Synthesis of modified oligonucleotides with increased stability

Abstract

The invention relates to synthesis of modified oligonucleotides with increased stability. The present disclosure relates to the synthesis of novel modified oligonucleotides. Synthesis of novel phosphoramidite is also provided.

Description

[0001] This application is a divisional application of Chinese patent application filed on March 26, 2021, with application number "202180037628.7" and invention title "Synthesis of Modified Oligonucleotides with Increased Stability". The original application was the Chinese national phase application of international application PCT / US2021 / 024425.

[0002] Cross-reference to related applications

[0003] This application claims the benefit of U.S. Provisional Application Serial No. 63 / 000,328, filed March 26, 2020, the entire disclosure of which is incorporated herein by reference.

[0004] Statement regarding federally funded research or development

[0005] This invention was made with government funding under grants NS104022 and OD020012 issued by the National Institutes of Health (NIH). The government holds certain rights to this invention. Technical Field

[0006] This disclosure relates to the synthesis of novel modified oligonucleotides and novel phosphoramides. Background Technology

[0007] Currently, the most common metabolically stabilizing backbone modification for complex therapeutic RNAs is phosphate thioester (PS) modification. While other available backbone modification alternatives, such as peptide nucleic acids (PNAs) and phosphoryldiamine morpholino oligonucleotides (PMOs), are effective as sterically blocking antisense oligonucleotides, these modifications are not tolerated in many promising RNA-based therapeutic strategies. These strategies include siRNAs, miRNAs, RNase H-dependent antisense oligonucleotides, and aptamer-based therapies. This poor tolerance is due to the inability of PNAs and PMOs to withstand biological mechanisms such as Argonaute proteins (siRNA / miRNAs) and RNase H, which strictly recognize RNA structures when forming “functional” RNA-protein complexes.

[0008] One of the most popular RNA-based therapeutic strategies is the use of metabolically stable, PS-modified RNA or PS / PO-modified chimeric oligonucleotides. However, a serious drawback of this strategy is the toxicity resulting from the non-specific binding of RNA to various proteins in vivo. Another drawback is that PS-modified, and more importantly, PS / PO-modified RNA, is degraded by endogenous nucleases. Therefore, the field of RNA therapy urgently needs additional scaffold modifications that provide greater metabolic stability without compromising drug efficacy.

[0009] Synthetic accessibility is also a crucial factor in the development of therapeutic oligonucleotides. Various other modified backbones (e.g., borate phosphates, aminophosphates, etc.) have been reported, but many of these backbones require specific synthetic procedures that are not always compatible with conventional phosphoramidine oligonucleotide synthesis cycles. This makes it difficult to freely synthesize / design chimeric backbones with these modifications in a manner similar to mixing PS / PO backbones with other sugar-modified backbones. This synthetic difficulty limits further diversification of design patterns for functional therapeutic oligonucleotides. Therefore, new chemical tools that are easy to synthesize and compatible with currently validated chemical modifications are in high demand in this field.

[0010] This paper presents a novel skeletal modification variant in which one or more carbon chains are inserted into the skeletal structure. The skeletal modifications presented herein are not expected to have a profound impact on the structure of RNA and therefore provide compatibility with a variety of RNA-binding biological mechanisms. Furthermore, these modifications are not expected to exhibit toxicity or nonspecific binding to proteins and therefore can be incorporated into a variety of therapeutic RNAs. Summary of the Invention

[0011] In one aspect, this disclosure provides a modified oligonucleotide comprising a 5' end, a 3' end, and at least one subunit of formula I:

[0012]

[0013] (I);

[0014] in:

[0015] B represents the base pairing portion;

[0016] W is O or O(CH2). n , where n is from 1 to 10;

[0017] X can be selected from H, OH, OR, F, SH, SR, NR. 2 2 and C 1-6 The group consisting of alkoxy groups;

[0018] Y chooses freely O – OH, OR, OR 2 NH – NH2, NR 2 2. BH3, S – R 1 The group consisting of SH;

[0019] Z represents O or O(CH2). n ;

[0020] R 1 It is alkyl, allyl, or aryl; and

[0021] R 2 It can be alkyl, allyl, or aryl.

[0022] In the implementation scheme of Formula I, Z is O(CH2). n n is 1, W is 0, and Y is 0. – .

[0023] In the implementation scheme of Formula I, Z is O and W is O(CH2). n n is 1, and Y is 0. – .

[0024] In the implementation scheme of Formula I, Z is O(CH2). n n is 1, W is 0, and Y is 0. – .

[0025] In the implementation scheme of Formula I, Z is O(CH2). n n is 1, and W is O(CH2). n And Y is O – .

[0026] In the implementation scheme of Formula I, Z is O(CH2). n n is not 1, and W is O(CH2). n And Y is O – .

[0027] In an embodiment of Formula I, the base pairing portion B is selected from the group consisting of adenine, guanine, cytosine, and uracil.

[0028] In another aspect, this disclosure provides a modified oligonucleotide comprising a 5' end, a 3' end, and at least one subunit of Formula II:

[0029]

[0030] (II)

[0031] in:

[0032] B represents the base pairing portion;

[0033] X can be selected from H, OH, OR, F, SH, SR, NR. 2 2 and C 1-6 The group consisting of alkoxy groups;

[0034] Y chooses freely O – OH, OR, OR 2 NH – NH2, NR 2 2. BH3, S – R1 The group consisting of SH;

[0035] R 1 It is alkyl, allyl, or aryl; and

[0036] R 2 It can be alkyl, allyl, or aryl.

[0037] In the implementation of Formula II, Y is 0.

[0038] In an embodiment of Formula II, the base pairing portion B is selected from the group consisting of adenine, guanine, cytosine, and uracil.

[0039] In another aspect, this disclosure provides a modified oligonucleotide comprising a 5' end, a 3' end, and at least one subunit of formula III:

[0040]

[0041] in:

[0042] B represents the base pairing portion;

[0043] R is alkyl, allyl, or aryl.

[0044] In the implementation of Formula III, Y is O.

[0045] In an embodiment of Formula III, the base pairing portion B is selected from the group consisting of adenine, guanine, cytosine, and uracil.

[0046] In another aspect, this disclosure provides a modified oligonucleotide comprising a 5' end, a 3' end, and at least one subunit of formula IV:

[0047]

[0048] in:

[0049] B represents the base pairing portion;

[0050] R is alkyl, allyl, or aryl.

[0051] In the implementation of Formula IV, the base pairing part B is selected from the group consisting of adenine, guanine, cytosine, and uracil.

[0052] In another aspect, this disclosure provides a modified oligonucleotide comprising a 5' end, a 3' end, and at least one subunit of formula V:

[0053]

[0054] in:

[0055] B represents the base pairing portion;

[0056] R is alkyl, allyl, or aryl.

[0057] In an embodiment of Formula V, the base pairing portion B is selected from the group consisting of adenine, guanine, cytosine, and uracil.

[0058] In another aspect, this disclosure provides a modified oligonucleotide comprising a 5' end, a 3' end, and at least one subunit of formula VI:

[0059]

[0060] in:

[0061] B represents the base pairing portion;

[0062] R is alkyl, allyl, or aryl.

[0063] In an embodiment of Formula VI, the base pairing portion B is selected from the group consisting of adenine, guanine, cytosine, and uracil.

[0064] In another aspect, this disclosure provides phosphorusamide derivatives of formula (VII):

[0065]

[0066] (VII)

[0067] in:

[0068] B represents the base pairing portion;

[0069] X can be selected from H, OH, OR, F, SH, SR, NR. 2 2. MOE, alkyl, allyl, aryl and C 1-6 The group consisting of alkoxy groups;

[0070] Z is either O or OCH2;

[0071] R stands for OMe or OCE (cyanoethyl);

[0072] R 1 It is alkyl, allyl, or aryl; and

[0073] R 2 It can be alkyl, allyl, or aryl.

[0074] In an embodiment of Formula VII, the base pairing portion B is selected from the group consisting of adenine, guanine, cytosine, and uracil.

[0075] In another aspect, this disclosure provides a phosphorusamide derivative of formula (VIII):

[0076]

[0077] (VIII)

[0078] in:

[0079] B represents the base pairing portion;

[0080] X can be selected from H, OH, OR, F, SH, SR, NR. 2 2. MOE, alkyl, allyl, aryl and C 1-6 The group consisting of alkoxy groups;

[0081] R 1 It is alkyl, allyl, or aryl; and

[0082] R 2 It can be alkyl, allyl, or aryl.

[0083] In an embodiment of formula (VIII), the base pairing portion B is selected from the group consisting of adenine, guanine, cytosine, and uracil.

[0084] In another aspect, this disclosure provides phosphoramide derivatives of formula (IX):

[0085]

[0086] (IX)

[0087] in:

[0088] B represents the base pairing portion;

[0089] X can be selected from H, OH, OR, F, SH, SR, NR. 2 2. MOE, alkyl, allyl, aryl and C 1-6 The group consisting of alkoxy groups;

[0090] R 1 It is alkyl, allyl, or aryl; and

[0091] R 2 It can be alkyl, allyl, or aryl.

[0092] In an embodiment of formula (IX), the base pairing portion B is selected from the group consisting of adenine, guanine, cytosine, and uracil.

[0093] In another aspect, this article provides a method for synthesizing modified oligonucleotides comprising inter-subunit bonds at the 5' end, 3' end, and at least one modified subunit, comprising:

[0094] (a) Providing a nucleoside having a 5'-protecting group attached to a solid support;

[0095] (b) Remove the protecting group;

[0096] (c) Combining the deprotected nucleoside with the phosphoramidide derivative of formula (VII) to form a phosphite triester;

[0097]

[0098] (VII)

[0099] (d) The capped triphosphite;

[0100] (e) Oxidation of the triphosphite;

[0101] (f) Repeat steps (b) to (e) using additional phosphorus amide; and

[0102] (g) Cutting from a solid support.

[0103] In another aspect, this disclosure provides for the use of phosphorusamide derivatives of formula (VII):

[0104]

[0105] (VII)

[0106] A method of coupling to the 5' end of a nucleoside or oligonucleotide, comprising adding the phosphoramidite derivative of formula (VII) to the nucleoside or oligonucleotide in an organic solvent containing an aromatic heterocyclic acid.

[0107] On the other hand, this disclosure provides a method for synthesizing exNA phosphoramide:

[0108] (a) Provide a nucleoside with a 3'-protecting group;

[0109] (b) Oxidating the 5'-hydroxy group of the nucleoside to a 5'-aldehyde group;

[0110] (c) Converting the 5'-aldehyde group of the nucleoside to a 5'-vinyl group by Vittigenelation;

[0111] (d) Hydroboration / oxidation of the 5'-vinyl group to produce a 6'-hydroxy group;

[0112] (e) Protect the 6'-hydroxy group with a DMTr group;

[0113] (f) Remove the 3'-protecting group of the nucleoside;

[0114] (g) Phosphorylation of the 3'-hydroxy group to produce 3'-phosphoramide. Attached Figure Description

[0115] The foregoing and other features and advantages of this disclosure will become more fully apparent from the following detailed description of illustrative embodiments taken in conjunction with the accompanying drawings. The patent or application document contains at least one color drawing. Upon request and payment of the necessary fees, our firm will provide a copy of the published patent or patent application with one or more color drawings.

[0116] Figure 1 This paper summarizes the modified inter-subunit connectors presented in this paper.

[0117] Figure 2 The synthesis of 2'-OMe-exNA phosphoramide 9a is provided.

[0118] Figure 3 The synthesis of 2'-F-exNA phosphoramide 9b is provided.

[0119] Figure 4 The synthesis of exNA-C phosphorous amide is provided.

[0120] Figure 5 The synthesis of exNA-G and exNA-A phosphorous amides is provided.

[0121] Figure 6 The synthesis of 5'-3'-bis-methylene-exNA phosphoramide is provided.

[0122] Figure 7 The synthesis of exNA-ribose-uridine phosphoramidide is provided.

[0123] Figure 8 The synthesis of exNA-ribose-cytosine phosphoramide is provided.

[0124] Figure 9 The synthesis of exNA-ribose-guanosine or exNA-ribose-adenine phosphoramide is provided.

[0125] Figure 10 The synthesis of phosphorusamide monomers is provided.

[0126] Figure 11 A general scheme for exNA conversion of sugar-modified nucleotides is provided.

[0127] Figure 12 The synthesis of oligonucleotides for incorporation into the exNA backbone is provided.

[0128] Figure 13 A chart of synthesized exNA-modified RNA nucleosides is provided.

[0129] Figure 14 It provides in vitro silencing efficacy of target mRNA with siRNA duplexes containing exNAs with inter-subunit bonds at various positions.

[0130] Figure 15 A model is provided that depicts the increase in 3' exonuclease stability of oligonucleotides with increasing number of exNA and phosphate thioester subunit bonds.

[0131] Figure 16 Results of the 3' exonuclease stability assay are provided. 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).

[0132] Figure 17 Results of 3' exonuclease assays for ex-NA subunit bonds in the context of polyuridine acyl sequences of oligonucleotides containing phosphodiester (PO) and phosphate thioester (PS) are provided. Oligonucleotides were tested with 1, 2, 3, 4, or 5 ex-NA subunit bonds.

[0133] Figure 18 Results of a 5'-phosphate-dependent 5'-exonuclease stability assay are provided. Each oligonucleotide was incubated at 2.5 µM (50 pmol) in RNase-free water or at 37°C in buffer A (EpiCentre, provided with Terminator). TM In the enzyme) with 3.3 units of Terminator TM (EpiCentre) exonuclease was incubated together.

[0134] Figure 19 Results of a non-5'-phosphate-dependent 5'-exonuclease stability assay are provided. Each oligonucleotide (10 µM) was incubated at 37 °C in RNase-free water or in 30 mM NaOAc (pH 6.0) buffer containing 0.25 U / mL bovine spleen phosphodiesterase II (BSP).

[0135] Figures 20A-20B Results are provided depicting the in vitro silencing activity of several siRNA duplexes containing one or more 3' exNA subunit bonds of the antisense strand. Antisense strands containing one, two, three, or four 3' exNA subunit bonds were used for dose-response profiling. Figure 20AThe percentage change in potency relative to the siRNA duplex control without exNA subunit bonds was also determined. Figure 20B ).

[0136] Figures 21A-21E Results were provided depicting the in vivo silencing activity of several siRNA duplexes containing one or more antisense strand 3' exNA subunit bonds. The siRNA duplexes were Di-siRNA formats targeting ApoE mRNA. Each siRNA duplex was administered to mice at 5 nmol via ICV injection, and ApoE mRNA levels were measured one month later. ApoE mRNA levels were measured in the following brain regions: medial cortex (…). Figure 21A ), striatum ( Figure 21B ), seahorse ( Figure 21C ),thalamus( Figure 21D ) and cerebellum ( Figure 21E ).

[0137] Figures 22A-22E Results were provided depicting the in vivo silencing activity of several siRNA duplexes containing one or more antisense strand 3' exNA subunit bonds. The siRNA duplexes target Htt mRNA. Each siRNA duplex was administered to mice at approximately 60 µg via ICV injection, and Htt mRNA levels were measured after 2 months. Htt mRNA levels were measured in the following brain regions: medial cortex (…). Figure 22A ), striatum ( Figure 22B ), seahorse ( Figure 22C ), frontal cortex ( Figure 22D ) and cerebellum ( Figure 22E The numbers 1-5 along the X-axis correspond to the siRNA chemical modification patterns depicted in Example 14.

[0138] Figures 23A-23E Results were provided depicting the in vivo silencing activity of several siRNA duplexes containing one or more antisense strand 3' exNA subunit bonds. The siRNA duplexes targeted Htt mRNA. Each siRNA duplex was administered to mice at approximately 60 µg via ICV injection, and Htt protein levels were measured after 2 months. Htt protein levels were measured in the following brain regions: medial cortex (…). Figure 22A ), striatum ( Figure 22B ), seahorse ( Figure 22C ), frontal cortex ( Figure 22D ) and thalamus ( Figure 22E The numbers 1-5 along the X-axis correspond to the siRNA chemical modification patterns depicted in Example 14. Detailed Implementation

[0139] Novel modified oligonucleotides and their synthesis are provided. Novel phosphorusamides and their synthesis are also provided.

[0140] Unless otherwise specified, the nomenclature described herein in conjunction with cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry, as well as hybridization, is that which is well known and commonly used in the art. Unless otherwise specified, the methods and techniques provided herein are performed according to conventional methods well known in the art and described in the various general and more specific references cited and discussed throughout this specification, unless otherwise stated. Enzymatic reactions and purification techniques are performed according to the manufacturer's instructions, as is commonly done in the art, or as described herein. The nomenclature and laboratory procedures and techniques used in conjunction with the analytical chemistry, synthetic organic chemistry, and pharmaceutical chemistry described herein are those well known and commonly used in the art. Standard techniques are used in chemical synthesis, chemical analysis, drug preparation, formulation, and delivery, as well as patient treatment.

[0141] Unless otherwise defined herein, the scientific and technical terms used herein have the meanings commonly understood by one of ordinary skill in the art. In the event of any potential ambiguity, the definitions provided herein take precedence over any dictionary or external definitions. Furthermore, unless the context requires otherwise, singular terms shall include plural and plural terms shall include singular. Unless otherwise stated, the use of “or” means “and / or”. The use of the term “including” and other forms such as “includes” and “included” is not restrictive.

[0142] To make this disclosure easier to understand, some terms are first defined.

[0143] The term "nucleoside" refers to a molecule having a purine or pyrimidine base covalently linked to ribose or deoxyribose. Exemplary nucleosides include adenosine, guanosine, cytidine, uridine, and thymidine. Other exemplary nucleosides include inosine, 1-methylinosine, pseudouridine, 5,6-dihydrouridine, nucleothymidine, 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 linked to a sugar moiety by an ester bond. Exemplary nucleotides include nucleosides with monophosphate, diphosphate, and triphosphate. The terms "polynucleotide" and "nucleic acid molecule" are used interchangeably herein and refer to a polymer of nucleotides linked together by a phosphodiester or thiophosphate bond between the 5' and 3' carbon atoms.

[0144] The term "RNA" or "RNA molecule" or "ribonucleic acid molecule" refers to a polymer of ribonucleotides (e.g., 2, 3, 4, 5, 10, 15, 20, 25, 30, or more ribonucleotides). The term "DNA" or "DNA molecule" or "deoxyribonucleic acid molecule" refers to a polymer of deoxyribonucleotides. DNA and RNA can be synthesized naturally (e.g., through DNA replication or DNA transcription, respectively). RNA can undergo post-transcriptional modification. DNA and RNA can also be chemically synthesized. 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 a single-stranded RNA that specifies the amino acid sequence of one or more polypeptide chains. During protein synthesis, this information is translated when ribosomes bind to mRNA.

[0145] As used herein, the term "small interfering RNA" ("siRNA") (also referred to in the art as "short interfering RNA") refers to RNA (or RNA analogs) containing about 10-50 nucleotides (or nucleotide analogs) capable of directing or mediating RNA interference. Preferably, the siRNA contains about 15-30 nucleotides or nucleotide analogs, more preferably about 16-25 nucleotides (or nucleotide analogs), even more preferably about 18-23 nucleotides (or nucleotide analogs), and even more preferably about 19-22 nucleotides (or nucleotide analogs) (e.g., 19, 20, 21, or 22 nucleotides or nucleotide analogs). The term "short" siRNA refers to siRNA containing about 21 nucleotides (or nucleotide analogs) (e.g., 19, 20, 21, or 22 nucleotides). The term "long" siRNA refers to siRNA containing about 24-25 nucleotides, such as 23, 24, 25, or 26 nucleotides. In some cases, short siRNAs may include fewer than 19 nucleotides, such as 16, 17, or 18 nucleotides, as long as the shorter siRNA retains the ability to mediate RNAi. Similarly, in some cases, long siRNAs may include more than 26 nucleotides, as long as the longer siRNA retains the ability to mediate RNAi without further processing (e.g., enzymatic processing) into the short siRNA.

[0146] The terms "nucleotide analog," "altered nucleotide," or "modified nucleotide" refer to non-standard nucleotides, including ribonucleotides or deoxyribonucleotides that are not naturally occurring. Exemplary nucleotide analogs are modified at any position to alter certain chemical properties of the nucleotide while retaining the ability of the nucleotide analog to perform its intended function. Examples of nucleotide positions from which this modification can occur include: position 5, such as 5-(2-amino)propyluridine, 5-bromouridine, 5-propynyluridine, 5-propenyluridine, etc.; position 6, such as 6-(2-amino)propyluridine; and position 8 of adenosine and / or guanosine, such as 8-bromoguanosine, 8-chloroguanosine, 8-fluoroguanosine, etc. Nucleotide analogs also include denitronucleotides, such as 7-denitroadenosine; O- and N-modified (e.g., alkylated, such as N6-methyladenosine, or as known in the art) nucleotides; and other heterocyclic modified nucleotide analogs, such as those described in Herdewijn, Antisense Nucleic Acid Drug Dev., Aug 2000 10(4):297-310.

[0147] Nucleotide analogs may also include 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 include those described in U.S. Patent Nos. 5,858,988 and 6,291,438.

[0148] The phosphate group of a nucleotide can also be modified, for example, by replacing one or more of the oxygen atoms in the phosphate group with sulfur (e.g., thiophosphate), or by making other substitutions that allow the nucleotide to perform its intended function, such as those described in, for example, Eckstein, Antisense Nucleic Acid Drug Dev. April 2000 10(2):117-21, Rusckowski et al. Antisense Nucleic Acid Drug Dev. October 2000 10(5):333-45, Stein, Antisense Nucleic Acid Drug Dev. October 2001 11(5):317-25, Vorobjev et al. Antisense Nucleic Acid Drug Dev. April 2001 11(2):77-85, and U.S. Patent No. 5,684,143. Some of the modifications mentioned above (e.g., phosphate group modifications) preferably reduce the rate of hydrolysis of, for example, polynucleotides containing the like in vivo or in vitro.

[0149] 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 altered or modified nucleotide compared to the corresponding unaltered or unmodified RNA, but retains the same or similar properties or functions as the corresponding unaltered or unmodified RNA. As discussed above, oligonucleotides can be linked by bonds, which results in a lower hydrolysis rate for RNA analogs compared to RNA molecules with phosphodiester bonds. For example, the nucleotides of the analog may contain methylene glycol, ethylene glycol, oxymethylthio, oxyethio, oxycarbonyloxy, phosphoryldiamine, phosphoramide, and / or thiophosphate bonds. Preferred RNA analogs include sugar and / or backbone-modified ribonucleotides and / or deoxyribonucleotides. Such alterations or modifications may also include the addition of non-nucleotide materials, such as addition to the ends or interior of the RNA (at one or more nucleotides of the RNA). The RNA analog only needs to be sufficiently similar to native RNA to have the ability to mediate RNA interference.

[0150] As used herein, the term “RNA interference” (“RNAi”) refers to the selective intracellular degradation of RNA. RNAi occurs naturally in cells to remove exogenous RNA (e.g., viral RNA). Natural RNAi occurs through fragments cut from free dsRNA that direct the degradation mechanism to other similar RNA sequences. Alternatively, RNAi can be artificially initiated, for example, by silencing the expression of a target gene.

[0151] An RNAi agent, such as an RNA silencing agent, is defined as having a strand that is sufficiently complementary to the target mRNA sequence to guide target-specific RNA interference (RNAi).

[0152] As used herein, the term “isolated RNA” (e.g., “isolated siRNA” or “isolated siRNA precursor”) refers to an RNA molecule that is substantially free from other cellular material or culture medium produced by recombinant technology, or substantially free from chemical precursors or other compounds produced by chemical synthesis.

[0153] As used herein, the term “RNA silencing” refers to a set of sequence-specific regulatory mechanisms mediated by RNA molecules (such as RNA interference (RNAi), transcriptional gene silencing (TGS), post-transcriptional gene silencing (PTGS), suppression, co-suppression, and translational repression) that lead to the suppression 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.

[0154] 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 without 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 some embodiments, the target polynucleotide sequence corresponds to a target gene, and the non-target polynucleotide sequence corresponds to a non-target gene. In some embodiments, the target polynucleotide sequence corresponds to a target allele, and the non-target polynucleotide sequence corresponds to a non-target allele. In some 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.

[0155] The term "in vitro" has its generally accepted meaning in the art, for example, referring to purified reagents or extracts, such as cell extracts. The term "in vivo" also has its generally accepted meaning in the art, for example, referring to living cells, such as immortalized cells, primary cells, cell lines, and / or cells within an organism.

[0156] As used herein, the term "transgenic" 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 transgenics may include genes that are partially or completely heterologous (i.e., exogenous) to the transgenic organism, or may represent genes homologous to the organism's endogenous genes. The term "transgenic" also means a nucleic acid molecule comprising one or more selected nucleic acid sequences, such as DNA, that encode one or more engineered RNA precursors to be expressed in a transgenic organism, such as an animal, wherein the sequences are partially or completely heterologous (i.e., exogenous) to the transgenic animal, or homologous to the transgenic animal's endogenous genes but designed to insert into a location in the animal's genome different from the natural gene. Transgenics include one or more promoters necessary for the expression of the selected nucleic acid sequence and any other DNA, such as introns, all of which are operatively linked to the selected sequence and may include enhancer sequences.

[0157] Genes “involved” in a disease or condition include genes whose normal or abnormal expression or function affects or causes a disease or condition or at least one symptom of the disease or condition.

[0158] As used herein, the term "gain-of-function mutation" refers to any mutation in a gene in which the protein encoded by the gene (i.e., the mutant protein) acquires a function not normally associated with the protein (i.e., the wild-type protein) and causes or contributes to a disease or condition. A gain-of-function mutation can be the deletion, addition, or substitution of one or more nucleotides in a gene, resulting in a change in the function of the protein encoding it. In one embodiment, a gain-of-function mutation alters the function of the mutant protein or causes it to interact with other proteins. In another embodiment, a gain-of-function mutation results in a reduction or removal of the normal wild-type protein, for example, through altered interaction between the mutant protein and the normal wild-type protein.

[0159] As used herein, the term "target gene" is a gene whose expression will be substantially suppressed or "silenced." This silencing can be achieved through RNA silencing, such as by cleaving the mRNA of the target gene or by translational repression of the target gene. The term "non-target gene" is a gene whose expression will not be substantially silenced. In one embodiment, the polynucleotide sequences of the target gene and non-target genes (e.g., the mRNA encoded by the target gene and non-target genes) may differ by one or more nucleotides. In another embodiment, the target gene and non-target genes may differ due to one or more polymorphisms (e.g., single nucleotide polymorphisms or SNPs). In another embodiment, the target gene and non-target genes may share less than 100% sequence identity. In yet another embodiment, the non-target gene may be a homolog of the target gene (e.g., an ortholog or paralog).

[0160] A "target allele" is an allele whose expression will be selectively suppressed or "silenced" (e.g., an SNP allele). This silencing can be achieved via RNA silencing, for example, by cleaving the mRNA of the target gene or the target allele with siRNA. The term "non-target allele" is an allele whose expression is not substantially silenced. In some embodiments, target and non-target alleles may correspond to the same target gene. In other embodiments, the target allele corresponds to or is associated with the target gene, and the non-target allele corresponds to or is associated with a non-target gene. In one embodiment, the polynucleotide sequences of the target and non-target alleles may differ by one or more nucleotides. In another embodiment, the target and non-target alleles may differ by one or more allele polymorphisms (e.g., one or more SNPs). In yet another embodiment, the target and non-target alleles may share less than 100% sequence identity.

[0161] As used herein, the term "polymorphism" refers to a variation (e.g., one or more deletions, insertions, or substitutions) in a gene sequence that is identified or detected when comparing identical gene sequences from different sources or subjects (but from the same organism). For example, polymorphism can be identified when comparing identical gene sequences from different subjects. Such identification of polymorphism is routine in the art, and the method is similar to those used to detect, for example, point mutations in breast cancer. For example, it can be identified from DNA extracted from the subject's lymphocytes, and then the polymorphic region can be amplified using primers specific to the polymorphic region. Alternatively, polymorphism can be identified when comparing two alleles of the same gene. In a particular embodiment, the polymorphism is a single nucleotide polymorphism (SNP).

