RNA silencing agents and methods of use

Abstract

An aspect of the present disclosure provides the nucleic acid for reducing the expression of target RNA.In some aspects, the present disclosure provides nucleic acid modification and base pairing configurations that are useful in the design of nucleic acid for RNA interference.The present disclosure provides the nucleic acid design strategy that can be useful in the design of RNA silencing agent, and relates to the discovery that the effective reduction of target RNA level can be achieved by using antisense strand that is configured to mediate wobble base pairing between its 14th nucleotide and its target RNA.

Description

[Technical field]

[0001] (CROSS REFERENCE TO RELATED APPLICATIONS) This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application No. 63 / 174,507, filed April 13, 2021, which is hereby incorporated by reference in its entirety.

[0002] (Reference to sequence listing submitted as a text file) This application contains a Sequence Listing, which has been submitted in ASCII format and is hereby incorporated by reference in its entirety. This ASCII copy, created on April 12, 2022, is named A127870007WO00-SEQ-JIB and is 10,192 bytes in size. [Background technology]

[0003] (background) The field of RNA interference (RNAi) has received considerable interest in recent years. This is because RNA silencing agents offer the ability to knock down the expression of specific proteins with a high degree of sequence specificity. RNAi has been useful in scientific research, for example, to study genetic and biochemical pathways, to elucidate the function of individual genes and gene products, and as a tool for target validation in the pharmaceutical industry. Furthermore, considerable efforts are being made with the goal of developing RNA silencing agents that can be mediated by RNA as a therapeutic strategy. Summary of the Invention [Means for solving the problem]

[0004] (Abstract) Among other aspects, the present disclosure provides nucleic acid design strategies that can be useful in designing RNA silencing agents.In some aspects, the present disclosure relates to the discovery that the effective reduction of target RNA levels can be achieved by using antisense strands that are configured to mediate wobble base pairing between the 14th nucleotide of the antisense strand and the target RNA.Thus, in some aspects, the present disclosure provides a nucleic acid comprising an antisense strand, which has a nucleotide at the 14th position from its 5' end that forms wobble base pairing with the target nucleotide at the corresponding position on the target mRNA.

[0005] In some aspects, the disclosure provides a nucleic acid for reducing expression of a target mRNA, the nucleic acid comprising an antisense strand 15 to 31 nucleotides in length having a sequence that is at least 90% complementary to a contiguous sequence of the target mRNA, the sequence of the antisense strand comprising an abasic site at position 14 from its 5' end, or the sequence of the antisense strand comprising a nucleotide at position 14 from its 5' end that does not form a canonical (e.g., Watson-Crick) base pair with a target nucleotide at the corresponding position on the contiguous sequence of the target mRNA.

[0006] In some embodiments, the nucleotide at position 14 on the antisense strand and the target nucleotide at the corresponding position on the target mRNA are mismatched (e.g., the nucleotide forms a mismatched base pair (e.g., a wobble base pair)). In some embodiments, the mismatched base pair is a wobble base pair. For example, in some embodiments, the nucleotide at position 14 on the antisense strand forms a wobble base pair with the target nucleotide. In some embodiments, the target nucleotide comprises either a cytidine or a guanosine. In some embodiments, the nucleotide at position 14 on the antisense strand comprises either an inosine or a uridine. In some embodiments, the wobble base pair is I:C or U:G. In some embodiments, if the target nucleotide comprises a cytidine, the nucleotide at position 14 on the antisense strand comprises an inosine. In some embodiments, if the target nucleotide comprises a guanosine, the nucleotide at position 14 on the antisense strand comprises a uridine.

[0007] In some embodiments, the antisense strand comprises at least one modified nucleotide and / or at least one modified internucleotide linkage. In some embodiments, the antisense strand comprises one or more nucleoside modifications selected from 2'-aminoethyl, 2'-fluoro, 2'-O-methyl, 2'-O-methoxyethyl, and 2'-deoxy-2'-fluoro-β-d-arabinonucleic acid. Further examples of nucleoside modifications are described elsewhere herein and include modified sugars (e.g., 2'-O substitutions on the sugar (e.g., ribose), including 2'-O-methoxyethyl sugars, 2'-fluoro sugar modifications (2'-fluoro), 2'-O-methyl sugars (2'-O-methyl), 2'-O-ethyl sugars, 2'-Cl, 2'-SH and substitutions thereof (e.g., 2'-SCH3), bicyclic sugar moieties) or substitutions (e.g., replacement of the 2'-O moiety with a lower alkyl or substitutions thereof (e.g., -CH3, -CF3), replacement of the 2'-O moiety with a 2'-amino or substitutions thereof, 2',3'-seconucleotide mimics, 2'-F-arabinonucleotides, inverted nucleotides, 2'-F-arabino ... Examples of suitable nucleotides include, but are not limited to, inverted nucleotides, inverted 2'-O-methyl nucleotides, 2'-O-deoxy nucleotides, substitution of 2'-O moieties with alkenyl, substitution of 2'-O moieties with alkynyl, substitution of 2'-O moieties with methoxyethyl (2'-O-MOE), substitution of 2'-O moieties with -H (as in DNA), or substitution of 2'-O moieties with other substituents. In some embodiments, the antisense strand comprises at least one phosphorothioate internucleotide linkage. In some embodiments, the sequence of the antisense strand is 100% complementary to the contiguous sequence of the target mRNA, except for the nucleotides that form the wobble base pair.

[0008] In some embodiments, the antisense strand is 15 to 25 nucleotides in length (e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length). In some embodiments, the antisense strand is 19 to 25 nucleotides in length. In some embodiments, the antisense strand is 21 nucleotides in length. In some embodiments, the sequence of the antisense strand is at least 80% identical to a nucleotide sequence in Table 1. In some embodiments, the sequence of the antisense strand is at least 85% identical (e.g., at least 90% identical, at least 95% identical, or 100% identical) to a nucleotide sequence in Table 1. In some embodiments, the sequence of the antisense strand is at least 80% identical to any one of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, and 50. In some embodiments, the sequence of the antisense strand is at least 85% identical (e.g., at least 90% identical, at least 95% identical, or 100% identical) to any one of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, and 50.

[0009] In some embodiments, the nucleic acid further comprises a sense strand that is 15 to 40 nucleotides in length (e.g., 15 to 35 nucleotides in length, 15 to 30 nucleotides in length, 15 to 25 nucleotides in length, 19 to 30 nucleotides in length, 19 to 25 nucleotides in length, or 25 to 30 nucleotides in length). In some embodiments, the sense strand forms a duplex region with the antisense strand. In some embodiments, the duplex region comprises canonical or non-canonical base pairing between the nucleotide on the sense strand and the nucleotide at position 14 on the antisense strand. In some embodiments, when the nucleotide at position 14 on the antisense strand comprises an inosine, the nucleotide on the sense strand comprises a cytidine, an adenosine, or a uridine. In some embodiments, when the nucleotide at position 14 on the antisense strand comprises a uridine, the nucleotide on the sense strand comprises an adenosine. In some embodiments, the sequence of the sense strand is at least 80% identical to any one of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, and 49. In some embodiments, the sequence of the sense strand is at least 85% identical (e.g., at least 90% identical, at least 95% identical, or 100% identical) to any one of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, and 49.

[0010] In some embodiments, the sense strand comprises at least one modified nucleotide and / or at least one modified internucleotide linkage. In some embodiments, the sense strand comprises one or more nucleoside modifications selected from 2'-aminoethyl, 2'-fluoro, 2'-O-methyl, 2'-O-methoxyethyl, and 2'-deoxy-2'-fluoro-β-d-arabinonucleic acid. Further examples of nucleoside modifications and modified nucleotides are described elsewhere herein. In some embodiments, the sense strand comprises at least one phosphorothioate internucleotide linkage. In some embodiments, the sense strand is linked to at least one N-acetylgalactosamine (GalNAc) moiety.

