Double-stranded nucleic acid inhibitor molecules containing triloops

Abstract

To provide double-stranded nucleic acid inhibitor molecules, to provide methods and compositions for reducing target gene expression, and to provide methods and compositions for treating a disease of interest.SOLUTION: Provided is a double-stranded nucleic acid inhibitor molecule, comprising: a sense strand comprising 20 to 65 nucleotides and having a specific region; an antisense strand comprising 15 to 40 nucleotides, the sense strand and antisense strand being separate strands; and a duplex formed by the specific region of the sense strand and the antisense strand, the specific region of the sense strand comprising a triloop and forming a duplex.SELECTED DRAWING: None

Description

[Background technology]

[0001] This application was filed on December 12, 2018, the entire contents of which are incorporated herein by reference. This application claims the benefit of, and is based on the filing date of, U.S. Provisional Patent Application No. 62 / 778,759. .

[0002] Oligonucleotides are polymers of nucleotides (RNA, DNA and their analogs). Nucleic acid inhibitor molecules are oligonucleotides that regulate intracellular RNA levels. It is a compound that has demonstrated initial promise in the treatment of cancer, viral infections, and genetic disorders. Nucleic acid inhibitor molecules act through a diverse set of mechanisms, including RNA interference (RNAi). RNA expression can be regulated by

[0003] RNAi is a conserved pathway found in many eukaryotic organisms, where bispecific A double stranded RNA molecule (dsRNA) is inserted into a target gene that has a sequence complementary to the dsRNA. In the typical RNAi pathway, long dsRNA molecules are cleaved by the Dicer enzyme, which inhibits expression. It is cleaved into short RNA duplexes called small interfering RNAs ("siRNAs"). siRNAs interact with Dicer, trans-activation response RNA-binding protein (TRBP), and and Argonaute 2 ("Ago2") to form the RNA-induced silencing complex (" It has been shown that they form a complex sometimes called RISC. Ago2 acts as a signaling pathway to direct the sequence specificity of the RISC complex toward cleavage of target mRNAs. The antisense strand of RNA (also called the guide strand) is used to cleave the target mRNA. It is an endonuclease.

[0004] A variety of double-stranded RNAi inhibitor molecular structures have been developed over the years. For example, initial research into RNAi inhibitor molecules was focused on mimicking natural siRNAs and their In this case, each strand is divided into smaller strands of 1 to 5 nucleotides in size, 19 to 25 nucleotides in length. The present study focused on double-stranded nucleic acid molecules with at least one 3'-overhang (e.g., (See US Patent No. 8,372,968). Subsequently, the RNAi inhibitor molecule is A long double-stranded R that is processed in vivo by the Dicer enzyme to become active NAi inhibitor molecules have been developed (see, e.g., U.S. Pat. No. 8,883,996). Later studies showed that one of the chains contains a thermodynamically stabilizing tetraloop structure. At least one terminus of at least one strand is connected to the double-stranded target region of the molecule, including the structure Extended double-stranded nucleic acid inhibitor molecules have been developed (e.g., and U.S. Pat. No. 8,513,207, each of which is incorporated herein by reference in its entirety. , U.S. Patent No. 8,927,705, WO2010 / 033225, and WO2016 / (See 100401).

[0005] In certain cases, under specific conditions, such as those experienced after in vivo administration, Chemically modified nucleotides are introduced into nucleic acid inhibitor molecules to introduce properties that may be desired in Such chemically modified nucleotides include, for example, those used in the construction of oligonucleotides. against nucleases or other enzymes that degrade or interfere with the structure or activity of to stabilize the oligonucleotide, to increase cellular uptake of the oligonucleotide, or These include those designed to improve the pharmacokinetic properties of oligonucleotides.

[0006] However, new double-stranded nucleic acid inhibitor molecules are being developed and / or Chemically modified nucleotides are introduced to impart desired properties to nucleic acid inhibitor molecules. The desire for structural and / or chemically modified nucleotides to enhance the activity of nucleic acid inhibitor molecules has been met. minimize any negative impacts that may be caused by the gene targeting the competing desire to minimize any reduction in efficacy or duration of drug downtime and It has to be balanced. Summary of the Invention

[0007] It has a sense strand with a stem-loop structure and another antisense strand, The double-stranded nucleic acid inhibitor molecule in which the loop portion of the loop structure contains a tri-loop is referred to herein as a As shown in the examples, double-stranded nucleic acid inhibitors containing triloops are stable. This tri-loop structure reduces target mRNA expression in vivo in a dose-dependent manner. The structure is removed from its naturally occurring context to form a chemically synthesized double-stranded nucleic acid inhibitor molecule. It was surprising that when incorporated into G, it can maintain a thermodynamically stable configuration. The conjugate of a ligand such as alNAc with the nucleotide in the triloop The thermodynamically stable configuration of the loop must not be disrupted, and the two GalNAc and triloop must be The conjugate with tetraloop is a tetraloop conjugated with three GalNAcs. Reduced potency in hepatocytes compared to traloop-containing double-stranded nucleic acid inhibitor molecules It is also significant that the results do not necessarily result in improved efficacy, but in certain specific cases have actually been found to result in improved efficacy. It was outside.

[0008] Additionally, the triloop-containing double-stranded nucleic acid inhibitor molecule contains a bicyclic nucleotide in the stem. It can be incorporated into the stem portion of the loop structure. International Publication WO2019 / 200124 As already demonstrated in m -Incorporation of ascending nucleotides into the stem duplex This is evidenced, in part, by enhanced duration of target mRNA knockdown in vivo. As shown, increased stability can be imparted to tetraloop-containing double-stranded nucleic acid inhibitor molecules. do.

[0009] Furthermore, the use of a triloop instead of a tetraloop and the use of a stem-loop structure The introduction of a bicyclic nucleotide into the stem portion of a double-stranded nucleic acid inhibitor molecule containing the nucleotide This allows for the use of shorter sense strands without reducing efficacy. The use of ethylene glycol fluoride provides manufacturing process advantages, reducing both time and costs. It also offers advantages in administration, as its low molecular weight allows for molecular-based It is possible to administer more triloop-containing double-stranded nucleic acid inhibitor molecules in one dose. This is because.

[0010] The double-stranded nucleic acid inhibitor molecule has a first region (R1) of the sense strand (S) and an antisense strand (A). The first duplex (D1) between the sense strand (AS) and the second region (R2) of the sense strand and a second duplex (D2) between the subregion (S1) and the second subregion (S2), In this case, S1 and S2 are connected by a triloop (triL). In addition, the stem portion of the stem-loop structure may, in certain embodiments, be at least At least one T m - elevated nucleotides, e.g., 4 to 12 T m-Elevated nucleotides, e.g. If so, 2 to 6 T m -Elevated nucleotide base pairs or 1 to 6 unpaired Ts m -Rising Nucleochi The stem-loop structure may be located at the 5' or 3' end of the sense strand. obtain.

[0011] In certain embodiments, the double-stranded nucleic acid inhibitor molecule comprises: comprising 20 to 65 nucleotides and a first region (R1) and a second region (R2); the sense strand; an antisense strand containing 15 to 40 nucleotides, the antisense strand, where the sense strand is a separate strand; a first region of the sense strand and an antisense strand, the first region being 15 to 40 base pairs long; the first duplex (D1) having a length of Includes; In this case, the second region (R2) of the sense strand is composed of the first sub-region (S1) and the second sub-region (S2). a tri-loop (triL) connecting the first and second regions; The first and second regions form a second duplex (D2).

[0012] In certain embodiments, the tri-loop has a nucleotide sequence of GAA.

[0013] In certain embodiments, the sense strand has between 22 and 65 nucleotides. In certain embodiments, the sense strand has 25 to 39 nucleotides. In an embodiment, the sense strand has 27 to 35 nucleotides.

[0014] In certain embodiments, the antisense strand has 20 to 24 nucleotides. In certain embodiments, the antisense strand has 20 to 22 nucleotides. .

[0015] In certain embodiments, the nucleotide immediately adjacent to the 5' end of the tri-loop is a C and the nucleotide immediately adjacent to the 3' end of the tri-loop is a G .

[0016] In certain embodiments, the antisense strand has 1 to 4 nucleotides at its 3' end. In certain embodiments, the single-stranded overhang The tag is 2 nucleotides in length.

[0017] In certain embodiments, the first duplex (D1) has a length of 18 to 30 base pairs. In a specific embodiment, the first duplex (D1) has a length of 18 to 24 base pairs. In a specific embodiment, the first duplex (D1) has a length of 20 to 22 base pairs. It has a length.

[0018] In certain embodiments, the second duplex (D2) has a length of 2 to 6 base pairs. In certain embodiments, the second duplex is m -Including elevated nucleotides and in certain embodiments, the second duplex does not have 4 to 10 T m -Rising Nuku It contains a nucleic acid sequence and is 2 to 5 base pairs in length.

[0019] In certain embodiments, the sense strand is 25 to 39 nucleotides in length, The antisense strand is 20 to 24 nucleotides in length, and the first duplex is 18 to 24 nucleotides in length. The first double strand has a length of 4 nucleotides, and the second double strand has a length of 2 to 6 base pairs. In certain embodiments, the sense strand is 27 to 35 nucleotides in length, and the The antisense strand is 20 to 22 nucleotides in length, and the first duplex is 18 to 22 nucleotides in length. The second double strand has a length of 2 to 3 base pairs.

[0020] In certain embodiments, the second duplex (D2) has a length of 2 base pairs. In certain embodiments, the second duplex (D2) has a length of 3 base pairs.

[0021] In certain embodiments, the first region (R1) of the sense strand is 20 nucleotides long. and the second region (R2) of the sense strand is 7 to 9 nucleotides in length; At this time, a first region formed by the first region of the sense strand and the antisense strand is The first strand (D1) has a length of 20 base pairs; The first subregion (S1) and the second subregion (S2) of the second region (R2) of the sense strand The second duplex (D2) formed by the above has a length of 2 or 3 base pairs and The duplex of m - containing elevated nucleotides; The antisense strand is 22 nucleotides in length and has two nucleotides at its 3' end. having a single-stranded overhang of ribonucleotide; and The tri-loop has a nucleotide sequence of GAA. D2 is 7 nucleotides in length and D2 is 2 base pairs in length. In an embodiment, R2 is 9 nucleotides in length and D2 has a length of 3 base pairs. .

[0022] In certain embodiments, each nucleotide in the second duplex (D2) is selected from the group consisting of T m-above In certain embodiments, the triloop-containing double-stranded nucleic acid inhibitor is a nucleotide. The bitter molecule does not contain any T other than the second duplex (D2). m -Contains elevated nucleotides do not.

[0023] In certain embodiments, the T m -The ascending nucleotides are bicyclic nucleotides, tricyclic nucleotides Formula nucleotides, G-clamps and their analogs, hexitol nucleotides, and modified modified nucleotides, wherein the modified nucleotides are selected from the group consisting of: The 2'-carbon is not modified with 2'-F or 2'-OMe. In some embodiments, the modified nucleotides are 5-bromouracil, 5-iodouracil, 5- Propynyl-modified pyrimidines, 2-aminoadenine, 2-thiouridine, 5Me-thiouridine Lysine or pseudouridine.

[0024] The triloop-containing double-stranded nucleic acid inhibitor molecule comprises at least one bicyclic nucleotide. In certain embodiments, at least one bicyclic nucleotide is represented by Formula I, I In certain embodiments, the at least one of the above has the structure I, III, IV, Va, or Vb. At least one bicyclic nucleotide is of formula Ia, Ib, Ic, Id, Ie, or If In certain embodiments, the at least one The bicyclic nucleotide may be one or more of the formula IIa, IIb, IIc, or IId. In certain embodiments, the at least one bicyclic nucleotide has a structure has the structure of Formula IIIa and / or IIIb. At least one bicyclic nucleotide has the structure of Formula IVa and / or IVb.

[0025] In certain embodiments, the at least one bicyclic nucleotide is one of the following: One or more: TIFF0007748500000001.tif51150 TIFF0007748500000002.tif210150 TIFF0007748500000003.tif210150 wherein B is a nucleobase, R2 is H or CH3, and W a and W b teeth each independently an H, an OH, a hydroxyl protecting group, a phosphorus moiety, or a bicyclic nucleotide to another nucleotide or to an internucleotide linkage that binds the oligonucleotide where W a or W b At least one of the bicyclic nucleotides is an oligonucleotide. [The internucleotide linking group is an internucleotide linking group attached to a nucleotide.]

[0026] In certain embodiments, the at least one bicyclic nucleotide is: TIFF0007748500000004.tif96170 wherein B, Wa, and Wb are as defined above, and R2 is CH3.

[0027] In certain embodiments, the at least one bicyclic nucleotide is a furanosyl. a first ring formed by linking the 2'-carbon and 4'-carbon of the furanosyl to form a second ring; This includes cross-linking that forms

[0028] In certain embodiments, a bridge connecting the 2'-carbon and the 4'-carbon of the furanosyl is teeth: a) 4'-CH2-ON(R)-2' and 4'-CH2-N(R)-O-2' (where , R, H, C1~C 12 Alkyl, or, for example, 4'-CH2-NH-O-2'(BNA NC Also known as 4'-CH2-N(CH3)-O-2' (BNA NC [NMe] and protecting groups including those known in the art); b) 4'-CH2-2';4'-(CH2)2-2';4'-(CH2)3-2';4'- (CH2)-O-2' (also known as LNA); 4'-(CH2)-S-2'; 4'-(C H2)2-O-2' (also known as ENA); 4'-CH(CH3)-O-2' (cEt and and 4'-CH(CH2OCH3)-O-2' (also known as cMOE), and its analogues; c) 4'-C(CH3)(CH3)-O-2' and its analogs; d) 4'-CH2-N(OCH3)-2' and its analogs; e) 4'-CH2-ON(CH3)-2' and its analogs; f) 4'-CH2-C(H)(CH3)-2' and analogs thereof; and g) 4'-CH2-C(=CH2)-2' and its analogues is selected from the group consisting of:

[0029] In certain embodiments, the tri-loop-containing double-stranded nucleic acid inhibitor molecule comprises a second In addition to the double strand, any T m -It also does not contain elevated nucleotides.

[0030] In certain embodiments, the tri-loop is linked to at least one ligand conjugate. In certain embodiments, the triloop comprises two ligands. In certain embodiments, the tri-loop comprises a tri-conjugated nucleotide. , and three ligand-conjugated nucleotides. The ligand is GalNAc. In certain embodiments, the GalNAc is The moiety is conjugated at the 2'-position to the nucleotide.

[0031] In certain embodiments, the tri-loop-containing double-stranded nucleic acid inhibitor molecule further comprises: The sense strand and / or the antisense strand contain a 5'-phosphate mimetic at the 5' end.

[0032] In certain embodiments, the triloop-containing double-stranded nucleic acid inhibitor molecule is prepared by the addition of a lipid nanotube. In certain embodiments, the lipid nanoparticles are formulated with a core lipid and and an envelope lipid, wherein the core lipid comprises a first cationic lipid and a first The envelope lipid comprises a PEGylated lipid, and the envelope lipid comprises a second cationic lipid, a neutral lipid, a sterol, In certain embodiments, the first cationic lipid comprises a PEGylated lipid, and a second PEGylated lipid. the first PEGylated lipid is DSG-mPEG, and the second PEGylated lipid is DL-048. The second cationic lipid is DL-103, the neutral lipid is DSPC, and the second cationic lipid is DL-103. The terol is cholesterol and the second PEGylated lipid is DSPE-mPEG is.

[0033] Another aspect is a therapeutically effective amount of a triloop-containing double-stranded nucleic acid interferon as described herein. Inhibitor molecules and pharmaceutically acceptable Possible excipients and a pharmaceutical composition comprising:

[0034] Another aspect is a method for reducing expression of a target gene in a subject, comprising: administering the triloop-containing double-stranded nucleic acid inhibitor molecule or pharmaceutical composition to a subject in need thereof. in an amount sufficient to reduce expression of said target gene. In certain embodiments, the administering step is intravenous, intramuscular, or subcutaneous. In certain embodiments, the subject is a human.

