Chemically Modified RNAi Constructs and Their Uses

Abstract

The present invention relates to a chemically modified RNAi construct for reducing the expression of a target gene. In particular, the present invention relates to a specific pattern of modified nucleotides incorporated into the RNAi construct to improve in vivo stability and efficacy. Additionally, a pharmaceutical composition comprising a chemically modified RNAi construct and a method for inhibiting target gene expression in vivo by administering the chemically modified RNAi construct, for example, to treat or improve various disease conditions are described.

Description

Technology Field

[0001] [Cross-reference of related applications]

[0002] This application claims the benefit of U.S. Provisional Application No. 62 / 777,677 filed on December 10, 2018, the entirety of which is incorporated herein by reference.

[0003] [Description of the text file submitted electronically]

[0004] The present application comprises a list of sequences submitted electronically in ASCII format, the entirety of which is incorporated herein by reference. A computer-readable copy of the list of sequences created on December 9, 2019, is named A-2327-WO-PCT_SeqList_ST25 and is 24.7 kilobytes in size.

[0005] [Technology Field]

[0006] The present invention relates to chemically modified RNAi constructs for reducing the expression of target genes in vivo. Specifically, the present invention relates to a specific pattern of modified nucleotides that imparts improved efficacy and stability to RNAi constructs in vivo. Such RNAi constructs are useful for inhibiting target gene expression for therapeutic purposes. Background Technology

[0007] RNA interference (RNAi) is a post-transcriptional gene silencing mechanism found in almost all organisms and is considered an evolutionarily conserved cellular defense mechanism (Fire et al ., Nature, Vol. 391; 806-811, 1998; Fire et al., Trends Genet, Vol. 15: 358-363, 1999; and Hamilton and Baulcombe, Science, Vol. 286, 950-952, 1999). Physiologically, the RNAi mechanism is initiated by the Dicer enzyme-mediated generation of a dimer consisting of 18 to 25 base pairs from longer non-coding RNA. These short RNA molecules are loaded into the RNA-induced silencing complex (RISC), where the sense strand or passenger strand is discarded and the antisense strand or guide strand hybridizes with a completely or partially complementary mRNA sequence (Nakanishi, Wiley Interdiscip. Rev. RNA, Vol. 7: 637-660, 2016). Subsequently, mRNA silencing is induced through Ago2-mediated degradation or translation repression (Bobbin and Rossi, Annu. Rev. Pharmacol. Toxicol., Vol. 56:103-122, 2016).

[0008] Advancements in RNAi technology and delivery methods have led to increasingly positive outcomes using RNAi-based therapies. These therapies represent a promising class of treatments, particularly against targets previously considered "undruggable" by small molecule or bioagent modalities. While significant progress has been made to overcome the inherent metabolic vulnerabilities of natural RNA through chemical modifications and the development of improved delivery methods, there is still a need in the field for RNAi agents with enhanced in vivo efficacy and stability suitable for therapeutic administration.

[0009] The present invention is based, in part, on the design of chemical modification patterns for RNAi constructs that improve the efficacy of the construct and / or the duration of gene silencing activity in vivo. The chemical patterns described herein can be universally applied to various RNAi constructs having different sequences and targets. RNAi constructs are useful, for example, for inhibiting target gene expression in vivo for therapeutic purposes.

[0010] Accordingly, the present invention provides an RNAi construct that inhibits the expression of a target gene sequence, wherein the RNAi construct comprises a sense strand and an antisense strand, wherein the antisense strand comprises a sequence complementary to the target gene sequence and the sense strand comprises a sequence sufficiently complementary to the sequence of the antisense strand to form a dimer region, and the RNAi construct comprises a structure represented by one of the formulas described herein. In certain embodiments, the RNAi construct of the present invention has a chemical modification pattern selected from one of the patterns designated as P1 to P30 as described herein.

[0011] In some embodiments, the RNAi construct comprises a structure represented by formula (A):

[0012] 5′-(N A ) x N L N L N L N L N L N L N F N L N F N F N F N F N L N L N M N L N M N L N T (n) y -3′

[0013] 3′-(N B )z N L N L N L N L N L N F N L N M N L N M N L N L N F N M N L N M N L N F N L -5′

[0014] (A)

[0015] In equation (A), the upper strand listed in the 5' to 3' direction is the sense strand and the lower strand listed in the 3' to 5' direction is the antisense strand; and each N F is a 2′-fluoro-modified nucleotide; and each N M represents a modified nucleotide independently selected from 2'-fluoro-modified nucleotides, 2'-O-methyl-modified nucleotides, 2'-O-methoxyethyl-modified nucleotides, 2'-O-alkyl-modified nucleotides, 2'-O-allyl-modified nucleotides, bicyclic nucleic acids (BNA), and deoxyribonucleotides; each N L represents a modified nucleotide independently selected from 2'-O-methyl modified nucleotide, 2'-O-methoxyethyl modified nucleotide, 2'-O-alkyl modified nucleotide, 2'-O-allyl modified nucleotide, BNA, and deoxyribonucleotide; N Trepresents a modified nucleotide selected from baseless nucleotides, inverted baseless nucleotides, inverted deoxyribonucleotides, 2'-O-methyl modified nucleotides, 2'-O-methoxyethyl modified nucleotides, 2'-O-alkyl modified nucleotides, 2'-O-allyl modified nucleotides, BNA, and deoxyribonucleotides. x may be an integer from 0 to 4, provided that if x is 1, 2, 3, or 4, N A One or more of the nucleotides are modified nucleotides independently selected from baseless nucleotides, inverted baseless nucleotides, inverted deoxyribonucleotides, 2'-O-methyl modified nucleotides, 2'-O-methoxyethyl modified nucleotides, 2'-O-alkyl modified nucleotides, 2'-O-allyl modified nucleotides, BNA, and deoxyribonucleotides. A One or more nucleotides may be complementary to nucleotides of the antisense strand. y may be an integer from 0 to 4, provided that if y is 1, 2, 3, or 4, one or more n nucleotides are modified or unmodified overhang nucleotides that do not form base pairs with nucleotides of the antisense strand. z may be an integer from 0 to 4, provided that if z is 1, 2, 3, or 4, N B One or more of the nucleotides are modified nucleotides independently selected from 2'-O-methyl modified nucleotides, 2'-O-methoxyethyl modified nucleotides, 2'-O-alkyl modified nucleotides, 2'-O-allyl modified nucleotides, BNA, and deoxyribonucleotides. N B When one or more nucleotides are present in the sense strand, N A It may be complementary to the nucleotide, or it may be an overhang nucleotide that does not form a base pair with the nucleotide of the sense strand.

[0016] In some embodiments, the RNAi construct comprises a sense strand of 19 to 23 nucleotide lengths and an antisense strand of 19 to 23 nucleotide lengths, the sequences of the antisense strand and the sense strand are sufficiently complementary to each other to form a dimer region of 19 to 21 base pairs, and the nucleotides at positions 2, 7, and 14 of the antisense strand (counted from the 5' end) are 2′-fluoro-modified nucleotides; the nucleotides of the sense strand at positions paired with positions 8 to 11 and 13 of the antisense strand (counted from the 5' end) are 2′-fluoro-modified nucleotides; and the sense strand and the antisense strand each do not have more than 7 total 2′-fluoro-modified nucleotides. The RNAi construct may have nucleotide overhangs at one or both of the 3' ends of the sense strand and the antisense strand. In certain embodiments, the RNAi construct has a nucleotide overhang at the 3' end of the antisense strand and a smooth end at the 5' end of the antisense strand.

[0017] In another embodiment of the present invention, the RNAi construct comprises a structure represented by formula (D):

[0018] 5′-(N A ) x N L N L N L N L N M N L N F N F N F N F N L N L N L N L N L N L N L N L N T (n) y -3′

[0019] 3′-(N B ) z NL N L N L N M N L N F N L N M N L N L N M N M N M N M N L N M N L N F N L -5′

[0020] (D)

[0021] In equation (D), the upper strand listed in the 5' to 3' direction is the sense strand and the lower strand listed in the 3' to 5' direction is the antisense strand; and each N F is a 2′-fluoro-modified nucleotide; and each N M represents a modified nucleotide selected from 2'-fluoro-modified nucleotide, 2'-O-methyl-modified nucleotide, 2'-O-methoxyethyl-modified nucleotide, 2'-O-alkyl-modified nucleotide, 2'-O-allyl-modified nucleotide, BNA, and deoxyribonucleotide; each N L represents a modified nucleotide independently selected from 2'-O-methyl modified nucleotide, 2'-O-methoxyethyl modified nucleotide, 2'-O-alkyl modified nucleotide, 2'-O-allyl modified nucleotide, BNA, and deoxyribonucleotide; N Trepresents a modified nucleotide selected from baseless nucleotides, inverted baseless nucleotides, inverted deoxyribonucleotides, 2'-O-methyl modified nucleotides, 2'-O-methoxyethyl modified nucleotides, 2'-O-alkyl modified nucleotides, 2'-O-allyl modified nucleotides, BNA, and deoxyribonucleotides. x may be an integer from 0 to 4, provided that if x is 1, 2, 3, or 4, N A One or more of the nucleotides are modified nucleotides independently selected from baseless nucleotides, inverted baseless nucleotides, inverted deoxyribonucleotides, 2'-O-methyl modified nucleotides, 2'-O-methoxyethyl modified nucleotides, 2'-O-alkyl modified nucleotides, 2'-O-allyl modified nucleotides, BNA, and deoxyribonucleotides. A One or more nucleotides may be complementary to nucleotides of the antisense strand. y may be an integer from 0 to 4, provided that if y is 1, 2, 3, or 4, one or more n nucleotides are modified or unmodified overhang nucleotides that do not form base pairs with nucleotides of the antisense strand. z may be an integer from 0 to 4, provided that if z is 1, 2, 3, or 4, N B One or more of the nucleotides are modified nucleotides independently selected from 2'-O-methyl modified nucleotides, 2'-O-methoxyethyl modified nucleotides, 2'-O-alkyl modified nucleotides, 2'-O-allyl modified nucleotides, BNA, and deoxyribonucleotides. N B When one or more nucleotides are present in the sense strand, N A It may be complementary to the nucleotide, or it may be an overhang nucleotide that does not form a base pair with the nucleotide of the sense strand.

[0022] In some embodiments of the present invention, the RNAi construct comprises a sense strand of 19 to 23 nucleotides in length and an antisense strand of 19 to 23 nucleotides in length, the sequences of the antisense strand and the sense strand are sufficiently complementary to each other to form a dichotomous region of 19 to 21 base pairs, and nucleotides at positions 2, 14, and 16 of the antisense strand (counted from the 5' end) are 2′-fluoro-modified nucleotides; nucleotides of the sense strand at positions paired with positions 10 to 13 of the antisense strand (counted from the 5' end) are 2′-fluoro-modified nucleotides; and the sense strand and the antisense strand each do not have more than 7 total 2′-fluoro-modified nucleotides. The RNAi construct may have a nucleotide overhang at the 3' end of the antisense strand and a smooth end at the 5' end of the antisense strand. Alternatively, RNAi constructs can have nucleotide overhangs at both the 3' ends of the sense strand and the antisense strand.

[0023] The RNAi construct of the present invention may include at least one backbone modification, such as a modified link between nucleotides or between nucleosides. In certain embodiments, the RNAi construct described herein includes at least one phosphorothioate link. In certain embodiments, the phosphorothioate link may be located at the 3' or 5' end of the sense and / or antisense strand.

[0024] The RNAi construct may further comprise a ligand that facilitates the delivery or uptake of the RNAi construct into a specific tissue or cell, e.g., hepatocytes. In some embodiments, the ligand targets the delivery of the RNAi construct to hepatocytes. In these and other embodiments, the ligand may comprise galactose, galactosamine, or N-acetyl-galactosamine (GalNAc). In certain embodiments, the ligand comprises a polyvalent galactose or polyvalent GalNAc moiety, e.g., a trivalent or tetravalent galactose or GalNAc moiety. The ligand may optionally be covalently attached to the 5' or 3' end of the sense strand of the RNAi construct via a linker. In some embodiments, the RNAi construct comprises a ligand and a linker having a structure according to any of Formulas I through IX described herein. In one embodiment, the RNAi construct comprises a ligand and a linker having a structure according to Formula VI. In another embodiment, the RNAi construct comprises a ligand and a linker having a structure according to Formula VII. In another embodiment, the RNAi construct comprises a ligand and a linker having a structure according to Formula IX.

[0025] The present invention also provides a pharmaceutical composition comprising any RNAi construct described herein and a pharmaceutically acceptable carrier, excipient, or diluent. Such pharmaceutical compositions are particularly useful for reducing or inhibiting the expression of a target gene in the cells (e.g., hepatocytes) of a subject, especially when the overexpression of the target gene product in the subject is associated with a pathological phenotype.

[0026] The present invention comprises a method for reducing or inhibiting the expression of a target gene in a cell, tissue, or subject. In one embodiment, the method comprises the step of bringing a cell or tissue into contact with any one of the RNAi constructs described herein. The cell or tissue may be in vitro or in vivo. In another embodiment, the method comprises the step of administering any one of the RNAi constructs described herein to a subject. The RNAi construct may be administered to the subject parenterally (e.g., intravenously or subcutaneously). Brief explanation of the drawing

[0027] Fig. 1 Figure 1 shows several representative embodiments of chemical modification patterns for RNAi constructs. In each schematic diagram, the upper strand represents the sense strand in the 5' to 3' direction, and the lower strand represents the antisense strand in the 3' to 5' direction. Black circles represent 2′-O-methyl(2′-OMe) modified nucleotides, striped circles represent 2′-fluoro(2′-F) modified nucleotides, and white circles represent inverted baseless nucleotides (invAb) or inverted deoxyribonucleotides (invdN). Light gray lines connecting the circles represent phosphodiester linkages, and black lines connecting the circles represent phosphothioate linkages. Black boxes indicate putative Ago2 cleavage sites within the RNAi constructs. Fig. 2 This is a bar graph showing the expression levels of the human PNPLA3 variant in the livers of mice injected with AAV encoding the human PNPLA3 variant and treated with a subcutaneous injection of 5 mg / kg of a labeled RNAi construct having a P1 or CM1 chemical modification pattern. Human PNPLA3 expression was measured by qPCR and reported as expression levels for vehicle-treated animals. Expression levels on day 8 after RNAi administration are presented. Fig. 3This is a bar graph showing the expression levels of human PNPLA3 variants in the livers of mice injected with AAV encoding the human PNPLA3 variant and treated with a subcutaneous injection of 5 mg / kg of a labeled RNAi construct having a P1, P2, P3, or P4 chemical modification pattern. Human PNPLA3 expression was measured by qPCR and reported as expression levels for vehicle-treated animals. Expression levels at day 15 after RNAi administration are presented. Figures 4a and 4b This is a line graph representing the total flux (photons per second) in mice receiving subcutaneous injection of the indicated RNAi construct or vehicle having a P9 chemical modification pattern at a dose of 1 mg / kg (Fig. 4a) or 3 mg / kg (Fig. 4b) for the number of weeks elapsed after RNAi construct injection. The total flux represents the signal from the luciferase reporter, which contains a sequence complementary to the sequence of the RNAi construct, expressed by the mice. A decrease in the total flux indicates a decrease in the expression of the luciferase reporter. Fig. 5 This is a bar graph showing the expression levels of human PNPLA3 variants in the livers of mice injected with AAV encoding the human PNPLA3 variant and treated with 3 mg / kg subcutaneous injections of the indicated RNAi constructs having chemical modification patterns of P9 (diploid numbers 7318 and 8709), CM2 (diploid number 8103), CM3 (diploid number 8104), or CM4 (diploid number 8105). Human PNPLA3 expression was measured by qPCR and reported as expression levels for vehicle-treated animals. Expression levels at day 28 after administration of the RNAi constructs are presented. Fig. 6This is a bar graph of mouse ASGR1 expression levels in the livers of mice treated with a subcutaneous injection of 5 mg / kg of the indicated ASGR1 RNAi construct. Mouse ASGR1 expression was measured by qPCR and reported as normalized expression levels based on Gapdh expression levels. Expression levels on days 4, 8, and 15 after administration of the RNAi construct or buffer (phosphate-buffered saline, PBS) are presented. Fig. 7 This is a line graph showing the percentage change in serum Lp(a) levels relative to baseline in double-transformed mice administered a subcutaneous injection of 0.5 mg / kg of the indicated LPA-targeted RNAi construct. The two RNAi constructs had identical sequences and differed only in the pattern of chemical modification. Double number 3632 had a modification pattern of CM1, and double number 3635 had a modification pattern of P1. The percentage change in serum Lp(a) levels at day 14 (D14) and day 28 (D28) after a single subcutaneous injection of the RNAi construct is presented. Specific details for implementing the invention

[0028] The present invention is based, in part, on the design of chemical modification patterns for RNAi constructs that result in potent and long-lasting knockdown of target gene expression in vivo across various sequences and targets. The chemically modified RNAi constructs described herein have been found to have improved efficacy and / or duration of gene silencing activity in vivo compared to previously described therapeutic RNAi agents having alternative chemical modification patterns. The modified RNAi constructs of the present invention are useful for inhibiting target gene expression in vivo, for example, for treating or improving various disease conditions. Accordingly, the present invention provides RNAi constructs that inhibit the expression of target gene sequences.

[0029] As used herein, the term “RNAi construct” refers to an agent comprising an RNA molecule capable of downregulating the expression of a target gene through an RNA interference mechanism when introduced into a cell. RNA interference is a method in which a nucleic acid molecule induces the cleavage and degradation of a target RNA molecule (e.g., messenger RNA or mRNA molecule) in a sequence-specific manner, for example, through the RNA-induced silencing complex (RISC) pathway. In some embodiments, the RNAi construct comprises a double-stranded RNA molecule comprising two antiparallel strands of adjacent nucleotides sufficiently complementary to each other to hybridize to form a dimer region. “Hybridizing” or “hybridization” typically refers to the pairing of complementary polynucleotides through hydrogen bonding between the complementary bases of two polynucleotides (e.g., Watson-Crick, Hoogsteen, or inverse Hoogsteen hydrogen bonds). A strand containing a region having a sequence substantially complementary to a target sequence (e.g., target mRNA) is referred to as an "antisense strand." A "sense strand" refers to a strand containing a region substantially complementary to a region of the antisense strand. In some embodiments, the sense strand may include a region having a sequence substantially identical to the target sequence.

[0030] Double-stranded RNA molecules may include chemical modifications to ribonucleotides, such as modifications to the ribose sugar, base, or backbone component of the ribonucleotide, e.g., those described herein or known in the art. Any such modification used in double-stranded RNA molecules (e.g., siRNA, shRNA, etc.) is included in the term "double-stranded RNA" for the purposes of this disclosure.

[0031] As used herein, if a polynucleotide containing a first sequence can be hybridized to a polynucleotide containing a second sequence to form a dimer region under certain conditions, e.g., physiological conditions, the first sequence is "complementary" to the second sequence. Other such conditions may include ordinary or strict hybridization conditions known to those skilled in the art. If a polynucleotide containing a first sequence base pair is paired with a polynucleotide containing a second sequence over the entire length of one or two nucleotide sequences without any mismatch, the first sequence is considered to be fully complementary (100% complementary) to the second sequence. If a sequence is at least about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% complementary to a target sequence, the sequence is "substantially complementary" to the target sequence. The percentage of complementarity can be calculated by dividing the number of bases of the first sequence that are complementary to the bases at the corresponding positions of the second or target sequence by the total length of the first sequence. Additionally, when two sequences are hybridized, if there are 5, 4, 3, or 2 or fewer mismatches for a 30-base pair dichotomous region, the sequence may be said to be substantially complementary to the other sequence. Generally, as defined herein, if any nucleotide overhang is present, the sequence of such overhang is not considered when determining the degree of complementarity between the two sequences. For example, a sense strand of 21 nucleotides and an antisense strand of 21 nucleotides in length, which are hybridized to form a 19-base pair dichotomous region having 2 nucleotide overhangs at the 3' ends of each strand, are considered to be fully complementary in the sense of the terms used herein.

[0032] In some embodiments, a region of the antisense strand comprises a sequence that is completely complementary to a region of the target gene sequence (e.g., target mRNA). In the above embodiments, the sense strand may comprise a sequence that is completely complementary to the sequence of the antisense strand. In other such embodiments, the sense strand may comprise a sequence that is substantially complementary to the sequence of the antisense strand having, for example, 1, 2, 3, 4, or 5 mismatches in the diady region formed by the sense and antisense strands. In certain embodiments, it is preferable that any mismatch occurs within the terminal region (e.g., within 6, 5, 4, 3, or 2 nucleotides at the 5' and / or 3' ends of the strand). In one embodiment, any mismatch in the diady region formed from the sense and antisense strands occurs within 6, 5, 4, 3, or 2 nucleotides at the 5' end of the antisense strand.

[0033] In certain embodiments, the sense strand and antisense strand of double-stranded RNA may be two separate molecules that are otherwise unconnected but hybridized to form a duplex region. Such double-stranded RNA molecules formed from two separate strands are referred to as "small interfering RNA" or "short interfering RNA" (siRNA). Accordingly, in some embodiments, the RNAi constructs of the present invention comprise siRNA.

[0034] In another embodiment, the sense strand and the antisense strand that hybridize to form a dichotomous region may be part of a single RNA molecule, that is, the sense and antisense strands are part of the self-complementary region of the single RNA molecule. In this case, the single RNA molecule comprises a dichotomous region (also referred to as a stem region) and a loop region. The 3' end of the sense strand is connected to the 5' end of the antisense strand by an adjacent sequence of unpaired nucleotides, which will form a loop region. The loop region typically has a length sufficient to allow the RNA molecule to fold itself so that the antisense strand can form base pairs with the sense strand to form a dichotomous or stem region. The loop region may contain about 3 to about 25, about 5 to about 15, or about 8 to about 12 unpaired nucleotides. Such RNA molecules having at least partially a self-complementary region are referred to as "short hairpin RNAs" (shRNAs). In certain embodiments, the RNAi constructs of the present invention comprise shRNAs. The length of a single, at least partially self-complementary RNA molecule may be about 40 to about 100 nucleotides, about 45 to about 85 nucleotides, or about 50 to about 60 nucleotides, and each includes a dichromatic region and a loop region having the lengths cited herein.

[0035] The RNAi construct of the present invention comprises a sense strand and an antisense strand, wherein the antisense strand comprises a region having a sequence substantially or completely complementary to the target gene sequence. The target gene sequence generally refers to a nucleic acid sequence comprising an incomplete or complete coding sequence for a polypeptide. The target gene sequence may also include a non-coding region, such as a 5' or 3' untranslated region (UTR). In certain embodiments, the target gene sequence is a messenger RNA (mRNA) sequence. The mRNA sequence refers to any messenger RNA sequence, including a splice variant, that encodes a protein, a protein variant, or an isoform from any species (e.g., mouse, rat, non-human primate, human). In one embodiment, the target gene sequence is an mRNA sequence that encodes a human protein. The target gene sequence may also be an RNA sequence other than an mRNA sequence, such as a tRNA sequence, a microRNA sequence, or a viral RNA sequence.

[0036] A region of the antisense strand of the RNAi construct may be substantially complementary or fully complementary to at least 15 consecutive nucleotides of the target gene sequence. In some embodiments, the target region of the gene sequence in which the antisense strand contains a complementary region may be in the range of about 15 to about 30 consecutive nucleotides, about 16 to about 28 consecutive nucleotides, about 18 to about 26 consecutive nucleotides, about 17 to about 24 consecutive nucleotides, about 19 to about 30 consecutive nucleotides, about 19 to about 25 consecutive nucleotides, about 19 to about 23 consecutive nucleotides, or about 19 to about 21 consecutive nucleotides.

[0037] The sense strand of an RNAi construct typically contains a sequence sufficiently complementary to the sequence of the antisense strand, so that the two strands hybridize under physiological conditions to form a diady region. A "double region" refers to a region of two complementary or substantially complementary polynucleotides that base pair with each other via Watson-Crick base pairing or other hydrogen bonding interactions to create a diady between the two polynucleotides. For example, by involving a Dicer enzyme and / or a RISC complex, the diady region of the RNAi construct must have sufficient length to allow the RNAi construct to enter the RNA interference pathway. For example, in some embodiments, the diady region is about 15 to about 30 base pairs long. Other lengths for the diady region within this range are also suitable, for example, about 15 to about 28 base pairs, about 15 to about 26 base pairs, about 15 to about 24 base pairs, about 15 to about 22 base pairs, about 17 to about 28 base pairs, about 17 to about 26 base pairs, about 17 to about 24 base pairs, about 17 to about 23 base pairs, about 17 to about 21 base pairs, about 19 to about 25 base pairs, about 19 to about 23 base pairs, or about 19 to about 21 base pairs. In one embodiment, the diady region is about 17 to about 24 base pairs long. In another embodiment, the diady region is about 19 to about 21 base pairs long. In a specific embodiment, the diady region is about 19 base pairs long. In another embodiment, the dichromatic region is about 21 base pairs long.

[0038] In the case of embodiments where the sense strand and the antisense strand are two distinct molecules (e.g., the RNAi construct comprises siRNA), the sense strand and the antisense strand do not need to be the same length as the disome region. For example, one or both strands may be longer than the disome region and have one or more unpaired nucleotides or mismatches adjacent to the disome region. Thus, in some embodiments, the RNAi construct comprises at least one nucleotide overhang. As used herein, "nucleotide overhang" refers to a nucleotide that extends beyond the unpaired nucleotide or disome region at the end of a strand. A nucleotide overhang is typically created when the 3' end of one strand extends beyond the 5' end of the other strand, or when the 5' end of one strand extends beyond the 3' end of the other strand. The length of the nucleotide overhang is generally 1 to 6 nucleotides, 1 to 5 nucleotides, 1 to 4 nucleotides, 1 to 3 nucleotides, 2 to 6 nucleotides, 2 to 5 nucleotides, or 2 to 4 nucleotides. In some embodiments, the nucleotide overhang comprises 1, 2, 3, 4, 5, or 6 nucleotides. In one specific embodiment, the nucleotide overhang comprises 1 to 4 nucleotides. In a specific embodiment, the nucleotide overhang comprises 2 nucleotides. In another specific embodiment, the nucleotide overhang comprises a single nucleotide.