[0011] In some aspects, the disclosure provides a nucleic acid for reducing expression of a target mRNA, the nucleic acid having formula (I): 5'-X 01 X 02 X 03 X 04 X 05 X 06 X 07 X 08 X 09 X 10 X 11 X 12 X 13 X 14 (X Y ) b N a -3'(I) Contains the antisense strand of N and X Y each is independently any type of nucleotide; a is an integer from 0 to 2; b is an integer from 1 to 17; X 01 ~X 13 are each independently any type of nucleotide, provided that X 01 ~(X Y ) b is at least 90% complementary to a contiguous nucleotide sequence of the target mRNA; 14 is an abasic site or X 14is a nucleotide that does not form a canonical (eg, Watson-Crick) base pair with a target nucleotide at a corresponding position on the contiguous nucleotide sequence of the target mRNA.

[0012] In some embodiments, the "X" nucleotide of formula (I) refers to a nucleotide that forms a region of complementarity with the target mRNA, as described elsewhere herein. In some embodiments, the "N" nucleotide of formula (I) refers to an optional nucleotide outside the region of complementarity. In some embodiments, when the nucleic acid further comprises a sense strand in a duplex with the antisense strand of formula (I), the "N" nucleotide refers to an optional nucleotide that forms an overhang, as described elsewhere herein.

[0013] In some embodiments, a is an integer from 1 to 2. In some embodiments, a is 0. In some embodiments, b is an integer from 1 to 11. In some embodiments, b is an integer from 5 to 11. In some embodiments, b is 7. In some embodiments, X 01 ~(X Y ) b The sequence of X is at least 80% identical to the nucleotide sequence of Table 1. 01 ~(X Y ) b is at least 85% identical (eg, at least 90% identical, at least 95% identical, or 100% identical) to a nucleotide sequence in Table 1.

[0014] In some embodiments, X 01 ~(X Y ) b The sequence of X is at least 95% complementary to a contiguous nucleotide sequence of the target mRNA. 01 ~(X Y ) b The array of X 14 (i) X 14contains an inosine and the target nucleotide at the corresponding position on the target mRNA contains a cytidine; or (ii) X 14 comprises a uridine and the target nucleotide comprises a guanosine. 01 ~X 21 The array of X 14 (i) X 14 contains an inosine and the target nucleotide contains a cytidine; or (ii) X 14 comprises a uridine and the target nucleotide comprises a guanosine. 01 ~(X Y ) b The sequence of X above is 14 Except X 01 100% complementary to the naturally occurring contiguous nucleotide sequence of the target mRNA, except for X 01 and the nucleotide at the corresponding position on the target mRNA contain a mismatched base pair.

[0015] In some embodiments, the target nucleotide comprises either a cytidine or a guanosine. 14 and the target nucleotide comprises a mismatched base pair. In some embodiments, the mismatched base pair is a wobble base pair. In some embodiments, X 14 is a nucleotide that forms a wobble base pair with the target nucleotide. 14 contains either inosine or uridine. In some embodiments, the wobble base pair is I:C or U:G. In some embodiments, when the target nucleotide contains a cytidine, X 14 In some embodiments, when the target nucleotide comprises guanosine, X 14 contains uridine.

[0016] In some embodiments, X 01and the nucleotide at the corresponding position on the target mRNA comprises a mismatched base pair. In some embodiments, the mismatched base pair is A:G or U:C. In some embodiments, X 01 contains either adenosine or uridine. In some embodiments, X 01 In some embodiments, X comprises an adenosine when the nucleotide at the corresponding position on the target mRNA comprises a cytidine. 01 contains uridine.

[0017] In some embodiments, the antisense strand of formula (I) comprises at least one modified nucleotide and / or at least one modified internucleotide bond. In some embodiments, the antisense strand comprises one or more nucleoside modifications selected from 2'-aminoethyl, 2'-fluoro, 2'-O-methyl, 2'-O-methoxyethyl, and 2'-deoxy-2'-fluoro-β-d-arabinonucleic acid. In some embodiments, the antisense strand comprises at least one phosphorothioate internucleotide bond. In some embodiments, the nucleic acid further comprises at least one targeting moiety (e.g., N-acetylgalactosamine (GalNAc)) attached to the antisense strand of formula (I). In some embodiments, at least one targeting moiety is attached to the antisense strand by a cleavable linker.

[0018] In some embodiments, the nucleic acid further comprises a sense strand between 15 and 40 nucleotides in length, the sense strand forming a duplex region with the antisense strand. In some embodiments, the duplex region is a region between nucleotides on the sense strand and X 14 In some embodiments, X 14When X comprises inosine, the nucleotides on the sense strand comprise cytidine, adenosine, or uridine. 14 contains a uridine, then the nucleotides on the sense strand contain adenosine. In some embodiments, the duplex region excludes any occurrence of N.

[0019] In some embodiments, the antisense strand has formula (II): 5'-X 01 X 02 X 03 X 04 X 05 X 06 X 07 X 08 X 09 X 10 X 11 X 12 X 13 X 14 X 15 X 16 X 17 X 18 X 19 X 20 X 21 -3'(II) is the antisense strand of X 01 ~X 13 and X 15 ~X 21 are each independently any type of nucleotide, provided that X 01 ~X 21 is at least 90% complementary to a contiguous nucleotide sequence of the target mRNA; 14 is a nucleotide containing inosine or uridine.

[0020] In some embodiments, the nucleic acid of the present disclosure is a small interfering RNA (siRNA). In some embodiments, the nucleic acid is a short hairpin RNA (shRNA).

[0021] In some aspects, the present disclosure provides compositions comprising the nucleic acids described herein and counterions. In some aspects, the present disclosure provides compositions comprising the nucleic acids described herein and a pharma- ceutically acceptable carrier.

[0022] In some aspects, the present disclosure provides a method for reducing the expression of a target mRNA in a cell. In some embodiments, the method comprises contacting the cell with a nucleic acid or composition of the present disclosure. In some embodiments, the cell is a mammalian cell. In some embodiments, the mammalian cell is a human cell or a non-human primate cell. In some embodiments, the cell is contacted with the nucleic acid or composition in vivo. In some embodiments, the cell is contacted with the nucleic acid or composition in vitro. In some embodiments, the target mRNA encodes a mutant protein. In some embodiments, the mutant protein comprises one or more mutations compared to a wild-type variant. In some embodiments, the target mRNA encodes a protein that is overexpressed in the cell. In some embodiments, the protein is overexpressed compared to a reference expression level (e.g., compared to a wild-type variant, compared to a normal healthy cell). In some embodiments, the target mRNA is a transcript of a gene selected from angiotensinogen (AGT), proprotein convertase subtilisin / kexin type 9 (PCSK9), complement factor B, diacylglycerol O-acyltransferase 2 (DGAT2), and microtubule-associated protein tau (MAPT). In some embodiments, the gene encodes a mutant protein compared to the corresponding wild-type sequence. In some embodiments, the gene encodes a wild-type protein.

[0023] In some aspects, the present disclosure provides a method of treating a subject. In some embodiments, the method comprises administering to the subject a nucleic acid of the present disclosure. In some embodiments, the subject is known to have or is suspected to have a disease or condition associated with the target mRNA of the nucleic acid. In some embodiments, the subject is known to have or is suspected to have the target mRNA. In some embodiments, the subject is a human. In some embodiments, the subject is a non-human animal (e.g., a mouse, a rat, a rabbit, a dog, a cat, a pig, or a non-human primate, such as a monkey or a chimpanzee). In some embodiments, the target mRNA encodes a mutant protein. In some embodiments, the mutant protein comprises one or more mutations compared to a wild-type variant. In some embodiments, the target mRNA encodes a protein that is overexpressed in the cell. In some embodiments, the protein is overexpressed compared to a reference expression level (e.g., compared to a wild-type variant, compared to a normal healthy cell). In some embodiments, the subject is known to have or is suspected to have a disease or condition associated with a gene selected from angiotensinogen (AGT), proprotein convertase subtilisin / kexin type 9 (PCSK9), complement factor B, diacylglycerol O-acyltransferase 2 (DGAT2), and microtubule-associated protein tau (MAPT). In some embodiments, the target mRNA is a transcript of the gene. In some embodiments, the gene encodes a mutant protein compared to the corresponding wild-type sequence. In some embodiments, the gene encodes a wild-type protein.

