes in the amino acid sequence are not encoded. Alternatively, a polymorphic sequence may encode different amino acids at a particular position, but the changes in amino acids do not affect the function of the protein. Polymorphic regions can also be found in the non-coding regions of a gene. In exemplary embodiments, polymorphisms are found in the coding region of a gene or in the uncoding region of a gene (e.g., 5'UTR or 3'UTR).

[0062] As used herein, the term “allele frequency” is a measure (e.g., a proportion or percentage) of the relative frequency of an allele (e.g., a SNP allele) at a single locus in an individual population. For example, if a population of individuals has n loci of a particular chromosomal locus (and the gene occupying that locus) within each somatic cell, the allele frequency of an allele is the proportion or percentage of the locus that the allele occupies in the population. In certain embodiments, the allele frequency of an allele (e.g., a SNP allele) is at least 10% (e.g., at least 15%, 20%, 25%, 30%, 35%, 40%, or more) in the sample population.

[0063] As used herein, the term “sample population” refers to a group of individuals that includes a statistically significant number of individuals. For example, a sample population may include 50, 75, 100, 200, 500, 1000, or more individuals. In certain embodiments, a sample population may include individuals that share at least a common disease phenotype (e.g., gain-of-function disorder) or mutation (e.g., gain-of-function mutation).

[0064] As used herein, the term “heterozygous” refers to the proportion of individuals in any given population who are heterozygous (i.e., possess two or more different alleles) at a particular locus (e.g., a SNP). Heterozygousness can be calculated for a sample population using methods well known to those skilled in the art.

[0065] The phrase "to examine the function of genes in cells or organisms" refers to examining or studying the resulting expression, activity, function, or phenotype.

[0066] As used herein, the term “RNA silencing agent” refers to RNA capable of inhibiting or “silencing” the expression of a target gene. In certain embodiments, RNA silencing agents can prevent the complete processing of mRNA molecules (e.g., complete translation and / or expression) through a post-transcriptional silencing mechanism. Examples of RNA silencing agents include small (<50 b.p.), non-coding RNA molecules, such as double-stranded RNAs containing paired strands, and precursor RNAs capable of generating such small non-coding RNAs. Exemplary RNA silencing agents include siRNA, miRNA, siRNA-like double-stranded RNAs, antisense oligonucleotides, GAPMER molecules, and dual-function oligonucleotides, as well as their precursors. In one embodiment, RNA silencing agents can induce RNA interference. In another embodiment, RNA silencing agents can mediate translational repression.

[0067] As used herein, the term “rare nucleotide” refers to naturally occurring nucleotides that occur infrequently, such as naturally occurring deoxyribonucleotides or ribonucleotides that do not occur infrequently, such as guanosine, adenosine, cytosine, or uridine. Examples of rare nucleotides, but not limited to, include inosine, 1-methylinosine, pseudouridine, 5,6-dihydrouridine, ribothymidine, 2N-methylguanosine, and 2,2N,N-dimethylguanosine.

[0068] The term "engineered" indicates that the precursor or molecule is not found in nature, in that all or part of the nucleic acid sequence of the precursor or molecule is created or selected by humans, as in the case of an engineered RNA precursor or engineered nucleic acid molecule. Once created or selected, the sequence is replicated, translated, transcribed, or otherwise processed by intracellular mechanisms. Therefore, an RNA precursor produced intracellularly from a transgene containing an engineered nucleic acid molecule is an engineered RNA precursor.

[0069] As used herein, the term “microRNA” (“miRNA”) also means “small temporal RNA” (“stRNA”) in the art, and refers to a small (e.g., 10 to 50 nucleotides) RNA that is genetically encoded (e.g., by the genome of a virus, mammal, or plant) and can direct or mediate RNA silencing. “miRNA disorder” refers to a disease or disorder characterized by abnormal expression or activity of miRNA.

[0070] As used herein, the term “dual-functional oligonucleotide” refers to an RNA silencing agent having the formula TL-μ, where T is the mRNA targeting portion, L is the linking portion, and μ is the miRNA recruitment portion. As used herein, the terms “mRNA targeting portion,” “targeting portion,” “mRNA targeting portion,” or “targeting portion” refer to a domain, portion, or region of a dual-functional oligonucleotide that is sufficiently sized and sufficiently complementary to a portion or region of mRNA selected or targeted for silencing (i.e., the portion has a sequence sufficient to capture the target mRNA). As used herein, the term “linking portion” or “linking portion” refers to a domain, portion, or region of an RNA silencing agent that covalently joins or links mRNA.

[0071] As used herein, the term “antisense strand” of an RNA silencing agent, e.g., siRNA or RNA silencing agent, refers to a strand substantially complementary to a section of approximately 10–50 nucleotides, e.g., approximately 15–30 nucleotides, 16–25, 18–23, or 19–22 nucleotides, of the mRNA of the targeted gene for silencing. The antisense strand, or first strand, has a sequence that is sufficiently complementary to the desired target mRNA sequence to direct target-specific silencing, e.g., sufficiently complementary to cause disruption of the desired target mRNA by an RNAi mechanism or process (RNAi interference), or sufficiently complementary to cause translational repression of the desired target mRNA.

[0072] The terms “sense strand” or “second strand” of an RNA silencing agent, such as siRNA, refer to a strand complementary to the antisense strand or first strand. The antisense strand and sense strand can also be referred to as the first strand or second strand, where the first strand or second strand is complementary to the target sequence, and each second strand or first strand is complementary to the first strand or second strand. A miRNA double-stranded intermediate or siRNA-like double-stranded complex includes a miRNA strand that is sufficiently complementary to a section of approximately 10–50 nucleotides of the mRNA of the gene targeted for silencing, and a miRNA* strand that is sufficiently complementary to form a double-stranded complex with the miRNA strand.

[0073] As used herein, the term “guide strand” refers to the strand of RNA silencing agent, such as a double-stranded siRNA or antisense strand of an siRNA sequence, that enters the RISC complex and directs the cleavage of the target mRNA.

[0074] As used herein, the term “asymmetry” refers to an unevenness in the binding strength or base pair strength between the ends of an RNA silencing agent (e.g., between the terminal nucleotides on the first strand or stem portion and the terminal nucleotides on the opposing second strand or stem portion), such as in the asymmetry of the double-stranded region of the RNA silencing agent (e.g., the stem of shRNA). This results in the 5' end of one strand of the double helix being more frequently in a transient unpaired state, e.g., a single-stranded state, than the 5' end of the complementary strand. This structural difference determines that one strand of the double helix is ​​preferentially incorporated into the RISC complex. The strand whose 5' end is less firmly paired with the complementary strand will preferentially be incorporated into RISC and mediate RNAi.

[0075] As used herein, the terms “bond strength” or “base pair strength” primarily refer to the strength of interactions between pairs of nucleotides (or nucleotide analogs) on opposing strands of an oligonucleotide double-strand (e.g., an siRNA double-strand), and between these nucleotides (or nucleotide analogs), such as through H bonds and van der Waals interactions.

[0076] As used herein, "5' end" refers to the 5' terminal nucleotide, for example, the 1 to approximately 5 nucleotides at the 5' end of an antisense strand, such as the 5' end of an antisense strand. As used herein, "3' end" refers to the region complementary to the 5' terminal nucleotide of a complementary antisense strand, for example, the 1 to approximately 5 nucleotide region, such as the 3' end of a sense strand.

[0077] As used herein, the term “destabilized nucleotide” refers to a first nucleotide or nucleotide analog that can form a base pair with a second nucleotide or nucleotide analog such that the base pair has a lower binding strength than a conventional base pair (i.e., a Watson-Crick base pair). In certain embodiments, the destabilized nucleotide can form a mismatch base pair with the second nucleotide. In other embodiments, the destabilized nucleotide can form a fluctuation base pair with the second nucleotide. In yet another embodiment, the destabilized nucleotide can form an ambiguous base pair with the second nucleotide.

[0078] As used herein, the term “base pair” refers to the interaction between pairs of nucleotides (or nucleotide analogs) on opposing strands of an oligonucleotide double helix (e.g., a double helix formed by the strands of an RNA silencing agent and a target mRNA sequence), primarily through nucleotide (or nucleotide analog) H bonds, van der Waals interactions, and other interactions. As used herein, the terms “bond strength” or “base pair strength” refer to the strength of a base pair.

[0079] As used herein, the term "mismatched base pair" refers to a base pair consisting of a non-complementary or non-Watson-Crick base pair that is not a normal complementary G:C, A:T, or A:U base pair. As used herein, the term "ambiguous base pair" (also known as a non-recognized base pair) refers to a base pair formed by a universal nucleotide.

[0080] As used herein, the term “universal nucleotide” (also known as “neutral nucleotide”) includes nucleotides (e.g., certain destabilized nucleotides) that have bases that do not significantly distinguish between bases on a complementary polynucleotide when forming base pairs (“universal bases” or “neutral bases”). Universal nucleotides are primarily hydrophobic molecules and can efficiently pack into antiparallel double-stranded nucleic acids (e.g., double-stranded DNA or RNA) through stacking interactions. The base portion of a universal nucleotide typically contains a nitrogen-containing aromatic heterocyclic moiety.

[0081] As used herein, the terms “sufficient complementarity” or “sufficient degree of complementarity” mean that the RNA silencing agent has sequences (e.g., in the antisense strand, mRNA targeting portion, or miRNA recruiting portion) sufficient to bind to the desired target and induce RNA silencing of the target mRNA, respectively.

[0082] As used herein, the term “translational repression” refers to the selective inhibition of mRNA translation. Spontaneous translational repression proceeds via miRNA cleaved from shRNA precursors. Both RNAi and translational repression are mediated by RISC. Both RNAi and translational repression occur spontaneously or can be initiated by humans, for example, to silence the expression of a target gene.

[0083] As used herein, the term "alkoxy" refers to an -O-alkyl group (alkyl is as defined herein). Examples of alkoxys include methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, sec-butoxy, and t-butoxy. In one embodiment, C1-C6 alkoxy groups are provided herein.

[0084] As used herein, the terms "halo" or "halogen" mean, unless otherwise specified, a fluorine, chlorine, bromine, or iodine atom, preferably fluorine, chlorine, or bromine, more preferably fluorine or chlorine, either alone or as part of another substituent.

[0085] As used herein, the term "hydroxy" means, either alone or as part of another substituent, an alcohol moiety having the formula -OH unless otherwise specified.

[0086] As used herein, the term "exNA" refers to an "extended nucleic acid" that includes an inter-subunit linkage containing one or more additional CH2 groups at the 3', 5', or both positions.

[0087] The preparation of linkers may involve the protection and deprotection of various chemical groups. The need for protection and deprotection, and the selection of appropriate protecting groups, can be readily determined by those skilled in the art. The chemistry of protecting groups is described, for example, in Greene, et al., Protective Groups in Organic Synthesis, 4d. Ed., Wiley & Sons, 2007, which is incorporated herein by reference in its entirety. The preparation of protecting groups and formation and cleavage methods described herein can be adjusted as necessary, taking into account various substituents.

[0088] Various methodologies of this disclosure include a step of comparing values, levels, features, properties, etc., with a “preferred control,” which is interchangeably referred to herein as “appropriate control.” A “preferred control” or “appropriate control” is any control or standard well known to those skilled in the art and useful for comparison purposes. In one embodiment, a “preferred control” or “appropriate control” is a value, level, feature, property, etc., determined before performing the RNAi methodology as described herein. For example, transcription rate, mRNA level, translation rate, protein level, biological activity, cellular characteristics or properties, genotype, phenotype, etc., can be determined before introducing the RNA silencing agent of this disclosure into cells or organisms. In another embodiment, a “preferred control” or “appropriate control” is a value, level, feature, property, etc., determined in cells or organisms, e.g., a control or, e.g., normal cells or organisms exhibiting normal traits. In yet another embodiment, a “preferred control” or “appropriate control” is a predefined value, level, feature, property, etc.

[0089] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which this disclosure pertains. Similar or equivalent methods and materials may be used in the implementation or testing of this disclosure, but suitable methods and materials are described below. All publications, patent applications, patents, and other references referenced herein are incorporated in their entirety by reference. In case of any conflict, this specification shall prevail, including definitions. In addition, materials, methods, and examples are illustrative and not intended to be limiting.

[0090] Various aspects of this disclosure are described in further detail in the following subsections.

[0091] I. Synthesis of Novel Modified Oligonucleotides This section describes a portfolio of synthesis procedures for oligonucleotides modified with novel skeletal modifications, specifically extended nucleic acids (exNA). This chemical modification of the skeleton significantly improves the metabolic stability of oligonucleotides. The chemical modification involves the insertion of one or more carbon atoms or chains at the 5', 3', or both positions of the skeleton. This structural modification creates a non-standard stretch / flexible structure on the oligo skeleton, protecting the oligonucleotide from cleavage by various nucleases.

[0092] Novel exNA modifications offer broad compatibility with siRNA scaffolds. Combinations of the exNA-PS scaffold enable significant improvements in metabolic stability (by 10–50 orders of magnitude compared to unmodified oligonucleotides) without compromising siRNA efficacy, as shown below (for example, the 5'-[exNA-PS]4-3' modification induces significantly higher exonuclease stability without negatively impacting siRNA efficacy). Therefore, these metabolically stabilizing exNA modifications significantly and robustly improve the performance of therapeutic oligonucleotide candidates in vivo.

[0093] This disclosure describes a synthesis protocol for exNA-modified oligonucleotides. Importantly, the synthesis of exNA monomer phosphoramidites can be achieved from commercially available nucleosides, and exNA-modified oligonucleotides can be prepared using conventional solid-phase oligonucleotide synthesis procedures in an automated oligonucleotide synthesizer.

[0094] This synthesis procedure offers the following notable advantages. For example, the conversion from conventional nucleosides to the "exNA format" is applicable to many diverse modified nucleosides. This thus expands the possibilities for synthesizing and creating a wider variety of modified oligonucleotides with chemical synthesis compatibility. Secondly, it does not require performing another specific synthesis procedure during the oligonucleotide synthesis cycle. This is a significant advantage in terms of the usability of these oligos, especially when using automated synthesizers where bottles of exNA phosphoramidite can be easily added to the machine. Thirdly, since exNA phosphoramidites and oligos are compatible with conventional deprotection conditions, no specific oligonucleotide deprotection conditions are required. Again, this is beneficial in terms of ease of synthesis and the use of automated synthesizers. Fourthly, it is possible to synthesize mixed-mer oligonucleotides that have both exNA and clinically validated modified nucleotides (e.g., 2'-OMe, 2'-F, phosphorothioates, various ligand conjugates, lipid conjugates, etc.).

[0095] In one embodiment, the present disclosure provides a modified oligonucleotide comprising a 5' end, a 3' end, and a linkage between at least one modified subunit of formula I: [ka] (In the formula, B is the base pair portion; W is O or O(CH2) n And n is between 1 and 10; X is H, OH, OR, F, SH, SR, NR 2 2 and C 1-6 - Selected from the group consisting of alkoxys; Y is O - OH, OR, OR 2 NH - NH2, NR 2 2, BH3, S - , R 1 Selected from the group consisting of , and SH; Z is O or O(CH2) n and; R 1 These are alkyl, allyl, or aryl compounds; R 2 (These are alkyl, allyl, or aryl compounds.)

[0096] In the embodiment of formula I, Z is O(CH2) n n is 1, W is O, and Y is O - That is the case.

[0097] In the embodiment of formula I, Z is O and W is O(CH2) n And n is 1, and Y is O - That is the case.

[0098] In the embodiment of formula I, Z is O(CH2) n n is 1, W is O, and Y is O - That is the case.

[0099] In the embodiment of formula I, Z is O(CH2) n Therefore, n is 1, and W is O(CH2) n And Y is O - That is the case.

[0100] In the embodiment of formula I, Z is O(CH2) n Therefore, n is not 1, and W is O(CH2) n And Y is O - That is the case.

[0101] In the embodiment of formula I, the base pair portion B is selected from the group consisting of adenine, guanine, cytosine, and uracil.

[0102] In another embodiment, the present disclosure provides a modified oligonucleotide comprising a 5' end, a 3' end, and a linkage between at least one modified subunit of formula II: [ka] (In the formula, B is the base pair portion; X is H, OH, OR, F, SH, SR, NR 2 2 and C 1-6 - Selected from the group consisting of alkoxys; Y is O - OH, OR, OR 2 NH - NH2, NR 2 2, BH3, S - , R 1 Selected from the group consisting of , and SH; R 1 These are alkyl, allyl, or aryl compounds; R 2 (These are alkyl, allyl, or aryl compounds.)

[0103] In one embodiment of formula II, Y is O.

[0104] In the embodiment of formula II, the base pair portion B is selected from the group consisting of adenine, guanine, cytosine, and uracil.

[0105] In another embodiment, the present disclosure provides a modified oligonucleotide comprising a 5' end, a 3' end, and at least one modified subunit linkage of formula III: [ka] (In the formula, B is the base pair portion; (R is alkyl, allyl, or aryl).

[0106] In one embodiment of Equation III, Y is O.

[0107] In the embodiment of formula III, the base pair portion B is selected from the group consisting of adenine, guanine, cytosine, and uracil.

[0108] In another embodiment, the present disclosure provides a modified oligonucleotide comprising a 5' end, a 3' end, and a linkage between at least one modified subunit of formula IV: [ka] (In the formula, B is the base pair portion; (R is alkyl, allyl, or aryl).

[0109] In the embodiment of formula IV, the base pair portion B is selected from the group consisting of adenine, guanine, cytosine, and uracil.

[0110] In another embodiment, the present disclosure provides a modified oligonucleotide comprising a 5' end, a 3' end, and a linkage between at least one modified subunit of formula V: [ka] (In the formula, B is the base pair portion; (R is alkyl, allyl, or aryl).

[0111] In the embodiment of formula V, the base pair portion B is selected from the group consisting of adenine, guanine, cytosine, and uracil.

[0112] In another aspect, the present disclosure provides a modified oligonucleotide comprising a 5' end, a 3' end, and a linkage between at least one modified subunit of formula VI: [ka] (In the formula, B is the base pair portion; (R is alkyl, allyl, or aryl).

[0113] In the embodiment of formula VI, the base pair portion B is selected from the group consisting of adenine, guanine, cytosine, and uracil.

[0114] In another aspect, the present disclosure provides a method for synthesizing a modified oligonucleotide comprising a 5' end, a 3' end, and at least one modified subunit linkage, the method is (a) To provide a nucleoside having a 5'-protecting group bonded to a solid support; (b) removing the protecting group; (c) Combining a deprotected nucleoside with a phosphoramidite derivative of formula (VII) to form a triester phosphite;

[0115] [ka] (d) capping with phosphite triester; (e) Oxidizing phosphite triesters; (f) Repeat steps (b) through (e) using additional phosphoramidite; (g) including cutting from a solid support.

[0116] II. Synthesis of Novel Phosphoramidite Derivatives This section describes a collection of synthetic procedures for novel phosphoramidite derivatives used to produce oligonucleotides modified with a novel skeletal modification, elongated nucleic acid (exNA). As shown in Figure 11, this modification is highly versatile and can be combined with many existing nucleosides to significantly increase the diversity of oligonucleotides with improved stability. In this embodiment, the disclosure provides a phosphoramidite derivative of formula (VII): [ka] (In the formula, B is the base pair portion; X is H, OH, OR, F, SH, SR, NR 2 2. MOE, alkyl, allyl, aryl, and C 1-6 - Selected from the group consisting of alkoxys; Z is either O or OCH2; R is either OMe or OCE (cyanoethyl): R 1 These are alkyl, allyl, or aryl compounds; R2 is alkyl, allyl or aryl).

[0117] In embodiments of Formula VII, the base pair moiety B is selected from the group consisting of adenine, guanine, cytosine, and uracil.

[0118] In another aspect, the present disclosure provides a phosphoramidite derivative of Formula (VIII):

Chemical formula

[0119] In embodiments of Formula (VIII), the base pair moiety B is selected from the group consisting of adenine, guanine, cytosine, and uracil.

[0120] In another aspect, the present disclosure provides a phosphoramidite derivative of Formula (IX):

Chemical formula

[0121] In the embodiment of formula (IX), the base pair portion B is selected from the group consisting of adenine, guanine, cytosine, and uracil.

[0122] In another aspect, the present disclosure provides a method for coupling a phosphoramidite derivative of formula (VII) to the 5' end of a nucleoside or oligonucleotide, [ka] This method involves adding a phosphoramidite derivative of formula (VII) to a nucleoside or oligonucleotide in an organic solvent containing an aromatic heterocyclic acid.

[0123] In another aspect, the present disclosure provides a method for synthesizing exNA phosphoamidites comprising: (a) To provide a nucleoside having a 3'-protecting group; (b) Oxidizing the 5'-hydroxyl group of a nucleoside to a 5'-aldehyde group; (c) Wittig olefinization converts the 5'-aldehyde group of the nucleoside to a 5'-vinyl group; (d) Hydroboration / oxidation of the 5'-vinyl group to produce a 6'-hydroxyl group; (e) Protecting the 6'-hydroxyl group with a DMTr group; (f) Removal of the 3'-protecting group of the nucleoside; (g) The process includes phosphytylating the 3'-hydroxyl group to produce a 3'-phosphoamidite.

[0124] III. Design of siRNA In some embodiments, the siRNA is designed as follows: First, a portion of the target gene is identified. The cleavage of mRNA at these sites eliminates the need for translation of the corresponding protein. The sense strand is designed based on the target sequence. In certain embodiments, the portion (and the corresponding sense strand) contains approximately 15–25 nucleotides, e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides. However, those skilled in the art will understand that siRNAs having lengths of less than 15 nucleotides or more than 25 nucleotides can also function to mediate RNAi. Thus, siRNAs of such lengths are also within the scope of this disclosure, as long as they retain the ability to mediate RNAi. It has been demonstrated that longer RNAi agents induce interferon or PKR responses in certain mammalian cells, which may be undesirable. In one embodiment, the RNAi agents of this disclosure do not induce a PKR response (i.e., are sufficiently short in length). However, longer RNAi agents may be useful, for example, in cell types that cannot produce a PKR response, or in situations where the PKR response is downregulated or suppressed by alternative means.

[0125] Sense strand sequences are designed so that the target sequence is essentially located in the center of the strand. Moving the target sequence to an off-center position may, in some cases, reduce the efficiency of siRNA cleavage. However, such compositions, i.e., less efficient compositions, may be desirable for use when off-silencing of wild-type mRNA is detected.

[0126] The antisense strand is typically the same length as the sense strand and contains complementary nucleotides. In one embodiment, the strands are perfectly complementary; that is, they have blunt ends when aligned or annealed. In another embodiment, the strands are aligned or annealed such that a 1, 2, 3, 4, 5, 6, or 7 nucleotide overhang is generated; that is, the 3' end of the sense strand is 1, 2, 3, 4, 5, 6, or 7 nucleotides longer than the 5' end of the antisense strand, and / or the 3' end of the antisense strand is 1, 2, 3, 4, 5, 6, or 7 nucleotides longer than the 5' end of the sense strand. The overhang may contain (or consist of) nucleotides corresponding to the target gene sequence (or its complement). Alternatively, the overhang may contain (or consist of) deoxyribonucleotides, e.g., dT, or nucleotide analogs, or other suitable non-nucleotide substances.

[0127] To facilitate the entry of the antisense strand into RISC (and thus increase or improve the efficiency of target cleavage and silencing), the base pair strength between the 5' end of the sense strand and the 3' end of the antisense strand may be modified (decreased or reduced). These are described in detail below and are incorporated by this reference into U.S. Patents 7,459,547, 7,772,203 and 7,732,593, titled “Methods and Compositions for Controlling Efficacy of RNA Silencing” (filed June 2, 2003) and U.S. Patents 8,309,704, 7,750,144, 8,304,530, 8,329,892 and 8,309,705, titled “Methods and Compositions for Enhancing the Efficacy and Specificity of RNAi” (filed June 2, 2003). In one embodiment, the base pair strength is low because the number of G:C base pairs between the 5' end of the first or antisense strand and the 3' end of the second or sense strand is less than the number of base pairs between the 3' end of the first or antisense strand and the 5' end of the second or sense strand. In another embodiment, the base pair strength is low due to at least one mismatched base pair between the 5' end of the first or antisense strand and the 3' end of the second or sense strand. In certain exemplary embodiments, the mismatched base pair is selected from the group consisting of: G:A, C:A, C:U, G:G, A:A, C:C, and U:U. In another embodiment, the base pair strength is low due to at least one fluctuating base pair, e.g., G:U, between the 5' end of the first or antisense strand and the 3' end of the second or sense strand. In another embodiment, the base pair strength is low due to at least one base pair containing a rare nucleotide, e.g., inosine(I). In certain exemplary embodiments, the base pairs are selected from the group consisting of I:A, I:U, and I:C. In yet another embodiment, the base pair strength is lower due to at least one base pair containing a modified nucleotide. In certain exemplary embodiments, the modified nucleotides are selected from the group consisting of 2-amino-G, 2-amino-A, 2,6-diamino-G, and 2,6-diamino-A.

[0128] The design of an siRNA suitable for targeting a specific target sequence is described in detail below. siRNAs can be designed for any other target sequence found in the target gene, following the exemplary teachings above. Furthermore, this technique is applicable to targeting any other target sequence, such as non-disease-causing target sequences.

[0129] To verify the effectiveness of siRNA in disrupting mRNA (e.g., mRNA expressed from a target gene), siRNA can be incubated with cDNA (e.g., cDNA corresponding to a target gene) in an in vitro mRNA expression system based on Drosophila melanogaster. 32 Newly synthesized mRNA (e.g., target mRNA) radiolabeled with 3P is detected by autoradiography on an agarose gel. The presence of cleaved mRNA indicates mRNA nuclease activity. Suitable controls include the omission of siRNA. Alternatively, a control siRNA is selected that has the same nucleotide composition as the selected siRNA but lacks significant sequence complementarity to the appropriate target gene. Such negative controls can be designed by randomly scrambling the nucleotide sequence of the selected siRNA. A homology search can be performed to confirm that the negative control lacks homology to any other gene in the appropriate genome. Furthermore, negative control siRNA can be designed by introducing one or more base mismatches into the sequence. An siRNA-mRNA complementation site that yields optimal mRNA specificity and maximum mRNA cleavage is selected.

[0130] IV. RNAi agents This disclosure includes, for example, siRNA molecules designed as described above. The siRNA molecules of this disclosure can be chemically synthesized, or transcribed in vitro from a DNA template, or in vivo from, for example, shRNA, or by cleaving a transcribed dsRNA template in vitro using recombinant human DICER enzyme to form a pool of 20, 21, or 23 bp double-stranded RNA-mediated RNAi. The siRNA molecules can be designed using any method known in the art.