[0035] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate certain embodiments. The present invention provides a method for producing a pharmaceutical composition comprising the steps of: It helps to explain certain principles that [Brief explanation of the drawings]

[0036] [Figure 1A] A diagram of an exemplary double-stranded nucleic acid inhibitor molecule is shown, comprising an antisense strand ("AS") and a sense strand ("S"), wherein the sense strand comprises a stem-loop structure and the loop is a tri-loop. [Figure 1B] The same illustrative diagram as in Figure 1A is shown. In Figure 1B, the sense strand is further divided into a first region (R1) that forms a duplex with the antisense strand (AS) and a second region (R2) that contains a tri-loop (triL) that connects the first subregion (S1) to a second subregion (S2), where S1 and S2 are sufficiently complementary to each other to form a duplex referred to herein as the "stem" or "stem duplex." [Figure 1C]The same exemplary diagram as in Figures 1A and 1B is shown. Figure 1C illustrates the first duplex (D1) and second duplex (D2) in the nucleic acid inhibitor molecule. The first duplex (D1) is formed between the first region (R1) of the sense strand and the antisense strand (AS). The second duplex (D2), or "stem," is formed between the first subregion (S1) and the second subregion (S2) of the second region (R2) of the sense strand. [Figure 1D] An exemplary double-stranded nucleic acid inhibitor molecule is depicted in which the second duplex (D2) is shorter than the second duplex depicted in Figure 1C. [Figure 2A] Illustrated is the structure of an exemplary double-stranded nucleic acid inhibitor molecule ("Construct 1") targeting a gene sequence of interest, as discussed in Example 1. The sense strand of Construct 1 comprises a six-base pair stem duplex and a tetraloop. Three of the four nucleotides of the tetraloop are conjugated to a single GalNAc molecule. [Figure 2B] Illustrated is the structure of an exemplary double-stranded nucleic acid inhibitor molecule ("Construct 2") targeting a gene sequence of interest as discussed in Example 1. The sense strand of Construct 2 comprises a six-base pair stem duplex and a tetraloop. The structure of Construct 2 is identical to that of Construct 1, except that only two of the four nucleotides of the tetraloop are conjugated to a single GalNAc molecule, rather than three of four as in Construct 1. [Figure 2C] The structure of an exemplary double-stranded nucleic acid inhibitor molecule ("Construct 3") targeting a gene sequence of interest, as discussed in Example 1, is shown. The sense strand of Construct 3 comprises a six-base pair stem duplex and a triloop. Two of the three nucleotides of the triloop are conjugated to a single GalNAc molecule. The structure of Construct 3 is identical to that of Construct 2, except that the loop portion of the stem-loop is a triloop instead of a tetraloop. [Figure 2D]The structure of an exemplary double-stranded nucleic acid inhibitor molecule ("Construct 4") targeting a gene sequence of interest, as discussed in Example 1, is depicted. The sense strand of Construct 4 comprises a three-base-pair stem duplex in which each of the nucleotides in the stem duplex is a bicyclic nucleotide, and a tri-loop. Two of the three nucleotides in the tri-loop are conjugated to a single GalNAc molecule. [Figure 2E] The structure of an exemplary double-stranded nucleic acid inhibitor molecule ("Construct 5") targeting a gene sequence of interest, as discussed in Example 1, is depicted. The sense strand of Construct 5 comprises a three-base-pair stem duplex in which each of the nucleotides in the stem duplex is LNA, and a tri-loop. Two of the three nucleotides in the tri-loop are conjugated to a single GalNAc molecule. [Figure 3A] 2A is a graph showing the percent of target gene mRNA remaining 4 days after administration of various dosages of Construct 1 (see FIG. 2A) to CD-1 mice as described in Example 1. The effective dose (ED50) was calculated to be 0.5726 mg / kg, as shown in FIG. 3A. [Figure 3B] 2B is a graph showing the percent of target gene mRNA remaining 4 days after administration of various dosages of Construct 2 (see FIG. 2B) to CD-1 mice as described in Example 1. The ED50 was calculated to be 0.3604 mg / kg, as shown in FIG. 3B. [Figure 3C] 2C is a graph showing the percent of target gene mRNA remaining 4 days after administration of various dosages of Construct 3 (see FIG. 2C) to CD-1 mice as described in Example 1. The ED50 was calculated to be 0.3144 mg / kg, as shown in FIG. 3C. [Figure 3D]2D is a graph showing the percent of target gene mRNA remaining 4 days after administration of various dosages of Construct 4 (see FIG. 2D) to CD-1 mice as described in Example 1. The ED50 was calculated to be 0.3012 mg / kg, as shown in FIG. 3D. [Figure 3E] 3E is a graph showing the percent of target gene mRNA remaining 4 days after administration of various dosages of Construct 5 (see FIG. 2E) to CD-1 mice as described in Example 1. The ED50 was calculated to be 0.2325 mg / kg, as shown in FIG. [Figure 4] 2A-E are graphs showing an overlay of the percent of target gene mRNA remaining 4 days after administration of various dosages of Constructs 1-5 (see FIGS. 2A-E) to CD-1 mice as described in Example 1. [Figure 5] 2A-E are bar graphs showing the percent of target gene mRNA remaining 4 days after administration of various dosages of Constructs 1-5 (see FIGS. 2A-E) to CD-1 mice as described in Example 1. [Figure 6A] The structure of an exemplary double-stranded nucleic acid inhibitor molecule ("Construct 6") targeting a gene sequence of interest, as discussed in Example 2, is depicted. The sense strand of Construct 6 comprises a six-base pair stem duplex and a tetraloop, two of the four nucleotides of which are conjugated to a single GalNAc molecule. [Figure 6B] Illustrated is the structure of an exemplary double-stranded nucleic acid inhibitor molecule ("Construct 7") targeting a gene sequence of interest as discussed in Example 2. The sense strand of Construct 7 contains a six-base pair stem duplex and a triloop. The structure of Construct 7 is identical to that of Construct 6, except that Construct 7 contains a triloop instead of a tetraloop. [Figure 6C]Illustrated is the structure of an exemplary double-stranded nucleic acid inhibitor molecule ("Construct 8") that targets a gene sequence of interest, as discussed in Example 2. The sense strand of Construct 8 comprises a three-base pair stem duplex in which each nucleotide in the stem portion of the stem-loop structure is a BNA, and a tetraloop. [Figure 6D] The structure of an exemplary double-stranded nucleic acid inhibitor molecule ("Construct 9") targeting a gene sequence of interest, as discussed in Example 2, is shown. The sense strand of Construct 9 contains a three-base pair stem duplex in which each nucleotide in the stem portion of the stem-loop structure is a BNA, and a triloop. The structure of Construct 9 is identical to that of Construct 8, except that Construct 9 contains a triloop instead of a tetraloop. [Figure 6E] Illustrated is the structure of an exemplary double-stranded nucleic acid inhibitor molecule ("Construct 10") that targets a gene sequence of interest, as discussed in Example 2. The sense strand of Construct 10 comprises a two-base-pair stem duplex in which each nucleotide in the stem portion of the stem-loop structure is a BNA, and a tetraloop. [Figure 6F] The structure of an exemplary double-stranded nucleic acid inhibitor molecule ("Construct 11") targeting a gene sequence of interest, as discussed in Example 2, is shown. The sense strand of Construct 11 contains a two-base pair stem duplex in which each nucleotide in the stem portion of the stem-loop structure is a BNA, and a triloop. The structure of Construct 11 is identical to that of Construct 10, except that Construct 11 contains a triloop instead of a tetraloop. [Figure 6G] Illustrated is the structure of an exemplary double-stranded nucleic acid inhibitor molecule ("Construct 12") that targets a gene sequence of interest, as discussed in Example 2. The sense strand of Construct 12 comprises a one-base-pair stem duplex in which both nucleotides in the stem portion of the stem-loop structure are BNAs, and a tetraloop. [Figure 6H]The structure of an exemplary double-stranded nucleic acid inhibitor molecule ("Construct 13") targeting a gene sequence of interest, as discussed in Example 2, is shown. The sense strand of Construct 13 contains a one-base pair stem duplex in which both nucleotides in the stem portion of the stem-loop structure are BNAs, and a triloop. The structure of Construct 13 is identical to that of Construct 12, except that Construct 13 contains a triloop instead of a tetraloop. [Figure 7] Shown are the percent of target gene mRNA remaining 4 days after administration of Construct 1 (see FIG. 2A) and Constructs 6-13 (see FIGS. 6A-H) to CD-1 mice as described in Example 2. The inclusion of a triloop in Constructs 7, 9, and 11 did not significantly reduce, and in certain cases actually improved, the potency of gene knockdown when compared to tetraloop Constructs 1, 6, 8, and 10, respectively. In contrast to Construct 12, which contains a tetraloop and a single base pair of bicyclic nucleotides in the stem duplex, which showed a strong reduction in target mRNA expression, Construct 13, which contains a triloop and a single base pair of bicyclic nucleotides in the stem duplex, did not reduce target mRNA expression. [Figure 8]

[0023] Figure 1 shows one non-limiting embodiment of a lipid nanoparticle (LNP) that can be used to formulate the double-stranded nucleic acid inhibitor molecule. The LNP comprises the following core lipids: DL-048 (cationic lipid) and DSG-mPEG (PEGylated lipid), and the following envelope lipids: DL-103 (cationic lipid), DSPC, cholesterol, and DSPE-mPEG (PEGylated lipid). DETAILED DESCRIPTION OF THE INVENTION

[0037] definition In order that this disclosure may be more readily understood, certain terms are first defined below. Additional definitions of terms and other terms may be found throughout the specification. If the definition of a term conflicts with a definition in an application or patent incorporated by reference, Please use the definitions set forth in the application to understand the meaning of the terms.

[0038] As used in this specification and the appended claims, the singular forms "a," "an," and "an" are used interchangeably. "The" includes plural references unless the context clearly indicates otherwise. For example, reference to "a method" refers to any method described herein and / or One or more methods of the kind that will be apparent to those skilled in the art upon reading this disclosure and the like, and and / or steps.

[0039] Administer: As used herein, "administering" a composition to a subject means administering that composition to a subject. Administering means giving, applying or contacting a composition to said subject. Many routes including, for example, topical, oral, subcutaneous, intramuscular, intraperitoneal, intravenous, intrathecal, and intradermal This can be achieved by either

[0040] Acyl: As used herein, the term "acyl" refers to alkylcarbonyl, It refers to cycloalkylcarbonyl and arylcarbonyl moieties.

[0041] Alkoxy: As used herein, the term "alkoxy" refers to a group substituted through an oxygen atom. refers to an alkyl group attached to a molecular moiety via a hydroxyl group.

[0042] Alkenyl: As used herein, the term “alkenyl” refers to at least one Straight or branched chains with two carbon-carbon double bonds and ranging from about 2 to about 20 carbon atoms "Substituted alkenyl" refers to a hydrocarbyl group that further has one or more substituents. As used herein, "lower alkenyl" refers to an alkenyl group having 2 to about 6 alkyl groups. refers to an alkenyl moiety having carbon atoms.

[0043] Alkyl: As used herein, the term “alkyl” refers to an alkyl group having 1 to about 20 carbon atoms. refers to a straight or branched chain hydrocarbyl group having 1 to 2 carbon atoms. Whenever possible, a numerical range is used, e.g., "C1-C6 alkyl" means that the alkyl group has one carbon atom. It may contain up to six carbon atoms, including two carbon atoms, three carbon atoms, etc. However, when no numerical range of carbon atoms is specified, the term "alkyl" is used. For example, the term "alkyl" includes C1-C 10 A subrange of (e.g., C1~C 6). "Substituted alkyl" refers to an alkyl moiety that has a substituent. When used, "lower alkyl" refers to an alkyl moiety having 1 to about 6 carbon atoms. vinegar.

[0044] Alkynyl: As used herein, "alkynyl" refers to an alkyl group having at least one carbon atom. A straight or branched chain hydrocarbon having a triple bond and ranging from about 2 to about 20 carbon atoms. "Substituted alkynyl" refers to an alkynyl group further bearing one or more substituents. As used herein, "lower alkynyl" refers to a group of about 2 to about 6 carbon atoms. It refers to an alkynyl moiety having an alkyl group.

[0045] Antisense strand: A double-stranded nucleic acid inhibitor molecule consists of two oligonucleotide strands, i.e. The antisense strand or a region thereof is a region of the target nucleic acid. In addition, the duplex is partially, substantially, or completely complementary to the corresponding region of The antisense strand of a nucleic acid inhibitor molecule or region thereof is a double-stranded nucleic acid inhibitor. Partially, substantially, or completely complementary to the sense strand of the molecule or a region thereof. In certain embodiments, the antisense strand contains no nucleotides that are non-complementary to the target nucleic acid sequence. The non-complementary nucleotides may be on either side of the complementary sequence or In certain embodiments, the antisense If the strand or region thereof is partially or substantially complementary to the sense strand or region thereof, In this case, the non-complementary nucleotides may be located between one or more regions of complementarity (e.g., The antisense strand of the double-stranded nucleic acid inhibitor molecule is Also called a guide strand.

[0046] Approximately: As used herein, refers to "approximately" as applied to one or more values ​​of interest. The term "" or "about" refers to a value similar to the stated reference value. In this document, the terms "approximately" or "about" are used unless otherwise stated or (unless otherwise clear from the context, in which case such a number would exceed 100% of the possible values) 25 in either direction (above or below) of the stated reference value, except %, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11 %, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less This refers to the range of values ​​that fall under.

[0047] Aryl: As used herein, the term "aryl" refers to an alkyl group having from 5 to up to 19 amino acids. "Substituted aryl" refers to an aromatic monocyclic or polycyclic group having carbon atoms in the range of It also refers to an aryl group having one or more substituents.

[0048] Bicyclic nucleotide: As used herein, the term "bicyclic nucleotide" refers to a nucleotide containing a bicyclic sugar moiety.

[0049] Bicyclic sugar moiety: As used herein, the term "bicyclic sugar moiety" refers to a 4- to 7-membered ring (including, but not limited to, furanosyl), and two atoms of the 4- to 7-membered ring Modified sugar moieties that include a bridge that links to form a second ring, thereby resulting in a bicyclic structure. Typically, the 4- to 7-membered ring is a sugar. In some embodiments, the 4- to 7-membered ring is In certain embodiments, the crosslink is at the 2'-carbon of the furanosyl. and the 4'-carbon.

[0050] Complementary: As used herein, the term "complementary" refers to a sequence in which two nucleotides are The structural relationship between two nucleotides that allows them to base pair with each other (e.g., (of two opposing nucleic acids or opposing regions of a single nucleic acid strand). For example, opposing The pyrimidine nucleotides of a nucleic acid and the purine nucleotides of a nucleic acid that are complementary to each other are hydrogen atoms. In some embodiments, complementary nucleotides may form bonds to form base pairs. in a Watson-Crick manner, or any other manner that allows for the formation of a stable duplex "Perfectly complementary" or 100% complementarity is when the first oligo Each nucleotide monomer of the nucleotide strand or segment of the first oligonucleotide strand Each of the second oligonucleotide strands or segments of the second oligonucleotide strands Refers to the ability to form base pairs with nucleotide monomers. Complementarity is the ability of two oligonucleotide strands (or two segments of two oligonucleotide strands) to interact with each other. Some, but not all, of the nucleotide monomers in a given fragment form base pairs with each other. "Substantial complementarity" refers to the situation where two origins have a complementarity of 90% or more. refers to a single oligonucleotide chain (or a segment of two oligonucleotide chains). "Complementary" refers to a target mRNA that results in a reduction in the amount of protein encoded by the target mRNA. Refers to the complementarity between the mRNA and the nucleic acid inhibitor molecule.

[0051] Complementary strand: As used herein, the term "complementary strand" refers to a strand that is partially complementary to another strand. Refers to the substantially or completely complementary strands of a double-stranded nucleic acid inhibitor molecule.

[0052] Cycloalkyl: As used herein, the term "cycloalkyl" refers to a group of 3 to 10 alkyl groups. 12 carbons, e.g., 3 to 8 carbons, e.g., 3 to 6 carbons, cyclic (i.e. "Substituted cycloalkyl" refers to a hydrocarbon group (containing a ring) that is further substituted with one or more substituents. It refers to a cycloalkyl group having a substituent.