[0039] The nucleotide in the overhang may be the ribonucleotide or modified nucleotide described herein. In some embodiments, the nucleotide in the overhang is a 2'-modified nucleotide (e.g., 2'-fluoromodified nucleotide, 2'-O-methylmodified nucleotide), a deoxyribonucleotide, an inverted nucleotide (e.g., inverted baseless nucleotide, inverted deoxyribonucleotide), or a combination thereof. For example, in one embodiment, the nucleotide in the overhang is a deoxyribonucleotide, e.g., deoxythymidine. In other embodiments, the nucleotide in the overhang is a 2'-O-methylmodified nucleotide, a 2'-fluoromodified nucleotide, a 2'-methoxyethyl modified nucleotide, or a combination thereof. In other embodiments, the overhang comprises a 5'-uridine-uridine-3'(5'-UU-3') dinucleotide. In the above embodiments, the UU dinucleotide may comprise a ribonucleotide or a modified nucleotide, for example, a 2'-modified nucleotide. In another embodiment, the overhang comprises a 5'-deoxythymidine-deoxythymidine-3' (5'-dTdT-3') dinucleotide. When the nucleotide overhang is present on the antisense strand, the nucleotides of the overhang may be complementary to the target gene sequence, may form a mismatch with the target gene sequence, or may comprise some other sequence (e.g., polypyrimidine or polypurine sequences, e.g., UU, TT, AA, GG, etc.).

[0040] Nucleotide overhangs may be located at the 5' end or the 3' end of one or both strands. For example, in one embodiment, the RNAi construct includes nucleotide overhangs at the 5' and 3' ends of the antisense strand. In another embodiment, the RNAi construct includes nucleotide overhangs at the 5' and 3' ends of the sense strand. In some embodiments, the RNAi construct includes nucleotide overhangs at the 5' end of the sense strand and the 5' end of the antisense strand. In other embodiments, the RNAi construct includes nucleotide overhangs at the 3' end of the sense strand and the 3' end of the antisense strand.

[0041] The RNAi construct may include a nucleotide overhang at one end of a double-stranded RNA molecule and a smooth end at the other end. "Smooth end" means that the sense strand and the antisense strand are fully base-paired at the ends of the molecule and there are no unpaired nucleotides extending beyond the disome region. In some embodiments, the RNAi construct includes a nucleotide overhang at the 3' end of the sense strand and smooth ends at the 5' end of the sense strand and the 3' end of the antisense strand. In other embodiments, the RNAi construct includes a nucleotide overhang at the 3' end of the antisense strand and smooth ends at the 5' end of the antisense strand and the 3' end of the sense strand. In certain embodiments, the RNAi construct includes smooth ends at both ends of the double-stranded RNA molecule. In the above embodiment, the sense strand and the antisense strand have the same length, and the dimer region has the same length as the sense and antisense strands (i.e., the molecule is a double strand along its entire length).

[0042] The sense strand and antisense strand of the RNAi construct of the present invention may each independently have a length of about 15 to about 30 nucleotides, about 19 to about 30 nucleotides, about 18 to about 28 nucleotides, about 19 to about 27 nucleotides, about 19 to about 25 nucleotides, about 19 to about 23 nucleotides, about 19 to about 21 nucleotides, about 21 to about 25 nucleotides, or about 21 to about 23 nucleotides. In certain embodiments, the sense strand and antisense strand are each independently about 18, about 19, about 20, about 21, about 22, about 23, about 24, or about 25 nucleotides. In some embodiments, the sense strand and the antisense strand are of equal length, but form a dichotomous region shorter than the strands so that the RNAi construct has two nucleotide overhangs. For example, in one embodiment, the RNAi construct comprises (i) a sense strand and an antisense strand each 21 nucleotides long, (ii) a dichotomous region 19 base pairs long, and (iii) two nucleotide overhangs of unpaired nucleotides at both the 3' end of the sense strand and the 3' end of the antisense strand. In another embodiment, the RNAi construct comprises (i) a sense strand and an antisense strand each 23 nucleotides long, (ii) a dichotomous region 21 base pairs long, and (iii) two nucleotide overhangs of unpaired nucleotides at both the 3' end of the sense strand and the 3' end of the antisense strand. In another embodiment, the sense strand and the antisense strand have the same length and form a doublet region along the entire length so as not to have nucleotide overhangs at both ends of the double-stranded molecule.In one such embodiment, the RNAi construct is a smooth end and comprises (i) a sense strand and an antisense strand, each 21 nucleotide long, and (ii) a dimer region 21 base pairs long. In another such embodiment, the RNAi construct is a smooth end and comprises (i) a sense strand and an antisense strand, each 23 nucleotide long, and (ii) a dimer region 23 base pairs long.

[0043] In another embodiment, the sense strand or the antisense strand is longer than the other strand, and the two strands form a dichotomous region having a length equal to the length of the short strand such that the RNAi construct includes at least one nucleotide overhang. For example, in one embodiment, the RNAi construct includes (i) a sense strand 19 nucleotides long, (ii) an antisense strand 21 nucleotides long, (iii) a dichotomous region 19 base pairs long, and (iv) a nucleotide overhang of two unpaired nucleotides at the 3' end of the antisense strand. In yet another embodiment, the RNAi construct includes (i) a sense strand 21 nucleotides long, (ii) an antisense strand 23 nucleotides long, (iii) a dichotomous region 21 base pairs long, and (iv) a nucleotide overhang of two unpaired nucleotides at the 3' end of the antisense strand.

[0044] The RNAi constructs of the present invention preferably comprise modified nucleotides. "Modified nucleotides" refers to nucleotides having one or more chemical modifications to a nucleoside, nucleobase, pentose ring, or phosphate group. As used herein, modified nucleotides do not include ribonucleotides containing adenosine monophosphate, guanosine monophosphate, uridine monophosphate, and cytidine monophosphate. However, RNAi constructs may comprise a combination of modified nucleotides and ribonucleotides. Incorporating modified nucleotides into one or both strands of a double-stranded RNA molecule can improve the in vivo stability of the RNA molecule, for example, by reducing the molecule's susceptibility to nucleases and other degradation processes. The efficacy of RNAi constructs for reducing the expression of target genes can also be enhanced by the incorporation of modified nucleotides, particularly when incorporated in a specific pattern as described more in detail herein.

[0045] In certain embodiments, the modified nucleotide has a modification of the ribose sugar. Such sugar modifications may include modifications at the 2' and / or 5' positions of the pentose ring as well as bicyclic sugar modifications. A 2'-modified nucleotide refers to a nucleotide having a pentose ring with a substituent at the 2' position other than OH. Such 2'-modifications include 2'-H (e.g., deoxyribonucleotides), 2'-O-alkyl (e.g., O-C1-C1). 10 or O-C1-C 10Substituted alkyl), 2'-O-allyl (O-CH2CH=CH2), 2'-C-allyl, 2'-deoxy-2'-fluoro (also referred to as 2'-F or 2'-fluoro), 2'-O-methyl (OCH3), 2'-O-methoxyethyl (O-(CH2)2OCH3), 2'-OCF3, 2'-O(CH2)2SCH3, 2'-O-aminoalkyl, 2'-amino (e.g., NH2), 2'-O-ethylamine, and 2'-azido, but are not limited thereto. Modifications at the 5' position of the pentose ring include, but are not limited to, 5'-methyl (R or S); 5'-vinyl, and 5'-methoxy.

[0046] "Cyclosylated sugar modification" refers to a modification of a pentose ring in which a bridge connects two atoms of the ring to form a second ring, thereby creating a cyclosylated sugar structure. In some embodiments, the cyclosylated sugar modification comprises a bridge between the 4' and 2' carbons of the pentose ring. A nucleotide comprising a sugar moiety having a cyclosylated sugar modification is referred herein as cyclosylated nucleic acid or BNA. Exemplary cyclosylated sugar modifications include α-L-methyleneoxy(4'-CH2-O-2') cyclosylated nucleic acid (BNA); β-D-methyleneoxy(4'-CH2-O-2') BNA (also referred to as locked nucleic acid or LNA); ethyleneoxy(4'-(CH2)2-O-2') BNA; aminooxy(4'-CH2-ON(R)-2') BNA; oxyamino(4'-CH2-N(R)-O-2') BNA; Methyl(methyleneoxy)(4'-CH(CH3)-O-2') BNA (also referred to as constrained ethyl or cEt); methylene-thio(4'-CH2-S-2') BNA; methylene-amino(4'-CH2-N(R)-2') BNA; methyl carbon-cyclic (4'-CH2-CH(CH3)-2') BNA; propylene carbon-cyclic (4'-(CH2)3-2') BNA; and methoxy(ethyleneoxy)(4'-CH(CH2OMe)-O-2') BNA (also referred to as constrained MOE or cMOE), but are not limited to these. These and other sugar-modified nucleotides that may be included in the RNAi constructs of the present invention are described in U.S. Patent No. 9,181,551, U.S. Patent Publication No. 2016 / 0122761, and Deleavey and Damha, Chemistry and Biology, Vol. 19: 937-954, 2012, the full text of which is incorporated herein by reference.

[0047] In some embodiments, the RNAi construct comprises one or more 2'-fluoro-modified nucleotides, 2'-O-methyl-modified nucleotides, 2'-O-methoxyethyl-modified nucleotides, 2'-O-alkyl-modified nucleotides, 2'-O-allyl-modified nucleotides, bicyclic nucleic acids (BNA), deoxyribonucleotides, or combinations thereof. In certain embodiments, the RNAi construct comprises one or more 2'-fluoro-modified nucleotides, 2'-O-methyl-modified nucleotides, 2'-O-methoxyethyl-modified nucleotides, or combinations thereof. In certain embodiments, the RNAi construct comprises one or more 2'-fluoro-modified nucleotides, 2'-O-methyl-modified nucleotides, or combinations thereof.

[0048] Both the sense and antisense strands of an RNAi construct may contain one or more modified nucleotides. For example, in some embodiments, the sense strand contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more modified nucleotides. In certain embodiments, all nucleotides in the sense strand are modified nucleotides. In some embodiments, the antisense strand contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more modified nucleotides. In other embodiments, all nucleotides in the antisense strand are modified nucleotides. In certain other embodiments, all nucleotides in the sense strand and all nucleotides in the antisense strand are modified nucleotides. In these and other embodiments, the modified nucleotides may be 2'-fluoro-modified nucleotides, 2'-O-methyl-modified nucleotides, or a combination thereof.

[0049] In certain embodiments, the modified nucleotides incorporated into one or both strands of the RNAi construct of the present invention have modifications to the nucleobases (also referred to herein as “bases”). “Modified nucleobases” or “modified bases” refer to bases other than the naturally occurring purine bases adenine (A) and guanine (G) and the pyrimidine bases thymine (T), cytosine (C), and uracil (U). Modified nucleobases may be synthetic and natural modifications, and include universal bases, 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine (X), hypoxanthine, 2-aminoadenine, 6-methyladenine, 6-methylguanine, and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyluracil and cytosine, 6-azouracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenine and guanine, 5-halo, particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracil and cytosine, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azadenine, 7-deazaguanine and 7-deazadenine and 3-deazaguanine and 3-deazadenine.

[0050] In some embodiments, the modified base is a universal base. "Universal base" refers to a base analog that indiscriminately forms base pairs with all natural bases in RNA and DNA without altering the double helix structure of the dimer region. Universal bases are known to those skilled in the art and include, but are not limited to, inosine, C-phenyl, C-naphthyl and other aromatic derivatives, azole carboxamides, and nitroazole derivatives, such as 3-nitropyrrole, 4-nitroindole, 5-nitroindole, and 6-nitroindole.

[0051] Other suitable modified bases that may be included in the RNAi constructs of the present invention are Herdewijn, Antisense Nucleic Acid Drug Dev., Vol. 10: 297-310, 2000 and Peacock et al Includes those described in ., J. Org. Chem., Vol. 76: 7295-7300, 2011, both of which are incorporated herein by reference in their entirety. Those skilled in the art are well aware that guanine, cytosine, adenine, thymine, and uracil may be replaced by other nucleotides, such as the modified nucleotides described above, without substantially altering the base pairing properties of the polynucleotide containing the nucleotide having these substituted nucleotides.

[0052] In some embodiments, the sense and antisense strands of an RNAi construct may contain one or more baseless nucleotides. A "baseless nucleotide" or "baseless nucleoside" is a nucleotide or nucleoside lacking a nucleobase at the 1' position of a ribose sugar. In certain embodiments, baseless nucleotides are incorporated into the ends of the sense and / or antisense strands of the RNAi construct. In one embodiment, the sense strand contains a baseless nucleotide as a terminal nucleotide at its 3' end, its 5' end, or both its 3' and 5' ends. In another embodiment, the antisense strand contains a baseless nucleotide as a terminal nucleotide at its 3' end, its 5' end, or both its 3' and 5' ends. In this embodiment, where the baseless nucleotide is a terminal nucleotide, it may be an inverted nucleotide—that is, it may be connected to an adjacent nucleotide via a 3'-3' nucleotide connection (when at the 3' end of the strand) or a 5'-5' nucleotide connection (when at the 5' end of the strand) instead of a natural 3'-5' nucleotide connection. Additionally, the baseless nucleotide may include a sugar modification, e.g., any of the sugar modifications mentioned above. In certain embodiments, the baseless nucleotide includes a 2'-modification, e.g., a 2'-fluoro modification, a 2'-O-methyl modification, or a 2'-H (deoxy) modification. In one embodiment, the baseless nucleotide includes a 2'-O-methyl modification. In another embodiment, the baseless nucleotide includes a 2'-H modification (i.e., it is a deoxy baseless nucleotide).

[0053] The inventors have discovered that when nucleotides modified into RNAi constructs according to a specific pattern are incorporated, RNAi constructs having improved gene silencing activity in vivo are produced. For example, in one embodiment, the RNAi construct of the present invention comprises a sense strand and an antisense strand comprising sequences sufficiently complementary to each other to form a dichotomous region of at least 15 base pairs, and

[0054] * The nucleotides at positions 2, 7, and 14 of the antisense strand (counted from the 5' end) are 2′-fluoro-modified nucleotides;

[0055] * The nucleotides of the sense strand at positions paired with positions 8 through 11 and 13 of the antisense strand (counted from the 5' end) are 2′-fluoro-modified nucleotides;

[0056] * The sense strand and the antisense strand do not each have more than 7 total 2′-fluoro-modified nucleotides.

[0057] In another embodiment, the RNAi construct of the present invention comprises a sense strand and an antisense strand having sequences sufficiently complementary to each other to form a dichotomous region of at least 19 base pairs, and

[0058] * The nucleotides at positions 2, 7, and 14 of the antisense strand (counted from the 5' end) are 2′-fluoro-modified nucleotides, the nucleotides at positions 4, 6, 10, and 12 (counted from the 5' end) are optionally 2′-fluoro-modified nucleotides, and all other nucleotides of the antisense strand are modified nucleotides that are not 2′-fluoro-modified nucleotides;

[0059] * The nucleotides of the sense strand at positions 8 through 11 and 13 paired with the antisense strand (counted from the 5' end) are 2′-fluoro-modified nucleotides, and the nucleotides of the sense strand at positions 3 and 5 paired with the antisense strand (counted from the 5' end) are optionally 2′-fluoro-modified nucleotides; and all other nucleotides of the sense strand are modified nucleotides that are not 2′-fluoro-modified nucleotides.

[0060] In these embodiments, the modified nucleotide other than the 2′-fluoro modified nucleotide may be selected from 2'-O-methyl modified nucleotide, 2'-O-methoxyethyl modified nucleotide, 2'-O-alkyl modified nucleotide, 2'-O-allyl modified nucleotide, BNA, and deoxyribonucleotide. In these and other embodiments, the terminal nucleotide at the 3' end, 5' end, or both the 3' end and 5' end of the sense strand may be a baseless nucleotide or a deoxyribonucleotide. In this embodiment, the baseless nucleotide or deoxyribonucleotide may be inverted—that is, instead of the natural 3'-5' nucleotide linkage, it may be connected to an adjacent nucleotide through a 3'-3' nucleotide linkage (when at the 3' end of the strand) or a 5'-5' nucleotide linkage (when at the 5' end of the strand).

[0061] In any of the aforementioned embodiments, nucleotides at positions 2, 7, 12, and 14 of the antisense strand (counted from the 5' end) are 2′-fluoro-modified nucleotides. In another embodiment, nucleotides at positions 2, 4, 7, 12, and 14 of the antisense strand (counted from the 5' end) are 2′-fluoro-modified nucleotides. In yet another embodiment, nucleotides at positions 2, 4, 6, 7, 12, and 14 of the antisense strand (counted from the 5' end) are 2′-fluoro-modified nucleotides. In yet another embodiment, nucleotides at positions 2, 4, 6, 7, 10, 12, and 14 of the antisense strand (counted from the 5' end) are 2′-fluoro-modified nucleotides. In an alternative embodiment, nucleotides at positions 2, 7, 10, 12, and 14 of the antisense strand (counted from the 5' end) are 2′-fluoro-modified nucleotides. In a specific other embodiment, nucleotides at positions 2, 4, 7, 10, 12, and 14 of the antisense strand (counted from the 5' end) are 2′-fluoro-modified nucleotides.

[0062] In any of the aforementioned embodiments, the nucleotides of the sense strand at positions 3, 8 to 11, and 13 of the antisense strand (counted from the 5' end) are 2′-fluoro-modified nucleotides. In some embodiments, the nucleotides of the sense strand at positions 5, 8 to 11, and 13 of the antisense strand (counted from the 5' end) are 2′-fluoro-modified nucleotides. In other embodiments, the nucleotides of the sense strand at positions 3, 5, 8 to 11, and 13 of the antisense strand (counted from the 5' end) are 2′-fluoro-modified nucleotides.

[0063] In a specific embodiment of the present invention, the RNAi construct comprises a sense strand and an antisense strand, wherein the antisense strand comprises a sequence complementary to a target gene sequence and the sense strand comprises a sequence sufficiently complementary to the sequence of the antisense strand to form a dichotomous region, and the RNAi construct comprises a structure represented by formula (A).

[0064] 5′-(N A ) x N L N L N L N L N L N L N F N L N F N F N F N F N L N L N M N L N M N L N T (n) y -3′

[0065] 3′-(N B ) z N L N L N L N L N L N F N L N M N L N M N L N L N F N M N L N M N L N F N L -5′

[0066] (A)

[0067] In the equation,

[0068] The upper strand listed in the 5' to 3' direction is the sense strand, and the lower strand listed in the 3' to 5' direction is the antisense strand;

[0069] Each N F is a 2′-fluoro-modified nucleotide;

[0070] Each N M represents a modified nucleotide independently selected from 2'-fluoro-modified nucleotide, 2'-O-methyl-modified nucleotide, 2'-O-methoxyethyl-modified nucleotide, 2'-O-alkyl-modified nucleotide, 2'-O-allyl-modified nucleotide, bicyclic nucleic acid (BNA), and deoxyribonucleotide;

[0071] Each N L represents a modified nucleotide independently selected from 2'-O-methyl modified nucleotide, 2'-O-methoxyethyl modified nucleotide, 2'-O-alkyl modified nucleotide, 2'-O-allyl modified nucleotide, BNA, and deoxyribonucleotide;

[0072] N T represents a modified nucleotide selected from baseless nucleotide, inverted baseless nucleotide, inverted deoxyribonucleotide, 2'-O-methyl modified nucleotide, 2'-O-methoxyethyl modified nucleotide, 2'-O-alkyl modified nucleotide, 2'-O-allyl modified nucleotide, BNA, and deoxyribonucleotide;

[0073] x is an integer from 0 to 4, provided that if x is 1, 2, 3, or 4, N A One or more of the nucleotides are modified nucleotides independently selected from baseless nucleotides, inverted baseless nucleotides, inverted deoxyribonucleotides, 2'-O-methyl modified nucleotides, 2'-O-methoxyethyl modified nucleotides, 2'-O-alkyl modified nucleotides, 2'-O-allyl modified nucleotides, BNA, and deoxyribonucleotides, and N A One or more nucleotides may be complementary to nucleotides of the antisense strand;

[0074] y is an integer from 0 to 4, provided that if y is 1, 2, 3, or 4, one or more n nucleotides are modified or unmodified overhang nucleotides that do not form base pairs with nucleotides of the antisense strand;

[0075] z is an integer from 0 to 4, provided that if z is 1, 2, 3, or 4, N B One or more of the nucleotides are modified nucleotides independently selected from 2'-O-methyl modified nucleotides, 2'-O-methoxyethyl modified nucleotides, 2'-O-alkyl modified nucleotides, 2'-O-allyl modified nucleotides, BNA, and deoxyribonucleotides, and N B When one or more nucleotides are present in the sense strand, N A It may be complementary to the nucleotide, or it may be an overhang nucleotide that does not form a base pair with the nucleotide of the sense strand.

[0076] In some embodiments in which the RNAi construct comprises a structure represented by formula (A), a nucleotide overhang exists at the 3' end of the sense strand—i.e., y is 1, 2, 3, or 4. In one such embodiment, y is 2. In an embodiment in which a 2-nucleotide overhang exists at the 3' end of the sense strand (i.e., y is 2), x is 0 and z is 2, or x is 1 and z is 2. In another embodiment in which the RNAi construct comprises a structure represented by formula (A), the RNAi construct comprises smooth ends at the 3' end of the sense strand and the 5' end of the antisense strand (i.e., y is 0). In an embodiment where there is no nucleotide overhang at the 3' end of the sense strand (i.e., y is 0), (i) x is 2 and z is 4, (ii) x is 3 and z is 4, (iii) x is 0 and z is 2, (iv) x is 1 and z is 2, or (v) x is 2 and z is 2. In any embodiment where x is greater than 0, N is the terminal nucleotide at the 5' end of the sense strand. A The nucleotide may be an inverted nucleotide, for example, an inverted base-free nucleotide or an inverted deoxyribonucleotide.

[0077] In a specific embodiment in which the RNAi construct comprises a structure represented by formula (A), N at positions 4 and 12 of the antisense strand calculated from the 5' end M are each 2′-fluoro-modified nucleotides. In another embodiment, N at positions 4, 6, and 12 of the antisense strand, calculated from the 5' end. M are each 2′-fluoro-modified nucleotides. In another embodiment, N at positions 4, 6, 10, and 12 of the antisense strand, counted from the 5' end. Mare each 2′-fluoro-modified nucleotides. In an alternative embodiment, in a specific embodiment in which the RNAi construct comprises a structure represented by formula (A), N at positions 10 and 12 of the antisense strand calculated from the 5' end. M are each 2′-fluoro-modified nucleotides. In the relevant embodiments, N at positions 4, 10, and 12 of the antisense strand, calculated from the 5' end. M are each 2′-fluoro-modified nucleotides. In another alternative embodiment in which the RNAi construct comprises a structure represented by formula (A), N at positions 4, 6, and 10 of the antisense strand calculated from the 5' end. M are each 2'-O-methyl modified nucleotides, and N at position 12 of the antisense strand calculated from the 5' end. M is a 2′-fluoro-modified nucleotide. In some embodiments in which the RNAi construct comprises a structure represented by formula (A), each N of the sense strand M is a 2'-O-methyl modified nucleotide. In another embodiment, each N of the sense strand M is a 2′-fluoro-modified nucleotide. In another embodiment in which the RNAi construct comprises a structure represented by formula (A), each N of both the sense strand and the antisense strand. M is a 2'-O-methyl modified nucleotide.

[0078] In any of the aforementioned embodiments in which the RNAi construct comprises a structure represented by formula (A), each N of both the sense strand and the antisense strand L may be a 2'-O-methyl modified nucleotide. In these embodiments and any of the embodiments described above, N of formula (A) T It may be an inverted baseless nucleotide, an inverted deoxyribonucleotide, or a 2'-O-methyl modified nucleotide.

[0079] In a specific embodiment of the present invention, the RNAi construct comprises a sense strand and an antisense strand, wherein the antisense strand comprises a sequence complementary to a target gene sequence and the sense strand comprises a sequence sufficiently complementary to the sequence of the antisense strand to form a dichotomous region, and the RNAi construct comprises a structure represented by formula (B).

[0080] 5′-(N A ) x N L N L N L N L N L N L N F N L N F N F N F N F N L N L N L N L N L N L N T (n) y -3′

[0081] 3′-(N B ) z N L N L N L N L N L N F N L N F N L N L N L N L N F N F N L N F N L N F N L -5′

[0082] (B)

[0083] In the equation,

[0084] The upper strand listed in the 5' to 3' direction is the sense strand, and the lower strand listed in the 3' to 5' direction is the antisense strand;

[0085] Each N F represents a 2′-fluoro-modified nucleotide;

[0086] Each N L represents a modified nucleotide independently selected from 2'-O-methyl modified nucleotide, 2'-O-methoxyethyl modified nucleotide, 2'-O-alkyl modified nucleotide, 2'-O-allyl modified nucleotide, BNA, and deoxyribonucleotide;

[0087] N T represents a modified nucleotide selected from baseless nucleotide, inverted baseless nucleotide, inverted deoxyribonucleotide, 2'-O-methyl modified nucleotide, 2'-O-methoxyethyl modified nucleotide, 2'-O-alkyl modified nucleotide, 2'-O-allyl modified nucleotide, BNA, and deoxyribonucleotide;

[0088] x is an integer from 0 to 4, provided that if x is 1, 2, 3, or 4, N A One or more of the nucleotides are modified nucleotides independently selected from baseless nucleotides, inverted baseless nucleotides, inverted deoxyribonucleotides, 2'-O-methyl modified nucleotides, 2'-O-methoxyethyl modified nucleotides, 2'-O-alkyl modified nucleotides, 2'-O-allyl modified nucleotides, BNA, and deoxyribonucleotides, and N A One or more nucleotides may be complementary to nucleotides of the antisense strand;

[0089] y is an integer from 0 to 4, provided that if y is 1, 2, 3, or 4, one or more n nucleotides are modified or unmodified overhang nucleotides that do not form base pairs with nucleotides of the antisense strand;

[0090] z is an integer from 0 to 4, provided that if z is 1, 2, 3, or 4, N BOne or more of the nucleotides are modified nucleotides independently selected from 2'-O-methyl modified nucleotides, 2'-O-methoxyethyl modified nucleotides, 2'-O-alkyl modified nucleotides, 2'-O-allyl modified nucleotides, BNA, and deoxyribonucleotides, and N B When one or more nucleotides are present in the sense strand, N A It may be complementary to the nucleotide, or it may be an overhang nucleotide that does not form a base pair with the nucleotide of the sense strand.