[0131] In one embodiment, instead of the RNAi agent being an interfering ribonucleic acid, such as the siRNA or shRNA described above, the RNAi agent may encode an interfering ribonucleic acid, such as the shRNA described above. In other words, the RNAi agent may serve as a transcription template for an interfering ribonucleic acid. Therefore, the RNAi agents of this disclosure may also include small hairpin RNA (shRNA) and expression constructs engineered to express shRNA. The transcription of shRNA is thought to be initiated by the polymerase III (pol III) promoter and terminated at position 2 of the 4-5-thymine transcription termination site. Upon expression, shRNAs are thought to fold into a stem-loop structure with a 3' UU overhang. Subsequently, the ends of these shRNAs are processed, converting them into siRNA-like molecules of approximately 21-23 nucleotides (Brummelkamp et al., 2002; Lee et al., 2002, above; Miyagishi et al., 2002; Paddison et al., 2002, above; Paul et al., 2002, above; Sui et al., 2002, above; Yu et al., 2002, above. Further information on shRNA design and use can be found on the internet at the following addresses: katandin.cshl.org:9331 / RNAi / docs / BseRI-BamHI_Strategy.pdf and katandin.cshl.org:9331 / RNAi / docs / Web_version_of_PCR_strategy1.pdf).

[0132] Expression constructs in this disclosure include, but are not limited to, any constructs suitable for use in a suitable expression system, as are known in the art, retroviral vectors, linear expression cassettes, plasmids, and viruses or virus-derived vectors. Such expression constructs may include one or more inducible promoters, RNA Pol III promoter systems, e.g., the U6 snRNA promoter or the H1 RNA polymerase III promoter, or other promoters known in the art. A construct may contain one or both strands of siRNA. An expression construct expressing both strands may also contain a loop structure linking both strands, or each strand may be transcribed separately from separate promoters within the same construct. Each strand may be transcribed from a separate expression construct (Tuschl, T., 2002, above).

[0133] Synthetic siRNA can be delivered to cells by methods known in the art, such as cationic liposome transfection and electroporation. One or more siRNAs can be expressed intracellularly from recombinant DNA constructs to obtain long-term suppression of target genes and to facilitate delivery under specific circumstances. Such methods for expressing double-stranded siRNA from recombinant DNA constructs intracellularly to enable longer-term target gene suppression are known in the art, such as mammalian Pol III promoter systems capable of expressing functional double-stranded siRNA (e.g., H1 or U6 / snRNA promoter systems (Tuschl, T., 2002, above) (Bagella et al., 1998; Lee et al., 2002, above; Miyagishi et al., 2002, above; Paul et al., 2002, above; Yu et al., 2002, above; Sui et al., 2002, above)). Transcription termination by T7 occurs at a sequence of four consecutive T residues within the DNA template, providing a mechanism to terminate the siRNA transcript at a specific sequence. siRNA is complementary to the target gene sequence in the 5'-3' and 3'-5' directions, and the two strands of siRNA can be expressed in the same construct or separate constructs. Hairpin siRNAs, driven by H1 or U6 snRNA promoters and expressed intracellularly, can inhibit the expression of target genes (Bagella et al., 1998; Lee et al., 2002, above; Miyagishi et al., 2002, above; Paul et al., 2002, above; Yu et al., 2002), above; Sui et al., 2002, above). Constructs containing siRNA sequences under the control of the T7 promoter also produce functional siRNA when co-transfected into cells with a vector expressing T7 RNA polymerase (Jacque et al., 2002, above). A single construct may contain multiple sequences encoding siRNA, such as multiple regions of a target gene targeting the same gene or multiple genes, and may be driven, for example, by another PolIII promoter site.

[0134] Animal cells express a series of non-coding RNAs called microRNAs (miRNAs), each approximately 22 nucleotides long. These can regulate gene expression at the post-transcriptional or translational level during animal development. One common characteristic of miRNAs is that they are entirely cleaved from a precursor RNA stem-loop of approximately 70 nucleotides, likely by the RNase III enzyme Dicer or its homolog. By substituting the stem sequence of the miRNA precursor with a sequence complementary to the target mRNA, vector constructs expressing the engineered precursor can be used to produce siRNA and initiate RNAi against specific mRNA targets in mammalian cells (Zeng et al., above, 2002). When expressed by a DNA vector containing a polymerase III promoter, microRNA-designed hairpins can perform gene expression silencing (McManus et al., 2002, above). MicroRNAs targeting polymorphisms may also be useful for blocking the translation of mutant proteins in the absence of siRNA-mediated gene silencing. Such applications may be useful, for example, in situations where a designed siRNA causes off-target silencing of wild-type proteins.

[0135] Viral delivery mechanisms can also be used to induce specific silencing of targeted genes by generating recombinant adenoviruses containing siRNA, for example, under the transcriptional control of the RNA Pol II promoter, via siRNA expression (Xia et al., 2002, see above). Infection of HeLa cells with these recombinant adenoviruses can reduce the expression of endogenous target genes. Injecting recombinant adenovirus vectors into transgenic mice expressing siRNA target genes results in in vivo reduction of target gene expression. Ibid. In animal models, synthetic siRNA can be efficiently delivered to transplanted mouse embryos by whole-embryonic electroporation (Calegari et al., 2002). In adult mice, efficient delivery of siRNA can be achieved by “high pressure” delivery techniques, rapidly injecting a large volume of siRNA-containing solution into the animal’s tail vein (within 5 seconds) (Liu et al., 1999, above; McCaffrey et al., 2002, above; Lewis et al., 2002). siRNA can also be delivered to animals using nanoparticles and liposomes. In certain exemplary embodiments, recombinant adeno-associated viruses (rAAVs) and their associated vectors can be used to deliver one or more siRNAs to cells, such as nerve cells (e.g., brain cells) (U.S. Patent Applications Nos. 2014 / 0296486, 2010 / 0186103, 2008 / 0269149, 2006 / 0078542, and 2005 / 0220766).

[0136] The nucleic acid compositions of this disclosure include both unmodified siRNA and modified siRNA known in the art, such as cross-linked siRNA derivatives or derivatives having non-nucleotide moieties linked to their 3' or 5' ends. By modifying siRNA derivatives in this way, it is possible to improve cellular uptake or enhance the cell targeting activity of the resulting siRNA derivatives compared to the corresponding siRNA, and this is useful for tracking siRNA derivatives within cells or improving the stability of siRNA derivatives compared to the corresponding siRNA.

[0137] As described herein, the introduction of engineered RNA precursors into cells or whole organisms results in the production of the desired siRNA molecules. Such siRNA molecules then associate with the endogenous protein components of the RNAi pathway, bind to specific mRNA sequences, and target them for cleavage and destruction. In this way, the mRNA targeted by the siRNA generated from the engineered RNA precursor is depleted from the cell or organism, thereby resulting in a decrease in the concentration of the protein encoded by that mRNA in the cell or organism. The RNA precursor is typically a nucleic acid molecule that either individually encodes one strand of the dsRNA or encodes the entire nucleotide sequence of the RNA hairpin loop structure.

[0138] The nucleic acid compositions of the present disclosure may not be conjugated or may be conjugated to another moiety such as a nanoparticle, thereby improving the properties of the composition, such as pharmacokinetic parameters such as absorption, efficacy, bioavailability and / or half-life. Conjugation can be achieved, for example, using methods known in the art, the following methods; Lambert et al., Drug Deliv. Rev.: 47(1), 99-112(2001) (describing nucleic acids attached to polyalkylcyanoacrylate (PACA) nanoparticles); Fattal et al., J. Control Release 53(1-3):137-43(1998) (describing nucleic acids bound to nanoparticles); Schwab et al., Ann. Oncol. 5 Suppl. 4:55-8(1994) (describing nucleic acids linked to intercalating agents, hydrophobic groups, polycations or PACA nanoparticles); and Godard et al., Eur. J. Biochem. 232(2):404-10(1995) (describing nucleic acids linked to nanoparticles).

[0139] The nucleic acid molecules of the present disclosure can also be labeled using any method known in the art. For example, the nucleic acid composition can be labeled with a fluorophore, such as Cy3, fluorescein, or rhodamine. Labeling can be performed using a kit, such as the SILENCER™ siRNA Labeling Kit (Ambion). Further, siRNA can be radiolabeled using, for example, 3 H, 32 P or another suitable isotope.

[0140] Furthermore, since RNAi is thought to proceed through at least one single-stranded RNA intermediate, those skilled in the art will understand that ss-siRNA (e.g., the antisense strand of ds-siRNA) can also be designed, generated (e.g., enzymatically generated) or expressed (e.g., from a vector or plasmid) and utilized according to the claimed methodology as described herein (e.g., for chemical synthesis). In addition, in invertebrates, RNAi can be effectively induced by long dsRNA (e.g., dsRNA about 100 - 1000 nucleotides in length, preferably about 200 - 500, such as about 250, 300, 350, 400 or 450 nucleotides in length, etc.) that acts as an effector of RNAi (Brondani et al., Proc Natl Acad Sci USA. 2001 Dec. 4;98(25):14428 - 33. Epub 2001 Nov. 27.)

[0141] V. RNA silencing agents In one embodiment, the present disclosure provides novel RNA silencing agents (e.g., siRNA and shRNA), methods of making RNA silencing agents, and methods (e.g., research and / or therapeutic methods) for using an improved RNA silencing agent (or a portion thereof) for RNA silencing of a target gene. The RNA silencing agent includes an antisense strand (or a portion thereof), and the antisense strand has sufficient complementarity to a heterozygous single nucleotide polymorphism to mediate an RNA-mediated silencing mechanism (e.g., RNAi).

[0142] In certain embodiments, siRNA compounds are provided having one or any combination of the following properties: (1) completely chemically stabilized (i.e., no unmodified 2'-OH residues); (2) asymmetric; (3) 11-16 base pair double helix; and (4) single-stranded, partially or completely phosphorothiolated 5-8 base tail. The number of phosphorothioate modifications varies from 6 to 17 in total in different embodiments. In certain embodiments, the siRNA comprises 8 total phosphorothioate modifications and at least one exNA nucleotide linkage. In certain embodiments, the siRNA comprises an antisense strand having 4 total phosphorothioate modifications and at least one exNA nucleotide linkage.

[0143] In certain embodiments, the siRNA compounds described herein can be conjugated to a variety of targeting agents, including but not limited to cholesterol, DHA, phenyltropane, cortisol, vitamin A, vitamin D, GalNac, and gangliozides. The cholesterol-modified form showed a 5- to 10-fold improvement in efficacy in vitro across a wide range of cell types (e.g., HeLa, neurons, hepatocytes, trophoblasts) compared to previously used chemical stabilization patterns (e.g., all purines modified instead of pyrimidines).

[0144] Certain compounds of this disclosure having the structural properties described above and herein may be referred to as “hsiRNA-ASP” (hydrophobically modified small interfering RNA characterized by a highly stabilized pattern). Furthermore, this hsiRNA-ASP pattern exhibits dramatically improved distribution via delivery from the brain and spinal cord to the liver, placenta, kidneys, spleen, and several other tissues, making it available for therapeutic intervention.

[0145] In the liver, hsiRNA-ASP is specifically delivered to endothelial cells and Kupffer cells, but not to hepatocytes; therefore, this chemical modification pattern is complementary to GalNac conjugates rather than competing technologies.

[0146] The compounds of this disclosure can be described in the following aspects and embodiments.

[0147] In a first embodiment, an oligonucleotide of at least 16 consecutive nucleotides is provided herein, the oligonucleotide having a 5' end, a 3' end and being complementary to a target, wherein: (1) the oligonucleotide comprises alternating 2'-methoxy-ribonucleotides and 2'-fluoro-ribonucleotides; (2) the nucleotides at positions 2 and 14 from the 5' end are not 2'-methoxy-ribonucleotides; (3) the nucleotides are linked via modified ligatures as shown in Figure 1.

[0148] a) Design of Ava molecules The siRNA molecules of this disclosure are double-stranded, consisting of a sense strand and a complementary antisense strand. In one embodiment, the siRNA molecule has a nucleotide length of about 10 to 50 or more, i.e., each strand contains 10 to 50 nucleotides (or nucleotide analogs). In another embodiment, the siRNA molecule has a nucleotide length of about 15 to 30 in each strand, for example, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30, where one of the strands is sufficiently complementary to the target region. In one embodiment, the strands are arranged such that at least 1, 2, or 3 bases are present at the ends of the strands that do not align (i.e., no complementary bases are produced in the opposing strands). This results in an overhang of 1, 2, or 3 residues at one or both ends of the double helix when the strands are annealed. In another embodiment, the siRNA molecule has a nucleotide length of about 10 to 50 or more, i.e., each chain contains 10 to 50 nucleotides (or nucleotide analogs). In yet another embodiment, the siRNA molecule has about 15 to 30, for example, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides per chain, with one chain substantially complementary to the target sequence and the other chain identical or substantially identical to the first chain.

[0149] Typically, siRNA can be designed using any method known in the art, for example, using the following protocol:

[0150] 2. The sense strand of the siRNA is designed based on the sequence of a selected target site. In one embodiment, the sense strand contains approximately 19–25 nucleotides, e.g., 19, 20, 21, 22, 23, 24, or 25 nucleotides. In another embodiment, the sense strand contains 21, 22, or 23 nucleotides. However, those skilled in the art will understand that siRNAs having a length of less than 19 nucleotides or a length of more than 25 nucleotides can also function to mediate RNAi. Thus, siRNAs of such lengths are also within the scope of this disclosure, insofar as they retain the ability to mediate RNAi. It has been demonstrated that longer RNA silencing agents induce interferon or protein kinase R (PKR) responses in certain mammalian cells, which may be undesirable. In one embodiment, the RNA silencing agents of this disclosure do not induce a PKR response (i.e., are sufficiently short in length). However, longer RNA silencing agents may be useful, for example, in cell types that cannot produce a PKR response, or in situations where the PKR response is downregulated or suppressed by alternative means.

[0151] The siRNA molecules described herein have sufficient complementarity with the target sequence so that the siRNA can mediate RNAi. Generally, siRNA containing a nucleotide sequence is sufficiently identical to the target sequence portion of the target gene in order to result in RISC-mediated cleavage of the target gene. Therefore, in one embodiment, the sense strand of the siRNA is designed to have a sequence sufficiently identical to a portion of the target. For example, the sense strand may have 100% identity with respect to the target site. However, 100% identity is not required. Identity greater than 80% between the sense strand and the target RNA sequence, e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even 100% identity, is used in one embodiment. The present disclosure has the advantage of allowing for specific sequence modifications to enhance the efficiency and specificity of RNAi. In one embodiment, the sense strand has 4, 3, 2, 1, or 0 mismatched nucleotides in a target region, such as a target region, where at least one base pair differs between the wild-type allele and the mutant allele. For example, the target region may contain a gain-of-function mutation, and the other strand may be identical or substantially identical to the first strand. Furthermore, siRNA sequences with small insertions or deletions of 1 or 2 nucleotides may also be effective in mediating RNAi. Alternatively, siRNA sequences with substitutions or insertions of nucleotide analogs may be effective in inhibition.

[0152] Sequence identity can be determined by sequence comparison and alignment algorithms known in the art. To determine the percentage of identity between two nucleic acid sequences (or two amino acid sequences), the sequences are aligned for optimal comparison (for example, gaps can be introduced into the first or second sequence for optimal alignment). Then, the nucleotides (or amino acid residues) at the corresponding nucleotide (or amino acid) positions are compared. If a position in the first sequence is occupied by the same residue as the corresponding position in the second sequence, the molecules are identical at that position. The percentage of identity between the two sequences corresponds to the number of identical positions shared by the sequences (i.e., % homology = number of identical positions / total number of positions x 100), and optionally, a penalty is given to the score for the number and / or length of introduced gaps.

[0153] The comparison of sequences and the determination of the percentage of identity between two sequences can be achieved using mathematical algorithms. In one embodiment, alignment is generated in specific parts of aligned sequences that have sufficient identity, but not in parts with a low degree of identity (i.e., local alignment). One non-restrictive example of a local alignment algorithm used for sequence comparison is the algorithm of Karlin and Altschul (1990) Proc.Natl.Acad.Sci.USA 87:2264-68, revised Karlin and Altschul (1993) Proc.Natl.Acad.Sci.USA 90:5873-77. Such an algorithm is incorporated into the BLAST program (version 2.0), Altschul, et al. (1990) J.Mol.Biol.215:403-10.

[0154] In another embodiment, the alignment is optimized by introducing appropriate gaps, and percentage identity is determined over the length of the aligned sequence (i.e., the gapped alignment). To obtain a gap alignment for comparison purposes, Gapped BLAST can be used as described in Altschul et al. (1997) Nucleic Acids Res. 25(17):3389-3402. In another embodiment, the alignment is optimized by introducing appropriate gaps, and percentage identity is determined over the entire length of the aligned sequence (i.e., global alignment). One non-restrictive example of a mathematical algorithm used for global sequence comparison is the Myers and Miller algorithm, CABIOS (1989). Such algorithms are incorporated into the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package. When using the ALIGN program to compare amino acid sequences, the PAM120 weight residue table, 12 gap length penalties, and 4 gap penalties can be used.

[0155] 3. The antisense or guide strand of an siRNA is routinely the same length as the sense strand and contains complementary nucleotides. In one embodiment, the guide and sense strands are perfectly complementary; that is, the strands are blunt-ended when aligned or annealed. In another embodiment, the siRNA strands may be paired to have 1-7 (e.g., 2, 3, 4, 5, 6, or 7) or 1-4 3' overhangs, e.g., 2, 3, or 4 nucleotides. The overhangs may contain (or consist of) nucleotides corresponding to the target gene sequence (or its complement). Alternatively, the overhangs may contain (or consist of) deoxyribonucleotides, e.g., dT, or nucleotide analogs, or other suitable non-nucleotide substances. Thus, in another embodiment, the nucleic acid molecule may have a 2-nucleotide 3' overhang, such as TT. The overhang nucleotides may be either RNA or DNA. As described above, it is desirable to select a target region where the mutant:wild-type mismatch is a purine:purine mismatch.

[0156] 4. Potential targets can be compared to appropriate genome databases (human, mouse, rat, etc.) using any method known in the art, potentially eliminating the need for any target sequence with significant homology to other coding sequences. One such sequence homology search method is known as BLAST and is available on the National Center for Biotechnology Information website.

[0157] 5. Select one or more sequences that meet the evaluation criteria.

[0158] General information regarding the design and use of siRNA can be found in "The siRNA User Guide," available on the website of The Max-Plank-Institut fur Biophysikalische Chemie.

[0159] Alternatively, siRNA can be functionally defined as a nucleotide sequence (or oligonucleotide sequence) that can hybridize to a target sequence (e.g., hybridize at 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, and 50 °C or 70 °C for 12 - 16 hours, and then wash). Additional hybridization conditions include hybridization at 70 °C in 1x SSC or 50 °C in 1x SSC, hybridization in 50% formamide, followed by washing at 70 °C in 0.3x SSC, or hybridization at 70 °C in 4x SSC or 50 °C in 4x SSC, hybridization in 50% formamide, followed by washing at 67 °C in 1x SSC. The hybridization temperature of a hybrid predicted to be less than 50 base pairs in length shall be 5 - 10 °C lower than the melting temperature (T m ). Here, T m is determined according to the following formula. For hybrids less than 18 base pairs in length, T m (°C) = 2(# of A + T bases) + 4(# of G + C bases). For hybrids 18 - 49 base pairs in length, T m (°C) = 81.5 + 16.6(log10[Na + ) + 0.41(%G + C) - (600 / N) (where N is the number of bases in the hybrid and [Na + is the concentration of sodium ions in the hybridization buffer (the [Na +=0.165 M). Additional examples of stringency conditions for polynucleotide hybridization are described in Sambrook, J., E. F. Fritsch, and T. Maniatis, 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., chapters 9 and 11, and Current Protocols in Molecular Biology, 1995, F. M. Ausubel et al., eds., John Wiley & Sons, Inc., sections 2.10 and 6.3-6.4, which are incorporated herein by reference.

[0160] Negative control siRNAs shall have the same nucleotide composition as the selected siRNA, but no significant sequence complementarity with the appropriate genome. Such negative controls can be designed by randomly scrambling the nucleotide sequence of the selected siRNA. A homology search can be performed to confirm that the negative control lacks homology with any other genes within the appropriate genome. Additionally, negative control siRNAs can be designed by introducing one or more base mismatches into the sequence.

[0161] 6. To verify the effectiveness of siRNA by destroying the target mRNA (e.g., wild-type or mutant mRNA), the siRNA can be incubated with the target cDNA in a Drosophila-based in vitro mRNA expression system. 32The newly synthesized target mRNA, radiolabeled with 3P, is detected by autoradiography on an agarose gel. The presence of cleaved target mRNA indicates mRNA nuclease activity. Suitable controls include the omission of siRNA and the use of non-target cDNA. Alternatively, a control siRNA is selected that has the same nucleotide composition as the selected siRNA but does not have significant sequence complementarity to the appropriate target gene. Such negative controls can be designed by randomly scrambling the nucleotide sequence of the selected siRNA. Homology searches can be performed to confirm that the negative control lacks homology to any other gene in the appropriate genome. Furthermore, negative control siRNA can be designed by introducing one or more base mismatches into the sequence.

[0162] siRNA can be designed to target any of the target sequences described above. siRNA comprises an antisense strand that is sufficiently complementary to the target sequence to mediate the silencing of the target sequence. In certain embodiments, the RNA silencing agent is siRNA.

[0163] In certain embodiments, the siRNA comprises a sense strand including the linkage shown in Figure 1, or an antisense strand including the linkage shown in Figure 1.

[0164] The siRNA-mRNA complementation site that provides optimal mRNA specificity and maximum mRNA cleavage is selected.

[0165] b) siRNA-like molecule The siRNA-like molecules of this disclosure have a sequence that is "sufficiently complementary" to the target sequence of mRNA in order to direct gene silencing by RNAi or translational repression (i.e., they have a sequence-containing strand). The siRNA-like molecules are designed in the same manner as siRNA molecules, but the degree of sequence identity between the sense strand and the target RNA approximates that is observed between miRNA and its target. Generally, when the degree of sequence identity between the miRNA sequence and the corresponding target gene sequence decreases, the tendency for post-transcriptional gene silencing to be mediated by translational repression rather than RNAi increases. Therefore, in another embodiment where post-transcriptional gene silencing by translational repression of the target gene is desired, the miRNA sequence has partial complementarity with the target gene sequence. In certain embodiments, the miRNA sequence has partial complementarity with one or more short sequences (complementary sites) dispersed within the target mRNA (e.g., within the 3'UTR of the target mRNA) (Hutvagner and Zamore, Science, 2002; Zeng et al., Mol. Cell, 2002; Zeng et al., RNA, 2003; Doench et al., Genes & Dev., 2003). Because the translational repression mechanism is cooperative, in certain embodiments, multiple complementary sites (e.g., 2, 3, 4, 5, or 6) may be targeted.

[0166] The ability of an siRNA-like double-stranded molecule to mediate RNAi or translational repression can be predicted by the distribution of non-identical nucleotides between the target gene sequence and the nucleotide sequence of the silencing agent in the complementary region. In one embodiment where gene silencing by translational repression is desired, at least one non-identical nucleotide is present in the central part of the complementary region such that the double helix formed by the miRNA guide strand and the target mRNA contains a central "bulge" (Doench JG et al., Genes & Dev., 2003). In another embodiment, 2, 3, 4, 5, or 6 consecutive or non-identical nucleotides are introduced. The non-identical nucleotides may be selected to form fluctuating base pairs (e.g., G:U) or mismatch base pairs (G:A, C:A, C:U, G:G, A:A, C:C, U:U). In a further embodiment, the "bulge" is centered on the nucleotides at positions 12 and 13 from the 5' end of the miRNA molecule.

[0167] c) Short hairpin RNA (shRNA) molecule In certain characteristic embodiments, the present disclosure provides shRNAs capable of mediating RNA silencing of target sequences with high selectivity. In contrast to siRNAs, shRNAs mimic the natural precursors of microRNAs (miRNAs) and enter the gene silencing pathway at the top. For this reason, shRNAs are thought to mediate gene silencing more efficiently by being supplied throughout the entire natural gene silencing pathway.

[0168] miRNAs are non-coding RNAs of approximately 22 nucleotides that can regulate gene expression at the post-transcriptional or translational level during plant and animal development. One common characteristic of miRNAs is that they are cleaved from a precursor RNA stem-loop of approximately 70 nucleotides, called pre-miRNA, likely by the RNase type III enzyme Dicer or its homolog. Naturally occurring miRNA precursors (pre-miRNAs) generally have a single strand that forms a double-stranded stem containing two complementary parts, and a loop connecting the two parts of the stem. In a typical pre-miRNA, the stem contains one or more bulges, e.g., extra nucleotides that create a single nucleotide “loop” within a part of the stem, and / or one or more unpaired nucleotides that create a gap in the hybridization of the two parts of the stem. The short hairpin RNAs or engineered RNA precursors of this disclosure are artificial constructs based on these naturally occurring pre-miRNAs but engineered to deliver a desired RNA silencing agent (e.g., siRNA of this disclosure). shRNA is formed by substituting the pre-miRNA stem sequence with a sequence complementary to the target mRNA. shRNA is processed throughout the cell's gene silencing pathway, thereby efficiently mediating RNAi.

[0169] The necessary elements of an shRNA molecule include a first and a second portion that have sufficient complementarity to anneal or hybridize to form a double-stranded or double-stranded stem portion. The two portions do not need to be completely or perfectly complementary. The first and second “stem” portions are joined by a portion having sequences that are not sufficiently sequence-complementary to anneal or hybridize to the other portion of the shRNA. This latter portion is called the “loop” portion within the shRNA molecule. The shRNA molecule is processed to produce siRNA. The shRNA may also contain one or more bulges, i.e., extra nucleotides that create small nucleotide “loops,” e.g., 1, 2, or 3 nucleotide loops, within the stem portion. The stem portions may be of the same length, or some may contain overhangs of, for example, 1 to 5 nucleotides. Overhang nucleotides may include, for example, uracil (U), e.g., all U. Such U is specifically encoded by thymidine (T) in the shRNA coding DNA, which signals the termination of transcription.