[0053] Deoxyribofuranosyl: As used herein, "deoxyribofuranosyl" means The term "antibody" refers to a nucleotide sequence found in naturally occurring DNA, as illustrated below, at the 2'-carbon atom. It refers to a furanosyl having a hydrogen group at the atom, as shown below. TIFF0007748500000005.tif64150

[0054] Deoxyribonucleotide: As used herein, "deoxyribonucleotide" means a The term "nucleotide" refers to a naturally occurring nucleotide (as defined herein) having a hydrogen group at the 2' position of the sugar moiety. "Nucleotides" refers to nucleotides (as defined herein) or modified nucleotides (as defined herein).

[0055] dsRNAi inhibitor molecule: As used herein, "dsRNAi inhibitor" refers to a The term "target molecule" refers to the sense strand (passenger) and the antisense strand (guide). wherein the antisense strand or a portion of the antisense strand is a target mRNA. Double-stranded, used by Argonaute 2 (Ago2) endonuclease in cleavage Refers to nucleic acid inhibitor molecules.

[0056] Double-stranded: As used herein, with respect to a nucleic acid (e.g., an oligonucleotide), The term "duplex" refers to the structure formed through complementary base pairing of two antiparallel sequences of nucleotides. This refers to the structure in which

[0057] excipients As used herein, " excipients The term "" refers to a composition, e.g. For example, it refers to a non-therapeutic agent that may provide or contribute to a desired consistency or stabilizing effect.

[0058] Furanosyl: As used herein, the term "furanosyl" refers to a four carbon atom. It refers to a structure containing a five-membered ring with one oxygen atom and one hydroxyl group.

[0059] Halo: As used herein, the terms "halo" and "halogen" are used interchangeably. and refers to an atom selected from fluorine, chlorine, bromine and iodine.

[0060] Heterocycle: As used herein, the term "heterocycle" or "heterocyclic" refers to a heterocycle that is Contains one or more heteroatoms (e.g., N, O, S, etc.) as part of the ring structure and non-aromatic cyclic (i.e., containing rings) having in the range of 3 up to 14 carbon atoms. "Substituted heterocyclic" or "substituted heterocycle" refers to a group that further contains one or more substituents. It refers to a heterocyclic group having a ring structure.

[0061] Internucleotide linkage group: As used herein, "internucleotide linkage group" or The term "internucleotide linkage" refers to a linkage capable of covalently joining two nucleoside moieties. Typically, the chemical group is a phosphorus containing phospho or phosphite group. The phospho-linking group is a phosphodiester bond, a phosphorodithioate bond, , phosphorothioate bond, phosphotriester bond, thionoalkylphosphonate bond , thione alkyl phosphotriester bond, phosphoramidite bond, phosphonate bond and and / or boranophosphate linkages. See, for example, U.S. Pat. No. 687,808; No. 4,469,863; No. 4,476,301; No. 5,0 No. 23,243; No. 5,177,196; No. 5,188,897; No. 5,26 No. 4,423; No. 5,276,019; No. 5,278,302; No. 5,286 ,717;Same No.5,321,131;Same No.5,399,676;Same No.5,405, No. 939; No. 5,453,496; No. 5,455,233; No. 5,466,6 No. 77; No. 5,476,925; No. 5,519,126; No. 5,536,82 No. 1; No. 5,541,306; No. 5,550,111; No. 5,563,253 No. 5,571,799; No. 5,587,361; No. 5,194,599 ; Same No. 5,565,555; Same No. 5,527,899; Same No. 5,721,218; As disclosed in US Pat. Nos. 5,672,697 and 5,625,050, many phosphorus-containing In another embodiment, the oligonucleotide comprises a phosphorus bond. One or more internucleotide linking groups containing no atoms, e.g., short chain alkyl or Cycloalkyl internucleotide linkages, mixed heteroatoms and alkyl or cycloalkyl internucleotide linkage, or one or more short chain heteroatoms or heterocyclic nucleotides Inter-polymer linkages include, but are not limited to, siloxane backbones; sulfonyl amide, sulfoxide and sulfone skeletons; formacetyl and thioformacetyl skeletons; methyl riboacetyl backbone; alkene-containing backbone ;Sulfamate backbone;Methyleneimino and methylenehydrazino backbone;Sulfonate and sulfonamide skeletons; and amide skeletons. No. 5,034,506; No. 5,166,315; No. 5,185,444; No. 5 ,214,134;Same No.5,216,141;Same No.5,235,033;Same No.5, No. 264,562; No. 5,264,564; No. 5,405,938; No. 5,4 No. 34,257; No. 5,466,677; No. 5,470,967; No. 5,48 No. 9,677; No. 5,541,307; No. 5,561,225; No. 5,596 ,086;No. 5,602,240;No. 5,610,289;No. 5,602, No. 240; No. 5,608,046; No. 5,610,289; No. 5,618,7 No. 04; No. 5,623,070; No. 5,663,312; No. 5,633,36 No. 0; No. 5,677,437; No. 5,792,608; No. 5,646,269 Non-phosphorus-containing linkages are known in the art, as disclosed in US Pat. Nos. 5,677,439 and 5,677,439. be.

[0062] Loop: As used herein, the term "loop" refers to a single strand of nucleic acid refers to the structure formed, in which the complementary regions adjacent to a particular single-stranded nucleotide region are , the single-stranded nucleotide region between the complementary regions is duplex formation or Watson-Crick The loop hybridizes in such a way that it is excluded from base pairing. A single-stranded nucleotide region. Examples of loops include hairpins, tetraloops, and triloops. This includes unpaired nucleotides present in structures such as loops.

[0063] Melting temperature: As used herein, "melting temperature" or "T m " is a double-stranded nucleic acid This is the temperature at which the two strands separate. T m is often a complementary nucleic acid or a portion thereof It is used as a measure of the duplex stability or binding affinity of the two strands of T m UV spec By using a spectrometry system to determine the formation and breakdown (melting) of hybridization, The base stacking that occurs during hybridization can be measured by U This is accompanied by a decrease in UV absorption (hypochromicity). m Show .

[0064] Modified nucleobase: As used herein, the term "modified nucleobase" refers to a base that is a nucleobase of a naturally occurring nucleic acid. Refers to any nucleobase that is not a base or a universal nucleobase. Suitable modified nucleobases These include diaminopurine and its derivatives, alkylated purines or pyrimidines, acylated purines, and pyrimidines. Other suitable purines or pyrimidines include thiolated purines or pyrimidines. Suitable modified nucleobases include analogues of purine and pyrimidine. including but not limited to 1-methyladenine, 2-methyladenine, N6-methyladenine , N6-isopentyladenine, 2-methylthio-N6-isopentyladenine, N,N -dimethyladenine, 8-bromoadenine, 2-thiocytosine, 3-methylcytosine, 5 -methylcytosine, 5-ethylcytosine, 4-acetylcytosine, 1-methylguanine, 2-methylguanine, 7-methylguanine, 2,2-dimethylguanine, 8-bromoguanine guanine, 8-chloroguanine, 8-aminoguanine, 8-methylguanine, 8-thioguanine uracil, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil uracil, 5-ethyluracil, 5-propyluracil, 5-methoxyuracil, 5-hydroxyuracil hydroxymethyluracil, 5-(carboxyhydroxymethyl)uracil, 5-(methylamino) 5-(carboxymethylaminomethyl)-uracil, 5-(carboxymethylaminomethyl)-uracil, 2-thiouracil uracil, 5-methyl-2-thiouracil, 5-(2-bromovinyl)uracil, uracil- 5-Hydroxyacetic acid, uracil-5-hydroxyacetic acid methyl ester, pseudouracil, 1-methyl Tyrpseudouracil, Queuosine, Hypoxanthine, Xanthine, 2-aminopurine , 6-hydroxyaminopurine, nitropyrrolyl, nitroindolyl and difluorotrimethylammonium nitrile 6-thiopurine and 2,6-diaminopurine, nitropyrrolyl, nitroindolyl and Typically, the nucleobase comprises a nitrogenous base. In this form, the nucleobase does not contain a nitrogen atom. See, for example, U.S. Patent Application Publication No. 200800004. See No. 274462.

[0065] Modified nucleoside: As used herein, the term "modified nucleoside" refers to a not bound to a phosphate group or modified phosphate group (as defined herein) and Bases (as defined herein), universal nucleobases (as defined herein) ) or modified sugar moieties (as defined herein), N-glycosidic bonds with, for example, deoxyribose or ribose or analogs thereof The above-mentioned modified or universal nucleobases (bases The 1'-adenosine analogue (also called an amide analogue) is generally located at the 1'-position of the sugar moiety of the nucleoside, and the 1'-adenosine analogue (also called an amide analogue) is located at the 1'-position of the sugar moiety of the nucleoside. It refers to nucleic acid bases other than cytosine, guanine, cytosine, thymine, and uracil. In some embodiments, the modified or universal nucleobase is a nitrogenous base. In some embodiments, the modified nucleobase does not contain a nitrogen atom. See U.S. Pat. No. 80274462. In certain embodiments, the modified nucleotides are , does not contain a nucleobase (non-basic). The acid-base or modified sugar is described herein.

[0066] Modified nucleotide: As used herein, the term "modified nucleotide" refers to a attached to a phosphate group or a modified phosphate group (as defined herein), and Groups (as defined herein), universal nucleobases (as defined herein) or a sugar (e.g., a saccharide) containing one or more of a modified sugar moiety (as defined herein). in an N-glycosidic bond with, for example, ribose or deoxyribose or its analogues The above-mentioned modified or universal nucleobases (as used herein, bases The nucleoside analogue is generally located at the 1' position of the nucleoside sugar moiety, and the 1' adenine , refers to nucleobases other than guanine, cytosine, thymine, and uracil. In certain embodiments, the modified or universal nucleobase is a nitrogenous base. In the above modified nucleobases, no nitrogen atoms are contained. For example, see U.S. Patent Application Publication No. 200800004. In certain embodiments, the modified nucleotide is a nucleic acid salt. modified or universal nucleobases suitable in the context of the present disclosure, Modified sugar moieties, or modified phosphate groups, are described herein.

[0067] Modified phosphate group: As used herein, the term "modified phosphate group" refers to a naturally occurring nucleic acid. This refers to modifications of the phosphate group that do not occur in protease inhibitors, including phosphate mimetics containing phosphorus atoms and phosphate-containing protease inhibitors. Naturally occurring phosphate mimetics as described herein, including anionic phosphate mimetics (e.g., acetate), Modified phosphate groups include phosphate mimetics that are not present in phosphate groups, as described herein, e.g., phosphate mimetics. Both phosphorus-containing internucleotide linking groups, including phosphorothioate, and non-phosphorus-containing linking groups are Also included are non-naturally occurring internucleotide linkage groups, including

[0068] Modified sugar moiety: As used herein, a "modified sugar moiety" refers to a substituted sugar moiety (as defined herein). "Sugar Analogs" refers to sugar analogs (as defined herein) or sugar analogs (as defined herein).

[0069] Naturally occurring nucleobases: As used herein, the term "naturally occurring nucleobases" refers to the five main The important naturally occurring heterocyclic nucleobases of RNA and DNA, namely the purine bases: adenine the amino acids α, β, and β-guanine (A) and γ, and the pyrimidine bases thymine (T), cytosine (C) and uracil (U).

[0070] Naturally occurring nucleosides: As used herein, the term "naturally occurring nucleosides" refers to N-glycosides with a natural sugar moiety (as defined herein) that is not attached to a phosphate group Refers to a naturally occurring nucleobase (as defined herein) in a bond.

[0071] Naturally occurring nucleotide: As used herein, the term "naturally occurring nucleotide" refers to a N-glycosidic bonds with natural sugar moieties (as defined herein) attached to the phosphate group "Nucleobase" refers to a naturally occurring nucleobase (as defined herein) in the presence of a nucleotide.

[0072] Naturally occurring sugar moiety: As used herein, the term "naturally occurring sugar moiety" refers to a ribofuranose aryl (as defined herein) or deoxyribofuranosyl (as defined herein) This refers to a ori.

[0073] Nucleic acid inhibitor molecule: As used herein, the term "nucleic acid inhibitor molecule" The term refers to a region in which an oligonucleotide molecule specifically targets a sequence in a target gene mRNA. refers to an oligonucleotide molecule that reduces or eliminates the expression of a target gene, including a region Typically, the target region of a nucleic acid inhibitor molecule is determined by the nucleic acid inhibitor's ability to bind to a specific target gene. A sequence that is sufficiently complementary to a sequence on the target gene mRNA to direct the effect of the bitter molecule. The nucleic acid inhibitor molecule includes a sequence of ribonucleotides, deoxyribonucleotides, , and / or modified nucleotides.

[0074] Nucleobase: As used herein, the term "nucleobase" refers to a naturally occurring nucleobase (Nucleobase). (as defined herein), modified nucleobases (as defined herein) or universal bases. refers to ape nucleobases (as defined herein).

[0075] Nucleoside: As used herein, the term "nucleoside" refers to a naturally occurring nucleoside. a nucleoside (as defined herein), or a modified nucleoside (as defined herein) (As shown)

[0076] Nucleotide: As used herein, the term "nucleotide" refers to a naturally occurring nucleotide. nucleotides (as defined herein) or modified nucleotides (as defined herein) This refers to a ori.

[0077] Overhang: As used herein, the term "overhang" refers to a bilayer A terminal non-base paired nucleotide ( In certain embodiments, the overhang refers to a first strand or region originates from one strand or region that extends beyond the ends of the complementary strands that form the duplex One of the two oligonucleotide regions that can form a double strand by hydrogen bonding of base pairs or both, at the 3' ends and / or at the ends of complementarity shared by two polynucleotides or regions. The double-stranded 3' end may have a 5' end and / or a 3' end that extends beyond the 5' end. The single-stranded region extending beyond the ends and / or 5' ends is called an overhang.

[0078] Pharmaceutical composition: As used herein, the term "pharmaceutical composition" refers to a pharmaceutical composition that is pharmacologically effective. an effective amount of a double-stranded nucleic acid inhibitor molecule and a pharmaceutically acceptable salt thereof; Possible excipients (as defined herein) (as applicable).

[0079] Pharmaceutically acceptable Possible excipients As used herein, "pharmaceutically acceptable" means Possible excipients The term " excipients However, there are cases where the efficacy is excessively high, commensurate with a reasonable benefit / risk ratio. without adverse side effects (e.g., toxicity, irritation, and allergic reactions) in humans and / or This means that it is suitable for use in animals.

[0080] Phosphomimetic: As used herein, the term "phosphomimetic" refers to a compound that mimics a phosphate group. It refers to a chemical moiety at the 5' end of an oligonucleotide that mimics the electrostatic and steric properties of Many phosphomimetics have been developed that can be attached to the 5' terminus of oligonucleotides (e.g. , U.S. Patent No. 8,927,513 (Prakash et al. Nucleic A See CIDs Res.2015,43(6):2993-3011). Typically, these 5' phosphate mimics contain a phosphatase-resistant linkage. Mimetics are disclosed in International Publication No. WO2018 / 045317, the entire contents of which are incorporated herein by reference. 5' phosphonates (e.g., 5' methylene phosphonates), as described in 5'-(E)-vinylphosphonate (5'MP) and 5'-(E)-vinylphosphonate (5'VP)), and The sugar moiety (e.g., ribose or deoxyribonucleotide) of the 5'-terminal nucleotide of a oligonucleotide 4' phosphate analogs (e.g., 4'-ribose, or analogs thereof) attached to the 4' carbon of Oxymethylphosphonate, 4'-thiomethylphosphonate or 4'-aminomethylphosphonate In certain embodiments, the 4'-oxymethyl phosphonate is , represented by the formula —O—CH—PO(OH) or —O—CH—PO(OR), wherein R is independently selected from H, CH, an alkyl group, or a protecting group. In one embodiment, the alkyl group is CH2CH3. More typically, R is independently H, CH 3 or CH2CH3. Other modifications at the 5' end of the oligonucleotide has been developed (see, for example, WO2011 / 133871).