[0024] Details of certain embodiments of the invention are set forth in the detailed description below. Other features, objects, and advantages of the invention will be apparent from the examples, drawings, and claims.

[0025] BRIEF DESCRIPTION OF THE DRAWINGS The accompanying drawings, which form a part of this specification, illustrate several non-limiting embodiments of the invention and, together with the description, serve to explain the principles of the invention. [Brief description of the drawings]

[0026] [Figure 1] FIG. 1 shows an exemplary nucleic acid structure in which the antisense strand is duplexed with the sense or target RNA strand.

[0027] [Diagram 2] FIG. 2 shows an exemplary formula for a nucleic acid having a sense strand (shown 5' to 3') and an antisense strand (shown 3' to 5').

[0028] [Figure 3A-3B] 3A-B show results from an in vivo study of siRNA (RD1354) in cynomolgus monkeys. Results for individual monkeys are shown in Figure 3A, and averaged results for the group are shown in Figure 3B. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0029] (Detailed Description) Aspects of the present disclosure relate to the discovery that effective reduction of target RNA levels can be achieved using antisense strands that are configured to mediate non-canonical interactions (e.g., mismatch interactions, e.g., wobble base pairing) between the 14th nucleotide and the target RNA. In some aspects, the present disclosure provides new strategies for the design of effective antisense molecules, complementing conventional guidelines to allow a greater number and variety of potential target mRNA sites than ever before without sacrificing efficiency.

[0030] The efficiency of small interfering RNA (siRNA) molecule depends on various factors, including the availability of target, the secondary structure of mRNA, the matching position of siRNA and mRNA, and the inherent characteristics of siRNA and mRNA.The precise design of siRNA is a crucial step due to the fact that only a few nucleotide changes in its sequence can change its function.

[0031] The present inventors have recognized and understood that conventional siRNA design strategies follow certain rules that may limit the number and types of possible RNA target sites. In some aspects, the present disclosure overcomes some of these limitations by providing a nucleic acid comprising an antisense strand that has an abasic site or a nucleotide at position 14 from its 5' end that does not form a canonical base pair with the target nucleotide at the corresponding position of the target RNA. In some embodiments, a non-canonical interaction (e.g., a wobble base pair) is formed between the 14th nucleotide and a G or C nucleotide on the target RNA. Thus, in some embodiments, the wobble base pair and other non-canonical interactions of the present disclosure provide an alternative design strategy to the traditional preference for A or U nucleotides at this position on the target molecule. In some aspects, the present disclosure relates to the surprising discovery that nucleic acids that form such wobble base pairs with the target RNA have resulted in a highly effective reduction of target RNA levels.

[0032] Figure 1 shows an exemplary nucleic acid structure, in which antisense strand (dotted shape) is in a duplex state with sense strand or target strand (solid shape).In some embodiments, the nucleic acid comprising antisense strand in a duplex state with sense strand can be generally referred to as RNA silencing agent herein.Examples of RNA silencing agent are provided elsewhere herein, and examples of RNA silencing agent include, but are not limited to, siRNA and shRNA.

[0033] The RNA silencing agent 100 is shown with an antisense strand (in dotted form) in a duplex with a sense strand (in plain form). As used herein, in some embodiments, the antisense strand of an RNA silencing agent refers to a strand that has a region of complementarity with a target strand (e.g., a target RNA, e.g., an mRNA). In some embodiments, the region of complementarity has a nucleotide sequence that is sufficiently complementary to the desired target strand to induce target-specific silencing (e.g., sufficiently complementary to induce destruction of the desired target strand by the RNAi mechanism or process (RNAi interference), or sufficiently complementary to induce translational suppression of the desired target mRNA).

[0034] As shown, the RNA silencing agent 100 comprises an inosine at nucleotide 14 on the antisense strand. As used herein, in some embodiments, nucleotide 14 refers to a nucleotide on the antisense strand that can form a non-canonical interaction (e.g., a wobble base pair) with a G or C nucleotide at the corresponding position on the target strand. In some embodiments, the nucleotide at nucleotide 14 on the antisense strand is numbered relative to its 5' end, and the 5'-most nucleotide on the antisense strand can be named nucleotide 1. In some embodiments, the nucleotide at nucleotide 14 on the antisense strand is numbered relative to its region of complementarity with the target strand, and the 5'-most nucleotide of the region of complementarity can be named nucleotide 1. For example, in some embodiments, the RNA silencing agent comprises an antisense strand having one or more nucleotides in the 5' overhang region relative to the sense strand. In this situation, the nucleotide at nucleotide 14 is numbered relative to the 5'-most nucleotide that is not present in the overhang region, and this latter 5'-most nucleotide can be named nucleotide 1.

[0035] As generally shown, the inosine at nucleotide 14 on the antisense strand of the RNA silencing agent 100 forms a wobble base pair with the cytidine at the corresponding position on the sense strand. As an example, the RNA silencing agent 100 shows a cytidine at the corresponding position on the sense strand, but other nucleosides can be utilized at this position. For example, inosine can form a wobble base pair with cytidine, adenosine, or uridine, so the nucleotide at the corresponding position on the sense strand can include any one of these nucleosides. However, as described herein, the nucleotide at nucleotide 14 can advantageously form a non-canonical interaction (e.g., a wobble base pair) with the corresponding position on the target RNA. Thus, it should be understood that in the context of the RNA silencing agent 100, the nucleotide at nucleotide 14 on the antisense strand does not need to form a wobble base pair with the nucleotide at the corresponding position on the sense strand. For example, in some embodiments, the nucleotide at the corresponding position on the sense strand of the RNA silencing agent comprises a nucleoside that does not base pair with the inosine at nucleotide position 14. Thus, in some embodiments, the corresponding position on the sense strand of the RNA silencing agent 100 can comprise any nucleoside (e.g., adenosine, guanosine, cytidine, uridine, thymidine, inosine, or analogs thereof) that may or may not base pair with the inosine at nucleotide position 14.

[0036] Target duplex 102 shows the antisense strand (dotted shape) of the RNA silencing agent 100 in a duplex with a target strand (solid shape). In some embodiments, the target strand is a target RNA (e.g., mRNA). As generally shown, the inosine at nucleotide 14 on the antisense strand forms a wobble base pair with a cytidine at the corresponding position on the target strand. In accordance with the present disclosure, the I:C wobble base pair provides an advantageous alternative to the otherwise unfavorable G:C base pair at this position. As described herein, in some embodiments, the antisense strand includes a region of complementarity, which refers to the nucleotides of the antisense strand that base pair with the nucleotides of the target strand.

[0037] In some embodiments, position 14 on the antisense strand may include an abasic site, or position 14 on the antisense strand may include a nucleotide that does not form a canonical base pair with the target nucleotide at the corresponding position on the target strand. Target duplex 102 shows an example where an inosine at position 14 on the sense strand forms a wobble base pair with a cytidine at the corresponding position on the target strand. It should be understood that in some embodiments, a wobble base pair is an example of a mismatch base pair according to the present disclosure. Thus, in some embodiments, when the target nucleotide includes a cytidine, the nucleotide at position 14 includes a nucleoside other than guanosine. For example, in some embodiments, the target nucleotide includes a cytidine and the nucleotide at position 14 includes an adenosine, a uridine, or a cytidine. However, in some embodiments, position 14 on the antisense strand includes an abasic site such that no nucleobase is present at this position.