[0170] In the shRNA (or engineered precursor RNA) of this disclosure, a portion of the double-stranded stem is a nucleic acid sequence complementary (or antisense) to the target sequence. Preferably, one strand of the stem portion of the shRNA is sufficiently complementary (e.g., antisense) to the target RNA (e.g., mRNA) sequence and mediates the degradation or cleavage of the target RNA via RNA interference (RNAi). Thus, the engineered RNA precursor comprises a double stem having two portions and a loop connecting the two stem portions. The antisense portion may be at the 5' or 3' end of the stem. The stem portion of the shRNA is preferably about 15 to about 50 nucleotides long. Preferably, the two stem portions are about 18 or 19 to about 21, 22, 23, 24, 25, 30, 35, 37, 38, 39, or 40 or more nucleotides long. In preferred embodiments, the length of the stem portion is 21 nucleotides or more. When used in mammalian cells, the stem length should be less than approximately 30 nucleotides to avoid inducing nonspecific responses such as the interferon pathway. In non-mammalian cells, the stem may exceed 30 nucleotides. In fact, the stem may contain much larger sections complementary to the target mRNA (up to the entire mRNA, and including the entire mRNA).

[0171] The two parts of a double stem must be sufficiently complementary to hybridize and form a double stem. Therefore, the two parts may, but do not, be completely or perfectly complementary. Furthermore, the two stem parts may be the same length, or one part may contain an overhang of 1, 2, 3, or 4 nucleotides. The overhanging nucleotide may contain, for example, uracil (U), or all U. Loops in shRNA or engineered RNA precursors may differ from the native pre-miRNA sequence by modifying the loop sequence to increase or decrease the number of paired nucleotides, or by replacing all or part of the loop sequence with a tetraloop or other loop sequence. Therefore, loops in shRNA or engineered RNA precursors may have 2, 3, 4, 5, 6, 7, 8, 9, or more nucleotide lengths, for example, 15 or 20 or more.

[0172] Loops within shRNA or engineered RNA precursors can differ from native pre-miRNA sequences by modifying the loop sequence to increase or decrease the number of paired nucleotides, or by replacing all or part of the loop sequence with tetraloops or other loop sequences. Therefore, loop portions within shRNA can be approximately 2 to approximately 20 nucleotides long, i.e., approximately 2, 3, 4, 5, 6, 7, 8, 9, or more, for example, 15 or 20 nucleotides, or more. Preferred loops consist of or contain "tetraloop" sequences. Exemplary tetraloop sequences, but not limited to these, include the sequences GNRA (where N is any nucleotide and R is a purine nucleotide), GGGG, and UUUU.

[0173] In certain embodiments, the shRNA of this disclosure comprises the sequence of the desired siRNA molecule described above. In other embodiments, the sequence of the antisense portion of the shRNA can be designed essentially as described above, or more generally, by selecting a sequence of 18, 19, 20, 21 nucleotides, or longer, from within the target RNA, for example, from a 100-200 or 300 nucleotide region upstream or downstream of the translation start site. Generally, the sequence can be selected from any portion of the target RNA (e.g., mRNA), such as the 5'UTR (untranslated region), coding sequence, or 3'UTR. This sequence can optionally follow immediately after a region of the target gene containing two adjacent AA nucleotides. The last two nucleotides of the nucleotide sequence can be selected to be UU. Using this approximately 21 nucleotide sequence, a portion of the double-stranded stem of the shRNA is constructed. This sequence can, for example, enzymatically replace the stem portion of the wild-type pre-miRNA sequence, or it can be included in the complete sequence that is synthesized. For example, DNA oligonucleotides can be synthesized that encode the entire stem-loop manipulated RNA precursor, or only the portion that is inserted into the double-stranded stem of the precursor, and a manipulated RNA precursor construct can be constructed from, for example, wild-type pre-miRNA using restriction enzymes.

[0174] The engineered RNA precursor contains approximately 21-22 nucleotide sequences of siRNA or siRNA-like double helix that are desired to be produced in vivo within the double-stranded stem. Therefore, the stem portion of the engineered RNA precursor contains at least 18 or 19 nucleotide pairs corresponding to the sequence of the exon portion of the gene whose expression is reduced or inhibited. Two 3' nucleotides adjacent to this region of the stem are selected to maximize siRNA production from the engineered RNA precursor and to maximize the effectiveness of the resulting siRNA when targeting the corresponding mRNA for RNAi-mediated translational repression or disruption in vivo and in vitro.

[0175] In certain embodiments, the shRNAs of this disclosure include miRNA sequences, optionally more preferably terminally modified miRNA sequences, to enhance entry into RISC. The miRNA sequences may be similar to or identical to any naturally occurring miRNA sequence (e.g., The miRNA Registry; Griffiths-Jones S, Nuc. Acids Res., 2004). To date, more than 1,000 naturally occurring miRNAs have been identified, and together they are thought to represent about 1% of all predicted genes in the genome. Many naturally occurring miRNAs are clustered together in the introns of pre-mRNA and can be identified in silico using homology-based searches (Pasquinelli et al., 2000; Lagos-Quintana et al., 2001; Lau et al., 2001; Lee and Ambros, 2001) or computer algorithms that predict the ability of candidate miRNA genes to form stem-loop structures with pre-mRNA (e.g., MiRScan, MiRSeeker) (Grad et al., Mol. Cell., 2003; Lim et al., Genes Dev., 2003; Lim et al., Science, 2003; Lai EC et al., Genome Bio., 2003). Online registries provide searchable databases of all publicly available miRNA sequences (The miRNA Registry, Sanger Institute website; Griffiths-Jones S, Nuc. Acids Res., 2004).Exemplary natural miRNAs include lin-4, let-7, miR-10, mirR-15, miR-16, miR-168, miR-175, miR-196 and their homologs, as well as other natural miRNAs of human origin and those of specific model organisms, such as Drosophila melanogaster, Caenorhabditis elegans, zebrafish, Arabidopsis thalania, Mus musculus, and Rattus norvegicus (described in PCT International Publication No. WO03 / 029459).

[0176] Naturally occurring miRNAs are expressed in vivo by endogenous genes and processed from hairpin or stem-loop precursors (pre-miRNA or pri-miRNA) by Dicer or other RNAses (Lagos-Quintana et al., Science, 2001; Lau et al., Science, 2001; Lee and Ambros, Science, 2001; Lagos-Quintana et al., Curr. Biol., 2002; Mourelatos et al., Genes Dev., 2002; Reinhart et al., Science, 2002; Ambros et al., Curr. Biol., 2003; Brennecke et al., 2003; Lagos-Quintana et al., RNA, 2003; Lim et al., Genes Dev., 2003; Lim et al., Science, 2003). miRNAs can exist transiently in vivo as double-stranded double helixes, but only one strand is incorporated into the RISC complex to direct gene silencing. Certain miRNAs, such as plant miRNAs, have complete or near-complete complementarity with their target mRNAs and therefore directly cleave the target mRNA. Other miRNAs have incomplete complementarity with their target mRNAs and therefore directly repress the translation of the target mRNA. The degree of complementarity between the miRNA and its target mRNA is thought to determine its mechanism of action. For example, complete or near-complete complementarity between the miRNA and its target mRNA predicts a cleavage mechanism (Yekta et al., Science, 2004), while incomplete complementarity predicts a translation repression mechanism. In certain embodiments, the miRNA sequence is a naturally occurring miRNA sequence whose abnormal expression or activity correlates with miRNA dysfunction.

[0177] d) Dual-function oligonucleotide tethering factors In other embodiments, the RNA silencing agents of this disclosure include dual-function oligonucleotide tethering factors useful for intercellular recruitment of miRNAs. Animal cells express a set of miRNAs, which are non-coding RNAs of about 22 nucleotides that can regulate gene expression at the post-transcriptional or translational level. Dual-function oligonucleotide tethering factors can suppress the expression of genes involved in processes such as atherosclerosis by binding miRNAs bound to RISC and recruiting them to target mRNAs. The use of oligonucleotide tethering factors offers several advantages over existing techniques for suppressing the expression of specific genes. First, in the methods described herein, endogenous molecules (often abundant), miRNAs, become capable of mediating RNA silencing. Thus, the methods described herein eliminate the need to introduce exogenous molecules (e.g., siRNA) to mediate RNA silencing. Second, the RNA silencing agents and, in particular, the ligation moieties (e.g., oligonucleotides, e.g., 2'-O-methyloligonucleotides) can be stabilized and made resistant to nuclease activity. As a result, the tethering factors of this disclosure can be designed for direct delivery, eliminating the need for indirect delivery (e.g., viruses) of precursor molecules or plasmids designed to produce the desired agent within the cell. Thirdly, the tethering factors and their respective portions can be designed to fit specific mRNA sites and specific miRNAs. The design may be cell and gene product specific. Fourthly, in the methods disclosed herein, the mRNA remains intact, allowing those skilled in the art to use the cell's own mechanisms to block protein synthesis with short pulses. Consequently, these RNA silencing methods are highly regulated.

[0178] The dual-function oligonucleotide tethering factors ("tethering factors") of this disclosure are designed to recruit miRNAs (e.g., endogenous cellular miRNAs) to target mRNAs to induce regulation of a gene of interest. In certain embodiments, the tethering factor has the formula TL-, where T is the mRNA targeting portion, L is the ligation portion, and is the miRNA recruitment portion. Any one or more portions may be double-stranded. However, preferably, each portion is single-stranded.

[0179] The portions within the tethering element can be positioned or ligated as shown in formula TL-μ (5' to 3' direction) (i.e., the 3' end of the targeting portion ligated to the 5' end of the ligation portion and the 3' end of the ligation portion ligated to the 5' end of the miRNA mobilization portion). Alternatively, these portions can be positioned or ligated within the tethering element as follows: μ-TL (i.e., the 3' end of the miRNA mobilization portion ligated to the 5' end of the ligation portion, and the 3' end of the ligation portion ligated to the 5' end of the targeting portion).

[0180] The mRNA targeting moiety described above can capture a specific target mRNA. According to this disclosure, since the expression of the target mRNA is undesirable, translational repression of the mRNA is desirable. The mRNA targeting moiety is sized to effectively bind to the target mRNA. The length of the targeting moiety varies considerably, partly depending on the length of the target mRNA and the degree of complementarity between the target mRNA and the targeting moiety. In various embodiments, the targeting moiety is less than about 200, 100, 50, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, or 5 nucleotides long. In certain embodiments, the targeting moiety is about 15 to about 25 nucleotides long.

[0181] As described above, the miRNA recruitment portion can associate with a miRNA. According to this disclosure, the miRNA can be any miRNA capable of repressing a target mRNA. Mammals have been reported to have more than 250 endogenous miRNAs (Lagos-Quintana et al. (2002) Current Biol. 12:735-739; Lagos-Quintana et al. (2001) Science 294:858-862; and Lim et al. (2003) Science 299:1540). In various embodiments, the miRNA can be any miRNA recognized in the art.

[0182] The ligation portion is any agent that can ligate the targeting portion so as to maintain the activity of the targeting portion. The ligation portion is preferably an oligonucleotide portion containing a sufficient number of nucleotides so that the targeting agent can adequately interact with each target. The ligation portion has little or no sequence homology to the cellular mRNA or miRNA sequence. Exemplary ligation portions include one or more 2'-O-methylnucleotides, such as 2'-β-methyladenosine, 2'-O-methylthymidine, 2'-O-methylguanosine, or 2'-O-methyluridine.

[0183] e) Gene silencing oligonucleotides In certain exemplary embodiments, gene expression (i.e., target gene expression) can be regulated using oligonucleotide-based compounds comprising two or more single-stranded antisense oligonucleotides linked via their 5' ends, allowing for the presence of two or more accessible 3' ends, in order to effectively inhibit or reduce target gene expression. Such linked oligonucleotides are also known as gene silencing oligonucleotides (GSOs) (see, for example, U.S. Patent No. 8,431,544, assigned to Idera Pharmaceuticals, Inc., which is incorporated herein by reference in its entirety for all purposes). Novel and improved GSOs and embodiments thereof, including inter-subunit linkages according to formula (I), are provided herein.

[0184] Linking at the 5' end of GSO is independent of other oligonucleotide linking and can occur directly via the 5', 3', or 2' hydroxyl group, or indirectly via a non-nucleotide linker or nucleoside, using either the 2' or 3' hydroxyl position of the nucleoside. Linking can also utilize a functionalized sugar or nucleic acid base of the 5' terminal nucleotide.

[0185] GSOs can contain two identical or different sequences conjugated at their 5'-5' ends via phosphodiesters, phosphorothioates, or non-nucleoside linkers. Such compounds may contain 15-27 nucleotides complementary to a specific portion of the target mRNA for downregulation of antisense of a gene product. GSOs containing identical sequences can bind to specific mRNAs via Watson-Crick hydrogen bond interactions and inhibit protein expression. GSOs containing different sequences can bind to two or more different regions of one or more mRNA targets and inhibit protein expression. Such compounds consist of heteronucleotide sequences complementary to the target mRNA, forming a stable double-strand structure via Watson-Crick hydrogen bonds. Under certain conditions, GSOs containing two free 3' ends (5'-5' attached antisense) can be more potent gene expression inhibitors than those with a single free 3' end or those without a free 3' end.

[0186] In some embodiments, the non-nucleotide linker is glycerol or formula HO-(CH2) o -CH(OH)-(CH2) p -OH is a glycerol homologue, where o and p are integers of 1 to about 6, 1 to about 4, or 1 to about 3, independently. In some other embodiments, the non-nucleotide linker is a derivative of 1,3-diamino-2-hydroxypropane. Some such derivatives are of the formula HO--(CH2) m --C(O)NH--CH2--CH(OH)--CH2--NHC(O)--(CH2) m --It has an OH group, where m is an integer between 0 and approximately 10, 0 and approximately 6, 2 and approximately 6, or 2 and approximately 4.

[0187] Some non-nucleotide linkers allow for the attachment of three or more GSO components. For example, the non-nucleotide linker glycerol has three hydroxyl groups to which GSO components can be covalently attached. Thus, some oligonucleotide-based compounds of this disclosure contain two or more oligonucleotides linked to a nucleotide or non-nucleotide linker. Such oligonucleotides according to this disclosure are referred to as "branched".

[0188] In certain embodiments, the GSO is at least 14 nucleotides long. In certain exemplary embodiments, the GSO is 15–40 nucleotides long or 20–30 nucleotides long. Thus, the component oligonucleotides of the GSO can independently be 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, or 40 nucleotides long.

[0189] These oligonucleotides can be prepared by methods recognized in the art, such as phosphoramidate or H-phosphonate chemistry, which can be carried out by manual or automated synthesizers. These oligonucleotides can also be modified in multiple ways without impairing their ability to hybridize to mRNA. Such modifications may include at least one internucleotide linkage of an oligonucleotide between the 5' end of one nucleotide and the 3' end of another nucleotide, which is an alkylphosphonate, phosphorothioate, phosphorodithioate, methylphosphonate, phosphate ester, alkylphosphonothioate, phosphoramidate, carbamate, carbonate, phosphate hydroxyl, acetamidate, or carboxymethyl ester, or a combination of these and other internucleotide linkages, where the phosphodiester linkage of the 5' nucleotide is substituted with any number of chemical groups.

[0190] VI. Modified RNA silencing agents In certain aspects of the present disclosure, the oligonucleotides, siRNAs, and RNA silencing agents (or any part thereof) of the present disclosure described above may be modified to further improve the activity of the agent. For example, the RNA silencing agent described in Section II above may be modified by any of the modifications described below. The modifications may act in part to further improve target recognition, to improve the stability of the agent (e.g., to prevent degradation), to promote cell uptake, to improve targeting efficiency, to improve the effectiveness of binding (e.g., to the target), to improve patient tolerance to the agent, and / or to reduce toxicity.

[0191] 1) Modifications to improve target identification In certain embodiments, the oligonucleotides, siRNAs, and RNA silencing agents of this disclosure may be substituted with destabilized nucleotides to improve single-nucleotide target recognition (see U.S. Patent Application No. 11 / 698,689, filed January 25, 2007, and U.S. Provisional Application No. 60 / 762,225, filed January 25, 2006, both of which are incorporated herein by reference). Such modifications may be sufficient to neutralize the specificity of the RNA silencing agent to non-target mRNA (e.g., wild-type mRNA) without any apparent effect on the specificity of the RNA silencing agent to target mRNA (e.g., gain-of-function mutant mRNA).

[0192] In preferred embodiments, the RNA silencing agent of this disclosure is modified by introducing at least one universal nucleotide into its antisense strand. The universal nucleotide comprises a base moiety that can indiscriminately base-pair with any of the four conventional nucleotide bases (e.g., A, G, C, U). Universal nucleotides are preferred because they have only a relatively small effect on the stability of the RNA double helix, or the stability of the double helix formed by the guide strand of the RNA silencing agent and the target mRNA. Exemplary universal nucleotides include those having an inosine base moiety or inosine-like base moiety selected from the group consisting of deoxyinosine (e.g., 2'-deoxyinosine), 7-deaza-2'-deoxyinosine, 2'-aza-2'-deoxyinosine, PNA-inosine, morpholino-inosine, LNA-inosine, phosphoramidate-inosine, 2'-O-methoxyethyl-inosine, and 2'-OMe-inosine. In a particularly preferred embodiment, the universal nucleotide is an inosine residue or a native analog thereof.

[0193] In certain embodiments, the RNA silencing agents of this disclosure are modified by introducing at least one destabilizing nucleotide within 5 nucleotides of a specificity-determining nucleotide (i.e., a nucleotide that recognizes a disease-associated polymorphism). For example, the destabilizing nucleotide may be introduced at a position within 5, 4, 3, 2, or 1 nucleotide from the specificity-determining nucleotide. In exemplary embodiments, the destabilizing nucleotide is introduced at a position 3 nucleotides away from the specificity-determining nucleotide (i.e., so that two stabilizing nucleotides are present between the destabilizing nucleotide and the specificity-determining nucleotide). In RNA silencing agents having two strands or strand portions (e.g., siRNA and shRNA), the destabilizing nucleotide may be introduced into a strand or strand portion that does not contain the specificity-determining nucleotide. In preferred embodiments, the destabilizing nucleotide is introduced into the same strand or strand portion that contains the specificity-determining nucleotide.

[0194] 2) Modifications to enhance efficacy and specificity In certain embodiments, the oligonucleotides, siRNAs, and RNA silencing agents of this disclosure can be modified in accordance with asymmetric design rules to easily enhance their efficacy and specificity in mediating RNAi (see U.S. Patents 8,309,704, 7,750,144, 8,304,530, 8,329,892, and 8,309,705). Such modifications facilitate the entry of the antisense strand of the siRNA (e.g., siRNA designed using the methods of this disclosure, or siRNA produced from shRNA) into RISC in a manner favorable to the sense strand. This allows the antisense strand to preferentially induce cleavage or translational repression of the target mRNA, thereby increasing or improving the efficiency of target cleavage and silencing. Preferably, the asymmetry of the RNA silencing agent is enhanced by reducing the base pair strength between the antisense strand 5' end (AS5') and the sense strand 3' end (S3') of the RNA silencing agent, relative to the binding strength or base pair strength between the antisense strand 3' end (AS3') and the sense strand 5' end (S'5) of the RNA silencing agent.

[0195] In one embodiment, the asymmetry of the RNA silencing agent of the Disclosure may be enhanced such that the number of G:C base pairs between the 5' end of the first or antisense strand and the 3' end of the sense strand portion is less than the number of G:C base pairs between the 3' end of the first or antisense strand and the 5' end of the sense strand portion. In another embodiment, the asymmetry of the RNA silencing agent of the Disclosure may be enhanced such that there is at least one mismatched base pair between the 5' end of the first or antisense strand and the 3' end of the sense strand portion. Preferably, the mismatched base pair is selected from the group consisting of: G:A, C:A, C:U, G:G, A:A, C:C, and U:U. In yet another embodiment, the asymmetry of the RNA silencing agent of the Disclosure may be enhanced such that there is at least one fluctuation base pair, for example, G:U, between the 5' end of the first or antisense strand and the 3' end of the sense strand portion. In another embodiment, the asymmetry of the RNA silencing agent of the Disclosure may be enhanced to include at least one base pair containing a rare nucleotide, such as inosine (I). Preferably, the base pair is selected from the group consisting of I:A, I:U, and I:C. In yet another embodiment, the asymmetry of the RNA silencing agent of the Disclosure may be enhanced to include at least one base pair containing a modified nucleotide. In a preferred embodiment, the modified nucleotide is selected from the group consisting of 2-amino-G, 2-amino-A, 2,6-diamino-G, and 2,6-diamino-A.

[0196] 3) RNA silencing agents with improved stability The RNA silencing agents of this disclosure may be modified to improve their stability in serum or growth media for cell culture. To improve stability, the 3'-residue may be stabilized against degradation, and in particular, it may be selected to consist of a purine nucleotide such as adenosine or guanosine nucleotide. Alternatively, substitution of pyrimidine nucleotides with modification analogs, such as substitution of uridine with 2'-deoxythymidine, is acceptable and does not affect the efficiency of RNA interference.

[0197] In one embodiment, the disclosure features an RNA silencing agent comprising first and second strands, wherein the second strand and / or the first strand are modified by substituting internal nucleotides with modified nucleotides to improve in vivo stability compared to the corresponding unmodified RNA silencing agent. As defined herein, “internal” nucleotides are those located at any position other than the 5' or 3' end of a nucleic acid molecule, polynucleotide, or oligonucleotide. Internal nucleotides may be located within a single-stranded molecule or within a double-stranded or double-stranded molecule. In one embodiment, the sense strand and / or antisense strand are modified by the substitution of at least one internal nucleotide. In another embodiment, the sense strand and / or antisense strand are modified by at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more internal nucleotides. In another embodiment, the sense strand and / or antisense strand are modified by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more internal nucleotides. In yet another embodiment, the sense strand and / or antisense strand are modified by all substitutions of internal nucleotides.

[0198] In one embodiment, the disclosure features an RNA silencing agent that is at least 80% chemically modified. In a preferred embodiment of the disclosure, the RNA silencing agent may be completely chemically modified, i.e., 100% of the nucleotides are chemically modified.

[0199] In preferred embodiments of this disclosure, the RNA silencing agent may comprise at least one modified nucleotide analog. The nucleotide analog may be positioned in a location where target-specific silencing activity, such as RNAi-mediated activity or translational repression activity, is substantially unaffected, for example, in a region at the 5' and / or 3' ends of the siRNA molecule. In particular, the incorporation of the modified nucleotide analog can stabilize the ends.

[0200] Exemplary nucleotide analogs include sugar- and / or skeletal-modified ribonucleotides (i.e., modifications to the phosphate sugar backbone). For example, phosphodiester linkages in native RNA can be modified to include at least one nitrogen or sulfur heteroatom. In exemplary skeletal-modified ribonucleotides, the phosphate ester group attached to an adjacent ribonucleotide is replaced by a modifying group, for example, a phosphothioate group. In exemplary sugar-modified ribonucleotides, the 2'OH- group is replaced by a group selected from H, OR, R, halo, SH, SR, NH2, NHR, NR2, or ON, where R is a C1-C6 alkyl, alkenyl, or alkynyl, and halo is F, Cl, Br, or I.

[0201] In certain embodiments, the modifications are 2'-fluoro, 2'-amino, and / or 2'-thio modifications. Particularly preferred modifications include 2'-fluorocytidine, 2'-fluorouridine, 2'-fluoroadenosine, 2'-fluoroguanosine, 2'-aminocytidine, 2'-aminouridine, 2'-aminoadenosine, 2'-aminoguanosine, 2,6-diaminopurine, 4-thiouridine, and / or 5-aminoallyluridine. In certain embodiments, the 2'-fluororibonucleotide is any uridine or cytidine. Additional exemplary modifications include 5-bromouridine, 5-iodouridine, 5-methylcytidine, ribothymidine, 2-aminopurine, 2'-aminobutyrylpyreneuridine, 5-fluorocytidine, and 5-fluorouridine. 2'-deoxynucleotides and 2'-Omenucleotides can also be used within the modified RNA silencing moieties of this disclosure. Additional modification residues include deoxy debases, inosine, N3-methyluridine, N6,N6-dimethyladenosine, pseudouridine, purine ribonucleosides, and ribavirin. In a particularly preferred embodiment, the 2' portion is a methyl group, such that the linking portion is a 2'-O-methyl oligonucleotide.

[0202] In exemplary embodiments, the RNA silencing agents of this disclosure include locked nucleic acids (LNAs). LNAs contain sugar-modified nucleotides that are resistant to nuclease activity (highly stable) and have single-nucleotide recognition for mRNA (Elmen et al., Nucleic Acids Res., (2005), 33(1):439-447; Braasch et al. (2003) Biochemistry 42:7967-7975, Petersen et al. (2003) Trends Biotechnol 21:74-81). These molecules have modifiable 2'-O, 4'-C-ethylene-bridged nucleic acids, such as 2'-deoxy-2''-fluorouridine. Furthermore, LNAs increase the specificity of oligonucleotides by restricting the sugar moiety to a 3'-endoconformation, thereby pre-organizing the nucleotides for base pairing and raising the melting temperature of the oligonucleotides by approximately 10°C per nucleotide.

[0203] In another exemplary embodiment, the RNA silencing agent of the present disclosure comprises peptide nucleic acids (PNAs). The PNAs contain modified nucleotides in which the sugar-phosphate moiety of the nucleotide is replaced with a neutral 2-aminoethylglycine moiety capable of forming a polyamide skeleton, thereby providing high resistance to nuclease digestion and conferring improved binding specificity to molecules (Nielsen, et al., Science, (2001), 254:1497-1500).

[0204] Nucleic acid base-modified ribonucleotides, i.e., ribonucleotides containing at least one non-naturally occurring nucleic acid base instead of naturally occurring nucleic acid bases, are also preferred. The bases can be modified to block the activity of adenosine deaminase. Examples of modified nucleic acid bases, but not limited to these, include uridine and / or cytidine modified at position 5, e.g., 5-(2-amino)propyluridine, 5-bromouridine; adenosine and / or guanosine modified at position 8, e.g., 8-bromoguanosine; deazanucleotides, e.g., 7-deaza-adenosine; and O- and N-alkylated nucleotides, e.g., N6-methyladenosine, which are preferred. It should be noted that the above modifications may be combined.