[0091] In some embodiments in which the RNAi construct comprises a structure represented by formula (B), a nucleotide overhang exists at the 3' end of the sense strand—i.e., y is 1, 2, 3, or 4. In one such embodiment, y is 2. In an embodiment in which a 2-nucleotide overhang exists at the 3' end of the sense strand (i.e., y is 2), x is 0 and z is 2, or x is 1 and z is 2. In another embodiment in which the RNAi construct comprises a structure represented by formula (B), the RNAi construct comprises smooth ends at the 3' end of the sense strand and the 5' end of the antisense strand (i.e., y is 0). In an embodiment where there is no nucleotide overhang at the 3' end of the sense strand (i.e., y is 0), (i) x is 2 and z is 4, (ii) x is 3 and z is 4, (iii) x is 0 and z is 2, (iv) x is 1 and z is 2, or (v) x is 2 and z is 2. In any embodiment where x is greater than 0, N is the terminal nucleotide at the 5' end of the sense strand. A The nucleotide may be an inverted nucleotide, for example, an inverted base-free nucleotide or an inverted deoxyribonucleotide.

[0092] In any of the aforementioned embodiments in which the RNAi construct comprises a structure represented by formula (B), each N of both the sense strand and the antisense strand Lmay be a 2'-O-methyl modified nucleotide. In these embodiments and any of the embodiments described above, N of formula (B) T It may be an inverted baseless nucleotide, an inverted deoxyribonucleotide, or a 2'-O-methyl modified nucleotide.

[0093] In some embodiments of the present invention, the RNAi construct comprises a sense strand and an antisense strand, wherein the antisense strand comprises a sequence complementary to a target gene sequence and the sense strand comprises a sequence sufficiently complementary to the sequence of the antisense strand to form a dichotomous region, and the RNAi construct comprises a structure represented by formula (C).

[0094] 5′-(Ab) x N L N L N L N L N L N L N L N L N F N L N F N F N F N F N L N L N M N L N M N L N T -3′

[0095] 3′-N L N L N L N L N L N L N L N L N L N F N L N F N L N L N L N L N F N L N L N M N L NF N L -5′

[0096] (C)

[0097] In the equation,

[0098] The upper strand listed in the 5' to 3' direction is the sense strand, and the lower strand listed in the 3' to 5' direction is the antisense strand;

[0099] Each N F is a 2′-fluoro-modified nucleotide;

[0100] Each N L represents a modified nucleotide independently selected from 2'-O-methyl modified nucleotide, 2'-O-methoxyethyl modified nucleotide, 2'-O-alkyl modified nucleotide, 2'-O-allyl modified nucleotide, BNA, and deoxyribonucleotide;

[0101] Each N M represents a modified nucleotide independently selected from 2'-fluoro-modified nucleotide, 2'-O-methyl-modified nucleotide, 2'-O-methoxyethyl-modified nucleotide, 2'-O-alkyl-modified nucleotide, 2'-O-allyl-modified nucleotide, BNA, and deoxyribonucleotide;

[0102] N T represents a modified nucleotide selected from baseless nucleotide, inverted baseless nucleotide, inverted deoxyribonucleotide, 2'-O-methyl modified nucleotide, 2'-O-methoxyethyl modified nucleotide, 2'-O-alkyl modified nucleotide, 2'-O-allyl modified nucleotide, BNA, and deoxyribonucleotide;

[0103] x is 0 or 1, and Ab is an inverted baseless nucleotide.

[0104] In certain embodiments in which the RNAi construct comprises a structure represented by formula (C), the NM of the antisense strand is a 2'-fluoro-modified nucleotide. In these and other embodiments, each NM of the sense strand is a 2'-O-methyl-modified nucleotide. In an alternative embodiment, each NM of the sense strand is a 2′-fluoro-modified nucleotide. In some embodiments in which the RNAi construct comprises a structure represented by formula (C), each NM of both the sense strand and the antisense strand is a 2'-O-methyl-modified nucleotide.

[0105] In any of the aforementioned embodiments in which the RNAi construct comprises a structure represented by formula (C), each N of both the sense strand and the antisense strand L may be a 2'-O-methyl modified nucleotide. In these embodiments and any of the embodiments described above, N of formula (C) T may be an inverted baseless nucleotide, an inverted deoxyribonucleotide, or a 2'-O-methyl modified nucleotide. For example, in one embodiment, N T is an inverted baseless nucleotide or an inverted deoxyribonucleotide, and x is 0. In another embodiment, N T is a 2'-O-methyl modified nucleotide, and x is 1. In another embodiment, N T is an inverted baseless nucleotide or an inverted deoxyribonucleotide, and x is 1.

[0106] In a specific embodiment, the RNAi construct of the present invention comprises a sense strand and an antisense strand, wherein the antisense strand comprises a sequence complementary to a target gene sequence and the sense strand comprises a sequence sufficiently complementary to the sequence of the antisense strand to form a dichotomous region, and the RNAi construct comprises a structure represented by formula (D).

[0107] 5′-(N A ) x NL N L N L N L N M N L N F N F N F N F N L N L N L N L N L N L N L N L N T (n) y -3′

[0108] 3′-(N B ) z N L N L N L N M N L N F N L N M N L N L N M N M N M N M N L N M N L N F N L -5′

[0109] (D)

[0110] In the equation,

[0111] The upper strand listed in the 5' to 3' direction is the sense strand, and the lower strand listed in the 3' to 5' direction is the antisense strand;

[0112] Each N F is a 2′-fluoro-modified nucleotide;

[0113] Each N Mrepresents a modified nucleotide independently selected from 2'-fluoro-modified nucleotide, 2'-O-methyl-modified nucleotide, 2'-O-methoxyethyl-modified nucleotide, 2'-O-alkyl-modified nucleotide, 2'-O-allyl-modified nucleotide, bicyclic nucleic acid (BNA), and deoxyribonucleotide;

[0114] Each N L represents a modified nucleotide independently selected from 2'-O-methyl modified nucleotide, 2'-O-methoxyethyl modified nucleotide, 2'-O-alkyl modified nucleotide, 2'-O-allyl modified nucleotide, BNA, and deoxyribonucleotide;

[0115] N T represents a modified nucleotide selected from baseless nucleotide, inverted baseless nucleotide, inverted deoxyribonucleotide, 2'-O-methyl modified nucleotide, 2'-O-methoxyethyl modified nucleotide, 2'-O-alkyl modified nucleotide, 2'-O-allyl modified nucleotide, BNA, and deoxyribonucleotide;

[0116] x is an integer from 0 to 4, provided that if x is 1, 2, 3, or 4, N A One or more of the nucleotides are modified nucleotides independently selected from baseless nucleotides, inverted baseless nucleotides, inverted deoxyribonucleotides, 2'-O-methyl modified nucleotides, 2'-O-methoxyethyl modified nucleotides, 2'-O-alkyl modified nucleotides, 2'-O-allyl modified nucleotides, BNA, and deoxyribonucleotides, and N A One or more nucleotides may be complementary to nucleotides of the antisense strand;

[0117] y is an integer from 0 to 4, provided that if y is 1, 2, 3, or 4, one or more n nucleotides are modified or unmodified overhang nucleotides that do not form base pairs with nucleotides of the antisense strand;

[0118] z is an integer from 0 to 4, provided that if z is 1, 2, 3, or 4, N B One or more of the nucleotides are modified nucleotides independently selected from 2'-O-methyl modified nucleotides, 2'-O-methoxyethyl modified nucleotides, 2'-O-alkyl modified nucleotides, 2'-O-allyl modified nucleotides, BNA, and deoxyribonucleotides, and N B When one or more nucleotides are present in the sense strand, N A It may be complementary to the nucleotide, or it may be an overhang nucleotide that does not form a base pair with the nucleotide of the sense strand.

[0119] In some embodiments in which the RNAi construct comprises a structure represented by formula (D), a nucleotide overhang exists at the 3' end of the sense strand—that is, y is 1, 2, 3, or 4. In one such embodiment, y is 2. In an embodiment in which a 2-nucleotide overhang exists at the 3' end of the sense strand (i.e., y is 2), x is 0 and z is 2, or x is 1 and z is 2. In another embodiment in which the RNAi construct comprises a structure represented by formula (D), the RNAi construct comprises smooth ends at the 3' end of the sense strand and the 5' end of the antisense strand (i.e., y is 0). In an embodiment where there is no nucleotide overhang at the 3' end of the sense strand (i.e., y is 0), (i) x is 2 and z is 4, (ii) x is 3 and z is 4, (iii) x is 0 and z is 2, (iv) x is 1 and z is 2, or (v) x is 2 and z is 2. In any embodiment where x is greater than 0, N is the terminal nucleotide at the 5' end of the sense strand. A The nucleotide may be an inverted nucleotide, for example, an inverted base-free nucleotide or an inverted deoxyribonucleotide.

[0120] In a specific embodiment in which the RNAi construct comprises a structure represented by formula (D), N at positions 4, 6, 8, 9, and 16 of the antisense strand calculated from the 5' end M are 2′-fluoro-modified nucleotides, respectively, and N at positions 7 and 12 of the antisense strand calculated from the 5' end. M are each 2'-O-methyl modified nucleotides. In another embodiment, N at positions 4 and 6 of the antisense strand, calculated from the 5' end. M are each 2′-fluoro-modified nucleotides, and N at positions 7 to 9 of the antisense strand calculated from the 5' end. Mare each 2'-O-methyl modified nucleotides. In another embodiment, N at positions 4, 6, 8, 9, and 16 of the antisense strand, counted from the 5' end. M are 2'-O-methyl modified nucleotides, respectively, and N at positions 7 and 12 of the antisense strand calculated from the 5' end. M are each 2′-fluoro-modified nucleotides. In an alternative embodiment in which the RNAi construct comprises a structure represented by formula (D), N at positions 4, 6, 8, 9, and 12 of the antisense strand calculated from the 5' end. M are 2′-O-methyl modified nucleotides, respectively, and N at positions 7 and 16 of the antisense strand calculated from the 5' end. M are each 2'-fluoro-modified nucleotides. In certain other embodiments in which the RNAi construct comprises a structure represented by formula (D), N at positions 7, 8, 9, and 12 of the antisense strand calculated from the 5' end. M are 2′-O-methyl modified nucleotides, respectively, and N at positions 4, 6, and 16 of the antisense strand calculated from the 5' end. M are each 2'-fluoro-modified nucleotides. In these and other embodiments in which the RNAi construct comprises a structure represented by formula (D), N of the sense strand M is a 2'-fluoro-modified nucleotide. In an alternative embodiment, N of the sense strand M is a 2'-O-methyl modified nucleotide.

[0121] In any of the aforementioned embodiments in which the RNAi construct comprises a structure represented by formula (D), each N of both the sense strand and the antisense strand L may be a 2'-O-methyl modified nucleotide. In these embodiments and any of the embodiments described above, N of formula (D) TIt may be an inverted baseless nucleotide, an inverted deoxyribonucleotide, or a 2'-O-methyl modified nucleotide.

[0122] RNAi constructs of the present invention may also include one or more modified internucleotide linkages. As used herein, the term “modified internucleotide linkage” refers to an internucleotide linkage other than a natural 3’ to 5’ phosphodiester linkage. In some embodiments, the modified internucleotide linkage is a phosphorus-containing internucleotide linkage, e.g., a phosphotriester, an aminoalkyl phosphotriester, an alkylphosphonate (e.g., methylphosphonate, 3’-alkylene phosphonate), a phosphinate, a phosphoramidate (e.g., 3’-aminophosphoramidate and aminoalkylphosphoramidate), a phosphorothioate (P=S), a chiral phosphorothioate, a phosphodithioate, a thionophosphoramidate, a thionoalkylphosphonate, a thionoalkylphosphotriester, and a boranophosphate. In one embodiment, the modified internucleotide linkage is a 2' to 5' phosphodiester linkage. In another embodiment, since the modified internucleotide linkage is a non-phosphorus-containing internucleotide linkage, it may be referred to as a modified internucleoside linkage. Such non-phosphorus-containing linkages include, but are not limited to, morpholino linkages (partially formed from the sugar portion of a nucleoside); siloxane linkages (-O-Si(H)2-O-); sulfide, sulfoxide, and sulfone linkages; formacetyl and thioformacetyl linkages; alkene-containing backbones; sulfamate backbones; methylenemethylimino (-CH2-N(CH3)-O-CH2-) and methylenehydrazino linkages; sulfonate and sulfonamide linkages; amide linkages; and other ones in which N, O, S, and CH2 component portions are mixed. In one embodiment, the modified internucleoside linkage is a peptide-based linkage (e.g., aminoethylglycine) to produce peptide nucleic acids or PNAs, such as those described in U.S. Patents No. 5,539,082; No. 5,714,331; and No. 5,719,262.Other suitable modified nucleotide-to-nucleoside linkages that can be used in RNAi compositions of the present invention are described in U.S. Patent No. 6,693,187, U.S. Patent No. 9,181,551, U.S. Patent Publication No. 2016 / 0122761, and Deleavey and Damha, Chemistry and Biology, Vol. 19: 937-954, 2012, the full text of which is incorporated herein by reference.

[0123] In certain embodiments, the RNAi construct of the present invention comprises one or more phosphorothioate nucleotide linkages. The phosphorothioate nucleotide linkages may be present on the sense strand, the antisense strand, or both strands of the RNAi construct. For example, in some embodiments, the sense strand comprises 1, 2, 3, 4, 5, 6, 7, 8, or more phosphorothioate nucleotide linkages. In other embodiments, the antisense strand comprises 1, 2, 3, 4, 5, 6, 7, 8, or more phosphorothioate nucleotide linkages. In yet another embodiment, both strands comprise 1, 2, 3, 4, 5, 6, 7, 8, or more phosphorothioate nucleotide linkages. The RNAi construct may comprise one or more phosphorothioate nucleotide linkages at the 3'-end, 5'-end, or both ends of the sense strand, antisense strand, or both strands. For example, in a specific embodiment, the RNAi construct comprises about 1 to about 6 or more (e.g., about 1, 2, 3, 4, 5, 6 or more) continuous phosphorothioate nucleotide linkages at the 3'-end of the sense strand, antisense strand, or both strands. In another embodiment, the RNAi construct comprises about 1 to about 6 or more (e.g., about 1, 2, 5, 4, 5, 6 or more) continuous phosphorothioate nucleotide linkages at the 5'-end of the sense strand, antisense strand, or both strands.

[0124] In some embodiments, the RNAi construct comprises a single phosphorothioate nucleotide linkage between the terminal nucleotides of the 3' end of the sense strand. In other embodiments, the RNAi construct comprises two consecutive phosphorothioate nucleotide linkages between the terminal nucleotides of the 3' end of the sense strand. In one embodiment, the RNAi construct comprises a single phosphorothioate nucleotide linkage between the terminal nucleotides of the 3' end of the sense strand and a single phosphorothioate nucleotide linkage between the terminal nucleotides of the 3' end of the antisense strand. In yet another embodiment, the RNAi construct comprises two consecutive phosphorothioate nucleotide linkages between the terminal nucleotides of the 3' end of the antisense strand (i.e., a phosphorothioate nucleotide linkage in the first and second nucleotide linkage at the 3' end of the antisense strand). In another embodiment, the RNAi construct comprises two consecutive phosphorothioate nucleotide linkages between the terminal nucleotides of both the 3' and 5' ends of the antisense strand. In another embodiment, the RNAi construct comprises two consecutive phosphorothioate nucleotide linkages between the terminal nucleotides of both the 3' and 5' ends of the antisense strand and two consecutive phosphorothioate nucleotide linkages at the 5' end of the sense strand. In another embodiment, the RNAi construct comprises two consecutive phosphorothioate nucleotide linkages between the terminal nucleotides of both the 3' and 5' ends of the antisense strand and two consecutive phosphorothioate nucleotide linkages between the terminal nucleotides of the 3' end of the sense strand.In another embodiment, the RNAi construct comprises two consecutive phosphorothioate nucleotide interlinks between terminal nucleotides at both the 3' and 5' ends of the antisense strand, and two consecutive phosphorothioate nucleotide interlinks between terminal nucleotides at both the 3' and 5' ends of the sense strand (i.e., phosphorothioate nucleotide interlinks between the first and second nucleotides at both the 5' and 3' ends of the antisense strand and phosphorothioate nucleotide interlinks between the first and second nucleotides at both the 5' and 3' ends of the sense strand). In another embodiment, the RNAi construct comprises two consecutive phosphorothioate nucleotide interlinks between the terminal nucleotides of both the 3' and 5' ends of the antisense strand and a single phosphorothioate nucleotide interlink between the terminal nucleotides of the 3' end of the sense strand. In any embodiment in which one or both strands comprise one or more phosphorothioate interlinks, the remaining internucleotide linkages within the strands may be natural 3' to 5' phosphodiester linkages. For example, in some embodiments, the internucleotide linkages of each of the sense and antisense strands are selected from phosphodiesters and phosphorothioates, wherein at least one internucleotide linkage is a phosphorothioate.

[0125] In an embodiment in which the RNAi construct comprises a nucleotide overhang, two or more unpaired nucleotides in the overhang may be connected by phosphorothioate nucleotide linkages. In a specific embodiment, all unpaired nucleotides within the nucleotide overhang at the 3' end of the antisense strand and / or sense strand are connected by phosphorothioate nucleotide linkages. In another embodiment, all unpaired nucleotides within the nucleotide overhang at the 5' end of the antisense strand and / or sense strand are connected by phosphorothioate nucleotide linkages. In yet another embodiment, all unpaired nucleotides in any nucleotide overhang are connected by phosphorothioate nucleotide linkages.

[0126] The RNAi construct of the present invention may have any one of the chemical modification patterns P1 to P30 illustrated in FIG. 1. For example, in some embodiments, the RNAi construct comprises a sense strand of length 19 to 23 nucleotides and an antisense strand of length 19 to 23 nucleotides, the sequences of the antisense strand and the sense strand are sufficiently complementary to each other to form a dichotomous region of 19 to 21 base pairs, and the nucleotides at positions 2, 7, and 14 of the antisense strand (counted from the 5' end) are 2′-fluoro-modified nucleotides; the nucleotides of the sense strand at positions paired with positions 8 to 11 and 13 of the antisense strand (counted from the 5' end) are 2′-fluoro-modified nucleotides; and the sense strand and the antisense strand each do not have a total of more than 7 2′-fluoro-modified nucleotides; RNAi constructs have nucleotide overhangs at the 3' ends of the sense strand and the antisense strand.

[0127] In one embodiment, the RNAi construct

[0128] (a) As a sense strand,

[0129] (i) 21 nucleotides long;

[0130] (ii) having 2′-fluoro-modified nucleotides at positions 7 and 9 to 12 (calculated from the 5' end) and 2′-O-methyl-modified nucleotides at positions 1 to 6, 8, and 13 to 21;

[0131] (iii) A sense strand having phosphorothioate internucleotide linkages between nucleotides at positions 19 and 20 (counted from the 5' end) and between nucleotides at positions 20 and 21;

[0132] and

[0133] (b) As an antisense strand,

[0134] (i) 21 nucleotides long;

[0135] (ii) having 2′-fluoro-modified nucleotides at positions 2, 4, 6, 7, 12 and 14 (calculated from the 5' end) and 2′-O-methyl-modified nucleotides at positions 1, 3, 5, 8 to 11, 13 and 15 to 21;

[0136] (iii) an antisense strand having phosphorothioate nucleotide linkages between nucleotides at positions 1 and 2 (counted from the 5' end), between nucleotides at positions 2 and 3, between nucleotides at positions 19 and 20, and between nucleotides at positions 20 and 21; comprising,

[0137] At this time, the RNAi construct has a nucleotide overhang containing two nucleotides at the 3' end of the sense strand and the 3' end of the antisense strand.

[0138] In another embodiment, the RNAi construct

[0139] (a) As a sense strand,

[0140] (i) 22 nucleotides long;

[0141] (ii) an inverted baseless nucleotide or an inverted deoxyribonucleotide at position 1 (calculated from the 5' end); 2′-fluoro-modified nucleotides at positions 8 and 10 to 13 and 2′-O-methyl-modified nucleotides at positions 2 to 7, 9 and 14 to 22;

[0142] (iii) A sense strand having phosphorothioate internucleotide linkages between nucleotides at positions 20 and 21 (counted from the 5' end) and between nucleotides at positions 21 and 22;

[0143] and

[0144] (b) As an antisense strand,

[0145] (i) 21 nucleotides long;

[0146] (ii) having 2′-fluoro-modified nucleotides at positions 2, 4, 6, 7, 12 and 14 (calculated from the 5' end) and 2′-O-methyl-modified nucleotides at positions 1, 3, 5, 8 to 11, 13 and 15 to 21;

[0147] (iii) an antisense strand having phosphorothioate nucleotide linkages between nucleotides at positions 1 and 2 (counted from the 5' end), between nucleotides at positions 2 and 3, between nucleotides at positions 19 and 20, and between nucleotides at positions 20 and 21; comprising,

[0148] In this case, the RNAi construct has a nucleotide overhang containing two nucleotides at the 3' end of the sense strand and a nucleotide overhang containing one or two nucleotides at the 3' end of the antisense strand.

[0149] In another embodiment, the RNAi construct

[0150] (a) As a sense strand,

[0151] (i) 21 nucleotides long;

[0152] (ii) having 2′-fluoro-modified nucleotides at positions 7 and 9 to 12 (calculated from the 5' end) and 2′-O-methyl-modified nucleotides at positions 1 to 6, 8, and 13 to 21;

[0153] (iii) A sense strand having phosphorothioate internucleotide linkages between nucleotides at positions 19 and 20 (counted from the 5' end) and between nucleotides at positions 20 and 21;

[0154] and

[0155] (b) As an antisense strand,

[0156] (i) 21 nucleotides long;

[0157] (ii) having 2′-fluoro-modified nucleotides at positions 2, 7, 10, 12 and 14 (calculated from the 5' end) and 2′-O-methyl-modified nucleotides at positions 1, 3 to 6, 8, 9, 11, 13 and 15 to 21;

[0158] (iii) an antisense strand having phosphorothioate nucleotide linkages between nucleotides at positions 1 and 2 (counted from the 5' end), between nucleotides at positions 2 and 3, between nucleotides at positions 19 and 20, and between nucleotides at positions 20 and 21; comprising,

[0159] At this time, the RNAi construct has a nucleotide overhang containing two nucleotides at the 3' end of the sense strand and the 3' end of the antisense strand.

[0160] In another embodiment, the RNAi construct

[0161] (a) As a sense strand,

[0162] (i) 21 nucleotides long;

[0163] (ii) having 2′-fluoro-modified nucleotides at positions 7 and 9 to 12 (calculated from the 5' end) and 2′-O-methyl-modified nucleotides at positions 1 to 6, 8, and 13 to 21;

[0164] (iii) A sense strand having phosphorothioate internucleotide linkages between nucleotides at positions 19 and 20 (counted from the 5' end) and between nucleotides at positions 20 and 21;

[0165] and

[0166] (b) As an antisense strand,

[0167] (i) 21 nucleotides long;

[0168] (ii) having 2′-fluoro-modified nucleotides at positions 2, 4, 6, 7, 10, 12 and 14 (counted from the 5' end) and 2′-O-methyl-modified nucleotides at positions 1, 3, 5, 8, 9, 11, 13 and 15 to 21;

[0169] (iii) an antisense strand having phosphorothioate nucleotide linkages between nucleotides at positions 1 and 2 (counted from the 5' end), between nucleotides at positions 2 and 3, between nucleotides at positions 19 and 20, and between nucleotides at positions 20 and 21; comprising,

[0170] At this time, the RNAi construct has a nucleotide overhang containing two nucleotides at the 3' end of the sense strand and the 3' end of the antisense strand.

[0171] In another specific embodiment, the RNAi construct

[0172] (a) As a sense strand,

[0173] (i) 21 nucleotides long;

[0174] (ii) having a 2′-fluoro-modified nucleotide at positions 7 and 9 to 12 (calculated from the 5' end), a 2′-O-methyl-modified nucleotide at positions 1 to 6, 8, and 13 to 20, and an inverted baseless nucleotide or inverted deoxyribonucleotide at position 21;

[0175] (iii) A sense strand having a phosphorothioate internucleotide link between nucleotides at positions 20 and 21 (counted from the 5' end);

[0176] and

[0177] (b) As an antisense strand,

[0178] (i) 21 nucleotides long;

[0179] (ii) having 2′-fluoro-modified nucleotides at positions 2, 7, 12 and 14 (calculated from the 5' end) and 2′-O-methyl-modified nucleotides at positions 1, 3 to 6, 8 to 11, 13, and 15 to 21;

[0180] (iii) an antisense strand having phosphorothioate nucleotide linkages between nucleotides at positions 1 and 2 (counted from the 5' end), between nucleotides at positions 2 and 3, between nucleotides at positions 19 and 20, and between nucleotides at positions 20 and 21; comprising,

[0181] At this time, the RNAi construct has a nucleotide overhang containing two nucleotides at the 3' end of the sense strand and the 3' end of the antisense strand.

[0182] In a specific embodiment, the RNAi construct comprises a sense strand of 19 to 21 nucleotide length and an antisense strand of 21 to 23 nucleotide length, wherein the sequences of the antisense strand and the sense strand are sufficiently complementary to each other to form a dichotomous region of 19 to 21 base pairs, and nucleotides at positions 2, 7, and 14 of the antisense strand (counted from the 5' end) are 2′-fluoro-modified nucleotides; nucleotides of the sense strand at positions paired with positions 8 to 11 and 13 of the antisense strand (counted from the 5' end) are 2′-fluoro-modified nucleotides; and the sense strand and the antisense strand each do not have a total of more than 7 2′-fluoro-modified nucleotides; RNAi constructs have a nucleotide overhang at the 3' end of the antisense strand and a smooth end at the 5' end of the antisense strand / 3' end of the sense strand.

[0183] In one embodiment, the RNAi construct

[0184] (a) As a sense strand,

[0185] (i) 21 nucleotides long;

[0186] (ii) 2′-fluoro-modified nucleotides at positions 9 and 11 to 14 (calculated from the 5' end); 2′-O-methyl-modified nucleotides at positions 1 to 8, 10, and 15 to 20; and having an inverted baseless nucleotide or an inverted deoxyribonucleotide at position 21;

[0187] (iii) A sense strand having a phosphorothioate internucleotide link between nucleotides at positions 20 and 21 (counted from the 5' end);

[0188] and

[0189] (b) As an antisense strand,

[0190] (i) 23 nucleotides long;

[0191] (ii) having 2′-fluoro-modified nucleotides at positions 2, 4, 6, 7, 12 and 14 (calculated from the 5' end) and 2′-O-methyl-modified nucleotides at positions 1, 3, 5, 8 to 11, 13 and 15 to 23;

[0192] (iii) an antisense strand having phosphorothioate nucleotide linkages between nucleotides at positions 1 and 2 (calculated from the 5' end), between nucleotides at positions 2 and 3, between nucleotides at positions 21 and 22, and between nucleotides at positions 22 and 23; comprising,

[0193] In this case, the RNAi construct has a nucleotide overhang containing two nucleotides at the 3' end of the antisense strand and a smooth end at the 5' end of the antisense strand.