[0162] Sequence variations between two alleles of the same gene in an organism are referred to herein as "allele polymorphisms". In some embodiments, allele polymorphisms correspond to SNP alleles. For example, an allele polymorphism may include a single nucleotide variation between the two alleles of an SNP. The polymorphism may be at a nucleotide within a coding region, but due to the degeneracy of the genetic code, the encoded amino acid sequence remains unchanged. Alternatively, the polymorphic sequence may encode a different amino acid at a specific position, but the change in amino acid does not affect protein function. Polymorphic regions can also be found in non-coding regions of a gene. In exemplary embodiments, polymorphisms are found in coding regions or untranslated regions of a gene (e.g., 5' UTR or 3' UTR).

[0163] As used herein, the term "allele frequency" is a measure (e.g., proportion or percentage) of the relative frequency of an allele (e.g., an SNP allele) at a single locus in an individual population. For example, if an individual population carries n loci (and the genes occupying said loci) of a particular chromosomal locus in each of their somatic cells, then the allele frequency is the fraction or percentage of loci occupied by the allele within said population. In a particular embodiment, the allele frequency of an allele (e.g., an SNP allele) is at least 10% (e.g., at least 15%, 20%, 25%, 30%, 35%, 40% or more) in the sample population.

[0164] 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 a particular embodiment, a sample population may include individuals who share at least a common disease phenotype (e.g., gain-of-function condition) or mutation (e.g., gain-of-function mutation).

[0165] As used herein, the term "heterozygosity" refers to the fraction of individuals in a population that are heterozygous (e.g., contain two or more distinct alleles) at a particular locus (e.g., at an SNP). Heterozygosity of a sample population can be calculated using methods well known to those skilled in the art.

[0166] The phrase “examining the function of genes in cells or organisms” refers to examining or studying the resulting expression, activity, function, or phenotype.

[0167] As used herein, the term "RNA silencer" refers to RNA capable of inhibiting or "silencing" the expression of a target gene. In some embodiments, RNA silencers are capable of preventing the complete processing (e.g., complete translation and / or expression) of mRNA molecules through a post-transcriptional silencing mechanism. RNA silencers include small (<50 bp), non-coding RNA molecules, such as RNA duplexes containing paired strands, and precursor RNAs from which such small non-coding RNAs can be generated. Exemplary RNA silencers include siRNA, miRNA, siRNA-like duplexes, antisense oligonucleotides, GAPMER molecules, and bifunctional oligonucleotides, and their precursors. In one embodiment, the RNA silencer is capable of inducing RNA interference. In another embodiment, the RNA silencer is capable of mediating translational repression.

[0168] As used herein, the term "rare nucleotide" refers to naturally occurring nucleotides that are not frequently found, including naturally occurring deoxyribonucleotides or ribonucleotides that are not frequently found, such as naturally occurring ribonucleotides that are not guanosine, adenosine, cytosine, or uridine. Examples of rare nucleotides include, but are not limited to, inosine, 1-methylinosine, pseudouridine, 5,6-dihydrouridine, nucleothymidine, 2N-methylguanosine, and 2,2N,N-dimethylguanosine.

[0169] The term "engineered" in engineered RNA precursors or engineered nucleic acid molecules indicates that the precursor or molecule does not exist in nature because all or part of its nucleic acid sequence was generated or selected by humans. Once the sequence is generated or selected, it can be replicated, translated, transcribed, or otherwise processed through intracellular mechanisms. Therefore, an RNA precursor generated intracellularly by a transgene including an engineered nucleic acid molecule is an engineered RNA precursor.

[0170] As used herein, the term “microRNA” (“miRNA”), also known in the art as “time-regulating small RNA” (“stRNA”), refers to a small (10–50 nucleotides) RNA that genetically encodes (e.g., through the genomes of viruses, mammals, or plants) and is capable of directing or mediating RNA silencing. “miRNA syndrome” should refer to a disease or condition characterized by the abnormal expression or activity of miRNAs.

[0171] As used herein, the term "bifunctional oligonucleotide" refers to an RNA silencing agent having the formula TL-μ, where T is the mRNA targeting moiety, L is the linking moiety, and μ is the miRNA recruitment moiety. As used herein, the terms "mRNA targeting moiety," "targeting moiety," "mRNA targeting portion," or "targeting portion" refer to a domain, portion, or region of a bifunctional oligonucleotide that is of sufficient size and sufficiently complementary to the mRNA moiety or region selected or targeted for silencing (i.e., the portion has a sequence sufficient to capture the target mRNA). As used herein, the term "linking moiety" or "linking portion" refers to a domain, portion, or region of an RNA silencing agent that is covalently linked or connected to mRNA.

[0172] As used herein, the term "antisense strand" in the context of RNA silencing agents, such as siRNA or RNA silencing agents, refers to a strand substantially complementary to a portion of approximately 10–50 nucleotides, such as approximately 15–30, 16–25, 18–23, or 19–22 nucleotides, of the mRNA targeting the gene used for silencing. The antisense strand, or first strand, has a sequence sufficiently complementary to the desired target mRNA sequence to guide target-specific silencing, for example, sufficient to trigger an RNAi mechanism or process (RNAi interference) to disrupt the complementarity of the desired target mRNA or sufficient to trigger translational repression of the desired target mRNA.

[0173] The term "sense strand" or "second strand" in RNA silencing agents, such as siRNA, refers to the strand complementary to the antisense strand or the first strand. Antisense and sense strands can also be referred to as the first or second strand, where the first or second strand is complementary to the target sequence, and the corresponding second or first strand is complementary to the first or second strand. miRNA duplex intermediates or siRNA-like duplexes consist of a miRNA strand sufficiently complementary to approximately 10-50 nucleotides of the mRNA targeting the silenced gene, and a miRNA* strand sufficiently complementary to form a duplex with the miRNA.

[0174] As used herein, the term “guide strand” refers to the strand of an RNA silencing agent, such as a double-stranded siRNA or an antisense strand of an siRNA sequence, which enters the RISC complex and guides the cleavage of the target mRNA.

[0175] As used herein, the term "asymmetry," such as in the asymmetry of the duplex region of an RNA silencing agent (e.g., the stem of shRNA), refers to an inequality in bond strength or base pairing strength between the ends of the RNA silencing agent (e.g., between the terminal nucleotides on the first strand or stem portion and the terminal nucleotides on the opposite second strand or stem portion), such that the 5' end of one strand of the duplex is more frequently in a transiently unpaired state, e.g., a single-stranded state, compared to the 5' end of the complementary strand. This structural difference determines that one strand of the duplex is preferentially incorporated into the RISC complex. The strand with a less tight 5' pairing with the complementary strand will preferentially be incorporated into the RISC and mediate RNAi.

[0176] As used herein, the term “bond strength” or “base pair strength” refers to the strength of the interaction between paired nucleotides (or nucleotide analogs) on opposite strands of an oligonucleotide duplex (e.g., siRNA duplex), primarily due to hydrogen bonds, van der Waals interactions, etc., between said nucleotides (or nucleotide analogs).

[0177] As used herein, “5’ end” in “as in the 5’ end of the antisense strand” refers to the 5’ end nucleotide of the antisense strand, such as one to about five nucleotides. As used herein, “3’ end” in “as in the 3’ end of the sense strand” refers to the region complementary to the nucleotide of the complementary antisense strand at the 5’ end, such as one to about five nucleotides.

[0178] As used herein, the term "destabilized nucleotide" refers to a first nucleotide or nucleotide analog capable of forming a base pair with a second nucleotide or nucleotide analog such that the bond strength of that base pair is lower than that of a conventional base pair (i.e., a Watson-Crick base pair). In some embodiments, the destabilized nucleotide is capable of forming a mismatched base pair with the second nucleotide. In other embodiments, the destabilized nucleotide is capable of forming a wobbly base pair with the second nucleotide. In still other embodiments, the destabilized nucleotide is capable of forming a fuzzy base pair with the second nucleotide.

[0179] As used herein, the term "base pair" refers to the interaction between paired nucleotides (or nucleotide analogs) on opposite strands of an oligonucleotide duplex (e.g., a duplex formed by a strand of an RNA silencer and a target mRNA sequence), primarily due to hydrogen bonds, van der Waals interactions, etc., between said nucleotides (or nucleotide analogs). As used herein, the term "bond strength" or "base pair strength" refers to the strength of a base pair.

[0180] As used herein, the term “mismatched base pair” refers to a base pair consisting of non-complementary or non-Watson-Crick base pairs, such as non-normally complementary G:C, A:T, or A:U base pairs. As used herein, the term “fuzzy base pair” (also known as an undiscriminating base pair) refers to a base pair formed from universal nucleotides.

[0181] As used herein, the term "universal nucleotide" (also known as "neutral nucleotide") includes nucleotides (e.g., certain destabilized nucleotides) that have a base that does not significantly distinguish the bases on complementary polynucleotides when forming base pairs ("universal base" or "neutral base"). Universal nucleotides are primarily hydrophobic molecules that can efficiently assemble into antiparallel double-stranded nucleic acids (e.g., double-stranded DNA or RNA) due to stacking interactions. The base portion of a universal nucleotide typically contains a nitrogen-containing aromatic heterocyclic moiety.

[0182] As used herein, the terms “sufficient complementarity” or “sufficient complementarity” refer to an RNA silencing agent having a sequence sufficient to bind to the desired target RNA and trigger RNA silencing of the target mRNA (e.g., in the antisense strand, the mRNA targeting portion, or the miRNA recruitment portion).

[0183] As used herein, the term "translation repression" refers to the selective repression of mRNA translation. Natural translation repression occurs via miRNA cleaved from shRNA precursors. Both RNAi and translation repression are RISC-mediated. Both RNAi and translation repression can occur naturally or can be artificially initiated, for example, by silencing the expression of target genes.

[0184] As used herein, the term "alkoxy" refers to an -O-alkyl group, wherein the alkyl group is as defined herein. Alkoxy groups include, for example, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, sec-butoxy, tert-butoxy, etc. In embodiments, C1-C6 alkoxy groups are provided herein.

[0185] As used herein, unless otherwise stated, the term "halo" alone or as part of another substituent means a fluorine, chlorine, bromine or iodine atom, preferably fluorine, chlorine or bromine, more preferably fluorine or chlorine.

[0186] As used herein, unless otherwise stated, the term "hydroxyl" alone or as part of another substituent means an alcohol moiety having the formula -OH.

[0187] As used herein, the term "exNA" refers to an "extended nucleic acid" containing intersubunit bonds that contain one or more additional CH2 groups at the 3', 5', or both positions.

[0188] The preparation of linkers can 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 can be found, for example, in Greene et al., Protective Groups in Organic Synthesis, 4th ed., Wiley & Sons, 2007, which is incorporated herein by reference in its entirety. The modification of protecting groups and the formation and cleavage methods described herein can be adjusted as needed according to various substituents.

[0189] The various methods disclosed herein include steps involving comparing values, levels, characteristics, properties, etc., with a “suitable control”, which is interchangeably referred to herein as a “suitable control”. A “suitable control” or “suitable control” is any control or standard familiar to those skilled in the art for comparative purposes. In one embodiment, a “suitable control” or “suitable control” is a value, level, characteristic, property, etc., determined prior to performing the RNAi method as described herein. For example, transcription rate, mRNA level, translation rate, protein level, biological activity, cellular characteristics or properties, genotype, phenotype, etc., may be determined prior to introducing the RNA silencing agent of this disclosure into cells or organisms. In another embodiment, a “suitable control” or “suitable control” is a value, level, characteristic, property, etc., determined in cells or organisms exhibiting, for example, normal characteristics (e.g., control or normal cells or organisms). In yet another embodiment, a “suitable control” or “suitable control” is a predefined value, level, characteristic, property, etc.

[0190] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. Although similar or equivalent methods and materials may be used in practice or testing of the methods and materials described herein, the following description of suitable methods and materials is preferred. All patents, patent applications, and other references cited herein are incorporated herein by reference in their entirety. In case of conflict, this specification (including definitions) shall prevail. Furthermore, the materials, methods, and examples described are illustrative only and not intended to be limiting.

[0191] Various aspects of this disclosure are described in more detail in the following subsections.

[0192] I. Synthesis of novel modified oligonucleotides

[0193] This section describes a synthetic procedure for oligonucleotides modified with a novel backbone, namely extended nucleic acid (exNA). This chemical backbone modification significantly enhances the metabolic stability of the oligonucleotides. The chemical modification involves inserting one or more carbon atoms or strands at the 5', 3', or both positions on the backbone. This structural regulation creates a non-canonical stretched / flexible structure on the oligonucleotide backbone, which protects the oligonucleotide from cleavage by various nucleases.

[0194] Novel exNA modifications are broadly compatible with siRNA scaffolds. The combination of exNA-PS backbones significantly enhances metabolic stability (by 10–50 orders of magnitude compared to unmodified oligonucleotides) without compromising siRNA efficacy (e.g., 5'-[exNA-PS]4–3' modifications do not negatively induce siRNA efficacy while inducing very high exonuclease stability), as described below. Therefore, this metabolically stable exNA modification broadly and robustly improves the in vivo performance of therapeutic oligonucleotide candidates.

[0195] In this disclosure, a synthetic scheme for exNA-modified oligonucleotides is described. Importantly, the exNA monomer phosphorous amide synthesis can be performed from commercially available nucleosides, and the exNA-modified oligonucleotides can be prepared using conventional oligonucleotide solid-phase synthesis procedures on an automated oligonucleotide synthesizer.

[0196] This synthetic procedure offers the following notable benefits. For example, converting conventional nucleosides to the “exNA format” is applicable to many different modified nucleosides. Therefore, this expands the possibility of synthesizing and producing more types of modified oligonucleotides with chemical synthesis compatibility. Second, no separate, specific synthetic procedure is required during the oligonucleotide synthesis cycle. This is a significant benefit to the ease of use of these oligonucleotides, especially for automated synthesizers where a vial of exNA phosphorusamide can be easily added to the machine. Third, specific oligonucleotide deprotection conditions are not required because exNA phosphorusamide and oligonucleotides are compatible with conventional deprotection conditions. Again, this facilitates easy synthesis and use with automated synthesizers. Fourth, mixed polymeric oligonucleotides with exNA and clinically validated modified nucleotides (e.g., 2'-OMe, 2'-F, phosphate thioesters, various ligand conjugates, lipid conjugates, etc.) can be synthesized.

[0197] In one aspect, this disclosure provides a modified oligonucleotide comprising a 5' end, a 3' end, and at least one subunit of formula I:

[0198]

[0199] (I);

[0200] in:

[0201] B represents the base pairing portion;

[0202] W is O or O(CH2). n , where n is from 1 to 10;

[0203] X can be selected from H, OH, OR, F, SH, SR, NR. 2 2 and C 1-6 The group consisting of alkoxy groups;

[0204] Y chooses freely O – OH, OR, OR 2 NH – NH2, NR 2 2. BH3, S – R 1 The group consisting of SH;

[0205] Z is O or O(CH) 2)n ;

[0206] R 1 It is alkyl, allyl, or aryl; and

[0207] R 2 It can be alkyl, allyl, or aryl.

[0208] In the implementation scheme of Formula I, Z is O(CH2). n n is 1, W is 0, and Y is 0. – .

[0209] In the implementation scheme of Formula I, Z is O and W is O(CH2). n n is 1, and Y is 0. – .

[0210] In the implementation scheme of Formula I, Z is O(CH2). n n is 1, W is 0, and Y is 0. – .

[0211] In the implementation scheme of Formula I, Z is O(CH2). n n is 1, and W is O(CH2). n And Y is O – .

[0212] In the implementation scheme of Formula I, Z is O(CH2). n n is not 1, and W is O(CH2). n And Y is O – .

[0213] In an embodiment of Formula I, the base pairing portion B is selected from the group consisting of adenine, guanine, cytosine, and uracil.

[0214] In another aspect, this disclosure provides a modified oligonucleotide comprising a 5' end, a 3' end, and at least one subunit of Formula II:

[0215]

[0216] (II)

[0217] in:

[0218] B represents the base pairing portion;

[0219] X can be selected from H, OH, OR, F, SH, SR, NR. 2 2 and C 1-6 The group consisting of alkoxy groups;

[0220] Y chooses freely O – OH, OR, OR 2 NH – NH2, NR 2 2. BH3, S – R 1 The group consisting of SH;

[0221] R 1 It is alkyl, allyl, or aryl; and

[0222] R 2 It can be alkyl, allyl, or aryl.

[0223] In the implementation of Formula II, Y is 0.

[0224] In an embodiment of Formula II, the base pairing portion B is selected from the group consisting of adenine, guanine, cytosine, and uracil.

[0225] In another aspect, this disclosure provides a modified oligonucleotide comprising a 5' end, a 3' end, and at least one subunit of formula III:

[0226]

[0227] in:

[0228] B represents the base pairing portion;

[0229] R is alkyl, allyl, or aryl.

[0230] In the implementation of Formula III, Y is O.

[0231] In an embodiment of Formula III, the base pairing portion B is selected from the group consisting of adenine, guanine, cytosine, and uracil.

[0232] In another aspect, this disclosure provides a modified oligonucleotide comprising a 5' end, a 3' end, and at least one subunit of formula IV:

[0233]

[0234] in:

[0235] B represents the base pairing portion;

[0236] R is alkyl, allyl, or aryl.

[0237] In the implementation of Formula IV, the base pairing part B is selected from the group consisting of adenine, guanine, cytosine, and uracil.

[0238] In another aspect, this disclosure provides a modified oligonucleotide comprising a 5' end, a 3' end, and at least one subunit of formula V:

[0239]

[0240] (V)

[0241] in:

[0242] B represents the base pairing portion;

[0243] R is alkyl, allyl, or aryl.

[0244] In an embodiment of Formula V, the base pairing portion B is selected from the group consisting of adenine, guanine, cytosine, and uracil.

[0245] In another aspect, this disclosure provides a modified oligonucleotide comprising a 5' end, a 3' end, and at least one subunit of formula VI:

[0246]

[0247] (VI)

[0248] in:

[0249] B represents the base pairing portion;

[0250] R is alkyl, allyl, or aryl.

[0251] In an embodiment of Formula VI, the base pairing portion B is selected from the group consisting of adenine, guanine, cytosine, and uracil.

[0252] In another aspect, this article provides a method for synthesizing modified oligonucleotides comprising inter-subunit bonds at the 5' end, 3' end, and at least one modified subunit, comprising:

[0253] (a) Providing a nucleoside having a 5'-protecting group attached to a solid support;

[0254] (b) Remove the protecting group;

[0255] (c) Combining the deprotected nucleoside with the phosphoramidide derivative of formula (VII) to form a phosphite triester;

[0256]

[0257] (VII)

[0258] (d) The capped triphosphite;

[0259] (e) Oxidation of the triphosphite;

[0260] (f) Repeat steps (b) to (e) using additional phosphorus amide; and

[0261] (g) Cutting from a solid support.

[0262] II. Synthesis of Novel Phosphamide Derivatives

[0263] A set of synthetic procedures for novel phosphoramide derivatives is described herein, which are used to prepare oligonucleotides modified with a novel backbone, namely extended nucleic acid (exNA). Figure 11 As shown, this modification is highly versatile and can be combined with many existing nucleosides to greatly enhance the diversity of oligonucleotides with enhanced stability. In this regard, this disclosure provides phosphoramidide derivatives of formula (VII):

[0264]

[0265] (VII)

[0266] in:

[0267] B represents the base pairing portion;

[0268] X can be selected from H, OH, OR, F, SH, SR, NR. 2 2. MOE, alkyl, allyl, aryl and C 1-6 The group consisting of alkoxy groups;

[0269] Z is either O or OCH2;

[0270] R stands for OMe or OCE (cyanoethyl);

[0271] R 1 It is alkyl, allyl, or aryl; and

[0272] R 2It can be alkyl, allyl, or aryl.

[0273] In an embodiment of Formula VII, the base pairing portion B is selected from the group consisting of adenine, guanine, cytosine, and uracil.

[0274] In another aspect, this disclosure provides a phosphorusamide derivative of formula (VIII):

[0275]

[0276] (VIII)

[0277] in:

[0278] B represents the base pairing portion;

[0279] X can be selected from H, OH, OR, F, SH, SR, NR. 2 2. MOE, alkyl, allyl, aryl and C 1-6 The group consisting of alkoxy groups;

[0280] R 1 It is alkyl, allyl, or aryl; and

[0281] R 2 It can be alkyl, allyl, or aryl.

[0282] In an embodiment of formula (VIII), the base pairing portion B is selected from the group consisting of adenine, guanine, cytosine, and uracil.

[0283] In another aspect, this disclosure provides phosphoramide derivatives of formula (IX):

[0284]

[0285] (IX)

[0286] in:

[0287] B represents the base pairing portion;

[0288] X can be selected from H, OH, OR, F, SH, SR, NR. 2 2. MOE, alkyl, allyl, aryl and C 1-6 The group consisting of alkoxy groups;

[0289] R 1 It is alkyl, allyl, or aryl; and

[0290] R 2 It can be alkyl, allyl, or aryl.

[0291] In an embodiment of formula (IX), the base pairing portion B is selected from the group consisting of adenine, guanine, cytosine, and uracil.

[0292] In another aspect, this disclosure provides for the use of phosphorusamide derivatives of formula (VII):

[0293]

[0294] (VII)

[0295] A method of coupling to the 5' end of a nucleoside or oligonucleotide, comprising adding the phosphoramidite derivative of formula (VII) to the nucleoside or oligonucleotide in an organic solvent containing an aromatic heterocyclic acid.

[0296] In another aspect, this disclosure provides a method for synthesizing exNA phosphoramide, comprising:

[0297] (a) Provide a nucleoside with a 3'-protecting group;

[0298] (b) Oxidating the 5'-hydroxy group of the nucleoside to a 5'-aldehyde group;

[0299] (c) Converting the 5'-aldehyde group of the nucleoside to a 5'-vinyl group by Vittigenelation;

[0300] (d) Hydroboration / oxidation of the 5'-vinyl group to produce a 6'-hydroxy group;

[0301] (e) Protect the 6'-hydroxy group with a DMTr group;

[0302] (f) Remove the 3'-protecting group of the nucleoside;

[0303] (g) Phosphorylation of the 3'-hydroxy group to produce 3'-phosphoramide.

[0304] III. siRNA Design

[0305] In some embodiments, siRNAs are designed as follows. First, a subset of target genes is identified. Cleavage of mRNA at these sites should eliminate the translation of the corresponding protein. A sense strand is designed based on the target sequence. In some embodiments, the subset (and the corresponding sense strand) comprises about 15 to 25 nucleotides, for example, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides. However, those skilled in the art will understand that siRNAs shorter than 15 or longer than 25 nucleotides can also mediate RNAi. Therefore, siRNAs of this length are also within the scope of this disclosure, as long as they retain the ability to mediate RNAi. Longer RNAi agents have been shown to elicit interferon or PKR responses in some mammalian cells, which may be undesirable. In one aspect, the RNAi agents of this disclosure do not elicit a PKR response (i.e., have a sufficiently short length). However, longer RNAi agents may be useful, for example, in cell types that do not produce a PKR response, or where the PKR response has been downregulated or inhibited by alternative methods.

[0306] The sense strand sequence is designed so that the target sequence is located essentially in the middle of the strand. In some cases, moving the target sequence off-center can reduce the cleavage efficiency of the siRNA. However, if a shutdown silencing of wild-type mRNA is detected, such a composition, i.e., a less efficient one, may be desirable.

[0307] The antisense strand is typically the same length as the sense strand and comprises complementary nucleotides. In one embodiment, the strands are perfectly complementary, i.e., the strands are blunt-ended upon alignment or annealing. In another embodiment, the strands are aligned or annealed to produce 1, 2, 3, 4, 5, 6, or 7 nucleotide overhangs, i.e., the 3' end of the sense strand extends 1, 2, 3, 4, 5, 6, or 7 nucleotides beyond the 5' end of the antisense strand and / or the 3' end of the antisense strand extends 1, 2, 3, 4, 5, 6, or 7 nucleotides beyond the 5' end of the sense strand. The overhangs may contain (or consist of) nucleotides corresponding to (or complementary to) the target gene sequence. Alternatively, the overhangs may contain deoxyribonucleotides, such as dT, or nucleotide analogs, or other suitable non-nucleotide material (or consist of).

[0308] To facilitate the entry of the antisense strand into the RISC (thereby increasing or enhancing 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 can be altered, for example, weakened or reduced, as detailed in U.S. Patent Nos. 7,459,547, 7,772,203, and 7,732,593 (filed June 2, 2003), entitled "Methods and Compositions for Controlling Efficacy of RNA Silencing," and U.S. Patent Nos. 8,309,704, 7,750,144, 8,304,530, 8,329,892, and 8,309,705 (filed June 2, 2003), entitled "Methods and Compositions for Enhancing the Efficacy and Specificity of RNAi," the contents of which are incorporated herein by reference in their entirety. In one embodiment, the base pair strength is lower because there are fewer G:C base pairs between the 5' end of the first or antisense strand and the 3' end of the second or sense strand compared to the G:C base pair between the 3' end of the first or antisense strand and the 5' end of the second or sense strand. In another embodiment, the lower base pair strength is 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 some 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 lower due to at least one wobbly base pair, such as 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 lower base pair strength is due to at least one base pair containing a rare nucleotide, such as inosine (I). In some exemplary embodiments, the base pair is selected from the group consisting of I:A, I:U, and I:C. In yet another embodiment, the lower base pair strength is due to at least one base pair containing a modified nucleotide. In some exemplary embodiments, 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.

[0309] The design of siRNAs suitable for targeting target sequences of interest is described in detail below. siRNAs can be designed for any other target sequences found in the target gene, based on the exemplary teachings described above. Furthermore, this technique is applicable to targeting any other target sequences, such as non-pathogenic target sequences.

[0310] To verify the effectiveness of siRNA in disrupting mRNA (e.g., mRNA expressed by the target gene of interest), siRNA can be incubated with cDNA (e.g., cDNA corresponding to the target gene of interest) in a Drosophila-based in vitro mRNA expression system. 32 Newly synthesized mRNAs (e.g., target mRNAs) radiolabeled with P are detected by autoradiography on agarose gels. The presence of cleaved mRNA indicates mRNA nuclease activity. Suitable controls include omitting the siRNA. Alternatively, a control siRNA with the same nucleotide composition as the selected siRNA but without significant sequence complementarity to the appropriate target gene is chosen. This negative control can be designed by randomly scrambling the nucleotide sequence of the selected siRNA; homology searches can be performed to ensure that the negative control lacks homology with any other gene in the appropriate genome. Furthermore, a negative control siRNA can be designed by introducing one or more base mismatches into the sequence. The siRNA-mRNA complementation site that results in optimal mRNA specificity and maximum mRNA cleavage is selected.

[0311] IV. RNAi agents

[0312] This disclosure includes siRNA molecules designed, for example, as described above. The siRNA molecules of this disclosure can be chemically synthesized, or transcribed in vitro from a DNA template, or transcribed in vivo from, for example, shRNA, or by using a recombinant human DICER enzyme to cleave an in vitro transcribed dsRNA template into pools of 20, 21, or 23 bp double-stranded RNA mediating RNAi. siRNA molecules can be designed using any method known in the art.

[0313] In one respect, the RNAi agent is not an interfering RNA, such as siRNA or shRNA as described above, but rather the RNAi agent can encode an interfering RNA, such as shRNA as described above. In other words, the RNAi agent can be a transcription template for interfering RNA. Therefore, the RNAi agents of this disclosure may also include small hairpin RNA (shRNA) and expression constructs engineered to express shRNA. Transcription of shRNA begins at the polymerase III (Pol III) promoter and is believed to terminate at position 2 of the 4-5-thymine transcription termination site. After expression, shRNAs are thought to fold into stem-loop structures with 3'UU protrusions; 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, ibid.); Miyagishi et al., 2002; Paddison et al., 2002, ibid.; Paul et al., 2002, ibid.; Sui et al., 2002, ibid.; Yu et al., 2002, ibid. More 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).

[0314] The expression constructs disclosed herein include any construct suitable for a suitable expression system, and include, 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 such as the U6 snRNA promoter or the H1 RNA polymerase III promoter, or other promoters known in the art. A construct may include one or two siRNA strands. An expression construct expressing two strands may also include a loop structure connecting the two strands, or each strand may be transcribed separately from different promoters in the same construct. Each strand may also be transcribed from a separate expression construct. (Tuschl, T., 2002, ibid.)