[0081] Protecting Group: As used herein, the term "protecting group" refers to a group that is capable of protecting a compound under certain conditions of a desired reaction. It is used in the conventional chemical sense as a group that reversibly renders a functional group unreactive under desired conditions. After this reaction, the protecting group can be removed to deprotect the protected functional group. All protecting groups must be removable under conditions that do not significantly degrade the molecule being synthesized. It must be.

[0082] Decrease (reduce): As used herein, "decrease (reduce)" The term " refers to its generally accepted meaning in the art. With respect to inhibitor molecules, the above terms generally refer to those observed in the absence of nucleic acid inhibitor molecules. A decrease in the expression of a gene or RNA molecule or one or more proteins below the normal range a decrease in the levels of proteins or equivalent RNA molecules that encode protein subunits, or refers to a decrease in the activity of one or more proteins or protein subunits .

[0083] Ribofuranosyl: As used herein, the term "ribofuranosyl" refers to a naturally occurring and has a hydrogen group at the 2' carbon, as shown below. refers to lanosil. TIFF0007748500000006.tif58150

[0084] Ribonucleotide: As used herein, the term "ribonucleotide" refers to a A naturally occurring nucleotide (as defined herein) having a hydroxyl group at the 2' position of the sugar moiety "Nucleotides" refers to nucleotides (as defined herein) or modified nucleotides (as defined herein).

[0085] Sense strand: A double-stranded nucleic acid inhibitor molecule consists of two oligonucleotide strands, i.e., The sense strand or a region thereof may be partially or substantially or completely complementary to the antisense strand or region thereof of the double-stranded nucleic acid inhibitor molecule. In certain embodiments, the sense strand is non-complementary to the antisense strand. The non-complementary nucleotides may be nucleotides located on either side of the complementary sequence. The sense strand or regions thereof may be flanked by complementary sequences. A particular sequence that is partially or substantially complementary to the antisense strand or a region thereof In embodiments, the non-complementary nucleotides are located between one or more regions of complementarity. The sense strand may contain a passenger Also called a chain.

[0086] Subject: As used herein, the term "subject" refers to mice, rabbits, and humans. "Individual" or "subject" refers to any mammal, including a mammalian animal. In one embodiment, the subject is a human. The term "patient" or "subject" is intended to be interchangeable with "subject."

[0087] Substituent or substituted: As used herein, the term "substituent" or "substituted" refers to a group of groups. The term "substituted" refers to the replacement of a hydrogen radical in a given structure with the radical of a substituent. More than one position in any given structure may be substituted with more than one substituent. Where available, the substituents may be the same or different at every position unless otherwise indicated. As used herein, the term "substituted" refers to any group that is compatible with organic compounds. The permissible substituents are contemplated to include acyclic and cyclic, branched, and unbranched alkyl groups. and unbranched, carbocyclic and heterocyclic, aromatic and non-aromatic substituents of organic compounds. This disclosure is not to be limited in any manner by the permissible substituents of organic compounds. It is not intended to be.

[0088] Substituted sugar moiety: As used herein, a "substituted sugar moiety" includes one or more modified Typically, the modification is at the 2', 3', 4' or 5' end of the sugar. In certain embodiments, the substituted sugar moiety occurs at the 2'-carbon position of a furanosyl. It is a bicyclic sugar moiety containing a bridge connecting the carbon to the 4-carbon.

[0089] Sugar analogs: As used herein, the term "sugar analogs" includes furanosyl refers to structures that can replace the naturally occurring sugar moiety of a nucleotide, resulting in The resulting nucleotides are (1) incorporation into the oligonucleotide and (2) the complementary nucleotide. Such structures are typically capable of hybridization to fragments of nucleotides. Relatively simple modifications to nosyl, such as rings containing different numbers of atoms (e.g., four-membered, six-membered, 1- or 7-membered ring; a non-oxygen atom (e.g., carbon, sulfur, or nitrogen) and a furanosyl oxygen or both a change in the number of atoms and a substitution of oxygen. Such structures are Sugar analogs may also contain substitutions corresponding to those described for the sugar. Sugar analogs include, but are not limited to, monosaccharide analogs, such as saccharide analogs of peptide nucleic acids. These include cyclohexyl, cyclohexyl, cyclohexyl, and cyclohexitol.

[0090] Sugar moiety: As used herein, the term "sugar moiety" refers to a naturally occurring sugar moiety or Refers to the modified sugar portion of a nucleotide or nucleoside.

[0091] Target site: As used herein, "target site," "target sequence," "target nucleic acid," "target" The terms "region" and "target gene" are used interchangeably, e.g., partially, substantially, or or its guide / antisense region, which is perfectly or sufficiently complementary to its target sequence. "Targeted" refers to cleavage mediated by an RNAi inhibitor molecule containing a sequence within "RNA or DNA sequence" refers to an RNA or DNA sequence that is "sequenced" to a specific sequence.

[0092] Tetraloop: As used herein, the term "tetraloop" refers to a loop of adjacent Stable secondary structures contribute to the stability of Watson-Crick hybridized nucleotides Without being bound by theory, it refers to a loop (single-stranded region) that forms a The base pairs can stabilize adjacent Watson-Crick base pairs through stacking interactions. In addition, the interactions between nucleotides in the tetraloop include, but are not limited to: However, non-Watson-Crick base pairing, stacking interactions, hydrogen bonds, and contact interactions (Cheong et al., Nature, 1990, 346(62 85):680-2, Heus and Pardi, Science, 1991, 25 3(5016):191-4). The tetraloop is a simple model loop consisting of random bases. This results in an increase in the melting temperature (Tm) of adjacent duplexes that is higher than predicted from the loop sequence. For example, a tetraloop is a double-stranded hairpin at least two base pairs long. , at least 50°C, at least 55°C, at least 56°C in 10 mM NaHPO4 , at least 58°C, at least 60°C, at least 65°C, or at least 75°C The tetraloop can be a ribonucleotide, a deoxyribonucleotide, In certain embodiments, the nucleotides may include tetranucleotides, ... A tetraloop consists of four nucleotides. In certain embodiments, the tetraloop is It consists of five nucleotides.

[0093] Examples of RNA tetraloops include the UNCG family of tetraloops (e.g., UUC G), GNRA family tetraloops (e.g., GAAA), and CUUG tetraloops tetraloop containing loops, including the CUYG family (Woese et al. ,PNAS,1990,87(21):8467-71,Antao et al.,N Ucleic Acids Res., 1991, 19(21):5901-5). RN Other examples of A tetraloops include GANC, A / UGNN, and UUUM tetraloop phages. Milley (Thapar et al., WILEY INTERDISCIP. REV RNA, 2014, 5(1):1-28) and GGUG, RNYA, and AGNN Tetraloop family (Bottaro et al., BIOPHYS J., 20 17, 113:257-67). Examples of DNA tetraloops include d(GNN A) Family of tetraloops (e.g., d(GTTA), d(GNRA)) family tetraloops of the d(GNAB) family, tetraloops of the d(CNNG) family and tetraloops of the d(TNCG) family (e.g., d(TTCG )) (Nakano et al. Biochemistry, 2002, 4 1(48):14281-14292. Shinji et al., Nippon K agakkai Koen Yokoshu,2000,78(2):731).

[0094] T m -elevated nucleotide: As used herein, "T m -elevated nucleotides The term is m - compared to an oligonucleotide duplex containing no elevated nucleotides , the melting temperature of the oligonucleotide duplex (T m ) refers to a nucleotide that increases T m - The elevated nucleotides include, but are not limited to, bicyclic nucleotides, tricyclic nucleotides, Modifications include nucleotides, G-clamps and their analogs, and hexitol nucleotides. Certain modified nucleotides having sugar moieties or modified nucleobases can be used in oligonucleotides. Double-stranded T m As used herein, " T m The term "2'-upper nucleotide" specifically refers to a 2'-O-nucleotide at the 2'-position of the sugar moiety. Nucleotides modified with Me or 2'-F are excluded.

[0095] Therapeutically effective amount: As used herein, a "therapeutically effective amount" or a "pharmacologically effective amount" is " refers to a double-stranded molecule effective to produce an intended pharmacological, therapeutic or prophylactic result. Refers to the amount of nucleic acid inhibitor molecule.

[0096] Tri-loop: As used herein, the term "tri-loop" refers to adjacent The hybridization of the nucleotides in the nucleotide sequence with the nucleotides forms a stable secondary structure that contributes to the stability of the nucleotides. It refers to a loop (single-stranded region) consisting of three nucleotides. Although not a triloop, the triloop is a non-Watson-Crick sequence of the nucleotides within the triloop. It can be stabilized by cross-type base pairing and base stacking interactions (Yoshizawa et al., Biochemistry 1997;36,4761-4767). The reloops also exhibit higher activity than predicted from a simple model loop sequence consisting of random bases. This can result in an increase in the melting temperature (Tm) of the adjacent duplex. nucleotides, deoxyribonucleotides, modified nucleotides, and combinations thereof. Examples of triloops include the GNA family triloops (e.g., GAA, GTA , GCA, and GGA) (Yoshizawa 1997). In one embodiment, the tri-loop has a nucleotide sequence of GAA.

[0097] Universal nucleobase: As used herein, a "universal nucleobase" refers to a nucleic acid It can pair with two or more of the bases typically found in naturally occurring nucleic acids, The salts refer to bases that can replace such naturally occurring bases in the main chain. The group need not be capable of pairing with each of the naturally occurring bases. For example, The bases pair exclusively or preferentially with purines or exclusively or preferentially with pyrimidines. The universal nucleobases may be in Watson-Crick or non-Watson-Crick phases. by forming hydrogen bonds through interactions (e.g., Hoogsteen interactions) Representative universal nucleobases include inosine and its derivatives. .

[0098] Detailed Description The present application provides a method for detecting a nucleic acid having a sense strand and an antisense strand, the method comprising: The present invention provides a double-stranded nucleic acid inhibitor molecule in which the loop portion of the stem-loop structure is a tri-loop. The double-stranded nucleic acid inhibitor molecule has a first region (R1) of the sense strand (S) and an amino acid sequence The first duplex (D1) between the antisense strand (AS) and the second region of the sense strand (R2) a second duplex (D2) between the first subregion (S1) and the second subregion (S2) of In this case, S1 and S2 are connected by a triloop (triL). See D. In addition, the stem portion of the stem-loop structure may, in certain embodiments, , at least one T m - elevated nucleotides, e.g., 4 to 12 T m -Elevated nucleotides , e.g., 2 to 6 T m The stem-loop structure may contain elevated nucleotide base pairs. can be located at the 5' or 3' end of the sense strand. Double-stranded nucleic acid inhibitor molecules containing the peptide are active in reducing target gene expression. Additionally, in certain embodiments, the tri-loop-containing double-stranded nucleic acid inhibitor molecule comprises: Increased potency of target gene expression compared to their tetraloop-containing counterparts It is possible.

[0099] In vitro or in vivo methods and compositions for treating diseases The triloop-containing polypeptides disclosed herein can be used to decrease the level or expression of a target gene in vivo. Methods of using the double-stranded nucleic acid inhibitor molecules and compositions containing same are also provided.

[0100] Triloop-containing nucleic acid inhibitor molecules The present application provides a method for detecting a nucleic acid having a sense strand and an antisense strand, the method comprising: The loop portion of the stem-loop structure is a tri-loop, and the sense strand and antisense strand The second strand has a 5' end and a 3' end, and thus adjacent oligonucleotides The present invention discloses double-stranded nucleic acid inhibitor molecules in which the separate strands do not form a stem / The loop-containing double-stranded nucleic acid inhibitor molecule comprises the sense strand ("S") and the antisense strand. ("AS") is highlighted in Figure 1A.

[0101] The sense strand may be further divided into antisense strands (AS strands) as shown in Figures 1B and 1C. a first region (R1) that forms a first duplex (D1) with the first subregion (S1 a second region (R2) comprising a loop (triL) connecting the first region (S1) to a second subregion (S2); S1 and S2 can be separated into two strands. ... 1C and 1D are sufficiently complementary to each other and are also referred to as the stem or stem duplex. As described herein, the loop is a triloop. In the present study, the triloop has the sequence GAA, but is a simple model loop consisting of random bases. This results in an increase in the melting temperature (Tm) of adjacent duplexes that is higher than predicted from the loop sequence. Other tri-loop sequences may also be used. The second duplex (D2) comprises at least one T m - a booster nucleotide, and in certain embodiments, the second duplex All of the nucleotides in strand (D2) are T m -It may be an elevated nucleotide. In general, the double-stranded nucleic acid inhibitor molecule does not contain any other double-stranded nucleic acid other than the second duplex (D2). Reno T m In certain embodiments, the double-stranded nucleic acid molecule does not contain any -associated nucleotides. The product is a dsRNAi inhibitor molecule.

[0102] In certain embodiments of the double-stranded nucleic acid inhibitor molecule, the sense strand comprises at least Together with T m -Stem duplex containing ascending nucleotides and a triloop (triL) (D2) and has a length of 20 to 65 nucleotides. The stem duplex is 2 to 6 base pairs in length. The strands are 15 to 40 nucleotides in length.

[0103] In certain embodiments, the sense strand comprises a stem duplex (D2) and a tri-loop (t riL), and has a length of 20 to 65 nucleotides, and the antisense strand is 15 In certain embodiments, the stem duplex (D2) and and an extension of the sense strand containing the triloop (triL) at the 3' end of the strand. In certain other embodiments, a nucleotide sequence comprising the stem (D2) and the triloop (triL) is provided. The sense strand extension is at the 5' end of the strand.

[0104] In certain embodiments, the double-stranded nucleic acid inhibitor molecule comprises a sense strand and an antisense strand. and a sense strand, wherein the sense and antisense strands are separate strands and are 18 to 200 μM in length. The first duplex (D1) of 24 base pairs is formed, while the sense strand is linked to the second duplex ( D2) and triloop (triL) and is 27 to 35 nucleotides in length, The antisense strand is 20 to 24 nucleotides in length. The sense strand is 27 to 35 nucleotides in length. In certain embodiments, the sense strand is 27 to 33 nucleotides in length. In certain embodiments, the second duplex (D In certain embodiments, the second duplex (D 2) has a length of 2 base pairs and is T m - containing elevated nucleotides. In this embodiment, the second duplex (D2) has a length of 3 base pairs and m -Rising Nucleo In certain embodiments, the second duplex (D2) comprises a length of 4 base pairs. Has and T m In certain embodiments, the second duplex comprises a -1 nucleotide. (D2) has a length of 5 base pairs and is m -Contains elevated nucleotides. In an embodiment, the second duplex (D2) has a length of 6 base pairs and does not contain any T m - In certain embodiments, the antisense strand does not contain 1, 2 It has a single-stranded overhang of 1, 3, or 4 nucleotides at its 3' end. Typically, the single-stranded overhang at the 3' end of the antisense strand is composed of two nucleotides. It consists of ochido.

[0105] In certain embodiments, the double-stranded nucleic acid inhibitor molecule comprises a sense strand and an antisense strand. and a sense strand, wherein the sense and antisense strands are separate strands and are 18 to 200 μM in length. A first duplex (D1) of 22 base pairs, e.g., 20 nucleotides, is formed, and the sense The strand contains a second duplex (D2) and a triloop (triL) and is 27-35 nucleotides long. The antisense strand is 20 to 24 nucleotides in length, for example, 22 In certain embodiments, D1 is 19 to 21 base pairs in length. In certain embodiments, the antisense strand has a length of 20 to 22 nucleotides. In certain embodiments, the sense strand is 29 to 33 nucleotides in length. In certain embodiments, the second duplex (D2) has a length of 2 to 6 base pairs. In certain embodiments, D2 has a length of 2 base pairs and m -Rising Nucleochi In certain embodiments, D2 has a length of 3 base pairs and includes T m -Rising Nu In certain embodiments, D2 has a length of 4 base pairs and includes a Tm In certain embodiments, D2 has a length of 5 base pairs, KatsuT m In certain embodiments, D2 is 6 base pairs in length. and any T m In certain embodiments, the nucleotide sequence is also free of up-converting nucleotides. The antisense strand has a single-stranded overhang of 1 to 5 nucleotides at its 3' end. In certain embodiments, the antisense strand has 1, 2, 3, or 4 It has a single-stranded overhang of nucleotides at its 3' end. The single-stranded overhang at the 3' end of the sense strand consists of two nucleotides.