[0194] In another embodiment, the RNAi construct

[0195] (a) As a sense strand,

[0196] (i) 22 nucleotides long;

[0197] (ii) an inverted baseless nucleotide or an inverted deoxyribonucleotide at position 1 (calculated from the 5' end); 2′-fluoro-modified nucleotides at positions 10 and 12 to 15 and 2′-O-methyl-modified nucleotides at positions 2 to 9, 11, and 16 to 22;

[0198] (iii) A sense strand having phosphorothioate internucleotide linkages between nucleotides at positions 20 and 21 (counted from the 5' end) and between nucleotides at positions 21 and 22;

[0199] and

[0200] (b) As an antisense strand,

[0201] (i) 23 nucleotides long;

[0202] (ii) having 2′-fluoro-modified nucleotides at positions 2, 4, 6, 7, 12 and 14 (calculated from the 5' end) and 2′-O-methyl-modified nucleotides at positions 1, 3, 5, 8 to 11, 13 and 15 to 23;

[0203] (iii) an antisense strand having phosphorothioate nucleotide linkages between nucleotides at positions 1 and 2 (calculated from the 5' end), between nucleotides at positions 2 and 3, between nucleotides at positions 21 and 22, and between nucleotides at positions 22 and 23; comprising,

[0204] In this case, the RNAi construct has a nucleotide overhang containing one or two nucleotides at the 3' end of the antisense strand and a smooth end at the 5' end of the antisense strand.

[0205] In another embodiment, the RNAi construct

[0206] (a) As a sense strand,

[0207] (i) 21 nucleotides long;

[0208] (ii) having 2′-fluoro-modified nucleotides at positions 9 and 11 to 14 (calculated from the 5' end) and 2′-O-methyl-modified nucleotides at positions 1 to 8, 10, and 15 to 21;

[0209] (iii) A sense strand having phosphorothioate internucleotide linkages between nucleotides at positions 19 and 20 (counted from the 5' end) and between nucleotides at positions 20 and 21;

[0210] and

[0211] (b) As an antisense strand,

[0212] (i) 23 nucleotides long;

[0213] (ii) having 2′-fluoro-modified nucleotides at positions 2, 4, 6, 7, 12 and 14 (calculated from the 5' end) and 2′-O-methyl-modified nucleotides at positions 1, 3, 5, 8 to 11, 13 and 15 to 23;

[0214] (iii) an antisense strand having phosphorothioate nucleotide linkages between nucleotides at positions 1 and 2 (calculated from the 5' end), between nucleotides at positions 2 and 3, between nucleotides at positions 21 and 22, and between nucleotides at positions 22 and 23; comprising,

[0215] In this case, the RNAi construct has a nucleotide overhang containing two nucleotides at the 3' end of the antisense strand and a smooth end at the 5' end of the antisense strand.

[0216] In another embodiment, the RNAi construct

[0217] (a) As a sense strand,

[0218] (i) 22 nucleotides long;

[0219] (ii) inverted baseless nucleotides or inverted deoxyribonucleotides at positions 1 and 22 (calculated from the 5' end); 2′-fluoro-modified nucleotides at positions 10 and 12 to 15 and 2′-O-methyl-modified nucleotides at positions 2 to 9, 11, and 16 to 21;

[0220] (iii) a sense strand having a phosphorothioate internucleotide link between nucleotides at positions 21 and 22;

[0221] and

[0222] (b) As an antisense strand,

[0223] (i) 23 nucleotides long;

[0224] (ii) having 2′-fluoro-modified nucleotides at positions 2, 4, 6, 7, 12 and 14 (calculated from the 5' end) and 2′-O-methyl-modified nucleotides at positions 1, 3, 5, 8 to 11, 13 and 15 to 23;

[0225] (iii) an antisense strand having phosphorothioate nucleotide linkages between nucleotides at positions 1 and 2 (calculated from the 5' end), between nucleotides at positions 2 and 3, between nucleotides at positions 21 and 22, and between nucleotides at positions 22 and 23; comprising,

[0226] In this case, the RNAi construct has a nucleotide overhang containing one or two nucleotides at the 3' end of the antisense strand and a smooth end at the 5' end of the antisense strand.

[0227] In one specific embodiment, the RNAi construct

[0228] (a) As a sense strand,

[0229] (i) 19 nucleotides in length;

[0230] (ii) having 2′-fluoro-modified nucleotides at positions 7 and 9 to 12 (calculated from the 5' end) and 2′-O-methyl-modified nucleotides at positions 1 to 6, 8, and 13 to 19;

[0231] (iii) A sense strand having phosphorothioate internucleotide linkages between nucleotides at positions 17 and 18 (counted from the 5' end) and between nucleotides at positions 18 and 19;

[0232] and

[0233] (b) As an antisense strand,

[0234] (i) 21 nucleotides long;

[0235] (ii) having 2′-fluoro-modified nucleotides at positions 2, 4, 6, 7, 12 and 14 (calculated from the 5' end) and 2′-O-methyl-modified nucleotides at positions 1, 3, 5, 8 to 11, 13 and 15 to 21;

[0236] (iii) an antisense strand having phosphorothioate nucleotide linkages between nucleotides at positions 1 and 2 (counted from the 5' end), between nucleotides at positions 2 and 3, between nucleotides at positions 19 and 20, and between nucleotides at positions 20 and 21; comprising,

[0237] In this case, the RNAi construct has a nucleotide overhang containing two nucleotides at the 3' end of the antisense strand and a smooth end at the 5' end of the antisense strand.

[0238] In another specific embodiment, the RNAi construct

[0239] (a) As a sense strand,

[0240] (i) 19 nucleotides in length;

[0241] (ii) having a 2′-fluoro-modified nucleotide at positions 7 and 9 to 12 (calculated from the 5' end); a 2′-O-methyl-modified nucleotide at positions 1 to 6, 8, and 13 to 18; and an inverted baseless nucleotide or an inverted deoxyribonucleotide at position 19;

[0242] (iii) A sense strand having phosphorothioate internucleotide linkages between nucleotides at positions 17 and 18 (counted from the 5' end) and between nucleotides at positions 18 and 19;

[0243] and

[0244] (b) As an antisense strand,

[0245] (i) 21 nucleotides long;

[0246] (ii) having 2′-fluoro-modified nucleotides at positions 2, 4, 6, 7, 12 and 14 (calculated from the 5' end) and 2′-O-methyl-modified nucleotides at positions 1, 3, 5, 8 to 11, 13 and 15 to 21;

[0247] (iii) an antisense strand having phosphorothioate nucleotide linkages between nucleotides at positions 1 and 2 (counted from the 5' end), between nucleotides at positions 2 and 3, between nucleotides at positions 19 and 20, and between nucleotides at positions 20 and 21; comprising,

[0248] In this case, the RNAi construct has a nucleotide overhang containing two nucleotides at the 3' end of the antisense strand and a smooth end at the 5' end of the antisense strand.

[0249] In another specific embodiment, the RNAi construct

[0250] (a) As a sense strand,

[0251] (i) 21 nucleotides long;

[0252] (ii) 2′-fluoro-modified nucleotides at positions 9 and 11 to 14 (calculated from the 5' end); 2′-O-methyl-modified nucleotides at positions 1 to 8, 10, and 15 to 20; and having an inverted baseless nucleotide or an inverted deoxyribonucleotide at position 21;

[0253] (iii) A sense strand having a phosphorothioate internucleotide link between nucleotides at positions 20 and 21 (counted from the 5' end);

[0254] and

[0255] (b) As an antisense strand,

[0256] (i) 23 nucleotides long;

[0257] (ii) having 2′-fluoro-modified nucleotides at positions 2, 7, 12 and 14 (calculated from the 5' end) and 2′-O-methyl-modified nucleotides at positions 1, 3 to 6, 8 to 11, 13, and 15 to 23;

[0258] (iii) an antisense strand having phosphorothioate nucleotide linkages between nucleotides at positions 1 and 2 (calculated from the 5' end), between nucleotides at positions 2 and 3, between nucleotides at positions 21 and 22, and between nucleotides at positions 22 and 23; comprising,

[0259] In this case, the RNAi construct has a nucleotide overhang containing two nucleotides at the 3' end of the antisense strand and a smooth end at the 5' end of the antisense strand.

[0260] In another embodiment, the RNAi construct

[0261] (a) As a sense strand,

[0262] (i) 22 nucleotides long;

[0263] (ii) an inverted baseless nucleotide or an inverted deoxyribonucleotide at position 1 (calculated from the 5' end); 2′-fluoro-modified nucleotides at positions 10 and 12 to 15 and 2′-O-methyl-modified nucleotides at positions 2 to 9, 11, and 16 to 22;

[0264] (iii) a sense strand having phosphorothioate internucleotide linkages between nucleotides at positions 20 and 21 and between nucleotides at positions 21 and 22;

[0265] and

[0266] (b) As an antisense strand,

[0267] (i) 23 nucleotides long;

[0268] (ii) having 2′-fluoro-modified nucleotides at positions 2, 7, 12 and 14 (calculated from the 5' end) and 2′-O-methyl-modified nucleotides at positions 1, 3 to 6, 8 to 11, 13, and 15 to 23;

[0269] (iii) an antisense strand having phosphorothioate nucleotide linkages between nucleotides at positions 1 and 2 (calculated from the 5' end), between nucleotides at positions 2 and 3, between nucleotides at positions 21 and 22, and between nucleotides at positions 22 and 23; comprising,

[0270] In this case, the RNAi construct has a nucleotide overhang containing one or two nucleotides at the 3' end of the antisense strand and a smooth end at the 5' end of the antisense strand.

[0271] In another embodiment, the RNAi construct

[0272] (a) As a sense strand,

[0273] (i) 21 nucleotides long;

[0274] (ii) 2′-fluoro-modified nucleotides at positions 9 and 11 to 14 (calculated from the 5' end); 2′-O-methyl-modified nucleotides at positions 1 to 8, 10, and 15 to 20; and having an inverted baseless nucleotide or an inverted deoxyribonucleotide at position 21;

[0275] (iii) A sense strand having a phosphorothioate internucleotide link between nucleotides at positions 20 and 21 (counted from the 5' end);

[0276] and

[0277] (b) As an antisense strand,

[0278] (i) 23 nucleotides long;

[0279] (ii) having 2′-fluoro-modified nucleotides at positions 2, 4, 7, 12 and 14 (calculated from the 5' end) and 2′-O-methyl-modified nucleotides at positions 1, 3, 5, 6, 8 to 11, 13 and 15 to 23;

[0280] (iii) an antisense strand having phosphorothioate nucleotide linkages between nucleotides at positions 1 and 2 (calculated from the 5' end), between nucleotides at positions 2 and 3, between nucleotides at positions 21 and 22, and between nucleotides at positions 22 and 23; comprising,

[0281] In this case, the RNAi construct has a nucleotide overhang containing two nucleotides at the 3' end of the antisense strand and a smooth end at the 5' end of the antisense strand.

[0282] In another specific embodiment, the RNAi construct

[0283] (a) As a sense strand,

[0284] (i) 21 nucleotides long;

[0285] (ii) 2′-fluoro-modified nucleotides at positions 9, 11 to 14, 17, and 19 (calculated from the 5' end); 2′-O-methyl-modified nucleotides at positions 1 to 8, 10, 15, 16, 18, and 20; and having an inverted baseless nucleotide or an inverted deoxyribonucleotide at position 21;

[0286] (iii) A sense strand having a phosphorothioate internucleotide link between nucleotides at positions 20 and 21 (counted from the 5' end);

[0287] and

[0288] (b) As an antisense strand,

[0289] (i) 23 nucleotides long;

[0290] (ii) having 2′-fluoro-modified nucleotides at positions 2, 4, 7, 12 and 14 (calculated from the 5' end) and 2′-O-methyl-modified nucleotides at positions 1, 3, 5, 6, 8 to 11, 13 and 15 to 23;

[0291] (iii) an antisense strand having phosphorothioate nucleotide linkages between nucleotides at positions 1 and 2 (calculated from the 5' end), between nucleotides at positions 2 and 3, between nucleotides at positions 21 and 22, and between nucleotides at positions 22 and 23; comprising,

[0292] In this case, the RNAi construct has a nucleotide overhang containing two nucleotides at the 3' end of the antisense strand and a smooth end at the 5' end of the antisense strand.

[0293] In another specific embodiment, the RNAi construct

[0294] (a) As a sense strand,

[0295] (i) 19 nucleotides in length;

[0296] (ii) having a 2′-fluoro-modified nucleotide at positions 7 and 9 to 12 (calculated from the 5' end); a 2′-O-methyl-modified nucleotide at positions 1 to 6, 8, and 13 to 18; and an inverted baseless nucleotide or an inverted deoxyribonucleotide at position 19;

[0297] (iii) A sense strand having phosphorothioate internucleotide linkages between nucleotides at positions 18 and 19 (counted from the 5' end) and optionally between nucleotides at positions 17 and 18;

[0298] and

[0299] (b) As an antisense strand,

[0300] (i) 21 nucleotides long;

[0301] (ii) having 2′-fluoro-modified nucleotides at positions 2, 7, 12 and 14 (calculated from the 5' end) and 2′-O-methyl-modified nucleotides at positions 1, 3 to 6, 8 to 11, 13, and 15 to 21;

[0302] (iii) an antisense strand having phosphorothioate nucleotide linkages between nucleotides at positions 1 and 2 (counted from the 5' end), between nucleotides at positions 2 and 3, between nucleotides at positions 19 and 20, and between nucleotides at positions 20 and 21; comprising,

[0303] In this case, the RNAi construct has a nucleotide overhang containing two nucleotides at the 3' end of the antisense strand and a smooth end at the 5' end of the antisense strand.

[0304] In another specific embodiment, the RNAi construct

[0305] (a) As a sense strand,

[0306] (i) 21 nucleotides long;

[0307] (ii) 2′-fluoro-modified nucleotides at positions 9 and 11 to 14 (calculated from the 5' end); 2′-O-methyl-modified nucleotides at positions 1 to 8, 10, and 15 to 20; and having an inverted baseless nucleotide or an inverted deoxyribonucleotide at position 21;

[0308] (iii) A sense strand having a phosphorothioate internucleotide link between nucleotides at positions 20 and 21 (counted from the 5' end);

[0309] and

[0310] (b) As an antisense strand,

[0311] (i) 23 nucleotides long;

[0312] (ii) having 2′-fluoro-modified nucleotides at positions 2, 4, 6, 7, 10, 12 and 14 (counted from the 5' end) and 2′-O-methyl-modified nucleotides at positions 1, 3, 5, 8, 9, 11, 13 and 15 to 23;

[0313] (iii) an antisense strand having phosphorothioate nucleotide linkages between nucleotides at positions 1 and 2 (calculated from the 5' end), between nucleotides at positions 2 and 3, between nucleotides at positions 21 and 22, and between nucleotides at positions 22 and 23; comprising,

[0314] In this case, the RNAi construct has a nucleotide overhang containing two nucleotides at the 3' end of the antisense strand and a smooth end at the 5' end of the antisense strand.

[0315] In another specific embodiment, the RNAi construct

[0316] (a) As a sense strand,

[0317] (i) 21 nucleotides long;

[0318] (ii) 2′-fluoro-modified nucleotides at positions 9 and 11 to 14 (calculated from the 5' end); 2′-O-methyl-modified nucleotides at positions 1 to 8, 10, and 15 to 20; and having an inverted baseless nucleotide or an inverted deoxyribonucleotide at position 21;

[0319] (iii) A sense strand having a phosphorothioate internucleotide link between nucleotides at positions 20 and 21 (counted from the 5' end);

[0320] and

[0321] (b) As an antisense strand,

[0322] (i) 23 nucleotides long;

[0323] (ii) having 2′-fluoro-modified nucleotides at positions 2, 7, 10, 12 and 14 (calculated from the 5' end) and 2′-O-methyl-modified nucleotides at positions 1, 3 to 6, 8, 9, 11, 13 and 15 to 23;

[0324] (iii) an antisense strand having phosphorothioate nucleotide linkages between nucleotides at positions 1 and 2 (calculated from the 5' end), between nucleotides at positions 2 and 3, between nucleotides at positions 21 and 22, and between nucleotides at positions 22 and 23; comprising,

[0325] In this case, the RNAi construct has a nucleotide overhang containing two nucleotides at the 3' end of the antisense strand and a smooth end at the 5' end of the antisense strand.

[0326] In another specific embodiment, the RNAi construct

[0327] (a) As a sense strand,

[0328] (i) 21 nucleotides long;

[0329] (ii) 2′-fluoro-modified nucleotides at positions 9, 11 to 14, 17, and 19 (calculated from the 5' end); 2′-O-methyl-modified nucleotides at positions 1 to 8, 10, 15, 16, 18, and 20; and having an inverted baseless nucleotide or an inverted deoxyribonucleotide at position 21;

[0330] (iii) A sense strand having a phosphorothioate internucleotide link between nucleotides at positions 20 and 21 (counted from the 5' end);

[0331] and

[0332] (b) As an antisense strand,

[0333] (i) 23 nucleotides long;

[0334] (ii) having 2′-fluoro-modified nucleotides at positions 2, 7, 10, 12 and 14 (calculated from the 5' end) and 2′-O-methyl-modified nucleotides at positions 1, 3 to 6, 8, 9, 11, 13 and 15 to 23;

[0335] (iii) an antisense strand having phosphorothioate nucleotide linkages between nucleotides at positions 1 and 2 (calculated from the 5' end), between nucleotides at positions 2 and 3, between nucleotides at positions 21 and 22, and between nucleotides at positions 22 and 23; comprising,

[0336] In this case, the RNAi construct has a nucleotide overhang containing two nucleotides at the 3' end of the antisense strand and a smooth end at the 5' end of the antisense strand.

[0337] In another specific embodiment, the RNAi construct

[0338] (a) As a sense strand,

[0339] (i) 21 nucleotides long;

[0340] (ii) 2′-fluoro-modified nucleotides at positions 9 and 11 to 14 (calculated from the 5' end); 2′-O-methyl-modified nucleotides at positions 1 to 8, 10, and 15 to 20; and having an inverted baseless nucleotide or an inverted deoxyribonucleotide at position 21;

[0341] (iii) A sense strand having a phosphorothioate internucleotide link between nucleotides at positions 20 and 21 (counted from the 5' end);

[0342] and

[0343] (b) As an antisense strand,

[0344] (i) 23 nucleotides long;

[0345] (ii) having 2′-fluoro-modified nucleotides at positions 2, 4, 7, 10, 12 and 14 (calculated from the 5' end) and 2′-O-methyl-modified nucleotides at positions 1, 3, 5, 6, 8, 9, 11, 13 and 15 through 23;

[0346] (iii) an antisense strand having phosphorothioate nucleotide linkages between nucleotides at positions 1 and 2 (calculated from the 5' end), between nucleotides at positions 2 and 3, between nucleotides at positions 21 and 22, and between nucleotides at positions 22 and 23; comprising,

[0347] In this case, the RNAi construct has a nucleotide overhang containing two nucleotides at the 3' end of the antisense strand and a smooth end at the 5' end of the antisense strand.

[0348] In some embodiments of the present invention, the RNAi construct comprises a sense strand of 19 to 23 nucleotide lengths and an antisense strand of 19 to 23 nucleotide lengths, wherein the sequences of the antisense strand and the sense strand are sufficiently complementary to each other to form a dimer region of 19 to 21 base pairs, and the nucleotides at positions 2, 14, and 16 of the antisense strand (counted from the 5' end) are 2′-fluoro-modified nucleotides; the nucleotides of the sense strand at positions paired with positions 10 to 13 of the antisense strand (counted from the 5' end) are 2′-fluoro-modified nucleotides; and the sense strand and the antisense strand each do not have more than 7 total 2′-fluoro-modified nucleotides. In this embodiment, the RNAi construct has a nucleotide overhang at the 3' end of the antisense strand and a smooth end at the 5' end of the antisense strand / 3' end of the sense strand. In an alternative embodiment, the RNAi construct has nucleotide overhangs at both the 3' ends of the sense strand and the antisense strand.

[0349] In one specific embodiment, the RNAi construct

[0350] (a) As a sense strand,

[0351] (i) 21 nucleotides long;

[0352] (ii) having a 2′-fluoro-modified nucleotide at positions 7 and 9 to 12 (calculated from the 5' end); a 2′-O-methyl-modified nucleotide at positions 1 to 6, 8, and 13 to 20; and an inverted baseless nucleotide or an inverted deoxyribonucleotide at position 21;

[0353] (iii) A sense strand having a phosphorothioate internucleotide link between nucleotides at positions 20 and 21 (counted from the 5' end);

[0354] and

[0355] (b) As an antisense strand,

[0356] (i) 23 nucleotides long;

[0357] (ii) having 2′-fluoro-modified nucleotides at positions 2, 4, 6, 8, 9, 14 and 16 (calculated from the 5' end) and 2′-O-methyl-modified nucleotides at positions 1, 3, 5, 7, 10 to 13, 15 and 17 to 23;

[0358] (iii) an antisense strand having phosphorothioate nucleotide linkages between nucleotides at positions 1 and 2 (calculated from the 5' end), between nucleotides at positions 2 and 3, between nucleotides at positions 21 and 22, and between nucleotides at positions 22 and 23; comprising,

[0359] In this case, the RNAi construct has a nucleotide overhang containing two nucleotides at the 3' end of the antisense strand and a smooth end at the 5' end of the antisense strand.

[0360] In another specific embodiment, the RNAi construct

[0361] (a) As a sense strand,

[0362] (i) 21 nucleotides long;

[0363] (ii) having a 2′-fluoro-modified nucleotide at positions 7 and 9 to 12 (calculated from the 5' end); a 2′-O-methyl-modified nucleotide at positions 1 to 6, 8, and 13 to 20; and an inverted baseless nucleotide or an inverted deoxyribonucleotide at position 21;

[0364] (iii) A sense strand having a phosphorothioate internucleotide link between nucleotides at positions 20 and 21 (counted from the 5' end);

[0365] and

[0366] (b) As an antisense strand,

[0367] (i) 23 nucleotides long;

[0368] (ii) having 2′-fluoro-modified nucleotides at positions 2, 7, 14 and 16 (calculated from the 5' end) and 2′-O-methyl-modified nucleotides at positions 1, 3 to 6, 8 to 13, 15 and 17 to 23;

[0369] (iii) an antisense strand having phosphorothioate nucleotide linkages between nucleotides at positions 1 and 2 (calculated from the 5' end), between nucleotides at positions 2 and 3, between nucleotides at positions 21 and 22, and between nucleotides at positions 22 and 23; comprising,

[0370] In this case, the RNAi construct has a nucleotide overhang containing two nucleotides at the 3' end of the antisense strand and a smooth end at the 5' end of the antisense strand.

[0371] In another specific embodiment, the RNAi construct

[0372] (a) As a sense strand,

[0373] (i) 21 nucleotides long;

[0374] (ii) having a 2′-fluoro-modified nucleotide at positions 7 and 9 to 12 (calculated from the 5' end); a 2′-O-methyl-modified nucleotide at positions 1 to 6, 8, and 13 to 20; and an inverted baseless nucleotide or an inverted deoxyribonucleotide at position 21;

[0375] (iii) A sense strand having a phosphorothioate internucleotide link between nucleotides at positions 20 and 21 (counted from the 5' end);

[0376] and

[0377] (b) As an antisense strand,

[0378] (i) 23 nucleotides long;

[0379] (ii) having 2′-fluoro-modified nucleotides at positions 2, 4, 6, 14 and 16 (calculated from the 5' end) and 2′-O-methyl-modified nucleotides at positions 1, 3, 5, 7 to 13, 15 and 17 to 23;

[0380] (iii) an antisense strand having phosphorothioate nucleotide linkages between nucleotides at positions 1 and 2 (calculated from the 5' end), between nucleotides at positions 2 and 3, between nucleotides at positions 21 and 22, and between nucleotides at positions 22 and 23; comprising,

[0381] In this case, the RNAi construct has a nucleotide overhang containing two nucleotides at the 3' end of the antisense strand and a smooth end at the 5' end of the antisense strand.

[0382] In another specific embodiment, the RNAi construct

[0383] (a) As a sense strand,

[0384] (i) 19 nucleotides in length;

[0385] (ii) having a 2′-fluoro-modified nucleotide at positions 5 and 7 to 10 (calculated from the 5' end); a 2′-O-methyl-modified nucleotide at positions 1 to 4, 6, and 11 to 18; and an inverted baseless nucleotide or an inverted deoxyribonucleotide at position 19;

[0386] (iii) A sense strand having a phosphorothioate internucleotide link between nucleotides at positions 18 and 19 (counted from the 5' end);

[0387] and

[0388] (b) As an antisense strand,

[0389] (i) 21 nucleotides long;

[0390] (ii) having 2′-fluoro-modified nucleotides at positions 2, 4, 6, 8, 9, 14 and 16 (calculated from the 5' end) and 2′-O-methyl-modified nucleotides at positions 1, 3, 5, 7, 10 to 13, 15 and 17 to 21;

[0391] (iii) an antisense strand having phosphorothioate nucleotide linkages between nucleotides at positions 1 and 2 (counted from the 5' end), between nucleotides at positions 2 and 3, between nucleotides at positions 19 and 20, and between nucleotides at positions 20 and 21; comprising,

[0392] In this case, the RNAi construct has a nucleotide overhang containing two nucleotides at the 3' end of the antisense strand and a smooth end at the 5' end of the antisense strand.

[0393] In another specific embodiment, the RNAi construct

[0394] (a) As a sense strand,

[0395] (i) 20 nucleotides long;

[0396] (ii) having an inverted baseless nucleotide or an inverted deoxyribonucleotide at position 1 (calculated from the 5' end); 2′-fluoro-modified nucleotides at positions 8 to 11; and 2′-O-methyl-modified nucleotides at positions 2 to 7 and 12 to 20;

[0397] (iii) A sense strand having phosphorothioate internucleotide linkages between nucleotides at positions 18 and 19 (counted from the 5' end) and between nucleotides at positions 19 and 20;

[0398] and

[0399] (b) As an antisense strand,

[0400] (i) 21 nucleotides long;

[0401] (ii) having 2′-fluoro-modified nucleotides at positions 2, 7, 14 and 16 (calculated from the 5' end) and 2′-O-methyl-modified nucleotides at positions 1, 3 to 6, 8 to 13, 15 and 17 to 21;

[0402] (iii) an antisense strand having phosphorothioate nucleotide linkages between nucleotides at positions 1 and 2 (counted from the 5' end), between nucleotides at positions 2 and 3, between nucleotides at positions 19 and 20, and between nucleotides at positions 20 and 21; comprising,

[0403] In this case, the RNAi construct has a nucleotide overhang containing one or two nucleotides at the 3' end of the antisense strand and a smooth end at the 5' end of the antisense strand.