[0205] In other embodiments, crosslinking can be used to alter the pharmacokinetics of RNA silencing agents, for example, to extend their half-life in the body. Therefore, the disclosure includes RNA silencing agents having two complementary strands of nucleic acid, wherein the two strands are crosslinked. The disclosure also includes RNA silencing agents that are conjugated or unconjugated (e.g., at their 3' end) to other parts (e.g., non-nucleic acid parts such as peptides) or organic compounds (e.g., dyes). Modifying siRNA derivatives in this way can improve cellular uptake or enhance the cellular targeting activity of the resulting siRNA derivatives compared to the corresponding siRNA, and is useful for tracking siRNA derivatives within cells or improving the stability of siRNA derivatives compared to the corresponding siRNA.

[0206] Other exemplary modifications include: (a) modifications at the 2' position, e.g., providing a 2'OMe portion on U in a sense or antisense chain, particularly in a sense chain, or providing a 2'OMe portion in a 3' overhang, e.g., at the 3' terminus (where the 3' terminus means the 3' atom or maximum 3' portion of the molecule, e.g., the most 3' P or 2' position as indicated by the context); (b) modifications of the phosphate skeleton, e.g., by substitution of O with S, e.g., providing phosphorothioate modifications to U or A or both in an antisense chain, e.g., by substitution of O with S; (c) substitution of U with a C5 aminolinker; (d) substitution of A with G (the sequence change is preferably located in the sense chain rather than the antisense chain); and (d) modifications at the 2', 6', 7', or 8' position. Exemplary embodiments are embodiments in which one or more of these modifications are present on the sense but not on the antisense chain, or embodiments in which the antisense chain has few such modifications. Further exemplary modifications include the use of methylated P in the 3' overhang, for example at the 3' terminus; combinations of 2' modifications, for example providing a 2'OMe moiety and modifying the skeleton, for example substituting O with S, for example providing a phosphorothioate modification; or the use of methylated P in the 3' overhang, for example at the 3' terminus; modification with a 3' alkyl group; modification with debasal pyrrolidone in the 3' overhang, for example at the 3' terminus; or modification with naproxen, ibuprofen, or other moieties that inhibit degradation at the 3' terminus.

[0207] 4) Modifications to enhance cellular uptake In other embodiments, RNA silencing agents may be modified with chemical moieties to enhance cellular uptake by target cells (e.g., nerve cells). Accordingly, the disclosure includes RNA silencing agents that are conjugated (e.g., at their 3' end) to other moieties (e.g., non-nucleic acid moieties such as peptides) or organic compounds (e.g., dyes), etc. Conjugation can be achieved, for example, using the methods known in this technology, or the following methods: Lambert et al., Drug Deliv. Rev.:47(1),99-112(2001) (described on nucleic acids attached to polyalkylcyanoacrylate (PACA) nanoparticles); Fattal et al., J. Control Release 53(1-3):137-43(1998) (described on nucleic acids bound to nanoparticles); Schwab et al., Ann. Oncol. 5 Suppl. 4:55-8(1994) (described on nucleic acids linked to intercalating agents, hydrophobic groups, polycations, or PACA nanoparticles); and Godard et al., Eur. J. Biochem. 232(2):404-10(1995) (described on nucleic acids linked to nanoparticles).

[0208] In certain embodiments, the RNA silencing agent of the disclosure is conjugated to a lipophilic moiety. In one embodiment, the lipophilic moiety is a ligand containing a cationic group. In another embodiment, the lipophilic moiety is attached to one or both strands of the siRNA. In an exemplary embodiment, the lipophilic moiety is attached to one end of the sense strand of the siRNA. In another exemplary embodiment, the lipophilic moiety is attached to the 3' end of the sense strand. In certain embodiments, the lipophilic moiety is selected from the group consisting of cholesterol, vitamin E, vitamin K, vitamin A, folic acid, or a cationic dye (e.g., Cy3). In an exemplary embodiment, the lipophilic moiety is cholesterol. Other lipophilic components include cholic acid, adamantane acetate, 1-pyrene butyric acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine.

[0209] 5) Tethering ligand Other entities may be tethered to the RNA silencing agent of this disclosure. For example, ligands tethered to the RNA silencing agent to improve stability, hybridization thermodynamics with target nucleic acids, targeting to specific tissues or cell types, or cell permeability, either by endocytosis-dependent or endocytosis-independent mechanisms. Ligands and associated modifications may also enhance sequence specificity, thereby reducing off-site targeting. Tethering factor ligands may include one or more modified bases or sugars that can function as intercalators. These are preferably located within internal regions, such as within the bulge of the RNA silencing agent / target double helix. Intercalators may be aromatic, e.g., polycyclic aromatic or heterocyclic aromatic compounds. Polycyclic intercalators may have stacking capabilities and may include systems having two, three, or four fused rings. Universal bases described herein may be included in ligands. In one embodiment, the ligand may include cleavage groups that contribute to the inhibition of target genes by cleaving the target nucleic acid. The cleavage group may be, for example, bleomycin (e.g., bleomycin-A5, bleomycin-A2, or bleomycin-B2), pyrene, phenanthroline (e.g., O-phenanthroline), polyamine, tripeptide (e.g., lys-tyr-lys tripeptide), or a metal ion chelating group. Examples of metal ion chelating groups include Lu(III) or EU(III) macrocyclic complexes, Zn(II) 2,9-dimethylphenanthroline derivatives, Cu(II) terpyridine, or acridine, which can promote the selective cleavage of target RNA at the bulge region by free metal ions such as Lu(III). In some embodiments, the peptide ligand can be tethered to an RNA silencing agent to promote the cleavage of target RNA, for example, at the bulge region. For example, 1,8-dimethyl-1,3,6,8,10,13-hexaazacyclotetradecane (Cycram) can be conjugated into peptides (e.g., by amino acid derivatives) to facilitate target RNA cleavage.The tethering ligand can be an aminoglycoside ligand, which can impart improved hybridization properties or improved sequence specificity to the RNA silencing agent. Exemplary aminoglycosides include glycosylated polylysine, galactosylated polylysine, neomycin B, tobramycin, kanamycin A, and acridine conjugates of aminoglycosides, such as Neo-N-acridine, Neo-S-acridine, Neo-C-acridine, Tobra-N-acridine, and KanaA-N-acridine. The use of acridine analogs can enhance sequence specificity. For example, neomycin B has high affinity for RNA compared to DNA, but lower sequence specificity. The acridine analog neo-5-acridine has high affinity for HIV Rev-response elements (RREs). In some embodiments, guanidine analogs (guanidinoglycosides) of aminoglycoside ligands are tethered to the RNA silencing agent. Within guanidinoglycosides, the amine group on the amino acid is replaced with a guanidine group. The attachment of a guanidine analog can increase the cellular permeability of RNA silencing agents. The tethering ligand may be a polyarginine peptide, peptoid, or peptide mimetic that can increase the cellular uptake of oligonucleotide agents.

[0210] Exemplary ligands are coupled to ligand-conjugate carriers, either directly or indirectly via intervening tethering factors, preferably by fetch binding. In exemplary embodiments, the ligand is attached to the carrier via intervening tethering factors. In exemplary embodiments, the ligand alters the distribution, targeting, or lifetime of the RNA silencing agent into which it is incorporated. In exemplary embodiments, the ligand provides, for example, higher affinity to selected targets, such as molecules, cells or cell types, compartments, such as cell or organ compartments, tissues, organs, or regions of the body, compared to species in which such ligands are absent.

[0211] Exemplary ligands can improve transport, hybridization, and specificity properties, and may also improve the nuclease resistance of polymer molecules and / or natural or modified ribonucleotides, including the resulting natural or modified RNA silencing agents or any combination of monomers described herein. Ligands can generally include, for example, therapeutic modifiers to increase uptake, e.g., diagnostic compounds or reporter groups to monitor distribution; crosslinkers; nuclease resistance-constituting moieties; and natural or aberrant nucleic acid bases. Common examples include lipophilic substances, lipids, steroids (e.g., ubaol, hecigenin, diosgenin), terpenes (e.g., triterpenes, e.g., sarsasapogenin, friederin, epifriederanol-deranolic acid-derived lithocholic acid), vitamins (e.g., folic acid, vitamin A, biotin, pyridoxal), carbohydrates, proteins, protein binders, integrin target molecules, polycations, peptides, polyamines, and peptide mimetic compounds. Ligands may include naturally occurring substances (e.g., human serum albumin (HSA), low-density lipoprotein (LDL), or globulin); carbohydrates (e.g., dextran, pullulan, chitin, chitosan, inulin, cyclodextrin, or hyaluronic acid); amino acids, or lipids. Ligands may also be recombinant or synthetic molecules, such as synthetic polymers, such as synthetic polyamino acids. Examples of polyamino acids include polylysine (PLL), poly-L-aspartic acid, poly-L-glutamic acid, styrene-maleic anhydride copolymer, poly(L-lactide-coglycolated) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacrylic acid), N-isopropylacrylamide polymer, or polyphosphatidine.Examples of polyamines include polyethyleneimine, polylysine (PLL), spermine, spermidine, polyamines, pseudopeptide-polyamines, peptide-mimicking polyamines, dendrimer polyamines, arginine, amidine, protamine, cationic lipids, cationic porphyrins, quaternary salts of polyamines, or alpha-helical peptides.

[0212] Ligands can also include target groups that bind to specific cell types, such as kidney cells, such as cell or tissue targeting agents, such as lectins, glycoproteins, lipids or proteins, such as antibodies. Target groups can also be thyroid-stimulating hormone, melanotropin, lectins, glycoproteins, surfactant protein A, mucin carbohydrates, polyvalent lactose, polyvalent galactose, N-acetylgalactosamine, N-acetylglucosamine, polyvalent mannose, polyvalent fucose, glycosylated polyamino acids, polyvalent galactose, transferrin, bisphosphonates, polyglutamates, polyaspartates, lipids, cholesterol, steroids, bile acids, folates, vitamin B12, biotin, or RGD peptides or RGD peptide mimetic compounds. Other examples of ligands include dyes, intercalating agents (e.g., acridine and substituted acridines), crosslinking agents (e.g., psoralen, mitomycin C), porphyrins (TPPC4, texaphylline, saffrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine, phenanthroline, pyrene), lys-tyr-lys tripeptides, aminoglycosides, guanidium aminoglycosides, artificial endonucleases (e.g., EDTA), lipophilic molecules (e.g., cholesterol and its thio analogues), cholic acid, cholanic acid, lithocholic acid, adamantane acetate, 1-pyrenebutyric acid, dihydrotestosterone, glycerol (e.g., esters (e.g., mono, bis, or tris fatty acid esters, e.g., C) 10 , C 11 , C 12 , C 13 , C 14 , C 15 , C 16 , C 17 , C 18 , C19 or C 20 fatty acids) and their ethers, for example, C 10 , C 11 , C 12 , C 13 , C 14 , C 15 , C 16 , C 17 , C 18 , C 19 , or C 20 Alkyl compounds (e.g., 1,3-bis-O(hexadecyl)glycerol, 1,3-bis-O(octadecyl)glycerol), geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, stearic acid (e.g., glyceryl distearate), oleic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine) and peptide conjugates (e.g., anthelmintic acid). Napedia peptides, Tat peptides), alkylating agents, phosphates, amino acids, mercaptos, PEGs (e.g., PEG-40K), MPEG, [MPEG]2, polyamino acids, alkyl groups, substituted alkyl groups, radiolabeled markers, enzymes, haptens (e.g., biotin), transport / absorption enhancers (e.g., aspirin, naproxen, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, bisimidazole, histamine, imidazole clusters, acridine-imidazole conjugates, Eu tetraazal macrorings) 3+ Examples include complexes, dinitrophenyl, HRP, or AP.

[0213] Ligands can be proteins such as glycoproteins, peptides such as molecules that have a specific affinity for the coligand, or antibodies such as antibodies that bind to specific cell types such as cancer cells, endothelial cells, or osteocytes. Examples of ligands include hormones and hormone receptors. They can also include non-peptide species, such as lipids, lectins, carbohydrates, vitamins, cofactors, polyvalent lactose, polyvalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine, polyvalent mannose, or polyvalent fucose. Examples of ligands include lipopolysaccharides, p38 MAP kinase activators, or NF-κB activators.

[0214] A ligand can be a substance, such as a drug, that can increase the uptake of RNA silencing agents into cells by disrupting the cytoskeleton of a cell, for example, by disrupting the microtubules, microfilaments, and / or intermediate filaments of a cell. Drugs may include, for example, taxone, vincristine, vinblastine, cytochalasin, nocodazole, japlakinolide, latranculine A, phalloidin, swinholide A, indanosine, or myoservin. Ligands can also increase the uptake of RNA silencing agents into cells by, for example, activating an inflammatory response. Exemplary ligands having such effects include tumor necrosis factor alpha (TNFα), interleukin-1 beta, or gamma interferon. In one embodiment, the ligand is a lipid or lipid molecule. Such a lipid or lipid molecule preferably binds to a serum protein, for example, human serum albumin (HSA). HSA-binding ligands allow for the distribution of conjugates to target tissues, such as non-renal target tissues of the body. For example, the target tissue may be the liver, such as hepatic parenchymal cells. Other molecules that can bind to HSA can also be used as ligands. For example, naproxen or aspirin can be used. Lipids or lipid ligands can be used to (a) improve the degradation tolerance of the conjugate, (b) increase targeting or transport to target cells or cell membranes, and / or (c) modulate binding to serum proteins, such as HSA. Lipid ligands can be used to modulate, for example, control the binding of the conjugate to target tissues. For example, lipids or lipid ligands that bind more strongly to HSA are less likely to target the kidney and therefore less likely to be excreted from the body. Lipids or lipid ligands that do not bind less strongly to HSA can be used to target the conjugate to the kidney. In a preferred embodiment, the lipid ligand binds to HSA. The lipid ligand can bind to HSA with sufficient affinity so that the conjugate is distributed to tissues other than the kidney, if preferred.However, it is preferable that the affinity is not so strong that it cannot reverse HSA-ligand binding. In another preferred embodiment, the lipid ligand binds weakly to HSA or does not bind at all, so the conjugate is preferably distributed to the kidney. Other parts that target kidney cells can also be used instead of, or in addition to, the lipid ligand.

[0215] In another embodiment, the ligand is a portion taken up by target cells, such as proliferating cells, e.g., a vitamin. These are particularly useful in treating undesirable cell proliferation, e.g., disorders characterized by malignant or non-malignant forms, e.g., cancer cells. Representative vitamins include vitamins A, E, and K. Other examples of vitamins include B vitamins, e.g., folic acid, B12, riboflavin, biotin, pyridoxal, or other vitamins or nutrients taken up by cancer cells. Similarly included are HSA and low-density lipoprotein (LDL).

[0216] In another embodiment, the ligand is preferably a cell permeabilizer, such as a helical cell permeator. Preferably, the agent is amphiphilic. Exemplary agents are peptides such as tat or antennopedia. If the agent is a peptide, it may be modified, including the use of peptidyl mimicry, inverted isomers, non-peptide or pseudopeptide bonds, and D-amino acids. The helical agent is preferably an alpha-helical agent, preferably having a lipophilic phase and an oleophobic phase.

[0217] The ligand may be a peptide or a peptide mimetic. A peptide mimetic (also referred to herein as an oligopeptide mimetic) is a molecule that can be folded into a specific three-dimensional structure similar to that of a natural peptide. The attachment of peptides and peptide mimetics to oligonucleotide agents can affect the pharmacokinetic distribution of RNA silencing agents, for example, by improving cellular recognition and absorption. The peptide or peptide mimetic moiety may be about 5 to 50 amino acids long, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long. The peptide or peptide mimetic may be, for example, a cell-permeable peptide, a cationic peptide, an amphiphilic peptide, or a hydrophobic peptide (for example, mainly consisting of Tyr, Trp, or Phe). The peptide moiety may be a dendrimeric peptide, a restrictive peptide, or a cross-linked peptide. The peptide moiety may be an L-peptide or a D-peptide. Alternatively, the peptide moiety may contain a hydrophobic membrane translocation sequence (MTS). Peptides or peptide mimetic compounds can be encoded by random DNA sequences, such as peptides identified from phage presentation libraries or one-bead-one-compound (OBOC) combinatorial libraries (Lam et al., Nature 354:82-84, 1991). In exemplary embodiments, the peptide or peptide mimetic compound tethered to an RNA silencing agent via an incorporated monomer unit is a cell-targeted peptide, such as an arginine-glycine-aspartate (RGD) peptide or RGD mimic. Some peptide moieties can range in length from about 5 to about 40 amino acids. Peptide moieties can have structural modifications, such as increasing stability or directing conformation. Any of the structural modifications described below may be used.

[0218] 6) Branched oligonucleotides If at least one of the oligonucleotides includes an inter-subunit linkage according to the embodiment of formula (I), two or more oligonucleotides may be linked to each other by one or more moieties independently selected from linkers, spacers, and branching points to form a branched compound. For example, a branched compound may contain two or more RNA silencing agents of the types shown above, thereby resulting in a new type of RNA silencing agent having a branched structure. In a typical embodiment, each oligonucleotide comprises an antisense strand (or a portion thereof), the antisense strand having sufficient complementarity to a heterozygous single nucleotide polymorphism to mediate an RNA-mediated silencing mechanism (e.g., RNAi).

[0219] In exemplary embodiments, the branched compound may have 2 to 8 RNA silencing agents attached via a linker. The linker may be hydrophobic. In typical embodiments, the branched oligonucleotide of the present application has 2 to 3 oligonucleotides. In one embodiment, the oligonucleotides independently have substantial chemical stability (e.g., at least 40% of the constituent bases are chemically modified). In specific embodiments, the oligonucleotides have complete chemical stability (i.e., all constituent bases are chemically modified). In some embodiments, the branched oligonucleotide comprises one or more single-stranded phosphorothioate tails, each having 2 to 20 nucleotides independently. In non-limiting embodiments, each single-stranded tail has 8 to 10 nucleotides.

[0220] In certain embodiments, branched compounds are characterized by three properties: (1) a branched structure, (2) complete metabolic stabilization, and (3) the presence of a single-stranded tail containing a phosphorothioate linker. In exemplary embodiments, branched oligonucleotides have two or three branches. Increasing the overall size of the branched structure promotes increased uptake. Also, although not bound by any particular theory regarding activity, multiple adjacent branches (e.g., two or three) are thought to allow each branch to act in coordination, thus dramatically improving the rates of internalization, transport, and release.

[0221] Branched compounds are provided in a variety of structurally diverse embodiments. In some embodiments, the nucleic acids attached at the branching point are single-stranded and include miRNA inhibitors, gapmers, mixmers, SSOs, PMOs, or PNAs. These single strands may be attached to the 3' or 5' end. Combinations of siRNA and single-stranded oligonucleotides can also be used for dual function. In another embodiment, short nucleic acids complementary to gapmers, mixmers, miRNA inhibitors, SSOs, PMOs, and PNAs are used to carry these active single-stranded nucleic acids and enhance their distribution and intracellular integration. The short double-stranded regions are used to rapidly dissociate when the branched structure is taken up into cells, using a low melting temperature (T m It has a temperature of approximately 37°C.

[0222] Di-siRNA compounds, i.e., branched oligonucleotides having two siRNAs and a linker, can contain a variety of chemically diverse conjugates. The conjugate bioactive ligands may be used to enhance cell specificity and promote membrane binding, internalization, and serum protein binding. Examples of bioactive moieties used for conjugation include DHAg2, DHA, GalNAc, and cholesterol. These moieties can be attached to the Di-siRNA via a connecting linker or spacer, or added via an additional linker or spacer attached to another free siRNA end.

[0223] While not bound by any specific theory, the presence of branched structures has been found to improve tissue retention levels in the brain by more than 100 times compared to unbranched compounds of the same chemical composition, suggesting a novel mechanism of cell retention and distribution. Branched oligonucleotides are unexpectedly uniformly distributed throughout the spinal cord and brain. Furthermore, branched oligonucleotides exhibit unexpectedly efficient systemic delivery to various tissues and very high levels of tissue accumulation.

[0224] The branched oligonucleotide may contain a variety of therapeutic nucleic acids, such as ASO, miRNA, miRNA inhibitors, splice switching, PMO, and PNA. In some embodiments, the branched oligonucleotide further contains a conjugated hydrophobic moiety, exhibiting unprecedented silencing and efficacy in vitro and in vivo.

[0225] Linker In one embodiment of the branched oligonucleotide compound, each linker is independently selected from ethylene glycol chains, alkyl chains, peptides, RNA, DNA, phosphates, phosphonates, phosphoramides, esters, amides, triazoles, and combinations thereof; where any carbon or oxygen atom of the linker is optionally substituted with a nitrogen atom, harbors a hydroxyl substituent, or harbors an oxo substituent. In one embodiment, each linker is an ethylene glycol chain. In another embodiment, each linker is an alkyl chain. In another embodiment, each linker is a peptide. In another embodiment, each linker is RNA. In another embodiment, each linker is DNA. In another embodiment, each linker is a phosphate. In another embodiment, each linker is a phosphonate. In another embodiment, each linker is a phosphoramide. In another embodiment, each linker is an ester. In another embodiment, each linker is an amide. In another embodiment, each linker is a triazole. VI.

[0226] In another embodiment, branched oligonucleotide compounds of formula (1) are provided herein: [ka] In the formula, L is selected from ethylene glycol chains, alkyl chains, peptides, RNA, DNA, phosphates, phosphonates, phosphoramidates, esters, amides, triazoles, and combinations thereof, and formula (1) optionally further comprises one or more branching points Bp and one or more spacers S; where Bp is, independently, a polyvalent organic species or a derivative thereof, in each occurrence, and S is, independently, selected from ethylene glycol chains, alkyl chains, peptides, RNA, DNA, phosphates, phosphonates, phosphoramidates, esters, amides, triazoles, and combinations thereof. N is an RNA double helix comprising a sense strand and an antisense strand, each of which independently comprises one or more chemical modifications, and n is 2, 3, 4, 5, 6, 7, or 8. In one embodiment, at least one N comprises a modified subunit linkage of formula (I).

[0227] In one embodiment, the compound of formula (1) has a structure selected from formulas (1-1) to (1-9) in Table 1. [Table 1]

[0228] In one embodiment, the compound of formula (1) is formula (1-1). In another embodiment, the compound of formula (1) is formula (1-2). In another embodiment, the compound of formula (1) is formula (1-3). In another embodiment, the compound of formula (1) is formula (1-4). In another embodiment, the compound of formula (1) is formula (1-5). In another embodiment, the compound of formula (1) is formula (1-6). In another embodiment, the compound of formula (1) is formula (1-7). In another embodiment, the compound of formula (1) is formula (1-8). In another embodiment, the compound of formula (1) is formula (1-9).

[0229] In embodiments of the compound of formula (1), each linker is independently selected from ethylene glycol chains, alkyl chains, peptides, RNA, DNA, phosphates, phosphonates, phosphoramides, esters, amides, triazoles, and combinations thereof; where any carbon or oxygen atom of the linker is optionally substituted with a nitrogen atom, has a hydroxyl substituent, or has an oxo substituent. In one embodiment of the compound of formula (1), each linker is an ethylene glycol chain. In another embodiment, each linker is an alkyl chain. In yet another embodiment of the compound of formula (1), each linker is a peptide. In yet another embodiment of the compound of formula (1), each linker is RNA. In yet another embodiment of the compound of formula (1), each linker is DNA. In yet another embodiment of the compound of formula (1), each linker is a phosphate. In yet another embodiment, each linker is a phosphonate. In yet another embodiment of the compound of formula (1), each linker is a phosphoramide. In another embodiment of the compound of formula (1), each linker is an ester. In another embodiment of the compound of formula (1), each linker is an amide. In another embodiment of the compound of formula (1), each linker is a triazole.

[0230] In one embodiment of the compound of formula (1), Bp is a polyvalent organic species. In another embodiment of the compound of formula (1), Bp is a derivative of a polyvalent organic species. In one embodiment of the compound of formula (1), Bp is a triol or tetrol derivative. In another embodiment, Bp is a tri or tetracarboxylic acid derivative. In another embodiment, Bp is an amine derivative. In another embodiment, Bp is a tri or tetraamine derivative. In another embodiment, Bp is an amino acid derivative.

[0231] A polyvalent organic species is a carbon atom with a moiety containing three or more valencies (i.e., an attachment site to a moiety such as S, L, or N as defined above). Non-limiting examples of polyvalent organic species include triols (e.g., glycerol, phloroglucinol), tetrols (e.g., ribose, pentaerythritol, 1,2,3,5-tetrahydroxybenzene), tricarboxylic acids (e.g., citric acid, 1,3,5-cyclohexanetricarboxylic acid, trimesic acid), tetracarboxylic acids (e.g., ethylenediaminetetraacetic acid, pyromellitic acid), tertiary amines (e.g., tripropargylamine, triethanolamine), triamines (e.g., diethylenetriamine), tetramines, and species containing combinations of hydroxyl, thiol, amino, and / or carboxyl moieties (e.g., amino acids such as lysine, serine, and cysteine).

[0232] In embodiments of the compound of formula (1), each nucleic acid comprises one or more chemically modified nucleotides. In embodiments of the compound of formula (1), each nucleic acid consists of chemically modified nucleotides. In specific embodiments of the compound of formula (1), more than 95%, more than 90%, more than 85%, more than 80%, more than 75%, more than 70%, more than 65%, more than 60%, more than 55%, or more than 50% of each nucleic acid comprises chemically modified nucleotides.

[0233] In one embodiment, each antisense chain independently contains a 5' terminal group R selected from the group in Table 2. [Table 2]

[0234] In one embodiment, R is R1. In another embodiment, R is R2. In another embodiment, R is R3. In another embodiment, R is R4. In another embodiment, R is R5. In another embodiment, R is R6. In another embodiment, R is R7. In another embodiment, R is R8.

[0235] Structure of equation (2) In one embodiment, the compound of formula (1) has the structure of formula (2): [ka] (wherein X is independently selected from adenosine, guanosine, uridine, cytidine, and their chemically modified derivatives for each occurrence, and Y is independently selected from adenosine, guanosine, uridine, cytidine, and their chemically modified derivatives for each occurrence; - represents a phosphodiester nucleoside bond; = represents a phosphorothioate nucleoside bond; and --- represents a base-pair interaction or mismatch, individually for each occurrence). Furthermore, at least one of the nucleoside links may be substituted with a modified subunit link of formula (I).

[0236] In certain embodiments, the structure of formula (2) does not contain mismatches. In one embodiment, the structure of formula (2) contains one mismatch. In another embodiment, the compound of formula (2) contains two mismatches. In another embodiment, the compound of formula (2) contains three mismatches. In yet another embodiment, the compound of formula (2) contains four mismatches. In one embodiment, each nucleic acid consists of a chemically modified nucleotide.