[0038] The RNA silencing agent 110 is shown with the antisense strand (in dotted form) in a duplex with the sense strand (in solid form), with nucleotide 14 comprising a uridine. In this example, the sense strand of the RNA silencing agent 110 comprises an adenosine at a position corresponding to the uridine at nucleotide 14. As described with respect to the RNA silencing agent 100, nucleotide complementarity at this position is not a requirement for the RNA silencing agent 110, since the advantages described herein relate to non-canonical interactions (e.g., wobble base pairs) formed at this position in the context of the target duplex. Thus, in some embodiments, the corresponding position on the sense strand of the RNA silencing agent 110 may comprise any nucleoside (e.g., adenosine, guanosine, cytidine, uridine, thymidine, inosine, or analogs thereof) that may or may not base pair with the uridine at nucleotide 14.

[0039] Target duplex 112 shows the antisense strand (dotted shape) of RNA silencing agent 110 in duplex with a target strand (solid shape). In some embodiments, the target strand is a target RNA (e.g., mRNA). As generally shown, a uridine at nucleotide 14 on the antisense strand forms a wobble base pair with a guanosine at the corresponding position on the target strand. In accordance with the present disclosure, the U:G wobble base pair provides an advantageous alternative to the otherwise unfavorable C:G base pair at this position.

[0040] Target duplex 112 shows an example where a uridine at position 14 on the sense strand forms a wobble base pair with a guanosine at the corresponding position on the target strand. It should be understood that in some embodiments, a wobble base pair is an example of a mismatch base pair according to the present disclosure. Thus, in some embodiments, when the target nucleotide comprises a guanosine, the nucleotide at position 14 comprises a nucleoside other than cytidine. For example, in some embodiments, the target nucleotide comprises a guanosine and the nucleotide at position 14 comprises an adenosine, guanosine, or uridine. However, in some embodiments, position 14 on the antisense strand comprises an abasic site such that no nucleobase is present at this position.

[0041] The nucleic acid structure in FIG. 1 is shown generally, and the nucleic acid structure in FIG. 1 should not be interpreted as a limitation to the present disclosure. For example, RNA silencing agents 100 and 110 are each shown as having an antisense strand that is 21 nucleotides long and is fully complementary to the sense strand. Similarly, target duplexes 102 and 112 are each shown as having an antisense strand that is 21 nucleotides long and is fully complementary to the target strand. It should be understood that these examples are provided for illustrative purposes, and that, as described elsewhere herein, the antisense strand or the sense strand can be longer or shorter than 21 nucleotides long, and the degree of complementarity of the RNA silencing agent or the target duplex can be less than 100%.

[0042] FIG. 2 shows an exemplary formula for an RNA silencing agent having a sense strand (shown 5' to 3') and an antisense strand (shown 3' to 5'). The variables N, X, and Z represent individual nucleotides, and the variables a and b are defined herein. In some embodiments, the RNA silencing agent is (Z Y ) b ~Z 14 The sense strand in (X Y ) b ~X 01In some embodiments, b is an integer between 1 and 17. By way of example, for a duplex region 21 nucleotides long (e.g., the duplex regions shown for RNA silencing agents 100 and 110), b is 7.

[0043] In some embodiments, (X Y ) b and X 13 ~X 01 are each independently any type of nucleotide, provided that (X Y ) b ~X 01 (Z Y ) b ~Z 14 At least 80% complementary to. Thus, in some embodiments, the duplex region of an RNA silencing agent refers to a sequence of a sense strand and a sequence of an antisense strand that are at least 80% complementary (e.g., at least 85% complementary, at least 90% complementary, at least 95% complementary, or 100% complementary).

[0044] In some embodiments, the RNA silencing agent is N aIn some embodiments, a is independently an integer between 0 and 2, such that the RNA silencing agent may optionally include at least one overhang of up to 2 nucleotides. As used herein, in some embodiments, an overhang refers to a terminal non-base paired nucleotide that originates from a strand or region and extends beyond the end of the complementary strand with which the strand or region forms a duplex. In some embodiments, the overhang comprises one or more unpaired nucleotides that extend from a duplex region at the 5' or 3' end of the RNA silencing agent. In some embodiments, the overhang is a 5' overhang or a 3' overhang on the antisense or sense strand of the RNA silencing agent. In some embodiments, the RNA silencing agent comprises a 5' overhang and a 3' overhang on the sense strand. In some embodiments, the RNA silencing agent comprises a 3' overhang on the sense strand and a 3' overhang on the antisense strand. In some embodiments, RNA silencing agent comprises 3' overhang on sense strand and 3' overhang on antisense strand, and does not comprise 5' overhang on sense strand or 5' overhang on antisense strand.Although not shown, it should be understood that in some embodiments, RNA silencing agent having 3' overhang on antisense strand can be configured so that its 3' overhang can be removed (e.g., cleaved) from its RNA silencing agent.

[0045] In some embodiments, the RNA silencing agent comprises at least one stem loop. In some embodiments, a is independently an integer between 0 and 30, such that the RNA silencing agent can optionally comprise at least one stem loop of up to 30 nucleotides. Thus, in some embodiments, an "N" nucleotide refers to an optional nucleotide at either or both ends of the nucleic acid that forms a stem loop. For example, in some embodiments, an N nucleotide at the 5' end of one strand and an N nucleotide at the 3' end of the other strand are covalently linked via a stem loop having a stem region and a loop region. In some embodiments, the stem region comprises a duplex between about 1 base and about 26 bases in length. In some embodiments, the loop region comprises a single-stranded portion between about 4 nucleotides and about 10 nucleotides in length.

[0046] In some embodiments, the RNA silencing agent is X 14 or the RNA silencing agent contains an abasic site, denoted by X in FIG. 14 As shown, Z includes nucleotides that do not form canonical base pairs with a target nucleotide at a corresponding position in a target strand (e.g., a target RNA, e.g., an mRNA), as indicated by 01 is an X on the antisense strand 14 In some embodiments, X is a nucleotide on the sense strand at a position corresponding to 14 is an abasic site. 14 is adenosine, inosine, or uridine. 01 is any type of nucleotide. 01 is guanosine, cytidine, adenosine, or uridine. 14 is inosine, and Z 01 is cytidine, adenosine, or uridine. 14 is uridine, and Z 01is adenosine or guanosine.

[0047] In some embodiments, the antisense strand of the RNA silencing agent in FIG. 2 is the antisense strand of formula (I) described elsewhere herein.

[0048] As described herein, in some embodiments, an RNA silencing agent refers to a nucleic acid comprising an antisense strand that has sufficient complementarity with a target strand (e.g., a target RNA sequence) to mediate an RNA-mediated silencing mechanism (e.g., RNAi). In some embodiments, the nucleic acid is a duplex molecule (or a molecule with a duplex-like structure) that comprises a sense strand and a complementary antisense strand (or a portion thereof). In some embodiments, the antisense strand comprises a nucleotide at position 14 from its 5' end that forms a wobble base pair with the nucleotide at the corresponding position on the target strand. In some embodiments, the nucleotide at position 14 comprises a nucleoside selected from inosine and uridine.