[0404] In another embodiment, the RNAi construct

[0405] (a) As a sense strand,

[0406] (i) 22 nucleotides long;

[0407] (ii) having an inverted baseless nucleotide or an inverted deoxyribonucleotide at position 1 (calculated from the 5' end); 2′-fluoro-modified nucleotides at positions 8 to 11; and 2′-O-methyl-modified nucleotides at positions 2 to 7 and 12 to 22;

[0408] (iii) A sense strand having phosphorothioate internucleotide linkages between nucleotides at positions 20 and 21 (counted from the 5' end) and between nucleotides at positions 21 and 22;

[0409] and

[0410] (b) As an antisense strand,

[0411] (i) 21 nucleotides long;

[0412] (ii) having 2′-fluoro-modified nucleotides at positions 2, 7, 14 and 16 (calculated from the 5' end) and 2′-O-methyl-modified nucleotides at positions 1, 3 to 6, 8 to 13, 15 and 17 to 21;

[0413] (iii) an antisense strand having phosphorothioate nucleotide linkages between nucleotides at positions 1 and 2 (counted from the 5' end), between nucleotides at positions 2 and 3, between nucleotides at positions 19 and 20, and between nucleotides at positions 20 and 21; comprising,

[0414] In this case, the RNAi construct has a nucleotide overhang containing two nucleotides at the 3' end of the sense strand and a nucleotide overhang containing one or two nucleotides at the 3' end of the antisense strand.

[0415] In a specific embodiment of the present invention, the RNAi construct comprises a sense strand of 19 to 23 nucleotide lengths and an antisense strand of 19 to 23 nucleotide lengths, wherein the sequences of the antisense strand and the sense strand are sufficiently complementary to each other to form a dichotomous region of 19 to 21 base pairs, and nucleotides at positions 2, 7, 12, and 14 of the antisense strand (counted from the 5' end) are 2′-fluoro-modified nucleotides; nucleotides of the sense strand at positions paired with positions 10 to 13 of the antisense strand (counted from the 5' end) are 2′-fluoro-modified nucleotides; the sense strand and the antisense strand each do not have more than 7 total 2′-fluoro-modified nucleotides; and the RNAi construct has nucleotide overhangs at the 3' ends of the sense strand and the antisense strand.

[0416] For example, in one embodiment, the RNAi construct

[0417] (a) As a sense strand,

[0418] (i) 21 nucleotides long;

[0419] (ii) 2′-fluoro-modified nucleotides at positions 7 to 10 (calculated from the 5' end); and 2′-O-methyl-modified nucleotides at positions 1 to 6 and 11 to 21;

[0420] (iii) A sense strand having phosphorothioate internucleotide linkages between nucleotides at positions 19 and 20 (counted from the 5' end) and between nucleotides at positions 20 and 21;

[0421] and

[0422] (b) As an antisense strand,

[0423] (i) 21 nucleotides long;

[0424] (ii) having 2′-fluoro-modified nucleotides at positions 2, 7, 12 and 14 (calculated from the 5' end) and 2′-O-methyl-modified nucleotides at positions 1, 3 to 6, 8 to 11, 13, and 15 to 21;

[0425] (iii) an antisense strand having phosphorothioate nucleotide linkages between nucleotides at positions 1 and 2 (counted from the 5' end), between nucleotides at positions 2 and 3, between nucleotides at positions 19 and 20, and between nucleotides at positions 20 and 21; comprising,

[0426] In this case, the RNAi construct has a nucleotide overhang containing two nucleotides at the 3' end of the sense strand and a nucleotide overhang containing two nucleotides at the 3' end of the antisense strand.

[0427] In another embodiment, the RNAi construct

[0428] (a) As a sense strand,

[0429] (i) 22 nucleotides long;

[0430] (ii) having an inverted baseless nucleotide or an inverted deoxyribonucleotide at position 1 (calculated from the 5' end); 2′-fluoro-modified nucleotides at positions 8 to 11; and 2′-O-methyl-modified nucleotides at positions 2 to 7 and 12 to 22;

[0431] (iii) A sense strand having phosphorothioate internucleotide linkages between nucleotides at positions 20 and 21 (counted from the 5' end) and between nucleotides at positions 21 and 22;

[0432] and

[0433] (b) As an antisense strand,

[0434] (i) 21 nucleotides long;

[0435] (ii) having 2′-fluoro-modified nucleotides at positions 2, 7, 12 and 14 (calculated from the 5' end) and 2′-O-methyl-modified nucleotides at positions 1, 3 to 6, 8 to 11, 13, and 15 to 21;

[0436] (iii) an antisense strand having phosphorothioate nucleotide linkages between nucleotides at positions 1 and 2 (counted from the 5' end), between nucleotides at positions 2 and 3, between nucleotides at positions 19 and 20, and between nucleotides at positions 20 and 21; comprising,

[0437] In this case, the RNAi construct has a nucleotide overhang containing two nucleotides at the 3' end of the sense strand and a nucleotide overhang containing one or two nucleotides at the 3' end of the antisense strand.

[0438] In a specific embodiment of the present invention, the RNAi construct comprises a sense strand of 19 to 21 nucleotide length and an antisense strand of 19 to 21 nucleotide length, wherein the sequences of the antisense strand and the sense strand are sufficiently complementary to each other to form a dichotomous region of 19 to 21 base pairs, and nucleotides at positions 2, 7, 12, and 14 of the antisense strand (counted from the 5' end) are 2′-fluoro-modified nucleotides; nucleotides of the sense strand at positions paired with positions 10, 11, and 13 of the antisense strand (counted from the 5' end) are 2′-fluoro-modified nucleotides; and the sense strand and the antisense strand each do not have a total of more than 7 2′-fluoro-modified nucleotides. In one such embodiment, the RNAi construct

[0439] (a) As a sense strand,

[0440] (i) 21 nucleotides long;

[0441] (ii) 2′-fluoro-modified nucleotides at positions 9 and 11 to 14 (calculated from the 5' end); and 2′-O-methyl-modified nucleotides at positions 1 to 8, 10, and 15 to 20 and an inverted baseless nucleotide or an inverted deoxyribonucleotide at position 21;

[0442] (iii) A sense strand having a phosphorothioate internucleotide link between nucleotides at positions 20 and 21 (counted from the 5' end);

[0443] and

[0444] (b) As an antisense strand,

[0445] (i) 21 nucleotides long;

[0446] (ii) having 2′-fluoro-modified nucleotides at positions 2, 7, 12 and 14 (calculated from the 5' end) and 2′-O-methyl-modified nucleotides at positions 1, 3 to 6, 8 to 11, 13, and 15 to 21;

[0447] (iii) an antisense strand having phosphorothioate nucleotide linkages between nucleotides at positions 1 and 2 (counted from the 5' end), between nucleotides at positions 2 and 3, between nucleotides at positions 19 and 20, and between nucleotides at positions 20 and 21; comprising,

[0448] In this case, the RNAi construct has two smooth ends.

[0449] In another such embodiment, the RNAi construct

[0450] (a) As a sense strand,

[0451] (i) 21 nucleotides long;

[0452] (ii) (calculated from the 5' end); having a 2′-fluoro-modified nucleotide at positions 9 to 12; and a 2′-O-methyl-modified nucleotide at positions 1 to 8 and 13 to 20 and an inverted baseless nucleotide or an inverted deoxyribonucleotide at position 21;

[0453] (iii) A sense strand having a phosphorothioate internucleotide link between nucleotides at positions 20 and 21 (counted from the 5' end);

[0454] and

[0455] (b) As an antisense strand,

[0456] (i) 21 nucleotides long;

[0457] (ii) having 2′-fluoro-modified nucleotides at positions 2, 7, 12 and 14 (calculated from the 5' end) and 2′-O-methyl-modified nucleotides at positions 1, 3 to 6, 8 to 11, 13, and 15 to 21;

[0458] (iii) an antisense strand having phosphorothioate nucleotide linkages between nucleotides at positions 1 and 2 (counted from the 5' end), between nucleotides at positions 2 and 3, between nucleotides at positions 19 and 20, and between nucleotides at positions 20 and 21; comprising,

[0459] In this case, the RNAi construct has two smooth ends.

[0460] In some embodiments of the present invention, the 5' end of the sense strand, antisense strand, or both the antisense and sense strands of an RNAi construct comprises a phosphate moiety. As used herein, the term “phosphate moiety” refers to a terminal phosphate group comprising an unmodified phosphate (-OP=O)(OH)OH) as well as a modified phosphate. A modified phosphate comprises a phosphate in which one or more O and OH groups are replaced by H, O, S, N(R), or an alkyl, wherein R is H, an amino protecting group, or an unsubstituted or substituted alkyl. Exemplary phosphate moietys include 5'-monophosphate; 5'-diphosphate; 5'-triphosphate; a (7-methylated or un-methylated) 5'-guanosine cap; a 5'-adenosine cap; or any other modified or unmodified nucleotide cap structure; 5'-monothiophosphate (phosphorothioate); 5'-monodithiophosphate (phosphorodithioate); 5'-alpha-thiotriphosphate; 5'-gamma-thiotriphosphate, 5'-phosphoramidate; 5'-vinylphosphate; 5'-alkylphosphonate (e.g., alkyl = methyl, ethyl, isopropyl, propyl, etc.); and 5'-alkyl etherphosphonate (e.g., alkyl ether = methoxymethyl, ethoxymethyl, etc.), but not limited thereto.

[0461] Modified nucleotides that can be incorporated into RNAi constructs of the present invention may have one or more chemical modifications described herein. For example, modified nucleotides may have modifications to the ribose sugar as well as modifications to the nucleobase. As an example, modified nucleotides may include a 2' sugar modification (e.g., 2'-fluoro or 2'-O-methyl) and a modified base (e.g., 5-methylcytosine or pseudouracil). In other embodiments, modified nucleotides may include sugar modifications combined with modifications to the 5' phosphate that create inter-nucleotide or inter-nucleoside linkages when the modified nucleotides are incorporated into a polynucleotide. For example, in some embodiments, modified nucleotides may include sugar modifications, such as 2'-fluoro modifications, 2'-O-methyl modifications, or cyclocyclic sugar modifications, as well as a 5' phosphorothioate group. Accordingly, in some embodiments, one or both strands of the RNAi construct of the present invention comprise a 2' modified nucleotide or a combination of linkages between BNA and phosphorothioate nucleotides. In certain embodiments, both the sense and antisense strands of the RNAi construct of the present invention comprise a combination of linkages between a 2'-fluoro modified nucleotide, a 2'-O-methyl modified nucleotide, and a phosphorothioate nucleotide.

[0462] In certain embodiments, the nucleotide at position 1 of the antisense strand, calculated from the 5' end of the RNAi construct, may include A, dA, dU, U, or dT. In some embodiments, at least one of the first three base pairs within the difid region from the 5' end of the antisense strand is an AU base pair. In one specific embodiment, the first base pair within the difid region from the 5' end of the antisense strand is an AU base pair.

[0463] The RNAi constructs of the present invention can be easily prepared using techniques known in the art, for example, conventional solid-phase nucleic acid synthesis methods. The polynucleotides of the RNAi constructs can be assembled in a suitable nucleic acid synthesizer using standard nucleotide or nucleoside precursors (e.g., phosphoramidite). Automated nucleic acid synthesizers are commercially available from several suppliers, including DNA / RNA synthesizers from Applied Biosystems (Foster City, CA), MerMade synthesizers from BioAutomation (Irving, TX), and OligoPilot synthesizers from GE Healthcare Life Sciences (Pittsburgh, PA). An exemplary method for synthesizing the RNAi constructs of the present invention is described in Example 1.

[0464] The 2' silyl protecting group can be used with the acid-unstable dimethoxytrityl (DMT) at the 5' position of ribonucleoside to synthesize oligonucleotides via phosphoramidite chemistry. The final deprotection condition is known not to significantly degrade the RNA product. All syntheses can be performed on a large, medium, or small scale using any automated or manual synthesizer. Syntheses can also be performed on multi-well plates, columns, or glass slides.

[0465] The 2'-O-silyl group can be removed by exposure to fluoride ions and may include any source of fluoride ions, for example, salts containing fluoride ions paired with inorganic counterions, for example, cesium fluoride and potassium fluoride, or salts containing fluoride ions paired with organic counterions, for example, tetraalkylammonium fluoride. In the deprotection reaction, a crown ether catalyst may be used with inorganic fluoride. Preferred sources of fluoride ions are tetrabutylammonium fluoride or aminohydrofluoride (for example, aqueous HF combined with triethylamine in a dipolar aprotic solvent, for example, dimethylformamide).

[0466] The selection of a protecting group for use in phosphite triesters and phosphotrysters can alter the stability of the triesters with respect to fluoride. Methyl protection of phosphotrysters or phosphite triesters can stabilize the affinity for fluoride ions and improve process yield.

[0467] Since ribonucleosides have reactive 2' hydroxyl substituents, it may be desirable to protect the reactive 2' position in RNA with a protecting group orthogonal to the 5'-O-dimethoxytrityl protecting group, for example, a protecting group that is stable to acid treatment. A silyl protecting group satisfies this criterion and can be easily removed in the final fluoride deprotection step, resulting in minimal RNA degradation.

[0468] Tetrazole catalysts can be used in standard phosphoramidite coupling reactions. Preferred catalysts include, for example, tetrazole, s-ethyl-tetrazole, benzylthiotetrazole, and p-nitrophenyltetrazole.

[0469] As will be understood by those skilled in the art, additional methods for synthesizing the RNAi constructs described herein will be apparent to those skilled in the art. Additionally, various synthetic steps may be performed in alternating sequences or orders to provide the desired compound. Other synthetic chemical modifications useful for synthesizing the RNAi constructs described herein, protecting groups (e.g., for hydroxyls, aminos, etc. present in bases), and protecting methodologies (protection and deprotection) are known in the art, for example, in the literature [R. Larock, Comprehensive Organic Transformations, VCH Publishers (1989); TW Greene and PGM Wuts, Protective Groups in Organic Synthesis, 2d. Ed., John Wiley and Sons (1991); L. Fieser and M. Fieser, Fieser and Fieser's Reagents for Organic Synthesis, John Wiley and Sons (1994); Includes those described in [and L. Paquette, ed., Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons (1995)] and subsequent editions thereof. Custom synthesis of RNAi agents is also available from several distributors including Dharmacon, Inc. (Lafayette, CO), AxoLabs GmbH (Kulmbach, Germany), and Ambion, Inc. (Foster City, CA).

[0470] RNAi constructs of the present invention may comprise a ligand. As used herein, "ligand" refers to any compound or molecule that may interact directly or indirectly with another compound or molecule. The interaction between the ligand and another compound or molecule may induce a biological response (e.g., initiating a signal transduction cascade or inducing receptor-mediated endocytosis) or may be a physical association. The ligand may modify one or more properties of the attached double-stranded RNA molecule, such as pharmacodynamic, pharmacokinetic, binding, uptake, cellular distribution, cellular uptake, charge, and / or removal properties of the RNA molecule.

[0471] Ligands are serum proteins (e.g., human serum albumin, low-density lipoprotein, globulin), cholesterol moiety, and vitamins (biotin, vitamin E, vitamin B12). 12It may include ), folate moiety, steroids, bile acids (e.g., choline acid), fatty acids (e.g., palmitic acid, myristic acid), carbohydrates (e.g., dextran, pullulan, chitin, chitosan, inulin, cyclodextrin, or hyaluronic acid), glycosides, phospholipids, or antibodies or binding fragments thereof (e.g., antibodies or binding fragments that target RNAi constructs in specific cell types, e.g., liver). Other examples of ligands include dyes, intercalators (e.g., acridine), crosslinking agents (e.g., psoralen, mitomycin C), porphyrins (TPPC4, texaphyrin, saphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g., EDTA), lipophilic molecules, e.g., adamantan acetic acid, 1-pyrene butyric acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, O3-(oleoyl)litocholic acid, O3-(oleoyl)cholic acid, dimethoxytrityl or phenoxazine), and peptides (e.g., Antenepedia peptide, Tat peptide, RGD Includes peptides), alkylating agents, polymers, such as polyethylene glycol (PEG) (e.g., PEG-40K), polyamino acids, and polyamines (e.g., spermine, spermidine).

[0472] In certain embodiments, the ligand has endosome-degrading properties. The endosome-degrading ligand promotes the lysis of endosomes and / or the translocation of the RNAi construct of the present invention or its components from the endosomes to the cytoplasm of the cell. The endosome-degrading ligand may be a polycationic peptide or peptidomimetic exhibiting pH-dependent membrane activity and carcinogenicity. In one embodiment, the endosome-degrading ligand assumes its active form at endosome pH. The "active" conformation is the conformation in which the endosome-degrading ligand promotes the lysis of endosomes and / or the translocation of the RNAi construct of the present invention or its components from the endosomes to the cytoplasm of the cell. An exemplary endosome-degrading ligand is a GALA peptide (Subbarao et al ., Biochemistry, Vol. 26: 2964-2972, 1987), EALA peptide (Vogel et al ., J. Am. Chem. Soc., Vol. 118: 1581-1586, 1996), and their derivatives (Turk et al Includes ., Biochem. Biophys. Acta, Vol. 1559: 56-68, 2002). In one embodiment, the endosome degrading component may contain a chemical group (e.g., an amino acid) that undergoes a change in charge or quantization in response to a change in pH. The endosome degrading component may be linear or branched.

[0473] In some embodiments, the ligand comprises a lipid or other hydrophobic molecule. In one embodiment, the ligand comprises a cholesterol moiety or another steroid. Cholesterol-conjugated oligonucleotides have been reported to be more active than their unconjugated counterparts (Manoharan, Antisense Nucleic Acid Drug Development, Vol. 12: 103-228, 2002). Ligands comprising a cholesterol moiety and other lipids for conjugation with nucleic acid molecules are also described in U.S. Patents No. 7,851,615; No. 7,745,608; and No. 7,833,992, all of which are incorporated herein by reference in their entirety. In another embodiment, the ligand comprises a folate moiety. Polynucleotides conjugated to a folate moiety may be absorbed by cells via receptor-mediated endocytosis pathways. Such folate-polynucleotide conjugates are described in U.S. Patent No. 8,188,247, the entirety of which is incorporated herein by reference.

[0474] The ligand can target the RNAi construct to a specific tissue or cell type to selectively inhibit the expression of the target gene in that specific tissue or cell type. In one embodiment, the ligand targets the delivery of the RNAi construct to hepatocytes using various approaches as described in more detail below. In a specific embodiment, the RNAi construct is targeted to hepatocytes by a ligand that binds to a surface-expressed asialoglycoprotein receptor (ASGR) or its components (e.g., ASGR1, ASGR2).

[0475] In some embodiments, the RNAi construct may specifically target the liver by using a ligand that binds to or interacts with a protein expressed on the surface of liver cells. For example, in certain embodiments, the ligand may comprise an antigen-binding protein (e.g., an antibody or a binding fragment thereof (e.g., Fab, scFv)) that specifically binds to receptors expressed on liver cells, such as the asialoglycoprotein receptor and the LDL receptor. In one specific embodiment, the ligand comprises an antibody or a binding fragment thereof that specifically binds to ASGR1 and / or ASGR2. In another embodiment, the ligand comprises a Fab fragment of an antibody that specifically binds to ASGR1 and / or ASGR2. The “Fab fragment” consists of one immunoglobulin light chain (i.e., a light chain variable region (VL) and a constant region (CL)) and one immunoglobulin heavy chain CH1 region and variable region (VH). In another embodiment, the ligand comprises a single-chain variable antibody fragment (scFv fragment) of an antibody that specifically binds to ASGR1 and / or ASGR2. The “scFv fragment” comprises VH and VL regions of the antibody, wherein these regions are present on a single polypeptide chain and optionally include a peptide linker between the VH and VL regions that allows Fv to form a desired structure for antigen binding. An exemplary antibody that specifically binds to ASGR1 and its binding fragments that can be used as ligands for targeting the liver in the RNAi constructs of the present invention are described in WIPO Publication No. 2017 / 058944, the full text of which is incorporated herein by reference. Other antibodies or their binding fragments that specifically bind to ASGR1, the LDL receptor, or other liver surface-expressed proteins suitable for use as ligands in the RNAi constructs of the present invention are commercially available.

[0476] In certain embodiments, the ligand comprises a carbohydrate. "Carbohydrate" refers to a compound composed of one or more monosaccharide units having at least six carbon atoms (which may be straight-chain, branched-chain, or cyclic) having an oxygen, nitrogen, or sulfur atom bonded to each carbon atom. Carbohydrates include, but are not limited to, sugars (e.g., monosaccharides, disaccharides, trisaccharides, tetrasaccharides, and oligosaccharides containing about 4, 5, 6, 7, 8, or 9 monosaccharide units) and polysaccharides, such as starch, glycogen, cellulose, and polysaccharide gums. In some embodiments, the carbohydrate is a monosaccharide selected from pentose, hexose, or heptose, and disaccharides and trisaccharides containing such monosaccharide units. In other embodiments, the carbohydrate incorporated into the ligand is an amino sugar, such as galactosamine, glucosamine, N-acetylgalactosamine, and N-acetylglucosamine.

[0477] In some embodiments, the ligand comprises hexose or hexosamine. Hexose may be selected from glucose, galactose, mannose, fucose, or fructose. Hexosamine may be selected from fructosamine, galactosamine, glucosamine, or mannosamine. In certain embodiments, the ligand comprises glucose, galactose, galactosamine, or glucosamine. In one embodiment, the ligand comprises glucose, glucosamine, or N-acetylglucosamine. In another embodiment, the ligand comprises galactose, galactosamine, or N-acetyl-galactosamine. In certain embodiments, the ligand comprises N-acetyl-galactosamine. Ligands comprising glucose, galactose, and N-acetyl-galactosamine (GalNAc) bind to ASGR expressed on the surface of hepatocytes, so these ligands are particularly effective for targeting compounds to hepatocytes. For example, see D'Souza and Devarajan, J. Control Release, Vol. 203: 126-139, 2015. Examples of GalNAc- or galactose-containing ligands that may be incorporated into the RNAi constructs of the present invention are described in U.S. Patents No. 7,491,805; No. 8,106,022; and No. 8,877,917; U.S. Patent Publication No. 20030130186; and WIPO Publication No. WO2013166155, all of which are incorporated herein by reference in their entirety.

[0478] In certain embodiments, the ligand comprises a polyvalent carbohydrate moiety. As used herein, "polyvalent carbohydrate moiety" refers to a moiety comprising two or more carbohydrate units capable of independently binding to or interacting with other molecules. For example, a polyvalent carbohydrate moiety comprises two or more binding domains composed of carbohydrates capable of binding to two or more different molecules or to two or more different sites on the same molecule. The valence of the carbohydrate moiety indicates the number of individual binding domains within the carbohydrate moiety. For example, in relation to carbohydrate moiety, the terms "monovalent," "divalent," "trivalent," and "tetravalent" refer to carbohydrate moiety having one, two, three, and four binding domains, respectively. The polyvalent carbohydrate moiety may include a polyvalent lactose moiety, a polyvalent galactose moiety, a polyvalent glucose moiety, a polyvalent N-acetyl-galactosamine moiety, a polyvalent N-acetyl-glucosamine moiety, a polyvalent mannose moiety, or a polyvalent fucose moiety. In some embodiments, the ligand includes a polyvalent galactose moiety. In other embodiments, the ligand includes a polyvalent N-acetyl-galactosamine moiety. In these and other embodiments, the polyvalent carbohydrate moiety may be divalent, trivalent, or tetravalent. In the above embodiments, the polyvalent carbohydrate moiety may be bi-antennary or tri-antennary. In one specific embodiment, the polyvalent N-acetyl-galactosamine moiety is trivalent or tetravalent. In another specific embodiment, the multivalent galactose moiety is trivalent or tetravalent. Exemplary trivalent and tetravalent GalNAc-containing ligands for incorporation into RNAi constructs of the present invention are described in detail below.

[0479] The ligand may be attached or conjugated directly or indirectly to the RNA molecule of the RNAi construct. For example, in some embodiments, the ligand is directly covalently attached to the sense or antisense strand of the RNAi construct. In other embodiments, the ligand is covalently attached to the sense or antisense strand of the RNAi construct through a linker. The ligand may be attached to a nucleobase, sugar moiety, or internucleotide linkage of the polynucleotide (e.g., sense strand or antisense strand) of the RNAi construct of the present invention. Conjugation or attachment to a purine nucleobase or its derivative may occur at any position including endocyclic and ectocyclic atoms. In certain embodiments, the 2-, 6-, 7-, or 8-position of the purine nucleobase is attached to the ligand. Conjugation or attachment to a pyrimidine nucleobase or its derivative may also occur at any position. In some embodiments, the 2-, 5-, and 6-positions of a pyrimidine nucleobase may be attached to a ligand. Conjugation or attachment to the sugar moiety of a nucleotide may occur at any carbon atom. Exemplary carbon atoms of the sugar moiety to which a ligand may be attached include the 2', 3', and 5' carbon atoms. The 1' position may also be attached to a ligand such as a baseless nucleotide. Internucleotide linkages may also support ligand attachment. In the case of phosphate-containing linkages (e.g., phosphodiesters, phosphorothioates, phosphodithiotates, phosphoromidates, etc.), the ligand may be attached directly to the phosphate atom or to an O, N, or S atom bonded to the phosphate atom. In the case of amine- or amide-containing nucleoside linkages (e.g., PNA), the ligand may be attached to the nitrogen atom or an adjacent carbon atom of the amine or amide.