[0237] In certain embodiments, more than 95%, more than 90%, more than 85%, more than 80%, more than 75%, more than 70%, more than 65%, more than 60%, more than 55%, or more than 50% of X' in the structure of formula (2) are chemically modified nucleotides. In other embodiments, more than 95%, more than 90%, more than 85%, more than 80%, more than 75%, more than 70%, more than 65%, more than 60%, more than 55%, or more than 50% of X' in the structure of formula (2) are chemically modified nucleotides.

[0238] Structure of equation (3) In one embodiment, the compound of formula (1) has the structure of formula (3): [ka] Here, X (underlined) is a nucleotide containing a 2'-deoxy-2'-fluoro modification, independently of each occurrence; X is a nucleotide containing a 2'-O-methyl modification, independently of each occurrence; Y (underline) Y is a nucleotide that independently contains a 2'-deoxy-2'-fluoro modification in each instance; Y is a nucleotide that independently contains a 2'-O-methyl modification in each instance.

[0239] In one embodiment, X is selected from the group consisting of 2'-deoxy-2'-fluoro-modified adenosine, guanosine, uridine, or cytidine. In one embodiment, X is selected from the group consisting of 2'-O-methyl-modified adenosine, guanosine, uridine, or cytidine. In one embodiment, Y is selected from the group consisting of 2'-deoxy-2'-fluoro-modified adenosine, guanosine, uridine, or cytidine. In one embodiment, Y is selected from the group consisting of 2'-O-methyl-modified adenosine, guanosine, uridine, or cytidine.

[0240] In certain embodiments, the structure of formula (3) does not contain any mismatches. In one embodiment, the structure of formula (3) contains one mismatch. In another embodiment, the compound of formula (3) contains two mismatches. In yet another embodiment, the compound of formula (3) contains three mismatches. In yet another embodiment, the compound of formula (3) contains four mismatches.

[0241] Structure of equation (4) In one embodiment, the compound of formula (1) has the structure of formula (4): [ka] (wherein X is independently selected from adenosine, guanosine, uridine, cytidine, and their chemically modified derivatives for each occurrence; Y is independently selected from adenosine, guanosine, uridine, cytidine, and their chemically modified derivatives for each occurrence; - represents a phosphodiester nucleoside bond; = represents a phosphorothioate nucleoside bond; and --- represents a base-pair interaction or mismatch for each occurrence). In addition, at least one of the nucleoside links may be substituted with a modified subunit link of formula (I).

[0242] In certain embodiments, the structure of formula (4) does not contain mismatches. In one embodiment, the structure of formula (4) contains one mismatch. In another embodiment, the compound of formula (4) contains two mismatches. In another embodiment, the compound of formula (4) contains three mismatches. In yet another embodiment, the compound of formula (4) contains four mismatches. In one embodiment, each nucleic acid consists of a chemically modified nucleotide.

[0243] In certain embodiments, more than 95%, more than 90%, more than 85%, more than 80%, more than 75%, more than 70%, more than 65%, more than 60%, more than 55%, or more than 50% of X' in the structure of formula (2) are chemically modified nucleotides. In other embodiments, more than 95%, more than 90%, more than 85%, more than 80%, more than 75%, more than 70%, more than 65%, more than 60%, more than 55%, or more than 50% of X' in the structure of formula (2) are chemically modified nucleotides.

[0244] Structure of equation (5) In some embodiments, the compound of formula (1) has the structure of formula (5): [ka] Here, X (underline) X is a nucleotide that independently contains a 2'-deoxy-2'-fluoro modification in each instance; X is a nucleotide that independently contains a 2'-O-methyl modification in each instance; Y (underline)Y is a nucleotide that independently contains a 2'-deoxy-2'-fluoro modification in each instance; Y is a nucleotide that independently contains a 2'-O-methyl modification in each instance.

[0245] In certain embodiments, X is selected from the group consisting of 2'-deoxy-2'-fluoro-modified adenosine, guanosine, uridine, or cytidine. In one embodiment, X is selected from the group consisting of 2'-O-methyl-modified adenosine, guanosine, uridine, or cytidine. In one embodiment, Y is selected from the group consisting of 2'-deoxy-2'-fluoro-modified adenosine, guanosine, uridine, or cytidine. In one embodiment, Y is selected from the group consisting of 2'-O-methyl-modified adenosine, guanosine, uridine, or cytidine.

[0246] In certain embodiments, the structure of formula (5) does not contain any mismatches. In one embodiment, the structure of formula (6) contains one mismatch. In another embodiment, the compound of formula (5) contains two mismatches. In yet another embodiment, the compound of formula (5) contains three mismatches. In yet another embodiment, the compound of formula (V) contains four mismatches.

[0247] Variable linker In embodiments of the compound of formula (1), L has structure L1. [ka]

[0248] In the L1 embodiment, R is R 3 Therefore, n is 2.

[0249] In the structural embodiment of formula (II), L has structure L1. In the structural embodiment of formula (III), L has structure L1. In the structural embodiment of formula (IV), L has structure L1. In the structural embodiment of formula (V), L has structure L1. In the structural embodiment of formula (VI), L has structure L1. In the structural embodiment of formula (VII), L has structure L1.

[0250] In embodiments of the compound of formula (1), L has structure L2. [ka]

[0251] In one embodiment of L2, R is R 3 And n is 2. In one embodiment of the structure of formula (2), L has structure L2. In an embodiment of the structure of formula (3), L has structure L2. In an embodiment of the structure of formula (4), L has structure L2. In an embodiment of the structure of formula (5), L has structure L2.

[0252] delivery system In another embodiment, a delivery system for therapeutic nucleic acids having the structure of formula (6) is provided herein: [ka] In the formula, L is selected from ethylene glycol chains, alkyl chains, peptides, RNA, DNA, phosphates, phosphonates, phosphoramidates, esters, amides, triazoles, and combinations thereof, and formula (6) optionally further comprises one or more branching points Bp and one or more spacers S; where Bp is, independently, a polyvalent organic species or a derivative thereof, in each occurrence, and S is, independently, a polyvalent organic species or a derivative thereof, in each occurrence, selected from ethylene glycol chains, alkyl chains, peptides, RNA, DNA, phosphates, phosphonates, phosphoramidates, esters, amides, triazoles, and combinations thereof. Each cNA is independently a carrier nucleic acid comprising one or more chemical modifications, where n is 2, 3, 4, 5, 6, 7, or 8. In one embodiment, at least one cNA comprises a modified subunit linkage of formula (I).

[0253] In one embodiment of the delivery system, L is an ethylene glycol chain. In another embodiment of the delivery system, L is an alkyl chain. In another embodiment of the delivery system, L is a peptide. In another embodiment of the delivery system, L is RNA. In another embodiment of the delivery system, L is DNA. In another embodiment of the delivery system, L is a phosphate. In another embodiment of the delivery system, L is a phosphonate. In another embodiment of the delivery system, L is a phosphoramide. In another embodiment of the delivery system, L is an ester. In another embodiment of the delivery system, L is an amide. In another embodiment of the delivery system, L is a triazole.

[0254] In one embodiment of the delivery system, S is an ethylene glycol chain. In another embodiment, S is an alkyl chain. In yet another embodiment of the delivery system, S is a peptide. In yet another embodiment, S is RNA. In yet another embodiment of the delivery system, S is DNA. In yet another embodiment of the delivery system, S is a phosphate. In yet another embodiment of the delivery system, S is a phosphonate. In yet another embodiment of the delivery system, S is a phosphoramide. In yet another embodiment of the delivery system, S is an ester. In yet another embodiment, S is an amide. In yet another embodiment, S is a triazole.

[0255] In one embodiment of the delivery system, n is 2. In another embodiment of the delivery system, n is 3. In yet another embodiment of the delivery system, n is 4. In yet another embodiment of the delivery system, n is 5. In yet another embodiment of the delivery system, n is 6. In yet another embodiment of the delivery system, n is 7. In yet another embodiment of the delivery system, n is 8.

[0256] In certain embodiments, each cNA contains more than 95%, more than 90%, more than 85%, more than 80%, more than 75%, more than 70%, more than 65%, more than 60%, more than 55%, or more than 50% chemically modified nucleotides.

[0257] In one embodiment, the compound of formula (6) has a structure selected from formulas (6-1) to (6-9) in Table 3. [Table 3]

[0258] In one embodiment, the compound of formula (6) has the structure of formula (6-1). In one embodiment, the compound of formula (6) has the structure of formula (6-2). In one embodiment, the compound of formula (6) has the structure of formula (6-3). In one embodiment, the compound of formula (6) has the structure of formula (6-4). In one embodiment, the compound of formula (6) has the structure of formula (6-5). In one embodiment, the compound of formula (6) has the structure of formula (6-6). In one embodiment, the compound of formula (6) has the structure of formula (6-7). In one embodiment, the compound of formula (6) has the structure of formula (6-8). In one embodiment, the compound of formula (6) has the structure of formula (6-9).

[0259] In one embodiment, the compound of formula (6) (for example, formulas (6-1) to (6-9), etc.) contains each cNA independently comprising at least 15 consecutive nucleotides. In one embodiment, each cNA independently consists of a chemically modified nucleotide.

[0260] In one embodiment, each NA hybridizes to at least one cNA. In one embodiment, at least one NA includes a modified inter-subunit linkage of formula (I). In certain embodiments, the compounds of the present disclosure are characterized by: (1) two or more branched oligonucleotides, e.g., with an equal number of 3' and 5' ends; (2) substantially chemically stabilized, e.g., more than 40%, optimally 100%, of the oligonucleotides being chemically modified (e.g., RNA absent, optionally DNA absent); (3) a phosphorothioate monooligonucleotide containing at least three, optionally 5 to 20 phosphorothioate links.

[0261] VII. Methods for introducing nucleic acids, vectors, host cells, and branched oligonucleotide compounds. The RNA silencing agents of this disclosure can be introduced directly into cells (e.g., nerve cells) (i.e., intracellularly), or they can be introduced from outside the cell into cavities, spaces, or the circulation of an organism, or they can be introduced orally, or by immersing cells or organisms in a solution containing nucleic acids. Blood vessels or extravascular circulation, the blood or lymphatic system, and cerebrospinal fluid are sites into which nucleic acids can be introduced.

[0262] The RNA silencing agents of this disclosure may be introduced using nucleic acid delivery methods known in the art, such as injection of a nucleic acid-containing solution, impact with nucleic acid-coated particles, immersion of cells or organisms in a nucleic acid solution, or electroporation of cell membranes in the presence of nucleic acids. Other methods known in the art for introducing nucleic acids into cells may also be used, such as lipid-mediated transport, chemical-mediated transport, and cationic liposome transfection, such as calcium phosphate. The nucleic acids may be introduced together with other components that perform one or more of the following activities: enhancing nucleic acid uptake by cells, or increasing inhibition of target genes.

[0263] Physical methods for introducing nucleic acids include injection of an RNA-containing solution, impact with RNA-coated particles, immersion of cells or organisms in an RNA solution, or electroporation of the cell membrane in the presence of RNA. Viral constructs packaged in viral particles achieve both efficient introduction of the expression construct into cells and transcription of the RNA encoded by the expression construct. Other methods known in the art for introducing nucleic acids into cells, such as lipid-mediated transport and chemical-mediated transport, such as calcium phosphate, may also be used. Thus, RNA can be introduced with components that perform one or more of the following activities: enhancement of RNA uptake by cells, inhibition of single-strand annealing, stabilization of single-strand annealing, or otherwise increased inhibition of the target gene.

[0264] RNA can be introduced directly into cells (i.e., into cells), or it can be introduced from outside cells into cavities, spaces, or the circulation of organisms, orally, or by immersing cells or organisms in a solution containing RNA. Blood vessels or extravascular circulation, the blood or lymphatic system, and cerebrospinal fluid are sites into which RNA can be introduced.

[0265] Cells containing the target gene may originate from germline or somatic cells, totipotent or pluripotent, mitotic or non-mitotic, parenchymal or epithelial tissue, immortalized or transformed cells, etc. The cells may be stem cells or differentiated cells. Differentiated cell types include adipocytes, fibroblasts, myocytes, cardiomyocytes, endothelial cells, neurons, glial cells, blood cells, megakaryocytes, lymphocytes, macrophages, neutrophils, eosinophils, basophils, mast cells, leukocytes, granulocytes, keratinocytes, chondrocytes, osteoblasts, osteoclasts, hepatocytes, and cells of endocrine or exocrine glands.

[0266] Depending on the specific target gene and the dose of the delivered double-stranded RNA material, this process can result in partial or complete loss of function of the target gene. Typical results include a reduction or loss of gene expression in at least 50%, 60%, 70%, 80%, 90%, 95%, or 99%, or more, of target cells. Inhibition of gene expression refers to the absence (or observable reduction) of levels of protein and / or mRNA products from the target gene. Specificity refers to the ability to inhibit the target gene without apparent effect on other genes in the cell. The results of inhibition can be confirmed by examining the external characteristics of the cell or organism (as shown in the examples below) or by biochemical methods, such as RNA solution hybridization, nuclease protection, Northern hybridization, reverse transcription, gene expression monitoring by microarray, antibody binding, enzyme-linked immunosorbent assay (ELISA), Western blotting, radioimmunoassay (RIA), other immunoassays, and fluorescence-activated cell sorting (FACS).

[0267] In the case of RNA-mediated inhibition in cell lines or whole organisms, gene expression can be conveniently assayed by using reporters or drug resistance genes whose protein products are readily assayable. Examples of such reporter genes include acetohydroxy acid synthase (AHAS), alkaline phosphatase (AP), beta-galactosidase (LacZ), beta-glucolonidase (GUS), chloramphenicol acetyltransferase (CAT), green fluorescent protein (GFP), horseradish peroxidase (HRP), luciferase (Luc), nopalin synthase (NOS), octopine synthase (OCS), and their derivatives. Multiple select markers conferring resistance to ampicillin, bleomycin, chloramphenicol, gentamicin, hygromycin, kanamycin, lincomycin, methotrexate, phosphinothricin, puromycin, and tetracycline are available. Depending on the assay, quantification of gene expression levels can determine inhibition levels greater than 10%, 33%, 50%, 90%, 95%, or 99% compared to cells not treated by this disclosure. If the dose of injected material is small and the time elapsed since RNAi agent administration is long, a smaller percentage of cells (e.g., at least 10%, 20%, 50%, 75%, 90%, or 95% of target cells) may be inhibited. Quantification of intracellular gene expression may indicate similar levels of inhibition at the level of target mRNA accumulation or target protein translation. For example, the efficiency of inhibition may be determined by evaluating the amount of intracellular gene products. mRNA can be detected by a hybridization probe having a nucleotide sequence outside the region used for the inhibitory double-stranded RNA, or the translated polypeptide can be detected by an antibody produced against the polypeptide sequence in that region.

[0268] RNA can be introduced in amounts that allow for the delivery of at least one copy per cell. Higher doses of material (e.g., at least 5, 10, 100, 500, or 1000 copies / cell) may result in more effective inhibition, while lower doses may be useful for specific applications.

[0269] In exemplary embodiments, the efficacy of the RNAi agents of this disclosure (e.g., siRNA targeting a target sequence of interest) is tested for their ability to specifically degrade mutant mRNA (e.g., production of target mRNA and / or target protein) in cells, particularly in neurons (e.g., neuronal clonal lines and / or primary neurons in the striatum or cortex). Other readily transfectable cells, such as HeLa cells or COS cells, are also suitable for cell-based validation assays. The cells are transfected with human wild-type or mutant cDNA (e.g., human wild-type or mutant target cDNA). Standard siRNA, modified siRNA, or a vector capable of generating siRNA from U-loop mRNA is co-transfected. The selective reduction of target mRNA and / or target protein is measured. The reduction of target mRNA or protein can be compared to the level of target mRNA or protein in the absence of the RNAi agent or in the presence of an RNAi agent that does not target the target mRNA. Exogenously introduced mRNA or protein (or endogenous mRNA or protein) can be assayed for comparison. When using nerve cells known to be somewhat resistant to standard transfection techniques, it may be desirable to introduce RNAi agents (e.g., siRNA) by passive uptake.

[0270] Recombinant adeno-associated viruses and vectors In certain exemplary embodiments, recombinant adeno-associated viruses (rAAVs) and their associated vectors can be used to deliver one or more siRNAs to cells, such as nerve cells (e.g., brain cells). AAVs can infect many different cell types, although their infectivity varies based on serotype determined by the sequence of their capsid proteins. Several native AAV serotypes have been identified, with serotypes 1–9 being the most commonly used for recombinant AAVs. AAV-2 is the most well-studied and publicly available serotype. Examples of the AAV-DJ lineage include serotypes AAV-DJ and AAV-DJ / 8. These serotypes are created by DNA shuffling of multiple AAV serotypes and produce AAVs containing hybrid capsids with improved transduction efficiency into various cells and tissues in vitro (AAV-DJ) and in vivo (AAV-DJ / 8).

[0271] In certain embodiments, broad central nervous system (CNS) delivery can be achieved by intravascular delivery of recombinant adeno-associated virus 7 (rAAV7), RAAV9, and rAAV10, or other suitable rAAVs (Zhang et al. (2011) Mol.Ther.19(8):1440-8.doi:10.1038 / mt.2011.98.Epub 2011 May24). rAAVs and their associated vectors are well known in the Art and are described in U.S. Patent Applications 2014 / 0296486, 2010 / 0186103, 2008 / 0269149, 2006 / 0078542, and 2005 / 0220766, each of which is incorporated herein by reference in whole for all purposes.

[0272] rAAV can be delivered to a subject in a composition according to any suitable method known in the art. rAAV can be suspended in a physiologically suitable carrier (i.e., a composition) and administered to a host animal, namely, a human, mouse, rat, cat, dog, sheep, rabbit, horse, cattle, goat, pig, guinea pig, hamster, chicken, turkey, or a non-human primate (e.g., macaque). In certain embodiments, the animal is a non-human host animal.

[0273] Delivery of one or more rAAVs to a mammalian subject can be, for example, by intramuscular injection or by administration into the bloodstream of the mammalian subject. Administration into the bloodstream may be by injection into a vein, artery, or any other vascular conduit. In certain embodiments, one or more rAAVs are administered into the bloodstream by isolated limb perfusion, a technique well known in surgical techniques, which essentially allows a person skilled in the art to isolate the limb from systemic circulation before administration of the rAAV virions. A variation of the isolated limb perfusion technique described in U.S. Patent No. 6,177,403 may also be used by a person skilled in the art to administer virions into the vascular structure of an isolated limb to potentially enhance transduction into muscle cells or tissues. Furthermore, in some cases, it may be desirable to deliver virions into the central nervous system (CNS) of the subject. "CNS" means all cells and tissues of the brain and spinal cord of vertebrates. Thus, this term includes, but is not limited to, nerve cells, glial cells, astrocytes, cerebrospinal fluid (CSF), interstitial space, bone, cartilage, etc. Recombinant AAVs can be delivered directly to the CNS or brain by injection into the ventricular region, as well as the striatum (e.g., the caudate nucleus or putamen of the striatum), the spinal cord and neuromuscular junction, or the cerebellar lobule, using neurosurgical techniques known in the art, such as stereotactic injection, with the use of needles, catheters, or related devices (e.g., Stein et al., J Virol 73:3424-3429, 1999; Davidson et al., PNAS 97:3428-3432, 2000; Davidson et al., Nat. Genet. 3:219-223, 1993; and Alisky and Davidson, Hum. Gene Ther. 11:2315-2329, 2000).

[0274] The compositions of this disclosure may comprise rAAV alone or in combination with one or more other viruses (e.g., a second rAAV encoding having one or more different transgenes). In certain embodiments, the composition comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different rAAVs, each having one or more different transgenes.

[0275] An effective dose of rAAV is sufficient to target an animal for infection and target the desired tissue. In some embodiments, an effective dose of rAAV is sufficient to create a stable somatic cell transgenic animal model. The effective dose may vary from animal to animal and tissue to tissue, depending primarily on factors such as the species, age, body weight, health status, and target tissue. For example, an effective dose of one or more rAAVs is generally about 10 9 ~10 16 This ranges from approximately 1 ml to approximately 100 ml of solution containing the genome copy. In some cases, approximately 10 11 ~10 12 The dosage of the rAAV genome copy is appropriate. In certain embodiments, 10 12 rAAV genome copies are effective for targeting cardiac, hepatic, and pancreatic tissues. In some cases, stable transgenic animals can be produced by multiple doses of rAAV.

[0276] In some embodiments, the rAAV composition is particularly suitable when high rAAV concentrations are present (for example, about 10%). 13 The formulation is designed to reduce the aggregation of AAV particles in the composition (at a rate of genome copies / mL or higher). Methods for reducing rAAV aggregation are well known in the art and include, for example, the addition of surfactants, pH adjustment, and salt concentration adjustment (e.g., Wright et al. (2005) Molecular Therapy 12:171-178, which is incorporated herein by reference).

[0277] A recombinant AAV (rAAV) vector comprises at least a transgene and its regulatory sequence, as well as 5' and 3' AAV reverse terminal repeats (ITRs). This recombinant AAV vector is packaged in a capsid protein and delivered to selected target cells. In some embodiments, the transgene is a nucleic acid sequence heterogeneous to the vector sequence, which encodes a polypeptide, protein, functional RNA molecule (e.g., siRNA) or other gene product of interest. The nucleic acid coding sequence is operably ligated to the regulatory component in a manner that enables transcription, translation, and / or expression of the transgene in cells of the target tissue.

[0278] The AAV sequence of a vector typically contains cis-acting 5' and 3' reverse-terminal repeat (ITR) sequences (see, e.g., B.J. Carter, "Handbook of Parvoviruses," ed., P. Tijsser, CRC Press, pp. 155-168 (1990)). ITR sequences are usually about 145 base pairs long. In certain embodiments, substantially the entire sequence encoding the ITR is used intramolecularly, although some minor modifications to these sequences are permissible. The ability to modify these ITR sequences is within the scope of those skilled in the art (see, e.g., text, Sambrook et al., "Molecular Cloning. A Laboratory Manual," 2nd ed., Cold Spring Harbor Laboratory, New York (1989); and K. Fisher et al., J Virol., 70:520-532 (1996)). Examples of such molecules used in this disclosure are “cis-acting” plasmids containing a transgene, wherein the selected transgene sequence and associated regulatory elements are adjacent to 5' and 3' AAV ITR sequences. The AAV ITR sequences can be obtained from any known AAV, such as the mammalian AAV types further described herein.

[0279] VIII. Treatment method As used herein, “treatment” or “to treat” is defined as the application or administration of a therapeutic agent (e.g., an RNA agent or vector or a transgene encoding it) to a patient, or the application or administration of a therapeutic agent to tissue or cell lines isolated from a patient, the patient having the disease or disorder, the symptoms of the disease or disorder or a predisposition to the disease or disorder, and the treatment being performed to treat, cure, reduce, alleviate, modify, repair, restore, improve or influence the disease or disorder, the symptoms of the disease or disorder or a predisposition to the disease or disorder.

[0280] In one embodiment, the Disclosure provides a method for preventing the aforementioned disease or disorder in a subject by administering a therapeutic agent (e.g., an RNAi agent or vector or a transgene encoding the same) to the subject. Subjects at risk of the disease can be identified, for example, by one or a combination of the diagnostic assays or prognostic assays described herein. Administration of the prophylactic agent can be performed before the appearance of symptoms characteristic of the disease or disorder, thereby preventing the disease or disorder or slowing its progression.

[0281] Another aspect of this disclosure relates to methods for therapeutically treating an object, i.e., methods for altering the onset of symptoms of a disease or disorder.

[0282] With regard to both preventive and therapeutic treatments, such treatments may be specifically adapted or modified based on knowledge derived from the field of pharmacogenomics. As used herein, “pharmacogenomics” refers to the application of genomics techniques, such as gene sequencing, statistical genetics, and gene expression analysis, to drugs in clinical development and on the market. More specifically, the term refers to the study of how a patient’s genes determine their response to a drug (e.g., a patient’s “drug response phenotype” or “drug response genotype”). Thus, another aspect of this disclosure provides a method for tailoring preventive or therapeutic treatments to an individual using either a target gene molecule or a target gene regulator of this disclosure according to the individual’s drug response genotype. Pharmacogenomics enables clinicians or physicians to target preventive or therapeutic treatments to patients who would benefit most from them, and to avoid treatments for patients who experience toxic drug-related side effects.

[0283] Therapeutic agents can be tested in appropriate animal models. For example, the efficacy, toxicity, or side effects of RNAi agent treatment can be determined using an animal model with an RNAi agent (or an expression vector or transgene encoding it) as described herein. Alternatively, the mechanism of action of such agents can be determined using an animal model with a therapeutic agent. For example, the efficacy, toxicity, or side effects of treatment with such agents can be determined using an animal model with a drug. Alternatively, the mechanism of action of such drugs can be determined using an animal model with a drug.

[0284] Pharmaceutical compositions containing the RNA silencing agents of this disclosure can be administered to any patient diagnosed with or at risk of developing a neurodegenerative disease. In one embodiment, the patient is diagnosed with a neurological disorder and is otherwise generally healthy. For example, the patient is not in the terminal stage and is likely to survive for at least two, three, five, or more years after diagnosis. The patient can receive treatment immediately after diagnosis, or treatment may be delayed until the patient experiences more debilitating symptoms, such as motor fluctuations and dyskinesis in patients with Parkinson's disease. In another embodiment, the patient has not reached a progressive stage of the disease.

[0285] RNA silencing agents modified to enhance uptake into nerve cells are available in unit doses of less than approximately 1.4 mg / kg body weight, or less than 10, 5, 2, 1, 0.5, 0.1, 0.05, 0.01, 0.005, 0.001, 0.0005, 0.0001, 0.00005 or 0.00001 mg / kg body weight, and RNA agents in doses of less than 200 nmole / kg body weight (e.g., approximately 4.4 × 10⁻⁶). 16 RNA silencing agents can be administered in unit doses of less than 1500 nmole / kg body weight, 750, 300, 150, 75, 15, 7.5, 1.5, 0.75, 0.15, 0.075, 0.015, 0.0075, 0.0015, 0.00075, or 0.00015 nmole / kg body weight. The unit dose can be administered, for example, by injection (e.g., intravenously or intramuscularly, subarachnoidally, or directly into the brain), by inhalation, or by topical application. Particularly preferred doses are less than 2 mg / kg body weight, 1, or 0.1 mg / kg body weight.