[0049] In some embodiments, the term nucleoside refers to a molecule having a purine or pyrimidine base covalently linked to a ribose or deoxyribose sugar. A nucleoside consists of a nucleic acid base (e.g., a nitrogenous base (e.g., a nucleic acid base)) and a pentose sugar (e.g., ribose). The pentose sugar can be either ribose or deoxyribose. Nucleosides are the biochemical precursors of nucleotides, which are the building blocks of RNA and DNA. The term "nucleotide," as used herein, refers to a nucleic acid base and a pentose sugar (i.e., a nucleoside) and one or more phosphate groups. In a nucleoside, the anomeric carbon is linked to the N9 of a purine or the N1 of a pyrimidine via a glycosidic bond. Examples of nucleosides and nucleobases include, but are not limited to, cytidine (C), uridine (U), adenosine (A), guanosine (G), thymidine (T), and inosine (I), but since a nucleoside comprises a nucleobase and a 5-carbon sugar, the term should also be understood to describe nucleosides resulting from modifications (as such term is defined herein).For example, nucleosides include natural nucleosides (e.g., deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine), nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolopyrimidine, 3-methyladenosine, 5-methylcytidine, C5 bromouridine, C5 fluorouridine, C5 iodouridine, C5 propynyluridine, C5 propynylcytidine, C5 methyl ur ... Cytidine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, 4-acetylcytidine, 5-(carboxyhydroxymethyl)uridine, dihydrouridine, methylpseudouridine, 1-methyladenosine, 1-methylguanosine, N6-methyladenosine, and 2-thiocytidine), chemically modified bases, biochemically modified bases (e.g., methylated bases), intercalated bases, rcalated bases, modified sugars (e.g., 2'fluororibose, ribose, 2'deoxyribose, 2'-O-methylcytidine, arabinose, and hexoses), or modified phosphate groups (e.g., phosphorothioate and 5'-N-phosphoramidite linkages), xanthine, hypoxanthine, nubularine, isoguanisine, tubercidin, 2-aminopurine, 2,6-diaminopurine, 3-deoxypurine, 4-aminopurine, 5-aminopurine, 6-aminopurine, 7-aminopurine, 8-aminopurine, 9-aminopurine, 10-aminopurine, 11-aminopurine, 12-aminopurine, 13-aminopurine, 14-aminopurine, 15-aminopurine, 16-aminopurine, 17-aminopurine, 18-aminopurine, 19-aminopurine, 20-aminopurine, 21-aminopurine, 22-aminopurine, 23-aminopurine, 24-aminopurine, 25-aminopurine, 26-aminopurine, 27-aminopurine, 28-aminopurine, 29-aminopurine, 30-aminopurine, 31-aminopurine, 32-aminopurine, 33-aminopurine, 34-aminopurine, 35-aminopurine, 36-aminopurine, 37-aminopurine, 38-aminopurine, 39-aminopurine, 40-aminopurine, 41-aminopurine, 42-aminopurine, 43-aminopurine, 44-aminopurine, 45-aminopurine, 46-aminopurine, 47-aminopurine, 48-aminopurine, 49-aminopurine, 50-aminopurine, The term nucleotide includes azaadenosine, 7-deazaadenosine, 7-methyladenosine, 8-azidoadenosine, 8-methyladenosine, 5-hydroxymethylcytosine, 5-methylcytidine, pyrrolocytidine, 7-aminomethyl-7-deazaguanosine, 7-deazaguanosine, 7-methylguanosine, 8-aza-7-deazaguanosine, thienoguanosine, inosine, 4-thio-uridine, 5-methoxyuridine, dihydrouridine, and pseudouridine. In some embodiments, the term nucleotide refers to a nucleoside having one or more phosphate groups attached to its sugar moiety by ester linkage. Examples of nucleotide include nucleoside monophosphate, nucleoside diphosphate, and nucleoside triphosphate.In some embodiments, the term nucleic acid refers to a polymer of nucleotides that are linked together by phosphodiester or phosphorothioate bonds between the 5' and 3' carbon atoms.As used herein, in some embodiments, nucleic acid can refer to a single-stranded molecule, or nucleic acid can refer to a double-stranded molecule (e.g., a sense strand in a duplex with an antisense strand).

[0050] In some embodiments, the nucleic acid of the present disclosure comprises an antisense strand that is at least 19 nucleotides long. For example, in some embodiments, the antisense strand is 19 to 31 nucleotides long (e.g., 19 to 25 nucleotides long, 19 to 21 nucleotides long, 21 to 31 nucleotides long, 21 to 25 nucleotides long, 19 nucleotides long, 20 nucleotides long, 21 nucleotides long, 22 nucleotides long, 23 nucleotides long, 24 nucleotides long, or 25 nucleotides long). In some embodiments, the antisense strand comprises a region of complementarity with a target strand (e.g., a target mRNA). In some embodiments, the region of complementarity refers to a nucleotide sequence of the antisense strand that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or 100%) complementary to a contiguous sequence of the target mRNA. In some embodiments, the region of complementarity is 19 to 31 nucleotides in length (e.g., 19 to 25 nucleotides in length, 19 to 21 nucleotides in length, 21 to 31 nucleotides in length, 21 to 25 nucleotides in length, 19 nucleotides in length, 20 nucleotides in length, 21 nucleotides in length, 22 nucleotides in length, 23 nucleotides in length, 24 nucleotides in length, or 25 nucleotides in length).

[0051] In some embodiments, the nucleic acid of the present disclosure includes a sense strand that forms a duplex region with the antisense strand. In some embodiments, the sense strand is at least 19 nucleotides in length. For example, in some embodiments, the sense strand is 19 to 40 nucleotides in length (e.g., 19 to 35 nucleotides, 19 to 30 nucleotides, 19 to 25 nucleotides, 19 to 21 nucleotides, 21 to 30 nucleotides, 25 to 30 nucleotides, or 30 to 40 nucleotides in length). In some embodiments, the duplex region refers to a structure formed through complementary base pairing of two antiparallel nucleotide sequences. In some embodiments, the duplex region formed between the sense strand and the antisense strand is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or 100%) complementary.

[0052] In some embodiments, the duplex region comprises at least one mismatched base pair (e.g., a nucleotide that does not base pair according to conventional Watson-Crick base pairing rules) of the duplex. In some embodiments, the mismatch of the at least one mismatched base pair comprises nucleotide at position 14 on the antisense strand, as described herein. For example, in some embodiments, the nucleotide at position 14 on the antisense strand can form a mismatched base pair with the corresponding nucleotide on the antisense strand (e.g., in the duplex region) and / or the target nucleotide at the corresponding position on the target strand. In some embodiments, the duplex region comprises more than one mismatch. In some embodiments, the duplex region comprises fewer than 30 mismatches. In some embodiments, the duplex region comprises more than one but fewer than 30 mismatches. In some embodiments, the duplex region comprises at least one but fewer than 11 mismatches. In some embodiments, the duplex region comprises at least one but fewer than six mismatches. In some embodiments, the duplex region comprises at least one but fewer than four mismatches. In some embodiments, when a duplex region contains more than one mismatch, the mismatches are contiguous (e.g., adjacent) in the nucleic acid. In some embodiments, when a duplex region contains more than one mismatch, the mismatches are not contiguous (e.g., non-adjacent) in the nucleic acid. In some embodiments, when a duplex region contains more than two mismatches, there is at least one group of two or more mismatches that are adjacent to each other. In some embodiments, when a duplex region contains more than two mismatches, there are no two or more mismatches that are adjacent to each other. In some embodiments, the duplex region does not contain any mismatches. In some embodiments, the mismatches in the duplex region comprise wobble base pairs.

[0053] In some embodiments, the double-stranded region comprises one or more wobble base pairs. In some embodiments, the wobble base pair of the one or more wobble base pairs comprises the 14th nucleotide on the antisense strand as described herein. For example, in some embodiments, the 14th nucleotide on the antisense strand can form a wobble base pair with the corresponding nucleotide on the antisense strand (e.g., in the double-stranded region) and / or the target nucleotide at the corresponding position on the target strand. In some embodiments, a wobble base pair is a term generally known in the art to refer to the base pairing (e.g., wobble base pair) of specific nucleotides, which are non-canonical in that they are not Watson-Crick base pairs (e.g., in the form of mismatched base pairs or a subset of mismatched base pairs). Specifically, the term wobble is used to describe the base pairing of hypoxanthine (inosine (I)) and uracil (U) (I:U base pair); guanine (G) and U (G:U base pair); I and adenine (A) (I:A base pair); and I and cytosine (C) (I:C base pair).