[0480] In certain embodiments, the ligand may be attached to the 3' or 5' end of the sense or antisense strand. In certain embodiments, the ligand is covalently attached to the 5' end of the sense strand. In these embodiments, the ligand is attached to the 5'-terminal nucleotide of the sense strand. In these and other embodiments, the ligand is attached to the 5'-position of the 5'-terminal nucleotide of the sense strand. In embodiments where an inverted baseless nucleotide or an inverted deoxyribonucleotide is the 5'-terminal nucleotide of the sense strand and is connected to an adjacent nucleotide via a 5'-5' nucleotide linkage, the ligand may be attached to the 3'-position of the inverted baseless nucleotide or the inverted deoxyribonucleotide. In other embodiments, the ligand is covalently attached to the 3' end of the sense strand. For example, in some embodiments, the ligand is attached to the 3'-terminal nucleotide of the sense strand. In these specific embodiments, the ligand is attached to the 3'-position of the 3'-terminal nucleotide of the sense strand. In an embodiment where an inverted baseless nucleotide or an inverted deoxyribonucleotide is the 3'-terminal nucleotide of the sense strand and is connected to an adjacent nucleotide via a 3'-3' nucleotide linkage, the ligand may be attached to the 5'-position of the inverted baseless nucleotide or the inverted deoxyribonucleotide. In an alternative embodiment, the ligand is attached near the 3' end of the sense strand but before one or more terminal nucleotides (i.e., before the 1, 2, 3, or 4 terminal nucleotides). In some embodiments, the ligand is attached to the 2'-position of the sugar of the 3'-terminal nucleotide of the sense strand. In some embodiments, the ligand is attached to the 2'-position of the sugar of the 5'-terminal nucleotide of the sense strand.

[0481] In certain embodiments, the ligand is attached to the sense or antisense strand via a linker. The “linker” is an atom or group of atoms that covalently bonds the ligand to the polynucleotide component of the RNAi construct. The linker may be about 1 to about 30 atomic lengths, about 2 to about 28 atomic lengths, about 3 to about 26 atomic lengths, about 4 to about 24 atomic lengths, about 6 to about 20 atomic lengths, about 7 to about 20 atomic lengths, about 8 to about 20 atomic lengths, about 8 to about 18 atomic lengths, about 10 to about 18 atomic lengths, and about 12 to about 18 atomic lengths. In some embodiments, the linker may comprise a difunctional linking moiety, which generally comprises an alkyl moiety having two functional groups. One of the functional groups is selected to bind to the compound of interest (e.g., the sense or antisense strand of the RNAi construct), and the other is selected to essentially bind any selected group, such as the ligand described herein. In certain embodiments, the linker comprises a chain structure or a repeating unit oligomer, such as an ethylene glycol or amino acid unit. Examples of functional groups that may typically be used in the difunctional linking moiety include, but are not limited to, electrophiles for reacting with the nucleophilic group and nucleophiles for reacting with the electrophilic group. In some embodiments, the difunctional linking moiety comprises amino, hydroxyl, carboxylic acid, thiol, unsaturated (e.g., double or triple bond), etc.

[0482] Linkers that can be used to attach a ligand to a sense or antisense strand in the RNAi constructs of the present invention are pyrrolidine, 8-amino-3,6-dioxoctanic acid, succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate, 6-aminohexanoic acid, and substituted C1-C 10 Alkyl, substituted, or unsubstituted C2-C 10 alkenyl or substituted or unsubstituted C2-C10 Preferred substituents for such linkers include, but are not limited to, alkynyl. Preferred substituents for such linkers include, but are not limited to, hydroxyl, amino, alkoxy, carboxyl, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl, and alkynyl.

[0483] In certain embodiments, the linker is cleavable. The cleavable linker is a linker that is sufficiently stable outside the cell but cleaves upon entry into the target cell, thereby releasing the two parts that are held together. In some embodiments, the cleavable linker is cleavable at least 10, 20, 30, 40, 50, 60, 70, 80, 90, or more, or at least 100 times faster, in the target cell or in the blood of the subject under a first reference condition (e.g., may be chosen to mimic or represent intracellular conditions) or under a second reference condition (e.g., may be chosen to mimic or represent conditions found in blood or serum).

[0484] Cleavage linkers are sensitive to the presence of cleavage agents, such as pH, redox potential, or degradable molecules. Generally, cleavage agents are found in cells at a more dominant or higher level or activity than in serum or blood. Examples of such degradable agents include redox agents that are selected for a specific substrate or lack substrate specificity, such as oxidative or reductive enzymes, or reducing agents present in cells, such as mercaptans, which can degrade redox cleavage linkers by reduction; esterases; endosomes or agonists that can create an acidic environment, for example, an environment causing a pH of 5 or lower; and enzymes that can hydrolyze or degrade acid cleavage linkers by acting as general acids, peptidases (which may be substrate-specific), and phosphatases.

[0485] Cleavable linkers may contain pH-sensitive moiety. The pH of human serum is 7.4, whereas the average intracellular pH is slightly lower, ranging from about 7.1 to 7.3. Endosomes have a more acidic pH in the range of 5.5 to 6.0, and lysosomes have a much more acidic pH of about 5.0. Some linkers will have a cleavable group that cleaves at a desired pH, thereby allowing RNA molecules to be released from ligands inside the cell or into a desired compartment of the cell.

[0486] Linkers may contain cleavable groups that can be cleaved by specific enzymes. The type of cleavable group included in the linker may depend on the cell to be targeted. For example, a liver-targeted ligand can be attached to an RNA molecule via a linker containing an ester group. Because liver cells are rich in esterases, the linker will be cleaved more efficiently in liver cells than in cell types that are not rich in esterases. Other types of cells rich in esterases include cells of the lungs, renal cortex, and testes. Linkers containing peptide bonds can be used when targeting peptidase-rich cells, such as liver cells and synovial cells.

[0487] Generally, the suitability of a candidate cleavable linker can be evaluated by testing the ability of a degrading agent (or condition) to cleave the candidate linker. Additionally, it would be desirable to test the candidate cleavable linker for its ability to resist cleavage when in contact with blood or non-target tissues. Thus, the relative sensitivity to cleavage between a first condition and a second condition can be determined, where the first condition is selected to represent cleavage in target cells, and the second is selected to represent cleavage in other tissues or biological fluids, e.g., blood or serum. Evaluations can be performed in cell-free systems, cells, cell cultures, organ or tissue cultures, or whole animals. It may be useful to perform an initial evaluation in cell-free or culture conditions and confirm it through further evaluation in whole animals. In some embodiments, a useful candidate linker is cleaved at least 2, 4, 10, 20, 50, 70, or 100 times faster in cells (or under in vitro conditions selected to mimic intracellular conditions) compared to blood or serum (or under in vitro conditions selected to mimic extracellular conditions).

[0488] In other embodiments, a redox cleavable linker is used. The redox cleavable linker is cleaved upon reduction or oxidation. An example of a reductive cleaver is a disulfide linker (-SS-). One or more of the methods described herein may be used to determine whether a candidate cleavable linker is a suitable "reductively cleavable linker," or whether it is suitable for use with, for example, a specific RNAi construct and a specific ligand. For example, the candidate linker may be evaluated by incubation with dithiothreitol (DTT) or other reducing agents known in the art, which mimics the cleavage rate observed in cells, for example, target cells. The candidate linker may also be evaluated under conditions selected to mimic blood or serum conditions. In certain embodiments, the candidate linker is cleaved by up to 10% in blood. In another embodiment, a useful candidate linker is degraded at least 2, 4, 10, 20, 50, 70, or 100 times faster in cells (or under in vitro conditions selected to mimic intracellular conditions) compared to blood (or under in vitro conditions selected to mimic extracellular conditions).

[0489] In another embodiment, a phosphate-based cleavable linker that is cleaved by an agent that degrades or hydrolyzes a phosphate group is used to covalently attach a ligand to the sense or antisense strand of an RNAi construct. An example of an agent that hydrolyzes a phosphate group in cells is an enzyme such as phosphatase in cells. Examples of phosphate-based cleavable groups are -OP(O)(ORk)-O-, -OP(S)(ORk)-O-, -OP(S)(SRk)-O-, -SP(O)(ORk)-O-, -OP(O)(ORk)-S-, -SP(O)(ORk)-S-, -OP(S)(ORk)-S-, -SP(S)(ORk)-O-, -OP(O)(Rk)-O-, -OP(S)(Rk)-O-, -SP(O)(Rk)-O-, -SP(O)(Rk)-O-, -SP(O)(Rk)-S-, and -OP(S)(Rk)-S-, where Rk can be hydrogen or alkyl. Specific embodiments include -OP(O)(OH)-O-, -OP(S)(OH)-O-, -OP(S)(SH)-O-, -SP(O)(OH)-O-, -OP(O)(OH)-S-, -SP(O)(OH)-S-, -OP(S)(OH)-S-, -SP(S)(OH)-O-, -OP(O)(H)-O-, -OP(S)(H)-O-, -SP(O)(H)-O-, -SP(S)(H)-O-, -SP(O)(H)-S-, and -OP(S)(H)-S-. Another specific embodiment is -OP(O)(OH)-O-. These candidate linkers can be evaluated using methods similar to those described above.

[0490] In other embodiments, the linker may include an acid-cleavable group that is cleaved under acidic conditions. In some embodiments, the acid-cleavable group is cleaved in an acidic environment where the pH is about 6.5 or lower (e.g., about 6.0, 5.5, 5.0 or lower), or by an agent such as an enzyme that can act as a general acid. In cells, certain low-pH organelles, such as endosomes and lysosomes, may provide a cleavage environment for the acid-cleavable group. Examples of acid-cleavable linkers include, but are not limited to, hydrazones, esters, and esters of amino acids. The acid-cleavable group may have the general chemical formula -C=NN-, C(O)O, or -OC(O). Specific embodiments are when the carbon attached to the oxygen of the ester (alkoxy group) is an aryl group, a substituted alkyl group, or a tertiary alkyl group, such as dimethyl, pentyl, or t-butyl. These candidates may be evaluated using methods similar to those described above.

[0491] In another embodiment, the linker may comprise an ester-based cleavable group, which is cleaved by enzymes, such as esterases and amidases in cells. Examples of ester-based cleavable groups include, but are not limited to, esters of alkylene, alkenylene, and alkynylene groups. The ester-cleavable group has the general chemical formula -C(O)O-, or -OC(O)-. These candidate linkers may be evaluated using methods similar to those described above.

[0492] In further embodiments, the linker may comprise a peptide-based cleavable group, which is cleaved in cells by enzymes, such as peptidases and proteases. The peptide-based cleavable group is a peptide bond formed between amino acids to produce oligopeptides (e.g., dipeptides, tripeptides, etc.) and polypeptides. The peptide-based cleavable group comprises an amide group (-C(O)NH-). The amide group may be formed between any alkylene, alkenylene, or alkynylene. A peptide bond is a special type of amide bond formed between amino acids to produce peptides and proteins. The peptide-based cleavable group is generally limited to peptide bonds (i.e., amide bonds) formed between amino acids to produce peptides and proteins. The peptide-based cleavable linker has the general chemical formula -NHCHR A C(O)NHCHR B It has C(O)-, where R A and R B is a side chain of two adjacent amino acids. These candidates can be evaluated using a method similar to that described above.

[0493] Other types of linkers suitable for attaching a ligand to a sense or antisense strand in the RNAi constructs of the present invention are known in the art and may include the linkers described in U.S. Patents No. 7,723,509; No. 8,017,762; No. 8,828,956; No. 8,877,917; and No. 9,181,551, all of which are incorporated herein by reference in their entirety.

[0494] In certain embodiments, a ligand covalently attached to the sense or antisense strand of an RNAi construct of the present invention comprises a GalNAc moiety, for example, a multivalent GalNAc moiety. In some embodiments, the multivalent GalNAc moiety is a trivalent GalNAc moiety and is attached to the 3' end of the sense strand. In other embodiments, the multivalent GalNAc moiety is a trivalent GalNAc moiety and is attached to the 5' end of the sense strand. In yet another embodiment, the multivalent GalNAc moiety is a tetravalent GalNAc moiety and is attached to the 3' end of the sense strand. In yet another embodiment, the multivalent GalNAc moiety is a tetravalent GalNAc moiety and is attached to the 5' end of the sense strand.

[0495] In certain embodiments, the RNAi construct of the present invention comprises a ligand having the following structure:

[0496]

[0497] In a preferred embodiment, a ligand having this structure is covalently attached to the 5' end of a sense strand through a linker, e.g., a linker described herein. In one embodiment, the linker is an aminohexyl linker.

[0498] Exemplary trivalent and tetravalent GalNAc moiety and linker capable of being attached to a double-stranded RNA molecule of the RNAi construct of the present invention are provided in structural formulas I to IX below. In the formulas listed herein, "Ac" represents an acetyl group.

[0499] In one embodiment, the RNAi construct comprises a ligand and a linker having the structure of the following formula I, wherein each n is independently 1 to 3, k is 1 to 3, m is 1 or 2, and j is 1 or 2, and the ligand is attached to the 3' end of the sense strand of a double-stranded RNA molecule (indicated by a wavy solid line):

[0500] [Chemical Formula I]

[0501]

[0502] In another embodiment, the RNAi construct comprises a ligand and a linker having the structure of the following formula II, wherein each n is independently 1 to 3, k is 1 to 3, m is 1 or 2, and j is 1 or 2, and the ligand is attached to the 3' end of the sense strand of a double-stranded RNA molecule (indicated by a wavy solid line):

[0503] [Chemical Formula II]

[0504]

[0505] In another embodiment, the RNAi construct comprises a ligand and a linker having the structure of the following formula III, and the ligand is attached to the 3' end of the sense strand of a double-stranded RNA molecule (indicated by a wavy solid line):

[0506] [Chemical Formula III]

[0507]

[0508] In another embodiment, the RNAi construct comprises a ligand and a linker having the structure of Formula IV below, and the ligand is attached to the 3' end of the sense strand of a double-stranded RNA molecule (indicated by a wavy solid line):

[0509] [Chemical Formula IV]

[0510]

[0511] In a specific embodiment, the RNAi construct comprises a ligand and a linker having the structure of the following formula V, wherein each n is independently 1 to 3 and k is 1 to 3, and the ligand is attached to the 5' end of the sense strand of a double-stranded RNA molecule (indicated by a wavy solid line):

[0512] [Chemical Formula V]

[0513]

[0514] In another embodiment, the RNAi construct comprises a ligand and a linker having the structure of the following formula VI, wherein each n is independently 1 to 3 and k is 1 to 3, and the ligand is attached to the 5' end of the sense strand of a double-stranded RNA molecule (indicated by a wavy solid line):

[0515] [Chemical Formula VI]

[0516]

[0517] In one specific embodiment, the RNAi construct comprises a ligand and a linker having the structure of the following formula VII, wherein X = O or S, and the ligand is attached to the 5' end of the sense strand of a double-stranded RNA molecule (indicated by the swivel line):

[0518] [Chemical Formula VII]

[0519]

[0520] In some embodiments, the RNAi construct comprises a ligand and a linker having the structure of the following formula VIII, wherein each n is independently 1 to 3, and the ligand is attached to the 5' end of the sense strand of a double-stranded RNA molecule (indicated by a wavy solid line):

[0521] [Chemical Formula VIII]

[0522]

[0523] In a specific embodiment, the RNAi construct comprises a ligand and a linker having the structure of the following formula IX, and the ligand is attached to the 5' end of the sense strand of a double-stranded RNA molecule (indicated by a wavy solid line):

[0524] [Chemical Formula IX]

[0525]

[0526] Phosphorothioate bonds can covalently link ligands and linkers to nucleic acid strands in place of phosphodiester bonds presented in any one of chemical formulas I to IX.

[0527] The present invention also comprises pharmaceutical compositions and formulations comprising the RNAi constructs described herein and pharmaceutically acceptable carriers, excipients, or diluents. Such compositions and formulations are useful for reducing the expression of target genes in subjects requiring them. When clinical application is considered, pharmaceutical compositions and formulations may be prepared in a form suitable for the intended application. Generally, this will involve the preparation of compositions that are essentially free of pyrogens as well as other impurities that may be harmful to humans or animals.

[0528] The phrases “pharmaceutical acceptable” or “pharmacologically acceptable” refer to molecular entities and compositions that do not produce adverse reactions, allergic reactions, or other side effects when administered to animals or humans. As used herein, “pharmaceutically acceptable carriers, excipients, or diluents” include solvents, buffers, solutions, dispersion media, coating agents, antimicrobial and antifungal agents, isotonic agents, and absorption retardants, etc., which are acceptable for use in formulating pharmaceuticals, such as pharmaceuticals suitable for administration to humans. The use of such media and agents for pharmaceutically active substances is well known in the art. Their use in therapeutic compositions is considered except where any conventional media or agent is incompatible with the RNAi construct of the present invention. Additional active ingredients may also be incorporated into the composition, provided that they do not inactivate the RNAi construct of the composition.

[0529] Compositions and methods for formulating pharmaceutical compositions rely on a number of criteria, including but not limited to the route of administration, the type and severity of the disease or disorder to be treated, or the dosage. In some embodiments, pharmaceutical compositions are formulated based on the intended delivery route. For example, in certain embodiments, pharmaceutical compositions are formulated for parenteral delivery. Parenteral delivery forms include intravenous, intra-arterial, subcutaneous, intrathecal, intraperitoneal, or intramuscular injection or infusion. In one embodiment, pharmaceutical compositions are formulated for intravenous delivery. In the above embodiment, pharmaceutical compositions may include lipid-based delivery vehicles. In another embodiment, pharmaceutical compositions are formulated for subcutaneous delivery. In the above embodiment, pharmaceutical compositions may include targeting ligands (e.g., GalNAc-containing or antibody-containing ligands described herein).

[0530] In some embodiments, the pharmaceutical composition comprises an effective amount of the RNAi construct described herein. An "effective amount" is an amount sufficient to produce a beneficial or desirable clinical result. In some embodiments, an effective amount is an amount sufficient to reduce target gene expression in a specific tissue or cell type of the subject (e.g., liver or hepatocytes).

[0531] Administration of the pharmaceutical composition of the present invention may be carried out via any common route as long as the target tissue is available through the said route. Such routes include, but are not limited to, parenteral (e.g., subcutaneous, intramuscular, intraperitoneal, or intravenous), oral, nasal, buccal, intradermal, transdermal, and sublingual routes, or direct injection into liver tissue or delivery via the portal vein. In some embodiments, the pharmaceutical composition is administered parenterally. For example, in certain embodiments, the pharmaceutical composition is administered intravenously. In other embodiments, the pharmaceutical composition is administered subcutaneously.

[0532] Lipid-based systems, including colloidal dispersion systems such as macromolecular complexes, nanocapsules, microspheres, beads, oil-in-water emulsions, micelles, mixed micelles, and liposomes, can be used as delivery vehicles for the RNAi constructs of the present invention. Commercially available lipid emulsions suitable for the delivery of nucleic acids of the present invention include Intralipid ® (Baxter International Inc.), Liposyn ® (Abbott Pharmaceuticals), Liposyn ® II (Hospira), Liposyn ®Includes III (Hospira), Nutrilipid (B. Braun Medical Inc.), and other similar lipid emulsions. A preferred colloidal system for use as a delivery vehicle in vivo is a liposome (i.e., an artificial membrane vehicle). The RNAi constructs of the present invention may be encapsulated within liposomes or may form a complex with liposomes, particularly cationic liposomes. Alternatively, the RNAi constructs of the present invention may be complexed with lipids, particularly cationic lipids. Suitable lipids and liposomes include neutral (e.g., dioleoylphosphatidylethanolamine (DOPE), dimyristoylphosphatidylcholine (DMPC), and dipalmitoylphosphatidylcholine (DPPC)), distearoylphosphatidylcholine), negative (e.g., dimyristoylphosphatidylglycerol (DMPG)), and cationic (e.g., dioleoyltetramethylaminopropyl (DOTAP) and dioleoylphosphatidylethanolamine (DOTMA)). The preparation and use of such colloidal dispersion systems are well known in the art. Exemplary formulations are also U.S. Patent No. 5,981,505; U.S. Patent No. 6,217,900; U.S. Patent No. 6,383,512; U.S. Patent No. 5,783,565; U.S. Patent No. 7,202,227; U.S. Patent No. 6,379,965; It is disclosed in U.S. Patent No. 6,127,170; U.S. Patent No. 5,837,533; U.S. Patent No. 6,747,014; and WO03 / 093449.

[0533] In some embodiments, the RNAi constructs of the present invention are completely encapsulated in a lipid formulation to form, for example, SNALPs or other nucleic acid-lipid particles. As used herein, the term “SNALP” refers to stable nucleic acid-lipid particles. SNALPs generally contain cationic lipids, non-cationic lipids, and lipids that prevent particle aggregation (e.g., PEG-lipid conjugates). SNALPs are highly useful for systemic application because they exhibit an extended circulatory lifetime after intravenous injection and accumulate at terminal sites (e.g., sites physically separated from the administration site). Nucleic acid-lipid particles typically have an average diameter of about 50 nm to about 150 nm, about 60 nm to about 130 nm, about 70 nm to about 110 nm, or about 70 nm to about 90 nm and are substantially non-toxic. Additionally, when present in nucleic acid-lipid particles, the nucleic acid is resistant to degradation by nucleases in aqueous solution. Nucleic acid-lipid particles and methods for producing the same are disclosed, for example, in U.S. Patents No. 5,976,567; No. 5,981,501; No. 6,534,484; No. 6,586,410; No. 6,815,432; and PCT Publication No. WO96 / 40964.

[0534] Pharmaceutical compositions suitable for injection include, for example, sterile aqueous solutions or dispersions and sterile powders for the immediate preparation of sterile injectable solutions or dispersions. Generally, these formulations are sterile and fluid to the extent that easy injection is possible. The formulations must be stable under manufacturing and storage conditions and must be preserved against the action of microorganisms such as bacteria and fungi. Suitable solvents or dispersion media may contain, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.), suitable mixtures thereof, and vegetable oils. Suitable fluidity may be maintained, for example, by the use of a coating such as lecithin, by maintaining the necessary particle size in the case of dispersions, and by the use of surfactants. Prevention of microbial action may be achieved by various antimicrobial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc. In many cases, it would be desirable to include an isotonic agent, for example, sugar or sodium chloride. Delayed absorption of the injectable composition may be caused by the use of absorption-delaying agents in the composition, for example, aluminum monostearate and gelatin.

[0535] Sterile injectable solutions can be prepared by incorporating an appropriate amount of an active compound into a solvent along with any other desired amount of components (e.g., as listed above) and then filtering and sterilizing. Generally, dispersions are prepared by incorporating various sterile active ingredients into a basic dispersion medium and a sterile vehicle containing other desired components, for example, those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, preferred manufacturing methods include vacuum-drying and freeze-drying techniques to produce powders of any additional desired components in addition to the active ingredient(s) from a solution that has been pre-sterilized and filtered.

[0536] The compositions of the present invention can generally be formulated in the form of neutral or salts. Pharmaceutically acceptable salts include, for example, acid addition salts (formed by free amino groups) derived from inorganic acids (e.g., hydrochloric acid or phosphoric acid) or organic acids (e.g., acetic acid, oxalic acid, tartaric acid, mandelic acid, etc.). Salts formed by free carboxyl groups may also be derived from inorganic bases (e.g., sodium, potassium, ammonium, calcium, or ferric hydroxide) or organic bases (e.g., isopropylamine, trimethylamine, histidine, procaine, etc.).

[0537] For parenteral administration in aqueous solutions, for example, the solution is generally adequately buffered, and the liquid diluent is first made isotonic, for example, with sufficient saline or glucose. Such aqueous solutions may be used for administration, for example, intravenously, intramuscularly, subcutaneously, and intraperitoneally. Preferably, sterile aqueous media known to those skilled in the art, particularly in light of the present invention, are used. For example, a single dose may be dissolved in 1 ml of isotonic NaCl solution and added to 1000 ml of subcutaneous solution or injected at a proposed injection site (see, for example, "Remington's Pharmaceutical Sciences" 15th Edition, pages 1035-1038 and 1570-1580). For human administration, the formulation must meet the standards of sterility, pyrogenicity, general safety, and purity required by FDA standards. In certain embodiments, the pharmaceutical composition of the present invention comprises or consists of a sterile saline solution and the RNAi construct described herein. In another embodiment, the pharmaceutical composition of the present invention comprises or consists of the RNAi construct described herein and sterile water (e.g., water for injection, WFI). In yet another embodiment, the pharmaceutical composition of the present invention comprises or consists of the RNAi construct described herein and phosphate-buffered saline (PBS).

[0538] In some embodiments, the pharmaceutical composition of the present invention is packaged with or stored in a delivery device. Devices for injectable formulations include, but are not limited to, injection ports, pre-filled syringes, automatic syringes, injection pumps, intra-body syringes, and injection pens. Devices for aerosolized or powder formulations include, but are not limited to, inhalers, blowers, suction devices, etc. Accordingly, the present invention comprises a delivery device comprising the pharmaceutical composition of the present invention for treating or preventing one or more diseases or disorders.

[0539] The present invention provides a method for reducing or inhibiting the expression of a target gene in a cell by contacting the cell with any one of the RNAi constructs described herein. The cell may be in vitro or in vivo. Target gene expression may be evaluated by measuring the amount or level of target mRNA, target protein, or another biomarker linked to the expression of the target gene. The reduction in target gene expression in cells or animals treated with the RNAi construct of the present invention may be determined in relation to the target gene expression in cells or animals not treated with the RNAi construct or treated with a control RNAi construct. For example, in some embodiments, a reduction or inhibition of target gene expression is evaluated by (a) measuring the amount or level of target mRNA in cells treated with the RNAi construct of the present invention, (b) measuring the amount or level of target mRNA in cells treated with a control RNAi construct (e.g., an RNAi agent targeting an RNAi construct having an RNA molecule not expressed in the cell or a nonsense or scrambled sequence), or in cells without the construct, and (c) comparing the target mRNA level measured from the treated cells in (a) with the target mRNA level measured from the control cells in (b). The target mRNA levels in the treated cells and the control cells may be normalized to RNA levels for the control gene (e.g., 18S ribosomal RNA or housekeeping genes) before comparison. Target mRNA levels may be measured by various methods including Northern blot analysis, nuclease protection analysis, fluorescence in situ hybridization (FISH), reverse transcriptase (RT)-PCR, real-time RT-PCR, quantitative PCR, microdroplet digital PCR, etc.

[0540] In another embodiment, the reduction or inhibition of target gene expression is evaluated by (a) measuring the amount or level of a target protein in cells treated with the RNAi construct of the present invention, (b) measuring the amount or level of a target protein in cells treated with a control RNAi construct (e.g., an RNAi agonist directed at an RNAi construct having an RNA molecule not expressed in the cell or a nonsense or scrambled sequence), or in cells without the construct, and (c) comparing the target protein level measured from the treated cells in (a) with the target protein level measured from the control cells in (b). Methods for measuring target protein levels are known to those skilled in the art and include Western blot, immunoassay (e.g., ELISA), and flow cytometry.