[0286] Direct delivery of RNA silencing agents to organs (e.g., directly to the brain) may be in doses of approximately 0.00001 mg to approximately 3 mg / organ, or preferably approximately 0.0001 to 0.001 mg / organ, approximately 0.03 to 3.0 mg / organ, approximately 0.1 to 3.0 mg / eye, or approximately 0.3 to 3.0 mg / organ. The dose may be an effective amount for treating or preventing neurodegenerative diseases or disorders, such as AD or ALS. In one embodiment, the unit dose is administered less than once daily, for example, every 2, 4, 8, or 30 days. In another embodiment, the unit dose is not administered at a constant frequency (e.g., not regularly). For example, the unit dose may be administered as a single dose. In one embodiment, the effective dose is administered in other conventional modes of treatment.

[0287] In one embodiment, the subject is administered an initial dose and one or more maintenance doses of the RNA silencing agent. The one or more maintenance doses are generally less than the initial dose, for example, half of the initial dose. The maintenance regimen may include treating the subject with one or more doses in the range of 0.01 μg to 1.4 mg / kg body weight / day, for example, 10, 1, 0.1, 0.01, 0.001, or 0.00001 mg / kg body weight / day. The maintenance dose is preferably administered for 5, 10, or less than 30 days. Furthermore, the treatment regimen may be continued for a period that varies depending on the nature of the specific disease, its severity, and the patient's overall condition. In a preferred embodiment, the dose may be delivered no more than once a day, for example, once every 24 hours, 36, 48 hours, or more, for example, once every 5 or 8 days. After treatment, the patient may be monitored for changes in the patient's condition and relief of symptoms of the disease. The dosage of the compound may be increased if the patient does not respond significantly to the current dosage level, or decreased if a reduction in symptoms of the condition is observed, the condition disappears, or undesirable side effects are observed.

[0288] The effective dose may be administered as a single dose or in two or more doses, where desired or deemed appropriate under specific circumstances. If facilitating repeated or frequent infusions is desirable, a delivery device such as a pump, a semi-permanent stent (e.g., intravenous, intraperitoneal, intracisional, or intrasacral), or the implantation of a reservoir may be recommended. In one embodiment, the pharmaceutical composition comprises multiple RNA silencing species. In another embodiment, the RNA silencing species have sequences that do not overlap or adjacency with respect to naturally occurring target sequences in other species. In another embodiment, the multiple RNA silencing species are specific to different naturally occurring target genes. In another embodiment, the RNA silencing agent is allele-specific. In another embodiment, the multiple RNA silencing species target two or more target sequences (e.g., 2, 3, 4, 5, 6, or more target sequences).

[0289] After successful treatment, it may be desirable to have patients receive maintenance therapy to prevent recurrence of the disease state, in which case the disclosed compound is administered in a maintenance dose ranging from 0.01 μg to 100 g / kg body weight (see U.S. Patent No. 6,107,094).

[0290] The concentration of the RNA silencing agent composition is sufficient to be effective in treating or preventing the disorder, or to modulate the physiological state in humans. The concentration or amount of RNA silencing agent administered depends on the parameters determined for the agent and the method of administration, e.g., nasally, in the cheek, or in the lungs. For example, nasal formulations tend to require much lower concentrations of some components to avoid nasal irritation or burning. Diluting oral formulations up to 10 to 100 times may be desirable to provide a suitable nasal formulation.

[0291] Certain factors may influence the dosage required to effectively treat a subject, including, but not limited to, the severity of the disease or disorder, previous treatments, the subject's overall health and / or age, and other pre-existing conditions. Furthermore, treatment of a subject with a therapeutically effective dose of the RNA silencing agent may consist of a single treatment or, preferably, a series of treatments. It will also be understood that the effective dose of the RNA silencing agent for treatment may increase or decrease during the course of a particular treatment. Changes in dosage may occur and become apparent from the results of the diagnostic assays described herein. For example, a subject may be monitored after administration of the RNA silencing agent composition. Based on the information from the monitoring, an additional dose of the RNA silencing agent composition may be administered.

[0292] The dosage depends on the severity and responsiveness of the disease being treated, and the course of treatment continues for several days to several months, or until a cure or reduction of the disease is achieved. The optimal dosage schedule can be calculated from measurements of drug accumulation in the patient's body. Those skilled in the art can easily determine the optimal dose, method of administration, and number of repetitions. The optimal dose may vary considerably depending on the relative potency of the individual oligomers and can generally be estimated based on the EC50, which has been shown to be effective in in vitro and in vivo animal models. In some embodiments, the animal model includes transgenic animals expressing genes that produce target RNA, such as human genes, e.g., RNA expressed in nerve cells. The transgenic animals may lack the corresponding endogenous RNA. In another embodiment, the composition for testing includes an RNA silencing agent that is at least internally complementary to a conserved sequence between the target RNA in the animal model and the target RNA in humans.

[0293] IX. Pharmaceutical composition and method of administration This disclosure relates to the use of the above-mentioned agents for prophylactic and / or therapeutic treatment as described below. Accordingly, the modulogenators of this application (e.g., branched oligonucleotides including RNA silencing agents) can be incorporated into pharmaceutical compositions suitable for administration. Such compositions typically include nucleic acid molecules, proteins, antibodies, or branched oligonucleotide compounds and pharmaceutically acceptable carriers. In this specification, the term “pharmaceutically acceptable carrier” is intended to include any and all solvents, dispersion media, coatings, antimicrobial and antifungal agents, isotonic and absorption retarders, etc., that are suitable for drug administration. The use of such media and agents for pharmaceutically active substances is well known in the art. Unless any conventional media or agent is incompatible with the active compound, their use in compositions is contemplated. Complementary active compounds can also be incorporated into compositions.

[0294] The pharmaceutical compositions of this disclosure are formulated to be compatible with their intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, intraperitoneal, intramuscular, oral (e.g., inhalation), transdermal (topical), and transmucosal administration. In certain exemplary embodiments, the pharmaceutical compositions of this disclosure are delivered to the cerebrospinal fluid (CSF) by routes of administration such as intrastriatal (IS) administration, intraventricular (ICV) administration, and intrathecal (IT) administration (e.g., via pump, infusion, etc.). Solutions or suspensions used for parenteral, intradermal, or subcutaneous application may include the following components: sterile diluents, e.g., water for injection, saline, fixative oil, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents; antimicrobial agents, e.g., benzyl alcohol or methylparaben; antioxidants, e.g., ascorbic acid or sodium bisulfite; chelating agents, e.g., ethylenediaminetetraacetic acid; buffers, e.g., acetic acid, citric acid, or phosphoric acid; and osmotic regulators, e.g., sodium chloride or dextrose. The pH can be adjusted with an acid or base such as hydrochloric acid or sodium hydroxide. Parenteral preparations can be sealed in glass or plastic ampoules, disposable syringes, or multi-dose vials.

[0295] Pharmaceutical compositions suitable for injection include sterile aqueous solutions (if water-soluble) or sterile dispersions and sterile powders for the immediate preparation of injectable sterile solutions or sterile dispersions. For intravenous, IS, ICV, and / or IT administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL® (BASF, Parsippany, NJ), or phosphate-buffered saline (PBS). In all cases, the composition must be sterile and fluid enough to allow for easy injection. It must also be stable under manufacturing and storage conditions and protected against microbial contamination such as bacteria or fungi. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. Adequate fluidity can be maintained, for example, by the use of coatings such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants. Prevention of microbial action can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, and thimerosal. In many cases, it is preferable to include isotonic agents, such as sugar and polyalcohols (mannitol, sorbitol, sodium chloride, etc.), in the composition. Sustained absorption of the injectable composition can be achieved by including absorption-delaying agents, such as aluminum monostearate and gelatin, in the composition.

[0296] Sterile injectable solutions can be prepared by incorporating the required amount of the active compound into a suitable solvent having, if necessary, one or a combination of the components listed above, followed by sterilization by filtration. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and other required components from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, preferred methods of preparation are vacuum drying and freeze-drying, from which a powder is obtained from a pre-sterilically filtered solution containing the active ingredient and any desired additional components.

[0297] Oral compositions generally contain an inert diluent or food carrier. They can be encapsulated in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, lozenges, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, in which case the compound in the fluid carrier is applied orally, rinsed, spat out, or swallowed. Pharmaceutically compatible binders and / or adjuvant materials may be included as part of the composition. Tablets, pills, capsules, lozenges, etc., may contain any of the following ingredients or compounds having similar properties: binders (e.g., microcrystalline cellulose, tragacanth gum, or gelatin), excipients (e.g., starch or lactose), dispersants (e.g., alginic acid, Primogel, or corn starch), lubricants (e.g., magnesium stearate or sterote), fluidizers (e.g., colloidal silicon dioxide), sweeteners (e.g., sucrose or saccharin), or flavorings (e.g., peppermint, methyl salicylate, or orange flavoring).

[0298] When administered by inhalation, the compound is delivered in the form of an aerosol spray from a pressurized container or dispenser containing a suitable propellant, such as a gas like carbon dioxide, or from a nebulizer.

[0299] Systemic administration may also be by mucosal or transdermal means. In the case of mucosal or transdermal administration, a penetrating agent suitable for the barrier to be penetrated is used in the formulation. Such penetrating agents are commonly known in the art and, for example, in the case of mucosal administration, include cleansing agents, bile salts, and fusidic acid derivatives. Mucosal administration can be carried out using nasal sprays or suppositories. For transdermal administration, the active compound is formulated in the form of an ointment, ointment, gel, or cream, as is commonly known in the art.

[0300] The compounds can also be prepared in the form of suppositories (e.g., including conventional suppository bases such as cocoa butter and other glycerides) or retaining enemas for rectal delivery.

[0301] RNA silencing agents can also be administered by transfection or infection using methods known in this technology. These methods include, but are not limited to, the following: McCaffrey et al. (2002), Nature, 418(6893), 38-9 (hydrodynamic transfection); Xia et al. (2002), Nature Biotechnol., 20(10), 1006-10 (viral-mediated delivery); or Putnam (1996), Am.J.Health Syst.Pharm. 53(2), 151-160, erratum at Am.J.Health Syst.Pharm. 53(3), 325 (1996).

[0302] RNA silencing agents can also be administered by any method suitable for administering nucleic acid agents such as DNA vaccines. These methods include needle-free methods such as gene guns, bioinjectors, skin patches, and the particulate DNA vaccine technology disclosed in U.S. Patent No. 6,194,389, as well as percutaneous needle-free vaccination of mammals using powdered vaccines disclosed in U.S. Patent No. 6,168,587. In addition, intranasal delivery is possible, as described in Hamajima et al. (1998), Clin. Immunol. Immunopathol., 88(2), 205-10. Liposomes (e.g., U.S. Patent No. 6,472,375), and microencapsulation can also be used. Biodegradable, targetable particulate delivery systems can also be used (e.g., as described in U.S. Patent No. 6,471,996).

[0303] In one embodiment, the active compound is prepared together with a carrier (e.g., a controlled-release formulation including implants and microencapsulation delivery systems) that protects the compound from rapid excretion from the body. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydride, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Methods for preparing such formulations will be apparent to those skilled in the art. These materials are commercially available from Alza Corporation and Nova Pharmaceuticals, Inc. Liposome suspensions (containing liposomes that target infected cells with monoclonal antibodies against viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, the method described in U.S. Patent No. 4,522,811.

[0304] For ease of administration and uniformity of dosage, it is particularly advantageous to formulate oral or parenteral compositions in unit dosage forms. As used herein, unit dosage forms refer to physically separate units suitable as unitary dosages for treating a subject, each unit containing a predetermined amount of the active compound calculated to produce the desired therapeutic effect in relation to the required pharmaceutical carrier. The specifications of the unit dosage forms in this disclosure are determined and directly depend on the inherent characteristics of the active compound, the specific therapeutic effect to be achieved, and the essential limitations of the art for formulating such active compound for treatment in an individual.

[0305] The toxicity and therapeutic efficacy of such compounds can be determined in cell cultures or experimental animals using standard pharmaceutical procedures, for example, to determine the LD50 (lethal dose for 50% of the population) and ED50 (therapeutably effective dose for 50% of the population). The dose ratio between the toxic effect and the therapeutic effect is the therapeutic index, which can be expressed as the LD50 / ED50 ratio. Compounds exhibiting a large therapeutic index are preferred. While compounds exhibiting toxic side effects may be used, care must be taken to design a delivery system that directs such compounds to the site of the affected tissue in order to minimize potential damage to uninfected cells and thereby reduce side effects.

[0306] Data obtained from cell culture assays and animal studies can be used to clearly state the dose range for use in humans. Doses of such compounds are preferably within the range of circulating concentrations including an ED50 with little to no toxicity. Doses may vary within this range depending on the dosage form adopted and the route of administration used. For any compound used in the methods of this disclosure, the therapeutically effective dose can first be estimated from a cell culture assay. Doses can be formulated in animal models to achieve a circulating plasma concentration range including an EC50 (i.e., the concentration of the test compound that achieves half of the maximum response) determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Plasma levels can be measured, for example, by high-performance liquid chromatography.

[0307] The pharmaceutical composition may be included in a container, pack, or dispenser, along with any instructions for administration.

[0308] As defined herein, the therapeutically effective dose (i.e., effective dose) of an RNA silencing agent depends on the RNA silencing agent selected. For example, if a plasmid encoding shRNA is selected, a single dose ranging from about 1 μg to 1000 mg may be administered. In some embodiments, 10, 30, 100, or 1000 μg may be administered. In some embodiments, 1 to 5 g of the composition may be administered. The composition may be administered at least once a day to at least once a week, for example, every other day. Those skilled in the art will understand that certain factors may influence the dose and timing required to effectively treat the subject, including, but not limited to, the severity of the disease or disorder, previous treatments, the subject's overall health and / or age, and other pre-existing diseases. Furthermore, treatment of a subject with a therapeutically effective dose of a protein, polypeptide, or antibody may consist of a single treatment or, preferably, a series of treatments.

[0309] The nucleic acid molecules of this disclosure can be inserted into expression constructs, for example, viral vectors, retroviral vectors, expression cassettes, or plasmid viral vectors, using methods known in this art, such as, for example but not limited to, those described in Xia et al., (2002) above. The expression constructs can be delivered to a target, for example, by inhalation, oral administration, intravenous injection, topical administration (see U.S. Patent No. 5,328,470), or stereotactic injection (see, for example, Chen et al. (1994), Proc. Natl. Acad. Sci. USA, 91, 3054-3057). The pharmaceutically appropriate preparation of the delivery vector may include the vector in an acceptable diluent, or it may include a sustained-release matrix into which the delivery vehicle is embedded. Alternatively, if the complete delivery vector can be produced intact from recombinant cells, such as a retroviral vector, the pharmaceutically appropriate preparation may include one or more cells that produce the gene delivery system.

[0310] The nucleic acid molecules of this disclosure may also include small hairpin RNA (shRNA) and expression constructs engineered to express shRNA. Transcription of shRNA is thought to be initiated by the polymerase III (pol III) promoter and terminated at position 2 of the 4-5-thymine transcription termination site. Upon expression, shRNA is thought to fold into a stem-loop structure with a 3' UU overhang. Subsequently, the ends of these shRNAs are processed, thereby converting them into approximately 21-nucleotide siRNA-like molecules. Brummelkamp et al. (2002), Science, 296, 550-553; Lee et al. (2002). See above; Miyagishi and Taira (2002), Nature Biotechnol., 20, 497-500; Paddison et al. (2002), See above; Paul (2002), See above; Sui (2002), See above; Yu et al. (2002), See above.

[0311] Expression constructs may be any construct suitable for use in a suitable expression system, but are not limited to, retroviral vectors, linear expression cassettes, plasmids, and viruses or virus-derived vectors known in the art. Such expression constructs may include one or more inducible promoters, RNA Pol III promoter systems, e.g., the U6 snRNA promoter or the H1 RNA polymerase III promoter, or other promoters known in the art. Constructs may include one or both strands of siRNA. Expression constructs expressing both strands may also include a loop structure linking both strands, or each strand may be transcribed separately from separate promoters within the same construct. Each strand may also be transcribed from another expression construct (Tuschl (2002), above).

[0312] In certain exemplary embodiments, compositions comprising the RNA silencing agents of this disclosure may be delivered to the target nervous system by various routes. Exemplary routes include delivery to the spinal cavity, parenchyma (e.g., the brain), nose, and eyes. The compositions may also be delivered systemically, for example, by intravenous, subcutaneous, or intramuscular injection, which is particularly useful for administering RNA silencing agents to peripheral nerve cells. Preferred delivery routes are to the brain, for example, to the ventricles or hypothalamus of the brain, or directly to the lateral or dorsal regions of the brain. RNA silencing agents for neuronal delivery may be incorporated into pharmaceutical compositions suitable for administration.

[0313] For example, a composition may contain one or more species of RNA silencing agents and a pharmaceutically acceptable carrier. The pharmaceutical compositions of this disclosure may be administered in multiple ways, depending on whether topical or systemic treatment is desired and the area to be treated. Administration may be topical (e.g., ocular, intranasal, transdermal), oral, or parenteral. Parenteral administration may include intravenous infusion, subcutaneous, intraperitoneal or intramuscular injection, intrathecal, or intraventricular (e.g., cerebral-ventricular) administration. In certain exemplary embodiments, the RNA silencing agents of this disclosure are delivered across the blood-brain barrier (BBB) ​​using various preferred compositions and methods described herein.

[0314] The delivery route may depend on the patient's condition. For example, a subject diagnosed with a neurodegenerative disease may receive the RNA silencing agent of this disclosure directly into the brain (e.g., the globus pallidus or striatum of the basal ganglia, and near medium spiny neurons in the striatum). In addition to the RNA silencing agent of this disclosure, the patient may receive a second treatment, such as palliative care and / or disease-specific treatment. The second treatment may be, for example, symptomatic treatment (e.g., to alleviate symptoms), neuroprotection (e.g., to slow or halt disease progression), or recovery (e.g., to reverse the disease process). Other treatments may include psychotherapy, physiotherapy, speech therapy, communication and memory aids, social support services, and dietary advice.

[0315] RNA silencing agents can be delivered to nerve cells in the brain. Delivery methods that do not require the composition to cross the blood-brain barrier can be utilized. For example, a pharmaceutical composition containing the RNA silencing agent of this disclosure can be delivered to a patient by direct injection into a region containing cells affected by the disease. For example, the pharmaceutical composition can be delivered by direct injection into the brain. The injection may be a stereotactic injection into a specific region of the brain (e.g., the substantia nigra, cortex, hippocampus, striatum, or globus pallidus). RNA silencing agents can be delivered to multiple regions of the central nervous system (e.g., multiple regions of the brain, and / or the spinal cord). RNA silencing agents can be delivered to diffuse regions of the brain (e.g., diffuse delivery to the cerebral cortex).

[0316] In one embodiment, the RNA silencing agent may be delivered by a cannula or other delivery device having one end implanted in a tissue, such as the brain, such as the substantia nigra, cortex, hippocampus, striatum, or globus pallidus. The cannula can be connected to a reservoir of the RNA silencing agent. Flow or delivery may be mediated by a pump, such as an osmotic pump or minipump, such as an Alzet pump (Durect, Cupertino, CA). In one embodiment, the pump and reservoir are implanted in a region away from the tissue, such as the abdomen, and delivery is carried out by a conduit leading from the pump or reservoir to the release site. Devices for delivery to the brain are described, for example, in U.S. Patents 6,093,180 and 5,814,014.

[0317] The RNA silencing agents of this disclosure can be further modified to allow them to cross the blood-brain barrier. For example, the RNA silencing agent can be conjugated to a molecule that allows the drug to cross the barrier. Such modified RNA silencing agents can be administered by any desired method, such as intraventricular or intramuscular injection, or pulmonary delivery.

[0318] In certain embodiments, exosomes are used to deliver the RNA silencing agents of this disclosure. The exosomes can pass through the BBB and, after systemic injection, specifically deliver siRNA, antisense oligonucleotides, chemotherapeutic agents, and proteins to neurons (Alvarez-Erviti L, Seow Y, Yin H, Betts C, Lakhal S, Wood MJ. (2011). Delivery of siRNA to the mouse brain by systemic injection of targeted exosomes. Nat Biotechnol. 2011 Apr;29(4):341-5.doi:10.1038 / nbt.1807; El-Andaloussi S, Lee Y, Lakhal-Littleton S, Li J, Seow Y, Gardiner C, Alvarez-Erviti L, Sargent IL, Wood MJ. (2011). Exosome-mediated delivery of siRNA in vitro and in vivo. Nat Protoc. 2012). Dec;7(12):2112-26.doi:10.1038 / nprot.2012.131;EL Andaloussi S,Mager I,Breakefield XO,Wood MJ.(2013).Extracellular vesicles: biology and emerging therapeutic opportunities.Nat Rev Drug Discov.2013 May;12(5):347-57.doi:10.1038 / nrd3978;El Andaloussi S, Lakhal S, Mager I, Wood MJ. (2013).Exosomes for targeted siRNA delivery across biological barriers.Adv Drug Deliv Rev. 2013 Mar;65(3):391-7.doi:10.1016 / j.addr.2012.08.008).

[0319] In certain embodiments, one or more lipophilic molecules are used to enable the RNA silencing agent of the present disclosure to be delivered across the blood-brain barrier (BBB) ​​(Alvarez-Ervit (2011)). The RNA silencing agent is then activated, for example, by sham lipophilic enzymatic degradation, releasing the drug into its active form.

[0320] In certain embodiments, one or more receptor-mediated permeabilization compounds can be used to increase blood-brain barrier (BBB) ​​permeability, thereby enabling delivery of the RNA silencing agents of this disclosure. These agents temporarily increase BBB permeability by increasing the osmotic pressure of the blood and loosening tight junctions between endothelial cells ((El-Andaloussi (2012))). By loosening tight junctions, the RNA silencing agents can be administered by conventional intravenous injection.

[0321] In certain embodiments, a nanoparticle-based delivery system is used to deliver the RNA silencing agent of this disclosure across the blood-brain barrier (BBB). As used herein, “nanoparticles” refers to polymer nanoparticles, which are typically solid and biodegradable colloidal systems, and are widely studied as drug or gene carriers (SPEgusquiaguirre, M. Igartua, R. Hernandez, and J. P. Edraz, “Nanoparticle delivery systems for cancer therapy: advances in clinical and preclinical research,” Clinical and Translational Oncology, vol. 14, no. 2, pp. 83-93, 2012). Polymer nanoparticles are classified into two main categories: natural polymers and synthetic polymers. Natural polymers for siRNA delivery include, but are not limited to, cyclodextrins, chitosan, and atelocollagen (Y. Wang, Z. Li, Y. Han, LHLiang, and A. Ji, "Nanoparticle-based delivery system for application of siRNA in vivo," Current Drug Metabolism, vol. 11, no. 2, pp. 182-196, 2010). Synthetic polymers, but not limited to, those that have been intensively studied include polyethyleneimine (PEI), poly(dl-lactide-co-glycolide) (PLGA), and dendrimers (X. Yuan, S. Naguib, and Z. Wu, "Recent advances of siRNA delivery by nanoparticles," Expert Opinion on Drug Delivery, vol. 8, no. 4, pp. 521-536, 2011).For a review of nanoparticles and other suitable delivery systems, see Jong-Min Lee, Tae-Jong Yoon, and Young-Seok Cho, "Recent Developments in Nanoparticle-Based siRNA Delivery for Cancer Therapy," BioMed Research International, vol. 2013, Article ID 782041, 10 pages, 2013. doi:10.1155 / 2013 / 782041 (the entire article is incorporated by reference).

[0322] The RNA silencing agents of this disclosure can be administered into the eye, for example, to treat retinal disorders such as retinopathy. For example, the pharmaceutical composition can be applied to the surface of the eye or nearby tissues, for example, the inside of the eyelid. They can be applied topically, for example by spray, by infusion, as an eyewash, or as an ointment. Ointments or droppable liquids can be delivered by ocular delivery systems known in the art, such as applicators or eyedroppers. Such compositions may contain mucomic mimics, for example hyaluronic acid, chondroitin sulfate, hydroxypropyl methylcellulose, or poly(vinyl alcohol), preservatives, for example sorbic acid, EDTA, or benzylcuronium chloride, and normal amounts of diluents and / or carriers. The pharmaceutical composition can also be administered inside the eye, and can be introduced by a needle or other delivery device that can introduce the pharmaceutical composition into a selected area or structure. Compositions containing RNA silencing agents can also be applied via ocular patches.

[0323] In general, the RNA silencing agents of this disclosure can be administered by any suitable method. As used herein, topical delivery may refer to the direct application of the RNA silencing agent to any surface of the body, such as the eyes, mucous membranes, body cavities, or any internal surface. Formulations for topical delivery may include transdermal patches, ointments, lotions, creams, gels, drops, sprays, and liquids. Conventional pharmaceutical carriers, aqueous, powder, or oily bases, thickeners, etc., may be required or desirable. Topical delivery can also be used as a means of selectively delivering the RNA silencing agent to the target epidermis or dermis, or a specific layer thereof, or to the underlying tissue.

[0324] Compositions for intrathecal or intracerebroventricular (e.g., cerebral / intracerebroventricular) administration may include sterile aqueous solutions that may also contain buffers, diluents, and other suitable additives. Preferably, compositions for intrathecal or intracerebroventricular administration do not contain transfection reagents or additional lipophilic moieties other than, for example, lipophilic moieties attached to the RNA silencing agent.

[0325] Parenteral administration formulations may include sterile aqueous solutions that may also contain buffers, diluents, and other suitable additives. Intracerebroventricular injection can be facilitated, for example, by an intracerebroventricular catheter attached to a reservoir. For intravenous use, the total concentration of the solute must be controlled to make the preparation isotonic.

[0326] The RNA silencing agents of this disclosure can be administered to a target by pulmonary delivery. The pulmonary delivery composition can be delivered by inhalation of the dispersion such that the composition in the dispersion reaches the lungs and is readily absorbed directly into the blood circulation from the alveolar region. Pulmonary delivery can be effective for both systemic and local delivery to treat lung diseases. In one embodiment, the RNA silencing agent administered by pulmonary delivery is modified to cross the blood-brain barrier.

[0327] Lung delivery can be achieved through various approaches, including the use of nebulization, aerosolization, micelles, and dry powder-based formulations. Delivery is possible using liquid nebulizers, aerosol-based inhalers, and dry powder dispersion devices. Quantitative devices are preferred. One advantage of using nebulizers or inhalers is that the possibility of contamination is minimized because the devices are standalone. Dry powder dispersion devices deliver drugs that can be readily formulated as dry powders, for example. RNA silencing agent compositions can be stored stably as lyophilized or spray-dried powders, either alone or in combination with suitable powder carriers. Delivery of compositions for inhalation can be mediated by a drug timing element that may include a timer, dose counter, time measuring device, or, if incorporated into the device, a time indicator that enables dose tracking, compliance monitoring, and / or administration to the patient during aerosol drug administration.