[0054] In some embodiments, the sense strand and / or the antisense strand comprises at least one modified nucleotide. In some embodiments, the modified nucleotide has one or more chemical modifications in its sugar, nucleobase and / or phosphate group. In some embodiments, the modified nucleotide has one or more chemical moieties attached to the corresponding reference nucleotide. Typically, the modified nucleotide imparts one or more desirable properties to the nucleic acid in which it is present. For example, the modified nucleotide may improve thermal stability, resistance to degradation, resistance to nuclease, solubility, bioavailability, bioactivity, reduced immunogenicity, etc. Examples of modified nucleotides include, but are not limited to, 2-amino-guanosine, 2-amino-adenosine, 2,6-diamino-guanosine, and 2,6-diamino-adenosine. Examples of nucleotide positions that can be derivatized include the 5-position, e.g., 5-(2-amino)propyluridine, 5-bromouridine, 5-propyne uridine, 5-propenyluridine, etc.; the 6-position, e.g., 6-(2-amino)propyluridine; the 8-position of adenosine and / or guanosine, e.g., 8-bromoguanosine, 8-chloroguanosine, 8-fluoroguanosine, etc. Nucleotide analogs also include deazanucleotides, e.g., 7-deaza-adenosine; O- and N-modified (e.g., alkylated, e.g., N6-methyladenosine, or as otherwise known in the art) nucleotides; and other heterocyclic modified nucleotide analogs known in the art. In some embodiments, the antisense strand comprises one or more nucleoside modifications selected from 2'-aminoethyl, 2'-fluoro, 2'-O-methyl, 2'-O-methoxyethyl, and 2'-deoxy-2'-fluoro-β-d-arabinonucleic acid.

[0055] Further examples of modified nucleotides according to the present disclosure include nucleotides having modified purine or pyrimidine nucleobases. The purine and / or pyrimidine nucleobases may be modified, for example, by amidation or deamination of the heterocyclic. Additionally, modified sugars (e.g., 2'-O substitutions on the sugar (e.g., ribose) including, but not limited to, 2'-O-methoxyethyl sugars, 2'-fluoro sugar modifications (2'-fluoro), 2'-O-methyl sugars (2'-O-methyl), 2'-O-ethyl sugars, 2'-Cl, 2'-SH, and substitutions thereof (e.g., 2'-SCH3), bicyclic sugar moieties) or substitutions (e.g., substitution of the 2'-O moiety with lower alkyl or substitutions thereof (e.g., -CH3, -CF3), 2'-amino or substitutions thereof) can be used to modify the nucleotides. Substitution of the 2'-O moiety by 2',3'-seconucleotide mimics, 2'-F-arabinonucleotides, inverted nucleotides, inverted 2'-O-methyl nucleotides, 2'-O-deoxynucleotides, substitution of the 2'-O moiety by alkenyl, substitution of the 2'-O moiety by alkynyl, substitution of the 2'-O moiety by methoxyethyl (2'-O-MOE), substitution of the 2'-O moiety by -H (as in DNA), or substitution of the 2'-O moiety by other substituents) may be introduced. Ribose mimics are also contemplated, such as, but not limited to, morpholino, glycol nucleic acid (GNA), UNA, cyclohexenyl nucleic acid (CeNA).

[0056] Other examples include 2'-4' sugar bridge variants, such as locked nucleic acids (LNAs), and 2'-O, 4'-C-ethylene bridged nucleic acids (ENAs). Locked nucleic acids are modified RNA nucleotides whose ribose sugar is modified by a bridge (often observed as a methylene bridge between the 2' oxygen and the 4' carbon) connecting the 2' oxygen and the 4' carbon. This bridge operably "locks" the ribose in a 3'-endo conformation. The conformation of the locked ribose sugar can enhance base stacking and backbone preorganization, which can affect (e.g., increase) its hybridization properties (e.g., thermal stability and hybridization specificity). Locked nucleic acids can be inserted into both RNA and DNA oligonucleotides to hybridize with DNA or RNA according to typical Watson-Crick base pairing rules (i.e., complementarity).

[0057] Other chemical reactions and modifications are known in the art of oligonucleotides that can be readily used in accordance with the present disclosure and are included within the definition of nucleic acid modification. For example, the term modification further includes any change, alteration, or manipulation that results in the formation of any nucleoside other than a naturally occurring nucleoside.

[0058] In some embodiments, the nucleic acid comprises more than one nucleoside modification. In some embodiments, the nucleic acid comprises more than two nucleoside modifications. In some embodiments, greater than 25% but less than or equal to 100% of the nucleosides in the nucleic acid comprise a nucleoside modification. In some embodiments, greater than 50% of the nucleosides in the nucleic acid comprise a nucleoside modification. In some embodiments, greater than 75% but less than or equal to 100% of the nucleosides in the nucleic acid comprise a nucleoside modification. In some embodiments, at least 75% (e.g., 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) of the nucleosides in the nucleic acid comprise a nucleoside modification. In some embodiments, at least 95% but less than 100% of the nucleosides in the nucleic acid comprise a nucleoside modification.

[0059] In some embodiments, the sense strand and / or the antisense strand comprises at least one modified internucleotide bond. As used herein, in some embodiments, modified internucleotide bond refers to an internucleotide bond that has one or more chemical modifications compared to a reference internucleotide bond that comprises a phosphodiester bond. In some embodiments, modified internucleotide bond is a bond that does not occur in nature. Typically, modified internucleotide bond imparts one or more desirable properties to the nucleic acid in which the modified internucleotide bond is present. For example, modified nucleotides can improve thermal stability, resistance to degradation, resistance to nuclease, solubility, bioavailability, bioactivity, reduced immunogenicity, and the like. In some embodiments, the antisense strand comprises at least one phosphorothioate internucleotide bond. Further modifications to the bond include amidation and peptide linker. Other examples include phosphodiesters, phosphotriesters, phosphoro(di)thioates, methylphosphonates, phosphoroamidate linkers, phosphonates, 3'-methylene phosphonates, 5'-methylene phosphonates, boranophosphates, etc. Additionally, the chirality of the isomers can be modified (e.g., Rp and Sp).

[0060] In some embodiments, the nucleic acid comprises more than two modified internucleotide linkages. In some embodiments, the nucleic acid comprises more than three modified internucleotide linkages. In some embodiments, more than 25% of the internucleotide linkages of the nucleic acid comprise a modification. In some embodiments, more than 50% of the internucleotide linkages of the nucleic acid comprise a modification. In some embodiments, more than 75% of the internucleotide linkages of the nucleic acid comprise a modification. In some embodiments, at least 75% (e.g., 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) of the internucleotide linkages of the nucleic acid comprise a modification. In some embodiments, at least 95% of the internucleotide linkages of the nucleic acid comprise a modification.

[0061] In some embodiments, the sense strand and / or the antisense strand is conjugated to at least one N-acetylgalactosamine (GalNAc) moiety.

[0062] In some embodiments, the present disclosure provides the nucleic acid for reducing the expression of target mRNA.In some embodiments, reducing the expression of target mRNA can be achieved by inducing target specific silencing, for example, by inducing the destruction of target mRNA by RNAi mechanism or process (RNAi interference) and / or by inducing the translation suppression of desired target mRNA. EXAMPLES

[0063] Example 1. AGT Gene Expression Assay for siRNA Knockdown The day before transfection, HepG2 cells were seeded in 96-well plates at 10,000 cells / well in antibiotic-free medium. AGT siRNA was diluted from a 10 mM stock solution to working stocks of 1 mM and 0.10 mM. Mixtures were prepared separately as shown below (amounts shown for triplicates), mixed gently, and incubated at room temperature for 5 minutes. [Table 3]

[0064] The mixtures were combined and incubated at room temperature for 20 minutes. During this incubation, the medium in the 96-well plate was replaced with 80 μL of antibiotic-free medium. A volume of 20 μL of the mixture was added to each well and the plate was gently tapped to mix the contents of the well. The cells were incubated at 37° C. in 5% CO2 for 24 hours (for mRNA analysis).