[0106] In certain embodiments, the double-stranded nucleic acid inhibitor molecule comprises a sense strand and an antisense strand. and a sense strand, wherein the sense and antisense strands are separate strands and forming a first duplex (D1) of 1 base pair, the sense strand consisting of 19 to 21 nucleotides; and connecting the first region (R1) and the first subregion (S1) to the second subregion (S2). and a second region (R2) of 7 to 15 nucleotides containing a triloop (triL), Each of S1 and S2 is 2 to 6 nucleotides in length and is a second duplex (D2) and the antisense strand is sufficiently complementary to form a 20-24 nucleotide sequence. In certain embodiments, the antisense strand is two nucleotides in length. In certain embodiments, S1 has a single-stranded overhang of nucleotide at its 3' end. and S2 are each 2 to 5 nucleotides in length. has a length of 2 base pairs and mIn certain embodiments, D2 has a length of 3 base pairs, and T m -Contains elevated nucleotides. In an embodiment, D2 has a length of 4 base pairs and m -Contains elevated nucleotides. In certain embodiments, D2 has a length of 5 base pairs and m -Contains elevated nucleotides In certain embodiments, D2 has a length of 6 base pairs and m -Rising Nucleo Does not contain chido.

[0107] In certain embodiments, the double-stranded nucleic acid inhibitor molecule comprises a sense strand and an antisense strand. wherein the sense and antisense strands are separate strands and comprise 20 base pairs. A pair of first duplexes (D1) is formed, the sense strand of which has a first region of 20 nucleotides. (R1) and a tri-loop ( a second region (R2) of 7 to 9 nucleotides containing the antisense oligonucleotide (a ... The base strand is 22 nucleotides long and has a single-stranded overhang of two nucleotides. at its 3' end, and each of S1 and S2 contains a bicyclic nucleotide. In certain embodiments, each of S1 and S2 is 3 nucleotides in length and In certain embodiments, the second duplex (D2) of base pairs is formed. Each is two nucleotides long and forms a two-base pair second duplex (D2). In certain embodiments, each nucleotide in the second duplex (D2) is m -Rising Nu and the double-stranded nucleic acid inhibitor molecule is a nucleotide other than the second duplex (D2). In any Tm -It also does not contain elevated nucleotides.

[0108] In certain embodiments of the triloop-containing double-stranded nucleic acid inhibitor molecules described herein, In certain embodiments, the second duplex (D2) has a length of 2 to 6 base pairs. In certain embodiments, D2 has a length of 2 to 4 base pairs. In certain embodiments, D2 has a length of 3 base pairs. In certain embodiments, D2 has a length of 4 base pairs. In certain embodiments, D2 has a length of 6 base pairs.

[0109] In certain embodiments of the triloop-containing double-stranded nucleic acid inhibitor molecules described herein, The second duplex (D2) contains 4 to 10 T m -Contains elevated nucleotides and 2 to 5 salts In certain embodiments, D2 has a length of 6 to 8 T m -Rising Nucleo In certain embodiments, D2 comprises six base pairs and has a length of 3 to 4 base pairs. T m - contains elevated nucleotides and has a length of 3 base pairs. D2 has four T m -contains elevated nucleotides and has a length of 2 base pairs. In this embodiment, each nucleotide in D2 is T m - is an elevated nucleotide.

[0110] In certain embodiments of the triloop-containing double-stranded nucleic acid inhibitor molecules described herein, The second duplex (D2) contains a single T m -Contains elevated nucleotides and 2 to 6 salts Some of the triloop-containing double-stranded nucleic acid inhibitor molecules described herein have a length of 1 base pair. In certain embodiments, the second duplex (D2) comprises 2 to 6 single T m -Rising Nucleochi and has a length of 2 to 6 base pairs, m -Elevated nucleotides Neither of them forms a base pair. For example, D2 is two T m -Contains elevated nucleotides and has a length of 2 to 6 base pairs, wherein the two T m -Elevated nucleotides , do not form base pairs. D2 has three T m -Contains elevated nucleotides and has 3 to 6 bases The length of the pair may be three T m -The ascending nucleotide forms a base pair No. D2 is a four-T m -contains elevated nucleotides and has a length of 4 to 6 base pairs In that case, the four T m -The ascending nucleotide does not form a base pair. , 5 T's m - may contain a nucleotide and have a length of 5 to 6 base pairs, In this case, the five T m -The ascending nucleotide does not form a base pair. D2 has six T m -above and may have a length of 6 base pairs, wherein the six T m - The ascending nucleotide does not form a base pair.

[0111] In certain embodiments, the tri-loop-containing double-stranded nucleic acid inhibitor molecule comprises either T m In certain embodiments, the double-stranded nucleic acid inhibitor does not contain any elevated nucleotides. The bitter molecule contains either T in the first region (R1) of the sense strand or the antisense strand.m In certain embodiments, the double-stranded nucleic acid inhibitors The molecule does not contain any T m -It also does not contain elevated nucleotides. In certain embodiments, the double-stranded nucleic acid inhibitor molecule comprises a second duplex (D2) In any T m -It also does not contain elevated nucleotides.

[0112] one or more T in the second duplex (D2) of the triloop-containing double-stranded nucleic acid molecule m - the elevated nucleotide is a T m - In certain embodiments, the double-stranded nucleic acid molecule may be any of the nucleotides. The molecule has at least two T m -contains elevated nucleotides, and Each T in the second duplex m - the ascending nucleotides are the same. In this embodiment, the double-stranded nucleic acid molecule has at least two different T m -Contains elevated nucleotides.

[0113] Any of the triloop-containing double-stranded nucleic acid molecules described herein may further comprise one or more of the T m - the elevated nucleotide is a dinucleotide described herein or otherwise available in the art The triloop-containing double-stranded nucleic acid described herein may be any of the cyclic nucleotides. In any of the molecules, at least one bicyclic nucleotide in the second duplex (D2) comprises a bicyclic sugar moiety, wherein the bicyclic sugar moiety is connected to the 2'-carbon of the furanosyl and and a substituted furanosyl containing a bridge connecting the 4'-carbon.

[0114] In any of the triloop-containing double-stranded nucleic acid inhibitor molecules described herein, the second At least one bicyclic nucleotide in the duplex (D2) is a nucleotide of formula I, II, III, I For example, at least one of the second double strands (D2) has the structure V, Va, or Vb. Another bicyclic nucleotide may have the structure of Formula I. At least one bicyclic nucleotide may also have the structure of Formula II. At least one bicyclic nucleotide in D2) may also have the structure of formula III. At least one bicyclic nucleotide in the second duplex (D2) may also have the structure of Formula IV At least one bicyclic nucleotide in the second duplex (D2) has the structure of formula Va: At least one bicyclic nucleotide in the second duplex (D2) may have the following structure: It may also have the structure of formula Vb:

[0115] At least one bicyclic nucleotide in the second duplex (D2) is represented by the formula Ia, Ib , Ic, Id, Ie, or If. At least one bicyclic nucleotide in the duplex (D2) is represented by the formula IIa, IIb, IIc In the second duplex (D2), the second duplex (D3) may have one or more of the following structures: At least one bicyclic nucleotide of the formula IIIa and / or IIIb may also have the structure At least one bicyclic nucleotide in the second duplex (D2) may be represented by Formula IV It may also have the structure IVa and / or IVb.

[0116] In any of the triloop-containing double-stranded nucleic acid molecules described herein, the second duplex (D 2) At least one bicyclic nucleotide (BN) in There are: (a) methyleneoxy BN, (b) ethyleneoxy BN, (c) aminooxy BN (d) oxyamino BN, (e) methyl(methyleneoxy) BN (constrained ethyl or c (also known as ET), (f) methylene-thio BN, (g) methyleneamino BN, (h) methyl (i) ethylene carbocyclic BN, and (ii) propylene carbocyclic BN. In one embodiment, at least Another BN is (a) methyleneoxy BN or (d) oxyamino BN, where R2 is CH3. For example, at least one BN in D2 is an oxyamino BN. N(d) where R2 is CH3.

[0117] Bicyclic nucleotides The triloop-containing double-stranded nucleic acid inhibitor molecules disclosed herein comprise a sense strand and an anti- a sense strand, and in certain embodiments, at least one bicyclic nucleotide , may be contained in the stem portion of a stem-loop structure present in the sense strand. typically contains a 4- to 7-membered ring (including, but not limited to, furanosyl), ~ a bicyclic structure by including a bridge connecting two atoms of a 7-membered ring to form a second ring In certain embodiments, the bridge has a sugar moiety that provides The 4'-carbon is linked to the 5'-carbon to form a second ring. Such bicyclic nucleotides are They have various names, including BNA and LNA, for bicyclic nucleic acid and locked nucleic acid, respectively. The synthesis of bicyclic nucleotides and their incorporation into nucleic acid compounds can be found, for example, in Singh et al., Chem. Co., 1999, 10, 1449-1452, which is incorporated herein by reference in its entirety. mmun.,1998,4,455-456;Koshkin et al.,Tetr ahedron,1998,54,3607-3630;Wahlestedt et al.,Proc.Natl.Acad.Sci.USA,2000,97,56 33-5638;Kumar et al.,Bioorg.Med.Chem.Let t.,1998,8,2219-2222;Singh et al.,J.Org.C hem., 1998, 63, 10035-10039; U.S. Patent No. 7,427,672 , No. 7,053,207, No. 6,794,499, No. 6,770,748, Nos. 6,268,490 and 6,794,499; U.S. Patent Application Publication No. 200402 No. 19565, No. 20040014959, No. 20030207841, No. 2 No. 0040192918, No. 20030224377, No. 20040143114 This has also been reported in the literature, including in US Pat. Nos. 20030082807 and 20030082807.

[0118] Typically, the bridge contains 2 to 8 atoms. In certain embodiments, the bridge In certain embodiments, the bridge contains 3 atoms. In certain embodiments, the bridge contains 4 atoms. In certain embodiments, the bridge comprises 5 atoms. The bridge comprises 6 atoms. In certain embodiments, the bridge comprises 7 atoms. In certain embodiments, the bridge comprises 8 atoms. The bridge contains more than eight atoms.

[0119] In certain embodiments, the bicyclic sugar moiety is a bicyclic sugar moiety having a 2'-carbon and a 4'-carbon of the furanosyl. - is a substituted furanosyl containing a bridge linking the -carbons to form a second ring. In one embodiment, the bicyclic nucleotide has the structure of Formula I: TIFF0007748500000007.tif48170 have [In the formula, B is a nucleobase; G is H, OH, NH2, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl substituted C1-C6 alkyl, substituted C2-C6 alkenyl, substituted C2-C6 alkynyl, acyl , substituted acyl, substituted amide, thiol, or substituted thio; X is O, S, or NR1, where R1 is H, C1-C6 alkyl, C1-C6 alkoxy, benzene, or pyrene; and W a and W b each independently represents H, OH, a hydroxyl protecting group, a phosphorus moiety, or a group represented by formula I to another nucleotide or oligonucleotide Internucleotide linking group, W a or W b At least one of the groups is represented by formula I The internucleotide linking group connects the nucleotide to be bound to the oligonucleotide.

[0120] In certain embodiments of formula I, G is H and X is NR1, where: R1 is benzene or pyrene. In certain embodiments of Formula I, G is H. , and X is S.

[0121] In certain embodiments of Formula I, G is H and X is O: TIFF0007748500000008.tif45150

[0122] In certain embodiments of formula I, G is H and X is NR1, where: R1 is H, CH3, or OCH3: TIFF0007748500000009.tif45150

[0123] In certain embodiments of Formula I, G is OH or NH and X is O .

[0124] In certain embodiments of Formula I, G is OH and X is O: TIFF0007748500000010.tif45150

[0125] In certain embodiments of Formula I, G is NH and X is O: TIFF0007748500000011.tif45150

[0126] In certain embodiments of formula I, G is CH or CHOCH and X is In certain embodiments of Formula I, G is CH3 and X is O: TIFF0007748500000012.tif45150

[0127] In certain embodiments of Formula I, G is CH2OCH3 and X is O: TIFF0007748500000013.tif45150

[0128] In certain embodiments, the bicyclic nucleotide has the structure of Formula II: TIFF0007748500000014.tif45150 [In the formula, B is a nucleobase; Q1 is CH2 or O; X is CH2, O, S, or NR1, where R1 is H, C1-C6 alkyl, C1-C6 alkoxy, benzene, or pyrene; When Q1 is O, X is CH2; When Q1 is CH2, X is CH2, O, S, or NR1, where R1 is H, C1-C6 alkyl, C1-C6 alkoxy, benzene or pyrene; W a and W b each independently represents H, OH, a hydroxyl protecting group, a phosphorus moiety, or a group represented by formula I Attaching a nucleotide represented by I to another nucleotide or oligonucleotide and W a or W b At least one of the groups is represented by Formula II. is an internucleotide linking group that connects a nucleotide to an oligonucleotide represented by ].

[0129] In certain embodiments of formula II, Q 1 is O and X is CH 2 . TIFF0007748500000015.tif45150

[0130] In certain embodiments of Formula II, Q1 is CH2 and X is O: TIFF0007748500000016.tif45150

[0131] In certain embodiments of Formula II, Q1 is CH2 and X is NR1, wherein R1 is H, CH3 or OCH3: TIFF0007748500000017.tif45150

[0132] In certain embodiments of Formula II, Q1 is CH2 and X is NH: TIFF0007748500000018.tif45150

[0133] In certain embodiments, the bicyclic nucleotide has the structure of Formula III: TIFF0007748500000019.tif45150 [In the formula, B is a nucleobase; Q2 is O or NR1, where R1 is H, C1-C6 alkyl, C1-C6 alkoxy koxy, benzene or pyrene; X is CH2, O, S, or NR1, where R1 is H, C1-C6 alkyl, C1-C6 alkoxy, benzene, or pyrene; When Q2 is O, X is NR1; When Q2 is NR1, X is O or S; W a and W b each independently represents H, OH, a hydroxyl protecting group, a phosphorus moiety, or a group represented by formula I Attaching the nucleotide represented by II to another nucleotide or oligonucleotide and W a or W b at least one of which is of formula II I is an internucleotide linking group that connects a nucleotide represented by I to an oligonucleotide. be].

[0134] In certain embodiments of Formula III, Q2 is O and X is NR1. Formula I In certain embodiments of II, Q2 is O and X is NR1, where R 1 is C1-C6 alkyl. In certain embodiments of Formula III, Q2 is O; and X is NR1 and R1 is H or CH3.

[0135] In certain embodiments of Formula III, Q2 is O and X is NR1, and R1 is CH3: TIFF0007748500000020.tif45150

[0136] In certain embodiments of Formula III, Q2 is NR1 and X is O. Formula I In certain embodiments of II, Q2 is NR1, where R1 is a C1-C6 alkyl. and X is O.

[0137] In certain embodiments of Formula III, Q2 is NCH3 and X is O: TIFF0007748500000021.tif45150

[0138] In certain embodiments, the bicyclic nucleotide has the structure of Formula IV: TIFF0007748500000022.tif45150 [In the formula, B is a nucleobase; P1 and P3 are CH2, P2 is CH2 or O, and P4 is O; or t W a and W b each independently represents H, OH, a hydroxyl protecting group, a phosphorus moiety, or a group represented by formula I Binding the nucleotide represented by V to another nucleotide or oligonucleotide It is an internucleotide linking group, W a or W b at least one of which is according to formula IV is the internucleotide linkage group that connects the represented nucleotide to the oligonucleotide].