[0328] Useful pharmaceutical excipients as carriers include stabilizers such as human serum albumin (HSA), bulking agents such as carbohydrates, amino acids, and polypeptides, pH adjusters or buffers, and salts such as sodium chloride. These carriers may be in crystalline or amorphous form, or a mixture of the two.

[0329] Particularly valuable bulking agents include suitable carbohydrates, polypeptides, amino acids, or combinations thereof. Suitable carbohydrates include monosaccharides such as galactose, D-mannose, and sorbose; disaccharides such as lactose and trehalose; cyclodextrins such as 2-hydroxypropyl-β-cyclodextrin; polysaccharides such as raffinose, maltodextrin, and dextran; and algitols such as mannitol and xylitol. The preferred group of carbohydrates includes lactose, trehalose, raffinose, maltodextrin, and mannitol. A suitable polypeptide is aspartame. Amino acids include alanine and glycine, with glycine being preferred.

[0330] Suitable pH adjusters or buffers include organic salts prepared from organic acids and bases, such as sodium citrate and sodium ascorbate. Sodium citrate is preferred.

[0331] The RNA silencing agents of this disclosure can be administered orally and nasally. For example, drugs administered via these membranes have a rapid onset of action, lead to therapeutic plasma levels, avoid the first-pass effect of hepatic metabolism, and avoid exposure of the drug to the adversarial gastrointestinal (GI) environment. An additional advantage is that easy access to the membrane site allows for easy application, localization, and removal of the drug. In one embodiment, the RNA silencing agent administered orally or nasally is modified to cross the blood-brain barrier.

[0332] In one embodiment, a unit dose or measured dose of a composition containing an RNA silencing agent is distributed by an implantable device. The device may include sensors to monitor parameters within the subject. For example, the device may include a pump, such as an osmotic pump, and optionally associated electronic equipment.

[0333] RNA silencing agents can be packaged in the natural capsid of a virus, or in a chemically or enzymatically produced artificial capsid or a structure derived therefrom.

[0334] It will be readily apparent to those skilled in the art that other suitable modifications and adaptations of the methods described herein can be made using suitable equivalents without departing from the scope of the embodiments disclosed herein. While specific embodiments have been described in detail so far, the same will be more clearly understood by referring to the following examples, which are included for illustrative purposes only and are not intended to limit the scope of the embodiments disclosed herein.

[0335] Example 1. Synthesis of 2'-OMe-exNA phosphoramidite Synthesis of compound 5a The following synthesis was completed according to Figure 2. IBX (5.53 g, 19.7 mmol) was added to an aqueous solution of CH3CN (80 mL) containing compound 3a (2.94 g, 7.89 mmol), and the mixture was stirred at 85°C for 2 hours. After cooling the mixture in an ice bath, the precipitate in the solution was filtered off through Celite. The collected eluent was evaporated and then evaporated three times together with anhydrous CH3CN under an argon atmosphere. Compound 4a, obtained as a white foam, was used without further purification. In a separate flask, tert-BuOK (2.57 g, 22.9 mmol) was added at 0°C to an anhydrous THF (80 mL) solution containing methyltriphenylphosphonium bromide (8.47 g, 23.7 mmol), and the mixture was stirred at 0°C for 30 minutes. To this solution, an anhydrous THF solution of compound 4a (80 mL) was added dropwise at 0°C (10 minutes), and the mixture was stirred at room temperature for 7 hours. After evaporating the excess THF, the resulting mixture was dissolved in excess ethyl acetate, washed with saturated aqueous solution of NH4Cl, dried over MgSO4, filtered, and evaporated. The obtained substance was dissolved in a minimum amount of CH2Cl2 and added dropwise to an excess solution of diethyl ether with vigorous stirring at 0°C. The precipitate in the solution was filtered through Celite, and the eluent was evaporated. The resulting crude substance was purified by silica gel column chromatography (hexane / ethyl acetate, 9:1 to 1:2) to obtain compound 5a as a white foam (2 steps, 2.19 g, 75%). 1 H NMR (500 MHz, CDCl3) δ 9.55 (br-s, 1H), 7.38 (d, 1H, J = 8.2 Hz), 5.89 (ddd, 1H, J = 17.1, 10.6, 6.6 Hz), 5.82 (d, 1H, J = 2.0 Hz), 5.77 (dd, 1H, J = 8.1, 1.5 Hz), 5.44 (dt, 1H, J = 17.2, 1.2 Hz), 5.34 (dt, 1H, J = 10.5, 1.1 Hz), 4.43-4.40 (m, 1H), 3.90 (dd, 1H, J = 7.7, 5.1 Hz), 3.71 (dd, 1H, J = 5.0, 2.0 Hz), 3.55 (s, 3H), 0.89 (s, 9H), 0.09 (s, 3H), 0.07 (s, 3H);13 C NMR (125 MHz, CDCl3) δ 163.4, 150.0, 139.7, 134.4, 119.2, 102.4, 89.7, 84.0, 83.5, 74.5, 58.7, 25.7, 18.2, -4.6, -4.7;HRMS (ESI) ,C 17 H 29 N2O5Si + [M + H] + The calculated value for m / z is 369.1840, and the measured value for m / z is 369.1838.

[0336] Synthesis of compound 6a A 158.3 mL solution of anhydrous THF containing compound 5a (7.29 g, 19.8 mmol) was added dropwise to a 237.4 mL solution of 9-BBN / THF (118.7 mmol) at 0°C for 10 minutes. The mixture was stirred at room temperature for 6 hours, then the solution was cooled on ice, methanol (65.4 mL) was added, and the mixture was stirred until no more foaming occurred. Next, H₂O (98.4 mL) was added dropwise for 10 minutes with vigorous stirring to avoid precipitation of the intermediate compound. NaBO₃·4H₂O (15.7 g, 102.0 mmol) was added all at once at 0°C, and the mixture was stirred overnight at room temperature. After evaporating the excess THF, the resulting crude mixture was dissolved in excess ethyl acetate and repeatedly washed with saturated aqueous NH₄Cl solution. After evaporating the organic layer, the resulting substance was dissolved in THF (450 mL) and H₂O (450 mL). To this solution, NaBO3·4H2O (15.7 g, 102.0 mmol) was added all at once at room temperature, and the mixture was stirred overnight at room temperature. After evaporating the excess THF, ethyl acetate was added to the mixture and extracted. The resulting organic layer was repeatedly washed with saturated aqueous NH4Cl solution, dried over MgSO4, filtered, and evaporated. The resulting crude material was purified by silica gel column chromatography (hexane / ethyl acetate, 7:3 to 0:10) to obtain compound 6a as a white foam (in two steps, 4.73 g, 62%). 1H NMR (500 MHz, CDCl3) δ 9.21(br-s, 1H), 7.35 (d, 1H, J = 8.1 Hz), 5.78-5.76 (m, 2H), 4.14-4.10 (m, 1H), 3.92-3.79 (m, 4H), 3.75 (dd, 1H, J = 5.2, 2.3 Hz), 2.06-2.00 (m, 1H), 1.90-1.82 (m, 1H), 0.91 (s, 9H), 0.11 (s, 3H), 0.10 (s, 3H); 13 HRMS (ESI), C 17 H 31 N2O5Si + [M + H] + The calculated value for m / z is 387.1946, and the measured value for m / z is 187.1944.

[0337] Synthesis of compound 8a To a 240 mL solution of anhydrous pyridine containing compound 6a (9.46 g, 24.5 mmol), DMTrCl (9.95 g, 29.4 mmol) was added and the mixture was stirred at room temperature for 2 hours. After quenching the reaction mixture with MeOH (20 mL), excess pyridine was evaporated, and the resulting substance was dissolved in excess ethyl acetate. The organic solution was washed with a saturated aqueous solution of NaHCO3, dried over MgSO4, filtered, evaporated, and then co-evaporated with toluene to remove the pyridine residue. This crude mixture containing compound 7 was dissolved in THF (330 mL), and a 1.0 M TBAF-THF solution (36.7 mL, 36.7 mmol) was added and the mixture was stirred at room temperature for 1 hour. After evaporating the excess THF and co-evaporating with CH2Cl2, the crude substance was purified by silica gel column chromatography to obtain compound 8a (13.15 g, 93%) in two steps. 11H NMR (500 MHz, CDCl3) δ 8.91 (br-s, 1H), 7.43 - 7.21 (m, 2H), 7.32 - 7.14 (m, 8H), 6.83 - 6.82 (m, 1H), 5.80 (d, 1H, J = 1.8 Hz), 5.69 (d, 1H, J = 8.2 Hz), 4.02 - 3.98 (m, 1H), 3.85 (dd, 1H, J = 6.7, 6.7 Hz), 3.79 (s, 6H), 3.72 (dd, 1H, J = 5.5, 1.9 Hz), 3.34 - 3.25 (m, 2H), 2.91 (br-s, 1H), 2.11 - 2.04 (m, 1H), 1.95 - 1.89 (m, 1H); 13 13C NMR (125 MHz, CDCl3) δ 163.2, 163.1, 158.4, 149.9, 114.8, 139.1, 136.1, 136.0, 129.92, 129.90, 128.0, 127.8, 126.8, 113.1, 102.5, 88.1, 86.6, 83.5, 81.3, 73.2, 60.1, 58.8, 55.2, 53.4, 33.4; HRMS (ESI), C 32 H 34 N2O8Na [M + Na] + Calculated m / z 597.2203 for [M + Na], found m / z 597.2153.

[0338] Synthesis of Compound 9a Compound 8a (9.57 g, 16.65 mmol) was anhydrous by repeated co-evaporation with anhydrous CH3CN, and then dissolved in anhydrous CH2Cl2 (150 mL). To this solution, N,N-diisopropylethylamine (7.6 mL, 62.4 mmol) and 2-cyanoethyl N,N-diisopropyl chlorophosphoramidite (4.85 mL, 25.0 mmol) were added at 0°C. After stirring at room temperature for 4 hours, CH2Cl2 (200 mL) and then saturated aqueous solution of NaHCO3 (350 mL) were added to the reaction mixture. The organic layer was repeatedly washed with saturated aqueous solution of NaHCO3, dried over MgSO4, filtered, and evaporated. The resulting crude material was purified by silica gel column chromatography (1% TEA-hexane-ethyl acetate, 80:20~30:70) to obtain compound 9a, which contains impurities such as phosphytylation reagent residue. To remove impurities, the obtained substance was dissolved in Et2O-ethyl acetate (1:1, v / v, 400 mL), and then repeatedly washed with a saturated aqueous solution of NaHCO3 to obtain compound 9a as a white solid (11.12 g, 86%). 31 P NMR (202 MHz, CDCl3) δ 150.0, 149.9;HRMS (ESI), C 41 H 52 N4O9P [M + H] + The calculated value for m / z is 775.3486, and the measured value for m / z is 775.3414.

[0339] Example 2. Synthesis of 2'-F-exNA phosphoramidite Synthesis of compound 5b The following synthesis was completed according to Figure 3. IBX (21.0 g, 75.0 mmol) was added to a 300 mL solution of anhydrous CH3CN containing compound 3b (10.8 g, 30.0 mmol), and the mixture was stirred at 85°C for 2 hours. After cooling the mixture in an ice bath, the precipitate in the solution was filtered off through Celite. The collected eluent was evaporated and then evaporated together with anhydrous CH3CN three times under an argon atmosphere. Compound 4b, obtained as a white foam, was used without further purification. In a separate flask, methyltriphenylphosphonium bromide (24.0 g, 68.1 mmol) was added all at once at 0°C to a 250 mL solution of anhydrous THF containing tert-BuOK (7.30 g, 65.1 mmol), and the mixture was stirred at 0°C for 1 hour. To this solution, a 150 mL solution of anhydrous THF containing compound 4a was added dropwise at 0°C (10 minutes), and the mixture was stirred overnight at room temperature. After evaporating the excess THF, the resulting mixture was dissolved in excess ethyl acetate, washed with saturated aqueous NH4Cl solution, dried over MgSO4, filtered, and evaporated. The obtained substance was dissolved in a minimum amount of CH2Cl2 and added dropwise to an excess diethyl ether solution with vigorous stirring at 0°C. The precipitate in the solution was filtered through Celite, and the eluent was evaporated. The resulting crude substance was purified by silica gel column chromatography (hexane / ethyl acetate, 8:2 to 6:4) to obtain compound 5b as a white foam (2 steps, 7.12 g, 67%). 1 H NMR (500 MHz, CDCl3) δ 11.4 (br-s, 1H), 7.65 (d, 1H, J = 8.1 Hz), 5.93 (ddd, 1H, J = 17.5, 10.4, 7.5 Hz), 5.82 (dd, 1H, J HF = 22.2 Hz, J HH = 1.3 Hz), 5.65 (d, 1H, J = 8.1 Hz), 5.42-5.38 (m, 1H), 5.33-5.31 (m, 1H), 5.15 (ddd, 1H, J HF = 53.4 Hz, J HH = 4.6, 1.2 Hz), 4.27 (ddd, 1H, J HF = 20.6 Hz, J HH= 8.4, 4.9 Hz), 4.18 (dd, 1H, J = 7.7, 7.7 Hz), 0.88 (s, 9H), 0.08 (s, 3H), 0.07 (s, 3H); 13 C NMR (125 MHz, CDCl3) δ 170.8, 183.7, 150.7, 142.5, 135.3, 120.2, 102.4, 92.9 (d, J CF = 186.2 Hz), 90.2 (d, J CF = 36.4 Hz), 83.4, 73.8 (d, J CF = 15.5 Hz), 60.2, 26.0, 21.2, 18.2, 14.6, -4.4, -4.5; 19 F NMR (470 MHz, DMSO-d6) δ -198.3 (ddd, J = 53.8, 20.8, 20.8 Hz).

[0340] Synthesis of compound 7b A 228 mL solution of anhydrous THF containing compound 5b (10.15 g, 28.5 mmol) was added dropwise to a 342 mL solution of 0.5 M 9-BBN / THF (171 mmol) at 0°C for 20 minutes. The mixture was stirred at room temperature for 4 hours, then the solution was cooled on ice, methanol (131 mL) was added, and the mixture was stirred until no more foaming occurred. Next, H₂O (197 mL) was added dropwise for 15 minutes with vigorous stirring to prevent precipitation of the intermediate compound. NaBO₃·4H₂O (21.9 g, 142.5 mmol) was added all at once at 0°C, and the mixture was stirred overnight at room temperature. After evaporating the excess THF, the resulting crude mixture was dissolved in excess ethyl acetate and repeatedly washed with saturated aqueous NH₄Cl solution. After evaporating the organic layer, the resulting substance was dissolved in THF (450 mL) and H₂O (450 mL). To this solution, NaBO3·4H2O (21.9 g, 142.5 mmol) was added all at once at room temperature, and the mixture was stirred overnight at room temperature. After evaporating the excess THF, ethyl acetate was added to the mixture and extracted. The resulting organic layer was repeatedly washed with saturated aqueous NH4Cl solution, dried over MgSO4, filtered, and evaporated. The resulting crude material was purified by silica gel column chromatography (CH2Cl2 / methanol, 100:0 to 93:7) to obtain compound 6b as a syrup (2.44 g, including reagent impurities); HRMS (ESI)C 16 H 28 FN2O5Si + [M + H] + The calculated m / z value for this compound was 375.1746, and the measured value was also 375.1746. Compound 6b, containing reagent impurities, was anhydrous by repeated co-evaporation with anhydrous pyridine under an argon atmosphere, and then dissolved in anhydrous pyridine (64 mL). DMTrCl (2.64 g, 7.79 mmol) was added to this solution, and the mixture was stirred at room temperature for 1 hour. After quenching the reaction with methanol (5 mL), the reaction mixture was diluted with ethyl acetate (300 mL), washed with saturated aqueous NaHCO3 solution, dried over MgSO4, filtered, evaporated, and co-evaporated three times with toluene to remove residual pyridine. The resulting crude substance was purified by silica gel column chromatography (hexane / ethyl acetate, 2:8 to 4:6) to obtain compound 7b as a white solid (2 steps, 2.25 g, 12%).1 1H NMR (500 MHz, CD3CN) δ 9.16 (broad singlet, 1H), 7.43 - 7.42 (multiplet, 2H), 7.31 - 7.28 (multiplet, 8H), 6.86 - 6.85 (multiplet, 4H), 5.75 (doublet of doublets, 1H, J HF = 20.0 Hz, J HH = 1.9 Hz), 5.59 (doublet, 1H, J = 8.1 Hz), 4.96 (doublet of doublets of doublets, J HF = 53.3 Hz, J HH = 4.6, 1.8 Hz), 4.06 - 3.98 (multiplet, 2H), 3.76 (singlet, 6H), 3.19 (doublet, 2H, J = 7.4, 5.6 Hz), 2.09 - 2.02 (multiplet, 1H), 1.89 - 1.82 (multiplet, 1H), 0.91 (singlet, 9H), 0.10 (singlet, 3H), 0.09 (singlet, 3H); 13 13C NMR (125 MHz, CD3CN) δ 163.9, 159.6, 151.1, 146.4, 141.9, 137.31, 137.26, 130.92, 130.89, 128.9, 128.8, 127.8, 114.0, 102.9, 93.7 (doublet, J CF = 188.0 Hz), 90.6 (doublet, J = 36.4 Hz), 87.0, 80.7, 74.6 (doublet, J = 15.4 Hz), 60.9, 55.9, 33.8, 26.1, 18.7, -4.5, -4.8; 19 19F NMR (470 MHz, CD3CN) δ -201.4 (doublet of doublets of doublets, J = 53.7, 19.1, 19.1 Hz); HRMS (ESI), C 37 19 45 19F N2O7Na [M + Na] + calculated m / z 699.2872 for, measured m / z 699.2866.

[0341] Synthesis of Compound 8b Compound 7a (2.24 g, 3.30 mmol) was dissolved in THF (36.0 mL), and 1.0 M TBAF-THF solution (4.0 mL, 4.0 mmol) was added. The mixture was stirred at room temperature for 30 minutes. The excess THF was evaporated, and after co-evaporation with CH2Cl2, the crude substance was purified by silica gel column chromatography [CH2Cl2 (1% TEA)-methanol 100:0 to 95:5] to obtain compound 8b (1.51 g, 81%). 1 H NMR (500 MHz, CDCl3) δ 9.20 (br-s, 1H), 7.45-7.43 (m, 2H), 7.32-7.13 (m, 8H), 6.87-6.85 (m, 4H), 5.77 (dd, 1H, JHF = 20.1 Hz, JHH = 1.5 Hz), 5.60 (d, 1H, J = 8.1 Hz), 4.98 (ddd, JHF= 51.0 Hz, JHH = 4.6, 1.6 Hz), 4.04-3.92 (m, 2H), 3.76 (s, 6H), 3.23-3.17 (m, 2H), 2.20 (br-s, 1H), 2.11-2.06 (m, 1H), 1.91-1.87 (m, 1H); 13 C NMR (125 MHz, CD3CN) δ 164.1, 159.7, 151.2, 146.4 141.7, 139.0, 137.33, 137.29, 131,00, 130.97, 129.3, 129.0, 128.9, 127.8, 126.3, 114.1, 103.0, 94.7 (d, J = 184.4 Hz), 90.3 (d, J = 35.4 Hz), 87.2, 80.5, 73.9 (d, J = 16.4 Hz), 61.0, 56.0, 33.9; 19 F NMR (470 MHz, CD3CN) δ -201.8 (ddd, J = 53.7, 20.8, 20.8 Hz).

[0342] Synthesis of compound 9b Compound 8 (1.5 g, 2.67 mmol) was anhydrous by repeated co-evaporation with anhydrous CH3CN, and then dissolved in anhydrous CH2Cl2 (30 mL). To this solution, N,N-diisopropylethylamine (1.76 mL, 10.1 mmol) and 2-cyanoethyl N,N-diisopropyl chlorophosphoramidite (0.90 mL, 4.01 mmol) were added at 0°C. After stirring at room temperature for 2 hours, CH2Cl2 (70 mL) and then saturated aqueous solution of NaHCO3 (100 mL) were added to the reaction mixture. The organic layer was repeatedly washed with saturated aqueous solution of NaHCO3, dried over MgSO4, filtered, and evaporated. The resulting crude material was purified by silica gel column chromatography (1% TEA-hexane-ethyl acetate, 80:20~20:80) to obtain compound 9a, which contains impurities such as phosphytylation reagent residue. To remove impurities, the obtained substance was dissolved in Et2O (100 mL) and then repeatedly washed with a saturated aqueous solution of NaHCO3 to obtain compound 9b as a white solid (1.47 g, 64%). 31 P NMR (202 MHz, CDCl3) δ 150.4 (d, J = 9.0 Hz), 149.9 (d, J = 10.0 Hz); 19 F NMR (470 MHz, CD3CN) δ -198.61, -198.63, -198.66, -198.68, -198.70, -198.73, -198.75, -198.77, -198.79, -198.82, -198.84, -199.04, -199.06, -199.08, -199.10, -199.12, -199.14, -199.15, -199.17, -199.20, -199.21, -199.24, -199.26.

[0343] Example 3. Synthesis of exNA-C phosphoramidite As shown in Figure 4, the starting material is first converted to a cytidine derivative (Kaura, M. et al. J. Org. Chem. 2014, 79, 6256-6268), and the 4-amino group of the resulting cytosine base is protected by an acyl protecting group such as acetyl. After deprotection of 3'-O-TBDMS, the resulting 3'-hydroxyl group is converted to a 3'-O-phosphoramidite. Each step is first quenched and extracted, and then purified by silica gel column chromatography.

[0344] Example 4. Synthesis of exNA-G and exNA-A phosphoramidites As shown in Figure 5, the 3'-O-TBDMS-protected starting material is first oxidized to an aldehyde using IBX, then subjected to Wittig olefination using an anhydrous THF solution containing methyltriphenylphosphonium bromide and tert-BuOK to produce a vinyl-substituted nucleoside derivative. This vinyl group reacts with 9-BBN to form a borylation intermediate, which proceeds to oxidation with sodium perborate to produce an exNA structure with a 6'-hydroxyl group. This hydroxyl group is first protected by a DMTr group, and the 3'-O-TBDMS group is deprotected with a 0.1 M TBAF-THF solution without silica gel column purification. The resulting 6'-O-DMTr nucleoside derivative is phosphytylated to produce a phosphoramidite. Except for the 6'-O-tritylation step, the product is first quenched and extracted, and then purified by silica gel column chromatography.

[0345] Example 5. Synthesis of 5'-3'-bis-methylene-exNA phosphoramidite As shown in Figure 6, the primary hydroxyl group of the starting material having a Nap-protected hydroxymethyl group (Betkekar, VVet al. Org. Lett. 2012, 14, 1, 198-201) is first selectively protected by a TBDPS group, followed by deoxygenation of the secondary alcohol (Prakash, TP et al. Nucleic Acids Res. 2015, 43, 2993-3011). Next, the TBDPS group is converted to a benzoyl (Bz) protecting group by deprotection in a 0.1 M TBAF-THF solution and benzoylation using pyridine containing benzoyl chloride. Then, the isopropylidene protecting group of the sugar is deprotected to produce a 1,2-bis-acetylated sugar, followed by conventional BSA / TMSOTf-mediated glycosylation of uracil to obtain a uridine nucleoside derivative. The Nap protecting group of the 3'-hydroxymethyl group is deprotected by DDQ. The resulting substance having a 6'-O-Bz-3'-hydroxymethyl group is converted to a 6'-O-DMTr-3'-TBDMS-protected hydroxymethyl compound having 2'-O-acetyl protection. After deprotection of the TBDMS group, the 3'-hydroxymethyl group is phosphylylated to obtain a 5'-3'-bis-exNA-phosphoramidite. Each step is first quenched and extracted, and then purified by silica gel column chromatography.

[0346] Example 6. Synthesis of exNA-ribo-uridine phosphoramidite The following synthesis was completed according to Figure 7. IBX (30.3 g, 108.2 mmol) was added to an anhydrous CH3CN solution (520 mL) containing compound 2 (15.4 g, 54.1 mmol), and the mixture was stirred at 85°C for 2 hours. After cooling the mixture in an ice bath, the precipitate in the solution was filtered off through Celite. The collected eluent was evaporated and then evaporated together with anhydrous CH3CN three times under an argon atmosphere. Compound 3, obtained as a white foam, was used without further purification. In a separate flask, methyltriphenylphosphonium bromide (43.3 g, 121.2 mmol) was added all at once at 0°C to an anhydrous THF solution (500 mL) containing tert-BuOK (13.2 g, 117.4 mmol), and the mixture was stirred at 0°C for 1 hour. To this solution, an anhydrous THF solution (150 mL) of compound 3 was added dropwise at 0°C (10 minutes), and the mixture was stirred at room temperature for 4 hours. After evaporating the excess THF, the resulting mixture was dissolved in excess ethyl acetate, washed with saturated NH4Cl aqueous solution, dried over MgSO4, filtered, and evaporated. The obtained substance was dissolved in the minimum amount of CH2Cl2 and added dropwise to an excess diethyl ether solution with vigorous stirring at 0°C. The precipitate in the solution was filtered through Celite, and the eluent was evaporated. The resulting crude substance was purified by silica gel column chromatography (hexane / ethyl acetate, 8:2-3:7) to obtain compound 4, which contained triphenylphosphine oxide impurities. Three-quarters of this crude substance was anhydrous by repeated co-evaporation with anhydrous CH3CN, and then dissolved in anhydrous THF (200 mL). To this solution, 0.5M9-BBN / THF (300 mL, 150.0 mmol) was added dropwise for 10 minutes, and the mixture was stirred overnight at room temperature. After confirming the disappearance of the starting material by TLC, the solution was cooled on ice, and methanol (200 mL) was added dropwise over 10 minutes. After the foaming stopped, H2O (300 mL) was added dropwise, followed by the addition of NaBO3·4H2O (19.2 g, 125.0 mmol) all at once. The solution was stirred overnight at room temperature. After evaporating the excess THF, the resulting crude mixture was dissolved in excess ethyl acetate and repeatedly washed with saturated NH4Cl aqueous solution. After evaporating the organic layer, the resulting substance was dissolved in THF (400 mL) and H2O (400 mL). To this solution, NaBO3·4H2O (19.2 g, 125.0 mmol) was added all at once at room temperature, and the mixture was stirred overnight at room temperature.After evaporating the excess THF, ethyl acetate was added to the mixture and extracted. The resulting organic layer was repeatedly washed with saturated NH4Cl aqueous solution, dried over MgSO4, filtered, and evaporated. The resulting crude material was purified by silica gel column chromatography (CH2Cl2-methanol, 100:0 to 93:7) to produce compound 5, which contained impurities from the reagent residue. TFA solution [TFA (85 mL) and H2O (9.2 mL)] was added to this substance and stirred at 0°C for 1 hour. After evaporation and co-evaporation with toluene four times, the crude material was purified by silica gel column chromatography (CH2Cl2-MeOH, 100:0 to 90:10) to obtain compound 6 (760 mg, 12%) in three steps. 1 H NMR (500 MHz, DMSO-d6) δ 11.4 (br-s, 1H), 7.58 (d, 1H, J = 5.0 Hz), 5.71 (d, 1H, J = 5.0 Hz), 5.64 (dd, 1H, J = 8.0, 2.2 Hz), 5.34 (d, 1H, J = 5.2 Hz), 5.09 (d, 1H, J = 4.7 Hz), 4.51 (br-s, 1H), 4.06, (dd, 1H, J = 9.8, 4.9 Hz), 3.80-3.78 (m, 1H), 3.53-3.45 (m, 2H), 1.84-1.70 (m, 2H); 13 C NMR (125 MHz, DMSO-d6) δ 163.5, 151.1, 141.6, 102.5, 89.0, 80.9, 73.5, 73.2, 58.0, 46.2, 36.8, 9.1;HRMS (ESI), C 10 H 14 N2O6Na [M + Na] + The calculated value for m / z is 281.0744, and the measured value for m / z is 281.0730.