[0065] After 24 hours, Lysis Solution was prepared by combining 49.5 μL of RT Lysis Solution per reaction and 0.5 μL of DNase I per reaction, and increased by the total number of reactions. The cell culture medium was aspirated and rinsed with 50 μL of cold PBS. A volume of 50 μL of Lysis Solution per well was added, mixed by pipetting, and then incubated at room temperature for 5 minutes. A volume of 5 μL of Stop Solution (room temperature) was added, mixed by pipetting, and then incubated at room temperature for 2 minutes. A Master Mix was prepared on ice as shown below. [Table 4]

[0066] On ice, a volume of 18 μL of Master Mix was added to each well of an optical 96-well PCR plate. A volume of 2 μL of lysate (or water for NTC) was added to each well. The plate was sealed with an optical adhesive cover, vortexed for 5-10 seconds, and briefly centrifuged to remove air bubbles. Reactions were set up to run on a QuantStudio3 qPCR instrument as described below. [Table 5]

[0067] The plate was placed in the qPCR instrument and the reaction was run. After the run was complete, the results were downloaded and the Cq values ​​(as well as Ct) were used to analyze the data according to the following: (i) Record the Ct values ​​for AGT and GAPDH for each sample; (ii) ΔΔCt=AGT Ct value-GAPDH Ct value; (iii) ΔΔCtRQ=ΔΔCt test sample-ΔΔCt untransfected test sample; (iv) RQ (fold change in expression)=2 -ΔΔCt ;(v)Remaining AGT%=2 -ΔΔCt × 100; (vi) % AGT ​​knockdown = 100 - % AGT ​​remaining.

[0068] Sequence information for the siRNAs evaluated in these experiments is provided below in Table 1. [Table 1-1] [Table 1-2] [Table 1-3]

[0069] The in vitro results from AGT knockdown experiments using siRNA molecules are shown below in Table 2. [Table 2]

[0070] The list of materials used in this example is as follows: HepG2 cells (ATCC Catalog No. HB-8065); AGT siRNA SMARTpool (Dharmacon Catalog No. L-010988-00-0005); Dharmafect 4 (Dharmacon Catalog No. T-2004-01); Cells-To-CT 1Step TaqMan Kit (Fisher Catalog No. A25603); AGT TaqMan Gene Expression Assay 250rxns-Hs01586213_m1 (Fisher Catalog No. 4331182); GAPDH TaqMan Gene Expression Assay 250rxns-Hs02786624_g1 (Fisher catalog no. 4331182); nuclease-free water; MicroAmp Optical 96-well Plates, 0.2 mL (10 plates) (Fisher catalog no. N8010560); MicroAmp Optical Adhesive Covers (100 sheets) (Fisher catalog no. 4311971).

[0071] Example 2. In vivo testing of RD1354 siRNA siRNA "RD1354" was evaluated in cynomolgus monkeys. Prior to the study, the monkeys were kept in isolation, during which the animals were observed daily for general health. Two cynomolgus monkeys were injected with a single subcutaneous dose of 3 mg / kg oligonucleotide on study day 1. The monkeys were observed daily for signs of illness or distress during the study. Animals were bled for serum analysis on day -6 and days 1 (pre-dose), 4, 8, 15, 22, 29, 36, and 43. Circulating AGT levels were quantified using an ELISA that is specific for human angiotensinogen (and cross-reactive with cynomolgus monkeys) according to the manufacturer's protocol (IBL America number 27412). Data were expressed as a percentage of baseline values ​​(pre-dose day 1) and shown as mean ± standard deviation. Results for individual monkeys are shown in Figure 3A, and averaged results for groups are shown in Figure 3B.

[0072] (Equivalents and Scope) In the claims, articles such as "a," "an," and "the" can mean one or more than one, unless indicated to the contrary or clear from the context. A claim or description including "or" between one or more elements of a group is considered to be satisfied if one, more than one, or all of the elements of the group are present in, used in, or otherwise relevant to a given product or process, unless indicated to the contrary or clear from the context. The invention includes embodiments in which exactly one element of the group is present in, used in, or otherwise relevant to a given product or process. The invention includes embodiments in which more than one or all of the elements of the group are present in, used in, or otherwise relevant to a given product or process.

[0073] Moreover, the present invention includes all variations, combinations, and permutations in which one or more limitations, elements, clauses, and descriptive terms from one or more of the enumerated claims are introduced into another claim. For example, any claim that is dependent on another claim may be modified to include one or more limitations found in any other claim that is dependent on the same base claim. Where elements are presented as enumerations, for example in Markush group format, each subgroup of the elements is also disclosed, and any element may be removed from the group. In general, when the invention or aspects of the invention are said to include certain elements and / or features, it should be understood that certain embodiments of the invention or aspects of the invention consist of or consist essentially of such elements and / or features. For the sake of brevity, those embodiments have not been specifically set forth in these terms herein.

[0074] The phrase "and / or" as used in this application in the specification and claims should be understood to mean "either or both" of the elements so conjoined (i.e., elements that are conjunctive in some cases and disjunctive in others). Multiple elements listed with "and / or" should be interpreted in the same manner (i.e., "one or more" of the elements so conjoined). Other elements other than the elements specifically identified by the "and / or" clause may optionally be present, whether related or unrelated to the elements specifically identified. Thus, as a non-limiting example, a reference to "A and / or B", when used in conjunction with an open-ended term such as "comprising", may in one embodiment refer to only A (optionally including elements other than B); in another embodiment, it may refer to only B (optionally including elements other than A); in yet another embodiment, it may refer to both A and B (optionally including other elements); and so forth.

[0075] As used in this application in the specification and claims, "or" should be understood to have the same meaning as "and / or" as defined above. For example, when separating items in a list, "or" or "and / or" is to be interpreted as being inclusive (i.e., including at least one of the number or recited elements, but also including more than one, including additional unrecited items as necessary). Terms expressly stated to the contrary (e.g., "only one of" or "exactly one of"), or "consisting of" only when used in the claims, refer to the inclusion of exactly one of the number or recited elements. In general, the term "or" as used herein shall only be construed as indicating exclusive alternatives (i.e., "one or the other, but not both") when followed by exclusive terms (e.g., "either," "one of," "only one of," or "exactly one of." "Consisting essentially of," when used in the claims, shall have its ordinary meaning as used in the field of patent law.

[0076] As used in this application in the specification and claims, the phrase "at least one" in reference to a list of one or more elements should be understood to mean that at least one element selected from any one or more of the elements in that list of elements does not necessarily have to include at least one of every element specifically listed within that list of elements, and does not exclude any combinations of elements in that list of elements. This definition also allows for elements other than those specifically identified within the list of elements to which the phrase "at least one" refers, whether related or unrelated to the elements specifically identified, may optionally be present. Thus, as a non-limiting example, "at least one of A and B" (or, equivalently, "at least one of A or B" or, equivalently, "at least one of A and / or B") can refer, in one embodiment, to at least one A (optionally including more than one A) and no B (optionally including elements other than B); in another embodiment, to at least one B ((optionally including more than one B) and no A (optionally including elements other than A); in yet another embodiment, to at least one A (optionally including more than one A) and at least one B (optionally including more than one B) (optionally including other elements); and so forth.

[0077] Unless expressly stated to the contrary, it is also to be understood that in any method claimed in this application that includes more than one step or action, the order of the method steps or actions is not necessarily limited to the order in which the method steps or actions are described.

[0078] In the claims and the above specification, all transitional phrases, such as "comprising," "including," "carrying," "having," "containing," "involving," "holding," "composed of," and the like, are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases "consisting of" and "consisting essentially of" are closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03. It should be understood that embodiments described herein using open-ended transitional phrases (e.g., "comprising") are also contemplated, in alternative embodiments, as "consisting of" and "consisting essentially of" the features described by the open-ended transitional phrase. For example, if the application describes a "composition comprising A and B," the application also contemplates the alternative embodiments "a composition consisting of A and B" and "a composition consisting essentially of A and B."