[0315] Synthetic siRNAs can be delivered into cells using methods known in the art, including cationic liposome transfection and electroporation. To achieve long-term repression of target genes and, in some cases, to facilitate delivery, one or more siRNAs can be expressed intracellularly from recombinant DNA constructs. Methods for expressing siRNA duplexes from recombinant DNA constructs within cells to allow for long-term repression of target genes in cells are known in the art, including mammalian Pol III promoter systems (e.g., H1 or U6 / snRNA promoter systems capable of expressing functional double-stranded siRNAs (Tuschl, T., 2002, ibid.); (Bagella et al., 1998; Lee et al., 2002, ibid.; Miyagishi et al., 2002, ibid.; Paul et al., 2002, ibid.; Yu et al., 2002, ibid.; Sui et al., 2002, ibid.). RNA Pol III transcriptional termination occurs during the execution of four consecutive T residues in the DNA template, providing a mechanism for ending the siRNA transcript at a specific sequence. The siRNA is complementary to the target gene sequence in the 5'-3' and 3'-5' directions, and the two strands of the siRNA can be expressed in the same construct or in different constructs. [The text then abruptly shifts to a seemingly unrelated topic:] ...by H1 or U6... Hairpin siRNAs driven by snRNA promoters and expressed in cells can inhibit target gene expression (Bagella et al., 1998; Lee et al., 2002, ibid.; Miyagishi et al., 2002, ibid.; Paul et al., 2002, ibid.; Yu et al., 2002, ibid.; Sui et al., 2002, ibid.). When co-transfected into cells with a vector expressing T7 RNA polymerase, constructs containing siRNA sequences controlled by the T7 promoter also produce functional siRNAs (Jacque et al., 2002, ibid.). A single construct may contain multiple sequences encoding siRNAs targeting the same or multiple genes, such as multiple regions of a target gene, and may be driven by, for example, a single PolIII promoter site.

[0316] Animal cells express a series of approximately 22-nucleotide non-coding RNAs known as microRNAs (miRNAs), which regulate gene expression at the post-transcriptional or translational level during animal development. A common characteristic of miRNAs is that they are all cleaved from a stem-loop of approximately 70 nucleotides of precursor RNA, likely by a type III RNase, Dicer, or its homolog. By replacing the stem sequence of the miRNA precursor with a sequence complementary to the target mRNA, vector constructs for expressing engineered precursors can be used to generate siRNAs to initiate RNAi targeting specific mRNAs in mammalian cells (Zeng et al., 2002, ibid.). When expressed by DNA vectors containing polymerase III promoters, microRNA-designed hairpins can silence gene expression (McManus et al., 2002, ibid.). In the absence of siRNA-mediated gene silencing, microRNAs targeting polymorphisms can also be used to block the translation of mutant proteins. Such applications can be useful in certain situations, for example, where designed siRNAs lead to off-target silencing of wild-type proteins.

[0317] Virus-mediated delivery mechanisms can also be used to induce specific silencing of target genes through siRNA expression, for example, by generating recombinant adenoviruses containing siRNA under the transcriptional control of the RNA Pol II promoter (Xia et al., 2002, ibid.). Infection of HeLa cells with these recombinant adenoviruses reduces the expression of endogenous target genes. Injection of recombinant adenovirus vectors into transgenic mice expressing siRNA target genes resulted in decreased in vivo expression of the target genes. Ibid. In animal models, whole-embryo electroporation can efficiently deliver synthetic siRNA into implanted mouse embryos (Calegari et al., 2002). In adult mice, efficient delivery of siRNA can be achieved via “high-pressure” delivery, i.e., rapid (within 5 seconds) injection of a large volume of siRNA-containing solution into the animal via the tail vein (Liu et al., 1999, ibid.; McCaffrey et al., 2002, ibid.; Lewis et al., 2002). Nanoparticles and liposomes can also be used to deliver siRNA into the animal. In some exemplary embodiments, recombinant adeno-associated virus (rAAV) and its associated vectors can be used to deliver one or more siRNAs into cells, such as nerve cells (e.g., brain cells) (US Patent Applications 2014 / 0296486, 2010 / 0186103, 2008 / 0269149, 2006 / 0078542, and 2005 / 0220766).

[0318] The nucleic acid compositions disclosed herein include unmodified siRNAs as known in the art and modified siRNAs, such as cross-linked siRNA derivatives or derivatives having non-nucleotide portions attached to, for example, their 3' or 5' ends. Modifying siRNA derivatives in this manner, compared to the corresponding siRNAs, can improve the cellular uptake of the resulting siRNA derivatives or enhance their cell-targeting activity, enabling them to be used for tracking siRNA derivatives in cells, or improving the stability of the siRNA derivatives compared to the corresponding siRNAs.

[0319] As described herein, the introduction of engineered RNA precursors into cells or the entire organism leads to the production of the desired siRNA molecules. These siRNA molecules then bind to endogenous protein components of the RNAi pathway to bind to and target specific mRNA sequences for cleavage and disruption. In this way, the mRNA targeted by the siRNA produced from the engineered RNA precursor is depleted from the cell or organism, resulting in a reduced concentration of the protein encoded by that mRNA in the cell or organism. RNA precursors are typically single-stranded nucleic acid molecules encoding dsRNA alone or entire nucleotide sequences encoding RNA hairpin loop structures.

[0320] The nucleic acid compositions disclosed herein may be unconjugated or may be conjugated to another part, such as nanoparticles, to enhance the properties of the composition, such as pharmacokinetic parameters, such as absorption, efficacy, bioavailability, and / or half-life. Conjugation may be accomplished by methods known in the art, such as those described in Lambert et al., Drug Deliv. Rev.:47(1), 99-112 (2001) (describing nucleic acids loaded onto polyalkyl cyanoacrylate (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 Supplement 4:55-8 (1994) (describing nucleic acids linked to intercalators, hydrophobic groups, polycations, or PACA nanoparticles); and Godard et al., Eur. J. Biochem. 232(2):404-10 (1995) (describing nucleic acids linked to nanoparticles).

[0321] The nucleic acid molecules disclosed herein can also be labeled using any method known in the art. For example, the nucleic acid compositions can be labeled with fluorophores such as Cy3, fluorescein, or rhodamine. Labeling can be performed using kits such as SILENCER. TM siRNA labeling kit (Ambion). Furthermore, siRNA can be radiolabeled, for example, using... 3H, 32 P or another suitable isotope.

[0322] Furthermore, since RNAi is believed to occur via 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 engineered (e.g., for chemical synthesis), generated (e.g., enzymatically generated), or expressed (e.g., from a vector or plasmid) as described herein and used according to the claimed methods. Additionally, in invertebrates, RNAi can be efficiently triggered by long dsRNAs (e.g., about 100-1000 nucleotides in length, preferably about 200-500, e.g., dsRNAs about 250, 300, 350, 400, or 450 nucleotides in length) acting as the effector of RNAi. (Brondani et al., Proc Natl Acad Sci USA. Dec 4, 2001; 98(25):14428-33. Epub Nov 27, 2001).

[0323] V. RNA silencers

[0324] In one embodiment, this disclosure provides novel RNA silencing agents (e.g., siRNA and shRNA), methods for preparing said RNA silencing agents, and methods for RNA silencing of target genes using said improved RNA silencing agents (or portions thereof) (e.g., research and / or therapeutic methods). The RNA silencing agent comprises an antisense strand (or a portion thereof) wherein the antisense strand is sufficiently complementary to a heterozygous single nucleotide polymorphism to mediate an RNA-mediated silencing mechanism (e.g., RNAi).

[0325] In some embodiments, the provided siRNA compound has one or any combination of the following properties: (1) completely chemically stable (i.e., without unmodified 2'-OH residues); (2) asymmetric; (3) an 11-16 base pair duplex; and (4) a 5-8 base single-stranded, partially or fully phosphorylated tail. In different embodiments, the total number of phosphate thioester modifications varies from 6 to 17. In some embodiments, the siRNA comprises 8 total phosphate thioester modifications and at least one exNA nucleotide inter-link. In some embodiments, the siRNA comprises an antisense strand having 4 total phosphate thioester modifications and at least one exNA nucleotide inter-link.

[0326] In some embodiments, the siRNA compounds described herein can be conjugated to a variety of targets, including but not limited to cholesterol, DHA, benzopyrene, cortisol, vitamin A, vitamin D, GalNac, and gangliosides. In a wide range of cell types (e.g., HeLa cells, neurons, hepatocytes, trophoblasts), the cholesterol-modified formulation exhibits a 5-10 fold improvement in efficacy in vitro compared to previously used chemically stable modifiers (e.g., where all purines but not pyrimidines are modified).

[0327] Certain compounds of this disclosure that possess the structural characteristics described herein may be designated as “hsiRNA-ASP” (small interfering RNA with advanced stable patterns of hydrophobic modification). Furthermore, this hsiRNA-ASP pattern has shown significantly improved distribution in the brain, spinal cord, liver, placenta, kidney, spleen, and several other tissues, making it suitable for therapeutic intervention.

[0328] In the liver, hsiRNA-ASP is specifically delivered to endothelial cells and kupper cells, rather than hepatocytes, which makes this chemical modification pattern complementary to, rather than competing with, GalNac conjugates.

[0329] The compounds disclosed herein can be described in the following aspects and embodiments.

[0330] In the first aspect, this document provides oligonucleotides of at least 16 consecutive nucleotides 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 of the 5' end are not 2'-methoxy-ribonucleotides; and (3) the nucleotides are arranged by means of... Figure 1 The modifier key connection is shown.

[0331] a) Design of siRNA molecules

[0332] The siRNA molecule disclosed herein is a duplex consisting of a sense strand and a complementary antisense strand. In one embodiment, the siRNA molecule has a length of about 10-50 or more nucleotides, i.e., each strand contains 10-50 nucleotides (or nucleotide analogs). In another embodiment, the siRNA molecule has a length of about 15-30 nucleotides per strand, for example, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides, wherein one strand is sufficiently complementary to the target region. In one aspect, the strands are aligned such that at least 1, 2, or 3 bases are misaligned at the ends of the strands (i.e., no complementary bases to said bases appear in the opposing strands), such that when the strands anneal, a protrusion of 1, 2, or 3 residues appears at one or both ends of the duplex. In another aspect, the siRNA molecule has a length of about 10-50 or more nucleotides, i.e., each strand contains 10-50 nucleotides (or nucleotide analogs). On another front, siRNA molecules have a length of about 15-30, for example, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides in each strand, one strand being substantially complementary to the target sequence and the other strand being the same as or substantially the same as the first strand.

[0333] Typically, siRNAs can be designed using any method known in the art, for example, by using the following scheme:

[0334] 2. The sense strand of the siRNA is designed according to the sequence of the selected target site. In one embodiment, the sense strand comprises about 19 to 25 nucleotides, such as 19, 20, 21, 22, 23, 24, or 25 nucleotides. In another embodiment, the sense strand comprises 21, 22, or 23 nucleotides. However, those skilled in the art will understand that siRNAs shorter than 19 nucleotides or longer than 25 nucleotides can also mediate RNAi. Therefore, siRNAs of this length are also within the scope of this disclosure, as long as they retain the ability to mediate RNAi. Longer RNA silencers have been shown to elicit interferon or protein kinase R (PKR) responses in some mammalian cells, which may be undesirable. In one aspect, the RNA silencers of this disclosure do not elicit PKR responses (i.e., have a sufficiently short length). However, longer RNA silencers may be useful, for example, in cell types that cannot produce PKR responses, or where PKR responses have been downregulated or inhibited by alternative methods.

[0335] The siRNA molecules described herein possess sufficient complementarity to the target sequence, enabling the siRNA to mediate RNAi. Generally, the siRNA containing the nucleotide sequence is sufficiently identical to the target sequence portion of the target gene to achieve RISC-mediated target gene cleavage. 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 the target site. However, 100% identity is not required. In one embodiment, greater than 80% identity between the sense strand and the target RNA sequence is utilized, such as 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even 100% identity. This disclosure has the advantage of tolerating certain sequence variations to improve the efficiency and specificity of RNAi. In one implementation, the sense strand has 4, 3, 2, 1, or 0 mismatched nucleotides with the target region, for example, in a target region where the wild-type and mutant alleles differ by at least one base pair, such as a target region containing a gain-of-function mutation, and the other strand is 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 nucleotide analog substitutions or insertions can be effectively repressed.

[0336] Sequence identity can be determined using sequence comparison and alignment algorithms known in the art. To determine the percentage identity of two nucleic acid sequences (or two amino acid sequences), the sequences are aligned for optimal comparison purposes (e.g., vacancies can be introduced in either the first or second sequence to achieve optimal alignment). The nucleotides (or amino acid residues) at the corresponding nucleotide (or amino acid) positions are then compared. When 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 two sequences is a function of the number of shared positions (i.e., % homology = number of shared positions / total number of positions x 100), with optional penalties for the number of introduced vacancies and / or the length of introduced vacancies.

[0337] Mathematical algorithms can be used to compare sequences and determine the percentage of identity between two sequences. In one implementation, alignments are generated on specific portions of the aligned sequences that have sufficient identity, but not on portions with low identity (i.e., local alignments). A non-limiting example of a local alignment algorithm for sequence comparison is Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264-68, modified to Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-77. This algorithm was incorporated into the BLAST procedure (version 2.0) of Altschul et al. (1990) J. Mol. Biol. 215:403-10.

[0338] In another implementation, alignment is optimized by introducing appropriate vacancies and determining the percentage of identity over the length of the aligned sequences (i.e., vacancy alignment). To obtain vacancy-containing alignments for comparison, GappedBLAST, as described by Altschul et al. (1997) Nucleic Acids Res. 25(17):3389-3402, can be used. In another implementation, alignment is optimized by introducing appropriate vacancies and determining the percentage of identity over the entire length of the aligned sequences (i.e., global alignment). A non-limiting example of a mathematical algorithm for global sequence comparison is the algorithm by Myers and Miller, CABIOS (1989). This algorithm was incorporated into the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package. When comparing amino acid sequences using the ALIGN program, a PAM120 weight residue table, a vacancy length penalty of 12, and a vacancy penalty of 4 can be used.

[0339] 3. The antisense or guide strand of siRNA is typically the same length as the sense strand and includes complementary nucleotides. In one embodiment, the guide and sense strands are perfectly complementary, i.e., the strands are blunt-ended upon alignment or annealing. In another embodiment, the strands of siRNA may be paired with 1 to 7 (e.g., 2, 3, 4, 5, 6, or 7) or 1 to 4, e.g., 2, 3, or 4 nucleotides, 3' overhangs. The overhangs may contain (or be composed of) nucleotides corresponding to the target gene sequence (or its complementary sequence). Alternatively, the overhangs may contain deoxyribonucleotides, such as dT, or nucleotide analogs, or other suitable non-nucleotide material (or be composed of). Thus, in another embodiment, the nucleic acid molecule may have a 2-nucleotide 3' overhang, such as TT. The overhanging nucleotides may be RNA or DNA. As described above, it is desirable to select a target region where the mutation: wild-type mismatch is a purine: purine mismatch.

[0340] 4. Using any method known in the art, potential targets can be compared with appropriate genomic databases (human, mouse, rat, etc.) and any target sequences that have significant homology with other coding sequences can be eliminated. One such method for homology searches of such sequences is called BLAST, which is available on the website of the National Center for Biotechnology Information.

[0341] 5. Select one or more sequences that meet the evaluation criteria.

[0342] More general information about the design and use of siRNA can be found in "The siRNA User Guide" on the Max-Plank-Institut furBiophysikalische Chemie website.

[0343] Alternatively, siRNA can be functionally defined as a nucleotide sequence (or oligonucleotide sequence) capable of hybridizing with a target sequence (e.g., hybridization at 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50 °C or 70 °C for 12–16 hours; followed by washing). Additional hybridization conditions include hybridization at 70 °C in 1xSSC or 50 °C in 1xSSC, 50% formamide, followed by washing at 70 °C in 0.3xSSC, or hybridization at 70 °C in 4xSSC or 50 °C in 4xSSC, 50% formamide, followed by washing at 67 °C in 1xSSC. For hybrids shorter than 50 base pairs, the hybridization temperature should be 5–10 °C lower than the melting temperature (Tm) of the hybrid, where Tm is determined according to the following equation: For hybrids shorter than 18 base pairs, Tm (°C) = 2(# of A + T bases) + 4(# of G + C bases). For hybrids with lengths between 18 and 49 base pairs, Tm(°C) = 81.5 + 16.6(log 10[Na]). + ])+0.41(% G+C)-(600 / N), where N is the number of bases in the hybrid, and [Na + [Na] is the concentration of sodium ions in the hybridization buffer (1xSSC of [Na]). + [=0.165 M]. Sambrook, J., EF Fritsch and T. Maniatis, 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Chapters 9 and 11 and Current Protocols in Molecular Biology, 1995, FM Ausubel et al., eds., John Wiley & Sons, Inc., Sections 2.10 and 6.3-6.4 provide other examples of stringent conditions for polynucleotide hybridization, which are incorporated herein by reference.

[0344] The negative control siRNA should have the same nucleotide composition as the selected siRNA, but without significant sequence complementarity to the appropriate genome. This negative control can be designed by randomly scrambling the nucleotide sequence of the selected siRNA. Homology searches can be performed to ensure that the negative control lacks homology with any other genes in the appropriate genome. Alternatively, the negative control siRNA can be designed by introducing one or more base mismatches into the sequence.

[0345] 6. To verify the effectiveness of siRNA in disrupting target mRNAs (e.g., wild-type or mutant mRNAs), siRNA can be incubated with cDNA in a Drosophila-based in vitro mRNA expression system. 32 Newly synthesized target mRNAs radiolabeled with P were detected by autoradiography on agarose gels. The presence of cleaved target mRNAs indicates mRNA nuclease activity. Suitable controls include omitting the siRNA and using a non-target cDNA. Alternatively, a control siRNA with the same nucleotide composition as the selected siRNA but without significant sequence complementarity to the appropriate target gene can be chosen. This negative control can be designed by randomly scrambling the nucleotide sequence of the selected siRNA. Homology searches can be performed to ensure that the negative control lacks homology with any other gene in the appropriate genome. Furthermore, negative control siRNAs can be designed by introducing one or more base mismatches into the sequence.

[0346] siRNAs can be designed to target any of the target sequences described above. The siRNA contains an antisense strand that is fully complementary to the target sequence to mediate the silencing of the target sequence. In some embodiments, the RNA silencing agent is siRNA.

[0347] In some embodiments, the siRNA comprises a sense strand, said sense strand comprising... Figure 1 The shown link may contain an antisense chain, which includes... Figure 1 The bonding shown.

[0348] Select the siRNA-mRNA complementary site that leads to optimal mRNA specificity and maximum mRNA cleavage.

[0349] b) siRNA-like molecules

[0350] The disclosed siRNA-like molecule has a sequence that is "fully complementary" to the target sequence of the mRNA (i.e., has a strand with the required sequence) to guide gene silencing via RNAi or translational repression. The siRNA-like molecule is designed in the same manner as the siRNA molecule, but the degree of sequence identity between the sense strand and the target RNA is close to that observed between miRNA and its target. Generally, as the degree of sequence identity between the miRNA sequence and the corresponding target gene sequence decreases, the tendency to mediate posttranscriptional gene silencing via translational repression rather than RNAi increases. Therefore, in another embodiment, where posttranscriptional gene silencing via translational repression of the target gene is required, the miRNA sequence is partially complementary to the target gene sequence. In some implementations, the miRNA sequence is partially complementary to 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 translational repression mechanisms are cooperative, multiple complementary sites (e.g., 2, 3, 4, 5, or 6) can be targeted in some implementations.

[0351] The ability of siRNA-like duplexes to mediate RNAi or translational repression can be predicted by the distribution of different nucleotides at complementary sites between the target gene sequence and the silencing agent nucleotide sequence. In one embodiment, where gene silencing via translational repression is required, the central portion of the complementary site contains at least one dissimilarity nucleotide such that the duplex formed by the miRNA guide strand and the target mRNA contains a central “bump” (Doench JG et al., Genes & Dev., 2003). In another embodiment, 2, 3, 4, 5, or 6 dissimilarity nucleotides, either consecutive or discontinuous, are introduced. The dissimilarity nucleotides can be chosen to form wobbly base pairs (e.g., G:U) or mismatched base pairs (G:A, C:A, C:U, G:G, A:A, C:C, U:U). In a further embodiment, the “bump” is located at nucleotide positions 12 and 13 at the 5' end of the miRNA molecule.

[0352] c) Short hairpin RNA (shRNA) molecules

[0353] In some specific embodiments, this disclosure provides shRNAs capable of mediating RNA silencing of target sequences with enhanced selectivity. Compared to siRNAs, shRNAs mimic the natural precursors of microRNAs (miRNAs) and enter the apex of the gene silencing pathway. For this reason, it is thought that shRNAs can mediate gene silencing more effectively through the entire natural gene silencing pathway.

[0354] miRNAs are approximately 22-nucleotide non-coding RNAs that regulate gene expression at the post-transcriptional or translational level during plant and animal development. A common characteristic of miRNAs is that they are excised from a precursor RNA stem-loop of approximately 70 nucleotides, known as premiRNAs, possibly by an RNase type III enzyme, Dicer, or a homolog thereof. Naturally occurring miRNA precursors (premiRNAs) have a single strand forming a double-stranded stem comprising two typically complementary parts and a loop connecting the two parts of the stem. In a typical premiRNA, the stem includes one or more protrusions, such as extra nucleotides that create a single nucleotide “loop” in one part of the stem, and / or one or more unpaired nucleotides that create vacancies when the two parts of the stem hybridize with each other. The short hairpin RNAs or engineered RNA precursors disclosed herein are artificial constructs based on these naturally occurring premiRNAs, but engineered to deliver the desired RNA silencing agent (e.g., siRNAs disclosed herein). shRNAs are formed by replacing the stem sequence of the premiRNA with a sequence complementary to the target mRNA. shRNAs are processed through the entire gene silencing pathway of the cell, thereby effectively mediating RNAi.

[0355] The essential elements of an shRNA molecule include a first part and a second part, which are sufficiently complementary to anneal or hybridize to form a double helix or double-stranded stem. These two parts do not need to be perfectly or completely complementary. The first and second “stem” parts are connected by a portion having a sequence whose sequence complementarity is insufficient to anneal or hybridize with the other parts of the shRNA. The latter part is referred to as the “loop” portion in the shRNA molecule. The shRNA molecule is processed to produce siRNA. shRNA may also include one or more protrusions, which are additional nucleotides that create small nucleotide “loops” within a portion of the stem, such as one, two, or three nucleotide loops. The stem portions can be of uniform length, or a portion may include, for example, a protrusion of 1-5 nucleotides. The protruding nucleotides may include, for example, uracil (Us), such as all Us. Such Us are particularly encoded by thymidines (Ts) in the DNA encoded by the shRNA, which signal the termination of transcription.

[0356] In the shRNA (or engineered precursor RNA) disclosed herein, a portion of the double-stranded stem is a nucleic acid sequence complementary to (or antisense) the target sequence. Preferably, one strand of the shRNA stem portion is sufficiently complementary to (e.g., antisense) the target RNA (e.g., mRNA) sequence to mediate the degradation or cleavage of the target RNA via RNA interference (RNAi). Thus, the engineered RNA precursor comprises a double-stranded 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 shRNA stem portion is preferably about 15 to about 50 nucleotides long. Preferably, the length of the two stem portions is about 18 or 19 to about 21, 22, 23, 24, 25, 30, 35, 37, 38, 39, or 40 or more nucleotides. In a preferred embodiment, the stem portion should be 21 nucleotides or longer. When used in mammalian cells, the stem portion should be less than about 30 nucleotides long to avoid eliciting nonspecific responses such as those from the interferon pathway. In non-mammalian cells, the stem can be longer than 30 nucleotides. In fact, the stem can include a larger portion (up to and including the entire mRNA) that is complementary to the target mRNA.

[0357] The two parts of a double-stranded stem must be sufficiently complementary to hybridize and form a double-stranded stem. Therefore, the two parts can, but do not need to, be completely or perfectly complementary. Furthermore, the two stem parts can have the same length, or one part can include a protruding end of 1, 2, 3, or 4 nucleotides. The protruding nucleotide can include, for example, uracil (Us), such as all Us. Loops in shRNA or engineered RNA precursors can differ from the natural premiRNA 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, the loop length in shRNA or engineered RNA precursors can be 2, 3, 4, 5, 6, 7, 8, 9, or more, for example, 15 or 20 or more nucleotides.

[0358] The loop in shRNA or engineered RNA precursors can be modified to increase or decrease the number of paired nucleotides, or replaced entirely or partially with a tetra-loop or other loop sequence to differ from the natural premiRNA sequence. Therefore, the length of the loop portion in shRNA can be from about 2 to about 20 nucleotides, i.e., about 2, 3, 4, 5, 6, 7, 8, 9 or more, for example, 15 or 20 or more nucleotides in length. Preferred loops consist of or contain a tetra-loop sequence. Exemplary tetra-loop sequences include, but are not limited to, the sequences GNRA, GGGG, and UUUU, where N is any nucleotide and R is a purine nucleotide.

[0359] In some embodiments, the shRNA disclosed herein 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 substantially as described above, or typically by selecting a sequence of 18, 19, 20, 21 nucleotides or longer from the target RNA (e.g., a region of 100 to 200 or 300 nucleotides upstream or downstream of the start of translation). Generally, this sequence can be selected from any portion of the target RNA (e.g., mRNA), including a 5' UTR (untranslated region), a coding sequence, or a 3' UTR. The sequence may optionally immediately follow a target gene region containing two adjacent AA nucleotides. The last two nucleotides of the nucleotide sequence may optionally be UU. This approximately 21 nucleotide sequence is used to generate a portion of the double-stranded stem in the shRNA. This sequence may, for example, enzymatically replace the stem portion of the wild-type premiRNA sequence, or be included in the synthesized complete sequence. For example, DNA oligonucleotides can be synthesized that encode the entire stem-loop engineered RNA precursor or only the portion to be inserted into the stem of the precursor duplex, and the engineered RNA precursor construct can be constructed using restriction endonucleases, such as from wild-type premiRNA.

[0360] Within the double-stranded stem, the engineered RNA precursor comprises approximately 21-22 nucleotide sequences of siRNA or siRNA-like double strands desired to be produced in vivo. Therefore, the stem portion of the engineered RNA precursor includes at least 18 or 19 nucleotide pairs corresponding to the exon sequences of the genes whose expression will be downgraded or repressed. Two 3' nucleotides located flanking this region of the stem are selected to maximize the production of siRNA from the engineered RNA precursor and to maximize the efficacy of the resulting siRNA in targeting the corresponding mRNA in vivo and in vitro for translational repression or disruption via RNAi.

[0361] In some embodiments, the shRNA disclosed herein includes a miRNA sequence, optionally with terminally modified miRNA sequences to enhance entry into RISC. The miRNA sequence may be similar to or identical to any naturally occurring miRNA sequence (see, for example, ThemiRNA Registry; Griffiths-Jones S, Nuc. Acids Res., 2004). To date, over one thousand natural miRNAs have been identified, which together are considered to comprise approximately 1% of all predicted genes in the genome. Many natural miRNAs cluster together in the introns of their pre-mRNAs and can be identified on computers using homology-based searches (Pasquinelli et al., 2000; Lagos-Quintana et al., 2001; Lau et al., 2001; Lee and Ambros, 2001) or computer algorithms (e.g., MiRScan, MiRSeeker) to predict the stem-loop structure of candidate miRNA genes forming primary mRNAs (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 a searchable database of all published miRNA sequences (miRNA Registry on the Sanger Institute website; Griffiths-Jones S, Nuc. AcidsRes., 2004). Exemplary natural miRNAs include lin-4, let-7, miR-10, miR-15, miR-16, miR-168, miR-175, miR-196 and their homologs, as well as other natural miRNAs from humans and certain model organisms, including Drosophila melanogaster, Caenorhabditis elegans, zebrafish, Arabidopsis thaliana, house mouse, and brown rat, as described in International PCT Publication WO 03 / 029459.

[0362] Naturally occurring miRNAs are expressed by endogenous genes in vivo and processed from hairpin or stem-loop precursors (premiRNAs or primary miRNAs) via 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 a double-stranded form, but only one strand is taken up by the RISC complex to guide gene silencing. Some miRNAs, such as plant miRNAs, have perfect or near-perfect complementarity with their target mRNAs, thus directly cleaving the target mRNA. Other miRNAs are not perfectly complementary to their target mRNAs, thus directly repressing translation. It is believed that the degree of complementarity between a miRNA and its target mRNA determines its mechanism of action. For example, perfect or near-perfect complementarity between a miRNA and its target mRNA predicts a cleavage mechanism (Yekta et al., Science, 2004), while imperfect complementarity predicts a translational repression mechanism. In specific implementations, the miRNA sequence is a naturally occurring miRNA sequence whose aberrant expression or activity is associated with miRNA-related disorders.