[0347] Synthesis of Compound 7 Compound 6 (760 mg, 2.94 mmol) was mixed with anhydrous pyridine (30 mL), followed by DMTr-Cl (1.3 g, 3.82 mmol). After stirring for 2 hours, the reaction mixture was first extracted with CH2Cl2, then with saturated aqueous NaHCO3 solution. The organic layer was dried over MgSO4, filtered, evaporated, and co-evaporated to remove pyridine. The resulting crude product was purified by silica gel column chromatography (CH2Cl2-MeOH 100:0~95:5) to obtain compound 7 (1.70 g, quantitative). HRMS (ESI), C 31 H 32 N2O8Na [M + Na] + The calculated value for m / z is 583.2051, and the measured value for m / z is 583.2025.

[0348] Synthesis of compound 8 To a 21 mL solution of anhydrous pyridine containing compound 7 (2.35 g, 4.19 mmol), imidazole (576.1 mg, 8.46 mmol) and TBDMSCl (1.10 g, 7.33 mmol) were added, and the mixture was stirred at room temperature for 2 hours. To this reaction mixture, CH2Cl2 (150 mL) and then saturated aqueous solution of NaHCO3 (150 mL) were added. The organic layer was washed with saturated aqueous solution of NaHCO3, dried over MgSO4, filtered, evaporated, and then co-evaporated with toluene to remove the pyridine residue. The resulting crude product containing compound 8, the 3'-O-TBDMS protected compound, and the 5'-3'-O-bis-TBDMS protected compound were separated by silica gel column chromatography [CH2Cl2 (1% TEA) - acetone 100:0 to 85:15] to obtain pure compound 8 (780 mg, 28%). 11H NMR (500 MHz, DMSO-d6) δ 11.4 (br-s, 1H), 7.53 - 7.52 (m, 2H), 7.39 - 7.22 (m, 8H), 6.89 - 6.88 (m, 4H), 5.72 (d, 1H, J = 5.0 Hz), 5.62 (d, 1H, J = 8.1, 2.0 Hz), 5.00 (d, 1H, J = 6.0 Hz), 4.19 (dd, 1H, J = 5.1, 5.1 Hz), 3.92 (ddd, 1H, J = 8.8, 8.8, 4.5 Hz), 3.78 - 3.73 (m, 7H), 3.07 - 3.03 (m, 2H), 2.05 - 1.83 (m, 2H), 0.83 (s, 9H), 0.05 (s, 3H), 0.01 (s, 3H); 13 13C NMR (125 MHz, DMSO-d6) δ 162.9, 158.0, 150.5, 145.1, 140.7, 135.8, 130.1, 129.4, 128.7, 128.3, 128.1, 127.1, 125.8, 113.6, 102.5, 88.8, 86.0, 81.5, 74.9, 73.4, 60.6, 55.5, 33.8, 26.1, 25.1, 18.4; HRMS (ESI), C 37 1 46 18H28N2O8Na [M + Na] + Calculated m / z 697.2916 for [M + Na], found m / z 697.2867.

[0349] Synthesis of Compound 9 Compound 8 (780 g, 1.16 mmol) was anhydrous by repeated co-evaporation with anhydrous CH3CN, and then dissolved in anhydrous CH2Cl2 (12 mL). To this solution, N,N-diisopropylethylamine (0.53 mL, 4.34 mmol) and 2-cyanoethyl N,N-diisopropyl chlorophosphoramidite (0.34 mL, 1.73 mmol) were added at 0°C. After stirring at room temperature for 4 hours, CH2Cl2 (90 mL) and then saturated aqueous solution of NaHCO3 (100 mL) were added to the reaction mixture. The organic layer was repeatedly washed with saturated aqueous solution of NaHCO3, dried over MgSO4, filtered, and evaporated. The resulting crude material was purified by silica gel column chromatography (1% TEA-hexane-ethyl acetate, 80:20 to 50:50) to obtain compound 9 (825.9 mg, 82%). 31 P NMR (202 MHz, CDCl3) δ 149.6, 149.1.

[0350] Example 7. Synthesis of exNA-ribo-cytosine phosphoramidite The starting material containing the vinyl-substituted uridine derivative is first converted to cytidine (Kaura, M. et al. J. Org. Chem. 2014, 79, 6256-6268), and the 4-amino group of the resulting cytosine base is protected by an acyl protecting group such as acetyl. After deprotection of 2'-3'-O-isopropylidene, the 6'-hydroxyl group is protected by DMTr and then by TBDMS. The silica gel column-separated 2'-O-TBDMS-protected compound is phosphytylated to obtain 3'-O-phosphoramidite. Each step is first quenched and extracted, and then purified by silica gel column chromatography.

[0351] Example 8. Synthesis of exNA-riboguanosine or exNA-ribo-adenine phosphoramidite According to Figure 9, the 2'-3'-O-bis-TBDMS-protected starting material is first oxidized to an aldehyde using IBX, then subjected to Wittig olefination using an anhydrous THF solution containing methyltriphenylphosphonium bromide and tert-BuOK to produce a vinyl-substituted nucleoside derivative. This vinyl group reacts with 9-BBN to form a borylation intermediate, which proceeds to oxidation with sodium perborate to produce an exNA structure with a 6'-hydroxyl group. This hydroxyl group is first protected by a DMTr group and then by TBDMS. The 2'-O-TBDMS-protected compound is separated by silica gel column chromatography and phosphytylated to obtain a 3'-O-phosphoramidite. Each step is first quenched and extracted, followed by purification by silica gel column chromatography. Without silica gel column purification, the 3'-O-TBDMS group is deprotected with a 0.1 M TBAF-THF solution. The resulting 6'-O-DMTr nucleoside derivative is phosphytylated to produce a phosphoramidite. Each step is first quenched and extracted, and then purified by silica gel column chromatography.

[0352] Example 9. Synthesis of exNA-ribo-uridine phosphoramidite According to Figure 10, the starting material protected with 5'-O-DMTr is first protected with TBDMS and then detritylated with 5'-O-. The resulting compound is then oxidized with IBX to an aldehyde and subjected to Wittig olefination using an anhydrous THF solution containing methyltriphenylphosphonium bromide and tert-BuOK to produce a vinyl-substituted nucleoside derivative. This vinyl group reacts with 9-BBN to form a borylation intermediate, which proceeds to oxidation with sodium perborate to produce an exNA structure with a 6'-hydroxyl group. This hydroxyl group is first protected with a DMTr group, and the 3'-O-TBDMS group is deprotected with a 0.1 M TBAF-THF solution without silica gel column purification. The resulting 6'-O-DMTr nucleoside derivative is phosphytylated to produce a methyl-protected phosphoramidite. Each step, with the exception of the initial 3'-O-TBDMS protection step, involves first quenching and extraction, followed by purification by silica gel column chromatography.

[0353] Example 10. Synthesis of oligonucleotides incorporating the exNA skeleton. A method for synthesizing modified oligonucleotides including a 5' terminus, a 3' terminus, and at least one linkage between modified subunits has been implemented according to Figure 12. This method involves (a) providing a nucleoside having a 5'-protecting group bound to a solid support; (b) removing the protecting group; and (c) combining the deprotected nucleoside with a phosphoramidite derivative of formula (VII) to form a triester phosphite. [ka] (VII) (d) capping the phosphite triester; (e) oxidizing the phosphite triester; (f) repeating steps (b) through (e) using an additional phosphoramidite; (g) cleaving from a solid support.

[0354] Figure 13 shows some examples of oligonucleotides synthesized by the above method having one or more exNA subunit linkages. The exNA subunit linkages are 5'-methylene-exNA-uridine and 2'-OH.

[0355] Example 11: Efficacy of in vitro silencing of target mRNA by siRNA double-stranded nucleotides containing exNA subunit linkages. ex-NA subunit linkages were used in oligonucleotide walking experiments, and each subunit linkage of the antisense and sense strands was modified with ex-NA subunit linkages. The ex-NA subunit linkages were one of the following: (ex_mU): 5'-methylene-exNA-uridine and 2'-OMe or (ex_fU) 5'-methylene-exNA-uridine and 2'-fluoro-ex-uridine. Tables 4-10 below show the antisense and sense strands used in this example, as well as the double strands formed by different combinations of antisense and sense strands. A novel synthetic scheme for generating ex-NA-containing oligonucleotides was also used, as shown in Figure 12. [Table 4-1] [Table 4-2] [Table 5] [Table 6] [Table 7] [Table 8] [Table 9] [Table 10]

[0356] The above siRNA double-stranded molecules were used in in vitro mRNA silencing experiments to determine their relative silencing effectiveness. Details of the experiment are described below.

[0357] In vitro screening.

[0358] 1.5 μM siRNA was passively delivered to cells. Cells were plated in Dulbecco's Modified Eagle Medium containing 6% FBS at a rate of 8,000 cells / well in 96-well cell culture plates. The siRNA was diluted to twice its final concentration in OptiMEM (Carlsbad, CA; 31985-088), and 50 μL of diluted siRNA was added to 50 μL of cells to achieve a final concentration of 3% FBS. Cells were incubated at 37°C and 5% CO2 for 72 hours.

[0359] Quantitative analysis of target mRNA. mRNA was quantified from cells using the QuantiGene 2.0 assay kit (Affymetrix, QS0011). Cells were lysed at 55°C for 30 minutes in 250 μL of a dilution lysate consisting of 1 part lysate (Affymetrix, 13228), 2 parts H2O, and 0.167 μg / μL of proteinase K (Affymetrix, QS0103). The cell lysates were thoroughly mixed, and 40 μL of each lysate was added to the wells of a capture plate containing 40 μL of a dilution lysate without proteinase K and 20 μL of a dilution probe set. The probe sets for human HTT and hypoxanthine phosphoribosyltransferase (HPRT) (Affymetrix; #SA-50339, SA-10030) were diluted and used according to the recommended protocol in the manufacturing description. The dataset was normalized to HPRT.

[0360] Cell processing: Reporter assay.

[0361] HeLa cells were grown and maintained in Gibco DMEM (reference #11965-092) containing 1% pen / strep and 10% thermally inactivated FBS. Three days before treatment, two 10 cm sheets were prepared. 2 2x10 in the dish 6 HeLa cells were plated. The following day, the DMEM was replaced with Gibco OptiMEM (reference #31985-070), and 6 μg of reporter plasmid was added to the cells using Invitrogen Lipofectamine 3000 (reference #L3000-015) according to the manufacturer's protocol. The cells were left in OptiMEM / lipofectamine overnight to maximize reporter plasmid transfection. The following day, the siRNA was diluted in OptiMEM and added three times to 96-well white-walled clear-bottom tissue culture plates for each reporter plasmid. HeLa cells transfected with reporter plasmids the previous night were placed in DMEM containing 6% heat-inactivated FBS (without pen / strep) in a 0.15 x 10⁶ solution. 6 The cells were resuspended at a concentration of 1 / mL and added to a plate containing siRNA.

[0362] Cells were treated with 1x Passive Lysis Buffer from the Dual-Luciferase Assay System Pack (Promega ref.#E1960) for 72 hours (until 100% confluence) and then lysed. After lysing, 50 μl of Luciferase Assay Reagent II (Promega ref.#E1960) was added, and luminescence was read. A second reading was then performed after adding 50 μL / well of Stop and Glow reagent (Promega ref.#E1960). Absorbance was normalized to the untreated control and graphed on a logarithmic scale.

[0363] As shown in Figure 14, all tested siRNA double strands effectively silenced the target HTT mRNA. Furthermore, numerous siRNA double strands silenced both the target mRNA and the control double-stranded siRNA. This data represents the first example of ex-NA nucleotide linkages incorporated into oligonucleotide strands.

[0364] Example 12: Nuclease stability of siRNA double strands containing exNA subunit linkages. It was hypothesized that ex-NA subunit linkages are useful in increasing the nuclease stability of oligonucleotides. This effect can be observed with ex-NA subunit linkages alone or in combination with phosphorothioate subunit linkages. Furthermore, multiple consecutive ex-NA subunit linkages in an oligonucleotide may have a greater impact on stability than a single ex-NA subunit linkage. There are two main ways in which stability may be increased: 1) by reducing the kinetics of nuclease cleavage due to abnormalities in the local scaffold structure of ex-NA, and 2) by reducing the nuclease binding affinity (3'-terminal region) due to multiple elongated scaffolds (Figure 15). To demonstrate this effect, several nuclease assays were performed using oligonucleotides containing one or more ex-NA subunit linkages.

[0365] 3'-exonuclease stability test.

[0366] Oligonucleotides with a different number of ex-NA subunit linkages at the 3' end were tested in a 3' exonuclease stability test. Oligonucleotides ex-21, ex-22, ex-23, ex-24, AS-0, and AS-2 (listed in Tables 4 and 6 above) at a concentration of 17.5 mM were incubated at 37°C in a buffer containing 10 mM Tris-HCl (pH 8.0), 2 mM MgCl2, and snake venom phosphodiesterase I (20 mU / mL). As shown in Figure 16, multiple ex-NA subunit linkages with a phosphorothioate subunit linkage (ex-24) significantly improved the 3'-exonuclease stability compared to AS-2, which had the same phosphorothioate content as found in clinically approved siRNA drugs. Furthermore, even a single ex-NA subunit linkage at the 3' end significantly improved stability (ex-21). Since 3'-exonuclease is dominant in serum, 3'ex-NA subunit linkages are useful for therapeutic oligonucleotides.

[0367] Additional 3' exonuclease tests were performed using ex-NA subunit linkages in the context of polyuridyl sequences containing oligonucleotides with phosphodiesters (PO) and phosphorothioates (PS). Oligonucleotides were tested with 1, 2, 3, 4, or 5 ex-NA subunit linkages. Table 11 below lists the polynucleotides used in this test. As shown in Figure 17, even the presence of a single ex-NA subunit linkage significantly improved the stability of the oligonucleotide. This was demonstrated for both PO and PS oligonucleotides. Furthermore, PO-containing oligonucleotides with 5 ex-NA subunit linkages achieved similar nuclease stability compared to PS-containing oligonucleotides without ex-NA subunit linkages (PS control). This result suggests that using ex-NA subunit linkages may reduce the number of PS-containing subunit linkages, thereby potentially reducing the toxicity associated with PS-containing oligonucleotides. [Table 11]

[0368] The fluorescein-labeled "FAM" used for oligonucleotides did not affect 3'-exonuclease activity and was used to monitor cleavage in stability testing.

[0369] 5'-exonuclease stability test.

[0370] Oligonucleotides having ex-NA subunit linkages at the 5' end were tested using two different 5' exonuclease stability tests.

[0371] The first test was a 5'-phosphate-dependent 5'-exonuclease stability test. The oligonucleotides used in this test are shown in Table 12 below. Oligonucleotides were used at 2.5 μM (50 pmol) and incubated with RNase-free water or with 3.3 units of Terminator® (EpiCentre) exonuclease in Buffer A (provided by EpiCentre with Terminator® enzyme) at 37°C. As shown in Figure 18, a single ex-NA subunit linkage (ON2) at the 5' end significantly improved the stability of the 5'-exonuclease compared to ON1, which contained a 5'-phosphodiester bond. Importantly, ON2 did not contain a phosphorothioate subunit linkage. This data indicates that a single ex-NA subunit linkage at the 5' end improves stability to a similar extent as multiple phosphorothioate subunit linkages (ON3) at the 5' end. Excessive phosphorothioate content in therapeutic oligonucleotides can be toxic. Using 5'ex-NA subunit linkages provides a mechanism to improve oligonucleotide stability, while reducing the phosphorothioate content.

[0372] The second 5'-exonuclease stability test was a 5'-phosphate-independent 5'-exonuclease stability test. The oligonucleotides used in this test are shown in Table 13 below. Oligonucleotides were used at 10 μM and incubated at 37°C with RNase-free water or 30 mM NaOAc (pH 6.0) buffer containing 0.25 U / mL bovine spleen phosphodiesterase II (BSP). As shown in Figure 19, a single ex-NA subunit linkage at the 5' end (ON4) exhibited similar 5'-exonuclease stability compared to ON5 containing multiple 5' phosphorothioate linkages. This data indicates that a single ex-NA subunit linkage at the 5' end improves stability to a similar degree as multiple phosphorothioate subunit linkages at the 5' end (ON5). Excessive phosphorothioate content in therapeutic oligonucleotides can be toxic. Using 5'ex-NA subunit linkages provides a mechanism to improve oligonucleotide stability, while reducing the phosphorothioate content. [Table 12] [Table 13]

[0373] Example 13 Activity of siRNA double strands containing interlinking of one or more antisense strand 3' terminal exNA subunits The in vitro silencing activity of several siRNA duplexes containing one or more antisense strand 3'-terminal exNA subunit ligatures was tested. Antisense strands containing one, two, three, or four 3'-terminal exNA subunit ligatures were used in dose-response curves, as shown in Figure 20A. The percentage change in potency compared to an siRNA duplex control without exNA subunit ligatures was also determined (Figure 20B). The data indicate that siRNA duplexes containing one, two, three, or four 3'-terminal exNA subunit ligatures possess higher silencing efficacy than siRNA duplexes containing antisense strands without exNA subunit ligatures.

[0374] Example 14: In vivo activity of siRNA double strands containing interlinks between one or more antisense strand 3' terminal exNA subunits. The in vivo silencing activity of several siRNA duplexes containing inter-exNA subunit linkages at the 3' end of one or more antisense strands was tested. As described above, the siRNA duplexes were in Di-siRNA format. The sequences and chemical modification patterns are shown in Table 14 below. Each siRNA targeted ApoE mRNA. 5 nmol of each Di-siRNA was administered to mice by ICV injection, and ApoE mRNA was quantified after 1 month. As shown in Figures 21A-21E, siRNAs containing inter-exNA subunit linkages were able to silence ApoE in several brain regions (medial cortex, striatum, hippocampus, thalamus, and cerebellum). The silencing efficacy of siRNA duplexes with low phosphorothioate (PS) content was largely maintained or improved by including inter-exNA subunit linkages. [Table 14]

[0375] Additional in vivo silencing activity experiments were performed using Di-sRNA double strands targeting Htt mRNA. The chemical modification patterns employed are listed below. Wild-type male mice were treated with approximately 60 μg of siRNA for 2 months, and Htt mRNA and protein levels were quantified in several brain regions (medial cortex, striatum, hippocampus, thalamus, and prefrontal cortex). siRNA double strands with an antisense strand containing one or two exNA nucleotide ligatures showed equivalent or superior silencing of Htt mRNA (Figures 22A-22E) and protein (Figures 23A-23E) expression compared to siRNA double strands without exNA nucleotide ligatures. ExNA nucleotide ligatures, which provide higher nuclease resistance than phosphorothioate modifications, can reduce toxic phosphorothioate modifications without sacrificing nuclease resistance or silencing effectiveness. The chemical modification patterns used in Figures 22-23 are as follows: 1-High PS: Antisense chain (5' to 3'): VP(mX)#(fX)#(mX)(fX)(fX)(fX)(mX)(fX)(mX)(fX)(mX)(fX)(mX)(fX)#(mX)#(fX)#(mX)#(mX)#(mX)#(fX)#(mX) Sense chain (5' to 3'): (mX)#(mX)#(mX)(fX)(mX)(fX)(mX)(fX)(mX)(fX)(mX)(mX)(mX)(fX)#(mX)#(mX) 2-Low PS fm: Antisense chain (5' to 3'): VP(mX)#(fX)#(mX)(fX)(fX)(fX)(mX)(fX)(mX)(fX)(mX)(fX)(mX)(fX)(mX)(fX)(mX)(mX)(mX)#(fX)#(mX) Sense chain (5' to 3'): (mX)#(mX)#(mX)(fX)(mX)(fX)(mX)(fX)(mX)(fX)(mX)(mX)(mX)(fX)#(mX)#(mX) 3-Low PS mf: Antisense chain (5' to 3'): VP(mX)#(fX)#(mX)(fX)(fX)(fX)(mX)(fX)(mX)(fX)(mX)(fX)(mX)(fX)(mX)(fX)(mX)(mX)(mX)#(mX)#(fX) Sense chain (5' to 3'): (mX)#(mX)#(mX)(fX)(mX)(fX)(mX)(fX)(mX)(fX)(mX)(mX)(mX)(fX)#(mX)#(mX) 4-Low PS mf 2 exNA: Antisense chain (5' to 3'): VP(mX)#(fX)#(mX)(fX)(fX)(fX)(mX)(fX)(mX)(fX)(mX)(fX)(mX)(fX)(mX)(fX)(mX)(mX)(mX)#(ex-mX)#(ex-fX) Sense chain (5' to 3'): (mX)#(mX)#(mX)(fX)(mX)(fX)(mX)(fX)(mX)(fX)(mX)(mX)(mX)(fX)#(mX)#(mX) 5-Low PS mf 1 exNA: Antisense chain (5' to 3'): VP(mX)#(fX)#(mX)(fX)(fX)(fX)(mX)(fX)(mX)(fX)(mX)(fX)(mX)(fX)(mX)(fX)(mX)(mX)(mX)#(mX)#(ex-fX) Sense chain (5' to 3'): (mX)#(mX)#(mX)(fX)(mX)(fX)(mX)(fX)(mX)(fX)(mX)(mX)(mX)(fX)#(mX)#(mX) In the case of the five chemical modification patterns above, "VP" corresponds to 5' vinyl phosphonate; "mX" corresponds to any nucleotide (A, U, G, or C) with 2'-O-methyl modification; "fX" corresponds to any nucleotide (A, U, G, or C) with 2'-fluoro modification; "#" corresponds to phosphorothioate modification; "ex-mX" corresponds to any nucleotide (A, U, G, or C) with 2'-O-methyl modification and exNA nucleotide linkage; "ex-fX" corresponds to any nucleotide (A, U, G, or C) with 2'-fluoro modification and exNA nucleotide linkage.

[0376] Reference The content of all references that may be cited throughout this application (including references to documents, patents, patent applications, and websites) is expressly incorporated by reference in their entirety for any purpose, just as the references described herein are expressly incorporated. Unless otherwise specified, this disclosure uses immunological, molecular biological, and cell biological techniques that are well known as prior art.

[0377] This disclosure also incorporates, by reference, a whole range of techniques well known in the fields of molecular biology and drug delivery. These techniques include, but are not limited to, those described in the following publications: Atwell et al.J.Mol.Biol.1997,270:26-35; Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993); Ausubel,FMet al.eds.,Short Protocols In Molecular Biology(4th Ed.1999)John Wiley&Sons,NY.(ISBN 0-471-32938-X); CONTROLLED DRUG BIOAVAILABILITY, DRUG PRODUCT DESIGN AND PERFORMANCE, SMOLEN AND BALL (EDS.), WILEY, NEW YORK (1984); Giege, R. and Ducruix, A. Barrett, Crystallization of Nucleic Acids and Proteins, a Practical Approach, 2nd ea., pp. 201-16, Oxford University Press, New York, New York, (1999); Goodson, in Medical Applications of Controlled Release, vol. 2, pp. 115-138 (1984); Hammerling, et al.,: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, N.Y., 1981; Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Kabat et al., Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987) and (1991); Kabat, E.A., et al. (1991) SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242; Kontermann and Dubel eds., ANTIBODY ENGINEERING (2001) Springer-Verlag. New York. 790 pp. (ISBN 3-540-41354-5). Kriegler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990); Lu and Weiner eds., CLONING AND EXPRESSION VECTORS FOR GENE FUNCTION ANALYSIS (2001) BioTechniques Press. Westborough, MA. 298 pp. (ISBN 1-881299-21-X). MEDICAL APPLICATIONS OF CONTROLLED RELEASE, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla. (1974); Old, R.W. & S.B. Primrose, PRINCIPLES OF GENE MANIPULATION: AN INTRODUCTION TO GENETIC ENGINEERING (3d Ed. 1985) Blackwell Scientific Publications, Boston. Studies in Microbiology;V.2:409 pp. (ISBN 0-632-01318-4). Sambrook, J. et al. eds., MOLECULAR CLONING: A LABORATORY MANUAL (2d Ed. 1989) Cold Spring Harbor Laboratory Press, NY. Vols. 1-3. (ISBN 0-87969-309-6). SUSTAINED AND CONTROLLED RELEASE DRUG DELIVERY SYSTEMS, JR Robinson, ed., Marcel Dekker, Inc., New York, 1978 Winnacker, EL FROM GENES TO CLONES: INTRODUCTION TO GENE TECHNOLOGY (1987) VCH Publishers, NY (translated by Horst Ibelgaufts). 634 pp. (ISBN 0-89573-614-4).

[0378] Equal parts This disclosure may be embodied in other specific forms without departing from its spirit or essential features. Therefore, the embodiments described above should be considered illustrative in all respects and not limiting to this disclosure. Accordingly, the scope of this disclosure is indicated not by the foregoing description but by the appended claims, and all modifications that fall within the meaning of the claims and the scope of equivalents are intended to be incorporated herein.

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