[0079] When ranges are given, the endpoints are included. Moreover, unless otherwise indicated or clear from the context and the understanding of one of ordinary skill in the art, values ​​expressed as ranges can, in various embodiments of the invention, take any particular value or subrange within the stated range to one tenth of the unit of the lower limit of that range, unless the context clearly indicates otherwise.

[0080] This application refers to various issued patents, published patent applications, journal articles, and other publications, all of which are incorporated herein by reference. In the event of a conflict between any of the incorporated references and this specification, this specification controls. Furthermore, any particular embodiment of the present invention that is within the scope of the prior art may be expressly excluded from any one or more of its claims. Such an embodiment may be excluded even if the exclusion is not expressly stated herein, since such an embodiment would be considered known to one of ordinary skill in the art. Any particular embodiment of the present invention may be excluded from any claim for any reason, whether related or unrelated to the existence of prior art.

[0081] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments described herein. The scope of the embodiments described herein is not intended to be limited to the above description, but rather is as set forth in the appended claims. Those skilled in the art will appreciate that various changes and modifications to the description can be made without departing from the spirit and scope of the present invention as defined in the appended claims.

[0082] The recitation of a listing of chemical groups in any definition of a variable herein includes the definition of that variable as any single group or combination of the listed groups. The recitation of an embodiment for a variable herein includes that embodiment in combination with any single embodiment or any other embodiment or portion thereof. The recitation of an embodiment herein includes that embodiment in combination with any single embodiment or any other embodiment or portion thereof.

Claims

1. A nucleic acid for reducing expression of a target mRNA, comprising: A nucleic acid comprising an antisense strand 15 to 31 nucleotides in length having a sequence that is at least 90% complementary to a contiguous sequence of the target mRNA, wherein the sequence of the antisense strand comprises an abasic site at position 14 from the 5' end, or the sequence of the antisense strand comprises a nucleotide at position 14 from the 5' end that does not form a canonical base pair with a target nucleotide at the corresponding position on the contiguous sequence of the target mRNA.

2. The nucleic acid of claim 1 , wherein the nucleotide at position 14 on the antisense strand forms a wobble base pair with the target nucleotide.

3. if the target nucleotide comprises a cytidine, then the nucleotide at position 14 on the antisense strand comprises an inosine; or if the target nucleotide comprises a guanosine, then the nucleotide at position 14 on the antisense strand comprises a uridine; The nucleic acid according to claim 2.

4. The nucleic acid of claim 3, wherein the antisense strand is 19 to 25 nucleotides in length.

5. 2. The nucleic acid of claim 1, further comprising a sense strand 15 to 40 nucleotides in length, said sense strand forming a duplex region with said antisense strand, said duplex region comprising canonical or non-canonical base pairing between a nucleotide on said sense strand and said nucleotide at position 14 on said antisense strand.

6. if the nucleotide at position 14 on the antisense strand comprises an inosine, then the nucleotide on the sense strand comprises a cytidine, an adenosine, or a uridine; or and when the nucleotide at position 14 on the antisense strand comprises a uridine, the nucleotide on the sense strand comprises an adenosine. The nucleic acid according to claim 5.

7. The nucleic acid of claim 5 , wherein the sense strand comprises at least one modified nucleotide and / or at least one modified internucleotide linkage.

8. 6. The nucleic acid of claim 5, wherein the sense strand comprises one or more nucleoside modifications selected from 2'-aminoethyl, 2'-fluoro, 2'-O-methyl, 2'-O-methoxyethyl, and 2'-deoxy-2'-fluoro-β-d-arabinoic acid.

9. The nucleic acid of claim 5 , wherein the sense strand contains at least one phosphorothioate internucleotide linkage.

10. 3. The nucleic acid of claim 2, wherein the sequence of the antisense strand is 100% complementary to the contiguous sequence of the target mRNA, except for the nucleotides that form the wobble base pair.

11. A nucleic acid for reducing expression of a target mRNA, comprising: Formula (I): 5’-X ０１ X ０２ X ０３ X ０４ X ０５ X ０６ X ０７ X ０８ X ０９ X １０ X １１ X １２ X １３ X １４ (X Ｙ ) ｂ N ａ -3’(I) Antisense strand of Including, N and X Ｙ each instance of is independently any type of nucleotide; a is an integer from 0 to 2, inclusive; b is an integer from 1 to 17, inclusive; X ０１ ~X １３ are each independently any type of nucleotide, provided that X ０１ ~ (X Ｙ ) ｂ is at least 90% complementary to a contiguous nucleotide sequence of the target mRNA; X １４ is an abasic site, or X １４ is a nucleotide that does not form a canonical base pair with a target nucleotide at a corresponding position on the contiguous nucleotide sequence of the target mRNA, Nucleic acid.

12. When the target nucleotide contains cytidine, X １４ contains inosine, or When the target nucleotide contains guanosine, X １４ contains uridine, The nucleic acid of claim 11.

13. The nucleic acid of claim 11, wherein the antisense strand comprises at least one modified nucleotide and / or at least one modified internucleotide bond.

14. 12. The nucleic acid of claim 11, wherein the antisense strand comprises one or more nucleoside modifications selected from 2'-aminoethyl, 2'-fluoro, 2'-O-methyl, 2'-O-methoxyethyl, and 2'-deoxy-2'-fluoro-β-d-arabinoic acid.

15. The nucleic acid of claim 11, wherein the antisense strand comprises at least one phosphorothioate internucleotide bond.

16. The nucleic acid of claim 11, wherein a is 0.

17. The nucleic acid of claim 11, wherein b is an integer between 1 and 11, inclusive.

18. The nucleic acid of claim 11, wherein b is an integer between 5 and 11, inclusive.

19. The nucleic acid of claim 11, wherein b is 7.

20. 12. The nucleic acid of claim 11, further comprising a sense strand 15 to 40 nucleotides in length, said sense strand forming a duplex region with said antisense strand, said duplex region comprising standard or non-standard base pairing between nucleotides on said sense strand and X14.

21. X １４ comprises an inosine, then the nucleotide on the sense strand comprises a cytidine, an adenosine, or a uridine; or X １４ comprises a uridine, and the nucleotide on the sense strand comprises an adenosine. The nucleic acid of claim 20.

22. 21. The nucleic acid of claim 20, wherein the double-stranded region is any N other than.

23. 21. The nucleic acid of claim 20, wherein the sense strand comprises at least one modified nucleotide and / or at least one modified internucleotide linkage.

24. 21. The nucleic acid of claim 20, wherein the sense strand comprises one or more nucleoside modifications selected from 2'-aminoethyl, 2'-fluoro, 2'-O-methyl, 2'-O-methoxyethyl, and 2'-deoxy-2'-fluoro-β-d-arabinoic acid.

25. 21. The nucleic acid of claim 20, wherein the sense strand comprises at least one phosphorothioate internucleotide linkage.

26. The nucleic acid of claim 20, wherein the sense strand is linked to at least one N-acetylgalactosamine (GalNAc) moiety.

27. The antisense strand has the formula (II): 5'-X ０１ X ０２ X ０３ X ０４ X ０５ X ０６ X ０７ X ０８ X ０９ X １０ X １１ X １２ X １３ X １４ X １５ X １６ X １７ X １８ X １９ X ２０ X ２１ -3'II)、 is the antisense strand of X ０１ ~X １３ and X １５ ~X ２１ are each independently any type of nucleotide, provided that X ０１ ~X ２１ is at least 90% complementary to said contiguous nucleotide sequence of said target mRNA; X １４ is a nucleotide containing inosine or uridine; The nucleic acid of claim 20.

28. A composition comprising a nucleic acid described in any one of claims 1 to 27 and a counterion.

29. A composition comprising a nucleic acid according to any one of claims 1 to 27 and a pharma- ceutically acceptable carrier.

30. A composition for use in a method for reducing expression of a target mRNA in a cell, the composition comprising a nucleic acid according to any one of claims 1 to 27, the method comprising: contacting said cell with said nucleic acid A composition comprising:

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