[0541] The present invention also provides a method for reducing or suppressing the expression of a target gene in a subject in need, comprising the step of administering any one of the RNAi constructs described herein to a subject. The RNAi constructs of the present invention may be used to treat or improve a pathological condition, disease, or disorder associated with abnormal target gene expression or activity, for example, where the overexpression of a gene product causes a pathological phenotype. An exemplary target gene is LPA , PNPLA3 , ASGR1 , F7 , F12 , FXI , APOCIII , APOB , APOL1 , TTR , PCSK9 , SCAP , KRAS , CD274 , PDCD1 , C5 , ALAS1 , HAO1 , LDHA , ANGPTL3 , SERPINA1 , AGT , HAMP , LECT2 , EGFR , VEGF , KIF11 , AT3 , CTNNB1 , HMGB1 , HIF1A , and STAT3 It includes, but is not limited to. Additionally, the target gene may include viral genes, such as hepatitis B and hepatitis C virus genes, human immunodeficiency virus genes, herpes virus genes, etc. In some embodiments, the target gene is a gene encoding human microRNA (miRNA).

[0542] In certain embodiments, the expression of a target gene is reduced by at least 50% in a cell or subject by the RNAi construct of the present invention. In some embodiments, the expression of a target gene is reduced by at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, or at least 85% in a cell or subject by the RNAi construct of the present invention. In other embodiments, the expression of a target gene is reduced by about 90% or more, e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more in hepatocytes by the RNAi construct of the present invention. The percentage reduction in target gene expression may be measured by any of the methods described herein as well as other methods known in the art.

[0543] The following examples, including experiments performed and results achieved, are provided for illustrative purposes only and should not be construed as limiting the scope of the appended claims.

[0544] Examples

[0545] Example 1. In vivo activity of PNPLA3 RNAi constructs with different chemical modification patterns

[0546] To evaluate the effects of different chemical modification patterns on the in vivo efficacy of RNAi constructs, RNAi constructs targeting the patatin-like phospholipase domain-containing 3 (PNPLA3) gene were synthesized with various patterns of 2′-fluoro-modified nucleotides and 2′-O-methyl-modified nucleotides and evaluated in a humanized mouse model expressing PNPLA3 as described in detail below.

[0547] RNAi constructs were synthesized using solid-state phosphoramidite chemistry. Synthesis was performed on a MerMade12 (Bioautomation) instrument.

[0548] ingredient

[0549] Acetonitrile (DNA synthesis grade, AXO152-2505, EMD)

[0550] Capping Reagent A (80:10:10 (v / v / v) Tetrahydrofuran / Lutidine / Acetic Anhydride, BIO221 / 4000, EMD)

[0551] Capping reagent B (16% 1-methylimidazole / tetrahydrofuran, BIO345 / 4000, EMD)

[0552] Activator solution (5-(ethylthio)-1H-tetrazole (ETT) in 0.25 M acetonitrile, BIO152 / 0960, EMD)

[0553] Detritylation reagent (3% dichloroacetic acid in dichloromethane, BIO830 / 4000, EMD)

[0554] Oxidizing reagent (0.02 M iodine in 70:20:10 (v / v / v) tetrahydrofuran / pyridine / water, BIO420 / 4000, EMD)

[0555] Diethylamine solution (20% DEA in acetonitrile, NC0017-0505, EMD)

[0556] Thiolization reagent (0.05 M 5-N-[(dimethylamino)methylene]amino-3H-1,2,4-dithiazole-3-thion (BIOSULII / 160K) in 40:60 (v / v) pyridine / acetonitrile)

[0557] 5′-aminohexyl linker phosphoramidite, phosphorylated phosphoramidite, 2′-deoxythymidine phosphoramidite, and 2′-methoxy and 2′-fluorophosphoramidites of adenosine, guanosine, cytosine, and uridine (Thermo Fisher Scientific), 0.10 M in acetonitrile on approximately 10 mL of molecular sieve (3 Å, JT Baker).

[0558] CPG support (Hi-Load Universal Support, 500A (BH5-3500-G1), 79.6 μmol / g, 0.126 g (10 μmol))

[0559] Ammonium hydroxide (concentrated form, JT Baker)

[0560] synthesis

[0561] The reagent solution, phosphoramidite solution, and solvent were attached to the MerMade12 instrument. Solid supports were added to each column (4 mL SPE tubes with top and bottom frits), and the columns were secured to the instrument. The columns were washed twice with acetonitrile. The phosphoramidite and reagent solution lines were purged. Synthesis was initiated using Poseidon software. Synthesis was achieved by repeating the deprotection / coupling / oxidation / capping synthesis cycle. Specifically, a detritylation reagent was added to the solid support to remove the 5'-dimethoxytrityl (DMT) protecting group. The solid support was washed with acetonitrile. After adding the phosphoramidite and activator solutions to the support, the system was incubated to couple incoming nucleotides to the free 5'-hydroxyl group. The support was washed with acetonitrile. Oxidizing or thiolizing reagents were added to the support to convert the phosphite tryster into the phosphate tryster or phosphorothioate. Capping reagents A and B were added to the support to block any unreacted oligonucleotide chains. The support was washed with acetonitrile. After the final reaction cycle, the resin was washed with a diethylamine solution to remove the 2-cyanoethyl protecting group. The support was washed with acetonitrile and vacuum dried.

[0562] GalNAc junction

[0563] A sense strand was prepared with a 5'-aminohexyl linker for conjugation to a trivalent N-acetyl-galactosamine (GalNAc) moiety (structure illustrated in Chemical Formula VII below). After automated synthesis, the column was removed from the instrument and transferred to a vacuum manifold inside a fume hood. The 5'-monomethoxytrityl (MMT) protecting group was removed from the solid support by serial processing with a 2 mL aliquot of 1% trifluoroacetic acid (TFA) in dichloromethane (DCM) accompanied by vacuum filtration. When orange / yellow was no longer observed in the eluent, the resin was washed with dichloromethane. The resin was then washed with 5 mL of 2% diisopropylethylamine in N,N-dimethylformamide (DMF). A solution of GalNAc3-Lys2-Ahx (67 mg, 40 μmol) in DMF (0.5 mL) in a separate vial (its structure and synthesis are described below) was prepared with 1,1,3,3-tetramethyluronium tetrafluoroborate (TATU, 12.83 mg, 40 μmol) and diisopropylethylamine (DIEA) (10.5 μL, 360 μmol). The activated coupling solution was added to the resin, the column was capped, and incubated overnight at room temperature. The resin was washed with DMF and DCM and vacuum dried.

[0564] cutting

[0565] The synthesis columns were removed from the synthesizer or vacuum manifold. The solid support was transferred from each column to a 10 mL vial. 4 mL of concentrated ammonium hydroxide was added to the solid support. The caps were securely fastened to the bottles, and the mixture was heated at 55°C for 4 hours. The bottles were transferred to a freezer and cooled in a fume hood for 20 minutes before opening. The mixture was filtered through an 8 mL SPE tube to remove the solid support. The vials and solid support were rinsed with 1 mL of 50:50 ethanol / water.

[0566] Analysis and purification

[0567] A portion of the combined filtrate was analyzed and purified by anion exchange chromatography. The collected fraction was desalted by size exclusion chromatography and analyzed by reverse-phase high-performance liquid chromatography-mass spectrometry (HPLC-MS). The collected fraction was freeze-dried to obtain a white amorphous powder.

[0568] Analytical Anion Exchange Chromatography (AEX):

[0569] Column: Thermo DNAPac PA200RS (4.6 x 50 mm, 4 μm)

[0570] Instrument: Agilent 1100 HPLC

[0571] Buffer A: 20 mM sodium phosphate, 10% acetonitrile, pH 8.5

[0572] Buffer B: 20 ​​mM sodium phosphate, 10% acetonitrile, pH 8.5, 1 M sodium bromide

[0573] Flow rate: 1 mL / min at 40℃

[0574] Gradient: 20–65% B within 6.2 minutes

[0575] Preparative anion exchange chromatography (AEX):

[0576] Column: Tosoh TSK Gel SuperQ-5PW, 21 x 150 mm, 13 μm

[0577] Instrument: Agilent 1200 HPLC

[0578] Buffer A: 20 mM sodium phosphate, 10% acetonitrile, pH 8.5

[0579] Buffer B: 20 ​​mM sodium phosphate, 10% acetonitrile, pH 8.5, 1 M sodium bromide

[0580] Flow rate: 8 mL / min

[0581] Injection volume: 5 mL

[0582] Gradient: 35–55% B over 20 minutes

[0583] Preparative Size Exclusion Chromatography (SEC):

[0584] Column: GE Hi-Prep 26 / 10

[0585] Device: GE AKTA Pure

[0586] 20% ethanol in water buffer solution

[0587] Flow rate: 10 mL / min

[0588] Injection volume: 15 mL using a sample loading pump

[0589] Ion-pair reverse phase (IP-RP) HPLC:

[0590] Column: Water Xbridge BEH OST C18, 2.5 μm, 2.1 x 50 mm

[0591] Instrument: Agilent 1100 HPLC

[0592] Buffer A: 15.7 mM DIEA, 50 mM hexafluoroisopropanol (HFIP) in water

[0593] Buffer B: 15.7 mM DIEA, 50 mM HFIP in 50:50 water / acetonitrile

[0594] Flow rate: 0.5 mL / min

[0595] Gradient: 10–30% B over 6 minutes

[0596] annealing

[0597] Small amounts of sense and antisense strands were weighed into individual vials. SiRNA reconstitution buffer (Qiagen) or phosphate-buffered saline (PBS) was added to the vials to a concentration of approximately 2 mM by dry weight. The actual sample concentration was measured using a NanoDrop One (ssDNA, extinction coefficient = 33 μg / OD260). Subsequently, the two strands were mixed in an equimolar ratio, the sample was heated in a 90°C incubator for 5 minutes, and allowed to cool slowly to room temperature. The sample was analyzed by AEX. The dimer was registered and submitted for in vivo testing as described in more detail below.

[0598] Preparation of GalNAc3-Lys2-Ahx

[0599] Chemical Formula VII

[0600]

[0601] In the equation, X = O or S. The wavy line represents the attachment point to the 5' terminal nucleotide of the sense strand of the RNAi construct.

[0602] Fmoc-Ahx-OH (1.13 g, 3.19 mmol) in DCM (30 mL) was added to a 50 mL Falcon tube, and DIEA (2.23 mL, 12.78 mmol) was added. The solution was added to 2-Cl trityl chloride resin (3.03 g, 4.79 mmol) in a 50 mL centrifuge tube and loaded onto a shaker for 2 hours. The solvent was decanted, and the resin was washed with a 17:2:1 mixture of DCM / MeOH / DIEA (30 ml x 2) and DCM (30 ml x 4) and dried. The loading was confirmed to be 0.76 mmol / g by UV spectrophotometric detection at 290 nm.

[0603] 3 g of loaded 2-Cl trityl resin was suspended in 20% 4-methylpiperidine in DMF (20 mL), and the solvent was drained after 30 minutes. This process was repeated once more, and the resin was washed with DMF (30 mL x 3) and DCM (30 mL x 3).

[0604] TATU (1.94 g, 6 mmol) was added to a solution of Fmoc-Lys(ivDde)-OH (3.45 g, 6 mmol) in DMF (20 mL), followed by DIEA (1.83 mL, 10.5 mmol). Then, the solution was added to the deprotected resin, and the suspension was placed in a shaker overnight. The solvent was drained, and the resin was washed with DMF (30 mL x3) and DCM (30 mL x3).

[0605] The resin was treated with 20% 4-methylpiperidine in DMF (15 mL), and the solvent was drained after 10 minutes. This process was repeated once more, and the resin was washed with DMF (15 mL x 4) and DCM (15 mL x 4).

[0606] TATU (1.94 g, 6 mmol) was added to a solution of Fmoc-Lys(Fmoc)-OH (3.54 g, 6 mmol) in DMF (20 mL), followed by DIEA (1.83 mL, 10.5 mmol). Subsequently, the solution was added to the deprotected resin, and the suspension was placed in a shaker overnight. The solvent was drained, and the resin was washed with DMF (30 mL x 3) and DCM (30 mL x 3).

[0607] The resin was treated with 5% hydrazine in DMF (20 mL), and the solvent was drained after 5 minutes. This process was repeated 4 more times, and the resin was washed with DMF (30 mL x 4) and DCM (30 mL x 4).

[0608] TATU (3.22 g, 10 mmol) was added to a solution of 5-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanoic acid (4.47 g, 10 mmol) in DMF (40 mL), and the solution was stirred for 5 minutes. DIEA (2.96 mL, 17 mmol) was added to the solution, and then the mixture was added to the resin. The suspension was kept at room temperature overnight, and the solvent was drained. The resin was washed with DMF (3 x 30 mL) and DCM (3 x 30 mL).

[0609] The resin was treated with 1% TFA in DCM (30 mL, containing 3% triisopropylsilane), and the solvent was decanted after 5 minutes. This process was repeated 3 more times, and the combined filtrate was vacuum concentrated. The residue was triturated with diethyl ether (50 mL), and the suspension was filtered and dried to obtain an unpurified product. The unpurified product was purified by reverse-phase chromatography and eluted with 0–20% MeCN in water. The fractions were combined and freeze-dried to obtain a product as a white solid.

[0610] Table 1 below illustrates the modification positions in the sense and antisense sequences for each of the modified PNPLA3 RNAi constructs. Nucleotide sequences are listed according to the following notation: dT, dA, dG, dC = corresponding deoxyribonucleotides; a, u, g, and c = corresponding 2'-O-methylribonucleotides; Af, Uf, Gf, and Cf = corresponding 2'-deoxy-2'-fluoro("2'-fluoro") ribonucleotides; Phos = terminal nucleotide having a monophosphate group at its 5' end; invAb = inverted baseless nucleotide (i.e., a baseless nucleotide connected to an adjacent nucleotide via a substituent at the 3' position (3'-3' linkage) if at the 3' end of the strand, or connected to an adjacent nucleotide via a substituent at the 5' position (5'-5' linkage) if at the 5' end of the strand); and invdX = inverted deoxyribonucleotide (i.e., a deoxyribonucleotide connected to an adjacent nucleotide via a substituent at the 3' position (3'-3' linkage) if at the 3' end of the strand, or via a substituent at the 5' position (5'-5' nucleotide linkage) if at the 5' end of the strand). The insertion of "s" in the sequence indicates that two adjacent nucleotides are connected by a phosphothiodiester group (e.g., phosphothioate linkage). Unless otherwise indicated, all other nucleotides are connected by a 3'-5' phosphodiester group. All RNAi constructs were conjugated to the GalNAc moiety illustrated in Formula VII through the 5' end of the sense strand. Additionally, Table 1 lists the pattern names and sequence family names for each RNAi construct. The pattern names are schematically shown in Figure 1. If an RNAi construct has the same sequence family name as another RNAi construct, the two constructs have the same core sequence but different chemical modification patterns.

[0611] [Table 1]

[0612] Exemplary modified PNPLA3 RNAi construct

[0613]

[0614]

[0615] In the initial experimental set, 13 different PNPLA3 RNAi constructs with different sequences were synthesized to have either a P1 chemical modification pattern or a CM1 control chemical modification pattern. siRNA molecules with the CM1 control chemical modification pattern have been reported to have potent and long-term gene silencing effects in vivo. Nair et al. See , J. Am. Chem. Soc., Vol. 136:16958-16961, 2014. The efficacy of chemically modified RNAi constructs in repressing PNPLA3 gene expression was evaluated in humanized mouse models expressing wild-type human PNPLA3 or variant forms of the human PNPLA3 gene. To generate the mouse models, 1 Χ 10⁻⁶ per animal was prepared in phosphate-buffered saline (Thermo Fisher Scientific, 14190-136). 12 By intravenously injecting associated adenovirus (AAV; serotype AAV8 or AAV7; endotoxin-free) diluted with canine viral particles into the tail vein of C57BL / 6NCrl male mice (Charles River Laboratories Inc.), human PNPLA3 , PNPLA3 rs738409 , or PNPLA3 rs738409-rs738408 Gene expression was induced. Mice were generally 10 to 12 weeks old, and each treatment group included animals with n of 4 to 6.

[0616] All RNAi constructs AAV- PNPLA3 , PNPLA3 rs738409 , and / or PNPLA3 rs738409-rs738408 Tested in mice injected with. At least two vehicle-treated controls: AAV-empt vector and vehicle-treated AAV- PNPLA3 , PNPLA3 rs738409 , or PNPLA3 rs738409-rs738408 ...was also included. Two weeks after AAV injection, mice were treated with a single dose of RNAi construct (0.5 mM) via subcutaneous injection at 0.5, 1.0, 3.0, or 5.0 milligrams per kilogram of animal, diluted with phosphate-buffered saline (Thermo Fisher Scientific, 14190-136). On days 8, 15, 22, 28, or 42 after RNAi construct injection, livers were collected from animals, snap-frozen in liquid nitrogen, and processed for purified RNA using the Qiagen QIACube HT instrument (9001793) and the Qiagen RNeasy 96 QIACube HT kit (74171) according to the manufacturer's instructions. Samples were analyzed using the QIAxpert system (9002340). RNA was treated with Promega RQ1 RNase-Free DNase (M6101) and prepared for real-time qPCR using the Applied Biosystems TaqMan™ RNA-to-CT™ Step 1 Kit (4392653). Real-time qPCR was performed on a QuantStudio real-time PCR machine. The results were obtained for mice. Gapdh (TaqMan from Invitrogen, respectively) TM Humans normalized in analysis, hs00228747_m1 and 4352932E) PNPLA3 Based on the gene expression of, compared to vehicle-treated control animals, humans PNPLA3 It is presented as a relative knockdown of mRNA expression.

[0617] Results from this initial set of experiments comparing RNAi constructs with P1 chemical modification patterns (double numbers 4544, 3552, 2393, 3464, 3918, 2390, 2391, 2392, 3465, 3467, 2394, 3539, and 3916) with those with CM1 control modification patterns (double numbers 2118, 2119, 2125, 2120, 2121, 2124, 2370, 2371, 2122, 2368, 2369, 2123, and 3558) RNAi constructs human PNPLA3 rs738409 When administered subcutaneously at 5 mg / kg to mice expressing the variant gene, the P1 pattern compound generally reduced PNPLA3 expression to a greater degree than the CM1 pattern compound when measured 8 days after injection, regardless of sequence.

[0618] Variations of the P1 modification pattern were prepared to modify strand length, terminal properties of the RNAi construct (i.e., overhang versus smooth ends), and / or to include inverted base-free nucleotides at the 5' or 3' ends of the sense strand, and were applied to RNAi constructs having the same core sequence. The improvement in in vivo efficacy of RNAi constructs with the new patterns was evaluated in humanized mouse models. Specifically, RNAi constructs with P1, P2, P3, or P4 chemical modification patterns (double numbers 3540, 5241, 5614, and 5615) were administered to humans at a dose of 5 mg / kg. PNPLA3 rs738409 The variant gene was administered subcutaneously to mice expressing the variant gene. The expression level of human PNPLA3 in the liver was evaluated 15 days after administration of the RNAi construct. The results are shown in Figure 3. RNAi constructs with P2, P3, or P4 patterns resulted in a greater mean decrease in PNPLA3 expression than RNAi constructs with P1 pattern.

[0619] Additional modifications of the P3 pattern were prepared to increase the efficacy and duration of mRNA knockdown in vivo. A P9 pattern was generated by replacing the 2′-fluoro-modified nucleotides at positions 4 and 6 of the antisense strand, calculated from the 5' end of the P3 pattern (Duplicate No. 6191), with 2′-O-methyl-modified nucleotides (Duplicate No. 6267). Additionally, an RNAi construct (Duplicate No. 7320) with a P9 pattern was synthesized, featuring an inverted adenosine deoxyribonucleotide instead of an inverted base-free nucleotide at the 3' end of the sense strand. All three constructs were evaluated in the aforementioned humanized mouse model. In animals treated with 5 mg / kg of dichromator number 6267, human PNPLA3 liver expression decreased by 97% on day 22 after administration, and in animals treated with 5 mg / kg of dichromator number 6191, human PNPLA3 liver expression decreased by 92% at the same time point. In animals treated with 3 mg / kg of dichromator number 7320, human PNPLA3 liver expression levels decreased by 95% on day 28 after administration, so dichromator number 7320 was more potent than dichromator numbers 6191 and 6267 and resulted in gene knockdown with a longer duration.

[0620] The P9 pattern was applied to PNPLA3 RNAi constructs (double numbers 7318, 7320, 7062, 8513, and 8709) having two different core sequences, and in vivo efficacy was evaluated in bioluminescent imaging assays at doses of 1 mg / kg and 3 mg / kg. For bioluminescent imaging assays, the relevant adenovirus (AAV) vector was designed to contain a murine cytomegalovirus promoter, the full sequence for firefly luciferase, and then, immediately downstream from the firefly luciferase stop codon, a synthesized string of mRNA sequences specific to the RNAi construct to be tested. Ten additional nucleotides were positioned at each end of the mRNA sequence. The vector "PP3A(DM)" was packaged with the AAV serotype AAVDJ8 (endotoxin-free). Before injection, PP3A(DM) was added to 5 x 10⁶ per animal in phosphate-buffered saline (Thermo Fisher Scientific, 14190-136). 11 The virus was diluted with can of virus particles and injected intravenously into the tail veins of BALB / c male mice (Charles River Laboratories Inc.). Mice were generally 10 to 12 weeks old, and n=5 animals were included per group.

[0621] Two weeks after AAV injection, RediJect D-luciferin bioluminescent substrate (PerkinElmer, 770504) was injected into mice according to the manufacturer's instructions. After a 10-minute pulse, mice were imaged on an IVIS spectrum in vivo imaging system (PerkinElmer). Mice were randomized into groups based on baseline total flux scores from a defined region of interest including the liver. After randomization, mice were treated with a single dose of RNAi construct (0.5 mM) via subcutaneous injection at 1.0 or 3.0 mg per kilogram of body weight, diluted with phosphate-buffered saline (Thermo Fisher Scientific, 14190-136), or with phosphate-buffered saline alone (indicated as "vehicle"). Mice were imaged weekly according to the same protocol with the same gating constraints applied to the total flux scores. Data are plotted as total flux (photons per second, y-axis) versus week (x-axis) after RNAi construct injection. A decrease in total flux indicates reduced expression of the luciferase reporter.

[0622] The results of this experiment are presented in Figures 4a and 4b. Signals from luciferase reporters in animals treated with different RNAi constructs having a P9 pattern were significantly reduced compared to signals from vehicle-treated animals for at least 3 weeks after a single dose of 1 mg / kg (Figure 4a) and for at least 5 weeks in the case of a single dose of 3 mg / kg RNAi construct (Figure 4b). For many RNAi constructs, a single dose of 3 mg / kg was sufficient to suppress luciferase reporter expression for up to 6 weeks.

[0623] These RNAi constructs (double numbers 7318, 7320, 7062, 8513, and 8709) were also evaluated in the aforementioned humanized mouse models. Specifically, the RNAi constructs were humanized at 0.5, 1, or 3 mg / kg PNPLA3 rs738409-rs738408 The variant gene was administered subcutaneously to mice expressing the variant. The expression levels of human PNPLA3 in the liver were evaluated by qPCR at 28 or 42 days after administration of the RNAi construct. The results compared human PNPLA3 with vehicle-treated control animals. PNPLA3 It is presented as a relative knockdown of mRNA expression and is shown in Table 2 below.

[0624] [Table 2]

[0625] In vivo efficacy of PNPLA3 RNAi construct

[0626]

[0627] RNAi constructs with the P9 variant pattern result in more potent and longer-lasting gene knockdown than previously tested patterns. When the RNAi construct was administered as a single dose of 0.5 mg / kg, human PNPLA3 liver expression decreased by approximately 50% 4 weeks after the single dose, whereas when the construct was administered as a single dose of 1 mg / kg, human PNPLA3 liver expression decreased by approximately 70% 4 weeks after the single dose. The 1 mg / kg dose was sufficient to maintain a reduction of more than 55% in PNPLA3 expression up to 6 weeks after the single dose. Administration of a single dose of 3 mg / kg of the RNAi construct reduced human PNPLA3 liver expression by more than 90% 4 weeks after the single dose. Human PNPLA3 liver expression was still reduced by more than approximately 75% 6 weeks after the administration of the 3 mg / kg dose. Improved efficacy and duration of gene knockdown were observed with RNAi constructs having two distinct sequences, showing that the P9 chemical modification pattern is effective in stabilizing RNAi constructs that are at least partially independent of the nucleobase sequence.

[0628] Next, the in vivo efficacy of PNPLA3 RNAi constructs with the P9 chemical modification pattern was compared with PNPLA3 RNAi constructs with one of three different control modification patterns. The CM2, CM3, and CM4 modification patterns have previously been reported to improve the efficacy and duration of gene silencing by increasing the metabolic stability of siRNA molecules. Foster et al See ., Molecular Therapy, Vol. 26: 708-717, 2018. All RNAi constructs had the same core nucleotide sequence in the sense and antisense strands, differing only in their chemical modification patterns. Two different constructs with the P9 modification pattern were synthesized. One had an inverted base-free group at the 3' end of the sense strand (Duplicate No. 7318), and the other had an inverted deoxythymidine at the 3' end of the sense strand (Duplicate No. 8709). RNAi constructs with one of the CM2, CM3, or CM4 modification patterns were also synthesized (Duplicate Nos. 8103, 8104, and 8105, respectively). Subsequently, each RNAi construct was humanized at a dose of 3 mg / kg PNPLA3 rs738409-rs738408 The variant gene was administered subcutaneously to mice expressing the variant gene. The expression level of human PNPLA3 in the liver was evaluated by qPCR 28 days after administration of the RNAi constructs. The results are shown in Figure 5. The RNAi construct with a P9 variant pattern containing an inverted base-free group at the 3' end of the sense strand (double number 7318) resulted in the greatest decrease in liver PNPLA3 expression among all constructs tested. The RNAi construct with a P9 variant pattern containing an inverted deoxythymidine at the 3' end of the sense strand (double number 8709) reduced liver PNPLA3 expression more significantly than the construct with the CM4 pattern (double number 8105) and showed a similar decrease in liver PNPLA3 expression as the constructs with the CM2 and CM3 patterns (double numbers 8103 and 8104, respectively).