[0363] d) Bifunctional oligonucleotide chains

[0364] In other embodiments, the RNA silencing agents of this disclosure comprise bifunctional oligonucleotide chains that can be used to recruit miRNAs intercellularly. Animal cells express a range of miRNAs, approximately 22 nucleotides of non-coding RNA, that regulate gene expression at the post-transcriptional or translational level during animal development. By binding to and recruiting RISC-bound miRNAs to target mRNAs, bifunctional oligonucleotide chains can suppress the expression of genes involved in processes such as atherosclerosis. The use of oligonucleotide chains offers several advantages compared to prior art methods for suppressing the expression of specific genes. First, the methods described herein allow endogenous (typically abundant) miRNAs to mediate 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 agent and, in particular, the linker portion (e.g., oligonucleotides such as 2'-O-methyl oligonucleotides) can be stabilized and resistant to nuclease activity. Therefore, the chains 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 reagents intracellularly. Third, the ligands and their respective portions can be designed to conform to specific mRNA sites and specific miRNAs. This design can be cell- and gene product-specific. Fourth, the methods disclosed herein preserve the integrity of the mRNA, allowing those skilled in the art to use the cell's own mechanisms to block protein synthesis in short pulses. Therefore, these RNA silencing methods are highly tunable.

[0365] The bifunctional oligonucleotide lineages (“lineages”) disclosed herein are designed to recruit miRNAs (e.g., endogenous cellular miRNAs) to target mRNAs, thereby inducing regulation of the gene of interest. In a preferred embodiment, the lineage has the formula TL-μ, where T is the mRNA targeting portion, L is the linking portion, and μ is the miRNA recruitment portion. Any one or more portions may be double-stranded. However, preferably, each portion is single-stranded.

[0366] The portions within the ligand can be arranged or connected (in the 5' to 3' direction), as shown in formula TL-μ (i.e., the 3' end of the targeting portion is connected to the 5' end of the connecting portion, and the 3' end of the connecting portion is connected to the 5' end of the miRNA recruitment portion). Alternatively, these portions can be arranged or connected in the ligand as follows: μ-TL (i.e., the 3' end of the miRNA recruitment portion is connected to the 5' end of the connecting portion, and the 3' end of the connecting portion is connected to the 5' end of the targeting portion).

[0367] As described above, the mRNA targeting moiety is capable of capturing a specific target mRNA. According to this disclosure, expression of the target mRNA is undesirable; therefore, translational repression of the mRNA is desirable. The size of the mRNA targeting moiety should be sufficient to effectively bind the target mRNA. The length of the targeting moiety varies considerably, depending in part 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 in length. In a particular embodiment, the length of the targeting moiety is about 15 to about 25 nucleotides.

[0368] As described above, the miRNA recruitment portion is capable of binding to the miRNA. According to this disclosure, the miRNA can be any miRNA capable of repressing the target mRNA. More than 250 endogenous miRNAs have been reported in mammals (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.

[0369] The linker is any reagent capable of connecting to the target moiety to maintain its activity. Preferably, the linker is an oligonucleotide moiety containing a sufficient number of nucleotides to allow the target agent to interact adequately with its respective target. The linker has little or no sequence homology with cellular mRNA or miRNA sequences. Exemplary linkers include one or more 2'-O-methylnucleotides, such as 2'-β-methyladenosine, 2'-O-methylthymidine, 2'-O-methylguanosine, or 2'-O-methyluridine.

[0370] e) Gene silencing oligonucleotides

[0371] In some exemplary embodiments, gene expression (e.g., target gene expression) can be regulated using oligonucleotide-based compounds comprising two or more single-stranded antisense oligonucleotides linked by their 5' ends, allowing for the presence of two or more accessible 3' ends to effectively inhibit or reduce target gene expression. Such linked oligonucleotides are also known as gene silencing oligonucleotides (GSOs). (See, for example, US 8,431,544, assigned to Idera Pharmaceuticals, Inc., which is incorporated herein by reference in its entirety for all purposes). This document provides novel and improved GSOs comprising subunit bonds according to formula (I) and embodiments thereof.

[0372] The 5' linker of GSO is independent of other oligonucleotide links and can be directly via the 5', 3', or 2' hydroxyl group, or indirectly via a non-nucleotide linker or nucleoside utilizing the 2' or 3' hydroxyl position of the nucleoside. Linkage can also utilize the functionalized sugar or nucleobase of the 5'-terminal nucleotide.

[0373] GSOs can contain two identical or different sequences conjugated at their 5'-5' ends by phosphodiester, thiophosphate, or non-nucleoside linkers. These compounds can contain 15 to 27 nucleotides complementary to specific portions of the target mRNA for antisense downregulation of the gene product. GSOs containing the same sequence can bind to specific mRNAs and repress protein expression via Watson-Crick hydrogen bond interactions. GSOs containing different sequences can bind to two or more different regions of one or more mRNA targets and repress protein expression. These compounds consist of heteronucleotide sequences complementary to the target mRNA, forming a stable double-stranded structure via Watson-Crick hydrogen bonds. Under certain conditions, GSOs containing two free 3' ends (5'-5' antisense links) may repress gene expression more effectively than those containing a single free 3' end or none at all.

[0374] In some implementations, the non-nucleotide linker is of the formula HO--(CH2). o --CH(OH)--(CH2) p --OH is a glycerol or glycerol homologue, wherein o and p are independently integers 1 to about 6, 1 to about 4, or 1 to about 3. In some other embodiments, the non-nucleotide linker is a derivative of 1,3-diamino-2-hydroxypropane. Some such derivatives have the molecular formula HO--(CH2). m --C(O)NH--CH2--CH(OH)--CH2--NHC(O)--(CH2) m--OH, where m is an integer from 0 to about 10, from 0 to about 6, from 2 to about 6, or from 2 to about 4.

[0375] Some nonnucleotide linkers allow the linkage of more than two GSO components. For example, the nonnucleotide linker glycerol has three hydroxyl groups, to which the GSO components can be covalently linked. Therefore, some oligonucleotide-based compounds of this disclosure contain two or more oligonucleotides linked to nucleotide or nonnucleotide linkers. Such oligonucleotides according to this disclosure are referred to as “branched”.

[0376] In some embodiments, the GSO is at least 14 nucleotides in length. In some exemplary embodiments, the GSO is 15 to 40 nucleotides or 20 to 30 nucleotides in length. Therefore, 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 in length.

[0377] These oligonucleotides can be prepared using methods recognized in the art, such as aminophosphate or H-phosphonate chemistry, which can be performed manually or by an automated synthesizer. These oligonucleotides can also be modified in a variety of ways without impairing their ability to hybridize with mRNA. Such modifications may include at least one internucleotide bond of the oligonucleotide, which is an alkylphosphonate, thiophosphate, dithiophosphate, methylphosphonate, phosphate, alkylthiophosphonate, aminophosphate, carbamate, carbonate, phosphate hydroxyl, acetamide, carboxymethyl ester, or a combination of these and other internucleotide bonds between the 5' end of one nucleotide and the 3' end of another nucleotide, wherein the 5' nucleotide phosphodiester bond has been substituted with any number of chemical groups.

[0378] VI. Modified RNA silencing agents

[0379] In certain aspects of this disclosure, the oligonucleotides, siRNAs, and RNA silencing agents (or any portion thereof) of this disclosure as described above may be modified to further enhance the activity of the reagent. For example, the RNA silencing agent described in Part II above may be modified with any of the modifications described below. Modifications may be used in part to further enhance target discrimination, enhance reagent stability (e.g., prevent degradation), promote cellular uptake, improve target efficiency, enhance binding efficacy (e.g., binding to the target), improve patient tolerability, and / or reduce toxicity.

[0380] 1) Modifications to enhance target recognition

[0381] In some embodiments, the oligonucleotides, siRNAs, and RNA silencing agents of this disclosure may be replaced with destabilizing nucleotides to enhance single nucleotide target discrimination (see U.S. Application Serial 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 modification may be sufficient to eliminate the RNA silencing agent's specificity for non-target mRNAs (e.g., wild-type mRNA) without significantly affecting the RNA silencing agent's specificity for target mRNAs (e.g., gain-of-function mutant mRNA).

[0382] In a preferred embodiment, 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 capable of base pairing with any of the four conventional nucleotide bases (e.g., A, G, C, U). Universal nucleotides are preferred because they have a relatively small impact on the stability of the RNA duplex or the duplex 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 an inosine analog 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, aminophosphate-inosine, 2'-O-methoxyethyl-inosine, and 2'-OMe-inosine. In a particularly preferred embodiment, the universal nucleotide is an inosine residue or a naturally occurring analogue thereof.

[0383] In some embodiments, the RNA silencing agent of this disclosure is modified by introducing at least one destabilizing nucleotide within 5 nucleotides of the specific determinant nucleotide (i.e., the nucleotide that recognizes the disease-related polymorphism). For example, the destabilizing nucleotide may be introduced within 5, 4, 3, 2, or 1 nucleotides of the specific determinant nucleotide. In an exemplary embodiment, the destabilizing nucleotide is introduced at a position 3 nucleotides away from the specific determinant nucleotide (i.e., such that there are 2 stabilizing nucleotides between the destabilizing nucleotide and the specific determinant nucleotide). In RNA silencing agents having two strands or strand portions (e.g., siRNA and shRNA), the destabilizing nucleotide may be introduced into the strand or strand portion that does not contain the specific determinant nucleotide. In a preferred embodiment, the destabilizing nucleotide is introduced into the same strand or strand portion that contains the specific determinant nucleotide.

[0384] 2) Modifications to improve efficacy and specificity

[0385] In some embodiments, the oligonucleotides, siRNAs, and RNA silencers of this disclosure can be modified according to asymmetric design rules to enhance the efficacy and specificity of RNAi-mediated RNAi (see U.S. Patent Nos. 8,309,704, 7,750,144, 8,304,530, 8,329,892, and 8,309,705). This modification favors the entry of the antisense strand of the siRNA (e.g., siRNA designed using the methods of this disclosure or siRNA generated from shRNA) into the RISC, favoring the sense strand, such that the antisense strand preferentially guides the 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 silencer is enhanced by reducing the base pair strength between the 5' end (AS 5') of the antisense strand and the 3' end (S 3') of the sense strand relative to the bond strength or base pair strength between the 3' end (AS 3') of the antisense strand and the 5' end (S' 5') of the sense strand.

[0386] In one embodiment, the asymmetry of the RNA silencer of this disclosure can be enhanced such that there are fewer G:C base pairs between the 5' end of the first or antisense strand and the 3' end of the sense strand compared to the 3' end of the first or antisense strand and the 5' end of the sense strand. In another embodiment, the asymmetry of the RNA silencer of this disclosure can 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. 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 another embodiment, the asymmetry of the RNA silencer of this disclosure can be enhanced such that there is at least one wobbly base pair, for example, G:U, between the 5' end of the first or antisense strand and the 3' end of the sense strand. In another embodiment, the asymmetry of the RNA silencer of this disclosure can be enhanced such that at least one base pair comprises a rare nucleotide, such as inosine (I). Preferably, the base pairs are 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 this disclosure can be enhanced such that at least one base pair comprises 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.

[0387] 3) RNA silencing agents with enhanced stability

[0388] The RNA silencing agents disclosed herein can be modified to improve their stability in serum or cell culture growth media. To enhance stability, the 3'-residues can be stabilized to prevent degradation; for example, they can be selected such that they consist of purine nucleotides, particularly adenosine or guanosine nucleotides. Alternatively, pyrimidine nucleotides can be substituted with modified analogs, such as uridine instead of 2'-deoxythymidine, which is acceptable and does not affect the efficiency of RNA interference.

[0389] In one aspect, this disclosure features an RNA silencer comprising first and second strands, wherein the second strand and / or the first strand is modified by replacing an internal nucleotide with a modified nucleotide, thereby enhancing in vivo stability compared to the corresponding unmodified RNA silencer. As defined herein, an "internal" nucleotide refers to a nucleotide present at any position other than the 5' or 3' end of a nucleic acid molecule, polynucleotide, or oligonucleotide. Internal nucleotides may be within a single-stranded molecule or within a strand of a duplex or double-stranded molecule. In one embodiment, the sense strand and / or antisense strand are modified by replacing at least one internal nucleotide. In another embodiment, the sense strand and / or antisense strand are modified by substituting 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 replacing at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more of the internal nucleotides. In yet another embodiment, the sense strand and / or antisense strand are modified by replacing all the internal nucleotides.

[0390] In one aspect, this disclosure is characterized by an RNA silencer that is at least 80% chemically modified. In a preferred embodiment of this disclosure, the RNA silencer may be completely chemically modified, i.e., 100% of the nucleotides are chemically modified.

[0391] In a preferred embodiment of this disclosure, the RNA silencing agent may comprise at least one modified nucleotide analog. The nucleotide analog may be located at a position where target-specific silencing activity (e.g., RNAi-mediated activity or translational repression activity) is substantially unaffected, for example, in the 5'-terminal and / or 3'-terminal regions of the siRNA molecule. In particular, the ends can be stabilized by incorporating a modified nucleotide analog.

[0392] Exemplary nucleotide analogs include sugar and / or backbone-modified ribonucleotides (i.e., including modifications to the phosphate sugar backbone). For example, the phosphodiester bonds of native RNA can be modified to include at least one of nitrogen or sulfur heteroatoms. In exemplary backbone-modified ribonucleotides, the phosphate ester group linked to an adjacent ribonucleotide is replaced by a modified group (e.g., a thiophosphate ester group). In exemplary sugar-modified ribonucleotides, the 2′-OH- group can be replaced by a group selected from H, OR, R, halogen, SH, SR, NH2, NHR, NR2, or ON, wherein R is C1-C6 alkyl, alkenyl, or alkynyl and the halogen is F, Cl, Br, or I.

[0393] In specific embodiments, the modification is a 2'-fluorine, 2'-amino, and / or 2'-sulfur modification. Particularly preferred modifications include 2'-fluoro-cytidine, 2'-fluoro-uridine, 2'-fluoro-adenosine, 2'-fluoro-guanosine, 2'-amino-cytidine, 2'-amino-uridine, 2'-amino-adenosine, 2'-aminoguanosine, 2,6-diaminopurine, 4-thiouridine, and / or 5-aminoallyluridine. In specific embodiments, the 2'-fluororibonucleotide is present in each uridine and cytidine. Other exemplary modifications include 5-bromo-uridine, 5-iodo-uridine, 5-methyl-cytidine, ribo-thymidine, 2-aminopurine, 2'-amino-butyryl-pyrene-uridine, 5-fluoro-cytidine, and 5-fluorouridine. 2'-Deoxynucleotides and 2'-Ome nucleotides may also be used for the RNA-silencing portion of the modifications disclosed herein. Other modified residues include deoxy-base-free, inosine, N3-methyl-uridine, N6,N6-dimethyl-adenosine, pseudouridine, purine ribonucleoside, and ribavirin. In a particularly preferred embodiment, the 2' portion is methyl, such that the linking moiety is a 2'-O-methyl oligonucleotide.

[0394] In an exemplary embodiment, the RNA silencing agent of this disclosure comprises locked nucleic acid (LNA). LNA comprises a sugar-modified nucleotide with anti-nuclease activity (highly stable) and single nucleotide differentiation of 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 2'-O,4'-C-ethylene-bridged nucleic acids and may have modifications such as 2'-deoxy-2''-fluorouridine. Furthermore, LNA increases the specificity of the oligonucleotide by restricting the sugar moiety to the 3'-inner conformation, thereby pre-organizing the nucleotide for base pairing and raising the melting temperature of the oligonucleotide by up to 10°C. / base.

[0395] In another exemplary embodiment, the RNA silencing agent of this disclosure comprises peptide nucleic acid (PNA). PNA comprises a modified nucleotide in which the sugar phosphate moiety of the nucleotide is replaced by a neutral 2-aminoethylglycine moiety that is capable of forming a polyamide backbone that is highly resistant to nuclease digestion and confers higher binding specificity to the molecule (Nielsen et al., Science, (2001), 254: 1497-1500).

[0396] Preferred ribonucleotides are those with modified nucleobases, i.e., ribonucleotides containing at least one non-naturally occurring nucleobase instead of a naturally occurring nucleobase. Bases can be modified to block the activity of adenosine deaminase. Exemplary modified nucleobases include, but are not limited to, uridine and / or cytidine modified at the 5-position, such as 5-(2-amino)propyluridine, 5-bromouridine; adenosine and / or guanosine modified at the 8-position, such as 8-bromoguanosine; denitronucleotides, such as 7-denitro-adenosine; and O- and N-alkylated nucleotides, such as N6-methyladenosine. It should be noted that the above modifications can be combined.

[0397] In other embodiments, cross-linking can be used to alter the pharmacokinetics of the RNA silencer, for example, by increasing its half-life in vivo. Therefore, this disclosure includes RNA silencers having two complementary nucleic acid strands, wherein the two strands are cross-linked. This disclosure also includes RNA silencers conjugated or unconjugated to another portion (e.g., a non-nucleic acid portion such as a peptide), an organic compound (e.g., a dye), or the like (e.g., at its 3' end). Modifying siRNA derivatives in this manner, compared to the corresponding siRNA, can improve the cellular uptake or enhance the cell-targeting activity of the resulting siRNA derivative, enabling it to be used for tracking siRNA derivatives in cells, or improve the stability of the siRNA derivative compared to the corresponding siRNA.

[0398] Other exemplary modifications include: (a) 2' modifications, such as providing a 2'OMe portion on the sense or antisense chain, but particularly on the sense chain, or providing a 2'OMe portion in a 3' overhang, such as at a 3' end (a 3' end refers to a 3' atom in the molecule or at the most 3' portion, for example, at the most 3' P or 2' position as indicated by the context); (b) modifications to the phosphate backbone, such as replacing O with S, for example on U or A or both, particularly providing a thiophosphate modification on the antisense chain; for example, replacing O with S; (c) replacing U with a C5 amino linker; (d) replacing A with G (the sequence change is preferably located on the sense chain rather than the antisense chain); and (d) modifications at the 2', 6', 7', or 8' positions. Exemplary embodiments are those in which one or more of these modifications are present on the sense chain but not on the antisense chain, or embodiments in which the antisense chain has fewer such modifications. Other exemplary modifications include the 3' protrusion, for example, using methylated P at the 3' end; 2' modifications, such as providing a 2'OMe moiety and a main chain modification, such as replacing O with S, for example, providing a thiophosphate modification, or a combination of using methylated P at the 3' protrusion, for example, at the 3' end; 3' alkyl modifications; modifications at the 3' protrusion, for example, using a debasing pyrrolidone; modifications with naproxen, ibuprofen, or other moieties that inhibit 3' end degradation.

[0399] 4) Modifications that enhance cellular uptake

[0400] In other embodiments, the RNA silencer can be chemically modified, for example, to enhance cellular uptake by target cells (e.g., neurons). Therefore, this disclosure includes RNA silencers conjugated or unconjugated to another portion (e.g., a non-nucleic acid portion, such as a peptide), an organic compound (e.g., a dye), etc. Conjugation can be accomplished by methods known in the art, such as those described in Lambert et al., Drug Deliv. Rev.: 47(1), 99-112 (2001) (describes nucleic acids loaded onto polyalkyl cyanoacrylate (PACA) nanoparticles); Fattal et al., J. Control Release 53(1-3):137-43 (1998) (describes nucleic acids bound to nanoparticles); Schwab et al., Ann. Oncol. 5 Supplement 4:55-8 (1994) (describes nucleic acids linked to intercalators, hydrophobic groups, polycations, or PACA nanoparticles); and Godard et al., Eur. J. Biochem. 232(2):404-10 (1995) (describes nucleic acids linked to nanoparticles).

[0401] In certain embodiments, the RNA silencing agent of this disclosure is conjugated to a lipophilic moiety. In one embodiment, the lipophilic moiety is a ligand comprising a cationic group. In another embodiment, the lipophilic moiety is attached to one or both strands of the siRNA. In one 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 some 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 moieties include cholic acid, adamantaneacetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis-O-(hexadecyl)glycerol, geraniol, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecanyl, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholic acid, dimethoxytriphenylmethyl, or phenoxazine.

[0402] 5) Tethering ligands

[0403] Other entities can be linked to the RNA silencing agent disclosed herein. For example, ligands may be linked to the RNA silencing agent to improve stability, hybridization thermodynamics with the target nucleic acid, targeting a specific tissue or cell type, or cell permeability, such as through endocytosis-dependent or independent mechanisms. Ligands and associated modifications can also increase sequence specificity, thereby reducing ectopic targeting. Etethered ligands may include one or more modified bases or sugars that can be used as intercalators. These are preferably located in internal regions, such as in the protrusions of the RNA silencing agent / target duplex. Intercalators may be aromatic compounds, such as polycyclic aromatic compounds or heterocyclic aromatic compounds. Polycyclic intercalators may have stacking capabilities and may include systems with 2, 3, or 4 fused rings. The universal bases described herein may be included on the ligand. In one embodiment, the ligand may include a cleaving group that facilitates the repression of the target gene by cleaving the target nucleic acid. The cleavage group can 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. The metal ion chelating group can include, for example, 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 by free metal ions (such as Lu(III)) at the protrusion site. In some embodiments, the peptide ligand can be linked to an RNA silencing agent to promote the cleavage of the target RNA, for example, in the protrusion region. For example, 1,8-dimethyl-1,3,6,8,10,13-hexaazacyclotetradecane (cyclam) can be conjugated to a peptide (e.g., via an amino acid derivative) to promote the cleavage of the target RNA. The tethering ligand can be an aminoglycoside ligand, which can impart improved hybridization properties or improved sequence specificity to the RNA silencer. Exemplary aminoglycosides include glycosylated polylysine, galactosylated polylysine, neomycin B, tobramycin, kanamycin A, and acridine conjugates of aminoglycosides, such as neo-N-acrididine, neo-S-acrididine, neo-C-acrididine, Tobra-N-acrididine, and KanaA-N-acrididine. The use of acridine analogs can increase sequence specificity. For example, neomycin B has a high affinity for RNA but low sequence specificity compared to DNA. The acridine analog neo-5-acrididine increases affinity for HIV Rev response elements (RREs). In some embodiments, a guanidine analog of the aminoglycoside ligand (guanidinoglycoside) is tethered to the RNA silencer. In guanidinoglycosides, the amino group on the amino acid is exchanged for a guanidino group. Attachment of guanidine analogs can enhance the cellular permeability of RNA silencing agents. Tethering ligands can be polyarginine peptides, peptide-like substances, or peptide-like compounds, which can enhance the cellular uptake of oligonucleotide reagents.

[0404] Exemplary ligands are coupled directly or indirectly (preferably covalently) to a ligand-conjugated vector via an inserted ligand. In an exemplary embodiment, the ligand is linked to the vector via an intervening ligand. In an exemplary embodiment, the ligand alters the distribution, targeting, or lifetime of the RNA silencing agent it incorporates. In an exemplary embodiment, the ligand provides enhanced affinity for selected targets, such as molecules, cells or cell types, compartments (e.g., cell or organ compartments), tissues, organs, or body regions, for example, compared to species without such ligands.

[0405] Exemplary ligands can improve transport, hybridization, and specificity properties, and can also improve the nuclease resistance of resulting natural or modified RNA silencing agents or polymer molecules containing any combination of monomers and / or natural or modified ribonucleotides described herein. Ligands can generally include therapeutic modifiers, such as those for enhancing absorption; diagnostic compounds or reporter groups, such as those for monitoring distribution; cross-linking agents; nuclease resistance-conferring moieties; and natural or unusual nucleobases. Common examples include lipophilic substances, lipids, steroids (e.g., urobilinogen, hecoside, diosgenin), terpenes (e.g., triterpenes, such as sarsaponin, limonene, epi-limonene alcohol-derived lithocholic acid), vitamins (e.g., folic acid, vitamin A, biotin, pyridoxal), carbohydrates, proteins, protein binders, integrin targeting molecules, polycations, peptides, polyamines, and peptide mimics. Ligands can include naturally occurring substances (e.g., human serum albumin (HSA), low-density lipoprotein (LDL), or globulins); carbohydrates (e.g., dextran, amylopectin, chitin, chitosan, inulin, cyclodextrin, or hyaluronic acid); amino acids; or lipids. Ligands can also be recombinant or synthetic molecules, such as synthetic polymers, like 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-co-glycolic acid) 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 polymers, or polyphosphazene. Examples of polyamines include: polyethyleneimine, polylysine (PLL), spermine, spermidine, polyamines, pseudopeptide-polyamines, pseudopeptide-polyamines, dendritic polyamines, arginine, amidine, protamine, cationic lipids, cationic porphyrins, quaternary salts of polyamines, or α-helical peptides.

[0406] Ligands may also include targeting groups that bind to specific cell types, such as kidney cells, for example, cell or tissue-targeting agents such as lectins, glycoproteins, lipids, or proteins, such as antibodies. Targeting groups may include thyroid-stimulating hormone, melanocyte-stimulating hormone, lectins, glycoproteins, surfactant protein A, mucin carbohydrates, polylactose, polygalactose, N-acetylgalactosamine, N-acetylglucosamine, polymannose, polyfucose, glycosylated polyamino acids, polygalactose, transferrin, bisphosphonates, polyglutamic acid, polyaspartic acid, lipids, cholesterol, steroids, bile acids, folic acid, vitamin B12, biotin, or RGD peptides or RGD peptide mimics. Other examples of ligands include dyes, intercalating agents (e.g., acridine and substituted acridine), cross-linking agents (e.g., psoralen, mitomycin C), porphyrins (TPPC4, texaphyrin, sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine, phenanthroline, pyrene), lys-tyr-lys tripeptides, aminoglycosides, guanidine aminoglycosides, artificial endonucleases (e.g., EDTA), lipophilic molecules such as cholesterol (and its thioanalytes), cholic acids, cholanonic acids, lithocholic acids, adamantaneacetic acid, 1-pyrenebutyric acid, dihydrotestosterone, glycerol (e.g., esters (e.g., mono-, di-, or tri-fatty acid esters, e.g., C), and esters (e.g., mono-, di-, or tri-fatty acid esters, e.g., C). 10 C 11 C 12 C 13 C 14 C 15 C 16 C 17 C 18 C 19 Or C 20 Fatty acids and their ethers, such as C 10 C 11 C 12 C 13 C 14 C 15 C 16 C 17 C 18 C 19 Or C 20Alkyl groups; for example, 1,3-bis-O-hexadecylglycerol, 1,3-bis-O-octadecylglycerol), geraniol, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecanyl, palmitic acid, stearic acid (e.g., glyceryl distearate), oleic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytriphenylmethyl or phenoxazine) and peptide conjugates (e.g., tentacles, Tat peptides), alkylating agents, phosphate esters, amino groups, thiol groups, PEG (e.g., PEG-40K), MPEG, [MPEG]2, polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes, haptens (e.g., biotin), transport / absorption promoters (e.g., aspirin, naproxen, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, diimidazole, histamine, imidazole clusters, acridine-imidazole conjugates, tetrazamacyclohexane Eu) 3+ (Complexes), dinitrophenyl, HRP or AP.

[0407] Ligands can be proteins, such as glycoproteins, or peptides, such as molecules with a specific affinity for a coligand, or antibodies, such as antibodies that bind to specific cell types, such as cancer cells, endothelial cells, or osteoblasts. Ligands may also include hormones and hormone receptors. They can also include non-peptide substances, such as lipids, lectins, carbohydrates, vitamins, cofactors, polylactose, polygalactose, N-acetylgalactosamine, N-acetylglucosamine, polymannose, or polyfucose. Ligands can be, for example, lipopolysaccharides, activators of p38 MAP kinase, or activators of NF-κB.