[0139] In certain embodiments of formula IV, P1, P2, and P3 are CH2, and P4 is O Is: TIFF0007748500000023.tif45150

[0140] In certain embodiments of Formula IV, P1 and P3 are CH2, P2 is O, and P4 is O: TIFF0007748500000024.tif45150

[0141] In certain embodiments, the bicyclic nucleotide has the structure of Formula Va or Vb: R: TIFF0007748500000025.tif65143 [In the formula, B is a nucleobase; r1, r2, r3, and r4 each independently represent H, halogen, C1 to C 12 Alkyl, Substitution C1~C 12 Alkyl, C2-C 12 Alkenyl, substituted C2-C 12 Alkenyl, C2-C 12 a Alkynyl; substituted C2-C 12 Alkynyl; C1-C 12 Alkoxy; substituted C1-C 12 Alkoxy , OT1, ST1, SOT1, SO2T1, NT1T2, N3, CN, C(=O)OT1, C(= O)NT1T2, C(=O)T1, O─C(=O)NT1T2, N(H)C(=NH)NT1 T2, N(H)C(=O)NT1T2 or N(H)C(=S)NT1T2, where Each of T1 and T2 is independently H, C1-C6 alkyl, or substituted C1-C16 Archi or r1 and r2 or r3 and r4 together are =C(r5)(r6), where r 5 and r6 each independently represent H, halogen, C1 to C 12 Alkyl or substituted C1-C 12 is alkyl; and W a and W b each independently represents H, OH, a hydroxyl protecting group, a phosphorus moiety, or a group of formula V to another nucleotide or oligonucleotide Internucleotide linking group, W a or W b At least one of the groups is represented by formula V The internucleotide linking group connects the nucleotide to be bound to the oligonucleotide.

[0142] In certain embodiments, the bicyclic sugar moiety is a bicyclic sugar moiety having a 2'-carbon and a 4'-carbon of the furanosyl. -carbons to form a second ring, wherein the furanosyl The bridge connecting the 2'-carbon and 4'-carbon of the syl includes, but is not limited to: a) 4'-CH2-ON(R)-2' and 4'-CH2-N(R)-O-2' (where , R, H, C1~C 12 Alkyl, or, for example, 4'-CH2-NH-O-2'(BNA NC also known as 4'-CH2-N(CH3)-O-2' (BNA NC As [NMe] (U.S. Pat. No. 7,499,499, which is incorporated herein by reference in its entirety) ,427,672); b) 4'-CH2-2';4'-(CH2)2-2';4'-(CH2)3-2';4'- (CH2)-O-2' (also known as LNA); 4'-(CH2)-S-2'; 4'-(C H2)2-O-2' (also known as ENA); 4'-CH(CH3)-O-2' (cEt and and 4'-CH(CH2OCH3)-O-2' (also known as cMOE), and analogs thereof (see U.S. Pat. No. 7,399,898, which is incorporated herein by reference in its entirety). as described in Issue 45); c) 4'-C(CH3)(CH3)-O-2' and its analogs, the entire contents of which are hereby incorporated by reference. As described in U.S. Patent No. 8,278,283, which is incorporated herein by reference; d) 4'-CH2-N(OCH3)-2' and analogs thereof (herein incorporated by reference in their entirety). As described in U.S. Patent No. 8,278,425, which is incorporated herein by reference; e) 4'-CH2-ON(CH3)-2' and analogs thereof (the entire contents of which are incorporated herein by reference). As described in U.S. Patent Publication No. 2004 / 0171570, which is incorporated herein by reference; f) 4'-CH2-C(H)(CH3)-2' and analogs thereof (the entire contents of which are hereby incorporated by reference). Chattopadhyaya et al., J. Org. Che, incorporated herein by reference. m., 2009, 74, 118-34); and g) 4'-CH2-C(=CH2)-2' and analogs thereof (the entire contents of which are incorporated herein by reference). (as described in U.S. Patent No. 8,278,426, incorporated herein by reference) Includes:

[0143] In certain embodiments, the bicyclic nucleotide (BN) is as shown below: (a) Methyleneoxy BN, (b) Ethyleneoxy BN, (c) Aminoxy BN; (d) oxyamino BN, (e) methyl(methyleneoxy) BN (constrained ethyl or cE (f) methylene-thio BN, (g) methyleneamino BN, (h) methylene and (i) propylene carbocyclic BN. TIFF0007748500000026.tif140150 TIFF0007748500000027.tif191150 TIFF0007748500000028.tif102150

[0144] In the bicyclic nucleotides (a) to (i) above, B is a nucleobase and R2 is H or or CH3, and W a and W b are each independently H, OH, a hydroxyl protecting group, The phosphorus moiety, or bicyclic nucleotide, can be attached to another nucleotide or oligonucleotide. The internucleotide linking group, W a or W b At least one of It is an internucleotide linking group that joins a nucleotide to an oligonucleotide.

[0145] In one embodiment of oxyaminoBN(d), R2 is CH3(BNA NC [N Me): TIFF0007748500000029.tif64150

[0146] In certain embodiments, bicyclic sugar moieties and bicyclic sugar moieties incorporating such bicyclic sugar moieties are Nucleotides are further defined by their isomeric configuration. In certain embodiments, the bicyclic sugar moiety or nucleotide is in the α-L configuration. The sugar moiety or nucleotide is in the β-D configuration. For example, in certain embodiments, The bicyclic sugar moiety or nucleotide has a 2'O,4'-C-methylene bridge in the α-L configuration. (2'-O-CH2-4')(α-L LNA). In certain embodiments, The bicyclic sugar moiety or nucleotide is in the R configuration. The sugar moiety or nucleotide is in the S configuration. For example, in certain embodiments, The bicyclic sugar moiety or nucleotide has a 4'-CH(CH3)-O-2' bridge in the S-configuration ( That is, cEt).

[0147] Tricyclic nucleotides In certain embodiments, the T m -The ascending nucleotide is a tricyclic nucleotide The synthesis of tricyclic nucleotides and their incorporation into nucleic acid compounds is also described, e.g. Steffens et al., J. Am.Chem.Soc.1997;119:11548-11549;Steffen s et al.,J.Org.Chem.1999;121(14):3249-32 55;Renneberg et al.,J.Am.Chem.Soc.2002;1 24:5993-6002;Ittig et al.,NUCLEIC ACIDS RES.2004;32(1):346-353;Scheidegger et al .,Chemistry 2006;12:8014-8023;Ivanova et. al., OLIGONUCLEOTIDES 2007;17:54-65 It has been reported in

[0148] In certain embodiments, the tricyclic nucleotides are, for example, those described herein, each of which is incorporated by reference. CJ, Bioorg. Med. Chem. 2002;10:841- 854 and U.S. Patent Application Publication Nos. 2015 / 0259681 and 2018 / 01 As discussed in US Pat. No. 6,2897, the 3'-carbon and 5'-carbon centers are Tricyclonucleotides (tricyclonucleotides) linked by ethylene fused to the isopropane ring In certain embodiments, the tricyclic nucleotide is The method is described, for example, in U.S. Patent Application Publication No. 2015 / 011205, which is incorporated herein by reference. As discussed in No. 5, the 2'-carbon and 4'-carbon of the furanosyl are linked together. a bridge forming a second ring, and a third fused ring connecting the 5'-carbon to the 2' carbon of the furanosyl a substituted flagellum resulting from a group linked to the methylene group of the bridge connecting the - and 4'-carbons Contains a nosyl ring.

[0149] Other T m -Elevated nucleotides In addition to bicyclic and tricyclic nucleotides, the nucleic acid inhibitor molecules described herein also include And other T m - Up-regulating nucleotides can be used. For example, in certain embodiments In this state, the above T m - The ascending nucleotide is a G-clamp, a guanidine G-clamp or and its analogues (Wilds et al., Chem, 2002;114:123 and W ilds et al., Chim Acta 2003;114:123), Hexyto nucleotides (Herdewijn, Chem. Biodiversity 201 0;7:1-59), or modified nucleotides. The modified nucleotides are, for example, , 5-bromo-uracil, 5-iodo-uracil, 5-propynyl-modified pyrimidines, or or 2-aminoadenine (also called 2,6-diaminopurine) (Deleavey et al., Chem. & Biol. 2012;19:937-54) or 2- and uridine, as described herein, including auridine, 5Me-thiouridine, and pseudouridine. Modified nucleotides can also have modified nucleobases, e.g., as described in As described in U.S. Pat. No. 8,975,389, which is incorporated herein by reference, or as described herein. As described in the document, except for the above T m -The ascending nucleotide is the 2'-carbon and having a modified sugar moiety that is not modified with 2'-F or 2'-OMe. can.

[0150] In certain embodiments, the T m -The ascending nucleotide is a bicyclic nucleotide In certain embodiments, the T m -The ascending nucleotide is a tricyclic nucleotide In certain embodiments, the T m - Elevated nucleotide, G-clamp, guanidine In certain embodiments, the T m -Rising Nucle In certain embodiments, the T m -rise The nucleotide is a bicyclic or tricyclic nucleotide. Record T m -The ascending nucleotide can be a bicyclic nucleotide, a tricyclic nucleotide, or a G-cyclic nucleotide. In certain embodiments, the antibody is a LAMP, a guanidine G-clamp, or an analog thereof. Above T m -The ascending nucleotides are bicyclic nucleotides, tricyclic nucleotides, G-clan nucleotides, guanidine G-clamps or analogs thereof, or hexitol nucleotides. do.

[0151] In certain embodiments, the T m - the elevated nucleotide is the nucleic acid inhibitor molecule T of the second duplex (D2) m Increase the temperature by at least 2°C per integration. In certain embodiments, the T m - Elevated nucleotide is D2 T m One built-in In certain embodiments, the temperature is increased by at least 3°C. m -Elevated nucleotides is D2 T m The temperature is increased by at least 4°C per incorporation. In this state, the above T m - Elevated nucleotide is D2 T m at least one built-in Increase the temperature by 5°C.

[0152] Other modifications The double-stranded nucleic acid inhibitor molecules described herein comprise at least one double-stranded nucleic acid inhibitor in the second duplex (D2). Another T m In addition to the -upper nucleotide, other nucleotide modifications may be included. Typically, multiple nucleotides of the double-stranded nucleic acid inhibitor molecule are bound to a nuclease. The molecules are modified to improve various characteristics, such as resistance to or reduced immunogenicity. For example, Bramsen et al. (2009), Nucleic Acids R See, e.g., 37, 2867-2881. Many nucleotide modifications are In the field of nucleotides, they have been used especially for nucleic acid inhibitor molecules. Modifications can be made to any of the above nucleotides, including sugar moieties, phosphodiester linkages, and nucleobases. Typical examples of nucleotide modifications include, but are not limited to, However, 2'-F, 2'-O-methyl ("2'-OMe" or "2'-OCH3") , and 2'-O-methoxyethyl ("2'-MOE" or "2'-OCH2CH2OC Modifications can be made at any of the above nucleosides, such as the 5'-carbon, as described herein. It can also occur at other positions in the sugar moiety of the thiamin.

[0153] In certain embodiments, the double-stranded nucleic acid inhibitor molecule is a compound known in the art. and as described herein, adenine, guanine, cytosine, thiamine ... The nucleic acid may also include one or more modified nucleobases other than amine and uracil. In certain embodiments, the modified or universal nucleobase is a nitrogenous base. In the above modified nucleobases, no nitrogen atoms are contained. For example, see U.S. Patent Application Publication No. 200800004. See US Pat. No. 274462. In certain embodiments, the modified nucleotides are It does not contain any acid or base (non-basic). A typical example of a modified nucleobase is 5'-methylcytosine. be.

[0154] The internucleotide linkages in naturally occurring RNA and DNA are 3'- and 5'-phosphodiesters. Modified phosphodiester bonds are known in the art and are used herein. As described in the specification, the internucleotide linkages containing phosphorus atoms and the internucleotide linkages not containing phosphorus atoms Typically, the internucleotide linkages are non-naturally occurring, including those containing non-naturally occurring internucleotide linkages. The double-stranded nucleic acid inhibitor molecule may comprise one or more linkers, as described herein. In another embodiment, the double-stranded nucleic acid inhibitor molecule comprises an alkyl-containing internucleotide linking group. One or more of the internucleotide linking groups of the oligonucleotides may be non-phosphorus-containing linking groups, as described herein. In certain embodiments, the double-stranded nucleic acid inhibitor molecule is one or more a number of phosphorus-containing internucleotide linkage groups and one or more non-phosphorus-containing internucleotide linkages Contains a group.

[0155] In certain embodiments, the double-stranded nucleic acid inhibitor molecule comprises at least one phosphatase. In certain embodiments, the duplex contains a holothioate internucleotide linking group. The nucleic acid inhibitor molecule may comprise fewer than 10, e.g., fewer than 5, phosphorothioate nucleosides. In certain embodiments, the double-stranded nucleic acid inhibitor molecule contains an inter-tide linking group. , containing four phosphorothioate internucleotide linking groups.

[0156] The 5' end of the sense and / or antisense strand of the double-stranded nucleic acid inhibitor molecule is It may contain naturally occurring substituents such as hydroxyl or phosphate groups. In certain embodiments, The hydroxyl groups may be used to form the sense and / or antisense double-stranded nucleic acid inhibitor molecules. In certain embodiments, the phosphate group is attached to the 5' end of the double-stranded nucleic acid. Typically, the inhibitor molecule is attached to the 5' end of the sense and / or antisense strand. In other embodiments, the phosphate is added to the monomer prior to oligonucleotide synthesis. In embodiments, 5'-phosphorylation is achieved after introduction of the nucleic acid inhibitor molecule into the cytosol, e.g., For example, this is naturally achieved by the cytosolic Clp1 kinase. The 5' terminal phosphate may be a phosphate group, e.g., 5'-monophosphate [(HO)2(O )PO-5'], 5'-diphosphate [(HO)2(O)POP(HO)(O) -O-5'] or 5'-triphosphate [(HO)2(O)PO-(HO)(O) POP(HO)(O)-0-5'].

[0157] The 5' ends of the sense and / or antisense strands of the double-stranded nucleic acid inhibitor molecule also For example, in some embodiments, the double-stranded nucleic acid inhibitor molecule can be modified The 5' ends of the sense and / or antisense strands of )Ρ-ΝΗ-5',(ΗΟ)(ΝΗ2)(O)Ρ-O-5']. a certain In this embodiment, the sense and / or antisense strands of the double-stranded nucleic acid inhibitor molecule The 5' end of the phosphates, such as 5'-methylenephosphonate (5'-MP), 5'-(E)-vinylphosphonate It contains 5'-VP. Lima et al., Cell, 2012, 1 50-883-94; WO2014 / 130607. Other suitable phosphate mimetics include As described in International Publication WO2018 / 045317, the entirety of which is incorporated herein by reference. The sugar moiety (e.g., ribose or decasaccharide) of the 5'-terminal nucleotide of the oligonucleotide It contains a 4-phosphate analogue attached to the 4'-carbon of ribose (oxyribose or its analogues). For example, in some embodiments, the sense and / or amino acid sequences of the double-stranded nucleic acid inhibitor molecules are or the 5' end of the antisense strand is bound to an oxymethylphosphonate, The oxygen atom of the oxymethyl group is attached to the 4'-carbon of the sugar moiety or its analogue. In this embodiment, the phosphate analog is a thiomethylphosphonate or an aminomethylphosphonate. In this case, the sulfur atom of the thiomethyl group or the nitrogen atom of the aminomethyl group is It is attached to the 4'-carbon of a nucleotide or its analogue.

[0158] In certain embodiments, the double-stranded nucleic acid inhibitor molecule comprises one or more deoxyribonucleotides. Typically, the double-stranded nucleic acid inhibitor molecule contains less than five ribonucleotides. In certain embodiments, the double-stranded nucleic acid inhibitor comprises at least 10 deoxyribonucleotides. The bitter molecule comprises one or more ribonucleotides. All of the nucleotides in the double-stranded nucleic acid inhibitor molecule are ribonucleotides.

[0159] In certain embodiments, the double-stranded nucleic acid intercalators described herein comprise a sugar moiety. one or more nucleotides outside the stem (second duplex or D2) of the inhibitor molecule The modified ring structures present in bicyclic or tricyclic nucleotides include, but are not limited to, and unlocked nucleic acids (“UNAs”) (see, e.g., Snead et al. al.(2013),Molecular Therapy-Nucleic Aci ds, 2, e103 (doi:10.1038 / mtna.2013.36) sea ​​bream).