[0629] In a separate experimental set, alternative modifications of the P3 modification pattern were designed, and their in vivo efficacy was evaluated in a humanized PNPLA3 mouse model. Modifications of the P3 pattern were applied to RNAi constructs with two different sequences. The sequences of the sense and antisense strands for each RNAi construct are presented in Table 1, and the modification patterns are schematically illustrated in Figure 1. RNAi constructs were humanized at a dose of 3 mg / kg PNPLA3 rs738409-rs738408 The RNAi constructs were administered subcutaneously to mice expressing the variant gene. The expression levels of human PNPLA3 in the liver were evaluated by qPCR 28 days after administration of the RNAi constructs. The results are shown in Table 3 below. All RNAi constructs reduced liver expression of human PNPLA3 by more than 90% 4 weeks after a single subcutaneous injection of 3 mg / kg.

[0630] [Table 3]

[0631] In vivo efficacy of PNPLA3 RNAi constructs with alternative chemical modification patterns

[0632]

[0633] Example 2. In vivo activity of ASGR1 RNAi constructs with different chemical modification patterns

[0634] As shown in Example 1, human PNPLA3The P1 chemical modification pattern applied to 13 different RNAi constructs having different sequences targeting mRNA improved the gene silencing efficacy of the constructs. To investigate whether the P1 chemical modification pattern enhances the efficacy of RNAi constructs targeting other liver genes, RNAi constructs with the P1 chemical modification pattern targeting asarloglycoprotein receptor 1 (ASGR1) mRNA were synthesized according to the method described in Example 1. RNAi constructs having the same sequence were synthesized using the CM1 control chemical modification pattern. The sequences of the RNAi constructs are provided in Table 4 below using the same notation described above for Table 1. A GalNAc moiety having the structure presented in Formula VII was conjugated to the 5' end of the sense strand of the RNAi construct designated as duplex number 1520, and a GalNAc moiety having the structure presented in Formula IX was conjugated to the 5' end of the sense strand of the RNAi construct designated as duplex number 1421. The conjugation of a GalNAc moiety to the sense strand of an RNAi construct was performed as described in Example 1, provided, however, that in the case of a GalNAc moiety having the structure presented in Formula IX, the GalNAc moiety was prepared as follows. TATU (3.22 g, 10 mmol) was added to a solution of 2-(2-(2-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)ethoxy)ethoxy)acetic acid (5.37 g, 10 mmol) in DMF (40 mL), and the solution was stirred for 5 minutes. DIEA (2.96 mL, 17 mmol) was added to the solution, and the mixture was then added to the resin described in Example 1 above. The suspension was kept at room temperature overnight, and the solvent was drained. The resin was washed with DMF (3 x 30 mL) and DCM (3 x 30 mL).

[0635] [Table 4]

[0636] Exemplary modified ASGR1 RNAi construct

[0637]

[0638] The in vivo efficacy of RNAi constructs in inhibiting liver mouse ASGR1 expression was evaluated by administering the RNAi constructs to C57BL / 6J mice. Wild-type C57BL / 6 animals (Charles River) aged 10 to 12 weeks were fed standard feed (2020X Teklad global soy protein-free extruded rodent feed; Harlan). On Day 0, mice received a subcutaneous injection of either the buffer or the labeled RNAi construct at a concentration of 5 mg / kg body weight in 0.25 ml of buffer (n = 9 per group). For further analysis, three animals were harvested on Day 4, three on Day 8, and three on Day 15. Total RNA from the livers of the harvested animals was processed for qPCR analysis. The efficacy of the RNAi constructs was evaluated by comparing the amount of Asgr1 mRNA in the liver tissue of animals treated with the RNAi constructs to the amount of Asgr1 mRNA in the liver tissue of animals injected with the buffer. The results show that animals administered an RNAi construct with a P1 modification pattern (double number 1520) exhibited a greater reduction in liver ASGR1 expression than animals administered an RNAi construct with a CM1 control modification pattern at all measured time points (Fig. 6). Similar to the results described in Example 1 using an RNAi construct targeting human PNPLA3 mRNA, the P1 chemical modification pattern improves the efficacy of the RNAi construct.

[0639] Example 3. In vivo activity of LPA RNAi constructs with different chemical modification patterns

[0640] To further evaluate the ability of RNAi constructs of the chemical modification patterns described herein to improve in vivo efficacy, a third liver gene LPARNAi constructs targeting a gene were synthesized and conjugated to a GalNAc moiety having the structure presented in Formula VII according to the method described in Example 1. The sequences of the RNAi constructs are provided in Table 5 below using the same notation described above for Table 1. Additionally, Table 5 lists the pattern names and sequence family names for each RNAi construct. The pattern names are schematically indicated in Figure 1. If an RNAi construct has the same sequence family name as another RNAi construct, the two constructs have the same core sequence but different chemical modification patterns.

[0641] [Table 5]

[0642] Exemplary modified LPA RNAi construct

[0643]

[0644] In initial experiments, RNAi constructs with identical nucleotide sequences were synthesized with either a CM1 control chemical modification pattern (double number 3632) or a P1 chemical modification pattern (double number 3635). The in vivo efficacy of the two constructs was evaluated in a double transgenic mouse model expressing fully functional human Lp(a) particles at average serum baseline Lp(a) levels of approximately 50–60 mg / dL. Lp(a) is a low-density lipoprotein composed of an LDL particle and a glycoprotein apolipoprotein (a) (apo(a)) linked to the apolipoprotein B of the LDL particle by disulfide bonds. Apo(a) LPA It is encoded by genes, and changes in serum Lp(a) levels LPA It reflects changes in gene expression. The whole human LPA Transgenic mouse (Frazer) expressing human apo(a) from a yeast artificial chromosome (YAC) containing a gene et al ., Nature Genetics, Vol. 9: 424-431, 1995) transgenic mice expressing human apoB-100 (Linton et al Double-transgenic mice were generated by crossing with (J. Clin. Invest., Vol. 92: 3029-3037, 1993). The LPA RNAi construct was administered as a single subcutaneous injection at a dose of 0.5 mg / kg. Serum samples were taken prior to injection, and then taken after injection on days 14 and 28. Lp(a) concentrations were measured in serum using an Lp(a) ELISA assay (Cat.# 10-1106-01, Mercodia AB, Uppsala, Sweden). The percentage change in Lp(a) levels for each animal at specific time points was calculated based on the animal's baseline Lp(a) level. The results are shown in Figure 7. Two weeks after injection, although not statistically significant, administration of duplex number 3635 with the P1 variant pattern resulted in a greater mean decrease (-49%) in serum Lp(a) levels compared to the control duplex number 3632 (-32%) with the CM1 variant pattern.

[0645] In a second series of experiments, LPA RNAi constructs having a P1 chemical modification pattern or a modification of that pattern were synthesized, targeting a region of LPA mRNA distinct from those of the first set of experiments. RNAi constructs with the new pattern were tested in vivo LPAImprovements in both the magnitude and duration of gene expression repression were evaluated in a double-transformed mouse model. Specifically, LPA RNAi constructs from three different sequence families containing one of the P1 variant pattern or pattern variants (e.g., P2, P4, P6, or P7 chemical modification patterns) were administered subcutaneously to the aforementioned double-transformed mice at a dose of 2 mg / kg. Serum Lp(a) levels were measured in the animals prior to injection and at weeks 1, 2, and 4 after administration of the LPA RNAi constructs to obtain baseline levels. The results of this experimental set are presented in Table 6 below. Across the three sequence families, RNAi constructs with P2, P4, P6, or P7 variant patterns resulted in a greater reduction and longer duration of repression of serum Lp(a) levels compared to RNAi constructs with the P1 variant pattern. RNAi constructs with P6 or P7 chemical modification patterns reduced serum Lp(a) levels by more than 80% for up to 4 weeks after a single subcutaneous injection of 2 mg / kg.

[0646] [Table 6]

[0647] In vivo efficacy of LPA RNAi constructs

[0648]

[0649] Next, alternative modifications to the chemical modification patterns were designed, and their in vivo efficacy was evaluated in a double-transgenic mouse model. Modifications to the chemical modification patterns were applied to RNAi constructs containing sequences from five different sequence families. The sequences of the sense and antisense strands for each RNAi construct are provided in Table 5, and the modification patterns are schematically illustrated in Figure 1. The RNAi constructs were administered subcutaneously to double-transgenic mice expressing human Lp(a) particles at a dose of 1 mg / kg. Serum Lp(a) levels in the animals were measured before injection and at weeks 2, 3, and 4 after administration of the LPA RNAi constructs to obtain baseline levels. The results are shown in Table 7 below. Several pattern modifications, such as P9, P19, P22, P24, P27, P28, and P29, reduced serum Lp(a) levels by more than 50% after 4 weeks of a single subcutaneous injection of 1 mg / kg. RNAi constructs with a P27 chemical modification pattern were particularly effective in suppressing Lp(a) serum levels, as they resulted in a sustained decrease of about 75% in Lp(a) levels 4 weeks after a single injection.

[0650] [Table 7]

[0651] In vivo efficacy of LPA RNAi constructs with alternative chemical modification patterns

[0652]

[0653] All publications, patents, and patent applications discussed and cited herein are incorporated herein by reference in their entirety. It is understood that the disclosed invention is not limited to the specific methods, protocols, and materials described, as such may vary. Furthermore, it is understood that the terms used herein are for the purpose of describing specific embodiments only and are not intended to limit the scope of the appended claims.

[0654] Those skilled in the art will recognize or be able to identify many equivalents to the specific embodiments of the invention described herein through ordinary experiment alone. Such equivalents are encompassed by the following claims.

Claims

Claim 1 An RNAi construct for repressing the expression of a target gene sequence, comprising a sense strand and an antisense strand, wherein the antisense strand comprises a sequence complementary to the target gene sequence and the sense strand comprises a sequence sufficiently complementary to the sequence of the antisense strand to form a dimer region, and the RNAi construct comprises a structure represented by formula (A). RNAi construct: 5'-(N A ) x N L N L N L N L N L N L N F N L N F N F N F N F N L N L N M N L N M N L N T (n) y -3' 3'-(N B ) z N L N L N L N L N L N F N L N M N L N M N L N L N F N M N L N M N L N F N L -5' (A) Here, the upper strand listed in the direction from 5' to 3' is the sense strand and the lower strand listed in the direction from 3' to 5' is the antisense strand; each N F is a 2'-fluoro-modified nucleotide; and each N M represents a modified nucleotide selected from 2'-fluoro-modified nucleotides and 2'-O-methyl-modified nucleotides independently; each N L represents a 2'-O-methyl modified nucleotide;N T represents a modified nucleotide selected from baseless nucleotides, inverted baseless nucleotides, inverted deoxyribonucleotides, 2'-O-methyl modified nucleotides, and deoxyribonucleotides; x is an integer from 0 to 4, provided that if x is 1, 2, 3, or 4, each N A The nucleotide is a modified nucleotide independently selected from baseless nucleotides, inverted baseless nucleotides, inverted deoxyribonucleotides, 2'-O-methyl modified nucleotides, and deoxyribonucleotides, and N A At least one of the nucleotides may be complementary to a nucleotide of the antisense strand; y is an integer from 0 to 4, provided that if y is 1, 2, 3, or 4, n nucleotides are modified or unmodified overhang nucleotides that do not independently form a base pair with a nucleotide of the antisense strand; z is an integer from 0 to 4, provided that if z is 1, 2, 3, or 4, each N B The nucleotide is a modified nucleotide independently selected from 2'-O-methyl modified nucleotides and deoxyribonucleotides, and N B When one or more nucleotides are present in the sense strand, N A It may be complementary to the nucleotide, or it may be an overhang nucleotide that does not form a base pair with a nucleotide of the sense strand. Claim 2 The RNAi construct according to claim 1, wherein the sense strand and the antisense strand are each independently 19 to 23 nucleotide long. Claim 3 In paragraph 1, (i) x is 0, y is 2, and z is 2, or (ii) x is 1 and N A is an inverted baseless nucleotide, where y is 2 and z is 2, or (iii) x is 2, y is 0, and z is 4, or (iv) x is 2, y is 0, and z is 2, or (v) x is 3 and the 5' end N A is an inverted baseless nucleotide, y is 0 and z is 4, or (vi) x is 0, y is 0 and z is 2, or (vii) x is 1 and N A RNAi construct in which is an inverted base-free nucleotide, y is 0, and z is 2. Claim 4 In paragraph 1, x is 2, and each N A is a 2'-O-methyl modified nucleotide, y is 0, z is 4, and each N B RNAi constructs, the nucleotide is a 2'-O-methyl modified nucleotide. Claim 5 In paragraph 1, N T RNAi constructs that are inverted baseless nucleotides, inverted deoxyribonucleotides, or 2'-O-methyl modified nucleotides. Claim 6 In claim 1, (i) N at positions 4 and 12 of the antisense strand calculated from the 5' end M are each 2'-fluoro-modified nucleotides; (ii) N at positions 4, 6, and 12 of the antisense strand calculated from the 5' end. M are each 2'-fluoro-modified nucleotides; (iii) N at positions 4, 6, 10, and 12 of the antisense strand calculated from the 5' end. M are each 2'-fluoro-modified nucleotides; (iv) N at positions 10 and 12 of the antisense strand calculated from the 5' end. M are each 2'-fluoro-modified nucleotides; or (v) N at positions 4, 10, and 12 of the antisense strand counted from the 5' end. M RNAi constructs, each being a 2'-fluoro-modified nucleotide. Claim 7 In claim 1, N at positions 4, 6, and 10 of the antisense strand calculated from the 5' end. M are each 2'-O-methyl modified nucleotides, and N at position 12 of the antisense strand calculated from the 5' end. M RNAi constructs, which are 2'-fluoro-modified nucleotides. Claim 8 In paragraph 7, an RNAi construct in which x is 2, y is 0, and z is 4. Claim 9 In Paragraph 8, each N A is a 2'-O-methyl modified nucleotide; and each N of the sense strand M is a 2'-O-methyl modified nucleotide;N T is an inverted base-free nucleotide; and each N B RNAi constructs that are 2'-O-methyl modified nucleotides. Claim 10 In paragraph 1, each N in both the sense and antisense strands M RNAi constructs that are 2'-O-methyl modified nucleotides. Claim 11 In paragraph 1, each N of the sense strand M RNAi constructs that are 2'-O-methyl modified nucleotides. Claim 12 In paragraph 1, each N of the sense strand M RNAi constructs, which are 2'-fluoro-modified nucleotides. Claim 13 An RNAi construct for inhibiting the expression of a target gene sequence, comprising a sense strand and an antisense strand, wherein the antisense strand comprises a sequence complementary to the target gene sequence and the sense strand comprises a sequence sufficiently complementary to the antisense strand to form a dimer region, and the RNAi construct comprises a structure represented by formula (B), RNAi construct:5'-(N A ) x N L N L N L N L N L N L N F N L N F N F N F N F N L N L N L N L N L N L N T (n) y -3' 3'-(N B ) z N L N L N L N L N L N F N L N F N L N L N L N L N F N F N L N F N L N F N L -5' (B) Here, the upper strand listed in the direction from 5' to 3' is the sense strand and the lower strand listed in the direction from 3' to 5' is the antisense strand; each N F is a 2'-fluoro-modified nucleotide; and each N L represents a 2'-O-methyl modified nucleotide;N T represents a modified nucleotide selected from baseless nucleotides, inverted baseless nucleotides, inverted deoxyribonucleotides, 2'-O-methyl modified nucleotides, and deoxyribonucleotides; x is an integer from 0 to 4, provided that if x is 1, 2, 3, or 4, each N A The nucleotide is a modified nucleotide independently selected from baseless nucleotides, inverted baseless nucleotides, inverted deoxyribonucleotides, 2'-O-methyl modified nucleotides, and deoxyribonucleotides, and N A At least one of the nucleotides may be complementary to a nucleotide of the antisense strand; y is an integer from 0 to 4, provided that if y is 1, 2, 3, or 4, n nucleotides are modified or unmodified overhang nucleotides that do not independently form a base pair with a nucleotide of the antisense strand; z is an integer from 0 to 4, provided that if z is 1, 2, 3, or 4, each N B The nucleotide is a modified nucleotide independently selected from 2'-O-methyl modified nucleotides and deoxyribonucleotides, and N B When one or more nucleotides are present in the sense strand, N A It may be complementary to the nucleotide, or it may be an overhang nucleotide that does not form a base pair with a nucleotide of the sense strand. Claim 14 In paragraph 13, (i) x is 0, y is 2, and z is 2; (ii) x is 0, y is 0, and z is 2; or (iii) x is 1 and N A is an inverted baseless nucleotide, where y is 2 and z is 2; (iv) x is 2, y is 0, and z is 4; or (v) x is 3 and the 5' end N A RNAi construct in which is an inverted base-free nucleotide, y is 0, and z is 4. Claim 15 In Paragraph 13, x is 2, and each N A The nucleotide is a 2'-O-methyl modified nucleotide, y is 0, z is 4, and each N B RNAi constructs, the nucleotide is a 2'-O-methyl modified nucleotide. Claim 16 In Paragraph 13, N T RNAi constructs that are inverted baseless nucleotides, inverted deoxyribonucleotides, or 2'-O-methyl modified nucleotides. Claim 17 RNAi construct for inhibiting the expression of a target gene sequence, comprising a sense strand and an antisense strand, wherein the antisense strand comprises a sequence complementary to the target gene sequence and the sense strand comprises a sequence sufficiently complementary to the sequence of the antisense strand to form a dimer region, and the RNAi construct comprises a structure represented by formula (C), RNAi construct:5'-(Ab) x N L N L N L N L N L N L N L N L N F N L N F N F N F N F N L N L N M N L N M N L N T -3' 3'-N L N L N L N L N L N L N L N L N L N F N L N F N L N L N L N L N F N L N L N M N L N F N L -5' (C) Here, the upper strand listed in the direction from 5' to 3' is the sense strand and the lower strand listed in the direction from 3' to 5' is the antisense strand; each N F is a 2'-fluoro-modified nucleotide; and each N L represents a 2'-O-methyl modified nucleotide; each N M represents a modified nucleotide selected from 2'-fluoro-modified nucleotides and 2'-O-methyl-modified nucleotides, and N T represents a modified nucleotide selected from baseless nucleotide, inverted baseless nucleotide, inverted deoxyribonucleotide, 2'-O-methyl modified nucleotide, and deoxyribonucleotide; x is 0 or 1, and Ab is an inverted baseless nucleotide. Claim 18 In paragraph 17, each N in both the sense and antisense strands M RNAi constructs that are 2'-O-methyl modified nucleotides. Claim 19 In Paragraph 18, N T RNAi constructs where is an inverted baseless nucleotide or an inverted deoxyribonucleotide and x is 0. Claim 20 In Paragraph 18, N T is a 2'-O-methyl modified nucleotide and is an RNAi construct where x is 1. Claim 21 In paragraph 17, N of the antisense strand M RNAi constructs, which are 2'-fluoro-modified nucleotides. Claim 22 In paragraph 21, each N of the sense strand M RNAi constructs that are 2'-O-methyl modified nucleotides. Claim 23 In paragraph 21, each N of the sense strand M RNAi constructs, which are 2'-fluoro-modified nucleotides. Claim 24 In Paragraph 21, N T RNAi constructs where is an inverted baseless nucleotide or an inverted deoxyribonucleotide and x is 0. Claim 25 An RNAi construct for inhibiting the expression of a target gene sequence, comprising a sense strand and an antisense strand, wherein the antisense strand comprises a sequence complementary to the target gene sequence and the sense strand comprises a sequence sufficiently complementary to the sequence of the antisense strand to form a dimer region, and the RNAi construct comprises a structure represented by formula (D), RNAi construct:5'-(N A ) x N L N L N L N L N M N L N F N F N F N F N L N L N L N L N L N L N L N L N T (n) y -3' 3'-(N B ) z N L N L N L N M N L N F N L N M N L N L N M N M N M N M N L N M N L N F N L -5' (D) Here, the upper strand listed in the direction from 5' to 3' is the sense strand and the lower strand listed in the direction from 3' to 5' is the antisense strand; each N F is a 2'-fluoro-modified nucleotide; and each N M represents a modified nucleotide selected from 2'-fluoro-modified nucleotides and 2'-O-methyl-modified nucleotides independently; each N L represents a 2'-O-methyl modified nucleotide;N T represents a modified nucleotide selected from baseless nucleotides, inverted baseless nucleotides, inverted deoxyribonucleotides, 2'-O-methyl modified nucleotides, and deoxyribonucleotides; x is an integer from 0 to 4, provided that if x is 1, 2, 3, or 4, each N A The nucleotide is a modified nucleotide independently selected from baseless nucleotides, inverted baseless nucleotides, inverted deoxyribonucleotides, 2'-O-methyl modified nucleotides, and deoxyribonucleotides, and N A At least one of the nucleotides may be complementary to a nucleotide of the antisense strand; y is an integer from 0 to 4, provided that if y is 1, 2, 3, or 4, n nucleotides are modified or unmodified overhang nucleotides that do not independently form a base pair with a nucleotide of the antisense strand; z is an integer from 0 to 4, provided that if z is 1, 2, 3, or 4, each N B The nucleotide is a modified nucleotide independently selected from 2'-O-methyl modified nucleotides and deoxyribonucleotides, and N B When one or more nucleotides are present in the sense strand, N A It may be complementary to the nucleotide, or it may be an overhang nucleotide that does not form a base pair with a nucleotide of the sense strand. Claim 26 In paragraph 25, the RNAi construct, wherein the sense strand and the antisense strand are each independently 19 to 23 nucleotide long. Claim 27 In paragraph 25, (i) x is 2, y is 0, and z is 4, or; (ii) x is 1 and N A is an inverted base-free nucleotide, y is 2, and z is 2 or; (iii) x is 1, and N A is an inverted base-free nucleotide, where y is 0 and z is 2; (iv) x is 0, y is 0 and z is 2; or (v) an RNAi construct where x is 2, y is 0 and z is 2. Claim 28 In paragraph 25, x is 2, and each N A The nucleotide is a 2'-O-methyl modified nucleotide, y is 0, z is 4, and each N B RNAi constructs, the nucleotide is a 2'-O-methyl modified nucleotide. Claim 29 In paragraph 28, N of the sense strand M is a 2'-fluoro-modified nucleotide;N T is an inverted baseless nucleotide; and N at positions 4, 6, 8, 9, and 12 of the antisense strand counted from the 5' end. M are 2'-O-methyl modified nucleotides, respectively, and N at positions 7 and 16 of the antisense strand calculated from the 5' end. M RNAi constructs, each being a 2'-fluoro-modified nucleotide. Claim 30 In paragraph 25, N T RNAi constructs that are inverted baseless nucleotides, inverted deoxyribonucleotides, or 2'-O-methyl modified nucleotides. Claim 31 In paragraph 25, N at positions 4, 6, 8, 9, and 16 of the antisense strand calculated from the 5' end. M are 2'-fluoro-modified nucleotides, respectively, and N at positions 7 and 12 of the antisense strand calculated from the 5' end. M RNAi constructs, each being a 2'-O-methyl modified nucleotide. Claim 32 In paragraph 25, N at positions 4, 6, 8, 9, and 16 of the antisense strand calculated from the 5' end. M are 2'-O-methyl modified nucleotides, respectively, and N at positions 7 and 12 of the antisense strand calculated from the 5' end. M RNAi constructs, each being a 2'-fluoro-modified nucleotide. Claim 33 In paragraph 25, N at positions 4, 6, 8, 9, and 12 of the antisense strand calculated from the 5' end. M are 2'-O-methyl modified nucleotides, respectively, and N at positions 7 and 16 of the antisense strand calculated from the 5' end. M RNAi constructs, each being a 2'-fluoro-modified nucleotide. Claim 34 In paragraph 25, N at positions 7, 8, 9, and 12 of the antisense strand calculated from the 5' end. M are 2'-O-methyl modified nucleotides, respectively, and N at positions 4, 6, and 16 of the antisense strand calculated from the 5' end. M RNAi constructs, each being a 2'-fluoro-modified nucleotide. Claim 35 In paragraph 25, the N of the sense strand M RNAi constructs, which are 2'-fluoro-modified nucleotides. Claim 36 In paragraph 25, the N of the sense strand M RNAi constructs that are 2'-O-methyl modified nucleotides. Claim 37 An RNAi construct according to any one of claims 1 to 36, wherein the sense strand, the antisense strand, or both the sense and antisense strands comprise a linkage between one or more phosphorothioate nucleotides. Claim 38 In paragraph 37, the RNAi construct comprises an antisense strand containing a link between two consecutive phosphorothioate nucleotides between the terminal nucleotides of both the 3' and 5' ends. Claim 39 In paragraph 37, the RNAi construct comprises a sense strand containing a single phosphorothioate internucleotide linkage between the terminal nucleotides of the 3' end. Claim 40 In paragraph 37, the RNAi construct comprises a sense strand containing a link between two consecutive phosphorothioate nucleotides between the terminal nucleotides of the 3' end. Claim 41 An RNAi construct comprising, in any one of claims 1 to 36, an additional ligand. Claim 42 In paragraph 41, the ligand is an RNAi construct comprising a cholesterol moiety, a vitamin, a steroid, a bile acid, a folate moiety, a fatty acid, a carbohydrate, a glycoside, or an antibody or an antigen-binding fragment thereof. Claim 43 In paragraph 41, the ligand is an RNAi construct comprising galactose, galactosamine, or N-acetyl-galactosamine. Claim 44 In paragraph 43, the ligand comprises a polyvalent galactose moiety or a polyvalent N-acetyl-galactosamine moiety, an RNAi construct. Claim 45 In paragraph 44, the RNAi construct, wherein the polyvalent galactose moiety or polyvalent N-acetyl-galactosamine moiety is trivalent or tetravalent. Claim 46 In paragraph 41, the RNAi construct is selectively covalently attached to the sense strand via a linker. Claim 47 In paragraph 46, the ligand is a RNAi construct covalently attached to the 5' end of the sense strand. Claim 48 A composition comprising an RNAi construct or a salt thereof according to any one of claims 1, 13, 17, and 25, and a carrier or excipient. Claim 49 In vitro (which inhibits the expression of a target gene in a cell, comprising the step of contacting a cell with an RNAi construct of any one of claims 1 to 36) in vitro ) method. Claim 50 delete Claim 51 delete Claim 52 delete Claim 53 delete Claim 54 delete Claim 55 delete Claim 56 delete Claim 57 delete Claim 58 delete Claim 59 delete Claim 60 delete Claim 61 delete Claim 62 delete Claim 63 delete Claim 64 delete Claim 65 delete Claim 66 delete Claim 67 delete Claim 68 delete Claim 69 delete Claim 70 delete Claim 71 delete

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