[0408] The ligand can be a substance, such as a drug, that can increase the uptake of an RNA silencer into the cell, for example, by disrupting the cell's cytoskeleton, such as by disrupting the cell's microtubules, microfilaments, and / or intermediate filaments. The drug can be, for example, taxa, vincristine, vinblastine, cytochalasin, nocodazole, japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, or myoservin. For example, the ligand can increase the uptake of an RNA silencer into the cell by activating an inflammatory response. Exemplary ligands with this effect include tumor necrosis factor α (TNFα), interleukin-1β, or gamma interferon. In one aspect, the ligand is a lipid or lipid-based molecule. This lipid or lipid-based molecule preferably binds to a serum protein, such as human serum albumin (HSA). HSA-binding ligands allow the conjugate to be distributed to target tissues, such as non-renal target tissues of the body. For example, the target tissue can be the liver, including the parenchymal cells of the liver. Other molecules that can bind HSA can also be used as ligands. For example, neproxin or aspirin can be used. Lipids or lipid-based ligands can (a) increase resistance to conjugate degradation, (b) increase targeting or transport to target cells or cell membranes, and / or (c) be used to modulate binding to serum proteins such as HSA. Lipid-based ligands can be used to modulate, for example, control the binding of the conjugate to target tissues. For example, lipids or lipid-based ligands that bind more strongly to HSA are less likely to target the kidneys and therefore less likely to be cleared from the body. Lipids or lipid-based ligands that bind less strongly to HSA can be used to target the conjugate to the kidneys. In a preferred embodiment, the lipid-based ligand binds to HSA. The lipid-based ligand can bind to HSA with sufficient affinity such that the conjugate will preferably be distributed to non-renal tissues. However, the affinity is preferably not strong enough to be irreversible in HSA-ligand binding. In a preferred embodiment, the lipid-based ligand binds weakly or not at all to HSA such that the conjugate will preferably be distributed to the kidneys. Targeting other parts of renal cells can also be used in place of or to supplement lipid-based ligands.

[0409] On the other hand, ligands are the portions absorbed by target cells, such as proliferating cells, and are like vitamins. These are particularly useful for treating diseases characterized by unwanted cell proliferation, such as malignant or non-malignant cell types, such as cancer cells. Exemplary vitamins include vitamins A, E, and K. Other exemplary vitamins include B vitamins, such as folic acid, B12, riboflavin, biotin, pyridoxal, or other vitamins or nutrients absorbed by cancer cells. Also included are HSA and low-density lipoprotein (LDL).

[0410] In another aspect, the ligand is a cell permeabilizer, preferably a helical cell permeabilizer. Preferably, the reagent is amphiphilic. Exemplary reagents are peptides, such as tat or tentacles. If the reagent is a peptide, it can be modified, including peptide mimics, transformants, non-peptide or pseudopeptide bonds, and the use of D-amino acids. The helical agent is preferably an α-helical agent, which preferably has a lipophilic phase and a lipophobic phase.

[0411] The ligand can be a peptide or a peptide mimic. A peptide mimic (also referred to herein as an oligopeptide mimic) is a molecule capable of folding into a defined three-dimensional structure similar to that of a natural peptide. The conjugation of peptides and peptide mimics to oligonucleotide reagents can influence the pharmacokinetic distribution of RNA silencing agents, for example, by enhancing cellular recognition and uptake. The peptide or peptide mimic moiety can be about 5–50 amino acids long, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long. The peptide or peptide mimic can be, for example, a cell-permeable peptide, a cationic peptide, an amphiphilic peptide, or a hydrophobic peptide (e.g., primarily composed of Tyr, Trp, or Phe). The peptide moiety can be a dendritic peptide, a bound peptide, or a cross-linked peptide. The peptide moiety can be an L-peptide or a D-peptide. In another alternative, the peptide moiety may include a hydrophobic membrane translocation sequence (MTS). Peptides or peptide mimics may be encoded by random sequences of DNA, such as peptides identified from phage display libraries or single-bead-one-compound (OBOC) combinatorial libraries (Lam et al., Nature 354:82-84, 1991). In exemplary embodiments, peptides or peptide mimics linked to RNA silencing agents via incorporated monomeric units are cell-targeting peptides, such as arginine-glycine-aspartic acid (RGD)-peptides or RGD mimics. The length of the peptide moiety can range from about 5 amino acids to about 40 amino acids. The peptide moiety may have structural modifications, such as to increase stability or direct conformational properties. Any structural modifications described below may be used.

[0412] 6) Branched oligonucleotides

[0413] Two or more oligonucleotides (wherein at least one of the oligonucleotides includes a subunit bond according to an embodiment of formula (I)) can be linked together by one or more portions independently selected from linkers, spacers, and branching points to form a branched compound. For example, the branched compound may contain two or more RNA silencers of the types described above, thereby producing novel RNA silencers with a branched structure. In a representative embodiment, each oligonucleotide contains an antisense strand (or a portion thereof), wherein the antisense strand is sufficiently complementary to the heterozygous single nucleotide polymorphism to mediate an RNA-mediated silencing mechanism (e.g., RNAi).

[0414] In exemplary embodiments, the branched compound may have two to eight RNA silencers linked by linkers. The linkers may be hydrophobic. In typical embodiments, the branched oligonucleotides of this application have two to three oligonucleotides. In one embodiment, the oligonucleotides independently possess significant chemical stability (e.g., at least 40% of the constituent bases are chemically modified). In certain embodiments, the oligonucleotides possess complete chemical stability (i.e., all constituent bases are chemically modified). In some embodiments, the branched oligonucleotides comprise one or more single-stranded phosphorothioate tails, each tail independently having two to twenty nucleotides. In non-limiting embodiments, each single-stranded tail has eight to ten nucleotides.

[0415] In some embodiments, the branched compound is characterized by three properties: (1) a branched structure, (2) complete metabolic stability, and (3) the presence of a single-chain tail containing a phosphate thioester linker. In exemplary embodiments, the branched oligonucleotide has two or three branches. The increased overall size of the branched structure promotes increased uptake. Furthermore, not bound by any particular activity theory, multiple adjacent branches (e.g., two or three) appear to allow each branch to act synergistically, thereby significantly increasing the rate of internalization, transport, and release.

[0416] Branching compounds are provided in various structurally different embodiments. In some embodiments, the nucleic acids linked at the branching point are single-stranded and include miRNA inhibitors, space bodies, hybrids, SSO, PMO, or PNA. These single strands can be linked at their 3' or 5' ends. Combinations of siRNA and single-stranded oligonucleotides can also be used for dual functionality. In another embodiment, short nucleic acids complementary to space bodies, hybrids, miRNA inhibitors, SSO, PMO, and PNA are used to carry these active single-stranded nucleic acids and enhance distribution and internalization. Short double-stranded regions have lower melting temperatures (T0). m (Approximately 37°C) is used for rapid dissociation when branching structures are internalized into cells.

[0417] "Di-siRNA" compounds, which are branched oligonucleotides containing two siRNAs and a linker, can comprise chemically diverse conjugates. The conjugated bioactive ligands can 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 linked to the Di-siRNA via a linker or spacer, or added via an additional linker or spacer attached to the end of another free siRNA.

[0418] Unbound by any particular theory, the presence of branched structures has been found to increase tissue retention levels in the brain by more than 100-fold compared to unbranched compounds with the same chemical composition, suggesting a novel mechanism for cell retention and distribution. Branched oligonucleotides are unexpectedly uniformly distributed throughout the spinal cord and brain. Furthermore, branched oligonucleotides exhibit surprisingly efficient systemic delivery to multiple tissues and very high levels of tissue accumulation.

[0419] Branched oligonucleotides can contain a variety of therapeutic nucleic acids, including ASO, miRNA, miRNA inhibitors, splicing conversion, PMO, and PNA. In some embodiments, branched oligonucleotides further contain conjugated hydrophobic moieties and exhibit unprecedented silencing and efficacy both in vitro and in vivo.

[0420] connector

[0421] In embodiments of the branched oligonucleotide compound, each linker is independently selected from ethylene glycol chains, alkyl chains, peptides, RNA, DNA, phosphate esters, phosphonates, aminophosphates, esters, amides, triazoles, and combinations thereof; wherein any carbon or oxygen atom of the linker is optionally substituted with a nitrogen atom, has a hydroxyl substituent, or has an oxygen 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 ester. In another embodiment, each linker is a phosphonate. In another embodiment, each linker is an aminophosphate ester. 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.

[0422] In another aspect, this paper provides branched oligonucleotide compounds of formula (1):

[0423] (1)

[0425] Wherein L is selected from ethylene glycol chains, alkyl chains, peptides, RNA, DNA, phosphate esters, phosphonates, aminophosphates, esters, amides, triazoles, and combinations thereof, wherein formula (1) optionally further comprises one or more branch points Bp and one or more spacers S; wherein Bp is independently a multivalent organic substance or a derivative thereof each time it appears; for each appearance, S is independently selected from ethylene glycol chains, alkyl chains, peptides, RNA, DNA, phosphate esters, phosphonates, aminophosphates, esters, amides, triazoles, and combinations thereof; N is an RNA duplex comprising a sense strand and an antisense strand, wherein the sense strand and the antisense strand each independently comprise one or more chemical modifications; and n is 2, 3, 4, 5, 6, 7, or 8. In an embodiment, at least one N comprises a subunit interunit bond of a modified formula (I).

[0426] In the embodiments, the compound of formula (1) has a structure selected from formulas (1-1)-(1-9) in Table 1.

[0427] Table 1

[0428]

[0429] 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).

[0430] In one embodiment of the compound of formula (1), each linker is independently selected from ethylene glycol chains, alkyl chains, peptides, RNA, DNA, phosphate esters, phosphonates, aminophosphates, esters, amides, triazoles, and combinations thereof; wherein any carbon or oxygen atom of the linker is optionally substituted with a nitrogen atom, has a hydroxyl substituent, or has an oxygen 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 another embodiment of the compound of formula (1), each linker is a peptide. In another embodiment of the compound of formula (1), each linker is RNA. In another embodiment of the compound of formula (1), each linker is DNA. In another embodiment of the compound of formula (1), each linker is a phosphate ester. In another embodiment, each linker is a phosphonate. In another embodiment of the compound of formula (1), each linker is an aminophosphate ester. 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.

[0431] In one embodiment of the compound of formula (1), Bp is a polyvalent organic substance. In another embodiment of the compound of formula (1), Bp is a derivative of a polyvalent organic substance. In one embodiment of the compound of formula (1), Bp is a triol or tetraol derivative. In another embodiment, Bp is a tricarboxylic acid or tetracarboxylic acid derivative. In another embodiment, Bp is an amine derivative. In another embodiment, Bp is a triamine or tetraamine derivative. In another embodiment, Bp is an amino acid derivative.

[0432] A multivalent organic substance is a moiety containing carbon and three or more valences (i.e., the junction with a moiety such as S, L, or N as defined above). Non-limiting examples of multivalent organic substances include triols (e.g., glycerol, phloroglucinol, etc.), tetraols (e.g., ribose, pentaerythritol, 1,2,3,5-tetrahydroxybenzene, etc.), tricarboxylic acids (e.g., citric acid, 1,3,5-cyclohexanetricarboxylic acid, pyromellitic acid, etc.), tetracarboxylic acids (e.g., ethylenediaminetetraacetic acid, pyromellitic acid, etc.), tertiary amines (e.g., triargylpropylamine, triethanolamine, etc.), triamines (e.g., diethylenetriamine, etc.), tetraamines, and substances containing combinations of hydroxyl, thiol, amino, and / or carboxyl moieties (e.g., amino acids such as lysine, serine, cysteine, etc.).

[0433] In one embodiment of the compound of formula (1), each nucleic acid comprises one or more chemically modified nucleotides. In one embodiment of the compound of formula (1), each nucleic acid is composed of chemically modified nucleotides. In certain embodiments of the compound of formula (1), each nucleic acid comprising >95%, >90%, >85%, >80%, >75%, >70%, >65%, >60%, >55%, or >50% of a chemically modified nucleotide.

[0434] In one implementation, each antisense strand independently contains a 5' terminal group R selected from the group in Table 2.

[0435] Table 2

[0436]

[0437] 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.

[0438] Structure of Equation (2)

[0439] In one embodiment, the compound of formula (1) has the structure of formula (2):

[0440] (2)

[0442] Wherein, X, for each occurrence, is independently selected from adenosine, guanosine, uridine, cytidine, and their chemically modified derivatives; Y, for each occurrence, is independently selected from adenosine, guanosine, uridine, cytidine, and their chemically modified derivatives; - represents a phosphodiester nucleoside bond; = represents a thiophosphate nucleoside bond; and --- each occurrence represents a base pairing interaction or mismatch, respectively. Furthermore, at least one nucleoside bond can be replaced by a subunit bond of formula (I).

[0443] In some 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 is composed of chemically modified nucleotides.

[0444] In some embodiments, the X' of the structure of formula (2) being >95%, >90%, >85%, >80%, >75%, >70%, >65%, >60%, >55%, or >50% is a chemically modified nucleotide. In other embodiments, the X' of the structure of formula (2) being >95%, >90%, >85%, >80%, >75%, >70%, >65%, >60%, >55%, or >50% is a chemically modified nucleotide.

[0445] Structure of Equation (3)

[0446] In one embodiment, the compound of formula (1) has the structure of formula (3):

[0447] (3)

[0449] in X For each occurrence, X is independently a nucleotide containing a 2'-deoxy-2'-fluorine modification; X is independently a nucleotide containing a 2'-O-methyl modification. Y For each occurrence, is independently a nucleotide containing a 2'-deoxy-2'-fluorine modification; Y, for each occurrence, is independently a nucleotide containing a 2'-O-methyl modification.

[0450] In one embodiment, X is selected from the group consisting of 2'-deoxy-2'-fluorine 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'-fluorine 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.

[0451] In some embodiments, the structure of formula (3) does not contain mismatches. In one embodiment, the structure of formula (3) contains one mismatch. In another embodiment, the compound of formula (3) contains two mismatches. In another embodiment, the compound of formula (3) contains three mismatches. In yet another embodiment, the compound of formula (3) contains four mismatches.

[0452] Structure of Equation (4)

[0453] In one embodiment, the compound of formula (1) has the structure of formula (4):

[0454] (4)

[0456] Wherein, X, for each occurrence, is independently selected from adenosine, guanosine, uridine, cytidine, and their chemically modified derivatives; Y, for each occurrence, is independently selected from adenosine, guanosine, uridine, cytidine, and their chemically modified derivatives; - represents a phosphodiester nucleoside bond; = represents a thiophosphate nucleoside bond; and --- each occurrence represents a base pairing interaction or mismatch, respectively. Furthermore, at least one nucleoside bond can be replaced by a subunit bond of formula (I).

[0457] In some 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 is composed of chemically modified nucleotides.

[0458] In some embodiments, the X' of the structure of formula (2) being >95%, >90%, >85%, >80%, >75%, >70%, >65%, >60%, >55%, or >50% is a chemically modified nucleotide. In other embodiments, the X' of the structure of formula (2) being >95%, >90%, >85%, >80%, >75%, >70%, >65%, >60%, >55%, or >50% is a chemically modified nucleotide.

[0459] Structure of Equation (5)

[0460] In some embodiments, the compound of formula (1) has the structure of formula (5):

[0461] (5)

[0463] in X For each occurrence, X is independently a nucleotide containing a 2'-deoxy-2'-fluorine modification; X is independently a nucleotide containing a 2'-O-methyl modification. Y For each occurrence, is independently a nucleotide containing a 2'-deoxy-2'-fluorine modification; Y, for each occurrence, is independently a nucleotide containing a 2'-O-methyl modification.

[0464] In some embodiments, X is selected from the group consisting of 2'-deoxy-2'-fluorinated adenosine, guanosine, uridine, or cytidine. In one embodiment, X is selected from the group consisting of 2'-O-methylinated adenosine, guanosine, uridine, or cytidine. In one embodiment, Y is selected from the group consisting of 2'-deoxy-2'-fluorinated adenosine, guanosine, uridine, or cytidine. In one embodiment, Y is selected from the group consisting of 2'-O-methylinated adenosine, guanosine, uridine, or cytidine.

[0465] In some embodiments, the structure of formula (5) does not contain mismatches. In one embodiment, the structure of formula (6) contains one mismatch. In another embodiment, the compound of formula (5) contains two mismatches. In another embodiment, the compound of formula (5) contains three mismatches. In yet another embodiment, the compound of formula (V) contains four mismatches.

[0466] Variable connector

[0467] In one embodiment of the compound of formula (1), L has the structure L1:

[0468] .

[0469] (L1)

[0470] In one implementation of L1, R is R 3 And n is 2.

[0471] In one embodiment of the structure of formula (II), L has the structure of L1. In one embodiment of the structure of formula (III), L has the structure of L1. In one embodiment of the structure of formula (IV), L has the structure of L1. In one embodiment of the structure of formula (V), L has the structure of L1. In one embodiment of the structure of formula (VI), L has the structure of L1. In one embodiment of the structure of formula (VII), L has the structure of L1.

[0472] In one embodiment of the compound of formula (1), L has the structure L2:

[0473] .

[0474] (L2)

[0475] In one implementation of L2, R is R 3And n is 2. In one embodiment of the structure of equation (2), L has the structure of L2. In one embodiment of the structure of equation (3), L has the structure of L2. In one embodiment of the structure of equation (4), L has the structure of L2. In one embodiment of the structure of equation (5), L has the structure of L2.

[0476] Delivery system

[0477] In another aspect, this paper provides a delivery system for therapeutic nucleic acids having the structure of formula (6):

[0478] (6)

[0480] Wherein L is selected from ethylene glycol chains, alkyl chains, peptides, RNA, DNA, phosphate esters, phosphonates, aminophosphates, esters, amides, triazoles, and combinations thereof, wherein formula (6) optionally further comprises one or more branch points Bp and one or more spacers S; wherein Bp is independently a multivalent organic substance or a derivative thereof each time it appears; S is independently selected from ethylene glycol chains, alkyl chains, peptides, RNA, DNA, phosphate esters, phosphonates, aminophosphates, esters, amides, triazoles, and combinations thereof each time it appears; each cNA is independently a vector nucleic acid containing one or more chemical modifications; n is 2, 3, 4, 5, 6, 7, or 8. In one embodiment, at least one cNA comprises a modified subunit interunit bond of formula (I).

[0481] 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 ester. In another embodiment of the delivery system, L is a phosphonate ester. In another embodiment of the delivery system, L is an aminophosphate ester. 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.

[0482] In one embodiment of the delivery system, S is an ethylene glycol chain. In another embodiment, S is an alkyl chain. In another embodiment of the delivery system, S is a peptide. In another embodiment, S is RNA. In another embodiment of the delivery system, S is DNA. In another embodiment of the delivery system, S is a phosphate ester. In another embodiment of the delivery system, S is a phosphonate ester. In another embodiment of the delivery system, S is an aminophosphate ester. In another embodiment of the delivery system, S is an ester. In another embodiment, S is an amide. In another embodiment, S is a triazole.

[0483] In one embodiment of the delivery system, n is 2. In another embodiment of the delivery system, n is 3. In another embodiment of the delivery system, n is 4. In another embodiment of the delivery system, n is 5. In another embodiment of the delivery system, n is 6. In another embodiment of the delivery system, n is 7. In another embodiment of the delivery system, n is 8.

[0484] In some implementations, each cNA contains >95%, >90%, >85%, >80%, >75%, >70%, >65%, >60%, >55%, or >50% of chemically modified nucleotides.

[0485] In one embodiment, the compound of formula (6) has a structure selected from formulas (6-1)-(6-9) in Table 3:

[0486] Table 3

[0487]

[0488] 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).

[0489] In one embodiment, the compounds of formula (6) (including, for example, any one of formulas (6-1)-(6-9)) each cNA independently comprises at least 15 consecutive nucleotides. In one embodiment, each cNA independently consists of chemically modified nucleotides.

[0490] In one embodiment, each NA hybridizes with at least one cNA. In one embodiment, at least one NA comprises a modified subunit interunit bond of formula (I). In some embodiments, the compounds of this disclosure are characterized by the following properties: (1) two or more branched oligonucleotides, for example, wherein unequal numbers of 3' and 5' ends are present; (2) substantially chemically stable, for example, wherein more than 40%, preferably 100%, of the oligonucleotides are chemically modified (e.g., without RNA and optionally without DNA); (3) a phosphate-thiophosphate monooligonucleotide containing at least 3, preferably 5-20 phosphate-thiophosphate bonds.

[0491] VII. Methods for introducing nucleic acids, vector host cells, and branched oligonucleotide compounds

[0492] The RNA silencing agent of this invention can be directly introduced into cells (e.g., nerve cells) (i.e., intracellularly); or introduced extracellularly into cavities, interstitial spaces, the circulation of an organism, or orally; or introduced by immersing cells or an organism in a solution containing nucleic acids. Blood vessels or extravascular circulation, the blood or lymphatic system, and cerebrospinal fluid are sites where nucleic acids can be introduced.

[0493] The RNA silencing agent of this disclosure can be introduced using nucleic acid delivery methods known in the art, including injecting a solution containing nucleic acid, bombarding with particles coated with nucleic acid, immersing cells or organisms in a nucleic acid solution, or electroporating the cell membrane in the presence of nucleic acid. Other methods known in the art for introducing nucleic acids into cells can be used, such as lipid-mediated carrier transport, chemically mediated transport, and cationic liposome transfection, such as calcium phosphate. The nucleic acid can be introduced together with other components that perform one or more of the following activities: enhancing cellular uptake of nucleic acids or otherwise increasing inhibition of target genes.

[0494] Physical methods for introducing nucleic acids include injecting a solution containing RNA, bombarding with particles coated with RNA, immersing cells or organisms in an RNA solution, or electroporating cell membranes in the presence of RNA. Viral constructs packaged into viral particles enable the efficient introduction of the expression construct into cells and transcription of RNA encoded by the expression construct. Other methods known in the art for introducing nucleic acids into cells can be used, such as lipid-mediated vector transport, chemically mediated transport such as calcium phosphate, etc. Therefore, RNA can be introduced together with components that perform one or more of the following activities: enhancing cellular uptake of RNA, inhibiting single-strand annealing, stabilizing single strands, or otherwise increasing repression of target genes.

[0495] RNA can be introduced directly into cells (i.e., intracellularly); or extracellularly into cavities, interstitial spaces, the body's circulation, or 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 where RNA can be introduced.

[0496] Cells carrying target genes can originate from germline or somatic cells, be totipotent or pluripotent, dividing or non-dividing, parenchymal or epithelial, immortalized or transformed, etc. Cells can 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 endocrine or exocrine gland cells.

[0497] Depending on the specific target gene and the dosage of the delivered double-stranded RNA material, this process may result in partial or complete loss of function of the target gene. Reduced or lost gene expression in at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% or more of the target cells is exemplary. Inhibition of gene expression refers to the absence (or observable reduction) of the level of protein and / or mRNA products from the target gene. Specificity refers to the ability to inhibit a target gene without significantly affecting other genes in the cell. The consequences of inhibition can be confirmed by examining extrinsic properties of the cell or organism (as illustrated in the examples below) or by biochemical techniques such as RNA solution hybridization, nuclease protection, Northern hybridization, reverse transcription, gene expression monitoring using microarrays, antibody binding, enzyme-linked immunosorbent assay (ELISA), Western blotting, radioimmunoassay (RIA), other immunoassays, and fluorescence-activated cell sorting (FACS).

[0498] For RNA-mediated repression in cell lines or the whole organism, gene expression can be conveniently measured using reporter genes or drug resistance genes whose protein products are readily measurable. Such reporter genes include acetylhydroxylase (AHAS), alkaline phosphatase (AP), β-galactosidase (LacZ), β-glucuronidase (GUS), chloramphenicol acetyltransferase (CAT), green fluorescent protein (GFP), horseradish peroxidase (HRP), luciferase (Luc), caustic solanine synthase (NOS), octopus solanine synthase (OCS), and their derivatives. A variety of optional markers are available to confer resistance to ampicillin, bleomycin, chloramphenicol, gentamicin, hygromycin, kanamycin, lincomycin, methotrexate, phosphatidylinosin, puromycin, and tetracycline. Quantification of gene expression levels, based on assays, allows for the determination of repression levels greater than 10%, 33%, 50%, 90%, 95%, or 99% compared to cells not treated according to this disclosure. Lower doses of injectable materials and longer durations of RNAi agent administration may result in inhibition of a smaller fraction of cells (e.g., at least 10%, 20%, 50%, 75%, 90%, or 95% of target cells). Quantification of gene expression in cells may show similar levels of inhibition at the accumulation of target mRNA or the translational level of target protein. For example, inhibitory efficiency can be determined by assessing the amount of gene product in cells; mRNA can be detected with hybridization probes having nucleotide sequences outside the region for repressive double-stranded RNA, or translated peptides can be detected with antibodies generated from peptide sequences targeting that region.

[0499] RNA can be introduced in amounts that allow delivery of at least one copy per cell. Higher doses (e.g., at least 5, 10, 100, 500, or 1000 copies per cell) of the material can produce more effective inhibition; lower doses may also be useful for specific applications.

[0500] In one exemplary aspect, the efficacy of the RNAi agents disclosed herein (e.g., siRNAs targeting a target sequence of interest) is tested to determine their ability to specifically degrade mutant mRNAs (e.g., the production of target mRNAs and / or target proteins) in cells, particularly in neurons (e.g., striatal or cortical neuronal clonal lines and / or primary neurons). Other readily transfectable cell types (e.g., HeLa cells or COS cells) are also suitable for cell-based validation analyses. 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 producing siRNA from U-loop mRNA is co-transfected. Selective reduction in target mRNAs and / or target proteins is measured. The reduction in target mRNAs or proteins can be compared to the levels of target mRNAs or proteins in the absence of an RNAi agent or in the presence of an RNAi agent that does not target the target mRNA. For comparative purposes, exogenously introduced mRNAs or proteins (or endogenous mRNAs or proteins) can be analyzed. When using neurons that are known to be resistant to standard transfection techniques, it may be necessary to introduce RNAi agents (e.g., siRNA) through passive uptake.

[0501] Recombinant adeno-associated virus and vector

[0502] In some exemplary embodiments, recombinant adeno-associated virus (rAAV) and its associated vectors can be used to deliver one or more siRNAs into cells such as nerve cells (e.g., brain cells). AAVs are capable of infecting many different cell types, although infection efficiency varies by serotype, which is determined by the sequence of the capsid protein. Several natural AAV serotypes have been identified, with serotypes 1–9 being the most commonly used for recombinant AAVs. AAV-2 is the most thoroughly studied and published serotype. The AAV-DJ system includes serotypes AAV-DJ and AAV-DJ / 8. These serotypes are generated by DNA shuffling of multiple AAV serotypes to produce AAVs with a mixed capsid that enhances transduction efficiency in various cells and tissues both in vitro (AAV-DJ) and in vivo (AAV-DJ / 8).

[0503] In certain embodiments, broad central nervous system (CNS) delivery can be achieved via 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 May 24, 2011). rAAVs and their associated vectors are well known in the art and 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 its entirety for all purposes.

[0504] rAAV can be delivered to a subject in compositional form using any suitable method known in the art. rAAV can be suspended in a physiologically compatible carrier (i.e., in the composition) and can be administered to a subject, i.e., a host animal, such as a human, mouse, rat, cat, dog, sheep, rabbit, horse, cow, goat, pig, guinea pig, hamster, chicken, turkey, or non-human primate (e.g., rhesus monkey). In some embodiments, the host animal is a non-human host animal.

[0505] One or more rAAVs can be delivered to a mammalian subject, for example, by intramuscular injection or by administration into the bloodstream of the mammalian subject. Administration into the bloodstream can be achieved by injection into a vein, artery, or any other vascular catheter. In some embodiments, one or more rAAVs are administered into the bloodstream via detached limb perfusion, a technique well-known in the surgical field that essentially allows a technician to detach a limb from the systemic circulation prior to the administration of the rAAV viral particles. A variant of the detached limb perfusion technique described in U.S. Patent No. 6,177,403 can also be used by a technician to administer viral particles to the vascular system of the detached limb to potentially enhance transduction to muscle cells or tissues. Furthermore, in some cases, it may be necessary to deliver viral particles to the central nervous system (CNS) of the subject. “CNS” refers to all cells and tissues of the brain and spinal cord in vertebrates. Therefore, the term includes, but is not limited to, neurons, glial cells, astrocytes, cerebrospinal fluid (CSF), interstitial spaces, bone, cartilage, etc. Recombinant AAVs can be delivered directly to the CNS or brain using neurosurgical techniques known in the art, such as stereotactic injection (see, for example, 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), using needles, catheters, or related devices, to areas such as the ventricles and striatum (e.g., the caudate nucleus or putamen of the striatum), spinal cord and neuromuscular junctions, or cerebellar lobules.

[0506] The compositions disclosed herein may contain rAAV alone or in combination with one or more other viruses (e.g., a second rAAV encoding having one or more different transgenes). In some embodiments, the compositions contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different rAAVs, each having one or more different transgenes.