[0160] In certain embodiments, one or two nucleotides of the double-stranded nucleic acid inhibitor molecule The glutathione is reversibly modified with a glutathione-sensitive moiety. The thione-sensitive moiety is located at the 2'-carbon of the sugar moiety and contains a sulfonyl group. In certain embodiments, the method of claim 1, further comprising administering to said patient a compound selected from the group consisting of: As described in US Pat. No. 5,997,107, the glutathione-sensitive moiety is a phosphoramidite. In certain embodiments, the double-stranded nucleic acid is compatible with conventional oligonucleotide synthesis methods. More than two nucleotides in the acid inhibitor molecule are reversible in the glutathione-sensitive portion In certain embodiments, the majority of nucleotides are modified with glutathione. In certain embodiments, the double-stranded nucleic acid inhibitor is reversibly modified with a reactive moiety. All or substantially all of the nucleotides in the target molecule are glutathione-sensitive moieties. It is reversibly modified.

[0161] The at least one glutathione-sensitive moiety typically comprises a double-stranded nucleic acid inhibitor. The 5'- or 3'-terminal nucleotides of the sense and / or antisense strands of the target molecule However, the at least one glutathione-sensitive moiety is located at the It may be located at any nucleotide of interest in the stranded nucleic acid inhibitor molecule.

[0162] In certain embodiments, the double-stranded nucleic acid inhibitor molecule is fully modified, In this case, all nucleotides of the sense strand and antisense strand are modified; Typically, all nucleotides are modified at the 2'-position of the sugar moiety. In certain embodiments, the fully modified nucleic acid inhibitor molecule does not include any reversible modifications. In this embodiment, at least one of the sense strands of the double-stranded nucleic acid inhibitor molecule, e.g. , at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 2 9, 30, 31, 32, 33, 34, 35, or 36 nucleotides are modified In some embodiments, at least one of the antisense strands of the double-stranded nucleic acid inhibitor molecule One, e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 nucleoside The thiol is modified.

[0163] In certain embodiments, the fully modified nucleic acid inhibitor molecule comprises one or more possible In certain embodiments, the duplex is modified with a glutathione-sensitive moiety. Substantially all of the nucleotides of the nucleic acid inhibitor molecule are modified. In embodiments, more than half of the nucleotides in the double-stranded nucleic acid inhibitor molecule are reversible. In certain embodiments, the double-stranded nucleic acid interface is modified by a chemical modification other than an organic modification. Fewer than half of the nucleotides in the inhibitor molecule are modified with chemical modifications other than reversible modifications Modifications can occur at groups on the nucleic acid inhibitor molecule, or at different modified nucleotides. There may be scattered

[0164] In certain embodiments of the double-stranded nucleic acid inhibitor molecule, from one to all nucleosides In certain embodiments, the double-stranded nucleic acid is modified at the 2'-carbon. The inhibitor molecule may be partially or completely composed of 2'-F, 2'-OMe, and / or 2'-MOE. or completely modified. In certain embodiments of the double-stranded nucleic acid inhibitor molecule described above, , one to all phosphorus atoms are modified, and one to all nucleotides have a sugar moiety is modified at the 2'-carbon of

[0165] In certain embodiments of the double-stranded nucleic acid inhibitor molecule, the sense and antisense All nucleotides in the strand are modified at the 2'-carbon of the sugar moiety. In certain embodiments of the double-stranded nucleic acid inhibitor molecule, the sense and antisense strands All nucleotides except for those in the second region (R2) of the sense strand are The 2'-carbon of the sugar moiety is modified with 2'-F or 2'-OMe. In certain embodiments of the double-stranded nucleic acid inhibitor molecule, the sense and antisense strands All nucleotides are T in the stem (second duplex or D2) m -Rising Nucleochi The 2'-carbon of the sugar moiety is modified with 2'-F or 2'-OMe, except for the In certain embodiments of the double-stranded nucleic acid inhibitor molecule, the sense and antisense strands are Every nucleotide in the sense strand is a T in the stem (second duplex or D2). m -rise A tolyl conjugated to a nucleotide and a ligand moiety, e.g., GalNAc Except for nucleotides in the loop, the 2'-carbon of the sugar moiety is 2'-F or 2'-O. It is modified with Me.

[0166] Methods for reducing target gene expression The triloop-containing double-stranded nucleic acid inhibitor molecules as described herein can be used to inhibit any desired double-stranded nucleic acid. The present invention can be used in a method for reducing target mRNA expression of a target gene. In general, the method for reducing mRNA expression involves administering to a sample or a subject in need thereof the The double-stranded nucleic acid inhibitor molecule as described herein is used to decrease mRNA expression of a target gene. The method may be in vitro or in vivo. It can be performed in vivo.

[0167] The level or activity of the target RNA may be determined by any method now known in the art or later developed. The expression of the target RNA and / or the target gene can be determined by an appropriate method. The methods used to measure "expression" are based on the properties of the target gene and its encoded RNA. For example, it may be understood that the target RNA sequence may depend on In this case, the term "expression" refers to the amount of expression of a target gene (either genomic or exogenously derived). It may refer to the protein or target RNA / transcript from which it is derived. In such cases, the target RNA Expression of the target RNA / transcript can be measured either by directly measuring the amount of the target RNA / transcript or by measuring the amount of the target RNA / transcript. It can be determined by measuring the amount of protein encoded by the transcript. Proteins can be detected by protein assays, for example, by staining or immunoblotting, or Or, if the protein catalyzes a reaction that can be measured, measuring the reaction rate. All such methods are known in the art and If target RNA levels are to be measured, a suitable probe for detecting RNA levels can be used. Art-recognized methods can be used for this purpose (e.g., RT -PCR, Northern blot, etc.) The above measurements can be performed on cells, cell extracts, tissues, tissue extracts, etc. The process can be carried out on starch or another suitable source material.

[0168] Pharmaceutical Composition The present disclosure provides a therapeutically effective amount of a triloop-containing double-stranded nucleic acid inhibitor as described herein. bitter molecules and pharmaceutically acceptable Possible excipients and a pharmaceutical composition comprising:

[0169] These pharmaceutical compositions may be sterilized by conventional sterilization techniques, or may be sterile filtered. The resulting aqueous solution can be packaged for immediate use or lyophilized. and the lyophilized formulation is then reconstituted in sterile aqueous solution prior to administration. excipients The pH of the formulation is Typically, 3 to 11, more preferably 5 to 9 or 6 to 8, most preferably 7 to 8, e.g. For example, it would be 7 to 7.5.

[0170] The pharmaceutical compositions of the present disclosure are adapted for therapeutic use. Accordingly, one aspect of the present disclosure is Subjects, including but not limited to humans suffering from a disease or condition, can be treated by administering the drug to said subject. can be used to treat by administering an effective amount of the pharmaceutical composition of the present disclosure In certain embodiments, the disease or condition is a compound of any of the compounds described herein. It is cancer as described above.

[0171] In certain embodiments, the present disclosure provides a therapeutically effective amount of a pharmaceutical composition as described herein. The present invention relates to the use of the composition of matter for the manufacture of a medicament for treating a subject in need thereof. In certain embodiments, the subject has cancer as described herein. do.

[0172] Pharmaceutically acceptable Possible excipients Pharmaceutically acceptable Possible excipients is typically conventional. W. Martin, Mack Publishing Co., Easton, PA, 15 th Remington's Pharmacy by Edition (1975) Medical Sciences is a company that specializes in the delivery of pharmaceutical compositions suitable for the pharmaceutical delivery of one or more therapeutic compositions. Compositions and formulations are described. Possible excipients Materials that can play a role as Some examples of ingredients include sugars, such as lactose, glucose, and sucrose; starch; , for example, corn starch and potato starch; cellulose and its derivatives, For example, sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate. ;malt;gelatin; excipients cocoa butter and suppository waxes; oils, such as lacquer Sesame, cottonseed, safflower, sesame, olive, corn, and soybean oils; buffers , for example, magnesium hydroxide and aluminum hydroxide; (isotonic saline, Ringer's solution); Ethyl alcohol; pH buffers; polyols, e.g., glycerol, propylene glycol ethanol, polyethylene glycol, etc.; and other non-toxic compatible substances used in pharmaceutical preparations Quality is included.

[0173] Dosage form The pharmaceutical composition may be administered in a suitable formulation for any intended route of administration, which may be selected in accordance with ordinary practice. Next excipients It can be formulated with

[0174] In one embodiment, the pharmaceutical composition comprises a tri-loop-containing duplex as described herein. containing nucleic acid inhibitor molecules and administered, for example, by subcutaneous, intramuscular, intravenous or epidural injection. Typically, the pharmaceutical compositions of the present disclosure are suitable for parenteral administration. Formulated in liquid form.

[0175] Suitable dosage forms for parenteral administration are typically, for example, sterile aqueous solutions, saline solutions, low molecular weight Alcohols, such as propylene glycol, polyethylene glycol, vegetable oils, gelatin and one or more suitable for parenteral administration, including hydroxybenzoates, fatty acid esters, e.g., ethyl oleate, and the like. The parenteral formulation may contain a plurality of vehicles, such as sugars, alcohols, antioxidants, buffers, bacteriostatic agents, and the like. Drugs, solutes that render the formulation isotonic with the blood of the intended recipient, or suspending agents, if any. Proper fluidity can be maintained, for example, by the use of surfactants. The liquid formulation containing the double-stranded nucleic acid inhibitor can be freeze-dried and used as a sterile injectable preparation. It can be stored for later use upon reconstitution with a liquid formulation.

[0176] The pharmaceutical compositions may also be administered topically or transdermally, rectally or vaginally using well-known techniques. may be manufactured for other routes of administration, including intranasal, intraocular, nasal, buccal, or sublingual administration. It can be formulated into a formulation.

[0177] Delivery Agent Triloop-containing double-stranded nucleic acid inhibitor molecules as described herein can be used, for example, as Liposomes and lipids, e.g., U.S. Pat. Nos. 6,815,432 and 6,586,410 No. 6,858,225, No. 7,811,602, No. 7,244,448 and those disclosed in U.S. Pat. Nos. 8,158,601; polymeric materials, e.g., those disclosed in U.S. Pat. Nos. 6,833,833; No. 5,393, No. 7,374,778, No. 7,737,108, No. 7,718 ,193, 8,137,695 and U.S. Patent Application Publication No. 2011 / 014343 No. 4, No. 2011 / 0129921, No. 2011 / 0123636, No. 201 1 / 0143435, 2011 / 0142951, 2012 / 002151 No. 4, No. 2011 / 0281934, No. 2011 / 0286957 and No. 20 08 / 0152661; capsids, capsoids, or other substances that can be ingested, distributed, or or mixed with other molecules, molecular structures, or compounds containing receptor targeting molecules that aid absorption. The molecule may be encapsulated, conjugated, or otherwise associated with a target molecule.

[0178] In certain embodiments, the triloop-containing double-stranded nucleic acid inhibitor molecule is prepared by the addition of a lipid nanotube. Lipid-nucleic acid nanoparticles are typically formulated as lipid-nucleic acid nanoparticles (LNPs). Depending on the desired particle size distribution, the resulting nanoparticle mixture The mixture is optionally extruded, for example, in a thermobarrel extruder, e.g., LIPEX® ) Polycarbonate membranes were extruded using an extruder (Northern Lipids, Inc.). (e.g., cutoff 100 nm). To prepare lipid nanoparticles, the solvent used to form the nanoparticles (e.g., ethanol) was used. It may be desirable to remove the solvent (ethanol) and / or exchange the buffer. This can be achieved, for example, by dialysis or tangential flow filtration. Methods for producing lipid nanoparticles are described, for example, in U.S. Patent Application Publication No. 2015 / 0374842 and 2014 / 0107178, and are known in the art.

[0179] In certain embodiments, the LNP comprises a cationic liposome and a PEGylated lipid. The LNP further comprises one or more envelope lipids, e.g., For example, cationic lipids, structural or neutral lipids, sterols, PEGylated lipids, or combinations thereof. It may comprise a mixture.

[0180] Cationic lipids for use in LNPs are described, for example, in U.S. Patent Application Publication No. 2015 / 0374 As discussed in US Pat. Nos. 842 and 2014 / 0107178, Typically, the cationic lipid is a lipid that has a net positive charge at physiological pH. In certain embodiments, the cationic liposomes are made of a material selected from the group consisting of DODMA, DOT MA, DL-048, or DL-103. In certain embodiments, the above structures or or neutral lipid is DSPC, DPPC, or DOPC. In certain embodiments, the sterol is cholesterol. DMPE-PEG, DSPE-PEG, DSG-PEG, DMPE-PEG2K, DSP In one embodiment, the PEG is E-PEG2K, DSG-PEG2K, or DSG-mPEG. wherein the cationic lipid is DL-048 and the pegylated lipid is DSG-mPEG. , wherein the one or more envelope lipids are DL-103, DSPC, cholesterol For example, the double-stranded nucleic acid inhibitor molecules can be formulated using See FIG. 8, which illustrates one non-limiting embodiment of an LNP that can be used to stomach.

[0181] In certain embodiments, the triloop-containing double-stranded nucleic acid inhibitor molecule comprises the covalently conjugated to a ligand that directs delivery of the oligonucleotide to the tissue of interest. Many such ligands have been explored. See, for example, the above two The stranded nucleic acid inhibitor molecule is attached to one or more sugar ligand moieties (e.g., N-acetylglucosamine). Conjugated to lactosamine (GalNAc) and incorporated into the above oligonucleotide The antibody can be directed to the liver. See, e.g., U.S. Patent No. 5,994,517; U.S. Patent No. See WO2016 / 100401. In this embodiment, the one or more ligands are linked to the trilobal structure of the double-stranded nucleic acid inhibitor molecule. The nucleotides are conjugated to one or more nucleotides in the fragment.

[0182] In certain embodiments, the double-stranded nucleic acid inhibitor molecule comprises a nucleotide sequence in the tri-loop. In one embodiment, the glycoprotein is conjugated to two or three sugar ligand moieties. Two of the nucleotides in the reloop are conjugated to sugar ligand moieties. In another embodiment, three of the nucleotides in the tri-loop are linked to a sugar ligand moiety. In certain embodiments, the sugar ligand moiety is GalNA. In one embodiment, the sugar ligand moiety is GalNAc and the triglyceride is carboxymethyl. In one embodiment, Ga is conjugated to two of the nucleotides in the loop. The lNAc is conjugated to the AA nucleotide of the GAA triloop. Other ligands that can be used include, but are not limited to, mannose-6-phosphatase. esters, cholesterol, folate, transferrin, and galactose (among others) For specific exemplary ligands, see, for example, WO2012 / 089352. I want to be.

[0183] The ligand may be capable of directing delivery of the oligonucleotide to a tissue of interest. and may be conjugated to any portion of the nucleotide. The ligand (e.g., GalNAc) is attached to the nucleotide at the 2'-position of the sugar moiety. It is conjugated.

[0184] Administration / Treatment Methods One embodiment is a method of treating a disorder, comprising administering to a subject a therapeutically effective amount of a compound described herein. and administering a pharmaceutical composition comprising the tri-loop-containing double-stranded nucleic acid inhibitor molecule as described above. The present invention relates to the above method, which includes:

[0185] In certain embodiments, the pharmaceutical compositions disclosed herein are directed to treating proliferative, inflammatory, autoimmune, Symptoms related to sexual neurological, ophthalmological, respiratory, metabolic, skin, hearing, liver, kidney disease, or infection In one embodiment, the present invention may be useful for treating or preventing proliferative, inflammatory, autoimmune, Methods for treating sexual, neurological, ophthalmic, respiratory, metabolic, skin, hearing, liver, kidney, or infectious diseases - Patents.com administering to the subject a therapeutically effective amount of a double-stranded nucleic acid inhibitor molecule as described herein The method comprises administering a pharmaceutical composition comprising:

[0186] In certain embodiments, the disorder is a rare disease, a chronic liver disease, a chronic kidney disease, a cardiovascular In certain embodiments, the disorder is a primary hypertension or a viral infection. Hyperoxaluria, including oxaluria (PH1, PH2, or PH3) or idiopathic hyperoxaluria In certain embodiments, the disorder is chronic kidney disease (CKD). In certain embodiments, the disorder is pyruvate dehydrogenase deficiency. In certain embodiments, the disorder is alpha-1 antitrypsin (A1AT) deficiency. do.