[0507] An effective amount of rAAV is an amount sufficient to target and infect an animal and the desired tissue. In some embodiments, an effective amount of rAAV is an amount sufficient to produce a stable somatic transgenic animal model. The effective amount will depend primarily on factors such as species, age, weight, the health status of the subject, and the tissue to be targeted, and therefore may vary from animal to tissue. For example, an effective amount of one or more rAAVs is typically in the range of about 1 ml to about 100 ml of solution containing about 10 9 Up to 10 1610 genome copies. In some cases, approximately 10 11 Up to 10 12 The dosage between rAAV genome copies is appropriate. In some implementations, 10 12 rAAV genome copies are effective at targeting heart, liver, and pancreatic tissues. In some cases, stable transgenic animals are produced by multiple doses of rAAV.

[0508] In some embodiments, the rAAV composition is formulated to reduce the aggregation of AAV particles in the composition, particularly in the presence of high rAAV concentrations (e.g., about 10). 13 (Genomic copies / mL or more). Methods for reducing rAAV aggregation are well known in the art and include, for example, adding surfactants, adjusting pH, adjusting salt concentration, etc. (see, for example, Wright et al. (2005) Molecular Therapy 12:171-178, the contents of which are incorporated herein by reference).

[0509] A “recombinant AAV (rAAV) vector” contains at least a transgene and its regulatory sequence, as well as 5' and 3' AAV inverted terminal repeats (ITRs). This recombinant AAV vector is packaged into a capsid protein and delivered to selected target cells. In some embodiments, the transgene is a nucleic acid sequence heterologous to the vector sequence, encoding a polypeptide, protein, functional RNA molecule (e.g., siRNA), or other gene product of interest. The nucleic acid coding sequence is operatively linked to the regulatory component in a manner that allows the transgene to be transcribed, translated, and / or expressed in cells of the target tissue.

[0510] The AAV sequence of the vector typically contains cis-acting 5' and 3' inverted terminal repeat (ITR) sequences (see, e.g., BJ Carter, "Handbook of Parvoviruses", edited by P. Tijsser, CRC Press, p. 155168 (1990)). The ITR sequence is typically about 145 base pairs in length. In some embodiments, a substantially complete sequence encoding the ITR is used in the molecule, although some degree of minor modification to these sequences is permitted. The ability to modify these ITR sequences is within the skill of the art (see, e.g., 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)). One example of such a molecule used in this disclosure is a transgenic "cis-acting" plasmid in which the selected transgenic sequence and associated regulatory element are flanked by 5' and 3' AAV ITR sequences. AAV ITR sequences can be obtained from any known AAV, including mammalian AAV types further described herein.

[0511] VIII. Treatment Methods

[0512] As used herein, “treatment” or “treating” is defined as the application or administration of a therapeutic agent (e.g., an RNA agent or vector or a transgene encoding them) to a patient, or to an isolated tissue or cell line from a patient suffering from a disease or condition, symptoms of a disease or condition, or susceptibility to a disease or condition, with the aim of curing, healing, alleviating, relieving, altering, remedying, improving, enhancing, or influencing the disease or condition, symptoms of a disease or condition, or disease predisposition.

[0513] In one aspect, this disclosure provides a method for preventing a disease or condition as described above in a subject by administering a therapeutic agent (e.g., an RNAi agent or vector or a transgene encoding them) to the subject. Subjects at risk of disease can be identified by, for example, any diagnostic or prognostic assay or a combination thereof described herein. Administration of the preventative agent can occur before the characteristic symptoms of the disease or condition manifest, thereby preventing the disease or condition or delaying its progression.

[0514] Another aspect of this disclosure relates to a method for therapeutically treating a subject, namely, altering the onset of symptoms of a disease or condition.

[0515] Regarding preventative and therapeutic approaches to treatment, such treatments can be specifically tailored or modified based on knowledge gained from the field of pharmacogenomics. As used herein, "pharmacogenomics" refers to the application of genomics technologies (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 studying how a patient's genes determine his or her response to a drug (e.g., a patient's "drug response phenotype" or "drug response genotype"). Therefore, another aspect of this disclosure provides methods for tailoring preventative or therapeutic treatments to an individual based on their drug response genotype, using target gene molecules or target gene modulators of this disclosure. Pharmacogenomics allows clinicians or physicians to target preventative or therapeutic treatments to patients who will benefit most from the treatment and avoid treating patients who will experience drug-related toxic side effects.

[0516] Therapeutic agents can be tested in appropriate animal models. For example, RNAi agents (or expression vectors or transgenes encoding them) as described herein can be used in animal models to determine the efficacy, toxicity, or side effects of treatment using the agent. Alternatively, therapeutic agents can be used in animal models to determine the mechanism of action of such agents.

[0517] Pharmaceutical compositions containing the RNA silencing agent disclosed herein 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 in good health. For example, the patient is not terminally ill and is likely to survive at least 2, 3, 5 years or more after diagnosis. The patient may receive treatment immediately after diagnosis or treatment may be delayed until the patient experiences more debilitating symptoms, such as motor fluctuations and dyskinesia in patients with Parkinson's disease. In another embodiment, the patient has not yet reached the late stage of the disease.

[0518] RNA silencing agents modified to enhance neuronal uptake can be administered at doses of less than approximately 1.4 mg per kilogram of 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 per kilogram of body weight, and less than 200 nanomoles of RNA reagent (e.g., approximately 4.4 x 10⁻⁶ mg per kilogram of body weight). 16The RNA silencing agent is administered in unit doses of 1500, 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 nanomoles per kilogram of body weight. For example, the unit dose can be administered by injection (e.g., intravenous or intramuscular injection, intrathecal injection, or direct injection into the brain), inhalation, or topical application. Particularly preferred doses are less than 2, 1, or 0.1 mg per kilogram of body weight.

[0519] RNA silencers can be delivered directly to organs (e.g., directly to the brain) at doses approximately 0.00001 mg to about 3 mg per organ, or preferably 0.0001-0.001 mg per organ, 0.03-3.0 mg per organ, 0.1-3.0 mg per eye, or 0.3-3.0 mg per organ. The dose can be an amount effective in treating or preventing neurodegenerative diseases or conditions (e.g., AD or ALS). In one embodiment, the unit dose is administered less frequently than once daily, such as less than once every 2, 4, 8, or 30 days. In another embodiment, the unit dose is not administered at a specific frequency (e.g., irregularly). For example, the unit dose may be administered as a single dose. In one embodiment, the effective dose is administered in conjunction with other conventional treatments.

[0520] In one implementation, an initial dose and one or more maintenance doses of the RNA silencing agent are administered to the subject. The maintenance dose is typically lower than the initial dose, for example, half the initial dose. The maintenance regimen may include treating the subject at a dose ranging from 0.01 μg to 1.4 mg per kilogram of body weight per day (e.g., 10, 1, 0.1, 0.01, 0.001, or 0.00001 mg per kilogram of body weight per day). The maintenance dose is preferably administered no more than once every 5, 10, or 30 days. Furthermore, the treatment regimen may continue for a period of time, which will vary 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 per day, for example, no more than once every 24, 36, 48, or more hours, or for example, no more than once every 5 or 8 days. After treatment, changes in the patient's condition and symptom relief of the disease state can be monitored. If the patient does not respond significantly to the current dose level, the dose of the compound may be increased, or if symptom relief of the disease state is observed, if the disease state has been eliminated, or if undesirable side effects are observed, the dose may be reduced.

[0521] If it is desired or deemed appropriate under specific conditions, the effective dose may be administered as a single, two, or more doses. If it is desired to facilitate repeated or frequent infusions, implantable delivery devices, such as pumps, semi-permanent stents (e.g., intravenous stents, intraperitoneal stents, intracisional stents, or intracapsular stents), or reservoirs may be recommended. In one embodiment, the pharmaceutical composition comprises multiple RNA silencing substances. In another embodiment, the RNA silencing substances have sequences that do not overlap or are not adjacent to another substance relative to a naturally occurring target sequence. In another embodiment, the multiple RNA silencing substances are specific to different naturally occurring target genes. In another embodiment, the RNA silencing agents are allele-specific. In yet another embodiment, the multiple RNA silencing substances target two or more target sequences (e.g., two, three, four, five, six, or more target sequences).

[0522] Following successful treatment, it may be desirable to subject the patient to maintenance therapy to prevent relapse of the disease state, wherein the compounds of this disclosure are administered at a maintenance dose ranging from 0.01 μg to 100 g per kilogram of body weight (see, for example, U.S. Patent No. 6,107,094).

[0523] The concentration of an RNA silencing agent composition is sufficient to effectively treat or prevent a condition or regulate a human physiological state. The concentration or amount of RNA silencing agent applied will depend on the determined reagent parameters and method of administration, such as nasal, buccal, or pulmonary administration. For example, nasal formulations tend to require much lower concentrations of some components to avoid irritation or burning of the nasal passages. Sometimes it is desirable to dilute oral formulations up to 10-100 times to provide a suitable nasal formulation.

[0524] Certain factors may affect the dosage required for effective treatment of a subject, including but not limited to the severity of the disease or condition, prior treatment, the subject's general health status and / or age, and any other pre-existing conditions. Furthermore, treatment of a subject with a therapeutically effective amount of RNA silencing agent may comprise a single treatment, or preferably, a series of treatments. It should also be understood that the effective dose of the RNA silencing agent used for treatment may increase or decrease during a particular treatment course. Dosage changes can arise from and become apparent from the results of diagnostic assays as described herein. For example, the subject may be monitored after administration of the RNA silencing agent composition. Based on information from the monitoring, additional amounts of the RNA silencing agent composition may be administered.

[0525] Dosing depends on the severity and responsiveness of the disease condition to be treated, and the treatment process may last from several days to several months, or until a cure is affected or a reduction in the disease condition is achieved. The optimal dosing regimen can be calculated from measurements of drug accumulation in the patient's body. An expert can readily determine the optimal dose, method of administration, and repetition rate. The optimal dose can vary depending on the relative potency of individual compounds and can generally be estimated based on EC50, which has been found to be effective in in vitro and in vivo animal models. In some embodiments, the animal model includes a transgenic animal expressing a human gene (e.g., a gene that produces target RNA (e.g., RNA expressed in nerve cells)). The transgenic animal may lack the corresponding endogenous RNA. In another embodiment, the composition used for testing includes an RNA silencer that is complementary at least in its internal region to a conserved sequence between the target RNA in the animal model and the target RNA in humans.

[0526] IX. Pharmaceutical Compositions and Administration

[0527] This disclosure relates to the use of the aforementioned reagents for preventive and / or therapeutic treatments as described below. Therefore, the modifiers of this application (e.g., branched oligonucleotides containing RNA silencing agents) can be incorporated into pharmaceutical compositions suitable for administration. Such compositions typically comprise nucleic acid molecules, proteins, antibodies, or branched oligonucleotide compounds and pharmaceutically acceptable carriers. As used herein, the phrase "pharmaceutically acceptable carrier" refers to any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, and absorption delay agents, etc., that are compatible with pharmaceutical administration. The use of such media and reagents for pharmaceutically active substances is well known in the art. Unless any conventional media or reagent is incompatible with the active compound, its use in the composition is contemplated. Complementary active compounds may also be incorporated into the composition.

[0528] The pharmaceutical compositions of this disclosure are formulated to be compatible with their intended route of administration. Examples of routes of administration include parenteral administration, such as intravenous, intradermal, subcutaneous, intraperitoneal, intramuscular, oral (e.g., inhalation), transdermal (topical), and transmucosal administration. In some exemplary embodiments, the pharmaceutical compositions of this disclosure are delivered to cerebrospinal fluid (CSF) via routes of administration including, but not limited to, intrastriatal (IS), intraventricular (ICV), and intrathecal (IT) administration (e.g., via pump, infusion, etc.). Solutions or suspensions for parenteral, intradermal, or subcutaneous application may contain the following components: sterile diluents, such as water for injection, saline solution, nonvolatile oils, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents; antibacterial agents, such as benzyl alcohol or methylparaben; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid; buffers, such as acetates, citrates, or phosphates; and agents for regulating tension, such as sodium chloride or dextran. pH can be adjusted using acids or bases (such as hydrochloric acid or sodium hydroxide). Parenteral preparations can be packaged in ampoules made of glass or plastic, disposable syringes, or multi-dose vials.

[0529] Pharmaceutical compositions suitable for injection include sterile aqueous solutions (in the water-soluble case) or dispersions and sterile powders for the ad hoc preparation of sterile injectable solutions or dispersions. Suitable carriers for intravenous, IS, ICV, and / or IT administration include physiological saline, antibacterial aqueous solutions, Cremophor EL, etc. TM (BASF, Parsippany, NJ) or phosphate-buffered saline (PBS). In all cases, the composition must be sterile and should be a fluid readily injectable. It must be stable under manufacturing and storage conditions and must be protected against contamination by microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.) and suitable mixtures thereof. Appropriate flowability can be maintained, for example, by using coatings such as lecithin, by maintaining the desired particle size in the case of dispersions, and by using surfactants. Microbial action can be prevented by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In many cases, it will be preferable to include isotonic agents in the composition, such as sugars, polyols (e.g., mannitol, sorbitol), and sodium chloride. The absorption of the injectable composition can be prolonged by including agents that delay absorption (e.g., aluminum monostearate and gelatin) in the composition.

[0530] Sterile injectable solutions can be prepared by incorporating the desired amount of the active compound with one or a combination of the ingredients listed above (as needed) into a suitable solvent, followed by filtration and sterilization. Typically, dispersions are prepared by incorporating the active compound into a sterile medium containing an alkaline dispersion medium and other desired ingredients listed above. In the case of sterile powders used to prepare sterile injectable solutions, preferred preparation methods include vacuum drying and freeze-drying, which yield powders of the active ingredient and any additional desired ingredients from a previously sterile filtered solution.

[0531] Oral compositions typically contain inert diluents or edible carriers. They may be encapsulated in gelatin capsules or compressed into tablets. For oral therapeutic administration, the active compound may be combined with excipients and used in tablet, lozenge, or capsule form. Oral compositions may also be prepared using fluid carriers for use as mouthwashes, wherein the compound in the fluid carrier is applied orally and rinsed and spat out or swallowed. Pharmaceutically compatible binder and / or adjuvant materials may be included as part of the composition. Tablets, pills, capsules, lozenges, etc., may contain any of the following or compounds with similar properties: binders, such as microcrystalline cellulose, tragacanth gum, or gelatin; excipients, such as starch or lactose; disintegrants, such as alginate, Primogel, or corn starch; lubricants, such as magnesium stearate or sterotes; gliding agents, such as colloidal silica; sweeteners, such as sucrose or saccharin; or flavoring agents, such as peppermint, methyl salicylate, or orange flavoring.

[0532] For administration by inhalation, the compound is delivered in the form of an aerosol spray from a pressure vessel or dispenser (containing a suitable propellant, such as a gas, like carbon dioxide) or a sprayer.

[0533] Systemic application can also be performed via mucosal or transdermal routes. For mucosal or transdermal application, a penetrant suitable for the permeability barrier is used in the formulation. Such penetrants are generally known in the art and include, for example (for mucosal application), detergents, bile salts, and fusidic acid derivatives. Mucosal application can be accomplished by using nasal sprays or suppositories. For transdermal application, the active compound is formulated into ointments, creams, gels, or creams known in the art.

[0534] The compounds can also be formulated as suppositories for rectal delivery (e.g., using conventional suppository bases such as cocoa butter and other glycerides) or retention enemas.

[0535] RNA silencing agents can also be administered via transfection or infection using methods known in the art, including but not limited to those described in McCaffrey et al. (2002), Nature, 418(6893), 38-9 (hydrodynamic transfection); Xia et al. (2002), Nature Biotechnol., 20(10), 1006-10 (virus-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).

[0536] RNA silencing agents can also be administered by any method suitable for administering nucleic acid reagents, such as DNA vaccines. These methods include gene guns, bioinjectors, and skin patches, as well as needle-free methods such as the microparticle DNA vaccine technology disclosed in U.S. Patent No. 6,194,389 and the transdermal needle-free vaccination of mammals using powdered vaccines disclosed in U.S. Patent No. 6,168,587. Furthermore, intranasal delivery is possible, particularly as described in Hamajima et al. (1998), Clin. Immunol. Immunopathol., 88(2), 205-10. Liposomes (e.g., as described in U.S. Patent No. 6,472,375) and microcapsules can also be used. Biodegradable, targeted microparticle delivery systems (e.g., as described in U.S. Patent No. 6,471,996) can also be used.

[0537] In one embodiment, the active compound is prepared together with a carrier that protects the compound from rapid elimination from the body, such as a controlled-release formulation comprising an implant and a microencapsulated delivery system. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydride, polyglycolic acid, collagen, polyorthoester, and polylactic acid can be used. Methods for preparing such formulations will be clear to those skilled in the art. These materials are also commercially available from Alza Corporation and Nova Pharmaceuticals, Inc. Liposome suspensions (including liposomes targeting 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, such as those described in U.S. Patent No. 4,522,811.

[0538] For ease of administration and dosage uniformity, it is particularly advantageous to formulate oral or parenteral compositions in unitary dosage form. As used herein, unitary dosage form refers to a physically discrete unit suitable as a single dose to a subject to be treated; each unit contains a predetermined amount of active compound calculated to produce the desired therapeutic effect, along with the required drug delivery system. The specifications of the unitary dosage forms disclosed herein are determined by and directly dependent on the unique properties of the active compound and the specific therapeutic effect to be achieved, as well as the inherent limitations in the field of formulating such active compound for the treatment of an individual.

[0539] The toxicity and therapeutic efficacy of such compounds can be determined using standard pharmaceutical procedures in cell cultures or laboratory animals, such as determining the LD50 (50% lethal dose) and ED50 (50% effective therapeutic dose). The dose ratio between toxicity and therapeutic effect is the therapeutic index, which can be expressed as the LD50 / ED50 ratio. Compounds exhibiting a large therapeutic index are preferred. Although compounds exhibiting toxic side effects can be used, caution should be exercised in designing delivery systems that target the compound to the affected tissue site to minimize potential damage to uninfected cells and thereby reduce side effects.

[0540] Data obtained from cell culture assays and animal studies can be used to formulate a range of dosage ranges for human use. The dosage of such compounds is preferably within a range including the ED50, having very low or no toxicity in circulating concentrations. The dosage may vary within this range depending on the dosage form used and the route of administration employed. For any compound used in the methods of this disclosure, the therapeutically effective dose can initially be estimated by cell culture assays. Doses can be formulated in animal models to achieve a range of circulating plasma concentrations including the EC50 (i.e., the concentration of the test compound that achieves a half-maximal response) as determined in cell culture. This information can be used to more accurately determine the useful dose in humans. Plasma levels can be measured, for example, by high-performance liquid chromatography.

[0541] The pharmaceutical composition may be included in a container, package, or dispenser along with optional instructions for use.

[0542] As defined herein, the therapeutically effective amount (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 in the range of approximately 1 μg to 1000 mg can be administered; in some embodiments, 10, 30, 100, or 1000 μg can be administered. In some embodiments, 1-5 g of the composition can be administered. The composition can be administered once or more daily to once or more weekly; including every other day. Those skilled in the art will understand that certain factors may affect the dose and duration required for effective treatment of a subject, including but not limited to the severity of the disease or condition, prior treatment, the subject's general health and / or age, and any other pre-existing conditions. Furthermore, treatment of a subject with a therapeutically effective amount of a protein, peptide, or antibody may comprise a single treatment, or preferably, a series of treatments.

[0543] The nucleic acid molecules disclosed herein can be inserted into expression constructs, such as viral vectors, retroviral vectors, expression cassettes, or plasmid viral vectors, for example using methods known in the art, including but not limited to those described above, as described by Xia et al. (2002). The expression constructs can be delivered to a subject by, for example, inhalation, oral administration, intravenous injection, local administration (see U.S. Patent No. 5,328,470), or by stereotactic injection (see, for example, Chen et al. (1994), Proc. Natl. Acad. Sci. USA, 91,3054-3057). Pharmaceutical formulations of the delivery vector may include the vector in an acceptable diluent, or may include a sustained-release matrix in which the delivery vector is embedded. Alternatively, when the complete delivery vector can be generated intact from recombinant cells (e.g., retroviral vectors), the pharmaceutical formulation may include one or more cells that generate the gene delivery system.

[0544] The nucleic acid molecules disclosed herein may also include small hairpin RNA (shRNA) and expression constructs engineered to express shRNA. Transcription of shRNA is initiated at the polymerase III (Pol III) promoter and is believed to terminate at position 2 of the 4-5-thymine transcription termination site. After expression, shRNA is believed to fold into a stem-loop structure with a 3'UU overhang; subsequently, the ends of these shRNAs are processed to convert shRNA into siRNA-like molecules of approximately 21 nucleotides. Brummelkamp et al. (2002), Science, 296, 550-553; Lee et al. (2002), ibid.; Miyagishi and Taira (2002), Nature Biotechnol., 20, 497-500; Paddison et al. (2002), ibid.; Paul (2002), ibid.; Sui (2002), ibid.; Yu et al. (2002), ibid.

[0545] Expression constructs can be any construct suitable for a suitable expression system, and include, 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 such as the U6 snRNA promoter or the H1 RNA polymerase III promoter, or other promoters known in the art. Constructs may include one or two siRNA strands. Expression constructs expressing two strands may also include a loop structure connecting the two strands, or each strand may be transcribed separately from different promoters in the same construct. Each strand may also be transcribed from a separate expression construct, Tuschl (2002), ibid.

[0546] In some exemplary embodiments, compositions including the RNA silencer of this disclosure can be delivered to the nervous system of a subject via a variety of routes. Exemplary routes include intrathecal, parenchymal (e.g., in the brain), nasal, and ocular delivery. The compositions can also be delivered systemically, for example, by intravenous, subcutaneous, or intramuscular injection, which is particularly useful for delivering RNA silencers to peripheral neurons. A preferred route of delivery is direct delivery to the brain, such as the ventricles or hypothalamus, or lateral or dorsal regions of the brain. RNA silencers for neuronal delivery can be incorporated into pharmaceutical compositions suitable for administration.

[0547] For example, the composition may include one or more of the RNA silencing agents of this disclosure and pharmaceutically acceptable carriers. The pharmaceutical compositions of this disclosure can be administered in a variety of ways, depending on whether local or systemic treatment is required and depending on the area to be treated. Administration may be local (including ocular, intranasal, transdermal), oral, or parenteral. Parenteral administration includes intravenous infusion, subcutaneous, intraperitoneal, or intramuscular injection, intrathecal, or intraventricular (e.g., intracerebral) administration. In some exemplary embodiments, the RNA silencing agents of this disclosure are delivered across the blood-brain barrier (BBB) ​​using a variety of suitable compositions and methods described herein.

[0548] The delivery route may vary depending on the patient's condition. For example, a subject diagnosed with a neurodegenerative disease may have the RNA silencing agent of this disclosure administered directly into the brain (e.g., into the globus pallidus or striatum of the basal ganglia, and near medium-sized polyspinous neurons in the striatum). In addition to the RNA silencing agent of this disclosure, secondary therapies, such as palliative care and / or disease-specific treatments, may be administered to the patient. Secondary therapies may be, for example, symptomatic treatment (e.g., for symptom relief), neuroprotective treatment (e.g., for slowing or stopping disease progression), or reversal treatment (e.g., for reversing disease progression). Other therapies may include psychotherapy, physical therapy, speech therapy, communication and memory assistance, social support services, and dietary advice.

[0549] RNA silencers 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 used. For example, a pharmaceutical composition containing an RNA silencer can be delivered to a patient by direct injection into a region containing disease-affected cells. For example, the pharmaceutical composition can be delivered into the brain by direct injection. Injection can be stereotactic to specific regions of the brain (e.g., substantia nigra, cortex, hippocampus, striatum, or globus pallidus). RNA silencers can be delivered to multiple regions of the central nervous system (e.g., multiple regions of the brain, and / or into the spinal cord). RNA silencers can be delivered to diffuse areas of the brain (e.g., diffuse delivery into the cerebral cortex).

[0550] In one embodiment, the RNA silencer can be delivered via a cannula or other delivery device, one end of which is implanted in tissue, such as the brain, including the substantia nigra, cortex, hippocampus, striatum, or globus pallidus. The cannula may be connected to a reservoir of the RNA silencer. Flow or delivery can be mediated by a pump (e.g., an osmotic pump or a micropump, such as the Alzet pump (Durect, Cupertino, CA)). In one embodiment, the pump and reservoir are implanted in an area remote from the tissue, such as in the abdomen, and delivery is influenced by a catheter leading from the pump or reservoir to the release site. Devices for delivery to the brain are described, for example, in U.S. Patent Nos. 6,093,180 and 5,814,014.

[0551] The RNA silencing agent disclosed herein can be further modified to enable it to cross the blood-brain barrier. For example, the RNA silencing agent can be conjugated to a molecule that enables the agent to cross the barrier. For example, such modified RNA silencing agents can be administered by any desired method, such as intraventricular or intramuscular injection, or delivery via the lungs.

[0552] In some implementations, exosomes are used to deliver the RNA silencing agent disclosed herein. Exosomes can cross the BBB after systemic injection and specifically deliver siRNA, antisense oligonucleotides, chemotherapeutic agents, and proteins to neurons (see 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 invitro and in vivo. Nat Protoc. 2012 Dec;7(12):2112-26. doi: El Andaloussi S, Lakhal S, Mäger 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).

[0553] In some embodiments, one or more lipophilic molecules are used to allow delivery of the RNA silencer of this disclosure via the BBB (Alvarez-Ervit (2011)). The RNA silencer is then activated, for example, by degradation by a lipophilically disguised enzyme, to release the drug in its active form.

[0554] In some embodiments, one or more receptor-mediated permeabilizing compounds may be used to increase the permeability of the BBB to allow delivery of the RNA silencing agent of this disclosure. These drugs temporarily increase the permeability of the BBB by increasing the osmotic pressure in the blood, which relaxes the tight junctions between endothelial cells (El-Andaloussi (2012)). By relaxing the tight junctions, normal intravenous injection of the RNA silencing agent can be performed.

[0555] In some embodiments, a nanoparticle-based delivery system is used to deliver the RNA silencing agent of this disclosure across the BBB. As used herein, “nanoparticle” refers to polymeric nanoparticles, typically solid, biodegradable colloidal systems, that have been extensively studied as drug or gene carriers (SP Egusquiaguirre, M. Igartua, RMHernandez and JL Pedraz, “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 fall into two main categories: natural polymers and synthetic polymers. Natural polymers used for siRNA delivery include, but are not limited to, cyclodextrin, chitosan, and determinated collagen (Y. Wang, Z. Li, Y. Han, LH Liang 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 include, but are not limited to, polyethyleneimine (PEI), poly(dl-lactide-co-glycolic acid) (PLGA), and dendritic polymers, which have been extensively studied (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 (incorporated in its entirety by reference).

[0556] The RNA silencers disclosed herein can be administered ocularly, for example, to treat retinal conditions such as retinopathy. For instance, the pharmaceutical compositions can be applied to the surface of the eye or nearby tissues, such as the inner eyelid. They can be applied topically, for example, by spray, drops, as an eye wash, or ointment. Ointments or drops can be delivered using ocular delivery systems known in the art, such as applicators or eye drops. Such compositions may include mucin mimics (such as hyaluronic acid, chondroitin sulfate, hydroxypropyl methylcellulose, or polyvinyl alcohol), preservatives (such as sorbic acid, EDTA, or benzyl chromium chloride), and commonly used amounts of diluent and / or carrier. The pharmaceutical compositions can also be administered intraocularly and can be introduced via a needle or other delivery device that can introduce the pharmaceutical composition into a selected area or structure. Compositions containing RNA silencers can also be applied via eye patches.

[0557] Generally, the RNA silencing agents disclosed herein can be administered by any suitable method. As used herein, local delivery can refer to the direct application of the RNA silencing agent to any surface of the body, including the eyes, mucous membranes, body cavity surfaces, or any internal surface. Formulations for local application can include transdermal patches, ointments, lotions, creams, gels, drops, sprays, and liquids. Conventional drug carriers, aqueous, powder, or oily bases, thickeners, etc., may be necessary or desired. Local application can also be used as a means of selectively delivering the RNA silencing agent to the epidermis or dermis of a subject, or to a specific layer or underlying tissue thereof.

[0558] Compositions intended for intrathecal or intraventricular (e.g., intracerebral) administration may include sterile aqueous solutions, which may also contain buffers, diluents, and other suitable additives. Compositions intended for intrathecal or intraventricular administration preferably do not include transfection reagents or additional lipophilic portions, except for lipophilic portions linked to RNA silencing agents.

[0559] Formulations intended for parenteral administration may include sterile aqueous solutions, which may also contain buffers, diluents, and other suitable additives. Intracardiac injection may be performed using, for example, an intracardiac catheter connected to a reservoir. For intravenous administration, the total solute concentration should be controlled to ensure the formulation isotonic.

[0560] The RNA silencer disclosed herein can be administered to a subject via pulmonary delivery. The pulmonary delivery composition can be delivered via an inhaled dispersion, allowing the composition in the dispersion to reach the lungs, where it can be readily absorbed directly into the bloodstream through the alveolar regions. Pulmonary delivery is effective for treating lung diseases for both systemic and local delivery. In one embodiment, the RNA silencer administered via pulmonary delivery has been modified to allow it to cross the blood-brain barrier.

[0561] Lung delivery can be achieved through various methods, including the use of nebulized, aerosolized, micellar-based, and dry powder formulations. Delivery can be achieved using liquid nebulizers, aerosol-based inhalers, and dry powder dispersion devices. Metering devices are preferred. One advantage of using nebulizers or inhalers is the minimization of the possibility of contamination, as these devices are independent. For example, dry powder dispersion devices can easily formulate drugs into dry powder form. RNA silencing agent compositions can be stored stably as lyophilized or spray-dried powders, alone or in combination with a suitable powder carrier. Delivery of compositions for inhalation can be mediated by a dosing timing element, which may include a timer, dose counter, time measuring device, or time indicator, enabling dose tracking, compliance monitoring, and / or dose triggering for the patient during aerosol drug administration when incorporated into a device.

[0562] Types of pharmaceutical excipients that can be used as carriers include stabilizers (such as human serum albumin (HSA)), fillers (such as carbohydrates), amino acids and peptides; pH adjusters or buffers; salts, such as sodium chloride; etc. These carriers can be in crystalline or amorphous form, or a mixture of both.

[0563] Particularly valuable fillers include compatible carbohydrates, peptides, amino acids, or combinations thereof. Suitable carbohydrates include monosaccharides such as galactose, D-mannose, sorbose, etc.; disaccharides such as lactose, trehalose, etc.; cyclodextrins such as 2-hydroxypropyl-β-cyclodextrin; and polysaccharides such as raffinose, maltodextrin, dextran, etc.; and sugar alcohols such as mannitol, xylitol, etc. Preferred carbohydrate groups include lactose, trehalose, raffinose, maltodextrin, and mannitol. Suitable peptides include aspartame. Amino acids include alanine and glycine, with glycine being preferred.

[0564] Suitable pH adjusters or buffers include organic salts prepared from organic acids and bases, such as sodium citrate, sodium ascorbate, etc.; sodium citrate is preferred.

[0565] The RNA silencers disclosed herein can be administered orally and nasally. For example, drugs administered via these membranes have a rapid onset of action, provide therapeutic plasma levels, avoid the first-pass effect of hepatic metabolism, and avoid exposure to the adverse gastrointestinal (GI) environment. Additional advantages include easy access to the membrane site, enabling easy application, localization, and removal of the drug. In one embodiment, the RNA silencer administered orally or nasally has been modified to cross the blood-brain barrier.

[0566] In one embodiment, a unit dose or measured dose of the composition containing the RNA silencing agent is administered via an implanted device. The device may include sensors that monitor parameters within the subject's body. For example, the device may include a pump (such as an osmotic pump) and optional associated electronics.

[0567] RNA silencing agents can be packaged in the natural capsid of a virus or in chemically or enzymatically produced artificial capsids or their derivative structures.

[0568] It will be apparent to those skilled in the art that other suitable modifications and adaptations can be made to the methods described herein using appropriate equivalents without departing from the scope of the embodiments disclosed herein. Certain embodiments have been described in detail, and a clearer understanding of the embodiments will be provided by referring to the following examples, which are included herein for illustrative purposes only and are not intended to limit the invention.

[0569] Example 1. Synthesis of 2'-OMe-exNA phosphoramide

[0570] Synthesis of compound 5a

[0571] according to Figure 2 The following synthesis was completed. IBX (5.53 g, 19.7 mmol) was added to anhydrous CH3CN (80 mL) of compound 3a (2.94 g, 7.89 mmol), and the mixture was stirred at 85 °C for 2 h. After cooling the mixture in an ice bath, the precipitate was filtered off through diatomaceous earth. The collected eluent was evaporated, co-evaporated three times with anhydrous CH3CN under an argon atmosphere, and compound 4a, a white foamy substance, was obtained and could be used without further purification. In a separate flask, tert-BuOK (2.57 g, 22.9 mmol) was added to anhydrous THF (80 mL) containing methyltriphenylphosphonium bromide (8.47 g, 23.7 mmol) at 0 °C, and the mixture was stirred at 0 °C for 30 min. Anhydrous THF solution (80 mL) of compound 4a was added dropwise to this solution at 0 °C (10 min), and the mixture was stirred at room temperature for 7 h. After evaporating excess THF, the resulting mixture was dissolved in excess ethyl acetate, washed with saturated NH4Cl aqueous solution, dried over MgSO4, filtered, and concentrated. The obtained material was dissolved in a minimal amount of CH2Cl2 and added dropwise to excess diethyl ether solution at 0°C with vigorous stirring. The precipitate in the solution was filtered through diatomaceous earth and the eluent was evaporated. The crude material obtained was purified by silica gel column chromatography (hexane / ethyl acetate, 9:1 to 1:2) to yield compound 5a as a white foamy substance (2.19 g, 75% in step 2). 1H 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 HRMS (ESI) C 17 H 29 N2O5Si + [M +H] + Calculated value m / z 369.1840, measured value m / z 369.1838.

[0572] Synthesis of compound 6a

[0573] At 0 °C, 0.5 M 9-BBN / THF solution (237.4 mL, 118.7 mmol) was added dropwise over 10 minutes to an aqueous solution of compound 5a (7.29 g, 19.8 mmol) in THF (158.3 mL). The mixture was stirred at room temperature for 6 hours, then the solution was frozen and methanol (65.4 mL) was added, with stirring until bubbling ceased. H₂O (98.4 mL) was then added dropwise over 10 minutes with vigorous stirring to prevent precipitation of the intermediate compound. NaBO₃⁻⁴H₂O (15.7 g, 102.0 mmol) was added in a single addition at 0 °C, and the mixture was stirred overnight at room temperature. After evaporation of excess THF, the resulting crude mixture was dissolved in excess ethyl acetate and repeatedly washed with saturated aqueous NH₄Cl solution. After evaporation of the organic layer, the resulting material was dissolved in THF (450 mL) and H₂O (450 mL). At room temperature, NaBO3-4H2O (15.7 g, 102.0 mmol) was added to this solution in a single addition, followed by stirring overnight at room temperature. After evaporating excess THF, ethyl acetate was added to the mixture, followed by extraction. The resulting organic layer was repeatedly washed with saturated NH4Cl aqueous solution, dried over MgSO4, filtered, and concentrated. The crude material obtained was purified by silica gel column chromatography (hexane / ethyl acetate, 7:3 to 0:10) to give compound 6a as a white foamy substance (4.73 g, 62% in step 2). 1 H 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 m / z is 387.1946, and the measured value m / z is 187.1944.

[0574] Synthesis of compound 8a

[0575] DMTrCl (9.95 g, 29.4 mmol) was added to compound 6a (9.46 g, 24.5 mmol) in an aqueous solution of pyridine (240 mL), 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 material 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 pyridine residues. This crude mixture containing compound 7a was dissolved in THF (330 mL), and 1.0 M TBAF-THF solution (36.7 mL, 36.7 mmol) was added, followed by stirring at room temperature for 1 hour. After evaporating excess THF and co-evaporating with CH2Cl2, the crude material was purified by silica gel column chromatography to give compound 8a (13.15 g, 93% in step 2). 1 H NMR (500 MHz, CDCl3) δ8.91 (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 CNMR (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 34N₂O₈Na [M + Na] + Calculated value m / z 597.2203, measured value m / z 597.2153.

[0576] Synthesis of compound 9a

[0577] 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). N,N-diisopropylethylamine (7.6 mL, 62.4 mmol) and 2-cyanoethyl N,N-diisopropylphosphonamide (4.85 mL, 25.0 mmol) were added to this solution at 0 °C. After stirring at room temperature for 4 hours, CH2Cl2 (200 mL) was added to the reaction mixture, followed by saturated NaHCO3 aqueous solution (350 mL). The organic layer was repeatedly washed with saturated NaHCO3 aqueous solution, dried over MgSO4, filtered, and then evaporated. The obtained crude material was purified by silica gel column chromatography (1% TEA-hexane-ethyl acetate, from 80:20 to 30:70) to give compound 9a with residual phosphite esterification reagent impurities. To remove impurities, the obtained material was dissolved in ethyl acetate (1:1, volume / volume, 400 mL), and then repeatedly washed with saturated NaHCO3 aqueous solution to give compound 9a as a white solid (11.12 g, 86%). 31 PNMR (202 MHz, CDCl3) δ 150.0, 149.9; HRMS (ESI) C 41 H 52 N4O9P [M + H] + The calculated value m / z is 775.3486, and the measured value m / z is 775.3414.

[0578] Example 2.2 Synthesis of '-F-exNA phosphoramide

[0579] Synthesis of compound 5b

[0580] according to Figure 3The following synthesis was completed. IBX (21.0 g, 75.0 mmol) was added to anhydrous CH3CN (300 mL) containing 10.8 g, 30.0 mmol, and stirred at 85 °C for 2 h. After cooling the mixture in an ice bath, the precipitate in the solution was filtered off through diatomaceous earth. The collected eluent was evaporated, co-evaporated three times with anhydrous CH3CN under an argon atmosphere, and the resulting compound 4b, a white foam, was ready for use without further purification. In a separate flask, methyltriphenylphosphonium bromide (24.0 g, 68.1 mmol) was added in a single step to anhydrous THF (250 mL) containing tert-BuOK (7.30 g, 65.1 mmol) at 0 °C, and stirred at 0 °C for 1 h. Anhydrous THF solution of compound 4b (150 mL) was added dropwise (10 min) to this solution at 0 °C, and stirred overnight at room temperature. After evaporating excess THF, the resulting mixture was dissolved in excess ethyl acetate, washed with saturated NH4Cl aqueous solution, dried over MgSO4, filtered, and concentrated. The obtained material was dissolved in a minimal amount of CH2Cl2 and added dropwise to excess diethyl ether solution at 0°C with vigorous stirring. The precipitate in the solution was filtered through diatomaceous earth and the eluent was evaporated. The crude material obtained was purified by silica gel column chromatography (hexane / ethyl acetate, 8:2 to 6:4) to give compound 5b as a white foamy substance (7.12 g, 67% in step 2). 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).

[0581] Synthesis of compound 7b

[0582] At 0 °C, a 0.5 M 9-BBN / THF solution (342 mL, 171 mmol) was added dropwise over 20 minutes to an aqueous solution of compound 5b (10.15 g, 28.5 mmol) in THF (228 mL). The mixture was stirred at room temperature for 4 hours, then the solution was frozen and methanol (131 mL) was added, with stirring until bubbling ceased. H₂O (197 mL) was then added dropwise over 15 minutes with vigorous stirring to prevent precipitation of the intermediate compound. NaBO₃⁻⁴H₂O (21.9 g, 142.5 mmol) was added in a single addition at 0 °C, and the mixture was stirred overnight at room temperature. After evaporating excess THF, the resulting crude mixture was dissolved in excess ethyl acetate and repeatedly washed with a saturated aqueous solution of NH₄Cl. After evaporating the organic layer, the resulting material was dissolved in THF (450 mL) and H₂O (450 mL). At room temperature, 21.9 g (142.5 mmol) of NaBO3-4H2O was added to this solution in a single addition, followed by stirring overnight at room temperature. After evaporation of excess THF, ethyl acetate was added to the mixture, followed by extraction. The resulting organic layer was repeatedly washed with saturated NH4Cl aqueous solution, dried over MgSO4, filtered, and concentrated. The crude material obtained was purified by silica gel column chromatography (CH2Cl2 / methanol, 100:0 to 93:7) to give compound 6b as a syrup (2.44 g with reagent impurities); HRMS (ESI) C 16 H 28 FN2O5Si + [M + H] +The calculated m / z value was 375.1746, and the measured m / z 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 by adding methanol (5 mL), the reaction mixture was diluted with ethyl acetate (300 mL), washed repeatedly with a saturated aqueous solution of NaHCO3, dried over MgSO4, filtered, evaporated, and then co-evaporated three times with toluene to remove residual pyridine. The crude material obtained was purified by silica gel column chromatography (hexane-ethyl acetate, 2:8 to 4:6) to give compound 7b as a white solid (2.25 g, 12% in step 2). 1 H NMR (500 MHz, CD3CN) δ 9.16 (br-s, 1H), 7.43-7.42 (m, 2H), 7.31-7.28 (m, 8H), 6.86-6.85 (m,4H), 5.75 (dd, 1H, J HF = 20.0 Hz, J HH = 1.9 Hz), 5.59 (d, 1H, J = 8.1 Hz), 4.96 (ddd, J HF = 53.3 Hz, J HH = 4.6, 1.8 Hz), 4.06-3.98 (m, 2H), 3.76 (s, 6H), 3.19 (dd, 2H, J = 7.4, 5.6 Hz), 2.09-2.02 (m, 1H), 1.89-1.82 (m, 1H), 0.91(s, 9H), 0.10 (s, 3H), 0.09 (s, 3H); 13 C 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 (d,J CF = 188.0 Hz), 90.6 (d, J = 36.4 Hz), 87.0, 80.7,74.6 (d, J = 15.4 Hz), 60.9, 55.9, 33.8, 26.1, 18.7, -4.5, -4.8; 19F NMR (470MHz, CD3CN) δ -201.4 (ddd, J = 53.7, 19.1, 19.1 Hz); HRMS (ESI) C 37 H 45 FN2O7Na[M + Na] + The calculated value m / z is 699.2872, and the measured value m / z is 699.2866.

[0583] Synthesis of compound 8b

[0584] Compound 7a (2.24 g, 3.30 mmol) was dissolved in THF (36.0 mL), followed by the addition of 1.0 MTBAF-THF solution (4.0 mL, 4.0 mmol), and the mixture was stirred at room temperature for 30 minutes. After evaporating excess THF and co-evaporating with CH2Cl2, the crude material was purified by silica gel column chromatography [CH2Cl2(1% TEA)-methanol, 100:0 to 95:5] to give 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 = 35.4Hz), 87.2, 80.5, 73.9 (d, J = 16.4 Hz), 61.0, 56.0, 33.9; 19F NMR (470 MHz, CD3CN) δ -201.8 (ddd, J = 53.7, 20.8, 20.8 Hz).

[0585] Synthesis of compound 9b

[0586] Compound 8b (1.5 g, 2.67 mmol) was anhydrous by repeated co-evaporation with anhydrous CH3CN and then dissolved in anhydrous CH2Cl2 (30 mL). N,N-diisopropylethylamine (1.76 mL, 10.1 mmol) and 2-cyanoethyl N,N-diisopropylphosphonamide (0.90 mL, 4.01 mmol) were added to this solution at 0 °C. After stirring at room temperature for 2 hours, CH2Cl2 (70 mL) was added to the reaction mixture, followed by saturated NaHCO3 aqueous solution (100 mL). The organic layer was repeatedly washed with saturated NaHCO3 aqueous solution, dried over MgSO4, filtered, and then evaporated. The obtained crude material was purified by silica gel column chromatography (1% TEA-hexane-ethyl acetate, 80:20 to 20:80) to give compound 9a with residual phosphite esterification reagent impurities. To remove impurities, the obtained material was dissolved in Et₂O (100 mL) and then repeatedly washed with a saturated NaHCO₃ aqueous solution to obtain compound 9b, 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.

[0587] Example 3. Synthesis of exNA-C phosphorousamide

[0588] according to Figure 4The starting material is first converted to a cytidine derivative (Kaura, M. et al., J. Org. Chem. 2014, 79, 6256−6268), and then the 4-amino group of the resulting cytosine base is protected with an acyl protecting group (such as an acetyl group). After deprotection by 3'-O-TBDMS, the resulting 3'-hydroxy group is converted to 3'-O-phosphamide. Each step is first quenched and extracted, and then purified by silica gel column chromatography.

[0589] Example 4. Synthesis of exNA-G and exNA-A phosphorous amides

[0590] according to Figure 5 First, the 3'-O-TBDMS-protected starting material was oxidized to an aldehyde using IBX, followed by to-Vitiger olefination using methyltriphenylphosphonium bromide and tert-BuOK in anhydrous THF solution to obtain a vinyl-substituted nucleoside derivative. This vinyl group reacts with 9-BBN to generate a borate intermediate, which is then oxidized by sodium perborate to give the exNA structure with a 6'-hydroxyl group. This hydroxyl group is first protected with a DMTr group without silica gel column purification, and then the 3'-O-TBDMS group is deprotected using 0.1 M TBAF-THF solution. The resulting 6'-O-DMTr nucleoside derivative is then phosphite-esterified to give phosphoramidite. Each step is first quenched and extracted, followed by purification by silica gel column chromatography, except for the 6'-O-triphenylmethylation step.

[0591] Example 5. Synthesis of 5'-3'-bis-methylene-exNA phosphoramide

[0592] according to Figure 6The primary hydroxyl group of the starting material with a Nap-protected hydroxymethyl group (Betkekar, VV et al., Org. Lett. 2012, 14, 1, 198-201) is first selectively protected by a TBDPS group, followed by secondary alcohol deoxygenation (Prakash, TP et al., Nucleic Acids Res. 2015, 43, 2993-3011). Next, the TBDPS group is deprotected in 0.1 M TBAF-THF solution and benzoylated using benzoyl chloride in pyridine to convert it to a benzoyl (Bz) protecting group. The isopropylidene protecting group of the sugar is then deprotected to yield a 1,2-bis-acetylated sugar, which is then subjected to conventional BSA / TMSOTf-mediated uracil glycosylation to produce a uridine nucleoside derivative. The Nap protecting group on the 3'-hydroxymethyl group is deprotected by DDQ. The obtained material with a 6'-O-Bz-3'-hydroxymethyl group will be converted into a 6'-O-DMTr-3'-TBDMS-protected hydroxymethyl compound with a 2'-O-acetyl group. After deprotection of the TBDMS group, the 3'-hydroxymethyl group is phosphite-esterified to give 5'-3'-bis-exNA-phosphite. Each step will first be quenched and extracted, followed by purification by silica gel column chromatography.

[0593] Example 6. Synthesis of exNA-ribose-uridine phosphoramidite

[0594] according to Figure 7The following synthesis was completed. IBX (30.3 g, 108.2 mmol) was added to anhydrous CH3CN (520 mL) containing 15.4 g, 54.1 mmol, and stirred at 85 °C for 2 hours. After cooling the mixture in an ice bath, the precipitate was filtered off through diatomaceous earth. The collected eluent was evaporated, co-evaporated three times with anhydrous CH3CN under an argon atmosphere, and the resulting compound 3, a white foam, was ready for use without further purification. In a separate flask, methyltriphenylphosphonium bromide (43.3 g, 121.2 mmol) was added in a single step to anhydrous THF (500 mL) containing tert-BuOK (13.2 g, 117.4 mmol) at 0 °C, and stirred at 0 °C for 1 hour. Anhydrous THF solution of compound 3 (150 mL) was added dropwise (over 10 minutes) to this solution at 0 °C, and stirred at room temperature for 4 hours. After evaporating excess THF, the resulting mixture was dissolved in excess ethyl acetate, washed with saturated NH4Cl aqueous solution, dried over MgSO4, filtered, and concentrated. The obtained material was dissolved in a minimal amount of CH2Cl2 and added dropwise to excess diethyl ether solution at 0°C with vigorous stirring. The precipitate in the solution was filtered through diatomaceous earth and the eluent was evaporated. The crude material was purified by silica gel column chromatography (hexane, ethyl acetate, 8:2 to 3:7) to give compound 4 with triphenylphosphine oxide impurities. This crude material was anhydrous by repeated co-evaporation with anhydrous CH3CN, and then dissolved in anhydrous THF (200 mL). 0.5 M 9-BBN / THF (300 mL, 150.0 mmol) was added dropwise to this solution over 10 minutes, and the mixture was stirred overnight at room temperature. After confirming the disappearance of the starting material by TLC, the solution was frozen, and then methanol (200 mL) was added dropwise over 10 minutes. After bubbling stopped, add 300 mL of H₂O dropwise, followed by a one-time addition of Na₂BO₃⁻⁴H₂O (19.2 g, 125.0 mmol). Stir the solution overnight at room temperature. After evaporating excess THF, dissolve the resulting crude mixture in excess ethyl acetate and wash repeatedly with saturated NH₄Cl aqueous solution. After evaporating the organic layer, dissolve the obtained material in 400 mL of THF and 400 mL of H₂O. Add 19.2 g, 125.0 mmol of Na₂BO₃⁻⁴H₂O to this solution one-time at room temperature, and stir overnight at room temperature. After evaporating excess THF, add ethyl acetate to the mixture and extract. Wash the resulting organic layer repeatedly with saturated NH₄Cl aqueous solution, dry to MgSO₄, filter, and concentrate.The crude product was purified by silica gel column chromatography (CH2Cl2-methanol, 100:0 to 93:7) to give compound 5 with reagent residue impurities. A TFA solution [TFA (85 mL) and H2O (9.2 mL)] was added to this obtained material and stirred at 0 °C for 1 hour. After evaporation, the mixture was co-evaporated four times with toluene. The crude product was purified by silica gel column chromatography (CH2Cl2-MeOH, 100:0 to 90:10) to give compound 6 (760 mg, 12% in step 3). 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 CNMR (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] + Calculated value m / z 281.0744, measured value m / z 281.0730.

[0595] Synthesis of Compound 7

[0596] Anhydrous pyridine (30 mL) was added to compound 6 (760 mg, 2.94 mmol), followed by the addition of DMTr-Cl (1.3 g, 3.82 mmol). After stirring for 2 hours, the reaction mixture was first extracted with CH2Cl2 and saturated NaHCO3 aqueous solution, and the organic layer was dried over MgSO4, filtered, evaporated, and co-evaporated to remove pyridine. The crude material obtained was purified by silica gel column chromatography (CH2Cl2-MeOH, 100:0 to 95:5) to give compound 7 (1.70 g, quantitative). HRMS (ESI) C 31 H 32 N₂O₈Na[M + Na]+ Calculated value m / z 583.2051, measured value m / z 583.2025.

[0597] Synthesis of Compound 8

[0598] Imidazole (576.1 mg, 8.46 mmol) and TBDMSCl (1.10 g, 7.33 mmol) were added to compound 7 (2.35 g, 4.19 mmol) in an aqueous solution of pyridine (21 mL), and the mixture was stirred at room temperature for 2 hours. CH2Cl2 (150 mL) was added to this reaction mixture, followed by saturated aqueous solution of NaHCO3 (150 mL). The organic layer was repeatedly washed with saturated aqueous solution of NaHCO3, dried over MgSO4, filtered, evaporated, and then co-evaporated with toluene to remove pyridine residues. The crude material containing compound 8, the 3'-O-TBDMS protected compound, and the 5'-3'-O-bis-TBDMS protected compound was separated by silica gel column chromatography [CH2Cl2 (1% TEA)-acetone, 100:0 to 85:15] to give pure compound 8 (780 mg, 28%). 1 H 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); 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 H 46 N₂O₈Na [M + Na] + The calculated value m / z is 697.2916, and the measured value m / z is 697.2867.

[0599] Synthesis of Compound 9

[0600] Compound 8 (780 g, 1.16 mmol) was anhydrous by repeated co-evaporation with anhydrous CH3CN and then dissolved in anhydrous CH2Cl2 (12 mL). N,N-diisopropylethylamine (0.53 mL, 4.34 mmol) and 2-cyanoethyl N,N-diisopropylphosphonamide (0.34 mL, 1.73 mmol) were added to this solution at 0 °C. After stirring at room temperature for 4 hours, CH2Cl2 (90 mL) was added to the reaction mixture, followed by saturated aqueous NaHCO3 solution (100 mL). The organic layer was repeatedly washed with saturated NaHCO3 solution, dried over MgSO4, filtered, and then evaporated. The obtained crude material was purified by silica gel column chromatography (1% TEA-hexane-ethyl acetate, 80:20 to 50:50) to give compound 9 (825.9 mg, 82%). 31 P NMR (202 MHz, CDCl3) δ 149.6, 149.1.

[0601] Example 7. Synthesis of exNA-ribose-cytosine phosphoramide

[0602] The starting material, containing vinyl-substituted uridine derivatives, is first converted to cytidine (Kaura, M. et al. J. Org. Chem. 2014, 79, 6256−6268). The 4-amino group of the resulting cytosine base is then protected with an acyl protecting group (such as an acetyl group). After deprotection of the 2'-3'-O-isopropylidene group, the 6'-hydroxy group is protected with DMTr, followed by TBDMS. The 2'-O-TBDMS-protected compound, separated by silica gel column chromatography, is then phosphite-esterified to yield 3'-O-phosphite. Each step is first quenched and extracted, followed by purification by silica gel column chromatography.

[0603] Example 8. Synthesis of exNA-ribose-guanosine or exNA-ribose-adenine phosphoramide

[0604] according to Figure 9 First, the 2'-3'-O-bis-TBDMS-protected starting material was oxidized to an aldehyde using IBX, followed by to-Vitiger olefination using methyltriphenylphosphonium bromide and tert-BuOK in anhydrous THF solution to yield a vinyl-substituted nucleoside derivative. This vinyl group reacted with 9-BBN to generate a borate intermediate, which was then oxidized by sodium perborate to give the exNA structure with a 6'-hydroxyl group. This hydroxyl group was first protected with a DMTr group, followed by TBDMS protection. The 2'-O-TBDMS-protected compound, separated by silica gel column chromatography, was phosphite-esterified to give 3'-O-phosphite. Each step involved quenching and extraction followed by purification by silica gel column chromatography, and deprotection of the 3'-O-TBDMS group was performed using 0.1 MTBAF-THF solution without silica gel column purification. The resulting 6'-O-DMTr nucleoside derivative was then phosphite-esterified to give phosphite. Each step involved quenching and extraction followed by purification by silica gel column chromatography.

[0605] Example 9. Synthesis of exNA-ribose-uridine phosphoramidite

[0606] according to Figure 10The starting material protected with 5'-O-DMTr is first protected with TBDMS, followed by 5'-O-detriphenylmethylation. The resulting compound is then oxidized to an aldehyde using IBX, followed by to-Vitiger olefination using methyltriphenylphosphonium bromide and tert-BuOK in anhydrous THF solution to yield a vinyl-substituted nucleoside derivative. This vinyl group reacts with 9-BBN to generate a borate intermediate, which is then oxidized with sodium perborate to give the exNA structure with a 6'-hydroxyl group. This hydroxyl group is first protected with a DMTr group without silica gel column purification, followed by deprotection of the 3'-O-TBDMS group using 0.1 MTBAF-THF solution. The resulting 6'-O-DMTr nucleoside derivative is then phosphite-esterified to give a methyl-protected phosphoramidite. Each step is first quenched and extracted, followed by purification by silica gel column chromatography, except for the first 3'-O-TBDMS protection step.

[0607] Example 10. Synthesis of oligonucleotides incorporated into the exNA backbone

[0608] according to Figure 12 A method for synthesizing modified oligonucleotides comprising a 5' end, a 3' end, and at least one modified intersubunit bond has been completed. The method comprises: (a) providing a nucleoside having a 5'-protecting group linked to a solid support; (b) removing the protecting group; and (c) combining the deprotected nucleoside with a phosphorous amide derivative of formula (VII) to form a phosphite triester.

[0609]

[0610] (VII)

[0611] (d) Capping the triphosphite; (e) Oxidizing the triphosphite; (f) Repeating steps (b) to (e) using additional phosphoramide; and (g) Cutting from the solid support.

[0612] Figure 13 Examples of oligonucleotides having one or more exNA-subunit interunit bonds synthesized by the above method are shown. The exNA-subunit interunit bond is a 5'-methylene-exNA-uridine with a 2'-OH group.

[0613] Example 11: In vitro silencing efficacy of target mRNA and siRNA duplex containing exNA subunit bonds.