[0187] In certain embodiments, the disorder is cancer. Non-limiting examples of such cancers include: , bile duct cancer, bladder cancer, transitional cell carcinoma, urothelial carcinoma, brain cancer, glioma, astrocytoma, breast cancer , metaplastic cancer, cervical cancer, squamous cell carcinoma of the cervix, rectal cancer, colorectal cancer, colon cancer, hereditary non-porous cancer. Liposis colon cancer, colorectal adenocarcinoma, gastrointestinal stromal tumor (GIST), endometrial cancer, endometrium Stromal sarcoma, esophageal cancer, esophageal squamous cell carcinoma, esophageal adenocarcinoma, intraocular melanoma, uveal melanoma, gallbladder cancer, Gallbladder adenocarcinoma, renal cell carcinoma, clear cell renal cell carcinoma, transitional cell carcinoma, urothelial carcinoma, Wilms' tumor, leukemia Acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), chronic myelomonocytic leukemia (CMML), liver cancer , liver carcinoma, hepatocellular carcinoma (hepatoma), hepatocellular carcinoma (hepatocellula r carcinoma), cholangiocarcinoma, hepatoblastoma, lung cancer, non-small cell lung cancer (NSCLC), mesothelial lymphoma, B-cell lymphoma, non-Hodgkin's lymphoma, diffuse large B-cell lymphoma, mantle cell lymphoma lymphoma, T-cell lymphoma, non-Hodgkin's lymphoma, precursor T-cell lymphoblastic lymphoma / leukemia disease, peripheral T-cell lymphoma, multiple myeloma, nasopharyngeal carcinoma (NPC), neuroblastoma, oropharynx Cancer, oral squamous cell carcinoma, osteosarcoma, ovarian cancer, pancreatic cancer, pancreatic ductal adenocarcinoma, pseudopapillary tumor, and acinar cell carcinoma These include: prostate cancer, prostate adenocarcinoma, skin cancer, melanoma, malignant melanoma, cutaneous melanoma, small intestine cancer, gastric cancer ), gastrointestinal stromal tumor (GIST), uterine cancer, or uterine sarcoma. Typically, the present disclosure provides a therapeutic The administration of a therapeutically effective amount of the pharmaceutical composition as described herein can be used to treat liver cancer, liver cancer, tumor, hepatocellular carcinoma (hepatoma), hepatocellular carcinoma (hepatocellular ca The present invention features methods for treating cholangiocarcinoma, cholangiocarcinoma, and hepatoblastoma.

[0188] In some embodiments, the present disclosure provides a method for reducing expression of a target gene in a subject. The method comprises administering to a subject in need thereof a pharmaceutical composition that reduces expression of the target gene. wherein the pharmaceutical composition comprises administering to a subject a therapeutically effective amount of a compound as described herein. and a tri-loop-containing double-stranded nucleic acid inhibitor molecule as described herein. Pharmaceutically acceptable Possible excipients and

[0189] The target gene can be a target gene from any mammal, for example, a human target gene. Any target gene can be silenced according to this method. In embodiments, for example, AGXT, GRHPR, HOGA1, HAO1, SERPINA The target genes, including LDHA, LDHA, and LDHA, are associated with chronic liver disease or chronic kidney disease. In certain embodiments, the target gene, which includes, for example, an HBV gene or an HCV gene, In certain embodiments, the target gene is associated with a viral infection. The target genes, including PCSK3 and PCSK9, are associated with cardiovascular disease. In embodiments, the target genes, including, for example, ALDH2, are involved in alcohol metabolism and liver function. Related to Noh.

[0190] Other exemplary target genes include, but are not limited to, KRAS, Factor VII, Eg5 , PCSK9, TPX2, apoB, SAA1, TTR, PDGF beta gene, Erb -B gene, Src gene, CRK gene, GRB2 gene, RAS gene, MEKK gene gene, JNK gene, RAF gene, Erk1 / 2 gene, PCNA (p21) gene, MYB gene, JUN gene, FOS gene, BCL-2 gene, cyclin D gene, VEGF gene, EGFR gene, cyclin A gene, cyclin E gene, WNT- 1 gene, beta-catenin gene, c-MET gene, PKC gene, NFKB gene , STAT3 gene, survivin gene, Her2 / Neu gene, topoisomerase I gene, topoisomerase II alpha gene, p73 gene, p21(WAF1 / CI P1) gene, p27(KIP1) gene, PPM1D gene, RAS gene, caveolith I gene, MIB I gene, MTAI gene, M68 gene, tumor suppressor gene These include mutations in the p53 tumor suppressor gene, and combinations thereof.

[0191] Dosage and Schedule Typically, the double-stranded nucleic acid inhibitor molecules are administered parenterally (e.g., intravenously, In other embodiments, the pharmaceutical composition is administered topically or intramuscularly. However, the pharmaceutical compositions disclosed herein can be delivered via, for example, The techniques include buccal, sublingual, rectal, vaginal, intraurethral, ​​topical, intraocular, intranasal, and / or intraauricular The administration can be carried out by any method known in the art, such as tablets, capsules, granules, etc. tablets, aqueous suspensions, gels, sprays, suppositories, salves, ointments ents) etc.

[0192] In certain embodiments, the double-stranded nucleic acid inhibitor molecule is administered to the recipient at least once a day. 20 micrograms to 10 milligrams per kilogram of body weight, 10 per kilogram 0 micrograms to 5 milligrams, 0.1 milligrams to 5.0 milligrams per kilogram Lamb, 0.25 milligrams to 5.0 milligrams per kilogram, or 1 kilogram Typically, the two drugs are administered at a dose of 0.2 to 3.0 milligrams per day. The interstranded nucleic acid inhibitor molecule is administered at a dose of approximately 0.25 milligrams per kilogram of recipient body weight per day. 10 mg to 2.0 mg per kilogram of recipient body weight per day, e.g., 0 mg per kilogram of recipient body weight per day 0.3 milligrams per kilogram of recipient body weight per day, and is administered at a dosage of 1 milligram per kilogram of recipient body weight per day.

[0193] The pharmaceutical compositions of the present disclosure can be administered daily or intermittently. Intermittent administration of stranded nucleic acid inhibitor molecules 1-6 days per week, 1-6 days per month, once per week, or every other week Once a month, once every two months, once every three months, or once or twice a year It can be administered once or divided into multiple annual, monthly, weekly, or daily doses. In some embodiments, intermittent dosing may include an initial administration of the double-stranded nucleic acid inhibitor molecule, a subsequent administration of the double-stranded nucleic acid inhibitor molecule, and a subsequent administration of the double-stranded nucleic acid inhibitor molecule. For up to 1 week, up to 1 month, up to 2 months, up to 3 months, or up to 6 months or more This may mean administration in cycles with a rest period without administration, or every other day, every other week, every other month, or every other day. It can mean administration every other day or every other year.

[0194] The therapeutically effective amount of the double-stranded nucleic acid inhibitor molecule will depend on the route of administration and the patient's physical characteristics, e.g. For example, the subject's size and weight, the extent of disease progression or invasion, the subject's age, health status, and may be gender dependent and may be adjusted, if necessary, depending on these and other factors. do. [Example]

[0195] Example 1: Synthesis of double-stranded nucleic acid inhibitor molecules containing triloops and tetraloops In vivo dose response Double-stranded nucleic acid inhibitors containing tetraloops and triloops in addition to various stem lengths have been developed. The bitter molecule was evaluated in a dose-response study. Female CD-1 mice were divided into study groups. Four female CD-1 mice were administered the test nucleic acid inhibitor molecule assigned to each group. Each of the five test nucleic acid inhibitor molecules was administered at 0.1 mg / kg, 0.2 mg / kg, and / kg, 0.4mg / kg, 0.8mg / kg, 1.6mg / kg, and 3.2mg / kg In addition, four control CD-1 mice received a placebo (PBS). The total sample size was 124 mice. Administration was subcutaneous and single dose, and 4 days after administration Mice were sacrificed at 0°C. Pharmacodynamic studies were performed and liver samples were taken for RT-qPCR. The tissue samples were lysed in QIAzol® Lysis reagent using the TissueLyser The mixture was homogenized using a RNase II (Qiagen, Valencia, CA). A was prepared according to the manufacturer's instructions (ThermoFisher Scientific, Waltham The cDNA was purified using MagMAX technology according to the High Capacity cDNA Reverse Transcription Kit (MA). (ThermoFisher Scientific, Waltham, MA) cDNA was prepared using the primers CFX384 for the target sequence. Real-Time PCR Detection System (Bio-Rad Laboratories, Inc., Hercules, CA).

[0196] The five test nucleic acid inhibitor molecules used in Example 1 (Constructs 1 to 5) ) are shown in Figures 2A-E. Conjugated to GalNAc and bicyclic nucleotides Except for the nucleotides in the loop that bind to the nucleic acid, all other nucleotides in the test nucleic acid inhibitor molecule Nucleotides can be either 2'-F or 2'-OMe at the 2'-position of the sugar moiety. The nucleic acid inhibitor molecules tested differed in the following respects: the loop region length (tetraloop vs. triloop); stem length (6 base pairs vs. 3 base pairs); the presence or absence of cyclic nucleotides; and the number of GalNAc in the loop (2 vs. 3). The nucleic acid inhibitor molecules in 2A-E are summarized in the following table: Table 1: Tested nucleic acid inhibitors in Figure 1 TIFF0007748500000030.tif57154

[0197] The long stem (6 base pairs) tetraloop constructs 1 and 2 are identical, with the only difference being is the number of GalNAc in the loop (3 vs. 2). System construct 2 is identical to construct 3, except that construct 2 has a tetraloop. Construct 3 has a tri-loop, whereas Construct 4 has a long stem. Construct 3 is identical to construct 4 except that construct 3 has 6 base pairs. Construct 4 has a base-paired stem duplex and no bicyclic nucleotides, while construct 5 has a base-paired stem duplex and no bicyclic nucleotides. , a three base pair stem duplex, in which all of the nucleotides in the stem duplex are bicyclic nucleotides Short stem constructs 4 and 5 are identical, the only difference being the The stem of Struct 4 has six BNAs NC [NMe] is present and construct 5 There are six LNA bicyclic nucleotides in the stem. The bicyclic nucleotide used in NC [NMe], wherein the bicyclic nucleoside The bridge connecting the 2'-carbon and 4'-carbon of the thiol is 4'-CH2-N(CH3)-O-2 The bicyclic nucleotide used in construct 5 is LNA, wherein: The bridge connecting the 2'-carbon and 4'-carbon of a bicyclic nucleotide is 4'-(CH2)- It's O-2'.

[0198] Nonlinear regression analysis (GraphPad Prism software) was used to calculate log[ mg / kg] compared to PBS, based on 50% percent target gene retention. Dose (ED 50 The tetraloop-long stem (6 base pairs) nucleic acid molecule was calculated using the triloop In addition, BNA NC [NMe]-containing triloop The short stem (3 base pair) nucleic acid inhibitor molecule is synthesized by combining the corresponding tri-loop short LNA-containing These were also compared to the triloop and stem nucleic acid molecules that do not contain bicyclic nucleotides. and tetraloop-length stem (6 base pairs in the stem) nucleic acid molecules. As shown in the figure, the corresponding tetramers containing either two or three GalNAc Compared to the loop nucleic acid molecules (constructs 1 and 2), the tri-loop-length stem nucleic acid inhibitor A bitter molecule (construct 3) and a triloop containing a bicyclic nucleotide in the short stem Both short stem nucleic acid molecules (constructs 4 and 5) exhibited similar efficacy in target gene knockdown. Showed down.

[0199] Example 2: Tetraloop- and triloop-containing polypeptides for targeted gene knockdown In vivo efficacy of double-stranded nucleic acid inhibitor molecules Female CD-1 mice were divided into study groups and administered the test nucleic acid inhibitors assigned to the groups. The test nucleic acid inhibitor molecules used in Example 2 (Constructs 6-1) were administered. 3) is shown in Figures 6A-H. Construct 1 (see Figure 2A) was also used. The nucleotides in the loop conjugated to GalNAc and bicyclic nucleotides Except for the nucleotide, all other nucleotides in the test nucleic acid inhibitor molecule have a sugar moiety At the 2'-position, it is modified with either 2'-OMe or 2'-F. The nucleic acid inhibitor molecules differed in the following respects: length of the loop portion (tetraloop vs. triloop); stem length (6 base pairs, 3 base pairs, 2 base pairs, and 1 base pair); and The nucleic acid inhibitor molecules in Figures 6A-H are classified as follows: Summarize in a table: Table 2: Tested nucleic acid inhibitors in Figure 6 TIFF0007748500000031.tif57142

[0200] The long stem (6 base pairs) constructs 6 and 7 are identical, with the only difference being the Construct 1 is the number of nucleotides (tetraloop vs. triloop) in the construct. Construct 1 is identical to Construct 6, except that Construct 1 is conjugated to the tetraloop Construct 6 contains three GalNAc residues constituting the tetraloop. Construct 8 (3 base pair stearyl amidophosphate) contains two GalNAc conjugated to the nucleotide sequence Construct 9 is identical to construct 9 (3 base pair stem), except that construct Construct 8 has a tetraloop, while construct 9 has a triloop. Constructs 10 and 11 (the 2 base pair stem) are identical, the only difference being the Construct 11 has a triloop while construct 12 has a tetraloop. Constructs 12 and 13 (1 base pair stem) are identical, the only difference being the Construct 12 has a tetraloop, while construct 13 has a triloop. The bicyclic nucleotides used in constructs 8 to 13 are BNA NC [NM e].

[0201] Animals were administered a single 0.5 mg / kg dose of the assigned test nucleic acid inhibitor molecule intradermally. The mice were sacrificed 4 days after administration. Liver tissue was biopsied using two 4 mm punches. The cells were collected by harvesting and then transferred to Invitrogen ( Trademark) RNAlater(TM) Solution (Thermo Fisher Scientif Tissue samples were stored in QIAzol® L (Waltham, MA). The lysis reagent was used in a TissueLyser II (Qiagen, Valencia, The RNA was then homogenized using the ThermoFish DNA polymerase (Cat. No. 1001226). MagMAX technology was used according to the manufacturer's instructions (Further Scientific, Waltham, MA). The cDNA was purified using a high-capacity cDNA reverse transcription kit (ThermoFisher Scientific cDNA was prepared using a PCR kit (Finific, Waltham, MA). Primers were used for PCR using the CFX384 Real-Time PCR Detector. tion System (Bio-Rad Laboratories, Inc., He It was used in the University of California, San Diego.

[0202] The tri-loop test nucleic acid inhibitor molecules (constructs 7, 9, 11, and 13) were prepared as follows: The corresponding tetraloop versions of the test nucleic acid molecules (constructs 1, 6, 8, 10, and 12). As demonstrated in Figure 7, the tri-loop-containing test Nucleic acid inhibitor molecules are available for 6 base pair, 3 base pair, and 2 base pair constructs: Similar knockdown of target gene mRNA compared to the corresponding tetraloop test nucleic acid molecule The nucleotide sequence shown is a tri-loop conjugated to two GalNAcs and Construct 11, which has two base pairs in the stem, is conjugated to three GalNAc. Construct containing a triloop supporting 6 base pairs in the stem 1. Constructs containing a triloop with one base pair in the stem 13 did not show knockdown of target gene mRNA, but Construct 12, which contains a single base pair in the showed.

Claims

1. 1. A double-stranded nucleic acid inhibitor molecule comprising: a sense strand comprising 20-65 nucleotides and having a first region (R1) and a second region (R2); an antisense strand comprising 15 to 40 nucleotides, wherein the sense strand and the antisense strand are separate strands; and a first duplex (D1) formed by the first region of the sense strand and the antisense strand, the first duplex having a length of 15 to 40 base pairs; Including; In this case, the second region of the sense strand comprises a first subregion (S1), a second subregion (S2), and a tri-loop (triL) connecting the first and second regions, and the first and second regions form a second duplex (D2), in the double-stranded nucleic acid inhibitor molecule.

2. 10. The double-stranded nucleic acid inhibitor molecule of claim 1, wherein the second duplex comprises at least one bicyclic nucleotide.

3. 3. The double-stranded nucleic acid inhibitor molecule of claim 1 or 2, wherein each nucleotide in the second duplex is a bicyclic nucleotide.

Images