[0614] In the oligonucleotide walking experiment, ex-NA subunit bonds were used, where each subunit bond in both the antisense and sense strands was modified by an ex-NA subunit bond. The ex-NA subunit bonds are (ex_mU): 5'-methylene-exNA-uridine with 2′-OMe or (ex_fU): 5'-methylene-exNA-uridine with 2′-fluoro-ex-uridine. Tables 4-10 below show the antisense and sense strands used in this embodiment, as well as the bistrands formed by different combinations of said antisense and sense strands. Figure 12 As shown, a novel synthetic scheme for generating oligonucleotides containing ex-NA was also employed.

[0615] Table 4 – Antisense strands with ex-NA subunit bonds

[0616]

[0617]

[0618] Table 5 – Sense chains with ex-NA subunit bonds

[0619]

[0620] Table 6 – Comparison of antisense chains

[0621]

[0622] Table 7 – Comparison of semantic chains

[0623]

[0624] Table 8 – siRNA duplexes with ex-NA modified antisense strands (D1-D20)

[0625]

[0626] Table 9 – siRNA duplexes with ex-NA modified sense strands (D25-D39)

[0627]

[0628] Table 10 – Control siRNA duplexes

[0629]

[0630] The siRNA duplexes listed above were used in in vitro mRNA silencing experiments to determine relative silencing efficacy. Experimental details are described below.

[0631] In vitro screening.

[0632] 1.5 μM siRNA was passively delivered to cells. Cells were plated at 8000 cells per well in Dürbeco Modified Igor Medium containing 6% FBS in 96-well cell culture plates. The siRNA was diluted twice to the final concentration in OptiMEM (Carlsbad, CA; 31985-088), and 50 µL of the diluted siRNA was added to 50 µL of cells to obtain 3% FBS. Cells were incubated at 37°C and 5% CO2 for 72 hours.

[0633] Quantitative analysis of target mRNA.

[0634] mRNA was quantified from cells using a QuantiGene 2.0 assay kit (Affymetrix, QS0011). Cells were lysed for 30 minutes at 55°C in a 250 μL diluted lysis mixture consisting of one part lysis mixture (Affymetrix, 13228), two parts H2O, and 0.167 μg / μL proteinase K (Affymetrix, QS0103). The cell lysates were thoroughly mixed, and 40 μL of each lysis lyse was added to each well of a capture plate containing 40 μL of diluted lysis lysate, excluding proteinase K, and 20 μL of diluted probe kit. Probe kits for human HTT and hypoxanthine phosphoribosyltransferase (HPRT) (Affymetrix; #SA-50339, SA-10030) were diluted and used according to the manufacturer's recommended protocol. The dataset was normalized to HPRT.

[0635] Cell treatment: reporter gene assay.

[0636] HeLa cells were grown and maintained in Gibco DMEM (ref. #11965-092) containing 1% penicillin / streptavidin and 10% heat-inactivated FBS. Two 10 cm cells were placed in the culture medium three days prior to treatment. 2 Petri dishes using 2x10 6 HeLa cells were plated. The next day, DMEM was replaced with Gibco OptiMEM (ref. #31985-070), and 6 µg of reporter plasmid was added to the cells using Invitrogen Lipofectamine 3000 (ref. #L3000-015) according to the manufacturer's protocol. The cells were left in OptiMEM / lipofectamine overnight to allow for maximum reporter plasmid transfection. The next day, siRNA was diluted in OptiMEM, and triplicate was added to 96-well clear-bottomed tissue culture plates for each reporter plasmid. The HeLa cells transfected with the reporter plasmid the previous night were plated at 0.15 x 10⁻⁶ cells per plate.6 Cells / mL were resuspended in DMEM containing 6% heat-inactivated FBS (penicillin / streptavidin-free) and added to plates containing siRNA.

[0637] Cells were lysed after treatment with 1x passive lysis buffer (Promega ref. #E1960) from the dual-sided luciferase assay system kit for 72 hours (100% confluence). Following lysis, fluorescence was read after adding 50 µl of luciferase assay reagent II (Promega ref. #E1960), followed by a second read after adding 50 µl / well of stop and luminescence reagent (Promega ref. #E1960). Absorbance was normalized to the untreated control and plotted logarithmically.

[0638] like Figure 14 As shown, all tested siRNA duplexes effectively silenced the target HTT mRNA. Furthermore, many siRNA duplexes silenced both the target mRNA and the control duplex siRNA. This data provides the first instance of ex-NA nucleotide bonds incorporated into oligonucleotide chains.

[0639] Example 12 Nuclease stability of siRNA duplexes containing exNA subunit bonds

[0640] It is hypothesized that ex-NA subunit bonds can be used to increase the nuclease stability of oligonucleotides. This effect can be observed when ex-NA subunit bonds are used alone or in combination with phosphate thioester subunit bonds. Furthermore, multiple consecutive ex-NA subunit bonds in an oligonucleotide may have a greater impact on stability than a single ex-NA subunit bond. Stability can be improved through two main mechanisms: 1) anomalous local backbone structures of ex-NA reduce nuclease cleavage kinetics, and 2) multi-elongated backbones reduce nuclease binding affinity (the effect of multi-elongation on the overall structure of the 3' end region). Figure 15 To demonstrate this effect, several nuclease assays were performed using oligonucleotides containing one or more ex-NA subunit bonds.

[0641] 3' Exonuclease stability test.

[0642] Oligonucleotides with varying numbers of ex-NA subunit bonds at the 3' end were tested in a 3' exonuclease stability assay. Oligonucleotides ex-21, ex-22, ex-23, ex-24, AS-0, and AS-2 (as listed above in Tables 4 and 6) were incubated at 17.5 mM in a buffer containing 10 mM Tris-HCl (pH 8.0), 2 mM MgCl2, and Snake Venom phosphodiesterase I (20 mU / mL) at 37 °C. Figure 16 As shown, compared to AS-2, which has the same phosphate thioester content in clinically approved siRNA drugs, multiple ex-NA subunit bonds to phosphate thioester subunit bonds (ex-24) significantly improved the stability of the 3'-exonuclease. Furthermore, even a single ex-NA subunit bond at the 3' end significantly improved stability (ex-21). Given the predominance of 3'-exonucleases in serum, 3' ex-NA subunit bonds could be used in therapeutic oligonucleotides.

[0643] In the context of a polyuridine acyl sequence containing an oligonucleotide with phosphodiester (PO) and phosphate thioester (PS), an additional 3' exonuclease assay was performed using ex-NA subunit bonds. Oligonucleotides were tested with 1, 2, 3, 4, or 5 ex-NA subunit bonds. Table 11 below lists the polynucleotides used in this assay. Figure 17 As shown, even the presence of a single ex-NA subunit bond significantly improves the stability of oligonucleotides. This was demonstrated in both PO and PS oligonucleotides. Furthermore, PO oligonucleotides with five ex-NA subunit bonds achieved similar nuclease stability compared to PS-containing oligonucleotides without ex-NA subunit bonds (PS control). This result suggests that the number of PS-containing subunit bonds may be reduced by using ex-NA subunit bonds, thereby decreasing the toxicity associated with PS-containing oligonucleotides.

[0644] Table 11 – Polyuridine oligonucleotides used for 3'-exonuclease stability testing

[0645]

[0646] The fluorescent label “FAM” used on oligonucleotides has no effect on 3' exonuclease activity and can be used to monitor cleavage in stability tests.

[0647] 5' Exonuclease stability test.

[0648] Oligonucleotides with ex-NA subunit bonds at the 5' end were tested in two different 5' exonuclease stability assays.

[0649] The first test was a 5'-phosphate ester-dependent 5'-exonuclease stability test. The oligonucleotides used in this test are shown in Table 12 below. The oligonucleotides were used at 2.5 µM (50 pmol) and in RNase-free water, or with 3.3 units of Terminator. TM (EpiCentre) exonuclease was incubated together at 37°C in buffer A (EpiCentre, provided with Terminator) with the exonuclease.TM Incubate in enzymes. Figure 18 As shown, a single ex-NA subunit bond at the 5' end (ON2) significantly improves the stability of the 5'-exonuclease compared to ON1, which contains a 5' phosphodiester bond. Importantly, ON2 does not contain a phosphate-thioester subunit bond. The data indicate that the degree to which a single ex-NA subunit bond at the 5' end improves stability is the same as that of multiple phosphate-thioester subunit bonds at the 5' end (ON3). Excessive phosphate-thioester content in therapeutic oligonucleotides can be toxic. The use of a 5' ex-NA subunit bond provides a mechanism to improve oligonucleotide stability while reducing phosphate-thioester content.

[0650] The second 5'-exonuclease stability assay is a non-5'-phosphate-dependent 5'-exonuclease stability assay. The oligonucleotides used in this assay are shown in Table 13 below. The oligonucleotides were used at 10 µM and incubated at 37°C in RNase-free water, or with 30 mM NaOAc (pH 6.0) buffer containing 0.25 U / mL bovine spleen phosphodiesterase II (BSP). Figure 19 As shown, a single ex-NA subunit bond at the 5' end (ON4) exhibits similar 5'-exonuclease stability compared to ON5, which contains multiple 5'-phosphothioester bonds. The data indicate that the single ex-NA subunit bond at the 5' end enhances stability to the same extent as the multiple phosphothioester subunit bonds at the 5' end (ON5). Excessive phosphothioester content in therapeutic oligonucleotides can be toxic. The use of a 5' ex-NA subunit bond provides a mechanism to improve oligonucleotide stability while simultaneously reducing phosphothioester content.

[0651] Table 12 – Oligonucleotides used in the 5'-phosphate-dependent 5'-exonuclease stability assay

[0652]

[0653] Table 13 – Oligonucleotides used for stability assays of non-5'-phosphate-dependent 5'-exonucleases

[0654]

[0655] Example 13 Activity of siRNA duplexes containing one or more antisense strand 3' exNA subunit bonds

[0656] The in vitro silencing activity of several siRNA duplexes containing one or more 3' exNA subunit bonds of the antisense strand was tested. Antisense strands containing one, two, three, or four 3' exNA subunit bonds were used for dose-response profiling, such as... Figure 20AThe description also included the determination of the percentage change in potency relative to the control of siRNA duplexes without exNA subunit bonds. Figure 20B Data show that siRNA duplexes with antisense strands containing one, two, three, or four 3' exNA subunit bonds have higher silencing efficacy than siRNA duplexes with antisense strands lacking exNA subunit bonds.

[0657] Example 14: In vivo activity of siRNA duplexes containing one or more antisense strand 3' exNA subunit bonds.

[0658] The in vivo silencing activity of several siRNA duplexes containing one or more antisense strand 3' exNA subunit bonds was tested. As mentioned above, the siRNA duplexes are in Di-siRNA format. Sequences and chemical modification patterns are listed in Table 14 below, and each siRNA targets ApoE mRNA. Mice were administered 5 nmol of each Di-siRNA via ICV injection, and ApoE mRNA was quantified after 1 month. Figures 21A-21E As shown, siRNAs containing exNA subunit bonds can silence ApoE in several brain regions (medial cortex, striatum, hippocampus, thalamus, and cerebellum). The silencing efficacy of siRNA duplexes containing low phosphate thioester (PS) content is largely maintained or improved when exNA subunit bonds are included.

[0659] Table 14 – Anti-ApoE siRNA sequences used in Example 14 and Figure 21

[0660]

[0661] Another in vivo silencing activity assay was performed in which the Di-sRNA duplex targeted Htt mRNA. The chemical modification modifiers used are listed below. Wild-type male mice were treated with approximately 60 µg of siRNA for 2 months, and then Htt mRNA and protein levels were quantified in several brain regions (medial cortex, striatum, hippocampus, thalamus, and frontal cortex). Compared to siRNA duplexes lacking exNA nucleotide internucleotide bonds, siRNA duplexes with antisense strands containing one or two exNA nucleotide internucleotide bonds showed the same or higher levels of Htt mRNA. Figures 22A-22E ) and protein ( Figures 23A-23E The expression is silenced. Compared with phosphate thioester modifications, the internucleotide bonds of exNA nucleotides confer stronger nuclease resistance, allowing for the reduction of toxic phosphate thioester modifications without sacrificing nuclease resistance or silencing efficacy.

[0662] Chemical modification patterns used in Figures 22 and 23:

[0663] 1 – High PS:

[0664] Antisense chain (5' to 3'):

[0665] VP(mX)#(fX)#(mX)(fX)(fX)(fX)(mX)(fX)(mX)(fX)(mX)(fX)(mX)(fX)#(mX)#(fX)#(mX)#(mX)#(mX)#(fX)#(mX)

[0666] There is a semantic chain (5' to 3'):

[0667] (mX)#(mX)#(mX)(fX)(mX)(fX)(mX)(fX)(mX)(fX)(mX)(mX)(mX)(fX)#(mX)#(mX)

[0668] 2 – Low PS fm:

[0669] Antisense chain (5' to 3'):

[0670] VP(mX)#(fX)#(mX)(fX)(fX)(fX)(mX)(fX)(mX)(fX)(mX)(fX)(mX)(fX)(mX)(fX)(mX)(mX)(mX)#(fX)#(mX)

[0671] There is a semantic chain (5' to 3'):

[0672] (mX)#(mX)#(mX)(fX)(mX)(fX)(mX)(fX)(mX)(fX)(mX)(mX)(mX)(fX)#(mX)#(mX)

[0673] 3 – Low PS mf:

[0674] Antisense chain (5' to 3'):

[0675] VP(mX)#(fX)#(mX)(fX)(fX)(fX)(mX)(fX)(mX)(fX)(mX)(fX)(mX)(fX)(mX)(fX)(mX)(mX)(mX)#(mX)#(fX)

[0676] There is a semantic chain (5' to 3'):

[0677] (mX)#(mX)#(mX)(fX)(mX)(fX)(mX)(fX)(mX)(fX)(mX)(mX)(mX)(fX)#(mX)#(mX)

[0678] 4 – Low PS mf 2 exNA:

[0679] Antisense chain (5' to 3'):

[0680] VP(mX)#(fX)#(mX)(fX)(fX)(fX)(mX)(fX)(mX)(fX)(mX)(fX)(mX)(fX)(mX)(fX)(mX)(mX)(mX)#(ex-mX)#(ex-fX)

[0681] There is a semantic chain (5' to 3'):

[0682] (mX)#(mX)#(mX)(fX)(mX)(fX)(mX)(fX)(mX)(fX)(mX)(mX)(mX)(fX)#(mX)#(mX)

[0683] 5 – Low PS mf 1 exNA:

[0684] Antisense chain (5' to 3'):

[0685] VP(mX)#(fX)#(mX)(fX)(fX)(fX)(mX)(fX)(mX)(fX)(mX)(fX)(mX)(fX)(mX)(fX)(mX)(mX)(mX)#(mX)#(ex-fX)

[0686] There is a semantic chain (5' to 3'):

[0687] (mX)#(mX)#(mX)(fX)(mX)(fX)(mX)(fX)(mX)(fX)(mX)(mX)(mX)(fX)#(mX)#(mX)

[0688] For the five chemical modification modes mentioned above, "VP" corresponds to 5' vinylphosphonate; "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'-fluorine modification; "#" corresponds to phosphate thioester modification; "ex-mX" corresponds to any nucleotide (A, U, G, or C) with 2'-O-methyl modification and exNA nucleotide inter-bond; and "ex-fX" corresponds to any nucleotide (A, U, G, or C) with 2'-fluorine modification and exNA nucleotide inter-bond.

[0689] Incorporated by reference

[0690] All sources cited throughout this application (including references, patents, patent applications, and websites) are hereby expressly incorporated by reference, especially the references cited herein. Unless otherwise stated, this disclosure employs conventional techniques of immunology, molecular biology, and cell biology well known in the art.

[0691] This disclosure also incorporates, by reference, 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:

[0692] Atwell et al., J. Mol. Biol. 1997, 270: 26-35;

[0693] Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993);

[0694] Ausubel, FM et al. (eds.), Short Protocols in Molecular Biology (4th edition, 1999), John Wiley & Sons, NY. (ISBN 0-471-32938-X);

[0695] Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984);

[0696] Giege, R. and Ducruix, A. Barrett, Crystallization of Nucleic Acids andProteins, a Practical Approach, 2nd edition, pp. 20 1-16, Oxford University Press, New York, New York, (1999);

[0697] Goodson, Medical Applications of Controlled Release, Vol. 2, pp. 115-138 (1984);

[0698] Hammerling et al., Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, NY, 1981;

[0699] Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd edition, 1988);

[0700] Kabat et al., Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987) and (1991);

[0701] Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Interest, 5th ed., US Department of Health and Human Services, NIHPublication No. 91-3242;

[0702] Kontermann and Dubel (eds.), Antibody Engineering (2001), Springer-Verlag, New York, p. 790 (ISBN 3-540-41354-5).

[0703] Kriegler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990);

[0704] Lu and Weiner, eds., Cloning and Expression Vectors for Gene Function Analysis (2001), BioTechniques Press, Westborough, MA. Page 298 (ISBN 1-881299-21-X).

[0705] Medical Applications of Controlled Release, Langer and Wise (eds.), CRCPres., Boca Raton, Fla. (1974);

[0706] Old, RW & SB Primrose, Principles of Gene Manipulation: AnIntroduction To Genetic Engineering (3rd ed. 1985) Blackwell Scientific Publications, Boston. Studies in Microbiology; V.2: p. 409 (ISBN 0-632-01318-4).

[0707] Sambrook, J. et al., eds., Molecular Cloning: A Laboratory Manual (2nd ed., 1989), Cold Spring Harbor Laboratory Press, NY. pp. 1-3. (ISBN 0-87969-309-6).

[0708] Sustained and Controlled Release Drug Delivery Systems, edited by JR Robinson, Marcel Dekker, Inc., New York, 1978

[0709] Winnacker, EL From Genes To Clones: Introduction To Gene Technology (1987), VCH Publishers, NY (translated by Horst Ibelgaufts). Page 634 (ISBN 0-89573-614-4).

[0710] Equivalent solution

[0711] This disclosure may be implemented in other specific forms without departing from its spirit or essential characteristics. Therefore, the above embodiments should be considered illustrative in all respects and not limiting of the invention. The scope of this disclosure is therefore indicated by the appended claims rather than by the foregoing description, and thus all variations within the meaning and equivalence of the claims are encompassed within the claims.

[0712] This application also relates to the following implementation schemes:

[0713] 1. A modified oligonucleotide comprising a 5' end, a 3' end, and at least one modified subunit of formula I:

[0714]

[0715] (I);

[0716] in:

[0717] B represents the base pairing portion;

[0718] W is O or O(CH2). n , where n is from 1 to 10;

[0719] X can be selected from H, OH, OR, F, SH, SR, NR. 2 2 and C 1-6 The group consisting of alkoxy groups;

[0720] Y chooses freely O – OH, OR, OR 2 NH – NH2, NR 2 2. BH3, S – R 1 The group consisting of SH;

[0721] Z represents O or O(CH2). n , where n is from 1 to 10;

[0722] R 1 It is alkyl, allyl, or aryl; and

[0723] R 2 It can be alkyl, allyl, or aryl.

[0724] 2. The modified oligonucleotide as described in Embodiment 1, wherein Z is O(CH2). n n is 1, W is 0, and Y is 0. - .

[0725] 3. The modified oligonucleotide as described in Implementation Scheme 1, wherein Z is O and W is O(CH2). n n is 1, and Y is 0. – .

[0726] 4. The modified oligonucleotide as described in Embodiment 1, wherein Z is O(CH2). n n is 1, W is 0, and Y is 0. - .

[0727] 5. The modified oligonucleotide as described in Embodiment 1, wherein Z is O(CH2). n n is 1, and W is O(CH2). n And Y is O – .

[0728] 6. The modified oligonucleotide as described in Embodiment 1, wherein Z is O(CH2). n n is not 1, and W is O(CH2). n And Y is O – .

[0729] 7. The modified oligonucleotide as described in Embodiment 1, wherein the base pairing portion B is selected from the group consisting of adenine, guanine, cytosine, and uracil.

[0730] 8. A modified oligonucleotide comprising a 5' end, a 3' end, and at least one modified subunit of formula II:

[0731]

[0732] (II)

[0733] in:

[0734] B represents the base pairing portion;

[0735] X can be selected from H, OH, OR, F, SH, SR, NR. 2 2 and C 1-6 The group consisting of alkoxy groups;

[0736] Y chooses freely O – OH, OR, OR 2 NH – NH2, NR 2 2. BH3, S – R 1 The group consisting of SH;

[0737] R 1 It is alkyl, allyl, or aryl; and

[0738] R 2 It can be alkyl, allyl, or aryl.

[0739] 9. The modified oligonucleotide as described in Embodiment 7, wherein Y is O.

[0740] 10. The modified oligonucleotide as described in embodiment 7, wherein the base pairing portion B is selected from the group consisting of adenine, guanine, cytosine, and uracil.

[0741] 11. A modified oligonucleotide comprising a 5' end, a 3' end, and at least one modified subunit of formula III:

[0742]

[0743] in:

[0744] B represents the base pairing portion;

[0745] R is alkyl, allyl, or aryl.

[0746] 12. The modified oligonucleotide as described in embodiment 10, wherein the base pairing portion B is selected from the group consisting of adenine, guanine, cytosine, and uracil.

[0747] 13. A modified oligonucleotide comprising a 5' end, a 3' end, and at least one modified subunit of formula IV:

[0748]

[0749] in:

[0750] B represents the base pairing portion;

[0751] R is alkyl, allyl, or aryl.

[0752] 14. The modified oligonucleotide as described in embodiment 12, wherein the base pairing portion B is selected from the group consisting of adenine, guanine, cytosine, and uracil.

[0753] 15. A modified oligonucleotide comprising a 5' end, a 3' end, and at least one modified subunit of formula V:

[0754]

[0755] in:

[0756] B represents the base pairing portion;

[0757] R is alkyl, allyl, or aryl.

[0758] 16. The modified oligonucleotide as described in embodiment 14, wherein the base pairing portion B is selected from the group consisting of adenine, guanine, cytosine, and uracil.

[0759] 17. A modified oligonucleotide comprising a 5' end, a 3' end, and at least one modified subunit of formula VI:

[0760]

[0761] in:

[0762] B represents the base pairing portion;

[0763] R is alkyl, allyl, or aryl.

[0764] 18. The modified oligonucleotide as described in embodiment 16, wherein the base pairing portion B is selected from the group consisting of adenine, guanine, cytosine, and uracil.

[0765] 19. A phosphorusamide derivative of formula (VII):

[0766]

[0767] (VII)

[0768] in:

[0769] B represents the base pairing portion;

[0770] X can be selected from H, OH, OR, F, SH, SR, NR. 2 2. MOE, alkyl, allyl, aryl and C 1-6 The group consisting of alkoxy groups;

[0771] Z is either O or OCH2;

[0772] R stands for OMe or OCE (cyanoethyl);

[0773] R 1 It is alkyl, allyl, or aryl; and

[0774] R 2 It can be alkyl, allyl, or aryl.

[0775] 20. The phosphoramidide derivative of formula (VII), wherein the base pairing portion B is selected from the group consisting of adenine, guanine, cytosine and uracil.

[0776] 21. A phosphorusamide derivative of formula (VIII):

[0777]

[0778] (VIII)

[0779] in:

[0780] B represents the base pairing portion;

[0781] X can be selected from H, OH, OR, F, SH, SR, NR. 2 2. MOE, alkyl, allyl, aryl and C 1-6 The group consisting of alkoxy groups;

[0782] R 1 It is alkyl, allyl, or aryl; and

[0783] R 2 It can be alkyl, allyl, or aryl.

[0784] 22. The phosphoramidide derivative of formula (VIII), wherein the base pairing portion B is selected from the group consisting of adenine, guanine, cytosine and uracil.

[0785] 23. A phosphorusamide derivative of formula (IX):

[0786]

[0787] (IX)

[0788] in:

[0789] B represents the base pairing portion;

[0790] X can be selected from H, OH, OR, F, SH, SR, NR. 2 2. MOE, alkyl, allyl, aryl and C 1-6 The group consisting of alkoxy groups;

[0791] R 1 It is alkyl, allyl, or aryl; and

[0792] R 2 It can be alkyl, allyl, or aryl.

[0793] 24. The phosphoramidide derivative of formula (IX), wherein the base pairing portion B is selected from the group consisting of adenine, guanine, cytosine and uracil.

[0794] 25. A method for synthesizing a modified oligonucleotide comprising a 5' end, a 3' end, and at least one modified subunit linkage, comprising:

[0795] (a) Providing a nucleoside having a 5'-protecting group attached to a solid support;

[0796] (b) Remove the protecting group;

[0797] (c) Combining the deprotected nucleoside with the phosphoramidide derivative of formula (VII) to form a phosphite triester;

[0798]

[0799] (VII)

[0800] (d) The capped triphosphite;

[0801] (e) Oxidation of the triphosphite;

[0802] (f) Repeat steps (b) to (e) using additional phosphorus amide; and

[0803] (g) Cutting from a solid support.

[0804] 26. A method for processing phosphorusamide derivatives of formula (VII)

[0805]

[0806] (VII)

[0807] A method of coupling to the 5' end of a nucleoside or oligonucleotide, comprising adding the phosphoramidite derivative of formula (VII) to the nucleoside or oligonucleotide in an organic solvent containing an aromatic heterocyclic acid.

[0808] 27. A method for synthesizing exNA phosphorous amide:

[0809] (a) Provide a nucleoside with a 3'-protecting group;

[0810] (b) Oxidating the 5'-hydroxy group of the nucleoside to a 5'-aldehyde group;

[0811] (c) Converting the 5'-aldehyde group of the nucleoside to a 5'-vinyl group by Vittigenelation;

[0812] (d) Hydroboration / oxidation of the 5'-vinyl group to produce a 6'-hydroxy group;

[0813] (e) Protect the 6'-hydroxy group with a DMTr group;

[0814] (f) Remove the 3'-protecting group of the nucleoside;

[0815] (g) Phosphorylation of the 3'-hydroxy group to produce 3'-phosphoramide.

Claims

1. A modified oligonucleotide comprising a 5' end, a 3' end, and at least one modified subunit of formula I: (I)； in: B represents the base pairing portion; W is O(CH2) n , where n is from 1 to 10 and where O(CH2) n The oxygen atom in formula (I) is bonded to the phosphorus atom in formula (I); X can be selected from H, OH, OR*, F, SH, SR*, NR. 2 2 and C 1-6 The group consisting of alkoxy groups; Y chooses freely O – OH, OR 2 NH – NH2, NR 2 2. BH3, S – R 1 The group consisting of SH; and Z is O; R* represents C with or without substitution. 1-6 Alkyl, alkenyl, ynyl, or aryl; R 1 It is alkyl, allyl, or aryl; and R 2 It can be alkyl, allyl, or aryl.

2. The modified oligonucleotide of claim 1, wherein n is 1 and Y is S. – Or O – .

3. The modified oligonucleotide of claim 1, wherein n is 1 and Y is 0. – .

4. The modified oligonucleotide of claim 1, wherein the base pairing portion B is selected from the group consisting of adenine, guanine, cytosine, and uracil.

5. A modified oligonucleotide comprising a 5' end, a 3' end, and at least one modified subunit of formula II: (II) in: B represents the base pairing portion; X can be selected from H, OH, OR*, F, SH, SR*, NR. 2 2 and C 1-6 The group consisting of alkoxy groups; Y chooses freely O – OH, OR 2 NH – NH2, NR 2 2. BH3, S – R 1 The group consisting of SH; R* represents a substituted or unsubstituted C1-6 alkyl, alkenyl, alkynyl, or aryl group; R 1 It is alkyl, allyl, or aryl; and R 2 It can be alkyl, allyl, or aryl.

6. The modified oligonucleotide of claim 5, wherein Y is S – Or O – .

7. The modified oligonucleotide of claim 5, wherein Y is O – .

8. The modified oligonucleotide of claim 5, wherein the base pairing portion B is selected from the group consisting of adenine, guanine, cytosine, and uracil.

9. The modified oligonucleotide of claim 5, wherein X is H, OH, OR* or F, and wherein R* is a C1 alkyl group.

10. A method for synthesizing the modified oligonucleotide as described in claim 1, the method comprising: (a) Provide a nucleoside with a 3'-protecting group; (b) Oxidating the 5'-hydroxy group of the nucleoside to a 5'-aldehyde group; (c) Converting the 5'-aldehyde group of the nucleoside to a 5'-vinyl group by Vittigenelation; (d) Hydroboration / oxidation of the 5'-vinyl group to produce a 6'-hydroxy group; (e) Protect the 6'-hydroxy group with a DMTr group; (f) Remove the 3'-protecting group of the nucleoside; (g) Phosphorylation of the 3'-hydroxy group to produce 3'-phosphoramide; Thus, the modified oligonucleotide is synthesized.

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